New disease surveillance tool

A new computational method called 'CATCH' designs molecular 'baits' for any virus known to infect humans and all their known strains, including those that are present in low abundance in clinical samples, such as Zika. The approach can help small sequencing centers around the globe conduct disease surveillance, which is crucial for controlling outbreaks.

Scientists have been able to detect some low-abundance viruses by analyzing all the genetic material in a clinical sample, a technique known as "metagenomic" sequencing, but the approach often misses viral material that gets lost in the abundance of other microbes and the patient's own DNA.

Another approach is to "enrich" clinical samples for a particular virus. To do this, researchers use a kind of genetic "bait" to immobilize the target virus's genetic material, so that other genetic material can be washed away. Scientists in the Sabeti lab had successfully used baits, which are molecular probes made of short strands of RNA or DNA that pair with bits of viral DNA in the sample, to analyze the Ebola and Lassa virus genomes. However, the probes were always directed at a single microbe, meaning they had to know exactly what they were looking for, and they were not designed in a rigorous, efficient way.

What they needed was a computational method for designing probes that could provide a comprehensive view of the diverse microbial content in clinical samples, while enriching for low-abundance microbes like Zika.

Short for "Compact Aggregation of Targets for Comprehensive Hybridization," CATCH allows users to design custom sets of probes to capture genetic material of any combination of microbial species, including viruses or even all forms of all viruses known to infect humans.

To run CATCH truly comprehensively, users can easily input genomes from all forms of all human viruses that have been uploaded to the National Center for Biotechnology Information's GenBank sequence database. The program determines the best set of probes based on what the user wants to recover, whether that's all viruses or only a subset. The list of probe sequences can be sent to one of a few companies that synthesize probes for research. Scientists and clinical researchers looking to detect and study the microbes can then use the probes like fishing hooks to catch desired microbial DNA for sequencing, thereby enriching the samples for the microbe of interest.

Tests of probe sets designed with CATCH showed that after enrichment, viral content made up 18 times more of the sequencing data than before enrichment, allowing the team to assemble genomes that could not be generated from un-enriched samples. They validated the method by examining 30 samples with known content spanning eight viruses. The researchers also showed that samples of Lassa virus from the 2018 Lassa outbreak in Nigeria that proved difficult to sequence without enrichment could be "rescued" by using a set of CATCH-designed probes against all human viruses. In addition, the team was able to improve viral detection in samples with unknown content from patients and mosquitos.

See: Hayden C. Metsky, Katherine J. Siddle, Adrianne Gladden-Young et al. Capturing sequence diversity in metagenomes with comprehensive and scalable probe design. Nature Biotechnology, 2019; 37 (2): 160 DOI: 10.1038/s41587-018-0006-x

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

New strategy to tackle “don’t eat me” signal on cancer cells

Myeloid immune cells kill cancer cells by eating them but cancer cells prevent this from happening by giving out a 'do not eat me' signal. Led by immunologists Ton Schumacher (Netherlands Cancer Institute and Oncode) and Ferenc Scheeren (Leiden University Medical Center), researchers from various research institutes have discovered a new method to inhibit the 'don’t eat me' signal, and have therefore found a new target for immunotherapy.

On 4 March 2019, the researchers published an article on this topic in the scientific journal Nature Medicine.

The “don’t eat me” signal

Different types of immune cells have different strategies to fight cancer cells. For example, some immune cells—myeloid cells—kill cancer cells by eating them. Cancer cells can prevent this by expressing proteins on their surfaces which give out inhibiting signals to the immune cells. One example is the 'don’t eat me' signal, officially called CD47, which ensures that the cancer cell stays alive.

Researchers around the world are now looking for medicines to block this “don’t eat me” signal. One method for doing so is to intervene on the surface of the cell, by covering the CD47 molecules on cancer cells with a specific antibody.

This method of blocking the CD47 signal from cancer cells is currently being clinically developed and is promising, but there are side effects, such as a decrease in red blood cells. On top of that, patients require a weekly IV to block the CD47 molecules on cancer cells adequately.

