Using light to turn yeast into biochemical factories

Researchers have used a combination of light and genetic engineering to controlling the metabolism, or basic chemical process, of a living cell. Building on techniques that already have transformed the field of neuroscience, the researchers used light to control genetically-modified yeast and increase its output of commercially valuable chemicals.

Yeast has been used for centuries to make bread, wine and beer. Through fermentation, yeast cells transform sugar into chemicals that make bread rise and turn grape juice into wine. Using their new technique, the Princeton researchers have now used fermentation and genetically-engineered yeast to produce other chemicals including lactic acid, used in food production and bioplastics, and isobutanol, a commodity chemical and an advanced biofuel.

Light played a key role in the experiment because it allowed the researchers to switch on genes that they had added to the yeast cells. These particular genes are sensitive to light, which can trigger or suppress their activity. In one case, turning on and off a blue light caused the special yeast to alternate between producing ethanol, a product of normal fermentation, and isobutanol, a chemical that normally would kill yeast at sufficiently high concentration.

The achievement of producing these chemicals was significant, but the researchers were intrigued by the development of light's broader role in metabolic research.The researchers started by putting a modified gene from a marine bacterium that is controllable by blue light into yeast's DNA. They then used light to turn on a chemical process that activates enzymes that naturally allow yeast to grow and multiply by eating glucose and secreting ethanol. But while those enzymes are active, ones that influence the production of isobutanol can't work. So the team turned to darkness to switch off the ethanol-producing enzymes to make room for the expression of their competitors.

READ MORE: Yeast helps hunt for new medicines

Scientists have developed a new way to predict potentially useful drugs from a pool of undefined chemicals. They were able to more quickly identify leads that could be used to treat a range of diseases, from infections, to cancer to Alzheimer's. The finding will also help better match drugs to a disease to maximize the benefit and reduce side-effects. See: Yeast news


Using light to control yeast's chemical production offers several advantages over techniques involving pure genetic engineering or chemical additives. For one, light is much faster and cheaper than most alternatives. It's also adjustable, meaning that turning it on and off can toggle the function of live cells on the spot at any point in the fermentation process (as opposed to chemicals, which generally can't be turned off once they are added.) Also, unlike chemical manipulators that diffuse throughout a cell, light can be applied to specific genes without affecting other parts of the cell.

Optogenetics, as the use of light to control genes is called, is already used in neuroscience and other fields, but this the first application of the technology to control cellular metabolism for chemical production. Gregory Stephanopoulos, an MIT chemical engineering professor who was not involved with Princeton's research, called it a turning point in the field of metabolic engineering.

See:

Evan M. Zhao, Yanfei Zhang, Justin Mehl, Helen Park, Makoto A. Lalwani, Jared E. Toettcher, José L. Avalos. Optogenetic regulation of engineered cellular metabolism for microbial chemical production. Nature, 2018; DOI: 10.1038/nature26141

Posted by Dr. Tim Sandle

Diagnostic innovation for childhood tuberculosis

Ahead of World TB Day 2018, the Foundation for Innovative New Diagnostics (FIND) has made two announcements:

Jointly with the South African Medical Research Council (SAMRC), FIND announced a new agreement to support diagnostic innovation for childhood tuberculosis (TB) in South Africa. The project is part of a global effort to improve childhood TB diagnosis, guide paediatric treatment, and reduce suffering, disease transmission and deaths from TB in babies and children.

Full release

FIND also announced that it has supported the World Health Organization (WHO) through the generation of critical evidence that has informed updated laboratory guidance for drug-resistant TB. The updated list of critical concentrations for drug susceptibility testing of medicines used in the treatment of drug-resistant TB is the result of a long collaboration between WHO and FIND on TB diagnostics and laboratory strengthening.

Full release

Detection of bacterial growth and Biofilm formation in pipelines

Bacteria embedded in the biofilm are more difficult and expensive to eliminate, than free-floating planktonic bacteria. Their presence in manufacturing pipelines can be responsible for a wide range of water quality and operational problems. By measuring bacteria bio-electrochemical activity (a phenomenon known as “ennoblement” or “cathodic depolarization”), the ALVIM technology detects biofilm formation since its first phases, online and in real time.

