Friday, 24 January 2020

Identifying harmful bacteria based on its DNA


A new bacterial identification method, called ON-rep-seq, examines selective, strain-specific fragments of the bacterial genome, allowing the generation of results that earlier required DNA sequencing of the entire bacterial genome or tedious approaches like pulsed field gel electrophoresis, which previously has been the golden the standard for strain-level typing of microorganisms. Hence, the method has the potential to change the approach utilized for investigating food-based disease outbreaks by making analysis much less time- and cost consuming.

Today, bacterial detection and identification based on bacterial DNA requires expensive instrumentation and many hours of work by highly trained specialists. Let's imagine, for example, there is a suspected Salmonella outbreak. Usually in order to locate its origin, not only will investigators have to analyze many samples, but the analysis has to be precise in order to distinguish one bacterial strain from another.

The new method is based on nanopore sequencing, which is a new, real-time DNA sequencing approach "that will definitely revolutionize the future of DNA sequencing" according to Lukasz Krych.

The research project was carried out in collaboration with the polish company GenXone S.A., which helped to set up a bioinformatics pipeline that is needed to perform fast and efficient analysis of the sequencing data.

The smallest ever sequencer offered by Oxford Nanopore Technologies, called MinION, is a $999 hand-held, USB-powered device that became commercially available in 2015. A year later it was taken to the International Space Station, where it achieved the first DNA sequencing in history performed in zero-gravity conditions. Despite the indisputable revolution in DNA sequencing offered by MinION, it quickly became clear that the data generated with the device are still not perfect due to e.g. sequencing errors while the analysis remained relatively expensive to perform (app. $150 per bacterium).

The scientists from the Department of Food Science at the University of Copenhagen have found a way to utilize this technology to analyze hundreds of bacteria at a time, cutting costs to less than $2 per bacterium, while at the same time increasing the accuracy to more than 99%.

At the moment, there are several companies testing the method to implement in their systems for establishing rapid screening programmes for thousands of strains.

Journal Reference:

Łukasz Krych, Josué L. Castro-Mejía, Laura M. Forero-Junco, Daniel N. Moesby, Morten B. Mikkelsen, Morten A. Rasmussen, Maciej Sykulski, Dennis S. Nielsen. DNA enrichment and tagmentation method for species-level identification and strain-level differentiation using ON-rep-seq. Communications Biology, 2019; 2 (1) DOI: 10.1038/s42003-019-0617-x

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Thursday, 23 January 2020

'Upgraded' CRISPR tool developed


Columbia scientists have captured the first images of a new gene editing tool that could improve upon existing CRISPR-based tools. The team developed the tool, called INTEGRATE, after discovering a unique "jumping gene" in Vibrio cholerae bacteria that could insert large genetic payloads in the genome without introducing DNA breaks.

In the new study the researchers harnessed a Nobel Prize-winning technique called cryo-electron microscopy to freeze the gene editing complex in action, revealing high-resolution details about how it works.

The researchers used a technique called cryo-electron microscopy, which involves flash freezing a sample of the gene editing complex in liquid nitrogen and bombarding it with electrons. They then used the images they captured with the electron microscope to generate atomic resolution models of the INTEGRATE system.

The structural model reveals that the complex is made up of two main sections that are arranged in a helical filament. The larger portion, called Cascade, winds around and carries a guide RNA that it uses to scan the cell for a matching sequence in DNA. Once it locates and binds the target sequence, it threads the DNA strand through the TniQ "transposition" proteins that sit on the end of the complex and recruit other enzymes that help modify the DNA.

The scanning mechanism of INTEGRATE appears to work in a similar way to other well-studied CRISPR systems, some of which also contain a Cascade complex with guide RNA. However, unlike other CRISPR systems that use Cascade to target DNA for cutting, the function of Cascade within INTEGRATE is to target DNA for highly accurate insertion of genetic payloads.