Are there any other ways to counteract the “don’t eat me” signal CD47? To investigate this, PhD student Meike Logtenberg, lead author of the article, set up a collaboration with experimental geneticist Thijn Brummelkamp, who uses a unique method to map the genetic regulation of any desired protein in a cell. "With this screening method you can potentially find new targets," says Meike Logtenberg.

Screening the CD47 molecule

Together with the immunologists, Brummelkamp screened CD47, which also plays a role in healthy cells as an immune system check, and found that the QPCTL enzyme is a crucial protein in forming the “don’t eat me” signal. QPCTL changes the structure of the CD47 protein and without any QPCTL activity, the CD47 molecules are no longer able to give off an inhibiting signal to myeloid cells.

Research leader Ton Schumacher: "In collaboration with the groups of Jeanette Leusen (UMC Utrecht) and Timo van den Berg (Sanquin Research), we then showed that as soon as we inhibited the activity of this enzyme, we instantly blocked the “don’t eat me” signal on tumor cells. Identifying this new target is especially relevant because the substances we can use to inhibit the QPCTL enzyme are likely to have some advantages over the strategies currently being clinically developed to inhibit the CD47 signal route."

Blocking the signal

With a QPCTL inhibitor, for example, it becomes easier to control how long you want to block the signal, and so-called small molecule inhibitors are easier to administer than antibodies. Moreover, the substance does not inhibit the CD47 molecules on the healthy red blood cells that a patient receives during a blood transfusion to fight anaemia.

Clinical studies

The researchers expect that QPCTL inhibitors will be available for testing in clinical studies in the coming years. First clinical trials are expected to take place in patients with blood cancer.

----

Logtenberg et al., ‘Glutaminyl cyclase is an enzymatic modifier of the CD47- SIRP axisand target for cancer immunotherapy’, Nature Medicine 4 March 2017 doi: 10.1038/s41591-019-0356-z


Corresponding author: Ton Schumacher
Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

EDQM and EU Commission discuss improving plasma supply management

The EDQM and the European Commission (EC) brought together stakeholders involved in the field of plasma to discuss ways forward for improving plasma supply management and donor protection, during a symposium organised in Strasbourg on 29 and 30 January 2019. As emphasised by those present at the meeting, this was the first time that all stakeholders in the sector had met to exchange their views on how to increase the supply of plasma for fractionation in Europe, while ensuring adequate protection of both donors and patients.

See:  https://www.edqm.eu/sites/default/files/press-release-plasma-symposium-february-2019.pdf

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Cleanroom facility of the future

Tim Sandle was featured in the American Pharmaceutical Review video ‘Clean facility of the future’.

Biopharmaceuticals have traditionally been produced in sterile environments as a means to ensure product quality and to reduce failed batches. In addition, as more scrutiny is put on the manufacture of all types of products, oral solid dosage manufacturers are investing in equipment, technologies and expertise to move their processes towards a cleaner manufacturing environment.

How do both sides of the industry ensure their products are produced in clean modern facilities? What are the latest tools, techniques, services and best practices to manufacture the highest quality products while minimizing or eliminating the possibility of batch failures or regulatory intervention?

The Clean Facility of the Future documentary will explore these issues and more by talking to many thought leaders on this topic including: regulatory, end-users, consultants, equipment/service providers.

People interviewed:

• Poonam Bhende
• Tony Cundell, Ph.D.
• Lori Daane, Ph.D.
• Sandra Gay, Ph.D.
• Lisa Graham, Ph.D.
• Patricia Hughes
• Ian Jennings, SVP
• David Jones, Ph.D.
• Michael Lindsay
• Michael Miller, Ph.D.
• Paula Peacos
• Tim Sandle, Ph.D.
• Allison Scott, Ph.D.
• Donald C. Singer
• Krista Spreng, Ph.D.
• Jeff Weber

See: https://www.americanpharmaceuticalreview.com/Clean-Facility-of-the-Future/

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Custom-made proteins may help create antibodies to fight HIV

A new way to create proteins that can sneak through HIV's protective coating may be a step toward understanding the key components needed for developing a vaccine for the virus, according to researchers.

Using computational modeling, a team of researchers led by Penn State designed and created proteins that mimicked different surface features of HIV. After being immunized with the proteins, rabbits developed antibodies that were able to bind with the virus.