The system allows the user to decide when to apply CIP, and check in real time if biofilm was actually removed. .… Read More

EU GMP Annex 1 presentation

A recording of Pharmig's response to draft EU GMP Annex 1, hosted by Tim Sandle

See: Annex 1 overview

Posted by Dr. Tim Sandle

Polyvinylidene Fluoride (PVDF)

PVDF (PVF2 or Polyvinylidene fluoride or polyvinylidene difluoride) is a semi-crystalline, high purity thermoplastic fluoropolymer.

News from Omnexus.

With service temperatures up to 150°C, PVDF displays good combination of properties such as:

• Exceptional chemical resistance
• High mechanical strength
• Piezoelectric and pyroelectric properties
• As well as good processability

Its highly desirable insolubility and electrical properties result from the polarity of alternating CH2 and CF2 groups on the polymer chain.

PVDF is readily melt-processible and can be fabricated into parts by injection and compression molding. As a result, it is commonly employed in chemical processing equipment such as pumps, valves, pipes, tubes and fittings; sensors and actuators etc.

It has many electronic applications, especially as jacketing materials for plenum-rated cable used in voice and video devices and alarm systems. The low flame spread and smoke generation of PVDF is a prime asset in these applications.

PVDF is gaining acceptance as a binder for cathodes and anodes in lithium-ion batteries, and as a battery separator in lithium-ion polymer systems.

Emerging applications of PVDF include fuel cell membranes, and components for aircraft interiors and office automation equipment.

PVDF (homopolymers and copolymers) is generally synthesized by the free radical polymerization of 1,1-difluoroethylene (CH2=CF2). The polymerization takes place in the suspension or emulsion from 10-150°C and pressure of 10-300 atm. The material obtained is then processed into film or sheets.

Suppression of the Test for Abnormal Toxicity from the European Pharmacopoeia

During its 159th plenary session, the European Pharmacopoeia Commission endorsed the complete suppression of the test for abnormal toxicity from the European Pharmacopoeia (Ph. Eur.).

As part of this exercise, 49 monographs revised to remove the test for abnormal toxicity were adopted by the Commission; notably, these included 36 monographs on vaccines for human use. In addition, as the general chapter Abnormal Toxicity (2.6.9) will no longer be referenced in any monograph, it will subsequently be rendered obsolete and will also be deleted from the Ph. Eur.

The Ph. Eur. Commission remains fully committed to the reduction of animal use wherever possible in pharmacopoeial testing, in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The decision to suppress the test for abnormal toxicity at the 159th session of the Ph. Eur. Commission is a strong illustration of this commitment.

See: abnormal toxicity

Posted by Dr. Tim Sandle

Decoy molecules target E. coli to treat UTI in mice

Researchers have designed sugar molecules that block E. coli bacteria from binding to urinary tissues, allowing the bacteria to be washed out of the urinary tract. The compounds represent a step toward treating UTIs without antibiotics.

The bacteria E. coli cause 80 percent of UTIs, leading to painful, burning urination. The bacteria then sometimes travel to the kidneys, causing back pain and fever. In rare cases, they spread to the blood, a potentially lethal complication.

Often, UTIs can be cleared up with antibiotics, but 10 to 20 percent of cases do not respond to current first-line drugs. Hultgren and his colleagues are working on an alternative that would prevent bacteria from causing disease, which may help reduce dependency on antibiotics.

E. coli's first step in causing UTIs is to latch onto sugars on the surface of the bladder with long, hairlike structures called pili. Hultgren and co-senior author James W. Janetka, PhD, an associate professor of biochemistry and molecular biophysics, previously created mannosides, modified forms of a sugar called mannose, that the bacteria favor over typical sugars on the bladder wall. When mice with UTIs were given the mannosides, the E. coli in their bladders grabbed hold of those molecules and were swept away.

Researchers have shown that E. coli also can latch onto galactose, another sugar molecule found on urinary tissues. A drug that undermines the bacteria's ability to stay in the body is less likely to drive resistance because, unlike antibiotics, it would not force bacteria to die or evolve resistance in order to survive, the researchers said.

The researchers also demonstrated that the galactoside prevented the bacteria's adhesive protein from sticking to human kidney tissue.