Many researchers around the world now use CRISPR-Cas9 to quickly and cheaply make precise modifications to the genome of a cell. However, most uses of CRISPR involve cutting both strands of the target DNA, and the DNA break must then be repaired by the host cell's own machinery. Controlling this repair process is still a major challenge in the field, and undesired gene edits are often introduced inadvertently in the genome. Additionally, existing tools often perform poorly at inserting large genetic payloads in a precise fashion. Improving the accuracy of gene editing is a priority for researchers and is critical for ensuring the safety of therapies developed with this technique.

The new INTEGRATE system developed by the Sternberg lab can accurately insert large DNA sequences without relying on the cell's machinery to repair the strands. As a result, INTEGRATE could prove to be a more accurate and efficient way of making certain gene modifications than the original CRISPR-Cas system that is widely in use. The new tool could also help scientists perform gene editing in cell types with limited DNA repair activity such as neurons, where attempts to use CRISPR have been comparatively less successful.


In addition to informing future engineering efforts, the structures highlight a possible proofreading checkpoint. Existing CRISPR technologies often suffer from so-called "off-target effects," in which unintended sequences are promiscuously modified. The new structures reveal how Cascade and TniQ work together to ensure that only the correct "on-target" sequences are marked for DNA insertion. The researchers plan to further explore this checkpoint while developing the tool for new therapeutic approaches to disease.

Journal Reference:

Tyler S. Halpin-Healy, Sanne E. Klompe, Samuel H. Sternberg, Israel S. Fernández. Structural basis of DNA targeting by a transposon-encoded CRISPR–Cas system. Nature, 2019; DOI: 10.1038/s41586-019-1849-0
Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Wednesday, 22 January 2020

Ranitidine containing medicinal products



At the request of the European Commission, the European Medicines EMA triggered a review of ranitidine medicines after tests showed that some of these products contained an impurity called Nnitrosodimethylamine (NDMA). NDMA is classified as a probable human carcinogen (a substance that could cause cancer) on the basis of animal studies.

Ranitidine medicines are used widely to reduce the production of stomach acid in patients with conditions such as heartburn and stomach ulcers. They are available over-the-counter and on prescription. Patients who have any questions about their current treatment can speak to their doctor or pharmacist. There are several other medicines used for the same conditions as ranitidine that could be used as an alternative.

Ranitidine belongs to a class of medicines known as H2 (histamine-2) blockers, which work by blocking histamine receptors in the stomach and reducing the production of stomach acid. It is used to treat and prevent conditions caused by excess acid in the stomach such as heartburn and stomach ulcers. Ranitidine-containing medicines are authorised by national authorities and are available as tablets and injectable formulations (EMA, 2019).

In 2018, NDMA and similar compounds known as nitrosamines were found in a number of blood pressure medicines known as ‘sartans’, leading to some recalls and to an EU review, which set strict new manufacturing requirements for these medicines. In 2019, a nitrosamine impurity has been detected in a few batches of pioglitazone from one company and in batches of ranitidine.

The chemical is present in some foods and in water supplies but is not expected to cause harm when ingested in very low levels.

The review of ranitidine medicines was initiated on 12 September 2019 at the request of the European Commission, under Article 31 of Directive 2001/83/EC. The review will be carried out by the Committee for Medicinal Products for Human Use (CHMP), responsible for questions concerning medicines for human use, which will adopt an opinion. The CHMP opinion will then be forwarded to the European Commission, which will issue a final legally binding decision applicable in all EU Member States. The EMA evaluated the data to assess whether patients using ranitidine are at any risk from NDMA and undertook to provide information about this (at the time of writing, no further information was made available) (EMA, 2019b).

The U.S. FDA has also been investigating NDMA and other nitrosamine impurities in blood pressure and heart failure medicines called Angiotensin II Receptor Blockers (ARBs) since last year. In the case of ARBs, the FDA has recommended numerous recalls as it discovered unacceptable levels of nitrosamines. According to the FDA, some ranitidine medicines, including some products commonly known as the brand-name drug Zantac, contain a nitrosamine impurity. The FDA is evaluating whether the low levels of NDMA in ranitidine pose a risk to patients (FDA, 2019a).

