MAY- JUNE- --2017 Scripts

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Clinical toxicities of nanocarrier systems Nano-silicon dioxide toxicological characterization on two human kidney cell lines Dangers of SiO2 nanoparticles Synthetic Biology--Toward Therapeutic Solutions Synthetic membranes created to mimic properties of living cells Mechanisms of Nanoparticle-Induced Oxidative Stress and Toxicity Sweet marjoram (Origanum majorana, Majorana hortensis) Chlorogenic Compounds from Coffee Beans Exert Activity against Respiratory Viruses

Brain blood vessel lesions tied to intestinal bacteria Gut microbes linked to brain structure in people with irritable bowel syndrome New Transmissible Vaccine Spreads Like Virus, No Consent Necessary Vitamin A deficiency is detrimental to blood stem cells Study describes how bacteria can remodel gene expression to infect intestines of host Nanocages dramatically facilitate structure formation of biomolecules Drinking coffee could lead to a longer life Coffee Drinking and Mortality in 10 European Countries-

Clinical toxicities of nanocarrier systems

• Karina R. Vega-Villa^a,
• Jody K. Takemoto^a,
• Jaime A. Yáñez^a,
• Connie M. Remsberg^{a, 1},
• M. Laird Forrest^b,
• Neal M. Davies^a,

 

Abstract

Toxicity of nanocarrier systems involves physiological, physicochemical, and molecular considerations. Nanoparticle exposures through the skin, the respiratory tract, the gastrointestinal tract and the lymphatics have been described. Nanocarrier systems may induce cytotoxicity and/or genotoxicity, whereas their antigenicity is still not well understood. Nanocarrier may alter the physicochemical properties of xenobiotics resulting in pharmaceutical changes in stability, solubility, and pharmacokinetic disposition. In particular, nanocarriers may reduce toxicity of hydrophobic cancer drugs that are solubilized. Nano regulation is still undergoing major changes to encompass environmental, health, and safety issues. The rapid commercialization of nanotechnology requires thoughtful environmental, health and safety research, meaningful, and an open discussion of broader societal impacts, and urgent toxicological oversight action.

• Nanocarrier;
• Polymeric micelles;
• Dendrimer;
• Nanosphere;

Silica

Stored

grains: beans, barley, grain crops, oats, rice, rye, sorghum, wheat, buckwheat, flax, corn, peas, seeds, soybeans Grain/cereal/flour bins

(empty/full), grain/ cereal flour storage areas (empty/full), food/

feed storage areas (empty/full), silo, household/ domestic dwelling indoor food handling areas, commercial transportation facilities,

food processing plant premise/equipment, eating establishments

food handling areas (food contact), eating establishment food

sewing areas (food contact), and food/grocery marketing/storage/

distribution facility premise

Indoor Non-Food

(including Commercial/Residential

Sites) : Kennels, pet sleeping quarters/veterinary,

cats (adult/kitten)*, dogs/canine (adult/puppies),

pet living/sleeping quarters, pet bedding, domestic

dwelling, household/domestic dwelling indoor premise, household/domestic dwelling content, human bedding/mattresses, refuse/solid waste containers (garbage cans)

SILICA GEL

Aquatic on-Food Site (Commercial)Sewage Systems Outdoor Sites

(including Commercial/Residential): Kennels/pet sleeping

quarters/veterinary, household/domestic dwelling (outdoor),

wood protection treatment to building/products (outdoor),

commercial/institutional/industrial areas (outdoor)

Indoor Food (including Cornmercial/Agricultural/ Residential) Grain crops, grain/cereal/flour bins (empty/ full), grain/cereal/flour elevators (empty/full), food/feed storage areas (empty/full), grain/ cereal/flour storage areas, dairy cattle, poultry, beef/range/feeder (cattle), hog/pig/swine, house- hold/domestic dwelling food indoor establishment, food processing plants premise/equipment, feed mills/feed processing

plants, flour mills, cereal plants, eating establishments food handling areas (contact), eating establishments food serving areas

(contact), food/grocery marketing/storage distribution

facility premise 000565 Indoor Non-Food (including Commercial/Agricultural/ Residential) Kennels/pet sleeping

quarters/veterinary, horses, animals (lab/research) ,

commercial transportation facility, eating establishments

non-food areas, commercial/institutional/industrial

premise/equipment (indoor), cats (adults/kittens), dogs/canines

(adult/puppies), monkeys, ferrets, birds, pet living/ sleeping quarters,

pet bedding, domestic dwellings, household/domestic dwellings (indoor), household/ domestic dwelling content, wood protection treatment

to building (indoor), human bedding/mattresses Indoor Medical:

Hospitals/medical institutions (human/veterinary

Exemption of Tolerances

silicon dioxide and silica gel (hydrated silica) have received

exemptions from tolerances and clearances for certain use patterns

associated with food commodities. These exemptions and clearances

are: -when applied as an inert ingredient, or occasionally as an active,

to growing crops and raw agricultural commodities (40 CFR 180.1001(c)

and (a)); -when applied as an inert, or occasionally as an active

to livestock (40 CFR 180.1001(e)); -when applied as an active ingredient

to growing crops, raw agricultural commodities after harvest and to livestock (40 CFR 180.1017). Current exemptions from tolerances in 40

CFR 180.1017, 185.1700 and 186.1700 are limited to the naturally

mined silicon dioxide-containing product diatomaceous earth.

Anhydrous silicon dioxide has a molecular weight of

60.09. Silica gel and other amorphous forms of silicon dioxide will have

a varying molecular weight, depending upon the extent of hydration. Diatomaceous earth consists of siliceous frustules and fragments

of various species of diatoms mined from the beds of former inland

lakes. It is composed of approximately 85% silica, other oxides

and organic materials. The natural grades are mined and then

dried, ground, sifted and bagged. Both forms used as pesticidal

active ingredients are generally white powders at room temperature which melt to a glassy consistency at high temperatures. Silicon

dioxide is practically insoluble in water, but is soluble in hydrofluoric acid. Heating with concentrated phosphoric acid may

slowly dissolve as well. Amorphous forms of silica may be dissolved by hot concentrated alkaline solutions, but crystalline forms generally are not soluble. Silica is not soluble in any organic solvent. The bulk density is in the range of 10-20 lb/ft 3 and the true density is approximately 2.2 g/cm 3. The pH of an aqueous suspension of silica gel can range from 2.3-7. All product chemistry requirements have been satisfied

Oral Administration

Rat: A group of 30 weanling Sprague-Dawley rats was administered 20

mg/day of diatomaceous earth in cottage cheese at a concentration of

5 mg/g cheese, in addition to a basal diet & libit were observed for

their life span (mean survival 840 days). Five malignant tumors (1

salivary gland carcinoma, 1 skin carcinoma, 2 sarcomas of the

uterus, 1 peritoneal mesothelioma) and 13 benign tumors (9 mammary fibro adenomas, 1 adrenal pheochromocytoma, 3 pancreatic adenomas) were

observed in treated animals. A control group of 27 rats with mean survival of 690 days had 3 carcinomas (1 each in lung, ovary and fore stomach) and 5 mammary fibro adenomas. k/

Mouse: Groups of 75 mice were exposed to various particulates including

0.5 g/day precipitated silica (particle size was reported to about 5

um or less in diameter) once an hour for 6 hours on 5 days/week for

1 year and observed for their lifespan. Survival at 600 days was 12/74

in the silica treated group and 17/75 in one control group and 13/73

in the second control group. The incidence of pulmonary adenomas

and adenocarcinomas in mice surviving 200 days or more was 5/63

and 5/52 in the control groups at 13/61 in the silica treated group.

I/ Rabbit: Inhalation of 40 mg/ml amorphous silica for up to to

1100 days was reported to produce Ildiffuse tissue reactions. a/

Dietary exposure

Dietary exposure to silicon dioxide and silica gel The

may occur from their application to certain crops and

in and around food handling and preparation areas. amount

of ingestion has not been quantified for this assessment because

they are exempt from tolerance requirements at all levels in food.

to be inconsequential because of the ubiquity of forms of silicon

dioxide in the environment

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Nano-silicon dioxide toxicological characterization on two human kidney cell lines

V Paget, J A Sergent and S Chevillard¹

Published under licence by IOP Publishing Ltd
Journal of Physics: Conference Series, Volume 304, Number 1

Abstract

Silicon dioxide nanoparticles (n-SiO₂) have recently encountered a wide variety of applications in medicine or engineering but their toxicological effects are poorly understood. In this study, we have used SiO₂-25 nm and SiO₂-100 nm mono-dispersed nanoparticles labeled with Rhodamine B and TMPyP respectively. These two fluorophores were incorporated during synthesis in order to track nanoparticles cell incorporation. Up-to-date, no evaluation of the toxicological effects of these nanoparticles upon human kidney has been published. As kidney is one of the major traditional retention organs, the aim of our study is to evaluate the potential toxicity of these nanoparticles on two human cell lines from proximal tubule (Caki-1 and Hek293). Our results report that the two cell lines do not show similar responses after 24 hours of exposure to SiO₂-nanoparticles disregarding a similar origin in the kidney. Interestingly, our results indicate that for both tested SiO₂-nanoparticles, Caki-1 cells present a higher sensitivity in terms of cytotoxicity and genotoxicity than Hek293 cells. Furthermore, our results show that for similar concentration of exposure, SiO₂-25 nm seems to be more cytotoxic and genotoxic than SiO₂-100nm for both tested cell lines.

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Dangers of SiO2 nanoparticles

the dangers of SiO2 nanoparticles has been studied pretty well.  I didn't realize how dangerous this compound is, and that the FDA allows it sprayed on unfinished food and not labeled.  In most people, it doesn't cause "acute" reactions, but it causes a.  After the obdy gets rid of the SiO2, and it's introduced into the continuous crystallization inside the body liquid crystalline matrix inside the body, it starts crystallizing to itself, forming larger crystals.  If you don't believe me, go take a vitamin pill containing amorphous SiO2, wait about 2 hours, then look at your blood under a darkfield microscope.  You'll see the crystals forming.  Some are as small as a little dot that will reflect the light), but others are as big as a red blood cell, and are obvious crystals.  When I did some darkfield work, I saw the crystals as well.  I asked what they were (not realizing anything in this email, below), and the guys said "artifacts."  He also noted that he has been seeing a LOT more artifacts in people's blood, and don't really know what they were, or where they are coming from.  Here's one extract from a book:

When it's sprayed on food, it acts as a drug delivery system, just like it says above.  It's properties allow it to coat proteins, gluten, amino acids, etc... to artificially transport them into the body.  Essentially it acts to force injections into us.  So when you said awhile ago that you speculated there might be forced vaccinations....it already is happening...through the food.  There's already evidence that TiO2 nano-particles cause iron uptake dysregulation in chicken intestinal cells, and amorphous SiO2 acts the same way.  SiO2 forces a breakdown in the gut regulation cells, and it does it through 'disconnecting' the cells from host. : SiO2 and TiO2 nanoparticles have what is known as 'light scattering' properties and electrical insulating properties.  When the SiO2 is ingested, it coats the outsides of the cell walls [the phosphatidylcholine, carbohydrate chains, and proteins (etc)].  When the insulating effect is began, the cell loses connection to what it's supposed to be doing.  This energy originates in the heart, and with every beat we have the heart putting out light energy and epigenetically changing DNA expression (almost instantaneously).  So you can see what happens when you insulate a certain set of cells from receiving that energy with TiO2 or SiO2 nanoparticles; they malfunction.  Also, SiO2 and TiO2, as I alread said, have 'light scattering' properties.  This is just another term for a re-direction or, changing the amplitude and wavelength of the energy being passed through it.  It does it very well.  So when our heart and nervous system emit informational instructions, these instructions are either not fully received (as I said before), or they are changed, so the cell gets bad information, and malfunctions.--I learned all this by studying the work and research of Dr. David Jeringan, Dr. Jerry Tennant, Dr. Hal Huggins,  (and from the researchers that they got their info from), and by reading about how you can extract the DNA of living cells, in tissue, and they do not malfunction (but obviously cannot reproduce).  They continue to operate normally until they are removed from their host, which says....the host is controlling the DNA expressions and forcing the cells to function correctly, so something else other than DNA is doing it.  Dr. Jernigan talks about how the light of the body controls DNA expression...so there it was, staring me in the face:  Disrupting energy flow by adding "inert" SiO2 and TiO2 nanoparticles to essentially all the food are the root issues with out food.  Not only that, nano-silicates have a immune-stimulating response when introduced to the immune system (just like the pico-sized aluminum particles in the vaccines), for the reasons I said above (the immune system recognizes a malfunctioning cell, figures out the cell wall is resonating "silicon signatures," and destroys it.  This is called cell mediated immunity to haptens (nanoparticles are the hapten" which induces autoimmunity, TNF-alpha, and interleukin, and cytokine increases).

From the University of Colorado Dept. of Microbiology, Immunology, and Pathology:  "anything implanted in bone will create an autoimmune response.  The only difference is the  time it takes."  ----guess what happened to this professor after he proved and released this information?  From the inside info I got, this news release (not the actual link) was unauthorized, and the Professor was "let go."  He went to Pepperdine after this, and continued research in this category.  I believe he started a business, or joined a business that is researching how to implant the patients own DNA into ceramic and composite compounds for future use inside the body.  Why??  LOL...we already "know" the body is going to form an autoimmunity to metallic implants into any bone structure!  It's just a matter of time until we find out our nano-silicon implants are really causing some issues.  If the body is "taught" that silica is an antigen, it will get rid of anything that is silicon-based or reject anything that is silicon-coated.  Hence, we get allergic to anything sprayed with nano-silica, or "learn" to get immune response to vitamins when mixed or coated with nano-silica (>90% of supplements).  --Now you see why Codex required capsule fill requirements, and silica was one of the approved fillers.  Also, essentially all the pharmaceutical companies are using one of three immune system modifiers: nano- silicon, alumina, or titanium.  MCC and polymers are carcinogenic, just as you said previously.  I looked up the GRAS report on those, and actually read it.  It seems the studies were "stopped" when an increase of organ weights were noticed, and then deemed "GRAS."  What a crock of dog shit! 

Results from realizing this:  I got off everything containing this junk, and perfect health returned.  Feel free to reiterate any of this to anyone you feel it's important to, or to post wherever you want.  I don't want any credit, just that these jokers get exposed for inducing rampant [and random] diseases in the population, and convincing younger people that their "genetics are broken," and "you need to take one of those genetic tests" and take supplements (containing junk that will break your genetics more) to target the"genetic mutations" they have.  Or for the older populations, that "they need to take [______] drug, because they have [______] disease."    The truth is, the body can NEVER fix the mutatuions with nanoparticles inside of it, blocking the cells from sensing what they need to do.  Well, I guess it's pretty ingenious plan if I wanted to steal the wealth from the babyboomers, and steal the future health of the younger generation away from them, get everyone on supplements of some sort (inducing money into the system), bring the entire population to their knees, turning them upside down and circle-jerking the money out of their pockets. 

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Brain blood vessel lesions tied to intestinal bacteria

Pre-clinical study links gut microbes, immune system to a genetic disorder that can cause stroke and seizures

A study in mice and humans suggests that bacteria in the gut can influence the structure of the brain's blood vessels, and may be responsible for producing malformations that can lead to stroke or epilepsy. The research, published in Nature, adds to an emerging picture that connects intestinal microbes and disorders of the nervous system. The study was funded by the National Institute of Neurological Disorders and Stroke (NINDS), a part of the National Institutes of Health (NIH).--Cerebral cavernous malformations (CCMs) are clusters of dilated, thin-walled blood vessels that can lead to seizures or stroke when blood leaks into the surrounding brain tissue. A team of scientists at the University of Pennsylvania investigated the mechanisms that cause CCM lesions to form in genetically engineered mice and discovered an unexpected link to bacteria in the gut. When bacteria were eliminated the number of lesions was greatly diminished.--"This study is exciting because it shows that changes within the body can affect the progression of a disorder caused by a genetic mutation," said Jim I. Koenig, Ph.D., program director at NINDS.--The researchers were studying a well-established mouse model that forms a significant number of CCMs following the injection of a drug to induce gene deletion. However, when the animals were relocated to a new facility, the frequency of lesion formation decreased to almost zero.--"It was a complete mystery. Suddenly, our normally reliable mouse model was no longer forming the lesions that we expected," said Mark L. Kahn, M.D., professor of medicine at the University of Pennsylvania, and senior author of the study. "What's interesting is that this variability in lesion formation is also seen in humans, where patients with the same genetic mutation often have dramatically different disease courses."--While investigating the cause of this sudden variability, Alan Tang, a graduate student in Dr. Kahn's lab, noticed that the few mice that continued to form lesions had developed bacterial abscesses in their abdomens -- infections that most likely arose due to the abdominal drug injections. The abscesses contained Gram-negative bacteria, and when similar bacterial infections were deliberately induced in the CCM model animals, about half of them developed significant CCMs.--"The mice that formed CCMs also had abscesses in their spleens, which meant that the bacteria had entered the bloodstream from the initial abscess site," said Tang. "This suggested a connection between the spread of a specific type of bacteria through the bloodstream and the formation of these blood vascular lesions in the brain."-The question remained as to how bacteria in the blood could influence blood vessel behavior in the brain. Gram-negative bacteria produce molecules called lipopolysaccharides (LPS) that are potent activators of innate immune signaling. When the mice received injections of LPS alone, they formed numerous large CCMs, similar to those produced by bacterial infection. Conversely, when the LPS receptor, TLR4, was genetically removed from these mice they no longer formed CCM lesions. The researchers also found that, in humans, genetic mutations causing an increase in TLR4 expression were associated with a greater risk of forming CCMs.--"We knew that lesion formation could be driven by Gram-negative bacteria in the body through LPS signaling," said Kahn. "Our next question was whether we could prevent lesions by changing the bacteria in the body."-The researchers explored changes to the body's bacteria (microbiome) in two ways. First, newborn CCM mice were raised in either normal housing or under germ-free conditions. Second, these mice were given a course of antibiotics to "reset" their microbiome. In both the germ-free conditions and following the course of antibiotics, the number of lesions was significantly reduced, indicating that both the quantity and quality of the gut microbiome could affect CCM formation. Finally, a drug that specifically blocks TLR4 also produced a significant decrease in lesion formation. This drug has been tested in clinical trials for the treatment of sepsis, and these findings suggest a therapeutic potential for the drug in the treatment of CCMs, although considerable research remains to be done.--"These results are especially exciting because they show that we can take findings in the mouse and possibly apply them at the human patient population," said Koenig. "The drug used to block TLR4 has already been tested in patients for other conditions, and it may show therapeutic potential in the treatment of CCMs, although considerable research still remains to be done."-Kahn and his colleagues plan to continue to study the relationship between the microbiome and CCM formation, particularly as it relates to human disease. Although specific gene mutations have been identified in humans that can cause CCMs to form, the size and number varies widely among patients with the same mutations. The group next aims to test the hypothesis that differences in the patients' microbiomes could explain this variability in lesion number.-Story Source-Materials provided by NIH/National Institute of Neurological Disorders and Stroke. -Journal Reference-Alan T. Tang, Jaesung P. Choi, Jonathan J. Kotzin, Yiqing Yang, Courtney C. Hong, Nicholas Hobson, Romuald Girard, Hussein A. Zeineddine, Rhonda Lightle, Thomas Moore, Ying Cao, Robert Shenkar, Mei Chen, Patricia Mericko, Jisheng Yang, Li Li, Ceylan Tanes, Dmytro Kobuley, Urmo Võsa, Kevin J. Whitehead, Dean Y. Li, Lude Franke, Blaine Hart, Markus Schwaninger, Jorge Henao-Mejia, Leslie Morrison, Helen Kim, Issam A. Awad, Xiangjian Zheng, Mark L. Kahn. Endothelial TLR4 and the microbiome drive cerebral cavernous malformations. Nature, 2017; 545 (7654): 305 DOI: 10.1038/nature22075 --NIH/National Institute of Neurological Disorders and Stroke. "Brain blood vessel lesions tied to intestinal bacteria: Pre-clinical study links gut microbes, immune system to a genetic disorder that can cause stroke and seizures." ScienceDaily. ScienceDaily, 18 May 2017. www.sciencedaily.com/releases/2017/05/170518140232.htm>.

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Gut microbes linked to brain structure in people with irritable bowel syndrome

A new study by researchers at UCLA has revealed two key findings for people with irritable bowel syndrome about the relationship between the microorganisms that live in the gut and the brain.--For people with IBS research shows for the first time that there is an association between the gut microbiota and the brain regions involved in the processing of sensory information from their bodies. The results suggest that signals generated by the brain can influence the composition of microbes residing in the intestine and that the chemicals in the gut can shape the human brain's structure. --Additionally, the researchers gained insight into the connections among childhood trauma, brain development and the composition of the gut microbiome. Previous studies performed in mice have demonstrated effects of gut microbiota on brain function and behavior, as well as the influence of the brain on the composition of microbes in the gut. However, to date, only one study performed in human subjects has confirmed the translatability of such findings to the human brain. Studies have also reported evidence for alterations in the composition of gut microbiota in people with irritable bowel syndrome, but there has been little consistency among studies regarding the specific microbial alterations and the relationship of such alterations with the cardinal symptoms of IBS, recurring abdominal pain and altered bowel habits. In relation to a person's history with childhood trauma, it has been shown to be associated with structural and functional brain changes; trauma in young children has also been shown to alter gut microbial composition. But how they are related has been unknown.--The UCLA researchers collected behavioral and clinical measures, stool samples and structural brain images from 29 adults diagnosed with IBS, and 23 healthy control subjects. They used DNA sequencing and various mathematical approaches to quantify composition, abundance and diversity of the gut microbiota. They also estimated the microbial gene content and gene products of the stool samples. Then the researchers cross-referenced these gut microbial measures with structural features of the brain.--Based on the composition of the microbes in the gut, the samples from those diagnosed with IBS clustered into two subgroups. One group was indistinguishable from the healthy control subjects, while the other differed. Those in the group with an altered gut microbiota had more history of early life trauma and longer duration of IBS symptoms.-The two groups also displayed differences in brain structure.--Analysis of a person's gut microbiota may become a routine screening test for people with IBS in clinical practice, and in the future, therapies such as certain diets and probiotics may become personalized based on an individual's gut microbial profile. At the same time, subgroups of people with IBS distinguished by brain and microbial signatures may show different responsiveness to brain-directed therapies such as mindfulness-based stress reduction, cognitive behavioral therapy and targeted drugs.--A history of early life trauma has been shown to be associated with structural and functional brain changes and to alter gut microbial composition. It is possible that the signals the gut and its microbes get from the brain of an individual with a history of childhood trauma may lead to lifelong changes in the gut microbiome. These alterations in the gut microbiota may feed back into sensory brain regions, altering the sensitivity to gut stimuli, a hallmark of people with IBS. Story Source-Materials provided by University of California, Los Angeles (UCLA), Health Sciences. Journal Reference-Jennifer S. Labus, Emily B. Hollister, Jonathan Jacobs, Kyleigh Kirbach, Numan Oezguen, Arpana Gupta, Jonathan Acosta, Ruth Ann Luna, Kjersti Aagaard, James Versalovic, Tor Savidge, Elaine Hsiao, Kirsten Tillisch, Emeran A. Mayer. Differences in gut microbial composition correlate with regional brain volumes in irritable bowel syndrome. Microbiome, 2017; 5 (1) DOI: 10.1186/s40168-017-0260-z University of California, Los Angeles (UCLA), Health Sciences. "Gut microbes linked to brain structure in people with irritable bowel syndrome." ScienceDaily. ScienceDaily, 5 May 2017. <www.sciencedaily.com/releases/2017/05/170505151656.htm>

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New Transmissible Vaccine Spreads Like Virus, No Consent Necessary

As governments move forward in their attempts to force their citizens into vaccination, whether they want it or not, researchers are now moving forward with what may become the future of forced vaccination – transmissible vaccines.--This is an area of research that scientists have been interested in for quite some time but that is only now becoming feasible.--The idea behind these vaccines are that they would themselves be infectious, passing along the vaccine from one person to another as if it were a virus.-Thus, it would be possible to vaccinate a small number of people manually, but as a result of the vaccine’s self-transmission, actually vaccinate a very high number of people.--According to PNAS (Proceeding of the National Academy of Sciences of the United States of America),several recombinant, transmissible vaccines are in development for wild animal populations, including one to protect wild rabbits against a fatal viral infection and another to prevent deer mice from carrying a virus responsible for a deadly human pulmonary disease.

But transmissible vaccines pose a special risk, essentially, they could become a virus themselves and spread rapidly amongst the population that was vaccinated. This has already happened with the oral polio vaccine and both the 1960s and the 2000s, but with a transmissible vaccine, the danger of spread of a disease would become even more dangerous since spread and transmission are what the vaccines are designed to do.--Scott Nuismer, a Professor of Biological Sciences and Mathematics at the University of Idaho and co-author of a study on transmissible vaccines published this year in the journal Proceedings of the Royal Society B, stated,--Obviously this is a controversial and potentially risky endeavor, so we wanted to figure out, is the potential benefit actually worth the risk?--Nuismer, along with colleagues at six other institutions, created a mathematical model mapping how a vaccine spreads through populations by altering one key element – the vaccine’s ability to spread – it was determined that the vaccine needed very little transmissibility in order to spread through the population. The potential risks of creating an epidemic and/or pandemic of the disease researchers allegedly want to stop, is of course not going to keep them down. As noted earlier, several transmissible vaccines are in development for wild animal populations and livestock.

So maybe these researchers will help protect the bunnies against a virus – or maybe they’ll just kill them all. It’s 50/50, but the researchers seem confidant, so what could go wrong?

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Synthetic Biology--Toward Therapeutic Solutions

Viktor Haellman 1 and Martin Fussenegger 1, 2

1 - Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland 2 - Faculty of Science, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland

Correspondence to Martin Fussenegger: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland. fussenegger@bsse.ethz.ch http://dx.doi.org/10.1016/j.jmb.2015.08.020 Edited by S. Chandrasegaran

Abstract

Higher multicellular organisms have evolved sophisticated intracellular and intercellular biological networks that enable cell growth and survival to fulfill an organism's needs. Although such networks allow the assembly of complex tissues and even provide healing and protective capabilities, malfunctioning cells can have severe consequences for an organism's survival. In humans, such events can result in severe disorders and diseases, including metabolic and immunological disorders, as well as cancer. Dominating the therapeutic frontier for these potentially lethal disorders, cell and gene therapies aim to relieve or eliminate patient suffering by restoring the function of damaged, diseased, and aging cells and tissues via the introduction of healthy cells or alternative genes. However, despite recent success, these efforts have yet to achieve sufficient therapeutic effects, and further work is needed to ensure the safe and precise control of transgene expression and cellular processes. In this review, we describe the biological tools and devices that are at the forefront of synthetic biology and discuss their potential to advance the specificity, efficiency, and safety of the current generation of cell and gene therapies, including how they can be used to confer curative effects that far surpass those of conventional therapeutics. We also highlight the current therapeutic delivery tools and the current limitations that hamper their use in human applications.

© 2015 Elsevier Ltd. All rights reserved.

Introduction

Eukaryotic cells possess a remarkable ability to continuously sense, integrate, and store relevant physiological and biological information related to the state of the organism. This ability gives cells inherent therapeutic capabilities that far surpass pharmaceuticals in terms of their ability to (i) sense a plethora of biological macromolecules, (ii) autonomously regulate dosage and bioavailability, and (iii) adapt the therapeutic effect depending on the tissue-specific environment and genetic variability. After decades of life science and medical research, scientists are poised to harness these intricate biological machines as therapeutic entities in the fight against current and future human diseases. The notion of harnessing cells for therapeutic purposes is not a new paradigm in medicine.