See:

Vasilios Kalas, Michael E. Hibbing, Amarendar Reddy Maddirala, Ryan Chugani, Jerome S. Pinkner, Laurel K. Mydock-McGrane, Matt S. Conover, James W. Janetka, Scott J. Hultgren. Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection. Proceedings of the National Academy of Sciences, 2018; 201720140 DOI: 10.1073/pnas.1720140115



Posted by Dr. Tim Sandle

Testing water for coliforms using a rapid method

Microbial control of pharmaceutical water systems is not only about numbers of microorganisms estimated to be present, as recovered through bioburden testing of a given volume of sampled water; microbiologists additionally need to know the types of organisms present within water.

With this a new article of interest has been published:

Marflitt, A. and Sandle, T. (2018): Evaluation of Readycult® Coliforms 100 Presence/Absence Test for the screening of coliforms and Escherichia coli in pharmaceutical water samples, European Journal of Parenteral and Pharmaceutical Science, 22 (4): 118-125

The abstract is:

The microbiological monitoring of water systems in pharmaceutical facilities requires an assessment of total microbial count, with an action level applicable to the water grade. It is also considered good practice in many facilities to assess water for the presence/absence of identified objectionable microorganisms. Included among these ‘objectionables’ are coliforms, as indicators of substandard water. The traditional approach for assessing for coliforms in water is using a specialised agar. This approach requires an incubation time within the region of 3 to 5 days. This paper assesses an alternative method, which provides a result within 18 to 24 hours. The method evaluated was the Readycult® Coliforms 100, which contains both a chromogenic substrate (to show the presence of coliforms) and a fluorogenic substrate (to show the presence of Escherichia coli). Through a series of experimental tests involving different grades of pharmaceutical water and different microorganisms, the Readycult® Coliforms 100 was shown to be suitable as a rapid microbiological method for screening water, with reactions produced comparable to conventional methods.

For further details, contact Tim Sandle

Posted by Dr. Tim Sandle

BSI Brexit Position Paper

British Standards Instiutte (BSI) has consulted its members and stakeholders about the possible implications of Brexit for standards. The BSI Brexit Position Statement sets out the eight key principles on which BSI’s post-Brexit position is based.

Since the UK voted to leave the EU in June 2016, BSI, in its capacity as the UK’s National Standards Body, has consulted its members and stakeholders about the possible implications of Brexit for standards.

As a result, BSI’s post-Brexit position is to continue to provide UK experts with the standards development framework to support trade in the UK, across Europe and globally. To enable this, our stakeholders are clear that BSI should remain a full member of the European Standards Organizations.

The Brexit Position Paper sets out the eight key principles on which our position is based. These principles are supported by statements from a range of BSI’s stakeholders, including industry associations and individual companies, consumer groups, users of standards and professional institutions.

Use this link to download aPDF of the paper now.

Social stress leads to changes in gut bacteria

Exposure to psychological stress in the form of social conflict alters gut bacteria in Syrian hamsters, according to a new study by Georgia State University.

It has long been said that humans have "gut feelings" about things, but how the gut might communicate those "feelings" to the brain was not known. It has been shown that gut microbiota, the complex community of microorganisms that live in the digestive tracts of humans and other animals, can send signals to the brain and vice versa.

In addition, recent data have indicated that stress can alter the gut microbiota. The most common stress experienced by humans and other animals is social stress, and this stress can trigger or worsen mental illness in humans. Researchers at Georgia State have examined whether mild social stress alters the gut microbiota in Syrian hamsters, and if so, whether this response is different in animals that "win" compared to those that "lose" in conflict situations.

Hamsters are ideal to study social stress because they rapidly form dominance hierarchies when paired with other animals. In this study, pairs of adult males were placed together and they quickly began to compete, resulting in dominant (winner) and subordinate (loser) animals that maintained this status throughout the experiment. Their gut microbes were sampled before and after the first encounter as well as after nine interactions. Sampling was also done in a control group of hamsters that were never paired and thus had no social stress.

Posted by Dr. Tim Sandle

U.S. E. coli outbreak

The CDC, the FDA, several states, and U.S. Department of Agriculture’s Food Safety and Inspection Service are investigating a multistate outbreak of Shiga toxin-producing E. coli O157:H7 infections.

The investigation started by the New Jersey Department of Health, working together with the CDC and FDA. In New Jersey, ill people included in the outbreak had test results showing the presence of E. coli bacteria. Laboratory testing is ongoing to link their illnesses to the outbreak using DNA fingerprinting.