In 2019, the FDA announced that a voluntary recall of 14 lots of prescription ranitidine capsules distributed by Sandoz Inc., used to decrease the amount of acid created by the stomach, had taken place in relation to N-nitrosodimethylamine (FDA, 2019b).

EMA (2019a) EMA to provide guidance on avoiding nitrosamines in human medicines, European Medicines Agency, at: https://www.ema.europa.eu/en/documents/press-release/ema-provide-guidance-avoiding-nitrosamines-human-medicines_en.pdf  (accessed 6th November 2019)


EMA (2019b) EMA to review ranitidine medicines following detection of NDMA. European Medicines Agency, at: https://www.ema.europa.eu/en/documents/referral/ranitidine-article-31-referral-ema-review-ranitidine-medicines-following-detection-ndma_en.pdf (accessed 6th November 2019)

FDA (2019a) Statement alerting patients and health care professionals of NDMA found in samples of ranitidine, US Food and Drug Administration at: https://www.fda.gov/news-events/press-announcements/statement-alerting-patients-and-health-care-professionals-ndma-found-samples-ranitidine (accessed 6th November 2019)

FDA (2019b) FDA announces voluntary recall of Sandoz ranitidine capsules following detection of an impurity, US Food and Drug Administration at: https://www.fda.gov/news-events/press-announcements/fda-announces-voluntary-recall-sandoz-ranitidine-capsules-following-detection-impurity (accessed 6th November 2019)

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Tuesday, 21 January 2020

Microbiologists investigate the cleanliness of hospital washers



Microbiologists have found that many washing machines in hospital setting as are reservoirs of multidrug-resistant bacteria. In one case study pathogens, were transmitted regularly to newborns in a neonatal intensive care unit at a children's hospital.

The bacterium involved was a single clone of Klebsiella oxytoca. The study showed how the organisms were transmitted repeatedly to new babies in a ward located in a children's hospital. The transmission of the organism halted only when a ‘smoking gun’ washing machine was disassembled and removed from the hospital.

Microbiologists, as reported by the American Society for Microbiology, have demonstrated how resistance genes, as well as many water-borne microorganisms, can persist in domestic washing machines at reduced temperatures.

Klebsiella oxytoca is a Gram-negative, rod-shaped bacterium that is closely related to K. pneumoniae. Outbreaks of antibiotic-resistant Klebsiella oxytoca have occurred in multiple hospitals and ICUs throughout the world

With the hospital case study, standard screening protocols demonstrated the presence of the Klebsiella organism on infants in the ICU. Genetic comparative testing traced the source of the bacterium to the washing machine. In the course of the investigation, both incubators (used for babies born prematurely) and healthcare workers were ruled out as the sources of the contamination.

It appears that clothes, like knitted caps and socks, and blankets washed in the machine transmitted K. oxytoca from the washer to the infants. The residual water on the rubber mantle of the washer together with the final rinsing process (where unheated water is used) were found to contain the contaminant.

The infants in the intensive care units (ICU) were colonized, but not infected by K. oxytoca. However, the potential for a serious public health risk exists unless action is taken.

Lead researcher Dr. Martin Exner states: “We have proven for the first time that a washing machine can also spread antibiotic-resistant bacteria to humans.”


The research also carries implications for household washers, as well as those located in hospitals. This may relate to factors associated with energy management and environmental concerns. The water temperatures used in many domestic washing machines have been declining, ostensibly to save energy (and money). This was driven temperatures to regularly to be below 60°C (140°F). At such temperatures the water is less effective in terms of killing vegetative bacteria.

The study recommended that changes in washing machine design and processing are needed in order to prevent the accumulation of residual water leading to conditions favourable for microbial growth.

The case study has been reported to the journal Applied and Environmental Microbiology, with the research paper headed “The washing machine as a reservoir for transmission of extended spectrum beta-lactamase (CTX-M-15)-producing Klebsiella oxytoca ST201 in newborns.”

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Monday, 20 January 2020

Variability and the LAL Assay for Bacterial Endotoxin Detection


With biological tests all measurements susceptible to variations in analytical conditions should be suitably controlled as far as is practicable. Here the LAL assay has a relatively elevated level of variability even for a biological assay. This variation derives from three principle sources: reagents, product, and method / instrumentation. This article examines some of the reasons for this variation in relation to the test and the test reagents. The article also examines the coefficient of variation which is one way to examine for test variation.
In relation to the LAL assay, Tim Sandle has written a paper assessing assay control.

This article examines some of the reasons for this variation in relation to the test and the test reagents. The article also examines the coefficient of variation which is one way to examine for test variation.







The reference is:

Sandle, T. (2019) Variability and the LAL Assay for Bacterial Endotoxin Detection, Journal of GxP Compliance, 23 (5): 1-10: http://www.ivtnetwork.com/article/variability-and-lal-assay-bacterial-endotoxin-detection

For further details, please contact Tim Sandle



Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Sunday, 19 January 2020

Collagen adhesion gene associated with bloodstream infections caused by MRSA



Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection.

A new paper of interest:

Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information.

WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria.

These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection.

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Saturday, 18 January 2020

Impact of vaccines on antimicrobial resistance


AMR is now considered a key threat to global health, leading to more mortality and increased healthcare costs threatening future conduct of routine medical procedures. Traditional approaches to address AMR include antibiotic stewardship, better hygiene/infection control, promoting antibiotic research and development, and restricting use for agricultural purposes.

A new article of interest:

Antibiotic use drives the development and spread of resistant bacterial infections. Antimicrobial resistance (AMR) has become a prolific global issue, due to significant increases in antibiotic use in humans, livestock and agriculture, inappropriate use (under-dosing and over-prescribing), and misuse of antibiotics (for viral infections where they are ineffective). Fewer new antibiotics are being developed.

While antibiotic development is declining, vaccine technology is growing. This review shows how vaccines can decrease AMR by preventing bacterial and viral infections, thereby reducing the use/misuse of antibiotics, and by preventing antibiotic-resistant infections. Vaccines are less likely to induce resistance. Some future uses and developments of vaccines are also discussed.

Vaccines, along with other approaches, can help reduce AMR by preventing (resistant) infections and reducing antibiotic use. Industry and governments must focus on the development of novel vaccines and drugs against resistant infections to successfully reduce AMR.

See: "Impact of vaccines on antimicrobial resistance."

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Friday, 17 January 2020

Ancient feces reveal how 'marsh diet' left Bronze Age Fen folk infected with parasites


New research published today in the journal Parasitology shows how the prehistoric inhabitants of a settlement in the freshwater marshes of eastern England were infected by intestinal worms caught from foraging for food in the lakes and waterways around their homes.

The Bronze Age settlement at Must Farm, located near what is now the fenland city of Peterborough, consisted of wooden houses built on stilts above the water. Wooden causeways connected islands in the marsh, and dugout canoes were used to travel along water channels.
The village burnt down in a catastrophic fire around 3,000 years ago, with artefacts from the houses preserved in mud below the waterline, including food, cloth, and jewellery. The site has been called "Britain's Pompeii."

Also preserved in the surrounding mud were waterlogged "coprolites" -- pieces of human faeces -- that have now been collected and analysed by archaeologists at the University of Cambridge. They used microscopy techniques to detect ancient parasite eggs within the faeces and surrounding sediment.

Very little is known about the intestinal diseases of Bronze Age Britain. The one previous study, of a farming village in Somerset, found evidence of roundworm and whipworm: parasites spread through contamination of food by human faeces.
The ancient excrement of the Anglian marshes tells a different story. "We have found the earliest evidence for fish tapeworm, Echinostoma worm, and giant kidney worm in Britain," said study lead author Dr Piers Mitchell of Cambridge's Department of Archaeology.
"These parasites are spread by eating raw aquatic animals such as fish, amphibians and molluscs. Living over slow-moving water may have protected the inhabitants from some parasites, but put them at risk of others if they ate fish or frogs."