Pioneering work in the effort to realize the potential of cell-based medicine dates back to the early 1900s and the first blood transfusions. Following the discovery of key factors that distinguish "self" from "non-self", medical professionals were able to advance the notion of cell-based medicine to whole organ transplantation. This advancement also shaped the modern concept of cell therapy when two patients suffering from genetic primary immunodeficiency disorders were cured in 1968 with bone marrow transplants that reconstituted their malfunctioning lymphoid lineages via cells from healthy donors [4]. More than two decades later, the same procedure also resulted in the birth of gene therapy when two patients suffering from primary immunodeficiency disorders were instead treated with their own ex vivo genetically corrected lymphocytes in 1990 [5]. Being able to provide treatment-J Mol Biol (2016) 428, 945962 946 options to patients suffering from previously incurable and potentially lethal diseases was a historic moment in medicine and offered a first hint of the curative capabilities of cells, thus prompting scientists to further explore the therapeutic potential of these two novel approaches. Today, these two largely overlapping research fields represent the "state of the art" in cell-based medicine; and cell therapy aspires to treat patients by replacing injured, diseased, and aging tissues with healthy cells, gene therapy aims to directly treat disease by introducing genes that can repair the effects of mis-regulated and malfunctioning genes. Although the realization of the potential of cell-based medicine for diseases other than primary immunodeficiency disorders has been hindered by failures and adverse events (e.g., inefficiency, graft rejection [6], graft-versus-host disease [7], and vector-mediated integrational mutagenesis and immune responses [8,9]), cell and gene therapies constitute indispensable resources in the pursuit of cures for genetic diseases and metabolic disorders. The expectations for gene- and cell-based therapies were dramatically boosted in 2012 as a result of the approval in the European Union of the gene therapy drug alipogene tiparvovec (Glybera; UniQure) for the treatment of patients suffering from reoccurring severe pancreatitis attacks, despite tight diet control, due to a genetic deficiency of the catabolic enzyme lipoprotein lipase. With a single dose capable of producing a sustained reduction of blood triglyceride levels and the incidence of pancreatitis through persistent gene expression in somatic tissue [10], alipogene tiparvovec set the stage for the next wave of gene therapy for the treatment of other genetic diseases such as Leber's congenital disease and Parkinson's disease [11,12], as well as multifactorial pathophysiological conditions such as cardiovascular disorders [13,14]. Similarly, cell therapy has made rapid progression into the clinical arena using ex vivo engineered autologous hematopoietic stem cells (HSCs) to treat monogenetic hematological and storage disorders [15,16], and adoptive immunotherapy using ex vivo engineered T cells has been shown to not only target and kill aggressive cancers such as acute myeloid leukemia [17,18] but also assist the host immune system in the identification of tumor cells [19,20]. Despite the promising potential of cell and gene therapies, these tools remain fundamentally limited by side effects and by their inability to achieve adequate therapeutic effects to completely treat many diseases [21,22]. To fully capitalize on cells' inherent therapeutic abilities, we need bioengineered tools to create safe and predicable systems that can regulate cellular behavior with high biological, spatial, and temporal precision. To this end, synthetic biology, which is a scientific field that interprets biology from an engineering perspective, presents interesting solutions for the precise control and modulation of both endogenous and exogenous genes and their products at the transcriptional, translational, and post-translational levels, from which robust autonomous biological networks can be engineered within cells to perform increasingly intricate tasks. In this review, we discuss the innovative biological tools and devices that have been created by synthetic biology and explain how these tools could be used to shape the future state of the art in cell and gene therapies. We first describe the vast biological toolbox generated by synthetic biology and biological engineering to confer the highest possible control and modularity of transcriptional, translational, and post-translational processes. We also highlight how synthetic-biology-inspired tools have allowed cell and gene therapies to surpass their initial limitations and how they can be further improved. Second, we present how synthetic biology has used these tools to convert cells into theranostic devices that are capable of diagnosing a disease state and trigger an autonomously regulated therapeutic response. Third, we present the major in vivo delivery platforms for therapeutic cells and genetic material and highlight the major limitations that currently hamper the advancement of cell and gene therapies. Finally, we summarize and discuss the future therapeutic prospects of cell and gene therapies, including the potential role of synthetic biology in realizing the full potential of cell-based medicine.

Building biological systems

Synthetic biology is based on the concept that biological components from different organisms can be combined and repurposed into artificial biological networks that can operate either in parallel or together with natural biological systems to confer useful biological functionalities to cells. Taking a rational approach to biology, synthetic biology defines biology by the underlying computational logic that dictates the functionality of macromolecules and networks with the aim of engineering biological systems in cells that can be precisely regulated with relevant triggers. Hence, to create increasingly intricate biological systems with potential therapeutic value, synthetic biology relies on the availability of well-characterized biological components. Transcriptional control Most of the current gene therapy and ex vivo cell engineering approaches employ either viral promoters or engineered tissue-specific and endogenous promoters to drive transgene expression. While viral promoters are able to achieve strong transgene A POI Ligand induced dimerization of pro-apoptotic protein domains Ligand molecule stabilizes fusion construct

Ribosome 3'UTR miRNAs guide endogenous protein complexes towards complementary RNA sequences to inhibit translation and degrade the transcript Exogenous proteins and molecules control ribozyme mediated cleavage and stability of transcripts Rewired signal transduction through engineered receptors and downstream response elements POI Destabilizing peptide domain fused to POI miRNA1 miRNA2

Predetermined cell response

Small molecule triggered OFF-system Small molecule regulated DNA-binding domain fused to transcriptional Pmin activator drives gene expression Inserted operator and promoter Trigger molecule abolish DNA binding of transcriptional activator and turning gene expression OFF ZF fused to transcriptional regulator GOI Pmin GOI TALE fused to transcriptional regulator gRNA dCas9 fused to transcriptional regulator Alternative gRNA can retarget Cas9 to mutiple DNA sequences--

Small molecule triggered ON-system Small molecule regulated DNA-binding domain fused to transcriptional PConst. repressor represses gene expression Inserted operator and promoter

Fig. 1. Synthetic biology tools to control transcriptional, translational, and post-translational events in mammalian cells: (a) Translational tools. Small noncoding RNA, for example, miRNA, can be used to control the translation of messenger RNA. By placing single and combinations of certain miRNA targeting sequences complementary to endogenous or synthetic miRNA in the 3 UTRs of transcripts, the presence of these miRNAs guides endogenous protein complexes to the complementary RNA sequence to inhibit translation and degrade the transcripts. Engineered ligand-regulated ribozymes can confer exogenous control of transcript stability and translation when placed in the 5 or 3 UTR of transcripts; (b) Customized cell signaling and post-translational tools. Signal transduction can be rewired through engineered receptors with an altered ligand-binding domain and receptor intracellular signaling domain or alternatively through downstream optimized synthetic transcriptional elements to express genes of interest (GOI). Exogenous control over post-translational events can be achieved through ligand-induced dimerization of effector proteins or by fusing the protein of interest to ligand-regulated protein domains. The examples shown demonstrate ligand-induced dimerization of pro-apoptotic effector proteins to confer trigger-mediated induction of apoptosis and a ligand-regulated degradation peptide that destabilizes the fused protein of interest (POI) in the absence of a stabilizing ligand; (c) Transcriptional tools. Prokaryotic trigger-inducible transcriptional regulators, for example, TetR, can regulate the activity of eukaryotic promoters, engineered to contain the corresponding DNA-binding sequence, in response to heterologous compounds. Programmable transcription factors based on ZFs, TALEs, and CRISPR/dead CRISPR-associated protein dCas9 can be engineered to regulate transcription of endogenous and exogenous genes from any promoter. Furthermore, CRISPR/ dCas9-based transcription factors can be easily retargeted to multiple DNA sequences simultaneously. Abbreviations: PConst., constitutive promoter; Pmin, minimal promoter. - Trigger molecule abolish DNA binding of transcriptional repressor and activating gene expression ON expression, they commonly produce genotoxicity effects [23] and are also prone to silencing in vivo [24]. On the contrary, tissue-specific and physiological promoters can achieve strong expression in desired cell lineages with reduced genotoxicity and cytotoxicity when their expression patterns are similar to those of the affected genes [25,26]. However, as endogenous promoters and regulatory elements are naturally rather large, suitable condensed tissue-specific and physiological promoters can be difficult to engineer [27], which can result in design-dependent cell-type variability within the same lineage [28] or failure to achieve sufficient transgene expression to reach a therapeutic effect [29]. In comparison, engineered small synthetic promoters that can flexibly switch gene expression ON and OFF and modulate transgene expression in response to a wide range of endogenous and exogenous triggers could offer an effective alternative approach to support the required dosages with appropriate spatiotemporal control.

948 Pioneering studies demonstrated that natural DNA-binding regulatory proteins from bacteria (e.g., LacI and TetR) could be converted into efficient gene expression regulators in mammalian cells when fused to transcriptional regulatory domains. For instance, when fused to a transcriptional activator domain (e.g., VP16), these synthetic transcription factors can activate transcription from a minimal eukaryotic promoter engineered to contain their corresponding DNA operator sequences [3032] (Fig. 1c). Alternatively, when fused to a transcriptional repressor domain (e.g., KRAB), they can block transcription from constitutive promoters with operator sequences placed adjacent to the promoter [33] (Fig. 1c). Because of continuous exposure to harsh environmental stimuli, bacteria have evolved a large number of trigger-regulated transcriptional response systems to ensure their survival [34]. Analogous to the tetracycline resistance gene regulation TetR system from Escherichia coli, most of these prokaryotic gene regulators function as transcriptional repressors that, in the absence of their trigger molecule, bind to their corresponding DNA operator sequence with high specificity and affinity. Upon exposure to the trigger, the trigger molecules bind to transcriptional regulators, which induce a conformational change of the DNA-binding domain and render them incapable of binding to DNA [35]. The extensive characterization and optimization of the allosteric regulation that dictates the DNA-binding ability of this class of transcriptional regulators have enabled synthetic biology to infer gene activation or repression in response to small diffusible molecules with near-binary precision, making them a particularly valuable engineering tool. By translating these small-molecule-controlled systems to mammalian transgene control devices, scientists have created an array of valuable model systems that include genetic switches [36,37], band-pass filters [38], time-delayed circuits [39,40], and oscillators [4143]. Trigger-inducible gene control devices engineered in this way have been programmed to respond not only to common cosmetic preservatives [44] and food additives [45,46] but also to biochemical inducers supplemented in drinking water [47], presenting potential biomedical applications to improve patient compliance to treatment regimes. Furthermore, similar devices pioneered a theranostic concept for biomedical research where the production and delivery of a therapeutic protein can be synchronized to a specific metabolic signature, for example, Kemmer et al., who converted a bacterial uric acid sensing protein into a mammalian theranostic uric acid homeostasis device used to attenuate the risks associated with uric acid accumulation in the blood system and organs leading to gouty arthritis [48]. By fusing the uric-acid-responsive repressor HucR to a KRAB protein domain, the authors were able to precisely control transgene expression from

Synthetic Biology and Solutions

a HucR DNA-binding motif linked to a synthetic promoter, which was used to efficiently modulate the uric acid concentration in hyperuricemic mice through the dose-dependent production of urate oxidase. In recent years, prokaryotic transcription factors as DNA-binding protein domains have gradually been replaced by zinc-finger (ZF) proteins, transcription activator-like effectors (TALEs), and clustered regular interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein Cas9-based regulators, which are not restricted to one specific DNA-binding sequence but can be flexibly reengineered to target virtually any DNA sequence (Fig. 1c). The first two programmable transcription systems are based on covalently assembled tandem arrays of Cys2-His2 ZF domains or short conserved 33- to 35-amino-acid repeats (TALEs) where each domain recognizes and binds three or single nucleotides, respectively [49,50]. If fused to a transcriptional regulator domain, these systems are able to target and regulate the transcription of virtually any exogenous or endogenous gene [51 57]. In comparison, CRISPR/Cas9-based transcriptional regulators rely on the WatsonCrick binding of an arbitrary programmable short noncoding guide RNAs to a 20-nucleotide target sequence [58]. Although initially used as a genome-editing tool due to the nuclease activity of Cas9 [59], catalytically inactive Cas9 retains its DNA-binding ability (dead Cas9), allowing scientists to repurpose CRISPR/Cas9 to interfere with transcription [60] or to efficiently regulate exogenous and endogenous gene expression when fused to transcription regulators [6163]. The relative ease of retargeting Cas9 with different guide RNAs, which only requires a 5-NGG protospacer adjacent motif sequence for successful recruitment of Cas9, makes CRISPR/ Cas9 a highly modular platform for targeting several genetic regions simultaneously and could become a potent tool for precise modulation of cell phenotypes, a concept that was recently implemented by Zalatan et al., who demonstrated that the regulatory function CRISPR/Cas9-based transcription factors can be directly encoded into the guide RNA by extending them with an effector protein-recruiting RNA scaffold [64]. This functionality made it possible to simultaneously recruit transcription activators or repressors to different DNA target sequences and redirect the metabolic flow of a distinct metabolic pathway in yeast, which could prove to be a viable tool for modulating malfunctioning metabolic pathways in mammals as well. Translational control The key regulatory role of functional RNA molecules in a wide range of biological systems has compelled scientists to elucidate their mechanistic behavior for 949 gene expression at the translational level using a ligand of choice when inserted in the 5 or 3 UTR of transgenes [8083] (Fig. 1a). The basic framework of these RNA regulators makes them a highly modular platform for creating ligand-regulated translational control devices [84,85], which can be incorporated into gene circuits to exogenously control cellular decisions and fates [86,87]. Introducing these relatively small control devices into gene and cell therapy platforms could be a simple approach to retain exogenous control over engineered cells, for example, in the case of cell therapy using ex vivo engineered HSCs in which the selective advantage of the engineered cells takes place through peripheral expansion rather than during thymic differentiation (e.g., WiskottAldrich syndrome [88]). Instead of using myeloablative conditioning and immunosuppressive regimes to eradicate the patient's own hematopoietic cells, an alternative could be selectively induced peripheral expansion with exogenous molecules and drugs. For example, this concept was conceptualized by Chen et al., who introduced a small drug-responsive ribozyme into the 3 UTR of a constitutively expressed interleukin-2-encoding transgene, thus enabling the precise proliferation control of engineered T cells [89]. Customized cell signaling and post-translational control Mammalian cells possess a remarkable ability to process and provide information and perform synchronized tasks in higher-order structures. Whereas transcription-factor-based sensors are mostly limited to a relatively small range of diffusible molecules, mammalian cells have a multitude of cellular receptors at their disposal that can sense a plethora of endogenous and exogenous molecules and stimuli. To capitalize on these abilities, synthetic biology and biological engineering have created a great number of systems that employ, modulate, and repurpose signaling networks through natural and engineered receptors, signal transducers, and downstream response elements. At the centre of the cells' immense ability to sense inputs, which range from physical inputs such as light to complex molecules such as odors and pheromones or proteins and peptides to small molecules, are the G-protein-coupled receptors (GPCRs), which transduce these extracellular signals through intracellular effector pathways to modulate diverse cellular responses [90]. Detailed structural and functional characterization of GPCRs has allowed scientists to engineer custom-designed GPCR to respond to synthetic ligands. These so-called RASSL (receptors activated solely by synthetic ligands) are rationally engineered endogenous GPCRs, whose receptor binding pocket has been modified by replacing its native ligand sensitivity by a the purpose of translating their functional properties into synthetic counterparts. The growing diversity and fast-acting characteristics of RNA-based regulators enable the precise and fine-tuned translational control of synthetic gene circuits via complementary base pairing of small noncoding RNA or catalytically active RNA-based regulators that can sense specific molecular cues and modulate translation accordingly. In eukaryotes, RNA interference (RNAi) is the best-characterized RNA-based regulatory system. RNAi is a translational inhibitory system mediated through small interfering RNAs and microRNAs (miRNAs) that guide endogenous protein complexes to the complementary messenger RNA transcripts, thus blocking translation and degrading the transcripts [65]. Because miRNA regulation appears to play a crucial role in maintaining tissue-specific lineages and functions with tissue-specific and developmental-stage-specific miRNA expression patterns [66,67], researchers have begun to capitalize on these tissue-specific patterns to reduce off-target effects in cell and gene therapies by introducing certain miRNA target sequences into the 3 untranslated regions (UTRs) of transgenes (Fig. 1a). In gene therapy, this approach has been used to develop viral vectors that selectively repress transgene expression in certain cells types [68], which can be extended to promote immunological tolerance to transgenes by preventing expression in antigen-presenting cells, as an example [69,70]. Similarly, cell therapy with ex vivo engineered HSCs explores developmental-stage and cell-lineage-specific miRNA expression patterns to reduce cytotoxic expression during HSC differentiation [71,72] and limit therapeutic transgene expression to specific HSC-derived cell lineages [73], including antigen-presenting cells, to promote immunological tolerance [74]. Although RNAi-mediated silencing has been proven as a valuable tool in both cell and gene therapies, synthetic biology demonstrates that RNAi-control devices using miRNA-mediated silencing can be expanded to create tight genetic switches [75], modular circuits that introduce timed expression of certain genes [76], and genetic circuits that regulate gene expression according to predetermined logic programs [77,78], including control devices capable of identifying diseased cells, such as cancer cells [79], based on cell-type-specific miRNA expression patterns, favorable characteristics that could be incorporated into cell and gene therapies in order to continuously modulate transgene expression according to the biological state of the cell. These characteristics can be further expanded to exogenous molecules through the rational engineering of functional RNA structures. By coupling an RNA aptamer sensor with RNA regulators, for example, ribozymes and riboswitches, we can engineer synthetic small-molecule and protein-responsive RNA regulators to control 950 synthetic ligand [91]. However, since the original endogenous GPCRs often show minor sensitivity to the synthetic ligands, the technology of receptors activated solely by synthetic ligand has often been associated with lack of specificity [92]. This issue has been successfully addressed by the follow-up technology known as DREADD (designer receptor exclusively activated by designer drugs), whereby directed molecular evolution linked to cell survival assays is used to generate designer GPCRs whose synthetic trigger compounds have no affinity to endogenous GPCRs [93]. Since the first reported DREADD receptor--engineered to from the human muscarinic acetylcholine M3 receptor to sense the biologically inert molecule clozapine (clozapine-N-oxide) [94]--the repertoire of DREADDs has rapidly developed into a powerful tool to study biological processes [95]. This growing number of custom-designed GPCRs makes DREADDs increasingly attractive orthogonal control tools for synthetic biologists to integrate into artificial gene networks. Additionally, with the possibility to create chimeric receptors with interchangeable intracellular effector domains [96] and artificial tethering synthetic transcription regulators to receptors [97], hence functionally rerouting the signal transduction of a decried receptor (Fig. 1b), GPCRs are becoming increasingly modular platforms for controlling specific cellular processes. By functionally rewiring the sensing and signaling ability of GPCRs to reliable transcriptional control devices, synthetic biologists have greatly expanded the available repertoire of tools used to remotely control cellular processes in response to almost any trigger of choice. This topic is discussed in further detail in the following section, which includes an explanation of how these constructs form the foundation for cell-based theranostic devices. Regulation at the post-translational level has the advantage of being able to rapidly modulate the desired cellular processes in a protein-based, trigger-induced fashion without relying on the transcription and translation of effector proteins, characteristics that are highly valuable to achieve more rapid response dynamics. For example, to enable precise control over apoptosis in engineered T cells via exogenous molecules, researchers have explored trigger-induced dimerization of different pro-apoptotic effector proteins using the human rapamycin-binding, FK506-binding protein FKBP12 engineered to bind the synthetic drug AP1903 [98101] (Fig. 1b). With a clearance rate of greater than 99% in preclinical in vivo studies [101], the trigger-induced caspase-9 apoptosis system is also able to remove more than 90% of engineered haploidentical T cells in patients within 30 min after induction [102], thus demonstrating the effectiveness of post-translational regulation of cellular processes and the value of this system as a safety switch to prevent graft-versus-host disease.

Synthetic Biology and Solutions

Another feature that can be achieved with posttranslational trigger-controlled elements is the rapid and reversible degradation of target proteins, for example, orthogonal degron systems, such as the auxin-controlled Aid/TIR1 protein degradation system from Arabidopsis thaliana [103] or novel engineered degradation tag from endogenous mammalian proteins, like a certain mutated variant of the FKBP12-rapamycin-binding domain [104] that can provide trigger-inducible degradation of fused proteins (Fig. 1b). Interestingly, the stability of this FKBP12-rapamycin-binding domain can be rescued together with any fused protein through ligand-triggered dimerization with endogenous FKBP12. Later iterations have further improved the system by using a synthetic-ligand-controlled full-length versions of FKBP12 that can self-stabilize in the presence of the ligand [105]. Functional as an exogenously controlled protein ON switch, this system has already demonstrated its applicability in preclinical in vivo studies in mice [106]. Optogenetics, the rational programming of cellular behavior by light, is gathering momentum in synthetic-biology-inspired cell engineering as it provides a traceless, precise, and orthogonal way to remote-control gene expression and cell function. From a biomedical perspective, optogenetic devices have the potential to improve patient compliance by replacing injection-based drug administration by simple illumination and to link therapeutic interventions to electronic devices that power-up and control the light source. To date, we have used light as a tool to modulate a diverse range of cellular processes, such as proteinprotein interactions [107], nuclear import [108,109], and secretion [110], including transcriptional activity [111]. To further develop and exploit light-triggered transgene expression, Folcher et al. pioneered the use of a near-infrared-activated diguanylate cyclase derived from Rhodobacter sphaeroides that is rewired to the endogenous stimulator of interferon gene pathway to mediate near-infrared light-regulated transgene expression in mammalian cells [112]. Similarly, Kim et al. capitalized on a blue-light-activated soluble guanylate cyclase to engineer an erectile optogenetic stimulator [113]. When introduced into the corpora cavernosa of rats, blue light was able to trigger a cGMP surge in the targeted tissue thereby controlling penile erection. This strategy could develop into a viable therapeutic alternative to treat individuals suffering from medical conditions that make them ineligible for erectile dysfunction drugs (e.g., Viagra®, Levitra®, and Cialis®).

Therapeutic designer cells

The ability to program mammalian cells to sense a wide range of stimuli, process information, and instigate well-coordinated predetermined tasks of developing stroke and aneurysm bleeding depending on their emotions [127]. To correlate the release of the neurotransmitter dopamine from reward- and pleasure-seeking behaviors of individuals with obesity, addiction, and drug abuse, the authors were able to create a synthetic gene circuit that can attenuate the risks associated with emotionally triggered high blood pressure in hypertensive individuals through the production and release of the antihypertensive atrial natriuretic peptide (ANP) [117] (Fig. 2a). To enable the continuous monitoring of dopamine and attenuation of hypertension, the authors rewired the cAMP-dependent PKA pathway from the human dopamine receptor D1 to drive the expression of ANP from a synthetic cAMP response-element-containing promoter. The implanted transgenic cells carrying the synthetic gene circuit were able to normalize the blood pressure in mice stimulated by food, sexual arousal, and addictive drugs. Theranostic cell implants to treat diabetes Diabetes mellitus is a complex disease characterized by chronically elevated blood glucose levels due to either an absolute loss of pancreatic -cells leading to insulin deficiency (type 1 diabetes) or an impaired insulin sensitivity in somatic cells that might result in other metabolic disorders such as obesity (type 2 diabetes). Late stages of type 1 diabetes are further associated with the excessive production of acidic ketone bodies in the liver as an alternative energy source to compensate for the limited glucose uptake. These ketone bodies enter into the blood stream, disrupting the body's delicate pH balance, resulting in acute diabetic ketoacidosis. In an attempt to use this physiological symptom as a trigger to treat reoccurring acidosis in type 1 diabetic mice, Ausländer et al. engineered a theranostic gene circuit that produces insulin in response to decreasing pH levels [118]. Using the GPCR T cell death associated gene 8, the authors were able to regulate the production of insulin from a cAMP responseelement-containing promoter through the cAMPdependent PKA pathway. This synthetic circuit demonstrated precise activation both in vitro and in vivo when the pH dropped below the physiological range. Additional, when implanted into type 1 diabetic mice, the designer cells were able to return both insulin and glucose levels to the same level as that of healthy mice. To show that the risk of hyperglycemia in type 2 diabetic mice can be attenuated and regulated with high spatiotemporal precision without requiring periodic invasive procedures, Ye et al. created an optogenetic synthetic gene circuit designed to remotely regulate blood glucose levels using blue light [119] (Fig. 2b). By rewiring the intracellular increase of calcium triggered by the human photo-makes them powerful diagnostic devices that can be employed to screen for systematic disease-related biomarkers (e.g., allergy profiling [114]), as well as novel drug candidates (e.g., phosphodiesterase-targeting drugs [115]), and even to trigger a therapeutic response, similar to a theranostic device. Implantation of such microencapsulated designer cells has been shown to successfully correct metabolic disorders such as hypertension [116,117], diabetes [118121], and obesity in mice [122], whereas the direct injection of ex vivo engineered T cells has demonstrated the ability to target and destroy cancer cells in humans [17,18,123,124]. Compared with most of the above discussed control devices that already offer the potential for integration into cell and gene therapy platforms for improved safety and specificity, these designer cells are engineered to harness the full capability of cells to treat more complex diseases. Metabolic disorders Unhealthy lifestyles and diets in the modern urbanized world characterized by a combination of increased intake of energy-dense foods (i.e., refined sugar and fat) and a lack of physical exercise have drastically increased the occurrence of obesity, glucose intolerance, hypertension, and dyslipidemia and the associated risk of developing heart disease, stroke, and diabetes [125]. In contrast to the high interrelationship of these metabolic disorders, conventional therapeutic strategies centre on separate diagnosis and treatment. In an attempt to address the high complexity of metabolic disorders, theranostic-inspired cell implants have demonstrated an impressive ability to treat metabolic disorders in vivo. A study from Ye et al. demonstrated the ability of synthetic biology to combine current drug-based therapy with gene therapy by employing a synthetic gene circuit triggered by a clinically approved hypertension drug, i.e., Guanabenz, to treat related metabolic syndromes in a combined manner [116]. Using a trace amine receptor (cTAAR1) to sense Guanabenz, the authors demonstrated that ligand-mediated activation of cTAAR1 was able to trigger the cAMP-dependent PKA pathway and drive the expression of a therapeutic fusion protein consisting of the glucagon-like peptide 1 (GLP-1) and leptin from a synthetic cAMP response-element-containing promoter. When implanted in mice developing metabolic syndrome, the coordinated administration of Guanabenz and the production of the GLP-1/leptin fusion protein were able to simultaneously alleviate obesity, hypertension, hyperglycemia, and dyslipidemia. A study by Rössger et al. further demonstrates the ability of theranostic cell implants to address more complex interdependences of metabolic diseases [117]. Because the blood pressure of humans fluctuates with our emotional state [126], patients suffering from hypertension have an increased risk

(a) Attenuate reward-triggered hypertension Dopamine

(b) Restoration of glucose homeostasis-Blue light

(c) Prevent diet-induced obesity Fatty acids

(d) Targeted destruction of cancer cells-Target cell

Tumour-associated antigen

ANP

GLP-1

Pramlintid

Cytotoxicity

DRD1

Melanopsin

ZAP70

Engineered T cell

Endogenous cAMP signalling

Endogenous NFAT signalling Endogenous co-repressors Fatty acids Pmin Pramlintid TtgR operator Lipid sensing receptor Pmin Pramlintid Fatty acids Endogenous co-activators

PLC }Endogenous signal transduction Pi CREB NFAT

Proliferation, survival, and cytokine production  Pmin ANP CRE operator

Pmin GLP-1 NFAT/CRE/SRE operator PhCMV TtgR PPAR PhCMV Melanopsin Exogenously controlled trigger-induced safety switch; Phloretin abolish TtgR DNA binding