See: Bio Exoert for further details

Posted by Dr. Tim Sandle

Feasibility of eliminating rabies in Africa

The Swiss Tropical and Public Health Institute, together with European and African collaborators (including the Institut Pasteur in Paris), carried out a mass dog vaccination in Chad and determined its effect on human rabies exposure. The study employed a bio-mathematical method for estimating the transmission dynamics of rabies. The researchers conclude that with political will and the necessary funding, elimination of rabies is possible in Africa.

Rabies is a viral disease that kills tens of thousands of people every year, predominantly in Africa and Asia. The disease is transmitted through the bites of infected dogs and foxes (read the disease fact sheet concerning rabies). In West- and Central Europe, rabies was eliminated some 20 years ago. Switzerland was declared free of rabies in 1999 after implementing a strategy targeting foxes.

The study is one of the first research projects to apply a rigorous phylodynamic method to dog rabies, and hence, it expands upon the normative phylogenetics (i.e. assessing the genetic relatedness of virus strains) with the dynamics of transmission over time. This made it possible to calculate the reproductive number of rabies among the dogs after the first mass dog vaccination in 2012.

See:

Jakob Zinsstag, Monique Lechenne, Mirjam Laager, Rolande Mindekem, Service Naïssengar, Assandi Oussiguéré, Kebkiba Bidjeh, Germain Rives, Julie Tessier, Seraphin Madjaninan, Mahamat Ouagal, Daugla D. Moto, Idriss O. Alfaroukh, Yvonne Muthiani, Abdallah Traoré, Jan Hattendorf, Anthony Lepelletier, Lauriane Kergoat, Hervé Bourhy, Laurent Dacheux, Tanja Stadler, Nakul Chitnis. Vaccination of dogs in an African city interrupts rabies transmission and reduces human exposure. Science Translational Medicine, 2017; 9 (421): eaaf6984 DOI: 10.1126/scitranslmed.aaf6984

Posted by Dr. Tim Sandle

What is NAD+ and Why Is It a Hot Topic in Longevity Science?

You may have repeatedly been hearing about NAD+ in health and nutrition circles and, honestly, wondering to yourself what is NAD+?

We’re here to help you understand the importance of NAD+, its functions, its relationship with aging, and why the longevity science community can't stop talking about it.

A guest post by Frank L. Jaksch Jr..

What is NAD+?

Nicotinamide adenine dinucleotide, or NAD+, is a coenzyme that can be found in every cell in your body. In a healthy body, all the necessary NAD+ is created by utilizing vitamin B3 (also known as niacin, or nicotinamide).
Why is NAD+ important?

To put it simply, NAD+ is important because it is required for the necessary biological processes that allow life to happen.

By assisting in the transfer of energy from fatty acids and glucose to the mitochondria (the "powerhouses of the cell") which, in turn, convert them to cellular energy, NAD+ plays a pivotal role in your cellular metabolism. Additionally, NAD+ plays a significant role in regulating how quickly your cells age.

Here are some of the other roles that NAD+ plays in the body:


● Unfolded Protein Response - a protein's shape dictates its function and NAD+ makes sure the form is correct

● Liver Health - your liver breaks down fats for energy and keeps your blood sugar balanced (among other things) and requires NAD+

● Sirtuins - a family of proteins that governs longevity in organisms and needs NAD+

● DNA Health - the systems that fix errors in our DNA require NAD+

● Circadian Rhythm - your sleeping cycle (and everything to do with it) requires NAD+

● Calcium Signaling - communication in the cell requires NAD+

● Stem Cell Maintenance - to have healthy stem cells you need NAD+

● Gene Expression - in response to changing conditions (such as nutrition and stress), your genes are always getting turned on and off, which (surprise!) require NAD+
What causes a decline in NAD+?

The levels of NAD+ decline as a result of the aging process, as well as due to a sedentary lifestyle and overeating. When NAD+ levels decline, the number and density of the mitochondria are also reduced which results in less efficiency or potential dysfunction.

The result of declining NAD+ levels can lead to adverse health consequences, including a slower metabolism. In other words, weaker mitochondria suggest the beginning of the end for your body.
What does NAD+ have to do with aging?

Good question! So, in addition to all the other jobs it has, NAD+ activates enzymes called “sirtuins” (SIRT1 and SIRT3), which help control your genes in ways that promote healthy aging.

The higher your NAD+ levels, the better activated your SIRT1 and SIRT3 enzymes are. This means you get to have healthier mitochondria essential to good health for your body. Makes sense?