"We were able to show that by using our designed proteins, the blood was able to spontaneously generate antibodies that can inhibit the infection of HIV in cellular models," said Cheng Zhu, a postdoctoral fellow at Penn State College of Medicine. "When we incubated the HIV virus, its infectivity was dramatically reduced by the rabbits' blood."

Zhu added that the study provides a novel way to design proteins for vaccines.

"The proteins -- or immunogens -- we developed aren't a finished product, but we were able to show evidence that it's possible to do," Zhu said. "Moreover, it's also very exciting that we were able to create a new method to tailor make proteins, which could open the door for developing vaccines for other infections, as well."

"Even if we develop an antibody for a particular strain of the virus, that antibody may not even notice the next strain of the virus," Dokholyan said. "In order to develop broadly neutralizing antibodies -- antibodies that neutralize multiple strains of a virus -- we need to find something that remains constant on the virus for those antibodies to latch onto."

According to Dokholyan, HIV uses a coating of carbohydrates to protect a protein on its surface called Env. While this protein could be a potential target for vaccines, the carbohydrate coating makes it difficult or impossible for antibodies to access and neutralize it.

But sometimes, holes naturally appear in this coating, exposing the Env protein to potential antibodies. Zhu said he and the other researchers wanted to find a way to target these holes.

"The idea would be to do molecular surgery to copy sections of the virus's surface and paste them onto different, benign proteins, so they would look but not act like the Env protein," Zhu said. "Hopefully, this would allow the immune system to recognize the virus and create antibodies to neutralize it in the future."

The researchers used computational models to design proteins that would mimic the conserved protein surface of different strains of HIV to be used in the vaccine. Dokholyan said that while usually proteins are engineered by changing one amino acid at a time, they wanted to try a different approach.

"Instead of changing one amino acid at a time, it's a large surface of the HIV strain that is cut and then plugged onto a different protein," Dokholyan said. "It's an important milestone be able to do these major molecular surgeries, and it's very exciting that the strategy worked with a very high accuracy."

After creating immunogens that used the new, HIV-mimicking proteins, the researchers immunized the rabbits and drew blood samples once a month. After analyzing the samples, the researchers found that the blood contained antibodies that were able to bind onto HIV.

The researchers said that while the findings are promising, there is still more work to be done.

"It's important that we were able to generate an immune response to HIV and show that it's possible as a proof of concept," Dokholyan said. "But, we still need to improve the antibodies' neutralization abilities and other aspects before it can become a viable vaccine."

Dokholyan said that in the future, the protein design method could potentially help create and personalize vaccines for different diseases in various areas in the world.

"Diseases can vary by location, for example, there are different strains of HIV in various countries or regions," Dokholyan said. "If we can easily customize proteins for vaccines, that's a good example of where personalized medicine is going to play a role."

The National Institutes of Health helped support this research.

See:

Cheng Zhu, Elena Dukhovlinova, Olivia Council, Lihua Ping, Edgar M. Faison, Shamit S. Prabhu, E. Lake Potter, Stephen L. Upton, Guowei Yin, James M. Fay, Laura P. Kincer, Ean Spielvogel, Sharon L. Campbell, S. Rahima Benhabbour, Hengming Ke, Ronald Sw

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Elemental impurities reference standards

The EDQM has updated the leaflets of its reference standards for elemental impurities (lead solution CRS, cadmium solution CRS, mercury solution CRS and arsenic solution CRS) with further information to facilitate their use and make traceability transparent. The mass fractions of the assigned element are traceable to the SI units (mole and kilogram) through an uninterrupted chain, similar to certified reference materials established by a national metrological institute.

More detailed information on the assigned value and the associated expanded uncertainty is now included in the leaflets accompanying the reference standards. In addition, the solvent composition is explicitly stated, as well as the density of the solution. The specific primary materials used to achieve the link to the SI units are also indicated.

Elemental impurities naturally occurring in the environment are amongst the greatest potential sources of contamination. Public health standards, such as the ICH Q3D Guideline for Elemental Impurities, require careful risk assessments of sources for elemental impurities. The changes are part of the EDQM’s efforts to provide efficient support to users of its pharmaceutical reference standards. These reference standards also underpin the European Pharmacopoeia (Ph. Eur.) chapter describing the determination of elemental impurities (2.4.20.).