Fig. 2. Engineered theranostic designer cells: (a) Reward-triggered high blood pressure regulator. The human dopamine receptor DRD1 functions as an emotional state sensor that, when stimulated by elevated dopamine levels, triggers the endogenous the cAMP-dependent PKA pathway to induce the expression of the antihypertensive ANP from a synthetic cAMP response-element-containing promoter, thus normalizing high blood pressure induced by food, sexual arousal, and addictive drugs (figure adapted from Ref. [117]); (b) Blue-light-regulated glucose homeostasis. The human photo-activated GPCR melanopsin functions as a blue-light sensor control device that, upon blue light stimulation, triggers the endogenous NFAT-dependent signaling pathway, inducing the expression of the GLP-1 from an NFAT response element-containing promoter, which restores glucose homeostasis (figure adapted from Ref. [119]); (c) A self-sufficient gene circuit prevents diet-induced obesity. The ligand-binding domain of human nuclear lipid receptor PPAR fused to the DNA-binding domain of the bacterial phloretin regulated repressor TtgR functions as a lipid sensor that, in the absence of fatty acids and phloretin, binds to TtgR-specific operator-containing promoters where PPAR recruits endogenous co-repressors. In response to increasing fatty acid levels, PPAR releases the co-repressors and recruits endogenous co-activators, thus inducing the expression of the pramlintide, which reduces the adsorption of glucose and other nutrients (e.g., fat). As a safety switch, phloretin can abolish the DNA-binding ability of the synthetic transcription factor by binding TtgR to trigger a conformational change in its DNA-binding domain (figure adapted from Ref. [122]); (d) Adoptive immunotherapy can target and destroy cancer cells. The CAR consists of a single-chain variable fragment of a tumor-associated antigen-specific monoclonal antibody fused to the intracellular signaling domains (CD3) of the TCR and co-receptor (CD8) function as a sensor for cancer cells for engineered cytotoxic T cells. Upon encountering a cell expressing the corresponding tumor-associated antigens on its surface, CAR antigen binding and dimerization triggers the ZAP70/PLC signaling pathway that mediates a cytotoxic immune response. Second- and third-generation CARs incorporate intracellular domains from co-stimulatory receptors, which additionally trigger cytokine production, including T-cell proliferation and survival. Abbreviations: CRE, cAMP response element; PhCMV, human Cytomegalovirus promoter; Pmin, minimal promoter; SRE, serum response element. Chimeric antigen receptor (CAR) Single-chain variable fragment CD8 co-stimulatory domain 2nd or 3rd generation CAR co-stimulatory domains CD3 intracellular signalling domain fatty acid levels. This of mice exposed to a fat-rich diet. Notably, the authors left the phloretin sensitivity of TtgR (an apple system was further able to significantly reduce bodyweight metabolite and cosmetic additive) intact, serving as a safety feature that can override the system by triggering the release of TtgR from the operator site and adding another layer of safety for the treatment of diet-induced obesity with the aid of theranostic cell implants. Theranostic cells that identify and destroy cancer The ability of cytotoxic T cells to distinguish among cells, seek out infected or damaged/dysregulated cells that display target antigens, and destroy the affected cells has made T cells an increasingly attractive tool to combat cancer. Part sensor and part effector cell, these natural theranostic cells already possess the tools required to become tumor-targeting killer cells. With improved understanding of the biological processes that regulate T-cell activation and targeting, researchers have been able to bypass the endogenous immune system's inability to recognize cancer cells as disease cells by engineering T-cell receptors (TCRs) that targets certain tumor-associated antigens [128]. Composed of the intracellular signaling domains from the TCR (CD3) and a co-receptor (CD8) fused to a single-chain variable fragment of a monoclonal antibody, these chimeric antigen receptors (CARs) enable cytotoxic T cells to recognize and destroy cancer cells (Fig. 2d). These tumor-associated antigen-specific T cells have shown great efficiency in targeting and killing a range of cancer cell types [17,18,123,124]. Later iterations of CARs also incorporate intracellular domains from various co-stimulatory receptors (e.g., CD28, CD134, and CD137) to additionally induce proliferation, cytokine production, and cell survival when encountering a cancer antigen to improve treatment efficacy of CAR T-cell immunotherapy [129]. However, the current generations of CAR-based cancer therapies that use single antigen targets can also be prone to on-target off-tumor effects [130,131]. To reduce nonspecific targeting, synthetic biologists have proposed certain interesting solutions that could increase the tumor specificity of CAR-based cancer therapy. For example, Wei et al. introduced a feedback loop in the TCR intracellular signaling pathway that led to novel activation patterns that can modulate the amplitude of the T-cell response [132], and Kloss et al. engineered a CAR-based logic AND gate that requires simultaneous stimuli from two tumor-associated antigens for activation, which further demonstrates the potential theranostic modularity of adaptive immunotherapy using ex vivo engineered T cells [133]. activated GPCR melanopsin to the calcium-dependent stimulation of calcineurin and calcineurin-mediated phosphorylation of the transcription factor nuclear factor of activated T cells (NFAT), the authors were able to remotely control the production of GLP-1 from an NFAT response-element-containing promoter. When implanted into type 2 diabetic mice, the designer cells were able to improve glucose tolerance and endogenous insulin regulation. An analogous strategy used to regulate blood glucose levels in mice developed by Stanley et al. [120] employed iron oxide nanoparticles to control the production of insulin through radio waves. Using a His-tag-modified, temperature-sensitive calcium channel TRPV1, the authors could specifically control the heat-mediated calcium influx using low-frequency radio waves that heated the anti-His iron oxide nanoparticles, which in turn activated the production of insulin from an NFAT response-element-containing promoter via the calcineurin-NFAT pathway. The authors went on to demonstrate control of TRPV1 with genetically encoded ferritin nanoparticles. In a follow-up study, Stanley et al. reported improved functionality of the system by adding a myristoylation signal to the genetically encoded nanoparticles to direct ferritin to the cell membrane [121]. Additionally, the authors demonstrated the functional delivery of the system in vivo using a replication-deficient adenovirus that, when subjected to low-frequency radio waves, was able to maintain blood glucose homeostasis in mice with eradicated -cells. Theranostic cell implants to treat obesity Recently, theranostic cell implants have also demonstrated interesting therapeutic solutions for regulation of the metabolic balance between food intake and energy expenditure to prevent diet-induced obesity. To this end, Rössger et al. engineered a closed-loop synthetic gene circuit to continuously balance blood fatty acid levels [122] (Fig. 2c). By fusing the ligand-binding domain of the human nuclear lipid receptor peroxisome proliferator-activated receptor (PPAR) to the bacterial DNA-binding repressor TtgR, the authors were able to create a binary lipid-sensing transcription factor. In its inactivated state, the synthetic transcription factor binds to the TtgR-specific operator-containing promoter, where PPAR attracts endogenous co-repressors, thus preventing activation of the transgene. When exposed to increasing levels of fatty acids, the co-repressors are released and replaced by endogenous co-activators to trigger the transcription of the transgene. When implanted into the mouse abdomen, the device was able to release the appetite-suppressing amylin derivative pramlintide (a licensed adjuvant for type 2 diabetes that reduces the adsorption of glucose and other nutrients such as fat) in a dose-dependent manner, thus reducing the blood

Delivery of Cell-Based and Gene-based Therapeutics

Despite our growing ability to engineer sophisticated therapeutic cell and gene systems, successful implementation of these systems is ultimately limited by four key factors: (i) delivery efficiency, (ii) persistence in the patient, (iii) therapeutic impact, and (iv) immunological properties. Although precise and efficient transcriptional, translational, and post-translational control devices can address some of these limitations, most are rooted in the current inability to safely and efficiently deliver therapeutic solutions to patients. Autologous stem-cell-based therapies have begun to overcoming some of these limitations and make headway in clinical applications, but the scarce nature of these cells still prompts the need for alternative cell sources or direct in vivo correction of the causes of the disease. Cell encapsulation To bypass the immunological obstacle of using allogeneic and xenogeneic cell sources, biologists and material scientists were brought together to engineer biocompatible containers that can shield their cellular content from components of the host immune system (e.g., antibodies) while allowing nutrients and oxygen to freely pass through the semipermeable membrane, preserving the viability and functionality of implanted cells. The foundation for creating such microdevices is the choice of suitable biocompatible/biodegradable materials with tunable biometric and mechanical properties. Currently, several clinical trials are underway to investigate the long-term feasibility of encapsulated insulin-producing cells for treatment of diabetes that use either the natural polymer alginate-poly- L -lysine or the synthetic-material-based polyethylene glycol [134]. In addition to providing a suitable scaffold for encapsulated cells at low cost, the superior biocompatibility of alginate makes it the preferred choice for in vivo applications [135]. However, the low tensile strength of alginate polymers poses a challenge for long-term implant stability, which may be overcome by the use of customizable synthetic polymers [136138]. However, despite the continuous improvements in biocompatible materials and implant design that have resulted in promising clinical trials to treat diabetes, the relatively short lifetime of active cells inside the implant still represents a limitation that remains to be resolved. Although future developments will surely address these issues, cell encapsulation remains a highly valuable tool that can be used to test prospective therapeutic solutions, as demonstrated in the previous section.--Viral delivery vectors In contrast to microcapsules that function autonomously without exposing the host to foreign genetic material, viral delivery platforms represent an invasive alternative for stably introducing and expressing genetic material in patients with the aim of achieving long-term therapeutic effects. The highly evolved biological machinery of viruses that efficiently gains access to a variety of host cells makes them a desirable platform for therapeutic gene transfer. The ideal therapeutic viral vector should exclusively enable efficient gene transfer without off-target expression, immunological side effects, and subsequent viral gene expression and replication in the host. Such recombinant viral gene transfer vectors are generally engineered by removing most viral genes except for certain viral elements that are essential for in vitro genome replication and packaging to encompass transgenes. For safe in vitro production, the necessary packaging and envelope genes are instead supplied in trans on separate plasmids or with helper viruses. The current state of the art in viral gene transfer is based on three different viral platforms: retroviruses, adenoviruses (Ads), or adeno-associated viruses (AAVs). Retroviruses, which are characterized by an RNA genome that is reverse-transcribed into the pro-viral complementary DNA, have been one of the longest preferred gene transfer vectors in clinical settings. Extensive studies and characterization of retroviruses of different origins and their ability to stably integrate the pro-viral DNA into target cell genome make retroviruses a favorable platform for stable long-term gene transfer. Current generations of retroviral gene therapy vectors are primarily based on lentiviruses, typically the human immunodeficiency virus, which can readily infect both dividing and nondividing cells [139]. To overcome the inherent limitations of retroviral vectors, for example, limited tissue tropism of individual retroviral strains and integration-triggered oncogene expression, vector engineering has made great efforts to improve the efficiency, specificity, and safety of retroviral vectors through pseudo-typing with heterologous glycoproteins from unrelated viruses to alter viral tropism [140] and through engineering so-called self-inactivating vectors in which enhancer and promoter elements contained in the long terminal repeats are deleted and transgene expression is instead controlled by internal promoters [141]. Ads and AAVs are DNA viruses that primarily reside as episomal structures in the nucleus of the host, thus reducing the risk of insertional mutagenesis that can infect both dividing and nondividing cells from a wide range of tissues with high efficiency. The initial generations of Ad vectors were typically based on either serotype 2 or serotype 5 (Ad2 or Ad5) due to their high transduction efficiency and broad --In contrast, AAV vectors can be relatively easily reengineered to bypass pre-existing immunity through the rational engineering of AAV vectors. By eliminating unmethylated CpG motifs contained in the vector genome and introducing specific capsid mutations that reduce proteolytic degradation and processing of the virions, recombinant AAV vectors can efficiently circumvent the TLR-9-mediated immune response and major histocompatibility complex class I presentation in antigen-presenting cells [158,159]. Additionally, directed evolution strategies of AAV vectors have proven a useful tool for engineering chimeric capsids to which innate neutralizing antibodies have a reduced affinity [160] and for creating vectors with altered tropism [161]. Alternative delivery vectors Although viral platforms using recombinant retroviruses, Ads, and AAVs have significantly advanced the field of therapeutic gene transfer, their inherent limitations relative to insertional mutagenesis, immunogenic potential, specificity, and limited DNA packaging capacity prompt the need for alternative gene transfer vectors. Since the transfer of large sequences of genomic DNA has been limited by the ability to transfer and retain the intact DNA, HSV-1 and baculovirus vectors with genomes upward of 150 kb are promising gene therapy vectors that can accommodate considerably larger nonviral DNA packages, which in theory could facilitate the transfer of multiple transgenes with appropriate genome regulatory regions to replace complete malfunctioning cascades and pathways. For example, with advances in amplicon-mediated production in bacteria, HSV-1 vectors that can accommodate and transduce nonviral DNA well over 100 kb, which if combined with viral elements from AAV can mediate site-specific integration for long-term expression, have been produced [162,163]. Similarly, Baculovirus has demonstrated the ability to efficiently transduce large nonviral DNA packages in both dividing and nondividing cells [164,165] and because Baculovirus is strictly an insect pathogen with no observed pre-existing immunity in humans [166], Baculovirus-based gene therapy vectors could prove to be a safe alternative to other viral vectors. Additionally, alternative nonviral delivery platforms have recently raised attention for direct delivery of RNA and circular DNA molecules through cationic polymers or lipids that condense negatively charged genetic material into spherical nanoparticles. However, nonviral vectors currently struggle with several limitations related to delivery efficiency, immunogenicity, and sustained effect due to RNA instability and rapid decreased plasmid expression in vivo. To alleviate certain of these issues, we are currently evaluating various polymers and lipids with novel properties for their ability to efficiently transduce tropism. To remove the replication ability of recombinant vectors, we introduce certain deletions in the early genes E3 and E1 together with mutations of E2 and/or E4 to reduce the toxicity and cytotoxic T-cell-mediated immune responses induced by viral gene expression [142]. The most promising advancements of Ad gene therapy vectors are the so-called gutted or helper-dependent constructs in which all coding viral genes except the inverted terminal repeats and the packaging signal are removed, thus providing increased nonviral DNA packaging capacity, decreased immunogenicity, and prolonged transgene expression [143]. However, since most of the viral components required for in vitro production are supplied in trans from an Ad helper virus, this platform carries the risk of contaminating the viral stock with particles containing the helper-virus genome. Later iterations of this vector system have been able to solve this issue by the use of non-Ad helper viruses but at the expense of production yield [143]. To promote tissue-specific targeting of the Ad vector, we have explored different approaches in altering the tropism of Ad (e.g., exchanging the fiber and knob domains [144], introducing interchangeable peptide [145,146], or introducing antibody domains that direct the virus to specific cell types [147]). In comparison to retroviruses and Ads, AAVs are relatively simple viral vectors composed solely of two genes, that is, rep and cap, which respectively encode the DNA replication and packaging machinery. Because AAVs are naturally replication deficient, recombinant AAV-based gene transfer vectors require the presence of Ad helper viruses in addition to the rep and cap genes in trans to facilitate in vitro production for a high production yield. Later iterations of AAV production systems have eliminated this requirement by supplying certain Ad genes on helper plasmids [148]. However, despite the full deletion of all viral genes from the viral vector, recombinant AAVs are limited to a maximal accommodation of upwards of 5-kb nonviral DNA without compromising the viral titres and infectivity [149]. The main limitations of the Ad and AAV gene therapy vectors are related to immunological memory because most humans have been previously exposed to at least one Ad or AAV serotype during their lives [150]. Attempts to bypass the pre-existing immunity to specific Ad serotypes require subsequent administration of vectors with alternative or chimeric Ad serotypes [151,152]. However, because both the major capsid protein hexon and the Ad fiber appear to contribute to specific immunity [153] and the immune system is able generate antibodies that are cross-reactive to several natural human Ad serotypes [154,155], this obstacle remains technically difficult to overcome. A promising alternative could be to replace the serotype with rare or animal Ad serotypes to which no pre-existing immunity has been observed [156,157]. genetic material [167], including the use of mini-circle DNA, which lacks a bacterial backbone and can attain significantly greater expression levels over longer periods of time compared with plasmid DNA [168].

Conclusions and future perspectives

In recent years, cell and gene therapies have made significant strides toward clinical application, providing novel therapeutic alternatives to previously incurable diseases. Although the initial generations of cell and gene therapy platforms suffered significant blowbacks, current generations are steadily improving due to viral vector engineering and synthetic-biology-inspired control devices. With increasingly advanced control devices becoming readily available, future cell and gene therapy platforms may incorporate such concepts as exogenous control elements that confer the ability to continuously control and modulate transgene expression and closed-loop transgene regulatory devices capable of autonomously modulating the dosage of therapeutic transgenes depending on patients' needs. However, because the current generation of gene transfer vectors is primarily limited by relative small tissue tropism and because cell therapy is limited to lineages and cell types that can easily be isolated, cultured, and engrafted back into the patient, further studies are needed on the efficient targeting of a broader range of tissues and cell types. To this end, we poise the rapidly progressing fields of stem cell biology and cell reprogramming to introduce the next stage in cell-based medicine. The ability to generate induced pluripotent stem cells from terminally differentiated cells could constitute an unlimited cell source for replacement of diseased, damaged, and aging cells in the future. This effort will require precise differentiation regimes to ensure efficient differentiation of pluripotent stem cells into desired cell lineages. However, even with the most efficient combination of chemicals, growth factors, and other effector molecules, the differentiation efficiency currently remains low, often resulting in undesired cell lineages. To this extent, stem cell differentiation could benefit greatly from temporally precise gene control devices created by synthetic biology to more accurately control the differentiation and lineage commitment of stem cells. With the addition of programmable transcription factors that can be retargeted to virtually any genetic sequence, scientists could set up artificial differentiation programs to mimic natural stimuli that determine cellular patterning and differentiation. Furthermore, these programmable DNA-targeting platforms have rapidly gained momentum as genome-editing platforms that have the potential to directly correct the primary cause of genetic diseases. Using programmable nucleases based on the ZF protein (ZFNs),  TALEs (TALENs), and CRISPR/Cas9, scientists have demonstrated the promising genome correction of several genetic diseases in induced pluripotent stem cells [169,170] and HSCs [171,172], including germlines and terminally differentiated tissues [173,174]. The novel ability to modify specific genetic sequences makes programmable nucleases interesting additions to gene transfer vectors to ensure long-term expression without the risk of insertional mutagenesis by targeting integration to a safe-harbor locus [175]. These steps open the door to direct the hardwiring of genes into stem cells to introduce novel characteristics into progeny cells that can potentially form fully functional tissues or artificial organs that perform novel tasks. Although still in its infancy, synthetic biology has extensively explored the concept of functional tissues in proof-of-principle studies using designer cells to treat a range of metabolic syndromes [116 122]. These immunoisolated xenogeneic designer cells demonstrate that the therapeutic potential of cells extends beyond replacement or correction of damaged tissues, which could provide a valuable addition to medicine in situations in which these designer cells could eliminate the need for continuous systematic administration of pharmaceuticals or other more invasive treatments. Hence, these cell implants could be viewed as removable "flash drive"-inspired plug-and-play devices that can temporarily "run" a theranostic "program" and autonomously attenuate disease-related symptoms. Additionally, with the emergence of remote-controlled gene circuits (such as light and radio-wave-induced systems [119121]), these designer cell implants could also become computer-interfaced devices, controlled by an electronic sensor (such as a blood glucose sensor) or directly by a medical professional, a concept that was recently further explored by Folcher et al., who developed a braincomputer-interfaced cell-based device, where the operator can modulate the output of the device with his or her thoughts [112], which in the future could be calibrated to pain-stimulated or epileptic brain waves in order to mediate precise production of corresponding therapeutics. However, due to the short-term applicability of these designer cell implants, future attempts to find novel cell-based therapeutic solutions will most likely start by considering alternative platforms to replace xenogeneic cell lines with more viable allogeneic of autogenic alternatives. 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Grassman, et al., Physiological promoters reduce gene therapy platforms as they become more advanced, precise, and dynamically complex. However, for more complex synthetic systems, success hinges on the daunting question of whether we can ensure that these systems do not interfere with endogenous human metabolic pathways and systems. However, what is clear is that synthetic biology has set the stage for future innovative therapeutic solutions, and we look forward with excitement to the medical advancements it might bring.

Acknowledgments

The authors thank Mingqi Xie and Pratik Saxena for generous advice and comments on the manuscript. Work in the laboratory of M.F. is supported by a European Research Council advanced grant (ProNet No. 321381) and, in part, by the National Centre of Competence in Research Molecular Systems Engineering.

Received 17 June 2015; Received in revised form 18 August 2015; Accepted 19 August 2015 Available online 1 September 2015 Keyword: synthetic biology Abbreviations used: HSC, hematopoietic stem cell; ZF, zinc finger; TALE, transcription activator-like effector; CRISPR, clustered regular interspaced short palindromic repeat; RNAi, RNA interference; GPCR, G-protein-coupled receptor; GLP-1, glucagon-like peptide 1; ANP, atrial natriuretic peptide; NFAT, nuclear factor of activated T cells; PPAR, peroxisome proliferator-activated receptor ; TCR, T-cell receptor; CAR, chimeric antigen receptor.

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Review: Synthetic Biology and Solutions

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Synthetic membranes created to mimic properties of living cells

Biochemists at the University of California San Diego have developed artificial cell membranes that grow and remodel themselves in a manner similar to that of living mammalian cells. -The achievement, detailed in a paper published in the Proceedings of the National Academy of Sciences, follows the successful design last year in the same laboratory of artificial, or synthetic, cell membranes capable of sustaining continual growth. The two developments now bring the researchers closer to mimicking all of the properties of living mammalian cell membranes with synthetic components. That's important because synthetic membranes that accurately mimic the behavior of living mammalian cell membranes could be used by biomedical researchers to develop more effective drugs that target membrane proteins and better understand the chemical changes that occur in dysfunctional membranes during disease. "While artificial membranes have been used to model the properties of native membranes, previous methods have not been able to mimic lipid membrane remodeling," said Neal Devaraj, an associate professor of chemistry and biochemistry at UC San Diego who headed the research team for both studies. "In our latest study, we show that reversible chemical reactions can be harnessed to achieve spontaneous remodeling of lipids in synthetic membranes." Living cells continually remodel their membranes to change their physical characteristics, a process that can affect the behavior of other biomolecules in the cell membrane. "Cells use lipid remodeling to respond to their environment and maintain membrane homeostasis or to carry out specific functions such as division and signaling," said Andrew Rudd, a co-author of the study and graduate student in the Devaraj lab. "Using phospholipid remodeling allows cells to generate new phospholipid species by recycling existing phospholipids instead of making them from scratch. This saves the cell time and energy." Devaraj explained that his team's latest development provides a way for biochemists to better understand the changes that occur in phospholipid membranes during lipid remodeling. "One exciting application would be to probe the behavior of bound and integral membrane proteins in response to shifts in membrane composition," explained Roberto Brea, a postdoctoral fellow in the Devaraj lab and the lead author of the study. "Integral membrane proteins are extremely important and common drug targets and we need a way to understand their behavior in lipid bilayers. This is one way to do that." Story Source-Materials provided by University of California - San Diego. Original written by Kim McDonald. Journal Reference-Roberto J. Brea, Andrew K. Rudd, and Neal K. Devaraj. Nonenzymatic biomimetic remodeling of phospholipids in synthetic liposomes. PNAS, July 2016 DOI: 10.1073/pnas.1605541113

University of California - San Diego. "Synthetic membranes created to mimic properties of living cells." ScienceDaily. ScienceDaily, 18 July 2016. <www.sciencedaily.com/releases/2016/07/160718160923.htm>.

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Vitamin A deficiency is detrimental to blood stem cells

Many specialized cells, such as in the skin, gut or blood, have a lifespan of only a few days. Therefore, steady replenishment of these cells is indispensable. They arise from so-called "adult" stem cells that divide continuously. In addition, there is a group of very special stem cells in the bone marrow that were first discovered in 2008 by a research team led by Andreas Trumpp, who is a division head at the DKFZ and director of HI-STEM. These cells remain in a kind of dormancy most of the time and only become active in an emergency such as bacterial or viral infections, heavy blood loss, or in the wake of chemotherapy. Once their work is done, the body sends its most potent stem cells back to sleep. The scientists assume that this protects them from dangerous mutations that may lead to leukemia.---The mechanisms that activate these special stem cells or make them go back to sleep after their work is done have remained elusive until now. The scientists have now identified retinoic acid, a vitamin A metabolite, as a crucial factor in this process. If this substance is absent, active stem cells are unable to return to a dormant state and mature into specialized blood cells instead. This means that they are lost as a reservoir. This was shown in studies with specially bred mice whose dormant stem cells are green fluorescent. "If we feed these mice on a vitamin A deficient diet for some time, this leads to a loss of the stem cells," said Nina Cabezas-Wallscheid, who is the first author of the publication. "Thus, we can prove for the first time that vitamin A has a direct impact on blood stem cells."--This finding not only enhances our understanding of the development of blood cells, it also sheds new light on prior studies that demonstrate that vitamin A deficiency impairs the immune system. "This shows how vitally important it is to have a sufficient intake of vitamin A from a balanced diet," Cabezas-Wallscheid emphasized. The body cannot produce its own vitamin A. -The scientists also have hopes for new prospects in cancer treatment. There is evidence that cancer cells, like healthy stem cells, also rest in a state of dormancy. When dormant, their metabolism is almost completely shut down -- and this makes them resistant to chemotherapy. "Once we understand in detail how vitamin A or retinoic acid, respectively, sends normal and malignant stem cells into dormancy, we can try to turn the tables," explained Trumpp. "If we could make cancer cells temporarily enter an active state, we could thus make them vulnerable to modern therapies."-In addition, in collaboration with colleagues from the European Bioinformatics Institute in Cambridge, the team performed genome-wide analyses of single cells and discovered that the transition from dormant to active stem cells and then on to progenitor cells is a continuous one and follows a different path for each individual cell. So far, scientists had assumed that specific cell types develop step by step in a defined pattern. This finding revolutionizes the previous concept of how cell differentiation in the body takes place. -Story Source-Materials provided by German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ). Journal Reference Nina Cabezas-Wallscheid, Florian Buettner, Pia Sommerkamp, Daniel Klimmeck, Luisa Ladel, Frederic B. Thalheimer, Daniel Pastor-Flores, Leticia P. Roma, Simon Renders, Petra Zeisberger, Adriana Przybylla, Katharina Schönberger, Roberta Scognamiglio, Sandro Altamura, Carolina M. Florian, Malak Fawaz, Dominik Vonficht, Melania Tesio, Paul Collier, Dinko Pavlinic, Hartmut Geiger, Timm Schroeder, Vladimir Benes, Tobias P. Dick, Michael A. Rieger, Oliver Stegle, Andreas Trumpp. Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy. Cell, 2017; DOI: 10.1016/j.cell.2017.04.018 German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ). "Vitamin A deficiency is detrimental to blood stem cells." ScienceDaily. ScienceDaily, 5 May 2017. <www.sciencedaily.com/releases/2017/05/170505112612.htm>.

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Mechanisms of Nanoparticle-Induced Oxidative Stress and Toxicity

Amruta Manke,1 Liying Wang,2 and Yon Rojanasakul1

1Department of Pharmaceutical Sciences and Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506, USA

2Physiology and Pathology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA

Received 9 May 2013; Accepted 16 July 2013

Academic Editor: Nikhat J. Siddiqi

Abstract

The rapidly emerging field of nanotechnology has offered innovative discoveries in the medical, industrial, and consumer sectors. The unique physicochemical and electrical properties of engineered nanoparticles (NP) make them highly desirable in a variety of applications. However, these novel properties of NP are fraught with concerns for environmental and occupational exposure. Changes in structural and physicochemical properties of NP can lead to changes in biological activities including ROS generation, one of the most frequently reported NP-associated toxicities. Oxidative stress induced by engineered NP is due to acellular factors such as particle surface, size, composition, and presence of metals, while cellular responses such as mitochondrial respiration, NP-cell interaction, and immune cell activation are responsible for ROS-mediated damage. NP-induced oxidative stress responses are torch bearers for further pathophysiological effects including genotoxicity, inflammation, and fibrosis as demonstrated by activation of associated cell signaling pathways. Since oxidative stress is a key determinant of NP-induced injury, it is necessary to characterize the ROS response resulting from NP. Through physicochemical characterization and understanding of the multiple signaling cascades activated by NP-induced ROS, a systemic toxicity screen with oxidative stress as a predictive model for NP-induced injury can be developed.