Disposal of human and animal waste into the water around the settlement likely prevented direct faecal pollution of the fenlanders' food, and so prevented infection from roundworm -- the eggs of which have been found at Bronze Age sites across Europe.

However, water in the fens would have been quite stagnant, due in part to thick reed beds, leaving waste accumulating in the surrounding channels. Researchers say this likely provided fertile ground for other parasites to infect local wildlife, which -- if eaten raw or poorly cooked -- then spread to village residents.


"The dumping of excrement into the freshwater channel in which the settlement was built, and consumption of aquatic organisms from the surrounding area, created an ideal nexus for infection with various species of intestinal parasite," said study first author Marissa Ledger, also from Cambridge's Department of Archaeology.

See:

Marissa L. Ledger, Elisabeth Grimshaw, Madison Fairey, Helen L. Whelton, Ian D. Bull, Rachel Ballantyne, Mark Knight, Piers D. Mitchell. Intestinal parasites at the Late Bronze Age settlement of Must Farm, in the fens of East Anglia, UK (9th century B.C.E.). Parasitology, 2019; 1 DOI: 10.1017/S0031182019001021

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Thursday, 16 January 2020

New Veterinary Medicines Regulation


A new Veterinary Medicines Regulation (Regulation (EU) 2019/6) has been introduced to modernise the existing rules on the authorisation and use of veterinary medicines in the European Union (EU). This becomes applicable on 28 January 2022.

The regulation contains new measures for increasing the availability and safety of veterinary medicines and enhances EU action against antimicrobial resistance. The European Medicines Agency (EMA) is working closely with the European Commission and other EU partners in preparation for the implementation of the new Regulation.

The main objectives of the new Regulation are to:


  • Simplify the regulatory environment and reduce administrative burden for pharmaceutical companies developing veterinary medicines, for example through streamlined pharmacovigilance rules;
  • Stimulate the development of innovative veterinary medicines, including products for small markets (minor use and minor species);
  • Improve the functioning of the internal market for veterinary medicines;
  • Strengthen EU action to fight antimicrobial resistance through specific measures ensuring prudent and responsible use of antimicrobials in animals, including reserving certain antimicrobials for the treatment of infections in people

See EMA at: https://www.ema.europa.eu/en/veterinary-regulatory/overview/implementation-new-veterinary-medicines-regulation

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Wednesday, 15 January 2020

Updates on Ethylene Oxide Sterilization of Medical Devices: Recent FDA Actions



The U.S. Food and Drug Administration (FDA) is providing information on recent actions responding to ongoing concerns about ethylene oxide in commercial operations and encouraging innovative approaches to medical device sterilization.

FDA innovation Challenges

On June 15, 2019, the FDA announced two Innovation Challenges to identify sterilization alternatives and reduce ethylene oxide emissions.

The FDA received 46 applications from companies large and small. After careful review using an established set of criteria, 12 challenge applicants have been selected to participate.

Ethylene Oxide Sterilization Master File Pilot Program

On Novembr 25, 2019, the FDA announced its Ethylene Oxide Sterilization Master File Pilot Program (EtO Pilot Program).

This voluntary program is intended to streamline the submission process, so that sterilization providers that sterilize single-use medical devices using fixed chamber sterilization processes may submit a Master File to the FDA when making certain changes between sterilization sites, or when making certain changes to sterilization processes that utilize reduced ethylene oxide concentrations, and PMA holders can reference such a Master File in a postapproval report instead of submitting a traditional PMA supplement.



General Hospital and Personal Use Devices Panel Advisory Committee Meeting


On November 6 and 7, 2019, the FDA held an advisory committee meeting to discuss ethylene oxide sterilization of medical devices and its role in maintaining public health. Based on panel discussions, the FDA is encouraging device manufacturers to move to electronic labeling and instructions for use in the near term and is committed to working with industry to make this change.