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

How fungi influence global plant colonization

The symbiosis of plants and fungi has a great influence on the worldwide spread of plant species. In some cases, it even acts like a filter. This has been discovered by an international team of researchers with participation from the University of Göttingen. The results appeared in the journal Nature Ecology & Evolution.

In the colonisation of islands by plant species, it isn't just factors like island size, isolation and geological development that play an important role, but also the interactions between species. The scientists found that the symbiosis of plant and fungus -- the mycorrhiza -- is of particular importance. The two organisms exchange nutrients via the plant's fine root system: the fungus receives carbohydrates from the plant; the plant receives nutrients that the fungus has absorbed from the soil.

"For the first time, new data on the worldwide distribution of plant species in 1,100 island and mainland regions allows us to investigate the influence of this interaction on a global scale," says Dr Patrick Weigelt from the University of Göttingen's Department of Biodiversity, Macroecology and Biogeography, who worked on the study. The results: mycorrhiza-plant interactions, which are naturally less frequent on islands because the two organisms rely on each other, mean that the colonisation of remote islands is hindered. The lack of this symbiotic relationship may act like a brake on the spread of the plants. This is not the case for plant species introduced by humans, as fungi and plants are often introduced together. Head of Department, Professor Holger Kreft, adds, "The proportion of plant species with mycorrhiza interactions also increases from the poles to the equator." One of the most prominent biogeographic patterns, the increase in the number of species from the poles to the tropics, is closely related to this symbiosis.

Dr Camille Delavaux, lead author from the University of Kansas (US), explains, "We show that the plant symbiotic association with mycorrhizal fungi is an overlooked driver of global plant biogeographic patterns. This has important consequences for our understanding of contemporary island biogeography and human-mediated plant invasions." The results show that complex relationships between different organisms are crucial for understanding global diversity patterns and preserving biological diversity. "The absence of an interaction partner can disrupt ecosystems and make them more susceptible to biological invasions," Weigelt stresses.

See:

Camille S. Delavaux, Patrick Weigelt, Wayne Dawson, Jessica Duchicela, Franz Essl, Mark van Kleunen, Christian König, Jan Pergl, Petr Pyšek, Anke Stein, Marten Winter, Peggy Schultz, Holger Kreft, James D. Bever. Mycorrhizal fungi influence global plant biogeography. Nature Ecology & Evolution, 2019; 3 (3): 424 DOI: 10.1038/s41559-019-0823-4

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Call for experts to join the Ph. Eur. Network

The EDQM is renewing its call for expressions of interest to join the European Pharmacopoeia (Ph. Eur.) as an independent scientific expert. Professionals from national authorities (e.g. pharmacopoeial authorities, official medicines control laboratories, licensing authorities and inspectorates), the private sector (pharmaceutical or chemical industries), academia, research organisations and other experts are encouraged to apply. This is an ideal opportunity to take part in the work of the Ph. Eur., to network with professionals with various backgrounds and from all over Europe and beyond, and to help shape Ph. Eur. texts, internationally-recognised quality standards for medicines.

Experts provide a vital and invaluable contribution to the elaboration and maintenance of Ph. Eur. texts. In return, they are given a unique opportunity to expand their knowledge of the Ph. Eur. and the European regulatory system, share information and experience, better understand the difficulties linked to the elaboration and revision of pharmacopoeial texts and network with their peers.

The terms of reference of the Ph. Eur. Groups of Experts and Working Parties for which candidates from non-Ph. Eur. member states may apply are provided in the Terms of reference and profile for members of Groups of Experts and Working Parties.

Applications will be examined by the Ph. Eur. Commission at its 165th session (26-27 November 2019) when all Ph. Eur. Groups of Experts and Working Parties will be (re)appointed. Potential candidates from Ph. Eur. Member States are invited to contact their National Pharmacopoeia Authorities.

For further details, see: https://www.edqm.eu/en/node/16716

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Yeast produce low-cost, high-quality cannabinoids

Synthetic biologists have created an enzymatic network in yeast that turns sugar into cannabinoids, including tetrahydrocannabinol and cannabidiol, but also novel cannabinoids not found in the marijuana plant itself. The yeast factories would be more environmentally friendly and less energy intensive than growing the plant and separating out the psychoactive and non-psychoactive ingredients. They may also yield cannabinoid derivatives with unexpected medical uses.