As your levels of NAD+ decline with aging, this also results in less engaged sirtuins, less healthy mitochondria, and an “older” body (think: less energy, achy joints, poor memory).
Why is it a hot topic in longevity science?

Despite first being discovered over 110 years ago, everyone is currently talking about NAD+ because many significant discoveries have emerged over the past couple of decades.

Recently, NAD+ has become a hot topic in longevity science thanks to an influential study published by David Sinclair, professor of genetics at Harvard Medical School. In the study, Sinclair and his colleagues bred mice with a defect in SIRT1. As a result of this defect, these mice aged rapidly and showed significant problems with their mitochondria.

However, once the mice turned 22 months old, they were given increased levels of NAD+, and the results were incredible. The mice had less insulin resistance, less inflammation, and less muscle wasting.

As a result of the increase of NAD+ levels, the mice began to appear as though they were merely six months old (for humans, that would be the equivalent of a 60-year-old’s cells shifting to being more like those that belong to a 20-year-old).

This study (and others like it) has led to the idea that increasing NAD+ levels could be a possible way to maintain long-term health in humans.
How do you increase NAD+ levels?

There is still a long path ahead for NAD+ research, but there are some ways you can increase your levels.

1. Exercise

When you work out, your body needs more NAD+ to produce more energy which puts your mitochondria into overdrive. The good news is that your body doesn't halt producing additional NAD+ when you finish exercising. So, by consistently working out, you will be able to increase your mitochondrial pumping protein.

2. Consume fermented foods and beverages

Fermented foods and drinks such as kimchi, kombucha (anything that undergoes the chemical process of fermentation by exposure to lactobacilli) receive, as a byproduct, a small amount of NAD+ which you then consume. Another reason to add kombucha to your morning routine!


3. Take an NAD+ booster

You can also take an NAD+ booster such as nicotinamide riboside (NR) which is a potent form of vitamin B3 that extends various health benefits, including endurance, performance, weight management, cardiovascular health, anti-aging, cognitive function and neuroprotection. If you opt to take an NR supplement (with your doctor's guidance), then take 100 to 250 mg each morning.

That being said, too much vitamin B can damage your liver and lead to other harmful effects.

There is still no conclusive evidence that NAD+ can reverse the aging process; however, by eating healthy, exercising regularly, wearing sunscreen, cutting down on alcoholic beverages, and taking vitamin D supplements, you are well on your way to staying young forever.

AUTHOR BIO

Frank L. Jaksch Jr. is the Chief Executive Officer and a co-founder of ChromaDex. He oversees research, strategy and operations for the Company with a focus on scientific and novel products for pharmaceutical and nutraceutical markets. Mr. Jaksch earned a B.S. in Chemistry and Biology from Valparaiso University.

The Problem of Biofilms and Pharmaceutical Water Systems

Biofilms are problematic to pharmaceutical water systems. If a biofilm develops then an out-of-control situation is likely to emerge. Such a situation can often only be detected from point-of-use samples and since several excursions are required to alert of the probability of a biofilm, the biofilm will most likely have become significantly established at this point in time. This article assesses biofilms and pharmaceutical water systems, considering prevention and control measures; looking at what can be meaningfully gleaned from monitoring; and measures required to address biofilms.

Tim Sandle has written an article for American Pharmaceutical Review. The reference is:

Sandle, T. (2017) The Problem of Biofilms and Pharmaceutical Water Systems, American Pharmaceutical Review, 20 (7): http://www.americanpharmaceuticalreview.com/Featured-Articles/345440-The-Problem-of-Biofilms-and-Pharmaceutical-Water-Systems/

The article can be accessed here: APR

Posted by Dr. Tim Sandle

US Food and Drug Administration Good ANDA submission practices

This draft guidance intended to assist applicants preparing to submit to FDA abbreviated new drug applications (ANDAs).

This guidance highlights common, recurring deficiencies that may lead to a delay in the approval of an ANDA. It also makes recommendations to applicants on how to avoid these deficiencies with the goal of minimizing the number of review cycles necessary for approval.

FDA is also issuing a Good ANDA Assessment Practices Manual of Policies and Procedures which establishes good ANDA assessment practices for the Office of Generic Drugs and the Office of Pharmaceutical Quality to increase their operational efficiency and effectiveness.

See: https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM591134.pdf


Posted by Dr. Tim Sandle