See:  https://www.fda.gov/media/132494/download?utm_campaign=2019-11-25%20Updates%20on%20Ethylene%20Oxide%20Sterilization%20of%20Medical%20Devices&utm_medium=email&utm_source=Eloqua

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Tuesday, 14 January 2020

Sleep and Energy: The Energy Consumed By Your Brain While Sleeping


Irrespective of all the research that has been going on throughout the years, there is a lot of important information that is not known regarding the human brain. Numerous mental diseases, as well as sleeping disorders, still exist and their causes along with no proper mental therapy are known. For any adult, the brain is responsible for taking up almost 20% of the total energy, especially when the adult is resting.

This can sound weird but the main objective of the brain is processing as well as transmitting information with the help of electrical signals. It is important to understand the energy consumed by the brain when an individual is sleeping. When you understand about sleep and energy, it will be easier for you to plan your regular activities. Also, to sleep properly, make sure that you are removing all the gadgets from your room, as stated by https://www.forbes.com.

A guest post by Silvia Watson.

The overview


An adult is responsible for using almost 20% energy of the brain constantly. When considered in detail, almost 15% of the cardiac output is responsible for going towards the activity of the human brain. Apart from that, 25% of the glucose from the entire body is also responsible for helping the brain. Glucose is undoubtedly one of the most important sources of energy within the body and it is normally stored in the skeletal muscles as well as liver as glycogen.

Depending on the sleep stage that you are in, the body is responsible for distributing energy in more than a single way. During stage 2 as well as stage 3, which mean light as well as deep sleep, the brain is not as active. One misconception that constantly floats around is that most of the energy is being used by the brain when any difficult task is being solved. However, it is important to understand that this is not true. This is why you should understand the important question, "How Much Energy Does the Brain Consume While Sleeping?"

What happens to the brain when you are sleeping?


Several studies have revealed that the energy expenditure of the human body is not responsible for changing between the different stages of sleep. There is one important example, which can help in proving that. While all the muscles are not that active when you are in the REM stage, the increased activity of the brain is responsible for making up for that, thereby, evening the usage of energy. On the other hand, the NREM stage is responsible for boosting high expenditure of energy and the muscles when the positions are being switched during sleeping, however, the brain is not as active.

Scientists have already found out that sleep deprivation is responsible for increasing the expenditure of energy even when you are sleeping. People who are dealing with problems of sleep deprivation also report feeling cold. However, it cannot be confirmed because fragmented sleep can leave you in the state of waking up when dealing with an unsuitable environment.

Conclusion

Therefore, it can be said that sleep and energy are highly related to one another. You need to understand how your brain is functioning when you are sleeping so that you can understand the conservation of energy within your body while you are in the resting phase.

(PMR: http://www.pharmamicroresources.com/)

Audit and Control for Healthcare Manufacturers (book)

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Monday, 13 January 2020

Audit and Control for Healthcare Manufacturers: A Systems-Based Approach

Compliance is an affirmative indication or judgement that the supplier of a product or service has met the requirements of the relevant specifications, contract or regulation; also the state of meeting the requirements. Compliance is something that meets both the text and the spirit of a requirement. A key way to assess compliance is through auditing. For further details, see the PDA Bookstore: https://www.pda.org/bookstore/product-detail/5014-audit-and-control-for-healthcare-manufacturers


Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Sunday, 12 January 2020

New toxin impedes bacterial growth



An international research collaboration has discovered a new bacteria-killing toxin that shows promise of impacting superbug infectious diseases.

The discovery of this growth-inhibiting toxin, which bacteria inject into rival bacteria to gain a competitive advantage is the result of teamwork by co-senior authors John Whitney, assistant professor of the Department of Biochemistry and Biomedical Sciences at McMaster University, and Mike Laub, professor of biology at the Massachusetts Institute of Technology (MIT).

Whitney and his PhD student Shehryar Ahmad at McMaster's Michael G. DeGroote Institute for Infectious Disease Research were studying how bacteria secrete antibacterial molecules when they came across a new toxin. This toxin was an antibacterial enzyme, one the researchers had never seen before.
After determining the molecular structure of this toxin, Whitney and Ahmad realized that it resembles enzymes that synthesize a well-known bacterial signalling molecule called (p)ppGpp. This molecule normally helps bacteria survive under stressful conditions, such as exposure to antibiotics.

Boyuan Wang, a postdoctoral researcher in the Laub lab who specializes in (p)ppGpp signaling, examined the activity of the newly discovered enzyme. He soon realized that rather than making (p)ppGpp, this enzyme instead produced a poorly understood but related molecule called (p)ppApp. Somehow, the production of (p)ppApp was harmful to bacteria.
The researchers determined that the rapid production of (p)ppApp by this enzyme toxin depletes cells of a molecule called ATP. ATP is often referred to as the 'energy currency of the cell' so when the supply of ATP is exhausted, essential cellular processes are compromised and the bacteria die.

"I find it absolutely fascinating that evolution has essentially "repurposed" an enzyme that normally helps bacteria survive antibiotic treatment and, instead, has deployed it for use as an antibacterial weapon," said Whitney.


The research conducted at McMaster University was funded by the Canadian Institutes for Health Research and is affiliated with the CIHR Institute for Infection and Immunity (CIHR-III) hosted at McMaster University with additional funding from the David Braley Centre for Antibiotic Discovery. The research at MIT was supported by the Howard Hughes Medical Institute and the U.S. National Institutes of Health.

See:

Shehryar Ahmad, Boyuan Wang, Matthew D. Walker, Hiu-Ki R. Tran, Peter J. Stogios, Alexei Savchenko, Robert A. Grant, Andrew G. McArthur, Michael T. Laub, John C. Whitney. An interbacterial toxin inhibits target cell growth by synthesizing (p)ppApp. Nature, 2019; DOI: 10.1038/s41586-019-1735-9

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Saturday, 11 January 2020

DNA is only one among millions of possible genetic molecules



Biology encodes information in DNA and RNA, which are complex molecules finely tuned to their functions. But are they the only way to store hereditary molecular information? Some scientists believe life as we know it could not have existed before there were nucleic acids, thus understanding how they came to exist on the primitive Earth is a fundamental goal of basic research.

The central role of nucleic acids in biological information flow also makes them key targets for pharmaceutical research, and synthetic molecules mimicking nucleic acids form the basis of many treatments for viral diseases, including HIV. Other nucleic acid-like polymers are known, yet much remains unknown regarding possible alternatives for hereditary information storage.

Using sophisticated computational methods, scientists explored the "chemical neighbourhood" of nucleic acid analogues. Surprisingly, they found well over a million variants, suggesting a vast unexplored universe of chemistry relevant to pharmacology, biochemistry and efforts to understand the origins of life. The molecules revealed by this study could be further modified to gives hundreds of millions of potential pharmaceutical drug leads.

Nucleic acids were first identified in the 19th century, but their composition, biological role and function were not understood by scientists until the 20th century. The discovery of DNA's double-helical structure by Watson and Crick in 1953 revealed a simple explanation for how biology and evolution function. All living things on Earth store information in DNA, which consists of two polymer strands wrapped around each other like a caduceus, with each strand being the complement of the other.

When the strands are pulled apart, copying the complement on either template results in two copies of the original. The DNA polymer itself is composed of a sequence of "letters," the bases adenine (A), guanine (G), cytosine (C) and thymine (T), and living organisms have evolved ways to make sure during DNA copying that the appropriate sequence of letters is almost always reproduced. The sequence of bases is copied into RNA by proteins, which then is read into a protein sequence. The proteins themselves then enable a wonderland of finely-tuned chemical processes which make life possible.

Small errors occasionally occur during DNA copying, and others are sometimes introduced by environmental mutagens. These small errors are the fodder for natural selection: some of these errors result in sequences which produce fitter organisms, though most have little effect, and many even prove lethal. The ability of new sequences to allow their hosts to better survive is the "ratchet" which allows biology to almost magically adapt to the constantly changing challenges the environment provides.

This is the underlying reason for the kaleidoscope of biological forms we see around us, from humble bacteria to tigers, the information stored in nucleic acids allows for "memory" in biology. But are DNA and RNA the only way to store this information? Or are they perhaps just the best way, discovered only after millions of years of evolutionary tinkering?
"There are two kinds of nucleic acids in biology, and maybe 20 or 30 effective nucleic acid-binding nucleic acid analogues. We wanted to know if there is one more to be found or even a million more. The answer is, there seem to be many, many more than was expected," says professor Jim Cleaves of ELSI.

Though biologists don't consider them organisms, viruses also use nucleic acids to store their heritable information, though some viruses use a slight variant on DNA, RNA, as their molecular storage system. RNA differs from DNA in the presence of a single atom substitution, but overall RNA plays by very similar molecular rules as DNA. The remarkable thing is, among the incredible variety of organisms on Earth, these two molecules are essentially the only ones biology uses.

Biologists and chemists have long wondered why this should be. Are these the only molecules that could perform this function? If not, are they perhaps the best, that is to say, other molecules could play this role, and perhaps biology tried them out during evolution?
The central importance of nucleic acids in biology has also long made them drug targets for chemists. If a drug can inhibit the ability of an organism or virus to pass its knowledge of how to be infectious on to offspring, it effectively kills the organisms or virus. Mucking up the heredity of an organism or virus is a great way to knock it dead. Fortunately for chemists, and all of us, the cellular machinery which manages nucleic acid copying in each organism is slightly different, and in viruses often very different.

Organisms with large genomes, like humans, need to be very careful about copying their hereditary information and thus are very selective about not using the wrong precursors when copying their nucleic acids. Conversely, viruses, which generally have much smaller genomes, are much more tolerant of using similar, but slightly different molecules to copy themselves.

This means chemicals that are similar to the building blocks of nucleic acids, known as nucleotides, can sometimes impair the biochemistry of one organism worse than another. Most of the important anti-viral drugs used today are nucleotide (or nucleoside, which are molecule differing by the removal of a phosphate group) analogues, including those used to treat HIV, herpes and viral hepatitis. Many important cancer drugs are also nucleotide or nucleoside analogues, as cancer cells sometimes have mutations that make them copy nucleic acids in unusual ways.

Since most scientists believe the basis of biology is heritable information, without which natural selection would be impossible, evolutionary scientists studying the origins of life have also focused on ways of making DNA or RNA from simple chemicals that might have occurred spontaneously on primitive Earth. Once nucleic acids existed, many problems in the origins of life and early evolution would make sense. Most scientists think RNA evolved before DNA, and for subtle chemical reasons which make DNA much more stable than RNA, DNA became life's hard disk.


However, research in the 1960s soon split the theoretical origins field in two: those who saw RNA as the simple "Occam's Razor" answer to the origins-of-biology problem and those who saw the many kinks in the armour of RNA's abiological synthesis. RNA is still a complicated molecule, and it is possible structurally simpler molecules could have served in its place before it arose.

Examining all of these basic questions, which molecule came first, what is unique about RNA and DNA, all at once by physically making molecules in the laboratory, is difficult. On the other hand, computing molecules before making them could potentially save chemists a lot of time.

See:

Henderson James Cleaves, Christopher Butch, Pieter Buys Burger, Jay Goodwin, Markus Meringer. One Among Millions: The Chemical Space of Nucleic Acid-Like Molecules. Journal of Chemical Information and Modeling, 2019; 59 (10): 4266 DOI: 10.1021/acs.jcim.9b00632

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Special offers