Cannabis and its extracts, including the high-inducing THC, or tetrahydrocannabinol, are now legal in 10 states and the District of Columbia, and recreational marijuana -- smoked, vaped or consumed as edibles -- is a multibillion-dollar business nationwide. Medications containing THC have been approved by the Food and Drug Administration to reduce nausea after chemotherapy and to improve appetite in AIDS patients.

CBD, or cannabidiol, is used increasingly in cosmetics -- so-called cosmeceuticals -- and has been approved as a treatment for childhood epileptic seizures. It is being investigated as a therapy for numerous conditions, including anxiety, Parkinson's disease and chronic pain.

But medical research on the more than 100 other chemicals in marijuana has been difficult, because the chemicals occur in tiny quantities, making them hard to extract from the plant. Inexpensive, purer sources -- like yeast -- could make such studies easier.

Turning yeast into chemical factories involves co-opting their metabolism so that, instead of turning sugar into alcohol, for example, yeast convert sugar into other chemicals that are then modified by added enzymes to produce a new product, such as THC, that the yeast secrete into the liquid surrounding them. The researchers ended up inserting more than a dozen genes into yeast, many of them copies of genes used by the marijuana plant to synthesize cannabinoids.

See:

Xiaozhou Luo, Michael A. Reiter, Leo d’Espaux, Jeff Wong, Charles M. Denby, Anna Lechner, Yunfeng Zhang, Adrian T. Grzybowski, Simon Harth, Weiyin Lin, Hyunsu Lee, Changhua Yu, John Shin, Kai Deng, Veronica T. Benites, George Wang, Edward E. K. Baidoo, Yan Chen, Ishaan Dev, Christopher J. Petzold & Jay D. Keasling. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature, 2019 DOI: 10.1038/s41586-019-0978-9 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Ph.Eur. micro determination (2.5.32.) revised

The chapter on micro determination (2.5.32.) has been revised in the Ph. Eur. Supplement 9.8. News from EDQM.

The revised part is reproduced here after:

“The instrument qualification is carried out according to established quality system procedures, for example using a suitable certified reference material. Sodium aminosalicylate dihydrate for equipment qualification CRS may be used when proceeding by direct or liquid sample introduction, whereas amoxicillin trihydrate for performance verification CRS may be used with the evaporation technique.”

The EDQM provides Sodium aminosalicylate dihydrate for equipment qualification CRS (catalogue code Y0001816) and amoxicillin trihydrate for performance verification CRS (catalogue code Y0001521), the latter should be used only for micro determination of water with oven.

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Cleaning and Cleanrooms e-book

Pharmaceutical Manufacturing: Understanding Your Process Series  - Cleaning and Cleanrooms (e-book)

In the past 15 years, PDA/DHI has published more than 1,000 practical scientific and regulatory chapters, written by global subject matter experts, which have been designed to help pharmaceutical and biotech manufacturers stay abreast, streamline processes and comply with regulators.

We have now collected bestsellers and added materials that have not published before in electronic book form covering three vital topics:

• Cleaning and Cleanrooms
• Sterilization
• Environmental Monitoring

These easily accessible, reasonably priced, informative collections offer background and hands-on applications that will help with a myriad of activities for manufacturers.

The Cleaning and Cleanrooms collection features a two-part history of cleaning and cleanrooms, classifications, supplies, sanitization and several other important topics. The book is edited by Tim Sandle and Jeanne Moldenhauer.