1. Introduction

The growing field of nanotechnology has transformed many sectors of the industrial field with their breakthrough applications in the areas of biotechnology, electronics, medicinal drug delivery, cosmetics, material science, aerospace engineering, and biosensors. Manufactured nanomaterials (NM) have gained commercial interest in a variety of consumer products. Their novel physicochemical, thermal, and electrical properties facilitate their application in clothing, medicine, and cosmetics thereby increasing the probability for human and environmental contact with these NM [1–3]. Of all the NM, carbon nanotubes (CNT) and metal-based nanoparticles (NP) have generated considerable commercial interest owing to their remarkable intrinsic properties such as high tensile strength and conductivity, which in turn meet the needs of the specific application for which these NP are designed [4, 5]. Their widespread use raises concerns of their inadvertent exposure in humans and the consequent deleterious health effects [6]. As compared to the growing commercial interest of NM, modest research effort has been invested in evaluating the potential adverse effects of these engineered NM. The sheer multiplicity of the physicochemical parameters of NM such as size, shape, structure, and elemental constituents makes the investigation of their toxic effects complex and challenging [7]. Some of the paradigms for NP-mediated toxicity include oxidative stress, inflammation, genetic damage, and the inhibition of cell division and cell death [8–11]. Most work to date has suggested that ROS generation (which can be either protective or harmful during biological interactions) and consequent oxidative stress are frequently observed with NP toxicity [3, 9]. The physicochemical characterization of NP including particle size, surface charge, and chemical composition is a key indicator for the resulting ROS response and NP-induced injury since many of these NP intrinsic properties can catalyze the ROS production [6]. NP-mediated ROS responses have been reported to orchestrate a series of pathological events such as genotoxicity, inflammation, fibrosis, and carcinogenesis. For instance, CNT-induced oxidative stress triggers cell signaling pathways resulting in increased expression of proinflammatory and fibrotic cytokines [12]. Some NP have been shown to activate inflammatory cells such as macrophages and neutrophils which can result in the increased production of ROS [13–15]. Other NP such as titanium dioxide (TiO2), zinc oxide (ZnO), cerium oxide (CeO2), and silver NP have been shown to deposit on the cellular surface or inside the subcellular organelles and induce oxidative stress signaling cascades that eventually result in oxidative stress to the cell [16]. The mechanism for ROS generation is different for each NP and to date the exact underlying cellular mechanism for ROS generation is incompletely understood and remains to be elucidated. Most of the metal-based NP elicit free radical-mediated toxicity via Fenton-type reactions [4, 17], whereas mitochondrial damage plays a major role in CNT-mediated ROS generation [18]. However, it is inaccurate to assume that ROS generation is a prerequisite to NP-induced toxicity since a few studies have reported the direct toxicity of NP without causing ROS [19]. Nevertheless, ROS generation is a major event during NP-induced injury that needs to be thoroughly characterized in order to predict NP-induced toxicity. This review will focus on oxidative stress as a mechanism for understanding NP-induced toxicity. For this paper, we have considered metal-based NP and CNT in the light of oxidative stress. The relationship between different NP characteristics and resulting oxidative stress is discussed.

1.1. Generation of ROS

ROS, key signaling molecules during cell signaling and homeostasis, are reactive species of molecular oxygen. ROS constitute a pool of oxidative species including superoxide anion ( ), hydroxyl radical (OH•), hydrogen peroxide (H2O2), singlet oxygen (1O2), and hypochlorous acid (HOCl). ROS are generated intrinsically or extrinsically within the cell. Molecular oxygen generates , the primary ROS via one-electron reduction catalyzed by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Further reduction of oxygen may either lead to H2O2 or OH• via dismutation and metal-catalyzed Fenton reaction, respectively [20, 21]. Some of the endogenous sources of ROS include mitochondrial respiration, inflammatory response, microsomes, and peroxisomes, while engineered NM, environmental pollutants act as exogenous ROS inducers. Physiologically, ROS are produced in trace amounts in response to various stimuli. Free radicals occur as essential byproducts of mitochondrial respiration and transition metal ion-catalyzed Fenton-type reactions [20]. Inflammatory phagocytes such as neutrophils and macrophages induce oxidative outburst as a defense mechanism towards environmental pollutants, tumor cells, and microbes. A variety of NP including metal oxide particles induce ROS as one of the principal mechanisms of cytotoxicity [22]. NP have been reported to influence intracellular calcium concentrations, activate transcription factors, and modulate cytokine production via generation of free radicals [12, 23].

1.2. Oxidative Stress

Antioxidant metabolite

Solubility

Concentration in human serum (μM)[79]

Concentration in liver tissue (μmol/kg)

Ascorbic acid (vitamin C)

Water

50 – 60[80]

260 (human)[81]

Glutathione

Water

4[82]

6,400 (human)[81]

Lipoic acid

Water

0.1 – 0.7[83]

4 – 5 (rat)[84]

Uric acid

Water

200 – 400[85]

1,600 (human)[81]

Carotenes

Lipid

β-carotene: 0.5 – 1[86] retinol (vitamin A): 1 – 3[87]

5 (human, total carotenoids)[88]

α-Tocopherol (vitamin E)

Lipid

10 – 40[87]

50 (human)[81]

Ubiquinol (coenzyme Q)

Lipid

5[89]

200 (human)[90]

Abundance of ROS can have potentially damaging biological responses resulting in oxidative stress phenomenon. It results from an imbalance between the production of ROS and a biological system’s ability to readily detoxify the reactive intermediates or repair the resulting damage. To overcome the excess ROS response, cells can activate enzymatic and nonenzymatic antioxidant systems [24]. The hierarchical model of oxidative stress was proposed to illustrate a mechanism for NP-mediated oxidative stress [4, 9]. According to this model, cells and tissues respond to increasing levels of oxidative stress via antioxidant enzyme systems upon NP exposure. During conditions of mild oxidative stress, transcriptional activation of phase II antioxidant enzymes occurs via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) induction. At an intermediate level, redox-sensitive mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain enhancer of activated Bcells (NF-κB) cascades mount a proinflammatory response. However, extremely toxic levels of oxidative stress result in mitochondrial membrane damage and electron chain dysfunction leading to cell death. Some of the key factors favoring the prooxidant effects of engineered NM include either the depletion of antioxidants or the increased production of ROS. Perturbation of the normal redox state contributes to peroxide and free radical production that has adverse effects on cell components including proteins, lipids, and DNA [23]. Given its chemical reactivity, oxidative stress can amount to DNA damage, lipid peroxidation, and activation of signaling networks associated with loss of cell growth, fibrosis, and carcinogenesis [16, 25, 26]. Besides cellular damage, ROS can result from interactions of NP with several biological targets as an effect of cell respiration, metabolism, ischemia/reperfusion, inflammation, and metabolism of various NM [22]. Most significantly, the oxidative stresses resulting from occupational NM exposures as well as experimental challenge with various NP lead to airway inflammation and interstitial fibrosis [27–30].

1.3. Nanoparticle-Induced Oxidative Stress

Nanomaterials of varying chemical composition such as fullerenes, CNT, and metal oxides have been shown to induce oxidative stress [20, 31]. The key factors involved in NP-induced ROS include (i) prooxidant functional groups on the reactive surface of NP; (ii) active redox cycling on the surface of NP due to transition metal-based NP; and (iii) particle-cell interactions [22, 25]. From a mechanistic point of view, we discuss the sources of ROS based on the physicochemical parameters and particle-cell interactions.--Several studies demonstrate the significance of reactive particle surface in ROS generation [20, 32]. Free radicals are generated from the surface of NP when both the oxidants and free radicals bound to the particle surface. Surface bound radicals such as SiO• and present on quartz particles are responsible for the formation of ROS such as OH• and [17, 25]. Ambient matter such as ozone and nitrogen dioxide (NO2) adsorbed on the particle surface is capable of inducing oxidative damage [16]. Reduced particle size results in structural defects and altered electronic properties on the particle surface creating reactive groups on the NP surface [27, 33]. Within these reactive sites, the electron donor or acceptor active sites interact with molecular O2 to form which in turn can generate additional ROS via Fenton-type reactions [3]. For instance, NP such as Si and Zn with identical particle size and shape lead to diverse cytotoxicity responses due to their surface properties. ZnO being more chemically active than SiO2, led to increased formation resulting in oxidative stress. Free radicals are either directly bound to the NP surface or may be generated as free entities in an aqueous suspension [17]. Dissolution of NP and subsequent release of metal ions can enhance the ROS response [25]. For instance, aqueous suspensions of quartz particles generate H2O2, OH•, and 1O2 [17, 20, 32].--Apart from surface-dependent properties, metals and chemical compounds on the NP surface accelerate the ROS response [34]. Transition metals including iron (Fe), copper (Cu), chromium (Cr), vanadium (V), and silica (Si) are involved in ROS generation via mechanisms such as Haber-Weiss and Fenton-type reactions [25]. Fenton reactions usually involve a transition metal ion that reacts with H2O2 to yield OH• and an oxidized metal ion. For example, the reduction of H2O2 with ferrous iron (Fe2+) results in the formation of OH• that is extremely reactive and toxic to biological molecules [21]. Cu and Fe metal NP have been reported to induce oxidative stress ( and OH•) via Fenton-type reaction [26], while the Haber-Weiss-type reaction involves a reaction between oxidized metal ion and H2O2 to induce OH• [21, 35]. NP including chromium, cobalt, and vanadium can catalyze both Fenton and Haber-Weiss-type reactions [26]. Glutathione reductase, an antioxidant enzyme, reduces metal NP into intermediates that potentiate the ROS response. In addition, some metal NP (Ar, Be, Co, and Ni) promote the activation of intercellular radical-inducing system such as the MAPK and NF-κB pathways [36].--In addition to the prooxidant effect of NP, ROS are also induced endogenously where the mitochondrion is a major cell target for NP-induced oxidative stress. Once NP gain access into the mitochondria, they stimulate ROS via impaired electron transport chain, structural damage, activation of NADPH-like enzyme system, and depolarization of the mitochondrial membrane [37, 38]. For instance, cationic polystyrene nanospheres induce mediated apoptosis in murine macrophages based on their ability to target mitochondria [38].--

Cellular internalization of NP has been shown to activate immune cells including macrophages and neutrophils, contributing to ROS/RNS [22, 25]. This process usually involves the activation of NADPH oxidase enzymes. In vivo particle exposures such as silica activate the rich pool of inflammatory phagocytes within the lung causing them to induce oxidative outburst [39]. NP with smaller particle size are reported to induce higher ROS owing to their unique characteristics such as high surface to volume ratio and high surface charge. Particle size determines the number of reactive groups/sites on the NP surface [34, 37, 40]. The pulmonary responses induced by inhaled NP are considered to be greater than those produced by micron-sized particles because of the increased surface area to particle mass ratio [28]. Larger surface area ensures that the majority of the molecules are exposed to the surface than the interior of the NM [3]. Accordingly, nano-sized SiO2 and TiO2 and MWCNT induce greater ROS as compared to their larger counterparts [41]. Additionally, a study with cobalt/chromium NP exposure demonstrated particle size dependent ROS-mediated genotoxicity [42].

2. Oxidant Generation via Particle-Cell Interactions

Besides being self-oxidative in nature, NP react with cells and induce their prooxidant effects via intracellular ROS generation involving mitochondrial respiration and activation of NADPH-like enzyme systems [43]. NP can activate the cellular redox system specifically in the lungs where immune cells including alveolar macrophages (AM) and neutrophils act as direct ROS inducers. Professional phagocytic cells including neutrophils and AM of the immune system induce substantial ROS upon internalization of NP via the NADPH oxidase enzyme system [44]. The phagocytic oxidative outburst is attributable to some of the NP physicochemical properties. In case of silica and quartz particles, inflammation-induced ROS was associated with the surface-based radical-generating properties of the particles [45]. Additionally, NP from the residual oily fly ash and diesel exhaust activate the pool of inflammatory phagocytes resulting in massive ROS release [46]. Furthermore, adsorption of chemicals such as organic matter onto the NP surface may drive the inflammation-induced oxidative stress [24].

2.1. Lung Injury Caused by Nanoparticle-Induced Reactive Nitrogen Species

Besides oxidative damage, NP exposure within the lung is reported to induce reactive nitrogen species (RNS). Particle deposition in the lung causes recruitment of inflammatory cells that generate ROS, clastogenic factors, and cytokines either harming or stimulating resident lung cells [31]. Inflammatory phagocytes are an important source of RNS/ROS generation within the lung. Owing to their inducible nitric oxide synthase (iNOS) activity, phagocytes can produce a large amount of genotoxic RNS, including nitric oxide (NO•) and the highly reactive peroxynitrite (ONOO−). ONOO− formed by the reaction of NO• and causes DNA fragmentation, lipid oxidation, and protein dysfunction consequently contributing to particle-induced lung injury [47]. In vivo exposure to SiO2 and quartz NP elicited an RNS response characterized by increased iNOS and NO• within the lung as a result of phagocyte influx [48, 49].

2.2. Mechanisms of ROS Production and Apoptosis within Metal Nanoparticles

Apoptosis has been implicated as a major mechanism of cell death caused by NP-induced oxidative stress [50–52]. Among the different apoptotic pathways, the intrinsic mitochondrial apoptotic pathway plays a major role in metal oxide NP-induced cell death since mitochondria are one of the major target organelles for NP-induced oxidative stress [38]. High levels of ROS in the mitochondria can result in damage to membrane phospholipids inducing mitochondrial membrane depolarization [53]. Small proportion of electrons escapes the mitochondrial chain and interacts with molecular oxygen to form which later gives rise to H2O2 or partially reduces to the damaging OH•. NP can catalyze the generation either by blocking the electron transport chain or accelerating electron transfer to molecular oxygen [54, 55]. Various metal oxide NP including Zn, Cu, Ti, and Si elicit ROS-mediated cell death via mitochondrial dysfunction [56–59].

3. Introduction to Transition Metals

Transition metal oxide particles have been used to revolutionize several fields including catalysis, sensors, optoelectronic materials, drug delivery, automobile, and material science engineering. Apart from industrial scale applications, metal NP are increasingly used in a variety of consumer products such as cosmetics, sunscreens, textiles, and food products. Among the transition metal oxides, titanium dioxide, cupric oxide, and zinc oxide have gained attention owing to their commercial usage [60]. Metal oxide particles can undergo surface modification for better stability and binding to other substrates. Such widespread applications are attributable to their electrochemical and physical properties reflecting their small sizes and reactive surfaces. For example, a relatively inert metal or metal oxide may become a highly effective catalyst when manufactured as NP. Their fixed particle mass, high aspect ratio, and particle surface bioreactivity tailor them to meet the needs of specific application. However, a high surface-to-volume ratio makes NP reactive and exposes them to environmental stressors, particularly free radical generation [61, 62]. Besides, the nanoscale dimensions enhance their cellular uptake and interaction with biological tissues. Metals can generate free radicals via the Fenton-type reactions that react with cellular macromolecules and induce oxidative stress [63]. The toxicity of metallic NP including Zn, Ti, Si, Fe, and Ce has been characterized by increased ROS generation and oxidative stress and apoptosis [61, 64–66]. The oxidative stress mediated outcomes of various metal NP are summarized in Table 1.

Table 1: List of studies describing the ROS-dependent effects of metal-based NP.

4. Prooxidant Effects of Metal Oxide Nanoparticles

To overcome the overwhelming ROS production, cells trigger either a defensive or an injurious response eliciting a chain of adverse biological responses. Free radicals are potentially damaging to cellular macromolecules including lipids, proteins, and nucleic acids. DNA is one of the major targets for oxidative stress and represents the first step involved in mutagenesis, carcinogenesis, and aging. ROS/RNS cause oxidative DNA damage in the form of DNA strand breaks, DNA protein cross-links, and alkali-labile sites [67, 68], and given their characteristic nature free radicals appear as one of the likely carcinogens [25, 69]. Testing the genotoxic potential is essential for carcinogenic risk assessment of NP. Genotoxic effects may be produced either by direct interaction of particles with genetic material or by secondary damage from particle-induced ROS. Transition metal NP induce chromosomal aberrations, DNA strand breaks, oxidative DNA damage, and mutations [70]. OH•, one of the highly potent radicals, is known to react with all components of DNA causing DNA single strand breakage via formation of 8-hydroxyl-2′-deoxyguanosine (8-OHdG) DNA adduct [71, 72]. 8-OHdG is a biomarker of OH•-mediated DNA lesions. NP exposure significantly elevated 8-OHdG levels both in vivo [73] and in vitro [74], demonstrating their mutagenic behavior. A recent study comparing metal oxide NP including Cu, Fe, Ti, and Ag reported ROS-mediated genotoxicity characterized by micronuclei and DNA damage in vivo [75].--Along with chromosomal damage, free radicals also interact with lipids and proteins, abundantly present in biomembranes, to yield lipid peroxidation products associated with mutagenesis. Polyunsaturated fatty acids are subject to oxidation giving rise to lipid hydroperoxides as the initial step in ROS generation [25, 76]. Prooxidant metals such as Cu and Fe react with these lipid hydroperoxides to induce DNA damaging end-products malondialdehyde (MDA) and 4-hydroxynonenal that act as inflammatory mediators and risk factors for carcinogenesis. Exposures to metal oxide NP of Ti, Cu, Si, and Fe were reported to induce tissue damage, abnormal cellular stress response via lipid peroxidation [77–79].---Alterations within the antioxidant defense system pose as a risk factor for carcinogenesis [68]. Glutathione, (GSH) a potent free-radical scavenger, is responsible for maintaining the cellular redox state and protecting cells from oxidative damage [80, 81]. NP-triggered free radicals reduce GSH into its oxidized form glutathione disulfide (GSSG), thereby contributing to oxidative stress, apoptosis, and sensitization to oxidizing stimuli [82, 83]. Apart from GSH, NP-induced ROS modulate the antioxidant activities of ROS-metabolizing enzymes including NADPH-dependent flavoenzyme, catalase, glutathione peroxidase, and superoxide dismutase [84].--It is well established that uncontrolled generation of ROS triggers a cascade of proinflammatory cytokines and mediators via activation of redox sensitive MAPK and NF-κB signaling pathways that control transcription of inflammatory genes such as IL-1β, IL-8, and TNF-α [21]. Oxidative stress plays a key role in NP-induced airway hypersensitivity and respiratory inflammation [85]. A study involving coexposure of metal oxide NP with a bacterial endotoxin demonstrated exaggerated lung inflammation and pulmonary edema [86]. Additionally, studies with different metal oxide NP have demonstrated ROS-mediated inflammatory response. For instance, SiO2 and TiO2 NP induce an elevated inflammatory response through the underlying mechanism of ROS generation [64, 85, 87]. Pulmonary inflammation may induce changes in membrane permeability, facilitating NP distribution beyond the lung and indirectly affecting cardiovascular performance [88].--Metal ion-induced free radicals can activate oncogenes such as Ras [25]. Excess amounts of NP have been associated with skin, bladder, liver, lung, and respiratory tract cancers [7]. Transition metals in trace amounts are introduced during the manufacture and preparation of CNT. Given their oxidizable nature, studies suggest that metals including Fe, Co, and Ni are more toxic and fibrogenic upon their interaction with CNT as compared to pure CNT [89–93]. Vanadium pentoxide (V2O5), a transition metal byproduct of petrochemicals, is associated with fibrosis via generation of H2O2 and other ROS [94]. Occupational exposures to combustion-derived NP such as welding fumes consisting of metals such as Fe, Mn, Si, Cr, and Ni induce fibrogenic responses [95]. Metal containing welding fume NP elicited ROS-dependent lipid peroxidation and inflammation in vivo [96, 97].

5. Cellular Signaling Affected by Metal Nanoparticles

The prooxidant effects of NP result in the activation of signaling pathways, transcription factors, and cytokine cascade contributing to a diverse range of cellular responses. The regulation of redox homeostasis entails signaling cascades such as HIF-1, NF-κB, PI3 K, and MAPK which control proliferation, metastasis, cell growth, apoptosis, survival, and inflammation [7, 12]. At an intermediate level of oxidative stress, proinflammatory pathways are activated in an attempt to maintain the redox equilibrium. The inflammatory cascade involves profibrotic mediators such as TNF-α, IL-1β, and TGF-β which have been implicated in the pathogenesis of fibrosis. Cells are known to counteract the overwhelming oxidative stress response via increased cytokine expression such as interleukins and TNF-α, activation of kinases, and inhibition of phosphatases thereby influencing the phosphorylation cascade. Protein phosphorylation is involved in the regulation of critical cellular responses including mitogenesis, cell adhesion, oncogenic transformation, and apoptosis. Thus, ROS response appears to be closely related to factors driving carcinogenesis [98].

5.1. NF-κB -The NF-κB group of proteins activates genes responsible for defense mechanisms against cellular stress and regulates miscellaneous functions such as inflammation, immune response, apoptosis, and cell proliferation. Prooxidant H2O2-mediated NF-κB activation through the classical IKK-dependent pathway is well established. ROS such as OH•, HOCl, and 1O2 and RNS such as ONOO− activate NF-κB via the release of IκBs resulting in the nuclear translocation of NF-κB [99, 100]. Once inside the nucleus, NF-κB induces transcription of proinflammatory mediators resulting in inflammation and oxidative stress. During NP-mediated lung injury, ROS activate NF-κB to modulate the production of proinflammatory TNF-α, IL-8, IL-2, and IL-6 from macrophages and lung epithelial cells [101]. Several metal oxide NP such as Zn, Cd, Si, and Fe exert their toxic effects via ROS-dependent NF-κB activation [62, 102, 103].

5.2. AP-1

Activator protein (AP)-1 is a transcription factor activated in response to oxidants, cytokines, growth factors, and bacterial and viral infections. It is responsible for regulation of cell proliferation, differentiation, and apoptosis, thereby it is a key factor in carcinogenesis [104]. Activation of AP-1 under oxidative conditions is believed to be mediated via phosphorylation of protooncogene c-jun [68]. Metal NP including Cr, Ni, and Fe have been shown to activate AP-1 via ROS generation [60]

.5.3. MAPK

MAPK are serine-threonine protein kinases that control a diverse range of cellular responses including proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis. MAPK consist of growth factor-regulated extracellular signal-related kinases (ERK) and the stress-activated MAPK, c-jun NH2-terminal kinases (JNK), and p38 MAPK. Once ROS production exceeds the capacity of the antioxidant proteins, free radicals may induce oxidative modification of MAPK signaling proteins (e.g., RTK and MAP3 K), thereby leading to MAPK activation. ROS may activate MAPK pathways via inhibition and/or degradation of MAPK phosphatases (MKP) [105, 106]. Finally, the site of ROS production and the concentration and kinetics of ROS production as well as cellular antioxidant pools and redox state are most likely to be important factors in determining the effects of ROS on activation of MAPK pathways [107]. Ag-NP activate JNK protein signaling and apoptosis in a variety of cells [50], whereas CeO2 NP trigger p38 MAPK signaling in bronchoalveolar cells [64].

5.4. PTP

Protein tyrosine phosphatases (PTP) are key regulatory components in signal transduction pathways involved in cell growth, differentiation, proliferation, and transformation. The highly reactive cysteine residues of PTP are predisposed to oxidative stress in the form of H2O2, free radicals or changes in intracellular thiol/disulfide redox state [98, 108]. Metal NP including Zn2+ and V5+ may be critical in redox regulation of PTP via the inhibition of MAPK and EGFR [109, 110].

5.5. Src

Src kinases belong to the nonreceptor tyrosine kinase family involved in the regulation of cell growth. Mild oxidative stress is sufficient to activate Src kinase which later triggers a cell signaling cascade [111]. This may explain the low dose of metal NP-induced lymphocyte cell death via ROS-dependent activation of Src kinases [112].

6. Carbon Nanotubes

One of the most promising materials in the field of nanotechnology is CNT, and their widespread applications are attributable to the diverse physical, chemical, and electrical characteristics they possess. CNT are high aspect ratio nanomaterials (HARN) having at least one of their dimensions in the order of 100 nm or less according to the British Standards Institute Report [113]. CNT are made of either single-walled (SW) or multiwalled (MW) graphite layers. With unique properties such as high tensile strength and conductivity, they have been explored in the areas of electronics, biotechnology, medicinal drug delivery, cosmetics, material science, and aerospace engineering. CNT structure facilitates their entry, deposition, and residence in the lungs and pleura, resulting in incomplete phagocytosis and clearance from the lungs [5]. Owing to their biopersistent and nonbiodegradable nature, and particularly their resemblance to needle-like asbestos fibers, CNT are believed to induce biologically harmful effects [89]. Physicochemical parameters such as particle size, surface modification, presence of metals, surface reactivity, and surface charge are responsible for the prooxidant effects of CNT. Frustrated phagocytosis of CNT has been implied in CNT-induced oxidative stress.

7. Carbon Nanotube-Induced Oxidative Stress

One of the most frequently reported toxicity endpoints for CNT is the formation of ROS which can be either protective or harmful during biological interactions. Oxidative stress may be caused directly by CNT-induced ROS in the vicinity or inside the cell or could arise more indirectly due to the effects of internalized CNT on mitochondrial respiration [114] or in depletion of antioxidant species within the cell [64]. Moreover, NADPH-mediated ROS are critical for SWCNT-induced pulmonary responses [91]. The most likely mechanism for CNT-induced oxidative stress and lung toxicity involves mitochondrial dysfunction. Incomplete phagocytosis of CNT, presence of transition metals and specific reactive groups on the CNT surface are key drivers of ROS generation. Metal impurities such as Fe, Co, and Ni introduced within the CNT during their synthesis are key factors driving CNT-mediated ROS response [115, 116]. CNT-induced oxidative stress mediates important cellular processes including inflammation, cell injury, apoptosis, and activation of cellular signaling pathways such as MAPK and NF-κB which are implicated in the pathogenesis of lung fibrosis [31, 117]. For instance, SWCNT dependent OH• generation leads to activation of molecular pathways MAPK, AP-1, NF-κB, and Akt associated with cell proliferation and tumor progression in vitro [93]. Several studies demonstrate SWCNT-induced oxidative stress [118–120]. Similarly, MWCNT exposures have been reported to induce ROS both in vitro and in vivo [18, 121–123]. Interestingly, oxidative stress is reported to be a mechanism for biodegradation of CNT. SWCNT undergoes oxidative biodegradation via myeloperoxidase, a prooxidant enzyme involved in host defense responses [120]. Table 2 summarizes the different studies that report ROS-dependent effects of CNT.

8. Role of ROS in CNT-Induced Inflammation

ROS and inflammation demonstrate an interdependent relationship in the case of exposure to NP. Inflammatory cells such as macrophages and neutrophils induce enormous ROS release in order to get rid of the NP. However, NP exposure-mediated oxidative stress leads to activation of RTK, MAPK, Akt, and NF-κB contributing to the proinflammatory cascade [124]. Accordingly, CNT-induced ROS were reported to elicit pro-inflammatory transcription factors such as NF-κB, AP-1 and MAPK in vivo. This was found to be an inflammation dependent response [93]. MWCNT treatment in macrophages mediates ROS-dependent activation of NF-κB pathway, thereby inducing the expression of chemokines and cytokines such as TNF-α, IL-1β, IL-6, IL-10, and MCP-1 [18]. Likewise, MWCNT-induced nitrosative stress in vivo is associated with pulmonary inflammation [125].

9. Role of ROS in CNT-Induced Genotoxicity

CNT elicit genotoxic effects through direct interaction with DNA or indirectly via CNT-induced oxidative stress and inflammatory responses. CNT-induced sustained oxidative stress can result in DNA damage and abnormal cell growth, possibly leading to carcinogenesis and fibrogenesis [126, 127]. A plethora of studies demonstrate the genotoxic potential of both MWCNT and SWCNT [128–131]. ROS can activate cellular signaling pathways resulting in cell cycle arrest and apoptosis. CNT induce a multitude of genotoxic responses including DNA strand breakage, oxidation, micronuclei induction, chromosomal aberrations, formation of γH2AX foci, and mutant frequencies

[132]. Oxidative stress-dependent DNA breakage and repair and activation of signaling pathways including poly-ADP-ribose polymerase (PARP), AP-1, NF-κB, p38, and Akt were reported in human mesothelial cells exposed to SWCNT [93]. CNT induce ROS-dependent lipid peroxidation both in vitro and in vivo [133, 134]. A number of studies account for mitochondrial membrane depolarization, damage, and oxidative stress upon CNT exposure [92, 135, 136]. Unlike the traditional prooxidant effect of NP, CNT have been reported to sequester ROS which in turn is associated with their structural defects [83]. This quenching is reported to be related to the genotoxic and inflammatory effects observed with CNT [137].

10. Role of ROS in CNT-Induced Fibrosis

Increased ROS has been implicated in lung inflammation and fibrosis. The inflammatory cascade is reported to contribute to oxidative stress mediated lung injury [138]. Exposure to CNT results in expression of genes responsible for inflammation and fibrosis via the activation of cell signaling pathways and transcription factors including NF-κB, STAT-1, MAPK, and RTK [31]. ROS-dependent p38-MAPK has been shown to be responsible for CNT-inducedcollagen and angiogenic responses [118]. Additionally, SWCNT induce fibrogenic effects via ROS-mediated NF-κB activation [139], whereas MWCNT induce fibroblast to myofibroblast differentiation via ROS-dependent NF-κB activation [18].

11. Oxidative Stress as an Underlying Mechanism for NP Toxicity

Findings from several studies have pointed out that ROS generation and oxidative stress occur as an early event leading to NP-induced injury. Oxidative stress corresponds with the physicochemical reactivity of NP including metal-based particles as well as the fibrous CNT. Oxidative stress related to NP exposure involves mitochondrial respiration, mitochondrial apoptosis, activation of NADPH oxidase system, alteration of calcium homeostasis, and depletion of antioxidant enzymes; all of which are associated with tissue injury. NP-driven ROS response contributes to activation of cell signaling pathways, inflammatory cytokine and chemokine expressions, and specific transcription factor activation. Activation of these cellular mechanisms is closely associated with transcription of genes involved in inflammation, genotoxicity, fibrosis, and cancer.-Thus, the pathological consequences observed during NP exposure could be attributable to ROS generation. It is essential to incorporate these adverse biological responses as a screening tool for toxic effects of NP. For instance, over-expression of antioxidant enzymes is indicative of the mild oxidative stress, whereas mitochondrial apoptosis occurs during conditions of toxic oxidative stress. The hierarchical model of ROS response provides a scale to gauge the adverse health effects upon NP exposures. A NP exposure study must collectively involve rigorous characterization of NP and assign in vitro and in vivo oxidative stress markers as toxicity endpoints as a predictive paradigm for risk assessment [6, 9, 12]. Figure 1 summarizes the key findings regarding the oxidative effects of NP and resulting toxicity. Prooxidant pathway for NP-induced toxicity: various NP exhibit oxidative stress dependent toxicity. Upon NP exposure, ROS generation is capable of inducing oxidative DNA damage, strand breaks, protein denaturation, and lipid peroxidation thereby demonstrating the mutagenic and carcinogenic characteristics associated with NP. Excess free radical production leads to mitochondrial membrane damage causing necrosis and cell death. Phagocytes including neutrophils and macrophages generate massive ROS upon incomplete phagocytosis of NP through the NADPH-oxidase enzyme system whereas NP-induced ROS triggers an inflammatory cascade of chemokine and cytokine expression via activation of cell signaling pathways such as MAPK, NF-κB, Akt, and RTK. Furthermore, oxidative stress mediated stimulation of these cellular mechanisms results in transcription of genes responsible for fibrosis, EMT, and carcinogenesis. NP-elicited ROS is at the center stage for majority of the ensuing adverse outcomes.

12. Conclusion

This paper reviews the cellular mechanisms of NP-induced oxidative stress and toxicity. We focus on the toxicity of metal oxide NP and CNT with respect to the oxidative stress paradigm. The principal factors for NP-induced oxidative stress involve (a) the oxidative properties of the NP themselves and (b) oxidant generation upon interaction of NP with cellular material. The direct prooxidant effects of NP are attributable to their physicochemical properties including surface reactivity, particle size, surface charge, chemical composition, and the presence of transition metals. Therefore, it is necessary to ensure extensive characterization of the physicochemical properties for safer design and manufacture of NP. Whereas, ROS mediated via NP-cell interaction involve mechanisms including immune cell activation, mitochondrial respiration, and NADPH oxidase system. Apart from ROS, NP also arbitrate RNS-mediated injury. Given their chemical reactivity, metal-based NP induce oxidative damage to cellular macromolecules such as proteins, lipids, and DNA via Fenton-type and Haber Weiss-type reactions. The key pathophysiological outcomes of oxidative insults during metal NP exposures involve cell membrane damage, lipid peroxidation, protein denaturation, and alteration of calcium homeostasis. Furthermore, the findings in the review article suggest that CNT-induced oxidative stress is indicative of the pulmonary toxicity of CNT. Metal-based NP and fibrous CNT-mediated ROS result in activation of cell signaling pathways, transcription factor activation, cytokine mediator release, and apoptosis. The persistent activation of these signaling cascades has some clinical ramifications. Redox imbalance via engineered NP exerts undesirable pathophysiological outcomes such as genotoxicity, inflammation, fibrosis, and carcinogenesis. It is of utmost importance to understand the molecular and cellular mechanisms of NP-induced oxidative stress which in turn will yield novel strategies to mitigate the toxicity of engineered NP. Moreover, it necessitates the establishment of stringent procedures for testing the oxidative potential of manufactured NP prior to their commercialization. Identifying the major cellular targets for NP-induced ROS will facilitate safer design and manufacture of NM in the market place.

Abbreviations

ROS: Reactive oxygen species

NP: Nanoparticles

NM: Nanomaterials

RNS: Reactive nitrogen species

CNT: Carbon nanotubes

H2O2: Hydrogen peroxide

: Superoxide anion

OH•: Hydroxyl radical

1O2: Singlet oxygen

HOCl: Hypochlorous acid

ONOO−: Peroxynitrite

AM: Alveolar macrophages

NADPH: Nicotinamide adenine dinucleotide phosphate

Nrf2: Nuclear factor (erythroid-derived 2)-like 2

MAPK: Mitogen activated protein kinase

NF-κB: Nuclear factor kappa-light-chain enhancer of activated B cells

iNOS: Inducible nitric oxide synthase

IL-1β: Interleukin-1beta

ERKs: Extracellular signal-related kinases

GSH: Glutathione

GSSG: Glutathione disulfide

8-OHdG: 8-Hydroxyl-2′-deoxyguanosine

AP-1: Activator protein-1

STAT-1: Signal transducer and activator of transcription-1

RTK: Receptor tyrosine kinases

PTP: Protein tyrosine phosphatases

HARN: High aspect ratio nanomaterials

PARP: Poly-ADP-ribose polymerase

TNF-α: Tumor necrosis factor-alpha

TGF-β: Transforming growth factor-beta

EMT: Epithelial-mesenchymal transition.

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Study describes how bacteria can remodel gene expression to infect intestines of host

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February 22, 2017 at 11:30 AM

Hebrew University researchers have described how infectious bacteria can sense they're attached to our intestinal cells, and then remodel their expression of specific genes, including those involved in virulence and metabolism, to exploit our cells and colonize our gut.--Infectious diarrhea, a common disease of children, is responsible for over 2 million infant deaths annually in developing countries alone. A primary cause of this and other devastating conditions is enteropathogenic bacteria, which attack the intestinal tract when contaminated food is consumed.--The infection process involves hundreds of genes and proteins, both in the infectious bacteria and the human host. However, the processes by which the pathogens establish themselves in our gut are poorly understood.--Now, a new study published in the prestigious journal Science, by researchers at the Hebrew University of Jerusalem's Faculty of Medicine, describes how pathogens sense their host, and tailor their gene expression to exploit their host to cause disease. The research was led by led by Prof. Ilan Rosenshine, the Etta Rosensohn Professor of Bacteriology at the Hebrew University.--Working with a pathogenic strain of E. coli, the researchers found that the bacteria can sense attachment to the human intestinal cells and activate gene expression in response.  This was demonstrated by engineering one of these genes to express a protein that stains the expressing bacteria to appear green under the microscope. Under microscopic examination, the researchers observed that only the attached bacteria fluoresce in bright green, whereas non-attached bacteria remain dark.--The researchers also deciphered how upon sensing that it has attached to intestinal cells, the pathogen reorganizes its gene expression, including genes involved in virulence and metabolism, to exploit the host cell. These findings may lead to the development of new strategies to combat bacterial infection.--"The next steps include mapping in detail the genes that change their expression upon attachment, and describing the precise effects of this expression remodeling," said Prof. Ilan Rosenshine. "Another important issue is testing whether similar regulation is involved in the infection processes of other pathogens." Source: http://new.huji.ac.il/en/article/33566

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Nanocages dramatically facilitate structure formation of biomolecules

Nano-size space help faster folding of molecules and stabilize the structure, which regulates enzyme reactions Macromolecules regularly fold and unfold themselves inside cells. Their diverse three-dimensional structures help determine their functions. Understanding molecule folding can shed light on complex physical processes that may influence diseases, cancers and allergies. G-quadruplexes are groups of guanine nucleic acids, the 'G's in the DNA sequence, that form specific shapes that look like futuristic, three-story office buildings. They were long dismissed as not having a biological function, but are now thought to help regulate gene expression, including for diseases. Understanding the physical processes that these compounds undergo when folding into extra-tight spaces may one day help develop drug treatments that target them. -Conducting experiments to learn about the folding process in confined areas is extremely challenging because it is easy to disturb not only the target molecule, which is just a few nanometers long, but also the surrounding infrastructure, which is just a fraction larger. -A team led by Hiroshi Sugiyama and Masayuki Endo of Kyoto University's Institute for Integrated Cell-Material Sciences (iCeMS) collaborating with the team from Kent State University designed structures and experimental system that successfully manipulates G-quadruplexes inside nanocages, which are also made of DNA. The team measured how different-sized spaces affect the thermodynamic stability and unfolding and folding kinetics of these molecules. -"Under the confined nano-space, the G-quadruplex structures revealed unprecedented fast folding kinetics with increased mechanical as well as thermodynamic stabilities, which directly supported theoretical predictions," the researchers conclude in their study published recently in the journal Nature Nanotechnology. The researchers built rectangle-shaped nanocages out of DNA that they wrapped around a G-quadruplex molecule. Tethers made of yet more DNA attached the molecule to two beads. The beads, controlled by lasers known as optical tweezers, exerted force on the molecule. This prompted the molecule to unfold and then refold. There was no interference between the nanocage and the target, because they both have negative charges and repel each other like magnets. -The team built small, medium and large nanocages. The molecules unfolded and refolded 100 times faster in the small and medium nanocages compared to molecules without nanocages The findings support predictions made by other researchers in the field, but this is the first demonstration with no interaction between the molecule and the cage. The researchers anticipate that the method can be applied for observation of other biomolecules such as proteins using more precisely designed nanocages. Story Source-Materials provided by Kyoto University. -Journal Reference-Prakash Shrestha, Sagun Jonchhe, Tomoko Emura, Kumi Hidaka, Masayuki Endo, Hiroshi Sugiyama, Hanbin Mao. Confined space facilitates G-quadruplex formation. Nature Nanotechnology, 2017; DOI: 10.1038/nnano.2017.29 -- Kyoto University. "Nanocages dramatically facilitate structure formation of biomolecules: Nano-size space help faster folding of molecules and stabilize the structure, which regulates enzyme reactions." ScienceDaily. ScienceDaily, 27 March 2017. <www.sciencedaily.com/releases/2017/03/170327131225.htm

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Sweet marjoram (Origanum majorana, Majorana hortensis)

AnticholinergicsAnticholinergics: Based on in vitro study, essential oils of various herbs, including marjoram, had inhibitory effects against

Marjoram/Drug Interactions:

AlcoholAlcohol: Co-administration of ethanol and the extracts of marjoram volatile oil in rats (Origanum majorana L., Lamiaceae) minimized the hazardous effects of ethanol toxicity on male fertility and on liver and brain tissues (49).

AntibioticsAntibiotics: Marjoram has been shown to exert antimicrobial effects in vitro (2; 3; 18; 19) against a variety of bacteria including Salmonella enterica and Shigella. Eugenol, a compound extracted from clove oil and marjoram, was shown to be active against Campylobacter jejuni, Listeria monocytogenes, and Salmonella enterica (4; 5; 6), and against one or more of six Bacillus species (Bacillus amyloliquefaciens ATCC 3842, Bacillus brevis FMC 3, Bacillus cereus FMC 19, Bacillus megaterium DSM 32, Bacillus subtilis IMG 22, and B. subtilis var. niger ATCC 10) (7).

Cholinesterase inhibitorsCholinesterase inhibitors: Based on in vitro study, essential oils of various herbs, including marjoram, had inhibitory effects against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) (70). According to in vitro study, ethanol extracts from Origanum majorana L. may inhibit acetylcholine (15).

Antiulcer agentsAntiulcer agents: Based on animal study, marjoram extracts had antiulcerogenic activity against stress-, indomethacin-, and necrotizing agent-induced ulcers (73). Basal gastric secretion and acid output were also reduced, and ethanol-induced depleted gastric wall mucus and nonprotein sulfhydryl (NP-SH) content were replenished

DiureticsDiuretics: Based on secondary sources, marjoram may have diuretic properties

IndomethacinIndomethacin: Based on animal study, marjoram extracts had antiulcerogenic activity against indomethacin-induced ulcers (73).

Marjoram/Herb/Supplement Interactions:

Alcohol--Alcohol: Co-administration of ethanol and the extracts of marjoram volatile oil in rats (Origanum majorana L., Lamiaceae) minimized the hazardous effects of ethanol toxicity on male fertility and on liver and brain tissues (49). According to in vitro study, ethanol extracts from Origanum majorana L. may inhibit acetylcholine (15).

Anticoagulants and antiplatelets Anticoagulants and antiplatelets: In in vitro study, a methanol extract of Origanum majorana inhibited platelet adhesion and affected platelet self-aggregation (35).

Antidiabetic agents Antidiabetic agents: In animal study, water extract of marjoram had hypoglycemic effects (56). Based on in vitro study, Origanum majorana may have antidiabetic and glucose homeostatic effects (36).

AntifungalsAntifungals: Based on in vitro research, oxygen uptake by the spores of various fungi, including Fusarium and Trichoderma species, increased in the presence of volatile substances extracted from marjoram (71). Volatile substances from marjoram reduced the spore germination of Mucor racemosus.

AntilipemicsAntilipemics: Based on animal study, in a diabetic rat model, marjoram extract decreased the elevated levels of triglycerides and total cholesterol (56).

AntineoplasticsAntineoplastics: Based on in vitro study, marjoram extracts had antiproliferative effects on the human lymphoblastic leukemia cell line Jurkat (26).

Antiprotozoals Antiprotozoals: Origanum majorana essential oil constituents have been shown to have ovicidal and adulticidal effects against insecticide-susceptible and pyrethroid/malathion-resistant human head lice (Pediculus humanus capitis) (72). Cineole, linalool, camphor, and terpineol monoterpenoid constituents were found to have the most activity, and were faster acting than d-phenothrin and pyrethrum.

Ambrosia maritimeAmbrosia maritime: Based on animal study, marjoram extract alone, or in combination with extract of Ambrosia maritime, had comparable hypoglycemic effects (56).

AntibacterialsAntibacterials: Marjoram has been shown to exert antimicrobial effects in vitro (2; 3; 18; 19) against a variety of bacteria including Salmonella enterica and Shigella. Eugenol, a compound extracted from clove oil and marjoram, was shown to be active against Campylobacter jejuni, Listeria monocytogenes, and Salmonella enterica (4; 5; 6), and against one or more of six Bacillus species (Bacillus amyloliquefaciens ATCC 3842, Bacillus brevis FMC 3, Bacillus cereus FMC 19, Bacillus megaterium DSM 32, Bacillus subtilis IMG 22, and B. subtilis var. niger ATCC 10) (7).

Anticholinergic herbs Anticholinergic herbs: Based on in vitro study, essential oils of various herbs, including marjoram, had inhibitory effects against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) (70). According to in vitro study, ethanol extracts from Origanum majorana L. may inhibit acetylcholine (15).

Anticoagulants and antiplatelets Anticoagulants and antiplatelets: In in vitro study, a methanol extract of Origanum majorana inhibited platelet adhesion and affected platelet self-aggregation (35).

AntifungalsAntifungals: Based on in vitro research, oxygen uptake by the spores of various fungi, including Fusarium and Trichoderma species, increased in the presence of volatile substances extracted from marjoram (71). Volatile substances from marjoram reduced the spore germination of Mucor racemosus.

AntilipemicsAntilipemics: Based on animal study, in a diabetic rat model, marjoram extract decreased the elevated levels of triglycerides and total cholesterol (56).

AntineoplasticsAntineoplastics: Based on in vitro study, marjoram extracts had antiproliferative effects on the human lymphoblastic leukemia cell line Jurkat (26).

AntioxidantsAntioxidants: Based on animal study, marjoram extract reduced malondialdehyde levels (73). In in vitro study, sweet marjoram was shown to retard lipid oxidation (24), and ursolic acid from sweet marjoram reduced Abeta-induced oxidative cell death (74).

AntiparasiticsAntiparasitics: Origanum majorana essential oil constituents have been shown to have ovicidal and adulticidal effects against insecticide-susceptible and pyrethroid/malathion-resistant human head lice (Pediculus humanus capitis) (72). Cineole, linalool, camphor, and terpineol monoterpenoid constituents were found to have the most activity, and were faster acting than d-phenothrin and pyrethrum.

Antiulcer herbs and supplementsAntiulcer herbs and supplements: Based on animal study, marjoram extracts had antiulcerogenic activity against stress-, indomethacin-, and necrotizing agent-induced ulcers (73). Basal gastric secretion and acid output were also reduced, and ethanol-induced depleted gastric wall mucus and nonprotein sulfhydryl (NP-SH) content were replenished.

DiureticsDiuretics: Based on secondary sources, marjoram may have diuretic properties.

HypoglycemicsHypoglycemics: In animal study, water extract of marjoram had hypoglycemic effects (56). Based on in vitro study, Origanum majorana may Have antidiabetic and glucose homeostatic effects (36).

UreaUrea: Based on animal study, in a diabetic rat model, marjoram extract decreased the elevated levels of urea (56).

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Sweet marjoram (Origanum majorana, Majorana hortensis)

AnticholinergicsAnticholinergics: Based on in vitro study, essential oils of various herbs, including marjoram, had inhibitory effects against

Marjoram/Drug Interactions:

AlcoholAlcohol: Co-administration of ethanol and the extracts of marjoram volatile oil in rats (Origanum majorana L., Lamiaceae) minimized the hazardous effects of ethanol toxicity on male fertility and on liver and brain tissues (49).

AntibioticsAntibiotics: Marjoram has been shown to exert antimicrobial effects in vitro (2; 3; 18; 19) against a variety of bacteria including Salmonella enterica and Shigella. Eugenol, a compound extracted from clove oil and marjoram, was shown to be active against Campylobacter jejuni, Listeria monocytogenes, and Salmonella enterica (4; 5; 6), and against one or more of six Bacillus species (Bacillus amyloliquefaciens ATCC 3842, Bacillus brevis FMC 3, Bacillus cereus FMC 19, Bacillus megaterium DSM 32, Bacillus subtilis IMG 22, and B. subtilis var. niger ATCC 10) (7).

Cholinesterase inhibitorsCholinesterase inhibitors: Based on in vitro study, essential oils of various herbs, including marjoram, had inhibitory effects against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) (70). According to in vitro study, ethanol extracts from Origanum majorana L. may inhibit acetylcholine (15). acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) (70).

Antiulcer agentsAntiulcer agents: Based on animal study, marjoram extracts had antiulcerogenic activity against stress-, indomethacin-, and necrotizing agent-induced ulcers (73). Basal gastric secretion and acid output were also reduced, and ethanol-induced depleted gastric wall mucus and nonprotein sulfhydryl (NP-SH) content were replenished

DiureticsDiuretics: Based on secondary sources, marjoram may have diuretic properties

IndomethacinIndomethacin: Based on animal study, marjoram extracts had antiulcerogenic activity against indomethacin-induced ulcers (73).

Marjoram/Herb/Supplement Interactions:

Alcohol--Alcohol: Co-administration of ethanol and the extracts of marjoram volatile oil in rats (Origanum majorana L., Lamiaceae) minimized the hazardous effects of ethanol toxicity on male fertility and on liver and brain tissues (49). According to in vitro study, ethanol extracts from Origanum majorana L. may inhibit acetylcholine (15).

Anticoagulants and antiplatelets Anticoagulants and antiplatelets: In in vitro study, a methanol extract of Origanum majorana inhibited platelet adhesion and affected platelet self-aggregation (35).

Antidiabetic agents Antidiabetic agents: In animal study, water extract of marjoram had hypoglycemic effects (56). Based on in vitro study, Origanum majorana may have antidiabetic and glucose homeostatic effects (36).

AntifungalsAntifungals: Based on in vitro research, oxygen uptake by the spores of various fungi, including Fusarium and Trichoderma species, increased in the presence of volatile substances extracted from marjoram (71). Volatile substances from marjoram reduced the spore germination of Mucor racemosus.

AntilipemicsAntilipemics: Based on animal study, in a diabetic rat model, marjoram extract decreased the elevated levels of triglycerides and total cholesterol (56).

AntineoplasticsAntineoplastics: Based on in vitro study, marjoram extracts had antiproliferative effects on the human lymphoblastic leukemia cell line Jurkat (26).

Antiprotozoals Antiprotozoals: Origanum majorana essential oil constituents have been shown to have ovicidal and adulticidal effects against insecticide-susceptible and pyrethroid/malathion-resistant human head lice (Pediculus humanus capitis) (72). Cineole, linalool, camphor, and terpineol monoterpenoid constituents were found to have the most activity, and were faster acting than d-phenothrin and pyrethrum.

Ambrosia maritimeAmbrosia maritime: Based on animal study, marjoram extract alone, or in combination with extract of Ambrosia maritime, had comparable hypoglycemic effects (56).

AntibacterialsAntibacterials: Marjoram has been shown to exert antimicrobial effects in vitro (2; 3; 18; 19) against a variety of bacteria including Salmonella enterica and Shigella. Eugenol, a compound extracted from clove oil and marjoram, was shown to be active against Campylobacter jejuni, Listeria monocytogenes, and Salmonella enterica (4; 5; 6), and against one or more of six Bacillus species (Bacillus amyloliquefaciens ATCC 3842, Bacillus brevis FMC 3, Bacillus cereus FMC 19, Bacillus megaterium DSM 32, Bacillus subtilis IMG 22, and B. subtilis var. niger ATCC 10) (7).

Anticholinergic herbs Anticholinergic herbs: Based on in vitro study, essential oils of various herbs, including marjoram, had inhibitory effects against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) (70). According to in vitro study, ethanol extracts from Origanum majorana L. may inhibit acetylcholine (15).

Anticoagulants and antiplatelets Anticoagulants and antiplatelets: In in vitro study, a methanol extract of Origanum majorana inhibited platelet adhesion and affected platelet self-aggregation (35).

AntifungalsAntifungals: Based on in vitro research, oxygen uptake by the spores of various fungi, including Fusarium and Trichoderma species, increased in the presence of volatile substances extracted from marjoram (71). Volatile substances from marjoram reduced the spore germination of Mucor racemosus.

AntilipemicsAntilipemics: Based on animal study, in a diabetic rat model, marjoram extract decreased the elevated levels of triglycerides and total cholesterol (56).

AntineoplasticsAntineoplastics: Based on in vitro study, marjoram extracts had antiproliferative effects on the human lymphoblastic leukemia cell line Jurkat (26).

AntioxidantsAntioxidants: Based on animal study, marjoram extract reduced malondialdehyde levels (73). In in vitro study, sweet marjoram was shown to retard lipid oxidation (24), and ursolic acid from sweet marjoram reduced Abeta-induced oxidative cell death (74).

AntiparasiticsAntiparasitics: Origanum majorana essential oil constituents have been shown to have ovicidal and adulticidal effects against insecticide-susceptible and pyrethroid/malathion-resistant human head lice (Pediculus humanus capitis) (72). Cineole, linalool, camphor, and terpineol monoterpenoid constituents were found to have the most activity, and were faster acting than d-phenothrin and pyrethrum.

Antiulcer herbs and supplementsAntiulcer herbs and supplements: Based on animal study, marjoram extracts had antiulcerogenic activity against stress-, indomethacin-, and necrotizing agent-induced ulcers (73). Basal gastric secretion and acid output were also reduced, and ethanol-induced depleted gastric wall mucus and nonprotein sulfhydryl (NP-SH) content were replenished.

DiureticsDiuretics: Based on secondary sources, marjoram may have diuretic properties.

HypoglycemicsHypoglycemics: In animal study, water extract of marjoram had hypoglycemic effects (56). Based on in vitro study, Origanum majorana may Have antidiabetic and glucose homeostatic effects (36).

UreaUrea: Based on animal study, in a diabetic rat model, marjoram extract decreased the elevated levels of urea (56).

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Gut microbes linked to brain structure in people with irritable bowel syndrome

A new study by researchers at UCLA has revealed two key findings for people with irritable bowel syndrome about the relationship between the microorganisms that live in the gut and the brain.--For people with IBS research shows for the first time that there is an association between the gut microbiota and the brain regions involved in the processing of sensory information from their bodies. The results suggest that signals generated by the brain can influence the composition of microbes residing in the intestine and that the chemicals in the gut can shape the human brain's structure. --Additionally, the researchers gained insight into the connections among childhood trauma, brain development and the composition of the gut microbiome. Previous studies performed in mice have demonstrated effects of gut microbiota on brain function and behavior, as well as the influence of the brain on the composition of microbes in the gut. However, to date, only one study performed in human subjects has confirmed the translatability of such findings to the human brain. Studies have also reported evidence for alterations in the composition of gut microbiota in people with irritable bowel syndrome, but there has been little consistency among studies regarding the specific microbial alterations and the relationship of such alterations with the cardinal symptoms of IBS, recurring abdominal pain and altered bowel habits. In relation to a person's history with childhood trauma, it has been shown to be associated with structural and functional brain changes; trauma in young children has also been shown to alter gut microbial composition. But how they are related has been unknown.--The UCLA researchers collected behavioral and clinical measures, stool samples and structural brain images from 29 adults diagnosed with IBS, and 23 healthy control subjects. They used DNA sequencing and various mathematical approaches to quantify composition, abundance and diversity of the gut microbiota. They also estimated the microbial gene content and gene products of the stool samples. Then the researchers cross-referenced these gut microbial measures with structural features of the brain.--Based on the composition of the microbes in the gut, the samples from those diagnosed with IBS clustered into two subgroups. One group was indistinguishable from the healthy control subjects, while the other differed. Those in the group with an altered gut microbiota had more history of early life trauma and longer duration of IBS symptoms.-The two groups also displayed differences in brain structure.--Analysis of a person's gut microbiota may become a routine screening test for people with IBS in clinical practice, and in the future, therapies such as certain diets and probiotics may become personalized based on an individual's gut microbial profile. At the same time, subgroups of people with IBS distinguished by brain and microbial signatures may show different responsiveness to brain-directed therapies such as mindfulness-based stress reduction, cognitive behavioral therapy and targeted drugs.--A history of early life trauma has been shown to be associated with structural and functional brain changes and to alter gut microbial composition. It is possible that the signals the gut and its microbes get from the brain of an individual with a history of childhood trauma may lead to lifelong changes in the gut microbiome. These alterations in the gut microbiota may feed back into sensory brain regions, altering the sensitivity to gut stimuli, a hallmark of people with IBS. Story Source-Materials provided by University of California, Los Angeles (UCLA), Health Sciences. Journal Reference-Jennifer S. Labus, Emily B. Hollister, Jonathan Jacobs, Kyleigh Kirbach, Numan Oezguen, Arpana Gupta, Jonathan Acosta, Ruth Ann Luna, Kjersti Aagaard, James Versalovic, Tor Savidge, Elaine Hsiao, Kirsten Tillisch, Emeran A. Mayer. Differences in gut microbial composition correlate with regional brain volumes in irritable bowel syndrome. Microbiome, 2017; 5 (1) DOI: 10.1186/s40168-017-0260-z University of California, Los Angeles (UCLA), Health Sciences. "Gut microbes linked to brain structure in people with irritable bowel syndrome." ScienceDaily. ScienceDaily, 5 May 2017. <www.sciencedaily.com/releases/2017/05/170505151656.htm>

JUNE 2017--TOP

Show for June 2017

Eggs can significantly increase growth in young children Removal of aging cells could extend human life CHELATES AND CHELATING AGENTS The Introduction of Diphtheria-Tetanus-Pertussis and Oral Polio Natural Experiment Crystals grown by light to create gold nanoparticle Historical FACTS On Dangers, Ineffectiveness Of Vaccines Nervous system manipulation by electromagnetic fields from monitors

The role of vitamin A in diabetes Low levels of vitamin A may fuel TB risk Scientists Find Simple Copper Complex Shuts Down Botulinum Neurotoxin Poisoning Common class of chemicals cause cancer by breaking down DNA repair mechanisms DNA double helix structures crystals-Method developed for DNA programmed material synthesis Self-assembled nanostructures can be selectively controlled Natural Anti-Biofilm Agents



Eggs can significantly increase growth in young children

Eggs significantly increased growth and reduced stunting by 47 percent in young children, finds a new study from a leading expert on child nutrition at the Brown School at Washington University in St. Louis. This was a much greater effect than had been shown in previous studies.--"Eggs can be affordable and easily accessible," said Lora Iannotti, lead author of the study.--"They are also a good source of nutrients for growth and development in young children," she said. "Eggs have the potential to contribute to reduced growth stunting around the world."--The study, "Eggs in Complementary Feeding and Growth," was published online June 6 in the journal Pediatrics.-Iannotti and her co-authors conducted a randomized, controlled trial in Ecuador in 2015. Children ages 6-9 months were randomly assigned to be given one egg per day for 6 months, versus a control group, which did not receive eggs.--Eggs were shown to increase standardized length-for-age score and weight-for-age score. Models indicated a reduced prevalence of stunting by 47 percent and underweight by 74 percent. Children in the treatment group had higher dietary intakes of eggs and reduced intake of sugar-sweetened foods compared to control.--"We were surprised by just how effective this intervention proved to be," Iannotti said. "The size of the effect was 0.63 compared to the 0.39 global average."--Eggs are a complete food, safely packaged and arguably more accessible in resource-poor populations than other complementary foods, specifically fortified foods, she said.--"Our study carefully monitored allergic reactions to eggs, yet no incidents were observed or reported by caregivers during the weekly home visits," Iannotti said. "Eggs seem to be a viable and recommended source of nutrition for children in developing countries."Story Source Materials provided by Washington University in St. Louis. Journal Reference-Lora L. Iannotti, Chessa K. Lutter, Christine P. Stewart, Carlos Andres Gallegos Riofrío, Carla Malo, Gregory Reinhart, Ana Palacios, Celia Karp, Melissa Chapnick, Katherine Cox, William F. Waters. Eggs in Early Complementary Feeding and Child Growth: A Randomized Controlled Trial. Pediatrics, 2017; e20163459 DOI: 10.1542/peds.2016-3459 --Washington University in St. Louis. "Eggs can significantly increase growth in young children: The effect was much greater than had been shown in previous studies." ScienceDaily. ScienceDaily, 7 June 2017. <www.sciencedaily.com/releases/2017/06/170607085615.htm>.

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The role of vitamin A in diabetes

There has been no known link between diabetes and vitamin A -- until now. A new study suggests that the vitamin improves the insulin producing β-cell´s function.-The researchers initially discovered that insulin-producing beta-cells contain a large quantity of a cell surface receptor for vitamin A.-"There are no unnecessary surface receptors in human cells. They all serve a purpose but which, in many cases, is still unknown and because of that they are called "orphan" receptors. When we discovered that insulin cells have a cell surface expressed receptor for vitamin A, we thought it was important to find out why and what the purpose is of a cell surface receptor interacting with vitamin A mediating a rapid response to vitamin A," explains Albert Salehi, senior researcher at the Lund University Diabetes Centre in Sweden.-The researchers believe that the purpose, in this particular case, is that vitamin A plays an important role for the development of beta-cells in the early stages of life, but also for a proper function during the remaining life especially during pathophysiological conditions, i.e some inflammatory conditions.-Albert Salehi and his research team, together with their colleagues at the University of Gothenburg, King's College (London) and the Oxford Centre for Diabetes, have mapped 220 different receptors on the surface of the beta cell, whose features are still not fully known. One of the findings is the cell surface expressed receptor for vitamin A.-In order to study the role of the vitamin in cases of diabetes, the researchers worked with insulin cells from mice and non-diabetic and type 2 diabetic donors. By partially blocking the vitamin A receptor and challenging the cells with sugar, they could see that the cells' ability to secrete insulin deteriorated. -"We saw close to a 30 per cent reduction," says Albert Salehi, adding that impaired cell survival and insulin secretion are key causes of type 2 diabetes.-The same tendency could be seen when comparing insulin cells from type 2 diabetic donors. Cells from patients with type 2 diabetes were less capable of insulin secretion compared with cells from people without diabetes.-The researchers also saw that the beta-cells' resistance to inflammation decreases in the absence of vitamin A. In case of a complete deficiency, the cells die. The discovery may also be significant for certain types of type 1 diabetes when the beta-cells are not sufficiently developed during the early stages of life.-"In animal experiments it is known that newborn mice need vitamin A to develop their beta-cells in a normal way. Most likely, the same applies to human beings. Children must absorb a sufficient amount of vitamin A through their diet," says Albert Salehi.-Vitamin A is found mainly in offal and dairy products. In Sweden, milk is enriched with vitamin A. There appears to be no vitamin A deficiency in Sweden in people who eat a standard variety of food, but vegetarians perhaps need to be aware of the problem.-Too much vitamin A is harmful and can lead to osteoporosis. However, there is no risk of excessive intake through food -- the risk lies in taking dietary supplements. Defects associated with vitamin A deficiency are, among other things, impaired night vision and reduced elasticity in the skin and mucous membranes.-In the event of a diabetes treatment based on the newly found cell surface receptor for vitamin A, Albert Salehi believes that the risk of excessive intake makes the vitamin A itself inappropriate.-"But we're trying to find substances such as small molecules or peptides that are similar to the vitamin A could activate the newly found receptor while lacking the unwanted effects" of vitamin A," he concludes.-Story Source-Materials provided by Lund University. Journal Reference-Stefan Amisten, Israa Mohammad Al-Amily, Arvind Soni, Ross Hawkes, Patricio Atanes, Shanta Jean Persaud, Patrik Rorsman, Albert Salehi. Anti-diabetic action of all-trans retinoic acid and the orphan G protein coupled receptor GPRC5C in pancreatic β-cells. Endocrine Journal, 2017; 64 (3): 325 DOI: 10.1507/endocrj.EJ16-0338 - vitamin A in diabetes." ScienceDaily. ScienceDaily, 13 June 2017. <www.sciencedaily.com/releases/2017/06/170613111649.htm>.

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Low levels of vitamin A may fuel TB risk

People with low levels of vitamin A living with individuals sick with tuberculosis may be 10 times more likely to develop the disease than people with high levels of the nutrient, according to research led by investigators at Harvard Medical School.--The findings, published May 20 in Clinical Infectious Diseases, are based on an analysis of blood drawn from more than 6,000 household contacts of people diagnosed with TB in Lima, Peru. The study findings do not prove a cause-and-effect relationship between vitamin A levels and TB disease, the researchers caution, but the potent link between the two suggests that vitamin A supplementation might be an important part of controlling the spread of TB -- one of the leading causes of death worldwide.--"This is one of the strongest risk factors reported in a large epidemiological study in years," said senior author Megan Murray, the Ronda Stryker and William Johnston Professor of Global Health at Harvard Medical School. "If the link is affirmed in a clinical trial of vitamin A supplementation, it would make a powerful case for using this approach to prevent TB in people at high risk of disease."-A 10-fold increase in risk is striking, the investigators said. To put it in perspective, smoking tobacco increases the risk for heart disease two to four times, according to the Centers for Disease Control and Prevention.-More than 1.8 million people died from TB in 2015. TB strikes hardest in low- and middle-income countries, where vitamin A deficiency can affect up to 30 percent of the population.-"It's exciting to think that something as simple and inexpensive as supplementing people's diets with vitamin A may be a powerful tool for preventing TB," Murray said.-Vitamin A, also known as retinol, is best known among public health experts for its association with blindness. Healthy levels of the nutrient have been defined as those needed to prevent damage to eyesight. Previous studies have suggested that vitamin A modulates the immune system and may ward off infection. However, just how vitamin A might affect the risk for TB has, up until now, remained unclear and a matter of debate.-In the Lima study, the researchers found that the protective effect of vitamin A grew stronger as levels of the nutrient increased. Protection continued to grow well above what has been considered the minimum healthy level.-Working with Mercedes Becerra, HMS professor of global health and social medicine, Murray designed the study to investigate bacterial and host determinants of TB infection and disease. The research was conducted through a cooperative agreement with the National Institutes of Health.-The study began with people who sought care in any one of 106 clinics around the city of Lima. When a patient was diagnosed with TB, his or her household contacts were asked whether they wanted to participate in the study. Those who agreed were asked to give a baseline blood sample.-Of the more than 6,000 participants who agreed to have their blood analyzed, 258 people developed TB disease. Among those, 192 became sick with TB after enrollment in the study. Researchers compared 180 blood samples obtained from people who developed TB disease during that time with blood samples obtained from household contacts who did not become sick. Participants were monitored regularly throughout the one-year follow-up for disease symptoms.-Vitamin A levels were a potent predictor of TB disease risk.-Vitamin A deficiency -- defined as less than 200 micrograms per liter of blood -- fueled the risk of developing TB disease 10-fold. That risk was 20 times higher among young people between the ages of 10 and 19. That finding, the researchers say, suggests that vitamin A may play an even greater role in immunity among younger people.-The researchers found that vitamin A levels in the baseline sample strongly predicted progression to TB disease, even after adjustment for socioeconomic status, body mass index and other conditions thought to increase TB disease risk or affect vitamin A levels.-Notably, the researchers analyzed blood samples before individuals became ill to rule out the possibility that low vitamin levels were a symptom of the disease known to interfere with appetite and lead to nutritional deficiencies.-Meanwhile, Murray and colleagues at Brigham and Women's Hospital and elsewhere have continued the research initiated on the original grant and are now seeking to identify different factors that affect the interplay between host and pathogen and that can alter infection risk and disease progression. Such factors include metabolic, genetic and immune factors. This ongoing work is part of the NIH-funded Tuberculosis Research Units program.-The work is part of a 20-year partnership between HMS researchers and Partners In Health, where Murray is director of research, PIH's sister organization Socios En Salud Sucursal Peru, and local and national community and government organizations in Peru.-This long-term collaboration has enabled Harvard Medical School scientists and others to develop the research capacity required to perform large, complex studies in resource-poor settings, including collecting tens of thousands of blood samples and processing them at local labs built by the research consortium.-Becerra noted that conducting research responsibly in these environments can not only deepen scientific and clinical knowledge, but also directly improve care. While following up with people who share a home with those sick with TB is a standard part of care in the United States and Europe, the practice is less common in resource-poor areas. To conduct this study, the Socios En Salud field team visited TB-exposed family members at home to see whether they became sick, referring them for treatment more promptly if they did, and thus improving the level of care they received, Becerra said.-Will vitamin A supplementation or improved diet to reduce the risk of TB? Answering that question will require a clinical trial, Murray said. The Tuberculosis Research Unit program collaborative includes colleagues who do basic science research using animal models, and Murray says that they have already begun building a guinea pig model for TB and vitamin A deficiency, a crucial first step in moving toward testing an intervention in humans.-"TB is a tough disease to live with, and a tough disease to treat," Murray said. "We'd love to keep people from getting sick in the first place."Story Source-Materials provided by Harvard Medical School. Original written by Jake Miller-Journal Reference-Omowunmi Aibana, Molly F. Franke, Chuan-Chin Huang, Jerome T. Galea, Roger Calderon, Zibiao Zhang, Mercedes C. Becerra, Emily R. Smith, Alayne G. Ronnenberg, Carmen Contreras, Rosa Yataco, Leonid Lecca, Megan B. Murray. Impact of Vitamin A and Carotenoids on the Risk of Tuberculosis Progression. Clinical Infectious Diseases, 2017; DOI: 10.1093/cid/cix476 -Harvard Medical School. "Low levels of vitamin A may fuel TB risk." ScienceDaily. ScienceDaily, 12 June 2017. <www.sciencedaily.com/releases/2017/06/170612135512.htm>.

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Removal of aging cells could extend human life

Clearance of SnCs by GCV reduces the development of post-traumatic OA

A recent study, led by an international team of researchers confirms that targeted removal of senescent cells (SnCs), accumulated in many vertebrate tissues as we age, contribute significantly in delaying the onset of age-related pathologies.-This breakthrough research has been led by Dr. Chaekyu Kim of the Johns Hopkins University School of Medicine, who is now at UNIST, and Dr. Ok Hee Jeon of the Johns Hopkins University School of Medicine in collaborations with the Mayo Clinic College of Medicine, the Buck Institute for Research on Aging, the University Medical Center Groningen, Unity Biotechnology, Inc., and the University of California, Berkeley.-In the study, the research team presented a novel pharmacologic candidate that alleviates age-related degenerative joint conditions, such as osteoarthritis (OA) by selectively destroying SnCs. Their findings, published April 24th in Nature Medicine (Impact Factor: 30.357), suggest that the selective removal of old cells from joints could reduce the development of post-traumatic OA and allow new cartilage to grow and repair joints.-Senescent cells (SnCs) accumulate with age in many vertebrate tissues and are present at sites of age-related pathlogy. Although these cells play an essential role in wound healing and injury repair, they may also promote cancer incidence in tissues. For instance, in articular joints, such as the knee and cartilage tissue, SnCs often are not cleared from the area after injury, thereby contributing to OA development.-To test the idea that SnCs might play a causative role in OA, the research team took both younger and older mice and cut their anterior cruciate ligaments (ACL) to minic injury. They, then, administered injections of an experimental drug, named UBX0101 to selectively remove SnCs after anterior cruciate ligament transection (ACLT) surgery.-Preclinical studies in mice and human cells suggested that the removal of SnCs significantly reduced the development of post-traumatic OA and related pain and created a prochondrogenic environment for new cartilage to grow and repair joints. Indeed, the research team reported that aged mice did not exhibit signs of cartilage regeneration after treatment with UBX0101 injections,-According to the research team, the relevance of their findings to human disease was validated using chondrocytes isolated from arthritic patients. The research team notes that their findings provide new insights into therapies targeting SnCs for the treatment of trauma and age-related degenerative joint disease.-Prior to this study, Johns Hopkins Technology Ventures (JHTV) granted UNITY Biotechnology Inc. the right to use the intellectual property around the senescent cell technology. UNITY is a company aiming to develop therapeutics that address age-related diseases.-Last October, the company announced $116 million in Series B funding from some of the big names in venture capital, including Amazon CEO Jeff Bezo's venture fund Bezos Expeditions, Mayo Clinic Ventures, Venrock, and ARCH Venture Partners.-UNITY has completed a rigorous screening and preclinical testing process of candidate drugs, discovered in this study, and is launching a new clinical trial to assess its first drug, for patients with osteoarthritis of the knee this year.-Ok Hee Jeon et al., "Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment," Nature Medicine, (2017).

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Scientists Find Simple Copper Complex Shuts Down Botulinum Neurotoxin Poisoning

By Bonnie Ward

Botulinum neurotoxin is probably best known to Americans as BOTOX, a cosmetic medicine, rather than as a cause of potentially dangerous food borne illnesses. Lesser known is that Clostridium botulinum, the bacterium that causes the neurointoxication, produces one of the most potent toxins on earth and is classified as a potential bioterrorism threat.--While no cure exists—and botulism treatment options are limited—a serendipitous discovery by scientists at The Scripps Research Institute (TSRI) may provide a new therapy that can stop the neurotoxin even in its more severe, advanced stages of action. The finding, based on rodent studies, was published recently in the Journal of the American Chemical Society.--Lead scientist Kim Janda, the Ely R. Callaway, Jr. Professor of Chemistry at TSRI, said he decided to explore botulism neurotoxin due to its debilitating and life-threatening effects, as well as its danger as a potential bioterrorism agent. "It’s on the same level as Anthrax, Plague, Ebola and other Category A priority pathogens," Janda said, referring to the Centers for Disease Control and Prevention’s (CDC) list of biological agents of highest concern. "Yet there is nothing even in phase I clinical trials."--Botulism is a rare but serious disorder that attacks the body’s ability to signal to muscles. Symptoms include blurry vision, slurred speech, muscle weakness and difficulty swallowing. It can lead to paralysis throughout the body, and even death by affecting the patient’s ability to breathe. According to the CDC, botulism is primarily transmitted through food or wounds infected by the botulism bacteria, which lives in the environment. In extremely small doses, the botulism toxin is injected for medical purposes, such as to relieve spasticity, and as a cosmetic wrinkle treatment.--To discover potential inhibitors of the toxin, Janda and his research team screened triazole compounds against the botulinum neurotoxin light chain, a proteolytic enzyme that disrupts neuronal signaling to muscles. The triazoles were synthesized using click chemistry—a method developed by TSRI Professor and Nobel laureate K. Barry Sharpless in the mid-1990s. Paul Bremer, a graduate student working in Janda’s laboratory and the study’s first author, said they hit upon a triazole compound provided by Sharpless’s laboratory that appeared to forcefully inhibit the toxin light chain in an enzymatic assay.--Further testing revealed a surprise. "We had found what we thought were active click compounds, but really they were only active because of the copper," Bremer said. Copper is used as a catalyst to accomplish click chemistry and trace amounts would not be anticipated to show activity in a bioassay, he explained. "Upon further experiments, it came as a complete surprise that copper was quite potently inhibiting the enzyme."

The scientists had accidentally landed upon a potential new therapy for type A of the neurotoxin, the most common and deadly cause of human botulism, using copper chloride, an inexpensive, readily available metal salt as the active ingredient.--Next, the researchers designed molecules called ligands to act as delivery vehicles for copper into neuronal cells, an essential step in translating the therapeutic action of copper to biological systems. The TSRI team then sent their ligand-copper complexes to their study collaborators at the University of Wisconsin-Madison, who administered it to mice. The compound extended the animals’ lives, even when they were given lethal doses of the toxin.

The researchers said further animal testing is needed to determine optimal dosage, dosing frequency and other factors. Janda said clinical trials to prove efficacy cannot be done in humans due to botulinum neurotoxicity dangers. However, the safety of the copper complex can be validated through several other clinical trials already underway for different uses, he added.

If found to be safe, Bremer said the copper therapeutic could provide a more effective therapy than existing approaches to botulism. Currently, botulism sufferers receive an anti-toxin medicine that can inactivate the toxin circulating in their system, thereby preventing further poisoning. However, the anti-toxin cannot reverse preexisting paralysis because the toxin acts inside cells. Consequently, disease recovery can be slow, and paralysis may take weeks or months to wear off.

"The anti-toxin is antibody-based, which means it only works outside the cells," said Janda. "This new therapy can readily enter cells where it can attack the etiological agent, a protease, which is responsible for paralysis seen from the neurotoxin."

The researchers also noted that the study further demonstrates the need to explore metals for therapeutic uses. Metals are not commonly used in drug design because of concerns about toxicity and specific targeting as compared to organic compounds. However, several metal-based therapies already exist. For instance, gold is used in therapies for certain cancers and rheumatoid arthritis, while other metal-based treatments are currently in clinical trials.

"These are kind of underappreciated medicinal agents," said Bremer. "Our work shows the need to explore their potential further."

The study, "Metal Ions Effectively Ablate the Action of Botulinum Neurotoxin A," was supported by the National Institutes of Health (grant R01A1119564.) In addition to Janda and Bremer, authors of the study include Lisa M. Eubanks of TSRI; Sabine Pellett, William H. Tepp and Eric A. Johnson of the University of Wisconsin-Madison; and James P. Carolan and Karen N. Allen of Boston University.

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CHELATES AND CHELATING AGENTS

Many essential biological chemicals are chelates. Chelates play important roles in oxygen transport and in photosynthesis. Furthermore, many biological catalysts (enzymes) are chelates. In addition to their significance in living organisms, chelates are also economically important, both as products in themselves and as agents in the production of other chemicals.--A chelate is a chemical compound composed of a metal ion and a chelating agent. A chelating agent is a substance whose molecules can form several bonds to a single metal ion. In other words, a chelating agent is a multidentate ligand. An example of a simple chelating agent is ethylenediamine.

ethylenediamine

A single molecule of ethylenediamine can form two bonds to a transition-metal ion such as nickel(II), Ni²⁺. The bonds form between the metal ion and the nitrogen atoms of ethylenediamine. The nickel(II) ion can form six such bonds, so a maximum of three ethylenediamine molecules can be attached to one Ni²⁺ ion.

chelate with one ethylenediamine ligand

chelate with two ethylenediamine ligands

chelate with three ethylenediamine ligands

In the two structures on the left, the bonding capacity of the Ni²⁺ ion is completed by water molecules. Each water molecule forms only one bond to Ni²⁺, so water is not a chelating agent. Because the chelating agent is attached to the metal ion by several bonds, chelates tend to be more stable than complexes formed with monodentate ligands such as water.--Porphine is a chelating agent similar to ethylenediamine in that it forms bonds to a metal ion through nitrogen atoms. Each of the four nitrogen atoms in the center of the molecule can form a bond to a metal ion. Porphine is the simplest of a group of chelating agents called porphyrins. Porphyrins have a structure derived from porphine by replacing some of the hydrogen atoms around the outside with other groups of atoms.

porphine

heme

One important porphyrin chelate is heme, the central component of hemoglobin, which carries oxygen through the blood from the lungs to the tissues. Heme contains a porphyrin chelating agent bonded to an iron(II) ion. Iron, like nickel, can form six bonds. Four of these bonds tie it to the porphyrin. One of iron's two remaining bonds holds an oxygen molecule as it is transported through the blood. Chlorophyll is another porphyrin chelate. In chlorophyll, the metal at the center of the chelate is a magnesium ion. Chlorophyll, which is responsible for the green color of plant leaves, absorbs the light energy that is converted to chemical energy in the process of photosynthesis.-- Another biologically significant chelate is vitamin B-12. It is the only vitamin that contains a metal, a cobalt(II) ion bonded to a porphyrin-like chelating agent. As far as is known, it is required in the diet of all higher animals. It is not synthesized by either higher plants or animals, but only by certain bacteria and molds. These are the sources of the B-12 found in animal products. Because vitamin B-12 is not found in higher plants, vegetarians must take care to include in their diets foods or supplements that contain the vitamin A chelating agent of particular economic significance is ethylenediaminetetraacetic acid (EDTA).

ethylenediaminetetraacetic acid (EDTA)

EDTA is a versatile chelating agent. It can form four or six bonds with a metal ion, and it forms chelates with both transition-metal ions and main-group ions. EDTA is frequently used in soaps and detergents, because it forms a complexes with calcium and magnesium ions. These ions are in hard water and interfere with the cleaning action of soaps and detergents. The EDTA binds to them, sequestering them and preventing their interference. In the calcium complex, [Ca(EDTA)]^2–, EDTA is a tetradentate ligand, and chelation involves the two nitrogen atoms and two oxygen atoms in separate carboxyl (-COO^–) groups. EDTA is also used extensively as a stabilizing agent in the food industry. Food spoilage is often promoted by naturally-occurring enzymes that contain transition-metal ions. These enzymes catalyze the chemical reactions that occur during spoilage. EDTA deactivates these enzymes by removing the metal ions from them and forming stable chelates with them. It promotes color retention in dried bananas, beans, chick peas, canned clams, pecan pie filling, frozen potatoes, and canned shrimp. It improves flavor retention in canned carbonated beverages, salad dressings, mayonnaise, margarine, and sauces. It inhibits rancidity in salad dressings, mayonnaise, sauces, and sandwich spreads. EDTA salts are used in foods at levels ranging from 33 to 800 ppm. In other applications, EDTA dissolves the CaCO₃ scale deposited from hard water without the use of corrosive acid. EDTA is used in the separation of the rare earth elements from each other. The rare earth elements have very similar chemical properties, but the stability of their EDTA complexes varies slightly. This slight variation allows EDTA to effectively separate rare-earth ions. EDTA is used as an anticoagulant for stored blood in blood banks; it prevents coagulation by sequestering the calcium ions required for clotting. As an antidote for lead poisoning, calcium disodium EDTA exchanges its chelated calcium for lead, and the resulting lead chelate is rapidly excreted in the urine. The calcium salt of EDTA, administered intravenously, is also used in the treatment of acute cadmium and iron poisoning. Dimercaprol (2,3-dimercapto-1-propanol) is an effective chelating agent for heavy metals such as arsenic, mercury, antimony, and gold. These heavy metals form particularly strong bonds to the sulfur atoms in dimercaprol

Dimercaprol was originally employed to treat the toxic effects of an arsenic-containing mustard gas called Lewisite [dichloro(2-chlorovinyl)arsine], which was used in World War I. The chelated metal cannot enter living cells and is rapidly excreted from the body. Since dimercaprol is water insoluble, it is dissolved in an oil base (often peanut oil) and injected intramuscularly.

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Common class of chemicals cause cancer by breaking down DNA repair mechanisms

A common class of chemicals found everywhere from car exhausts, smoke, building materials and furniture to cosmetics and shampoos could increase cancer risk because of their ability to break down the repair mechanisms that prevent faults in our genes, according to a study published today in the journal Cell. Aldehydes are a class of chemicals made in our own bodies in small quantities but increasingly found everywhere in our environment. Exposure to these chemicals has previously been linked with cancer, but the reasons for the link remain unclear --New research led by Professor Ashok Venkitaraman, Director of the Medical Research Council Cancer Unit at the University of Cambridge, has used genetically-engineered human cells and cells from patients bearing a faulty copy of the breast cancer gene BRCA2 to identify the mechanism by which exposure to aldehydes could promote cancer .Damage to our DNA, which arises frequently as cells in our bodies divide, can lead to the development of cancers, but our body has its own defence mechanism that helps repair this damage. However, Professor Venkitaraman and colleagues found that aldehyde exposure breaks down this defence mechanisms even in normal healthy cells, but people who have inherited a faulty copy of BRCA2 are particularly sensitive to such damage. Everyone is born with two copies of most genes. People who inherit a single faulty copy of the BRCA2 gene are susceptible to cancer. The reason why is not fully understood, because their cells should be able to repair DNA using the lower -- but still adequate -- levels of BRCA2 protein made from the remaining, intact copy of the gene. This new study shows that aldehydes trigger the degradation of BRCA2 protein in cells. In people who inherit one faulty copy of the BRCA2 gene, this effect pushes down BRCA2 protein levels below the amount required for adequate DNA repair, breaking down the normal mechanisms that prevent mutations, which could promote cancer formation. Around one in 100 people may carry a faulty BRCA2 gene, putting them at risk of developing breast, ovarian, prostate and pancreatic cancer. Exposure to aldehydes could increase their chances of developing these cancers. "Our study shows how chemicals to which we are increasingly exposed in our day-to-day lives may increase the risk of diseases like cancer," says Professor Venkitaraman. "It also helps to explain why 'the faults in our exposure to environmentally unsafe chemicals can exacerbate a weakened genome creating the faulty genes we are born with -- could make some people particularly sensitive to the cancer-causing effects of these chemicals. "An important implication of our work is that it may be aldehyde exposure that triggers cancer susceptibility in people who inherit one faulty copy of the BRCA2 gene. This may help us in future to prevent or treat cancer in such people." One common potential source of aldehydes is alcohol: our body converts the alcohol that we drink into acetaldehyde, one such chemical. Ordinarily, this is broken down by a natural enzyme known as acetaldehyde dehydrogenase, but over 500 million people mainly from countries such as Japan, China and Korea inherit a faulty gene, ALDH2, that inactivates this enzyme. This is why many Asian people develop flushes when they drink, but could mean they are also particularly sensitive to the cancer-promoting effect. This new research shows that aldehyde accumulation in such people could trigger cancer susceptibility by degrading BRCA2, compromising DNA repair, whether or not they inherit a faulty copy of BRCA2. An estimated 30-60% of people from Japan, Korea and China carry the faulty ALDH2 and may therefore be at risk from cancer through this new mechanism. -Story Source-Materials provided by University of Cambridge. The original story is licensed under a Creative Commons License. Journal ReferenceTan, SLW et al. A class of environmental and endogenous toxins induces BRCA2 haploinsufficiency and genome instability. Cell, 2017 DOI: 10.1016/j.cell.2017.05.010 University of Cambridge. "Common class of chemicals cause cancer by breaking down DNA repair mechanisms." ScienceDaily. ScienceDaily, 1 June 2017. <www.sciencedaily.com/releases/2017/06/170601151916.htm>.

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The Introduction of Diphtheria-Tetanus-Pertussis and Oral Polio Vaccine Among Young Infants in an Urban African Community- A Natural Experiment

Highlights

• •When DTP and OPV were introduced in Guinea-Bissau in 1981, allocation by birthday resulted in a natural experiment of being vaccinated early or late.
• •Between 3 and 5 months of age, children who received DTP and OPV early had 5-fold higher mortality than still unvaccinated children.
• •In the only two studies of the introduction of DTP and OPV, co-administration of OPV with DTP may have reduced the negative effects of DTP.

Few studies have examined what happened to child survival when DTP and OPV were introduced in low-income countries. These vaccines were introduced in 1981 in an urban community in Guinea-Bissau from 3 months of age in connection with 3-monthly weighing sessions. Children were therefore allocated by birthday to receive vaccines early or late between 3 and 5 months of age. In this natural experiment vaccinated children had 5-fold higher mortality than not-yet-DTP-vaccinated children. DTP-only vaccinations were associated with higher mortality than DTP + OPV vaccinations. Hence, DTP may be associated with a negative effect on child survival.

Abstract

Background

We examined the introduction of diphtheria-tetanus-pertussis (DTP) and oral polio vaccine (OPV) in an urban community in Guinea-Bissau in the early 1980s.

Methods

The child population had been followed with 3-monthly nutritional weighing sessions since 1978. From June 1981 DTP and OPV were offered from 3 months of age at these sessions. Due to the 3-monthly intervals between sessions, the children were allocated by birthday in a ‘natural experiment’ to receive vaccinations early or late between 3 and 5 months of age. We included children who were <6 months of age when vaccinations started and children born until the end of December 1983. We compared mortality between 3 and 5 months of age of DTP-vaccinated and not-yet-DTP-vaccinated children in Cox proportional hazard models.

Results

Among 3–5-month-old children, having received DTP (±OPV) was associated with a mortality hazard ratio (HR) of 5.00 (95% CI 1.53–16.3) compared with not-yet-DTP-vaccinated children. Differences in background factors did not explain the effect. The negative effect was particularly strong for children who had received DTP-only and no OPV (HR = 10.0 (2.61–38.6)). All-cause infant mortality after 3 months of age increased after the introduction of these vaccines (HR = 2.12 (1.07–4.19)).

Conclusion

DTP was associated with increased mortality; OPV may modify the effect of DTP.

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Crystals grown by light to create gold nanoparticle

A team of University of Florida researchers has figured out how gold can be used in crystals grown by light to create nanoparticles, a discovery that has major implications for industry and cancer treatment and could improve the function of pharmaceuticals, medical equipment and solar panels.-Nanoparticles can be "grown" in crystal formations with special use of light, in a process called plasmon-driven synthesis. However, scientists have had limited control unless they used silver, but silver limits the uses for medical technology. The team is the first to successfully use gold, which works well within the human body, with this process.---"How does light actually play a role in the synthesis? [This knowledge] was not well developed," said David Wei, an associate professor of chemistry who led the research team. "Gold was the model system to demonstrate this."--Gold is highly desired for nanotechnology because it is malleable, does not react with oxygen and conducts heat well. Those properties make gold an ideal material for nanoparticles, especially those that will be placed in the body.--When polyvinylpyrrolidone, or PVP, a substance commonly found in pharmaceutical tablets, is used in the plasmon-driven synthesis, it enables scientists to better control the growth of crystals.--In Wei's research, PVP surprised the team by showing its potential to relay light-generated "hot" electrons to a gold surface to grow the crystals.--The research describes the first plasmonic synthesis strategy that can make high-yield gold nanoprisms. Even more exciting, the team has demonstrated that visible-range and low-power light can be used in the synthesis. -Combined with nanoparticles being used in solar photovoltaic devices, this method can even harness solar energy for chemical synthesis, to make nanomaterials or for general applications in chemistry.--Wei has spent the last decade working in nanotechnology. He is intrigued by its applications in photochemistry and biomedicine, especially in targeted drug delivery and photothermal therapeutics, which is crucial to cancer treatment. His team includes collaborators from Pacific Northwest National Laboratory, where he has worked as a visiting scholar, and Brookhaven National Laboratory.--In addition, the project has provided an educational opportunity for chemistry students: one high school student (through UF's Student Science Training Program), two University scholars who also funded by the Howard Hughes Medical Institute, five graduate students and two postdocs.

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DNA double helix structures crystals-Method developed for DNA programmed material synthesis

Engineers of Friedrich-Alexander Universität Erlangen Nürnberg (FAU) have succeeded in producing complex crystal lattices, so-called clathrates, from nanoparticles using DNA strands. The programmed synthesis of clathrates represents a template for the precision modelling of novel nanomaterials. DNA is the blueprint of biological life: it contains all hereditary information and the arrangement of its base pairs determines the structure of amino acids and ultimately the whole organism. For some years now, scientists have been using the structuring potential of DNA in other disciplines such as computer science or for creating new materials on the nano scale. In collaboration with the world's leading nanotechnology experts from the University of Michigan and North Western University, FAU engineers have opened up a new era in DNA programmed material synthesis. The team has succeeded in reordering pyramid-shaped gold crystals to form complex clathrate compounds.

DNA determines lattice structure

For the synthesis process, the 250 nanometre gold crystals -- which in the experiment represent atoms that can form clathrates -- are held in a suspension which is supplemented with artificial DNA. 'The DNA strands attach to the gold particles and move them into a certain position during a self-assembling process,' explains Professor Michael Engel, member of the Institute for Multiscale Simulation. 'Depending on the length of the DNA sequences and the arrangement of the base pairs, different three-dimensional lattice structures form. Through DNA programming we can more or less determine the structure of the crystal lattice in a very precise manner.'

Clathrates -- nuclear cages with a wide range of applications

Clathrates are of particular interest in the field of materials research because they are composed of nuclear cages in which other substances, usually gases, can be embedded. 'The controlled production of colloidal clathrates opens up a wide range of possible applications,' says Michael Engel. 'Materials could be used for recognising proteins or viruses and targeted manipulation of certain parameters of the crystal lattice can lead to material properties which are not achievable in simpler colloidal crystals.'-Story Source-Materials provided by University of Erlangen-Nuremberg. University of Erlangen-Nuremberg. "DNA double helix structures crystals: Method developed for DNA programmed material synthesis." ScienceDaily. ScienceDaily, 4 April 2017. <www.sciencedaily.com/releases/2017/04/170404104732.htm>.

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Historical FACTS On Dangers, Ineffectiveness Of Vaccines

http://www.vaccinationdebate.com/web2.html

8-28-2009

* In 1871-2, England, with 98% of the population aged between 2 and 50 vaccinated against smallpox, it experienced its worst ever smallpox outbreak with 45,000 deaths. During the same period in Germany, with a vaccination rate of 96%, there were over 125,000 deaths from smallpox. http://www.soilandhealth.org/02/0201hyglibcat/020119hadwin/020119hadwin.toc.html

The Hadwen Documents

* In Germany, compulsory mass vaccination against diphtheria commenced in 1940 and by 1945 diphtheria cases were up from 40,000 to 250,000. (Don't Get Stuck, Hannah Allen)

* In the USA in 1960, two virologists discovered that both polio vaccines were contaminated with the SV 40 virus which causes cancer in animals as well as changes in human cell tissue cultures. Millions of children had been injected with these vaccines. (Med Jnl of Australia 17/3/1973 p555)

* In 1967, Ghana was declared measles free by the World Health Organisation after 96% of its population was vaccinated. In 1972, Ghana experienced one of its worst measles outbreaks with its highest ever mortality rate. (Dr H Albonico, MMR Vaccine Campaign in Switzerland, March 1990)

In the UK between 1970 and 1990, over 200,000 cases of whooping cough occurred in fully vaccinated children. (Community Disease Surveillance Centre, UK)

In the 1970's a tuberculosis vaccine trial in India involving 260,000 people revealed that more cases of TB occurred in the vaccinated than the unvaccinated. (The Lancet 12/1/80 p73)

* In 1977, Dr Jonas Salk who developed the first polio vaccine, testified along with other scientists, that mass inoculation against polio was the cause of most polio cases throughout the USA since 1961. (Science 4/4/77 "Abstracts" )

* In 1978, a survey of 30 States in the US revealed that more than half of the children who contracted measles had been adequately vaccinated. (The People's Doctor, Dr R Mendelsohn)

* In 1979, Sweden abandoned the whooping cough vaccine due to its ineffectiveness. Out of 5,140 cases in 1978, it was found that 84% had been vaccinated three times! (BMJ 283:696-697, 1981)

* The February 1981 issue of the Journal of the American Medical Association found that 90% of obstetricians and 66% of pediatricians refused to take the rubella vaccine.

* In the USA, the cost of a single DPT shot had risen from 11 cents in 1982 to $11.40 in 1987. The manufacturers of the vaccine were putting aside $8 per shot to cover legal costs and damages they were paying out to parents of brain damaged children and children who died after vaccination. (The Vine, Issue 7, January 1994, Nambour, Qld)

* In Oman between 1988 and 1989, a polio outbreak occurred amongst thousands of fully vaccinated children. The region with the highest attack rate had the highest vaccine coverage. The region with the lowest attack rate had the lowest vaccine coverage. (The Lancet, 21/9/91)

* In 1990, a UK survey involving 598 doctors revealed that over 50% of them refused to have the Hepatitis B vaccine despite belonging to the high risk group urged to be vaccinated. (British Med Jnl, 27/1/1990)

* In 1990, the Journal of the American Medical Association had an article on measles which stated " Although more than 95% of school-aged children in the US are vaccinated against measles, large measles outbreaks continue to occur in schools and most cases in this setting occur among previously vaccinated children." (JAMA, 21/11/90)

* In the USA, from July 1990 to November 1993, the US Food and Drug Administration counted a total of 54,072 adverse reactions following vaccination. The FDA admitted that this number represented only 10% of the real total, because most doctors were refusing to report vaccine injuries. In other words, adverse reactions for this period exceeded half a million! (National Vaccine Information Centre, March 2, 1994)

* In the New England Journal of Medicine July 1994 issue a study found that over 80% of children under 5 years of age who had contracted whooping cough had been fully vaccinated.

* On November 2nd, 2000, the Association of American Physicians and Surgeons (AAPS) announced that its members voted at their 57th annual meeting in St Louis to pass a resolution calling for an end to mandatory childhood vaccines. The resolution passed without a single "no" vote. http://www.wellnesschiro.com/physicians_group_end_mandatory_vaccines.htm

(Report by Michael Devitt)

http://www.vaccinationdebate.com/web2.html

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Self-assembled nanostructures can be selectively controlled

Plasmonic nanoparticles exhibit properties based on their geometries and relative positions. Researchers have now developed an easy way to manipulate the optical properties of plasmonic nanostructures that strongly depend on their spatial arrangement.-- The plasmonic nanoparticles can form clusters, plasmonic metamolecules, and then interact with each other. Changing the geometry of the nanoparticles can be used to control the properties of the metamolecules. --"The challenge is to make the structures change their geometry in a controlled way in response to external stimuli. In this study, structures were programmed to modify their shape by altering the pH," tells Assistant Professor Anton Kuzyk from Aalto University.

Utilization of programmable DNA locks

In this study plasmonic metamolecules were functionalized with pH-sensitive DNA locks. DNA locks can be easily programmed to operate at a specific pH range. Metamolecules can be either in a "locked" state at low pH or in relaxed state at high pH. Both states have very distinct optical responses. This in fact allows creating assemblies of several types of plasmonic metamolecules, with each type designed to switch at different a pH value. -The ability to program nanostructures to perform a specific function only within a certain pH window could have applications in the field of nanomachines and smart nanomaterials with tailored optical functionalities. -This active control of plasmonic metamolecules is promising for the development of sensors, optical switches, transducers and phase shifters at different wavelengths. In the future, pH-responsive nanostructures could also be useful in the development of controlled drug delivery.--Story Source-Materials provided by Aalto University. Journal Reference-Anton Kuzyk, Maximilian J. Urban, Andrea Idili, Francesco Ricci, Na Liu. Selective control of reconfigurable chiral plasmonic metamolecules. Science Advances, 2017; 3 (4): e1602803 DOI: 10.1126/sciadv.1602803

Aalto University. "Self-assembled nanostructures can be selectively controlled." ScienceDaily. ScienceDaily, 24 April 2017. <www.sciencedaily.com/releases/2017/04/170424094048.htm>.

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Nervous system manipulation by electromagnetic fields from monitors

Abstract

Physiological effects have been observed in a human subject in response to stimulation of the skin with weak electromagnetic fields that are pulsed with certain frequencies near ½ Hz or 2.4 Hz, such as to excite a sensory resonance. Many computer monitors and TV tubes, when displaying pulsed images, emit pulsed electromagnetic fields of sufficient amplitudes to cause such excitation. It is therefore possible to manipulate the nervous system of a subject by pulsing images displayed on a nearby computer monitor or TV set. For the latter, the image pulsing may be imbedded in the program material, or it may be overlaid by modulating a video stream, either as an RF signal or as a video signal. The image displayed on a computer monitor may be pulsed effectively by a simple computer program. For certain monitors, pulsed electromagnetic fields capable of exciting sensory resonances in nearby subjects may be generated even as the displayed images are pulsed with subliminal intensity.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the electromagnetic field that emanates from a monitor when the video signal is modulated such as to cause pulses in image intensity, and a nearby subject who is exposed to the field.

FIG. 2 shows a circuit for modulation of a composite video signal for the purpose of pulsing the image intensity.

FIG. 3 shows the circuit for a simple pulse generator.

FIG. 4 illustrates how a pulsed electromagnetic field can be generated with a computer monitor.

FIG. 5 shows a pulsed electromagnetic field that is generated by a television set through modulation of the RF signal input to the TV.

FIG. 6 outlines the structure of a computer program for producing a pulsed image.

FIG. 7 shows an extrapolation procedure introduced for improving timing accuracy of the program of FIG. 6.

FIG. 8 illustrates the action of the extrapolation procedure of FIG. 7.

FIG. 9 shows a subject exposed to a pulsed electromagnetic field emanating from a monitor which is responsive to a program running on a remote computer via a link that involves the Internet.

FIG. 10 shows the block diagram of a circuit for frequency wobbling of a TV signal for the purpose of pulsing the intensity of the image displayed on a TV monitor.

FIG. 11 depicts schematically a recording medium in the form of a video tape with recorded data, and the attribute of the signal that causes the intensity of the displayed image to be pulsed.

FIG. 12 illustrates how image pulsing can be embedded in a video signal by pulsing the illumination of the scene that is being recorded.

FIG. 13 shows a routine that introduces pulse variability into the computer program of FIG. 6.

FIG. 14 shows schematically how a CRT emits an electromagnetic field when the displayed image is pulsed.

FIG. 15 shows how the intensity of the image displayed on a monitor can be pulsed through the brightness control terminal of the monitor.

FIG. 16 illustrates the action of the polarization disc that serves as a model for grounded conductors in the back of a CRT screen.

FIG. 17 shows the circuit for overlaying image intensity pulses on a DVD output.

FIG. 18 shows measured data for pulsed electric fields emitted by two different CRT type monitors, and a comparison with theory

Images (10)

BACKGROUND OF THE INVENTION

The invention relates to the stimulation of the human nervous system by an electromagnetic field applied externally to the body. A neurological effect of external electric fields has been mentioned by Wiener (1958), in a discussion of the bunching of brain waves through nonlinear interactions. The electric field was arranged to provide "a direct electrical driving of the brain". Wiener describes the field as set up by a 10 Hz alternating voltage of 400 V applied in a room between ceiling and ground. Brennan (1992) describes in U.S. Pat. No. 5,169,380 an apparatus for alleviating disruptions in circadian rythms of a mammal, in which an alternating electric field is applied across the head of the subject by two electrodes placed a short distance from the skin. -A device involving a field electrode as well as a contact electrode is the "Graham Potentializer" mentioned by Hutchison (1991). This relaxation device uses motion, light and sound as well as an alternating electric field applied mainly to the head. The contact electrode is a metal bar in Ohmic contact with the bare feet of the subject, and the field electrode is a hemispherical metal headpiece placed several inches from the subject's head.--In these three electric stimulation methods the external electric field is applied predominantly to the head, so that electric currents are induced in the brain in the physical manner governed by electrodynamics. Such currents can be largely avoided by applying the field not to the head, but rather to skin areas away from the head. Certain cutaneous receptors may then be stimulated and they would provide a signal input into the brain along the natural pathways of afferent nerves. It has been found that, indeed, physiological effects can be induced in this manner by very weak electric fields, if they are pulsed with a frequency near ½ Hz. The observed effects include ptosis of the eyelids, relaxation, drowziness, the feeling of pressure at a centered spot on the lower edge of the brow, seeing moving patterns of dark purple and greenish yellow with the eyes closed, a tonic smile, a tense feeling in the stomach, sudden loose stool, and sexual excitement, depending on the precise frequency used, and the skin area to which the field is applied. The sharp frequency dependence suggests involvement of a resonance mechanism.--It has been found that the resonance can be excited not only by externally applied pulsed electric fields, as discussed in U.S. Pat. Nos. 5,782,874, 5,899,922, 6,081,744, and 6,167,304, but also by pulsed magnetic fields, as described in U.S. Pat. Nos. 5,935,054 and 6,238,333, by weak heat pulses applied to the skin, as discussed in U.S. Pat. Nos. 5,800,481 and 6,091,994, and by subliminal acoustic pulses, as described in U.S. Pat. No. 6,017,302. Since the resonance is excited through sensory pathways, it is called a sensory resonance. In addition to the resonance near ½ Hz, a sensory resonance has been found near 2.4 Hz. The latter is characterized by the slowing of certain cortical processes, as discussed in the '481, '922, '302, '744, '944, and '304 patents.--The excitation of sensory resonances through weak heat pulses applied to the skin provides a clue about what is going on neurologically. Cutaneous temperature-sensing receptors are known to fire spontaneously. These nerves spike somewhat randomly around an average rate that depends on skin temperature. Weak heat pulses delivered to the skin in periodic fashion will therefore cause a slight frequency modulation (fm) in the spike patterns generated by the nerves. Since stimulation through other sensory modalities results in similar physiological effects, it is believed that frequency modulation of spontaneous afferent neural spiking patterns occurs there as well. It is instructive to apply this notion to the stimulation by weak electric field pulses administered to the skin. The externally generated fields induce electric current pulses in the underlying tissue, but the current density is much too small for firing an otherwise quiescent nerve. However, in experiments with adapting stretch receptors of the crayfish, Terzuolo and Bullock (1956) have observed that very small electric fields can suffice for modulating the firing of already active nerves. Such a modulation may occur in the electric field stimulation under discussion.-- Further understanding may be gained by considering the electric charges that accumulate on the skin as a result of the induced tissue currents. Ignoring thermodynamics, one would expect the accumulated polarization charges to be confined strictly to the outer surface of the skin. But charge density is caused by a slight excess in positive or negative ions, and thermal motion distributes the ions through a thin layer. This implies that the externally applied electric field actually penetrates a short distance into the tissue, instead of stopping abruptly at the outer skin surface. In this manner a considerable fraction of the applied field may be brought to bear on some cutaneous nerve endings, so that a slight modulation of the type noted by Terzuolo and Bullock may indeed occur

How you get Hit

I claim:

1. A method for manipulating the nervous system of a subject located near a monitor, the monitor emitting an electromagnetic field when displaying an image by virtue of the physical display process, the subject having a sensory resonance frequency, the method comprising:

creating a video signal for displaying an image on the monitor, the image having an intensity;

modulating the video signal for pulsing the image intensity with a frequency in the range 0.1 Hz to 15 Hz; and

setting the pulse frequency to the resonance frequency.

2. A computer program for manipulating the nervous system of a subject located near a monitor, the monitor emitting an electromagnetic field when displaying an image by virtue of the physical display process, the subject having cutaneous nerves that fire spontaneously and have spiking patterns, the computer program comprising:

a display routine for displaying an image on the monitor, the image having an intensity;

a pulse routine for pulsing the image intensity with a frequency in the range 0.1 Hz to 15 Hz; and

a frequency routine that can be internally controlled by the subject, for setting the frequency;

whereby the emitted electromagnetic field is pulsed, the cutaneous nerves are exposed to the pulsed electromagnetic field, and the spiking patterns of the nerves acquire a frequency modulation.

3. The computer program of claim 2, wherein the pulsing has an amplitude and the program further comprises an amplitude routine for control of the amplitude by the subject.

4. The computer program of claim 2, wherein the pulse routine comprises:

a timing procedure for timing the pulsing; and

an extrapolation procedure for improving the accuracy of the timing procedure.

5. The computer program of claim 2, further comprising a variability routine for introducing variability in the pulsing.

6. Hardware means for manipulating the nervous system of a subject located near a monitor, the monitor being responsive to a video stream and emitting an electromagnetic field when displaying an image by virtue of the physical display process, the image having an intensity, the subject having cutaneous nerves that fire spontaneously and have spiking patterns, the hardware means comprising:

pulse generator for generating voltage pulses;

means, responsive to the voltage pulses, for modulating the video stream to pulse the image intensity;

whereby the emitted electromagnetic field is pulsed, the cutaneous nerves are exposed to the pulsed electromagnetic field, and the spiking patterns of the nerves acquire a frequency modulation.

7. The hardware means of claim 6, wherein the video stream is a composite video signal that has a pseudo-dc level, and the means for modulating the video stream comprise means for pulsing the pseudo-dc level.

8. The hardware means of claim 6, wherein the video stream is a television broadcast signal, and the means for modulating the video stream comprise means for frequency wobbling of the television broadcast signal.

9. The hardware means of claim 6, wherein the monitor has a brightness adjustment terminal, and the means for modulating the video stream comprise a connection from the pulse generator to the brightness adjustment terminal.

10. A source of video stream for manipulating the nervous system of a subject located near a monitor, the monitor emitting an electromagnetic field when displaying an image by virtue of the physical display process, the subject having cutaneous nerves that fire spontaneously and have spiking patterns, the source of video stream comprising:

means for defining an image on the monitor, the image having an intensity; and

means for subliminally pulsing the image intensity with a frequency in the range 0.1 Hz to 15 Hz;

whereby the emitted electromagnetic field is pulsed, the cutaneous nerves are exposed to the pulsed electromagnetic field, and the spiking patterns of the nerves acquire a frequency modulation.

11. The source of video stream of claim 10 wherein the source is a recording medium that has recorded data, and the means for subliminally pulsing the image intensity comprise an attribute of the recorded data.

12. The source of video stream of claim 10 wherein the source is a computer program, and the means for subliminally pulsing the image intensity comprise a pulse routine.

13. The source of video stream of claim 10 wherein the source is a recording of a physical scene, and the means for subliminally pulsing the image intensity comprise:

pulse generator for generating voltage pulses;

light source for illuminating the scene, the light source having a power level; and

modulation means, responsive to the voltage pulses, for pulsing the power level.

14. The source of video stream of claim 10, wherein the source is a DVD, the video stream comprises a luminance signal and a chrominance signal, and the means for subliminal pulsing of the image intensity comprise means for pulsing the luminance signal.

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Natural Anti-Biofilm Agents

Posted by drwillipMarch 13, 2015

Introduction

Biofilms are gelatinous masses of microorganisms capable of attaching to virtually any surface. According to the NIH, they factor into nearly 80% of all bacterial infections [Schachter, 2003] and are inherently resistant to antibiotics. Biofilms are what keep wounds from healing, and bladder infections recurring. They may also be why lyme disease lingers. Biofilms are at the heart (and lung) of bacterial pneumonia, and are the death of cystic fibrosis kids and burn patients. Biofilms cause tooth decay, gum disease, sinusitis, ear infections, and Legionnaires’ disease. Biofilms glom onto medical devices (e.g., heart valves, catheters, joint replacements) where they are deadly, or difficult to eradicate. Biofilms plague hospitals, and contribute greatly to our health care burden. [Hall-Stoodley et al., 2004] -Biofilms are also good for us. They line the digestive tract, especially the lower intestines, and the skin. Healthy biofilms contain many different species of bacteria working together to benefit humans. Many trillions of organisms protect us from pathogens and toxins, help boost our immune defenses, keep our plumbing working, steer us away from obesity, and may even make us think and feel better [Pollan, 2013; Rose, 2011]. An imbalance of bacteria in the gut – particularly from antibiotic usage, stress, or lack of fiber in the diet – leaves us susceptible to disease.--There are likely a number of stealthy biofilms that adversely affect the body in unknown ways. The list may include fetal development, autism, depression, chronic fatigue, Lyme disease, cognitive impairment, etc. [Janossy, 2015] Chronic Lyme disease may play a role in the development of dementia and Alzheimer’s. [McDonald, 2012] Many stealthy infectious agents have been identified (e.g., Borrellia, Mycoplasma, Bartonella, Babesia, Rickettsia), but some are still unknown or poorly understood. Chronic inflammation from biofilm infection can lead to cancer, cardiovascular disease, dementia, and other debilitating conditions. [Esser et al., 2015]---Recently, new anti-biofilm agents have been developed as adjuncts or alternatives to classical antibiotic treatment. Many of these novel agents show "resistance" to the emergence of antimicrobial resistance, and even enhance the activity of conventional antibiotics. Anti-biofilm substances may be synergistic with other antimicrobials to overcome persistent infectious threats. [Wu et al., 2004]  The following is a quick review of many of the natural anti-biofilm agents currently under study.

Biofilm-Disrupting Enzymes

Enzymes, like DNase I, α-amylase and DspB are biofilm-dispersing agents that degrade the biofilm matrix, permitting increased penetration of antibiotics. DNase I cleavage of extracellular DNA leads to alterations in biofilm architecture, which permits increased antibiotic penetration. [Tetz et al., 2009] A DNA-dissolving drug (Pulmozyme) has been used in cystic fibrosis patients to help disrupt the biofilm. α-amylase is a proven anti-biofilm agent against Staphylococcus aureus, Vibrio cholerae and Pseudomonas aeruginosa, not only inhibiting biofilm formation, but also degrading preformed mature biofilms [Kalpana et al., 2012]. DspB is a soluble β-N-acetylglucosaminidase with broad-spectrum activity to dissolve the biofilm matrix, and shows synergy with other antimicrobials [Darouiche et al., 2009].---Proteolytic enzymes like serrapeptase help the body break down protein involved in inflammation and mucous. It may also help disrupt the outer layers of biofilms and uncover hidden microbes.-Unfortunately, the high cost of industrial enzyme production makes their large-scale application as anti-biofilm agents unfeasible. [Sun et al., 2013]--Bacteriophages are viruses that produce a number of enzymes that negate the protection afforded by biofilms. Phages degrade the biofilm matrix and lyse bacteria, while leaving friendly bacteria unharmed. Phage modification of biofilm architecture also increases susceptibility to antibiotics. However, phage-resistant bacteria can evolve rapidly. [Sun et al., 2013]

Quorum-Sensing Inhibitors

Quorum-Sensing (QS) is a form of communication bacteria use to cooperatively build biofilm communities. Most bacteria produce QS signals, as well as QS inhibitors. Usnic acid, a lichen metabolite, possesses inhibitory activity against bacterial and fungal biofilms via QS interference. QS inhibitors can increase the susceptibility of biofilms to antibiotics. QS Inhibitors are generally regarded as safe in humans. [Sun et al., 2013]

Garlic inhibits the expression of several genes that control bacterial QS. The star in garlic’s arsenal is ajoene, the sulfur-containing compound produced when garlic is crushed. Ajoene inhibits production of rhamnolipid, which shields biofilms from white blood cells. Over 90% of biofilm bacteria were killed with a combination of ajoene and the antibiotic tobramycin. Garlic also has anti-viral, anti-fungal, and anti-protozoal properties, and benefits the cardiovascular and immune systems. [Jakobsen et al., 2012] These sulfur compounds from garlic quickly lose their activity upon exposure to oxygen. A willow bark extract, hamamelitannin, also inhibits QS. [Morgan, 2015]

Antioxidants

The anticancer, antioxidant, and anti-inflammatory effects of flavonoids are well established. Yet, their biofilm disrupting function is practically unknown. Flavonoids appear to suppress the formation of biofilms via a non-specific QS inhibition [Vikram et al., 2010]. The flavonoid phloretin inhibited biofilm formation in E. coli O157:H7, and ameliorated colon inflammation in rats without harming beneficial biofilms [Lee et.al., 2011]. Naturally occurring flavanols in cocoa may reverse memory decline significantly. [Brickman, et al., 2014] Their ability to inhibit QS might provide a clue for their action.--The anti-aging antioxidant resveratrol, associated with red wine, is produced by plants when under attack by pathogens. Resveratrol demonstrated significant antimicrobial properties on periodontal pathogens [O’Connor et al., 2011]--Cranberry has a reputation for keeping bacteria from sticking to surfaces. The red pigments in cranberries have been shown to inhibit biofilm formation. These proanthocyanidins [PACs] have been reported to possess antimicrobial, anti-adhesion, antioxidant, and anti-inflammatory properties. [Bodet et al., 2006] They prevent the attachment of pathogens to host tissues, and can inhibit the formation of biofilms in the mouth and urinary tract. [Labrecque et al., 2006] Cranberry PACs stopped the gum disease pathogen, Porphyromonas gingivitis, from adhering and forming biofilm, which markedly reduced its invasiveness. [La et al., 2010] These unique PACs also prevented adherence and biofilm formation by Candida albicans, the causative agent of thrush and yeast infections. [Feldman et al., 2012]  Cranberry juice extract, at low micromolar levels, inhibited tissue-destroying enzymes made by bacteria [La et al., 2010] and humans. [Bodet et al., 2007] Cranberry PACs also prevented dental plaque, by inhibiting biofilm-forming enzymes, [Steinberg et al., 2004] and keeping bacteria from aggregating. [Weiss et al., 1998; Yamanaka et al., 2004] Daily use of a cranberry-containing mouthwash for 6 weeks significantly reduced levels of mutans streptococci in human saliva. [Weiss et al., 2004] The anti-adhesive benefits of cranberry for urinary tract infections may be substantially increased by increasing the alkalinity of urine (https://thescienceofnutritiondotnet.wordpress.com/2015/07/17/beat-urinary-tract-infections-with-nutrition/).

Chlorogenic acids (CGA), largely from coffee, are cinnamic acid derivatives with important antioxidant and anti-inflammatory activities. [Farah et al., 2008] In vitro antibacterial and anti-biofilm activities of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia resistant to trimethoprim/sulfamethoxazole (TMP/SMX) was investigated. The MIC and MBC values ranged from 8 to 32 μg/mL. In vitro antibiofilm testing showed a 4-fold reduction in biofilm viability at 4x MIC. [Karunanidhi et al., 2012]

Boswellic acids are pentacyclic triterpenes, produced in plants belonging to the genus Boswellia, with potent anti-biofilm properties. Acetyl-11-keto-β-boswellic acid, which exhibited the most potent antibacterial activity, was effective against all 112 pathogenic gram positive bacteria tested (MIC range, 2-8 μg/ml). It inhibited biofilms formed by S. aureus and S. epidermidis, and could also disrupt preexisting biofilms. Disruption of bacterial membranes is the likely mode of action. [Raja et al., 2011]

The leaf extract of Pongamia pinnata showed significant antibiofilm activity [Karlapudi et al., 2012]. The antimicrobial activity of the plant extract is attributed to the presence of phenolic compounds, such as alkaloids, flavonoids, terpenoids and polyacetylenes. [Shan et al., 2007]--Five Indonesian medical plant extracts were shown to inhibit Pseudomonas aeruginosa and Staphylococcus aureus biofilm formation at concentrations as low as 0.12 mg/mL. [Pratiwi et al., 2015]

Farnesol and xylitol were shown to possess antibiofilm and antibacterial effects when used in root canal irrigants. [Alves et al., 2013] Xylitol is a low-carb sweetener found in toothpaste and diet sodas. When bacteria incorporate xylitol into the biofilm, it makes for a flimsy structure. [Morgan, 2015]

Aspirin and many other naturally-occurring salicylates have been shown to inhibit the macromolecules that make up the biofilm matrix [Domenico et al, 1990; Muller et al., 1998]. Salicylates are produced by many plants in response to infection.

Pro-oxidants can also be effective against biofilms. Oxidative agents are microbicidal, and offer possibilities for reducing the pathogenic activities of biofilms, especially those with an anaerobic component. In one study, 85% improvement was seen among 66 chronic Lyme disease patients with hyperbaric oxygen therapy, together with antibiotics. [Huang et al., 2014] Oxygen-supercharged Kaqun water, which has exhibited anticancer properties, may also prove useful. In contrast, nitric oxide, a signaling molecule involved in the immune system, promotes biofilm formation. [Plate & Marietta, 2012]

Fatty Acid Inhibitors

Several Salvia (Sage) species widely used as spices were evaluated for their antimicrobial activities, including their anti-adhesive and anti-biofilm effects. Salvia triloba extract demonstrated significant bacteriocidal activity against MRSA. Its volatile oil was active against all tested microorganisms except P. aeruginosa. S. triloba extract and volatile oil were active against biofilms, demonstrating anti-adhesion and anti-biofilm activities, respectively. The antimicrobial activities of other Salvia species were negligible. [Al-Bakri et al., 2010]

Short- and medium-chain fatty acids exhibit antimicrobial activity. Formic, capric, and lauric acids are broadly inhibitory for bacteria. Undecylenic acid is another medium chain fatty acid known for its anti-biofilm ability – including the disruption of troubling biofilms of Candida albicans. [McLain et al., 2000] These fatty acid inhibitors contribute to the formation and interaction of species within biofilms. [Huang et al., 2011]

Bacterial Anti-biofilm Inhibitors

Not surprisingly, bacteria compete with one another for turf. Certain substances on the surface of one bacteria work to inhibit biofilms from another. Extracellular polysaccharides (EPS)  are the essential building blocks for the biofilm matrix of most microorganisms. Thus, EPS is the stuff of biofilms, but can also inhibit their neighbors’ biofilms, from initial adhesion, dispersion, cell to cell communication, to matrix degradation. [Rendueles et al.,2013] One example of this EPS anti-biofilm activity is in Actinobacillus pleuropneumoniae serotype 5. The EPS from these bacteria inhibits cell-to-cell and cell-to-surface interactions of other bacteria, preventing them from forming or maintaining biofilms. This is one of a growing number of natural bacterial polysaccharides that exhibit broad-spectrum, non-biocidal anti-biofilm activity. [Karwacki et al., 2013]-Numerous bacteria produce anti-biofilm agents. Extracts of a coral-associated bacteria induced a reduction in S. aureus and Serratia marcescens biofilm formation. A novel natural product, 4-phenylbutanoic acid, from the marine bacterium Bacillus pumilus, shows inhibitory activity against biofilms from a broad range of bacteria. [Nithya et al., 2011] Ethyl acetate extracts of the bacterium, Bacillus firmus—a coral-associated bacterium–show antibiofilm activity against biofilms formed by multidrug resistant S. aureus. [Gowrishankar et al., 2012]

Streptococcus salivarius, a non-biofilm, harmless inhabitant of the human mouth, uses two enzymes to inhibit the formation of dental biofilms, otherwise known as plaque. These enzymes were identified as fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA), which affected a decrease in EPS production. The large quantities of FruA that S. salivarius produces may play an important role in microbial interactions for sucrose-dependent biofilm formation in the mouth. [Ogawa et al., 2011]

Minerals that Affect Biofilm

Many enzymes in the body are metallo-enzymes that rely on iron, zinc, selenium, manganese, magnesium  and other minerals for activity. Toxic heavy metals (mercury, lead, cadmium) can displace the metal component of these metallo-enzymes and render them ineffective or non-functional. Some of these enzymes (e.g., SOD, catalase, glutathione reductase) play key roles in our antioxidant defenses. Toxic metal elimination and good mineral nutrition from safe-grown foods and dietary supplements can enhance our immune capabilities substantially to reduce the risk of infection.

Silver is an important antimicrobial agent used as a coating to reduce bacterial adhesion to biomaterials and prevent infections. Silver ions increase bacterial membrane permeability, induce de-energization of cells, leakage of cellular content, and disruption DNA replication. [Marambio-Jones & Hoek, 2010] Many studies support an anti-biofilm component of silver. However, a recent study suggests that silver may indirectly promote bacterial adhesion [Carvalho et al., 2013].

Iron promotes EPS production and biofilm formation in many pathogenic, biofilm-producing bacteria. By tying up iron, lactoferrin could conceivably show anti-biofilm activity. Lactoferrin shows powerful anti-candida and anti-bacterial properties. [DePas et al., 2012]

Bismuth is an element in the earth’s crust that has been shown to possess anti-biofilm activity. Bismuth appears to work largely by inhibiting bacterial EPS [Domenico et al., 1991, 1992] via competitive interference with iron metabolism. [Domenico et al., 1996] Interestingly, Pepto-Bismol is comprised of two independent and additive anti-biofilm agents, bismuth and salicylate [Domenico et al, 1991, 1992]. The likely main action of Pepto-Bismol is to dampen overgrowth of biofilm in the gut.- Bismuth is a mild agent, but its potency can be enhanced up to 1000-fold with lipophilic thiols. [Domenico et al., 1997] Some thiols used to potentiate bismuth are naturally-occurring, and some are synthetic. Each possesses a unique antibacterial spectrum that adds to the utility of these novel anti-biofilm compounds. Bismuth-thiols (BTs) have potent, broad spectrum activity, even against antibiotic resistant bacteria.  Additionally BTs prevent and eradicate microbial biofilms at low micromolar concentrations. [Domenico et al., 1999; Folsom et al., 2011] Topical BT administration to infected open fracture wounds potentiated the effect of systemically administered antibiotics, reduced infection rate and bacteria quantity associated with bone and orthopaedic implants. [Penn-Barwell 2015] Microbion Corporation’s lead compound, BisEDT, has been granted FDA Qualified Infectious Disease Product (QIDP) status, and is in Phase 2 clinical studies for treatment of orthopedic wound infections and chronic wounds.-The low toxicity of bismuth makes it quite different than most heavy metals, which weaken immunity and create an environment for unhealthy biofilms. Chelation therapy with EDTA removes many of these heavy metals and shows anti-biofilm effects. Chelating agents show biofilm dispersing qualities because

the biofilm matrix is held together largely by minerals like calcium, magnesium, and iron. Phosphate is involved also, to solidify the biofilm structure. EDTA weakens the structure of biofilms to allow the immune system or antibiotics to gain access to the microbes hiding deep within biofilm community.  EDTA may supercharge antibiotics by 1000-fold. [Finnegan & Percival, 2014]

Another metal chelator, N-acetyl-L-cysteine (NAC), at low milligram levels, was found to decrease biofilm formation by a variety of bacteria and reduced the production of EPS matrix, while promoting biofilm disruption [Pézer- Giraldo et al., 1997]. Synergy of NAC with ciprofloxacin was shown against biofilm production and pre-formed mature biofilms from many pathogenic microbes on ureteral stent surfaces. NAC increased ciprofloxacin action by degrading the EPS matrix of biofilms. [El-feky et al., 2009]

A healthy gut maintains healthy biofilm communities that support the absorption of nutrients. Using detox agents like charcoal to mop up certain poisons and toxic metabolites, may conceivably protect the gut biofilm, and ward off pathogenic biofilms. Charcoal shows life-extending effects in laboratory rats.[Frolkis et al., 1989]

Other Modalities that Inhibit Biofilms

Antimicrobial Peptides (AMPs) are cationic, amphipathic substances that are part of the innate immunity in animals, plants, and some microbes. AMPs bind to and disrupt bacterial membranes, and efficiently kill biofilms.  AMPs from sea urchins, sea cucumbers and echinoderms have all been shown to disrupt biofilms. Their drawbacks are a sensitivity to salt, ionic strength, pH and proteolytic activity in body fluids. Synthetic AMPs have recently emerged as attractive anti-biofilm agents. Specifically targeted AMPs (STAMPs) are fusion peptides that target single pathogens, and are relatively stable under a range of physiological conditions. STAMPs can selectively eliminate the biofilm-forming, tooth-decay pathogen Streptococcus mutans from a mixed-species environment. [Sun, 2013]

Low-frequency ultrasound treatment in combination with antibiotics is promising for biofilm removal. [He et al., 2011] Ultrasound facilitates transport of antibiotics across biofilms, and increases sensitivity of biofilm-growing bacteria to antibiotics. [Carmen et al., 2005; Dong et al., 2013] It has been used as a treatment for chronic rhinosinusitis. [Bartley & Young, 2009] Ultrasound could conceivably be used in tandem with any one or more anti-biofilm agents.

Taurolidine is active against a wide range of microorganisms, including antibiotic-resistant bacteria, fungi, and mycobacteria.[Watson et al., 1995; Torres-Viera et al., 2000]  Taurolidine and its derivatives react with the bacterial cell wall, cell membrane, and endotoxins. Microbes are killed and the resulting toxins are inactivated, which reduces the inflammatory effect. Taurolidine is also used in the prevention and treatment of catheter related infections. [Zweich et al., 2013; Handrup et al., 2012; Chu et al., 2012; Diamanti et al., 2014] Taurolidine decreases bacterial adherence to host cells by destroying fimbriae (appendages used to stick to surfaces), which prevents biofilm formation. Bacterial resistance against taurolidine has yet to be observed. No systemic side effects have been identified. However, high concentrations (up to 5 mg/mL) are required for activity.

Derivatives of 2-aminoimidazoles have recently been developed as molecules that both inhibit biofilm formation and disperse bacterial biofilms [Richards & Melander, 2009]. Examples of natural products in this class include oroidin, ageliferin, and mauritiamine. [Mourabit & Potier, 2001; Huigens et al., 2007, 2008] These naturally occurring secondary metabolites are produced by marine plants and animals (e.g., sponges, coral) to keep them slime free [Kelly et al., 2003; Tsukamoto et al., 1996; Yamada, 1997]. 2-aminoimidazole/triazole conjugates (effective range, 50-150 µM) eliminate biofilm colonization, augment the action of conventional antibiotics, suppress multidrug resistance, and are not hemolytic at active concentrations. [Huigens et al., 2009] Unfortunately, the activity of these agents may be significantly impaired in vivo by calcium or manganese ions [Rogers et al., 2009; Rogers et al., 2010].

Baking Soda (sodium bicarbonate) is one of the most useful health tools around. It’s alkalizing effects notwithstanding, antibiofilm activity may be one of the important reasons for its wide ranging benefits.[Gawande, 2008] Bicarbonates also work well with bismuth thiols, which show optimum effects at alkaline pHs. [Domenico et al., 1997] Potassium bicarbonate may be the preferred oral form of baking soda, since potassium offsets the ill effects of a high-sodium diet, helps build bones, lowers blood pressure, etc. It also raises the pH of urine to significantly improve host defenses against biofilms in the urinary tract (see my blog on how to prevent urinary tract infections: https://thescienceofnutrition.me/2015/07/17/beat-urinary-tract-infections-with-nutrition/).

Mucus is also beneficial In the fight against bacteria. Polymers called mucins adhere to bacteria and prevent them from sticking together on a surface, making them harmless. [Caldara et al., 2012]  Mucin complexity makes the commercial production of synthetic mucin impractical. However, mucins from jellyfish may be a commercially viable source in the future. [Ohta et al., 2009]

All major religions promote fasting, and medical research is just beginning to appreciate the benefits of intermittent fasting, from normalizing hormone levels, improved mental clarity, to fighting infection. Fasting can starve microbes while improving immunity, and may help combat biofilms. [Janossy, 2015]

The overconsumption of sugar and refined, processed food has altered our gut biofilms in unhealthy ways, which predisposes us to disease. [http://www.nutraingredients.com/Research/Gut-microbiota-shifts-could-predict-diabetes-risk-suggests-study] Artificial sweeteners also alter the gut microflora in negative ways. [Suez et al., 2014] Sugar promotes the growth of pathogenic yeast and other fungal biofilms. [http://www.thecandidadiet.com/causes.htm]

On the other hand, sugar has been shown to boost the effectiveness of antibiotics against biofilms. Administering sugar with gentamicin cured mice with chronic urinary tract infections, and kept the bacteria from spreading to their kidneys. Perhaps by jump starting the germs’ metabolism with sugar, they can be coaxed out of the biofilm mode. [Allison et al., 2011]

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Drinking coffee could lead to a longer life, research says

Whether it's caffeinated or decaffeinated, coffee is associated with lower mortality, which suggests the association is not tied to caffeine Here's another reason to start the day with a cup of joe: Scientists have found that people who drink coffee appear to live longer. Drinking coffee was associated with a lower risk of death due to heart disease, cancer, stroke, diabetes, and respiratory and kidney disease for African-Americans, Japanese-Americans, Latinos and whites. People who consumed a cup of coffee a day were 12 percent less likely to die compared to those who didn't drink coffee. This association was even stronger for those who drank two to three cups a day -- 18 percent reduced chance of death- Lower mortality was present regardless of whether people drank regular or decaffeinated coffee, suggesting the association is not tied to caffeine, said Veronica W. Setiawan, lead author of the study and an associate professor of preventive medicine at the Keck School of Medicine of USC. "We cannot say drinking coffee will prolong your life, but we see an association," Setiawan said. "If you like to drink coffee, drink up! If you're not a coffee drinker, then you need to consider if you should start."The study, which will be published in the July 11 issue of Annals of Internal Medicine, used data from the Multiethnic Cohort Study, a collaborative effort between the University of Hawaii Cancer Center and the Keck School of Medicine.The ongoing Multiethnic Cohort Study has more than 215,000 participants and bills itself as the most ethnically diverse study examining lifestyle risk factors that may lead to cancer. Lower mortality was present regardless of whether people drank regular or decaffeinated coffee, suggesting the association is not tied to caffeine, said Veronica W. Setiawan, The ongoing Multiethnic Cohort Study has more than 215,000 participants and bills itself as the most ethnically diverse study examining lifestyle risk factors that may lead to cancer. "Until now, few data have been available on the association between coffee consumption and mortality in nonwhites in the United States and elsewhere," the study stated. "Such investigations are important because lifestyle patterns and disease risks can vary substantially across racial and ethnic backgrounds, and findings in one group may not necessarily apply to others." Since the association was seen in four different ethnicities, Setiawan said it is safe to say the results apply to other groups. "This study is the largest of its kind and includes minorities who have very different lifestyles," Setiawan said. "Seeing a similar pattern across different populations gives stronger biological backing to the argument that coffee is good for you whether you are white, African-American, Latino or Asian."

Benefits of drinking coffee

Previous research by USC and others have indicated that drinking coffee is associated with reduced risk of several types of cancer, diabetes, liver disease, Parkinson's disease, Type 2 diabetes and other chronic diseases. Setiawan, who drinks one to two cups of coffee daily, said any positive effects from drinking coffee are far-reaching because of the number of people who enjoy or rely on the beverage every day. "Coffee contains a lot of antioxidants and phenolic compounds that play an important role in cancer prevention," Setiawan said. "Although this study does not show causation or point to what chemicals in coffee may have this 'elixir effect,' it is clear that coffee can be incorporated into a healthy diet and lifestyle." About 62 percent of Americans drink coffee daily, a 5 percent increase from 2016 numbers, reported the National Coffee Association. As a research institution, USC has scientists from across disciplines working to find a cure for cancer and better ways for people to manage the disease. The Keck School of Medicine and USC Norris Comprehensive Cancer Center manage a state-mandated database called the Los Angeles Cancer Surveillance Program, which provides scientists with essential statistics on cancer for a diverse population. Researchers from the USC Norris Comprehensive Cancer Center have found that drinking coffee lowers the risk of colorectal cancer. But drinking piping hot coffee or beverages probably causes cancer in the esophagus, according to a World Health Organization panel of scientists that included Mariana Stern from the Keck School of Medicine.

Hearing from the WHO

In some respects, coffee is regaining its honor for wellness benefits. After 25 years of labeling coffee a carcinogen linked to bladder cancer, the World Health Organization last year announced that drinking coffee reduces the risk for liver and uterine cancer. "Some people worry drinking coffee can be bad for you because it might increase the risk of heart disease, stunt growth or lead to stomach ulcers and heartburn," Setiawan said. "But research on coffee have mostly shown no harm to people's health."

Coffee by the numbers

Setiawan and her colleagues examined the data of 185,855 African-Americans (17 percent), Native Hawaiians (7 percent), Japanese-Americans (29 percent), Latinos (22 percent) and whites (25 percent) ages 45 to 75 at recruitment. Participants answered questionnaires about diet, lifestyle, and family and personal medical history. They reported their coffee drinking habits when they entered the study and updated them about every five years, checking one of nine boxes that ranged from "never or hardly ever" to "4 or more cups daily." They also reported whether they drank caffeinated or decaffeinated coffee. The average follow-up period was 16 years.

Sixteen percent of participants reported that they did not drink coffee, 31 percent drank one cup per day, 25 percent drank two to three cups per day and 7 percent drank four or more cups per day. The remaining 21 percent had irregular coffee consumption habits. Over the course of the study, 58,397 participants -- about 31 percent -- died. Cardiovascular disease (36 percent) and cancer (31 percent) were the leading killers. The data was adjusted for age, sex, ethnicity, smoking habits, education, preexisting disease, vigorous physical exercise and alcohol consumption.Setiawan's previous research found that coffee reduces the risk of liver cancer and chronic liver disease. She is currently examining how coffee is associated with the risk of developing specific cancers. Researchers from the University of Hawaii Cancer Center and the National Cancer Institute contributed to this study. The study used data from the Multiethnic Cohort Study, which is supported by a $19,008,359 grant from the National Cancer Institute of the National Institutes of Health.-Story Source-Materials provided by University of Southern California. -Journal References-Marc J. Gunter et al. Coffee Drinking and Mortality in 10 European Countries: A Multinational Cohort Study. Annals of Internal Medicine, 2017 DOI: 10.7326/M16-2945 -Song-Yi Park et al. Association of Coffee Consumption With Total and Cause-Specific Mortality Among Nonwhite Populations. Annals of Internal Medicine, 2017 DOI: 10.7326/M16-2472 --University of Southern California. "Drinking coffee could lead to a longer life, scientist says: Whether it's caffeinated or decaffeinated, coffee is associated with lower mortality, which suggests the association is not tied to caffeine." ScienceDaily. ScienceDaily, 10 July 2017. <www.sciencedaily.com/releases/2017/07/170710172118.htm>.

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Chlorogenic Compounds from Coffee Beans Exert Activity against Respiratory Viruses.

Planta Med. 2017 May;83(7):615-623

Authors: Sinisi V, Stevaert A, Berti F, Forzato C, Benedetti F, Navarini L, Camps A, Persoons L, Vermeire K

Abstract
Chlorogenic acids are secondary metabolites in diverse plants. Some chlorogenic acids extracted from traditional medicinal plants are known for their healing properties, e.g., against viral infections. Also, green coffee beans are a rich source of chlorogenic acids, with 5-O-caffeoylquinic acid being the most abundant chlorogenic acid in coffee. We previously reported the synthesis of the regioisomers of lactones, bearing different substituents on the quinidic core. Here, 3,4-O-dicaffeoyl-1,5-γ-quinide and three dimethoxycinnamoyl-γ-quinides were investigated for in vitro antiviral activities against a panel of 14 human viruses. Whereas the dimethoxycinnamoyl-γ-quinides did not show any antiviral potency in cytopathogenic effect reduction assays, 3,4-O-dicaffeoyl-1,5-γ-quinide exerted mild antiviral activity against herpes simplex viruses, adenovirus, and influenza virus. Interestingly, when the compounds were evaluated against respiratory syncytial virus, a potent antiviral effect of 3,4-O-dicaffeoyl-1,5-γ-quinide was observed against both subtypes of respiratory syncytial virus, with EC50 values in the submicromolar range. Time-of-addition experiments revealed that this compound acts on an intracellular post-entry replication step. Our data show that 3,4-O-dicaffeoyl-1,5-γ-quinide is a relevant candidate for lead optimization and further mechanistic studies, and warrants clinical development as a potential anti-respiratory syncytial virus drug.-PMID: 27806409 [PubMed - indexed for MEDLINE

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Coffee Drinking and Mortality in 10 European Countries- A Multinational Cohort Study

Marc J Gunter, Neil Murphy, Amanda J Cross, Laure Dossus, Laureen Dartois, Guy Fagherazzi, Rudolf Kaaks, Tilman Kühn, Heiner Boeing, Krasimira Aleksandrova, Anne Tjønneland, Anja Olsen, Kim Overvad, Sofus Christian Larsen, Maria Luisa Redondo Cornejo, Antonio Agudo, María José Sánchez Pérez, Jone M Altzibar, Carmen Navarro, Eva Ardanaz, Kay-Tee Khaw, Adam Butterworth, Kathryn E Bradbury, Antonia Trichopoulou, Pagona Lagiou, Dimitrios Trichopoulos, Domenico Palli, Sara Grioni, Paolo Vineis, Salvatore Panico, Rosario Tumino, Bas Bueno-de-Mesquita, Peter Siersema, Max Leenders, Joline W J Beulens, Cuno U Uiterwaal, Peter Wallström, Lena Maria Nilsson, Rikard Landberg, Elisabete Weiderpass, Guri Skeie, Tonje Braaten, Paul Brennan, Idlir Licaj, David C Muller, Rashmi Sinha, Nick Wareham, Elio Riboli

Annals of Internal Medicine 2017 July 11

Background: The relationship between coffee consumption and mortality in diverse European populations with variable coffee preparation methods is unclear.

Objective: To examine whether coffee consumption is associated with all-cause and cause-specific mortality.

Design: Prospective cohort study.

Setting: 10 European countries.

Participants: 521 330 persons enrolled in EPIC (European Prospective Investigation into Cancer and Nutrition).

Measurements: Hazard ratios (HRs) and 95% CIs estimated using multivariable Cox proportional hazards models. The association of coffee consumption with serum biomarkers of liver function, inflammation, and metabolic health was evaluated in the EPIC Biomarkers subcohort (n = 14 800).

Results: During a mean follow-up of 16.4 years, 41 693 deaths occurred. Compared with nonconsumers, participants in the highest quartile of coffee consumption had statistically significantly lower all-cause mortality (men: HR, 0.88 [95% CI, 0.82 to 0.95]; P for trend < 0.001; women: HR, 0.93 [CI, 0.87 to 0.98]; P for trend = 0.009). Inverse associations were also observed for digestive disease mortality for men (HR, 0.41 [CI, 0.32 to 0.54]; P for trend < 0.001) and women (HR, 0.60 [CI, 0.46 to 0.78]; P for trend < 0.001). Among women, there was a statistically significant inverse association of coffee drinking with circulatory disease mortality (HR, 0.78 [CI, 0.68 to 0.90]; P for trend < 0.001) and cerebrovascular disease mortality (HR, 0.70 [CI, 0.55 to 0.90]; P for trend = 0.002) and a positive association with ovarian cancer mortality (HR, 1.31 [CI, 1.07 to 1.61]; P for trend = 0.015). In the EPIC Biomarkers subcohort, higher coffee consumption was associated with lower serum alkaline phosphatase; alanine aminotransferase; aspartate aminotransferase; γ-glutamyltransferase; and, in women, C-reactive protein, lipoprotein(a), and glycated hemoglobin levels.

Limitations: Reverse causality may have biased the findings; however, results did not differ after exclusion of participants who died within 8 years of baseline. Coffee-drinking habits were assessed only once.

Conclusion: Coffee drinking was associated with reduced risk for death from various causes. This relationship did not vary by country.Primary Funding Source: European Commission Directorate-General for Health and Consumers and International Agency for Research on Cancer.

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