Contents:

• The Development of Cleanrooms: An Historical Review Part 1: From Civil War to Safe Surgical Practice by Tim Sandle(New)
• The Development of Cleanrooms: An Historical Review Part 2: The Path Towards International Harmonization by Tim Sandle (New)
• Understanding Cleanroom Classifications by Jeanne Moldenhauer. (Chapter excerpted from Contamination Control in Healthcare Product Manufacturing, Volume 3, Chapter 12 published 2014.)
• Cleanroom Supplies by Jeanne Moldenhauer. (Chapter excerpted from Environmental Monitoring: A Comprehensive Handbook, Volume 1, Chapter 11 published 2005.)
• Practical Aspects of Cleaning, Sanitizing and Disinfecting Rooms and Surfaces by Jeanne Moldenhauer. (Chapter excerpted from Environmental Monitoring: A Comprehensive Handbook, Volume 1, Chapter 12 published 2005.)
• Cleaning Validation: Process Life Cycle Approach by Paul Lopolito and Elizabeth Rivera. (Chapter excerpted from Contamination Control in Healthcare Product Manufacturing, Volume 3, Chapter 10 published 2014.)

Available to download. Prior to purchase please view the download instructions and Terms of Usage.
Format: PDF (1 file 1.72 MB)

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

How bacteria communicate

Researchers at Binghamton University, State University of New York have uncovered the unique way in which a type of Gram-negative bacterium delivers the toxins that make us sick. Understanding this mechanism may help design better ways to block and eventually control those toxins.

Assistant Professor Xin Yong and graduate student Ao Li from the Department of Mechanical Engineering, along with Associate Professor Jeffrey W. Schertzer from the Department of Biological Sciences, published their findings in the Journal of Biological Chemistry.

The study looked at how bacteria communicate via the transportation of small molecules. Yong and Schertzer explained that communication molecules stimulate the production of outer membrane vesicles. These small packages then bud off from the surface of the bacterium and contain highly concentrated toxins.

Originally, it was hypothesized that the communication molecule induced vesicle production by controlling gene expression, but that's not what's going on.

Yong and Schertzer decided to work together on a model to understand more about how the communication molecule inserts itself into the membrane of bacteria in order to physically stimulate the production of these toxin delivery vehicles.

Yong's model allowed them to look at the details of the molecule and understand more about how it interacted with the membrane on a very short timescale. Schertzer and Yong explained that the communication molecule has both a head and a tail that are known to be flexible, but they did not expect this type of change. In the future, they hope to test what would change in the interaction when the tail is removed or the head is modified.

READ MORE: ISS is not causing bacteria to mutate into dangerous, antibiotic-resistant superbugs

While the study may sound fairly specific, it has some wider implications for all Gram-negative bacteria. Learning more about how Gram-negative bacteria communicate with each other can help researchers build a stronger understanding of multispecies interactions and how to eventually control these types of high-risk infections.

See:

Ao Li, Jeffrey W. Schertzer, Xin Yong. Molecular conformation affects the interaction of the Pseudomonas quinolone signal with the bacterial outer membrane. Journal of Biological Chemistry, 2019; 294 (4): 1089 DOI: 10.1074/jbc.AC118.006844

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Insight into how bacteria move on surfaces

Understanding bacteria motility would not only expand our understanding of their behavior, but would also help us fight certain aggressive pathogens. However, the question has gone unanswered because microbiologists have lacked the tools to visualize bacterial filaments directly.

Lorenzo Talà, a PhD student in the lab of Alexandre Persat at EPFL's Institutes of Bioengineering and Global Health has developed a microscopy method that can directly observe the structures many bacteria use to crawl.

"Bacterial surfaces are decorated with protein filaments involved in motility, adhesion, signaling and pathogenicity, which ultimately rule how bacteria interact with their environments" says Talà. "However, they are so small that observing them in live cells is extremely complex. So we are left with little knowledge of their dynamic activities."

This is especially true for structures known as "type IV pili": nanometer-wide filaments that extend and retract from the surface of many bacteria, helping them walk in a way known as "twitching motility." The term might not sound very serious, but it mechanically activates virulence in certain pathogens -- meaning that it is a prime target for fighting them. The scientists studied the bacterium Pseudomonas aeruginosa.

But do single bacteria orchestrate type IV pili motion to power their motility? "In our studies of type IV pili and mechano-activation of virulence in Pseudomonas aeruginosa, one technical paradox has been a source of frustration: pili, but also fimbriae, flagella, and injection systems permanently extend outside single cells, so why can't we directly visualize them?"

To overcome this, the scientists explored an emerging microscopy method pioneered by their collaborator Philipp Kukura at Oxford University. Using a technique called interferometric scattering microscopy (iSCAT), they were able to see these nanometers-wide filaments in live cells, without any chemical labels, at high speed, and in three dimensions.

"iSCAT represents a major technological advance in microbiology," says Persat. "We recently described the visualization technique and received extensive positive feedback among scientists across a variety of disciplines simply because we could finally dynamically observe pili in live bacteria straight out of culture."

To understand the coordination of type IV pili movements, the scientists focused on precisely timing the succession of surface attachment, retraction, and cell body displacements using iSCAT. The approach revealed three key events that lead to successful and energetically efficient movement across surfaces.

First, contact of the pilus tip with the surface activates a molecular motor that initiates retraction. Second, this retraction enhances the attachment of the pilus to the surface, increasing the bacterium's displacement. Finally, a second, stronger molecular motor enforces the bacterium's displacement under high friction.

This sequence shows that pili act as sensors, and reveals a new mechanism by which bacteria interact with surfaces. It also reveals that bacteria use sensory mechanisms to coordinate the dynamic motion of their motility machineries, in a striking analogy to the way higher organisms, including humans, move their limbs to generate displacement.

"The human central nervous system processes mechanosensory signals to sequentially engage motor components, thus triggering muscle contraction and resulting in gait," explains Talà. "Our work shows that in the same manner, bacteria use a sense of touch to sequentially engage molecular motors, generating cycles of pili extension and retraction that result in a walk pattern."

See:

Lorenzo Talà, Adam Fineberg, Philipp Kukura, Alexandre Persat. Pseudomonas aeruginosa orchestrates twitching motility by sequential control of type IV pili movements. Nature Microbiology, 2019; DOI: 10.1038/s41564-019-0378-9

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Why Are Prescription Drug Prices Soaring?

You can't watch the news these days without seeing at least one story about rising prescription drug prices. Whether it's a personal story about a family that has to choose between insulin and groceries or one about the treatment for a rare neuromuscular disorder that went from the low price of free to the mind-blowing amount of $375,000 a year, these tales are all over. You might even know someone who's personally affected by these soaring drug prices.

A guest post by Megan Ray Nichols

So why are prescription drug prices climbing much faster than the rate of inflation?

Drug Prices vs. Inflation

Researchers just published a study that's been following prescription drug prices since 2008. Between 2008 and 2016, the cost of brand drugs oral medication increased by nine percent a year. Injectable drugs went up by 15 percent annually, and even generic drugs saw some price increases.

For comparison, annual inflation is only about two percent. That means prescription drug prices are rising between five and eight times faster than the cost of your weekly groceries or gas. What's sending the cost of these drugs into the stratosphere? 

A Lack of Competition

When a new product hits the market outside of the pharmaceutical industry, its price is based on the cost of similar competitive products. The amount will rise and fall as manufacturers change their prices to compete with one another, which provides the consumer with a variety of options to choose from.

That competition doesn't exist in the pharmaceutical industry. When the FDA approves a drug and it hits the market, the company that created it has a lengthy exclusivity patent — no one else is allowed to make different versions of that drug, so you won't see any generic versions available until the patent runs out.

The price of the drug also has minimal effect on the demand for it. If a cell phone or a new computer is in high demand, the manufacturer can increase the price, and while some consumers will still spend the money, the high price will reduce the number of sales.

With drugs, there's no difference in demand when sellers drive up the price. Someone who needs insulin when it costs $50 a bottle will still need insulin when it costs $500 a bottle — they'll have to choose buying their medication over feeding themselves for the week, but they'll still buy it.

What's the Real Price?

Imagine going to the grocery store and shopping for food the same way you shop for prescription medication. You're given a food plan and only allowed to buy the foods on that plan — and what you pay for a gallon of milk might be more or less than the guy behind you. You can't shop around, and for some products, there are no cost-saving generic alternatives. No store brands. No buying two percent milk instead of whole because you're trying to watch your weight.

That's precisely what it's like to shop for prescription drugs — and even then, you'll probably never know the real price of your prescription medications. The pharmaceutical manufacturer doesn't set the amount that you cough up — it's arbitrarily set by the insurance company, which is why there's so much variation in what people pay for their prescriptions. You may even be paying more than the guy behind you because the insurance company doesn't want to spend any more for a vial of insulin or an epi-pen than they have to.

With the average American spending more than $1,200 a year on prescriptions, this massive inflation is leaving people with an untenable choice — pay bills and buy food, or make sure they have another month of their prescription medication. 

A Drive for Profit

There are a lot of greedy people in Big Pharma, especially if you listen to mainstream media. There are some examples of this, such as former hedge fund manager Martin Shkreli, dubbed Pharma Bro, who bought the rights to AIDS treatment Daraprim and increased the price from $13.50 a pill to over $750.

The pharmaceutical industry is a very profitable one. In 2017, the global pharmaceutical market was worth $934 billion, and that's expected to climb to over $1.1 trillion by 2021. For many in the industry, it's not about helping people — it's about padding their wallets. Since 1998, the industry has spent more than $3 billion hiring lobbyists to gently — or not so gently — encourage Congress to do their bidding. They hired so many lobbyists in 2016 that they could assign 14 of them to each person in the House and Senate.

This number doesn't even take into account the hundreds of millions of dollars that they've spent over the years in the form of political campaign contributions.

Between 40 and 70 percent of our prescription drugs are imported from other countries, but the average patient isn't allowed to take advantage of the lower prices those countries charge for their medications. 

Moving Forward

Drug prices are quickly heading skyward, increasing up to 15 percent every year. While some of it can be attributed to new drugs and treatments entering the marketplace, that doesn't account for the established drugs and generic alternatives that are also climbing. Greed plays a large part, and so does the lack of transparency in the pricing of these medications. When people are choosing between paying for their prescriptions and paying for their daily necessities, you know there's a problem that needs to be fixed.

Monocyte activation test - live webinar

Monocyte activation test: a powerful tool to assess pyrogenic risk in the pharmaceutical process

Microbial risk in the pharmaceutical manufacturing process cannot be limited to viable microorganisms. Even if drug substances are manufactured in clean conditions and final drug products are sterilized, some subcellular microbial components may remain at the end of the manufacturing process.

To minimize the risk of subcellular microbial components remaining in the final drug product, a risk assessment approach of the whole manufacturing process can be applied, as described by Friedrich von Wintzingerode¹.

These subcellular contaminants often include Pathogen Associated Molecular Pattern (the so-called PAMPs) that can trigger the human immune system leading to inflammatory response and constitute a pyrogenic risk for patients. That’s why ensuring the absence of such components in the final drug product before batch release is key for product quality and patient safety.

In any case, testing for endotoxins in the final drug product before batch release is currently a minimum requirement from regulations. To reinforce the risk management approach, a new recommendation was added to the European Pharmacopeia chapter 5.1.10 « Guidelines for using the test for bacterial endotoxins » requiring users to carefully evaluate the risk for pyrogens (i.e. endotoxins and non-endotoxin pyrogens) before implementing the Bacterial Endotoxin Test (BET) as the sole pyrogenicity test. This is because the BET is designed to detect endotoxins only, leaving room for missing non-endotoxin pyrogens that could be responsible for fever reaction in patients.

The EP chapter 5.1.10 also indicates that “To rule out the presence of non-endotoxin pyrogens in substances or products, the use of the monocyte-activation test (2.6.30) is recommended at release or during development of the production process”. Indeed, the Monocyte Activation Test (MAT), mimics the human immune reaction to pyrogens by detecting all kinds of pyrogens that trigger the monocytes through the toll-like receptor (TLR) pathway, making it a powerful tool to assess pyrogenic risk in pharmaceutical process.

The PyroMAT™ System, our ready-to-use MAT kit using a monocytic cell line, has demonstrated the ability to detect a wide range of pyrogens. Each batch of PyroMAT™ cells is qualified for the expression of all the surface TLRs to ensure the detection of both endotoxins and non-endotoxin pyrogens. With our PyroMAT™ System, we provide a new solution for sensitive, robust, and easy-to-perform pyrogen testing.

New tools to assess the risk of microbial impurities in the pharmaceutical manufacturing process

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¹ von Wintzingerode F. Biologics Production: Impact of Bioburden Contaminations of Non-Sterile Process Intermediates on Patient Safety and Product Quality.  Am Pharm Rev. 2017 Apr;20(3)

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology