GN/ Giant bacteria found in Guadeloupe mangroves challenge traditional concepts
Genetics biweekly vol.31, 15th June — 29th June
TL;DR
- Researchers describe the morphological and genomic features of a ‘macro’ microbe’ — a giant filamentous bacterium composed of a single cell discovered in the mangroves of Guadeloupe. Using various microscopy techniques, the team also observed novel, membrane-bound compartments that contain DNA clusters dubbed ‘pepins.’
- Researchers have identified a molecule in the blood that is produced during exercise and can effectively reduce food intake and obesity in mice.
- A full DNA analysis of mites that live in the hair follicles of all humans reveals explanations for their bizarre mating habits, body features and evolutionary future. Inbreeding and isolation means they have shed genes and cells and are moving closer to a permanent existence with us.
- The deletion of the Wt1 gene during the early stages of the embryonic reproductive organ formation leads to differences in sex development in adult mice, according to a new study.
- Researchers have developed a unique 3D printed system for harvesting stem cells from bioreactors.
- Scientists have determined the process for incorporating selenium — an essential trace mineral found in soil, water and some foods that increases antioxidant effects in the body — to 25 specialized proteins, a discovery that could help develop new therapies to treat a multitude of diseases from cancer to diabetes.
- Researchers have identified how the bacterium that causes tuberculosis (TB) can evolve rapidly in response to new environments.
- Scientists have studied the human immune response after immunization with the malaria pathogen Plasmodium falciparum. Their findings could explain why natural infections, to which people in endemic areas are constantly exposed, offer little protection against new diseases with other strains, and why the effect of the vaccination available to date lasts only a short time.
- Thirteen genetic variants associated with disease in cats are present in more pedigreed breeds than previously thought, according to the largest ever DNA-based study of domestic cats. However, these variants are declining in frequency in breeds that are regularly screened for the genetic markers.
- A new statistical tool for predicting protein function could help with tasks ranging from producing biofuels to improving crops to developing new disease treatments. Not only could it help with the difficult job of altering proteins in practically useful ways, but it also works by methods that are fully interpretable — an advantage over conventional AI.
- And more!
Overview
Genetic technology is defined as the term which includes a range of activities concerned with the understanding of gene expression, advantages of natural genetic variation, modifying genes and transferring genes to new hosts. Genes are found in all living organisms and are transferred from one generation to the next. Gene technology encompasses several techniques including marker-assisted breeding, RNAi and genetic modification. Only some gene technologies produce genetically modified organisms.
Modern genetic technologies like genome editing would not be possible without all the previous generations of genetic technologies that have enabled scientists to discover what genes are, what they do and how DNA can be modified to add, remove or replace genes. You can find major genetic technologies development milestones via the link.
Gene Technology Market
- The valuation of the genetic engineering market is projected to escalate to USD 6.90 MN by the end of 2027.
- Global Genetic Engineering Market is projected to grow at 12.48% CAGR during the assessment period (2017–2027).
- North America holds the largest share in the global genetic engineering market, followed by Europe and the Asia Pacific, respectively.
- Another research provider, MarketsandMarkets, forecasts the genome editing, genome engineering market to grow from USD 3.19 billion in 2017 to USD 6.28 billion by 2022, at a compounded annual growth rate (CAGR) of 14.5% during the forecast period. The key factors propelling market growth are rising government funding and growth in the number of genomics projects, high prevalence of infectious diseases (like COVID-19) and cancer, technological advancements, increasing production of genetically modified (GM) crops, and growing application areas of genomics.
Latest News & Research
A centimeter-long bacterium with DNA contained in metabolically active membrane-bound organelles
by Volland JM, Gonzalez-Rizzo S, Gros O, et al. in Science
At first glance, the slightly murky waters in the tube look like a scoop of stormwater, complete with leaves, debris, and even lighter threads in the mix. But in the Petri dish, the thin vermicelli-like threads floating delicately above the leaf debris are revealed to be single bacterial cells, visible to the naked eye.
The unusual size is notable because bacteria aren’t usually visible without the assistance of microscope. “It’s 5,000 times bigger than most bacteria. To put it into context, it would be like a human encountering another human as tall as Mount Everest,” said Jean-Marie Volland, a scientist with joint appointments at the U.S. Department of Energy (DOE) Joint Genome Institute (JGI), a DOE Office of Science User Facility located at Lawrence Berkeley National Laboratory (Berkeley Lab) and the Laboratory for Research in Complex Systems (LRC) in Menlo Park, Calif. Volland and colleagues, including researchers at the JGI and Berkeley Lab, LRC, and at the Université des Antilles in Guadeloupe, described the morphological and genomic features of this giant filamentous bacterium, along with its life cycle.
For most bacteria, their DNA floats freely within the cytoplasm of their cells. This newly discovered species of bacteria keeps its DNA more organized. “The big surprise of the project was to realize that these genome copies that are spread throughout the whole cell are actually contained within a structure that has a membrane,” Volland said. “And this is very unexpected for a bacterium.”
The bacterium itself was discovered by Olivier Gros, a marine biology professor at the Université des Antilles in Guadeloupe, in 2009. Gros’ research focuses on marine mangrove systems, and he was looking for sulfur-oxidizing symbionts in sulfur-rich mangrove sediments not far from his lab when he first encountered the bacteria. “When I saw them, I thought, ‘Strange,’” he said. “In the beginning I thought it was just something curious, some white filaments that needed to be attached to something in the sediment like a leaf.” The lab conducted some microscopy studies over the next couple of years, and realized it was a sulfur-oxidizing prokaryote.
Silvina Gonzalez-Rizzo, an associate professor of molecular biology at the Université des Antilles and a co-first author on the study, performed the 16S rRNA gene sequencing to identify and classify the prokaryote. “I thought they were eukaryotes; I didn’t think they were bacteria because they were so big with seemingly a lot of filaments,” she recalled of her first impression. “We realized they were unique because it looked like a single cell. The fact that they were a ‘macro’ microbe was fascinating!”
“She understood that it was a bacterium belonging to the genus Thiomargarita,” Gros noted. “She named it Ca. Thiomargarita magnifica.”
“Magnifica because magnus in Latin means big and I think it’s gorgeous like the French word magnifique,” Gonzalez-Rizzo explained. “This kind of discovery opens new questions about bacterial morphotypes that have never been studied before.”
Volland got involved with the giant Thiomargarita bacteria when he returned to the Gros lab as a postdoctoral fellow. When he applied to the discovery-based position at the LRC that would see him working at the JGI, Gros allowed him to continue research on the project.
At the JGI, Volland began studying Ca. T. magnifica in Tanja Woyke’s Single Cells Group to better understand what this sulfur-oxidizing, carbon fixing bacterium was doing in the mangroves. “Mangroves and their microbiomes are important ecosystems for carbon cycling. If you look at the space that they occupy on a global scale, it’s less than 1% of the coastal area worldwide. But when you then look at carbon storage, you’ll find that they contribute 10–15% of the carbon stored in coastal sediments,” said Woyke, who also heads the JGI’s Microbial Program and is one of the article’s senior authors. The team was also compelled to study these large bacteria in light of their potential interactions with other microorganisms. “We started this project under the JGI’s strategic thrust of inter-organismal interactions, because large sulfur bacteria have been shown to be hot spots for symbionts,” Woyke said. “Yet the project took us into a very different direction,” she added.
Volland took on the challenge to visualize these giant cells in three dimensions and at relatively high magnification. Using various microscopy techniques, such as hard x-ray tomography, for instance, he visualized entire filaments up to 9.66 mm long and confirmed that they were indeed giant single cells rather than multicellular filaments, as is common in other large sulfur bacteria. He was also able to use imaging facilities available at Berkeley Lab, such as confocal laser scanning microscopy and transmission electron microscopy (TEM) to visualize the filaments and the cell membranes in more details. These techniques allowed him to observe novel, membrane-bound compartments that contain DNA clusters. He dubbed these organelles “pepins,” after the small seeds in fruits. DNA clusters were plentiful in the single cells.
The team learned about the cell’s genomic complexity. As Volland noted, “The bacteria contain three times more genes than most bacteria and hundreds of thousands of genome copies (polyploidy) that are spread throughout the entire cell.” The JGI team then used single cell genomics to analyze five of the bacterial cells on the molecular level. They amplified, sequenced and assembled the genomes. In parallel, Gros’ lab also used a labeling technique known as BONCAT to identify areas involved in protein-making activities, that confirmed that the entire bacterial cells were active.
“This project has been a nice opportunity to demonstrate how complexity has evolved in some of the simplest organisms,” said Shailesh Date, founder and CEO of LRC, and one of the article’s senior authors. “One of the things we’ve argued is that there is need to look at and study biological complexity in much more detail than what is being done currently. So organisms that we think are very, very simple might have some surprises.”
The LRC provided funding for Volland through grants from the John Templeton Foundation and the Gordon and Betty Moore Foundation. “This groundbreaking discovery highlights the importance of supporting fundamental, creative research projects to advance our understanding of the natural world,” added Sara Bender of the Gordon and Betty Moore Foundation. “We look forward to learning how the characterization of Ca. Thiomargarita magnifica challenges the current paradigm of what constitutes a bacterial cell and advances microbial research.”
For the team, characterizing Ca. Thiomargarita magnifica has paved the way for multiple new research questions. Among them, is the bacterium’s role in the mangrove ecosystem. “We know that it’s growing and thriving on top of the sediment of mangrove ecosystem in the Caribbean,” Volland said. “In terms of metabolism, it does chemosynthesis, which is a process analogous to photosynthesis for plants.” Another outstanding question is whether the new organelles named pepins played a role in the evolution of the Thiomargarita magnifica extreme size, and whether or not pepins are present in other bacterial species. The precise formation of pepins and how molecular processes within and outside of these structures occur and are regulated also remain to be studied.
Gonzalez-Rizzo and Woyke both see successfully cultivating the bacteria in the lab as a way to get some of the answers. “If we can maintain these bacteria in a laboratory setting, we can use techniques that are not feasible right now,” Woyke said. Gros wants to look at other large bacteria. “You can find some TEM pictures and see what look like pepins so maybe people saw them but did not understand what they were. That will be very interesting to check, if the pepins are already present everywhere.”
Human follicular mites: Ectoparasites becoming symbionts
by Gilbert Smith, Alejandro Manzano Marín, Mariana Reyes-Prieto,et al in Molecular Biology and Evolution
Microscopic mites that live in human pores and mate on our faces at night are becoming such simplified organisms due to their unusual lifestyles that they may soon become one with humans, new research has found.
The mites are passed on during birth and are carried by almost every human, with numbers peaking in adults as the pores grow bigger. They measure around 0.3mm long, are found in the hair follicles on the face and nipples, including the eyelashes, and eat the sebum naturally released by cells in the pores. They become active at night and move between follicles looking to mate.
The first ever genome sequencing study of the D. folliculorum mite found that their isolated existence and resulting inbreeding is causing them to shed unnecessary genes and cells and move towards a transition from external parasites to internal symbionts.
Dr Alejandra Perotti, Associate Professor in Invertebrate Biology at the University of Reading, who co-led the research, said: “We found these mites have a different arrangement of body part genes to other similar species due to them adapting to a sheltered life inside pores. These changes to their DNA have resulted in some unusual body features and behaviours.”
The in-depth study of the Demodex folliculorum DNA revealed: Due to their isolated existence, with no exposure to external threats, no competition to infest hosts and no encounters with other mites with different genes, genetic reduction has caused them to become extremely simple organisms with tiny legs powered by just 3 single cell muscles. They survive with the minimum repertoire of proteins — the lowest number ever seen in this and related species. This gene reduction is the reason for their nocturnal behaviour too. The mites lack UV protection and have lost the gene that causes animals to be awakened by daylight. They have also been left unable to produce melatonin — a compound that makes small invertebrates active at night — however, they are able to fuel their all-night mating sessions using the melatonin secreted by human skin at dusk.
Their unique gene arrangement also results in the mites’ unusual mating habits. Their reproductive organs have moved anteriorly, and males have a penis that protrudes upwards from the front of their body meaning they have to position themselves underneath the female when mating, and copulate as they both cling onto the human hair.
One of their genes has inverted, giving them a particular arrangement of mouth-appendages extra protruding for gathering food. This aids their survival at young age. The mites have many more cells at a young age compared to their adult stage. This counters the previous assumption that parasitic animals reduce their cell numbers early in development. The researchers argue this is the first step towards the mites becoming symbionts. The lack of exposure to potential mates that could add new genes to their offspring may have set the mites on course for an evolutionary dead end, and potential extinction. This has been observed in bacteria living inside cells before, but never in an animal.
Some researchers had assumed the mites do not have an anus and therefore must accumulate all their faeces through their lifetimes before releasing it when they die, causing skin inflammation. The new study, however, confirmed they do have anuses and so have been unfairly blamed for many skin conditions.
An exercise-inducible metabolite that suppresses feeding and obesity
by Veronica L. Li, Yang He, Kévin Contrepois, Hailan Liu, et al in Nature
Researchers at Baylor College of Medicine, Stanford School of Medicine and collaborating institutions report that they have identified a molecule in the blood that is produced during exercise and can effectively reduce food intake and obesity in mice. The findings improve our understanding of the physiological processes that underlie the interplay between exercise and hunger.
“Regular exercise has been proven to help weight loss, regulate appetite and improve the metabolic profile, especially for people who are overweight and obese,” said co-corresponding author Dr. Yong Xu, professor of pediatrics- nutrition and molecular and cellular biology at Baylor. “If we can understand the mechanism by which exercise triggers these benefits, then we are closer to helping many people improve their health.”
“We wanted to understand how exercise works at the molecular level to be able to capture some of its benefits,” said co-corresponding author Jonathan Long, MD, assistant professor of pathology at Stanford Medicine and an Institute Scholar of Stanford ChEM-H (Chemistry, Engineering & Medicine for Human Health). “For example, older or frail people who cannot exercise enough, may one day benefit from taking a medication that can help slow down osteoporosis, heart disease or other conditions.”
Xu, Long and their colleagues conducted comprehensive analyses of blood plasma compounds from mice following intense treadmill running. The most significantly induced molecule was a modified amino acid called Lac-Phe. It is synthesized from lactate (a byproduct of strenuous exercise that is responsible for the burning sensation in muscles) and phenylalanine (an amino acid that is one of the building blocks of proteins).
In mice with diet-induced obesity (fed a high-fat diet), a high dose of Lac-Phe suppressed food intake by about 50% compared to control mice over a period of 12 hours without affecting their movement or energy expenditure. When administered to the mice for 10 days, Lac-Phe reduced cumulative food intake and body weight (owing to loss of body fat) and improved glucose tolerance.
The researchers also identified an enzyme called CNDP2 that is involved in the production of Lac-Phe and showed that mice lacking this enzyme did not lose as much weight on an exercise regime as a control group on the same exercise plan. Interestingly, the team also found robust elevations in plasma Lac-Phe levels following physical activity in racehorses and humans. Data from a human exercise cohort showed that sprint exercise induced the most dramatic increase in plasma Lac-Phe, followed by resistance training and then endurance training. “This suggests that Lac-Phe is an ancient and conserved system that regulates feeding and is associated with physical activity in many animal species,” Long said.
“Our next steps include finding more details about how Lac-Phe mediates its effects in the body, including the brain,” Xu said. “Our goal is to learn to modulate this exercise pathway for therapeutic interventions.”
Deletion of Wt1 during early gonadogenesis leads to differences of sex development in male and female adult mice
by Alejo Torres-Cano, Rosa Portella-Fortuny, Claudia Müller-Sánchez, Sonia Porras-Marfil, Marina Ramiro-Pareta, You-Ying Chau, Manuel Reina, Francesc X. Soriano, Ofelia M. Martínez-Estrada in PLOS Genetics
The deletion of the Wt1 gene during the early stages of the embryonic reproductive organ formation leads to differences in sex development in adult mice, according to an article led by the lecturer Ofelia Martínez-Estrada, from the Faculty of Biology and the Biomedicine Research Institute (IRBio) of the University of Barcelona.
Among the participants in the article are the experts Francesc X. Soriano, from the Department of Cell Biology, Physiology and Immunology, and the Institute of Neurosciences of the UB (UBNeuro), and Manuel Reina, from the same Department and the Research Group Celltec UB.
The Wt1 gene or Wilms tumour gene is expressed during the embryonic development of mammals in many organs and tissues (urogenital system, spleen, heart, diaphragm, etc.). In scientific literature, the mutations of the Wt1 gene are related to some pathologies — syndromes such as Denys-Drash, Frasier and Meacham’s — which include genitourinary defects and differences in the sex development (such as ambiguous genitalia or abnormal development of the gonads). These differences in the sexual development are congenital disorders in which the development of the chromosomal, gonadal or anatomic sex is atypical. Despite the efforts to understand the genetic factors that cause these alterations, the origin is unknown in many cases and it is hard to offer a precise diagnosis to the affected people.
Murine models with modifications in the expression of key genes in the sex development are shaped as decisive elements for studying this complex process in mammals. Therefore, in recent years, new genetic tools have been generated in mutant mice models to study different aspects of the biology of the WT1 gene.
As part of the study, the team presents a new genetically modified mouse model (Wt1KO) which revealed the importance of the Wt1 gene in the initial differentiation of the embryonic gonad at early stages and its impact in the formation of the reproductive system of adult mice. According to the conclusions, female and male Wt1KO mutant mice — unable to express the Wt1 gene in reproductive organs from the early formation stages- showed ambiguous genital tracts and their gonads remained at an undifferenced stage.
“In this study, we state that the Wt1 gene is necessary for activating the pathways that determine the development of the male and female sex, since embryonic mutant gonads do not express the specific genes for each genetic program,” notes lecturer Ofelia Martínez-Estrada, from the Department of Cell Biology, Physiology and Immunology of the UB.
To date, it has been hard to assess the functions of the WT1 transcription factor — coded by the mentioned gene- during the early differentiation of the gonad and its impact on adult sex development. The lack of development in gonads or in the genital tract (gonadal agenesis) and the embryonic lethality shown in Wt1KO mutant mice hindered the progress of research to elucidate the role of this gene in these development processes.
“Based on the obtained results, we propose that this murine model could contribute to improve the knowledge on the functions of the WT1 gene in some progenitor cell populations in different organs and tissues, as well as the importance of these cell populations in the formation of organs in adults,” concludes lecturer Ofelia Martínez-Estrada.
Structure of the mammalian ribosome as it decodes the selenocysteine UGA codon
by Tarek Hilal, Benjamin Y. Killam, Milica Grozdanović, Malgorzata Dobosz-Bartoszek, Justus Loerke, Jörg Bürger, Thorsten Mielke, Paul R. Copeland, Miljan Simonović, Christian M. T. Spahn in Science
A Rutgers scientist is part of an international team that has determined the process for incorporating selenium — an essential trace mineral found in soil, water and some foods that increases antioxidant effects in the body — to 25 specialized proteins, a discovery that could help develop new therapies to treat a multitude of diseases from cancer to diabetes.
The research includes the most in-depth description yet of the process by which selenium gets to where it needs to be in cells, which is crucial for many aspects of cell and organismal biology. First, selenium is encapsulated within selenocysteine (Sec), an essential amino acid. Then, Sec is incorporated into 25 so-called selenoproteins, all of them key to a host of cellular and metabolic processes. Understanding the workings of these vital mechanisms in such a detailed manner is critical to the development of new medical therapies, according to researchers including Paul Copeland, a professor in the Department of Biochemistry and Molecular Biology at Rutgers Robert Wood Johnson Medical School.
“This work revealed structures that had never before been seen, some of which are unique in all of biology,” said Copeland, an author of the study.
Copeland and the team were able to visualize the cell mechanisms by using a specialized cryo-electron microscope, which uses beams of electrons rather than light to form three-dimensional images of complex biological formations at nearly atomic resolution. The process uses frozen samples of molecular complexes and then applies sophisticated image processing — employing today’s vast computing power to string together thousands of images to produce three-dimensional cross-sections and even stop-motion animation conveying a sense of motion within the biomolecules. As a result, scientists can view representations of the intricate structure of proteins and other biomolecules and even how these structures move and change as they function as cellular “machines.”
The incorporation of selenium takes place deep within an individual cell’s intricate machinery. Scientists already knew which proteins and molecules of RNA — a nucleic acid present in all cells involved in the production of proteins — enabled the process. However, they were not able to discern the critical step of how these factors worked in tandem to complete the cycle, dictating the function of the cell’s ribosome — a large macromolecular machine that binds RNA to make more proteins. What they found was that the processes that occur are not like any understood to take place anywhere else in the human body.
“This amino acid gets attached to a unique RNA molecule and that has to be carried to the ribosome via a unique protein factor,” said Copeland, whose lab has spent the past 20 years working to understand how these biomolecules function on a biochemical level. “And all of this evolved in humans specifically to allow selenium to be incorporated into this handful of proteins.”
Once Sec is ensconced in the selenoproteins, the proteins perform a wide range of vital functions necessary for growth and development. They produce nucleotides, the building blocks of DNA. They break down or store fat for energy. They create cell membranes. They produce the thyroid hormone, which controls the human body’s metabolism. And they respond to what is known as oxidative stress by detoxifying chemically reactive byproducts in cells. Diseases and disorders such as cancer, heart disease, male infertility, diabetes and hypothyroidism can arise when the production of selenoproteins is disrupted.
“Understanding the mechanism by which Sec is incorporated is a foundational part of developing new therapies for a multitude of disease states,” Copeland said.
Rapid adaptation of a complex trait during experimental evolution of Mycobacterium tuberculosis
by Madison A Youngblom, Tracy M Smith, John F Kernien, Mohamed A Mohamed, Sydney S Fry, Lindsey L Bohr, Tatum D Mortimer, Mary B O’Neill, Caitlin S Pepperell in eLife
Researchers have identified how the bacterium that causes tuberculosis (TB) can evolve rapidly in response to new environments, says a study.
As with other species of bacteria, Mycobacterium tuberculosis (M. tuberculosis) is able to form complex structures called biofilms which allow bacterial cells to resist stressors such as antibiotics and immune cells. For this study, the research team evolved populations of M. tuberculosis in the lab and found that it could form thick biofilms due to mutations in genetic regions that cause multiple changes to happen at once. These findings could inform the development of antibiotics targeted at biofilm growth. As the second leading cause of death due to infectious disease globally, TB is a major threat to public health and there is an urgent need for new strategies for diagnosing, treating and controlling the infection.
“TB remains a challenging infection to treat due to the bacterium’s ability to persist in the face of antibiotic and immune pressure, and to acquire novel drug resistances,” explains Madison Youngblom, Graduate Student at senior author Caitlin Pepperell’s lab, University of Wisconsin-Madison, US, and co-first author of the study alongside Tracy Smith, New York Genome Center, New York City, US. “To better treat and control TB, we need to understand the sources of the bacterium’s robustness and identify its vulnerabilities. We wanted to learn more about how it is able to form biofilms by discovering the genes and genetic regions involved in biofilm growth, as well as how the bacterium evolves in response to changes in its environment.”
To do this, the team used experimental evolution of M. tuberculosis — a powerful tool for illuminating the strengths and vulnerabilities of the bacterium that has led to important insights on the fundamental processes that guide its adaptation. They evolved six closely related M. tuberculosis strains under selective pressure to grow as a biofilm. At regular intervals, they photographed the biofilm and described its growth according to four criteria: how much liquid surface the biofilm covered, its attachment to and growth up the sides of the dish, how thick the biofilm grew and the continuity of growth (compared to discontinuous patches of growth).
Their work revealed that each strain was able to adapt rapidly to environmental pressure, with the growth of a thicker and therefore more robust biofilm. The genetic regions that mutated during the experiment, causing this biofilm growth, were mostly regulators such as regX3, phoP, embR and Rv2488c. “These regulators control the activity of multiple genes, meaning a single mutation can cause many changes to occur in one go,” Youngblom explains. “This is an efficient process that we observed when we looked at the different characteristics of the bacteria, such as their cell size and growth rate.”
Additionally, the team found evidence suggesting that the genetic background of the parent strain of M. tuberculosis had an impact on the enhanced growth of the biofilms. This means that interactions between genetic factors could play an important role in the adaptation of the M. tuberculosis to changing environments.
“Bacteria are prone to growing as biofilms in many contexts, including the infection of humans and other hosts, and during colonisation of natural and built environments,” says senior author Caitlin Pepperell, Principal Investigator at the University of Wisconsin-Madison. “In a medical context, the insights gained from our work could be used to explore potential new antibiotics that are better able to attack bacteria that grow this way. We imagine such biofilm-directed therapies for TB would likely be add-ons to conventional therapy to help shorten and simplify current treatment strategies.”
A modular 3D printed microfluidic system: a potential solution for continuous cell harvesting in large-scale bioprocessing
by Lin Ding, Sajad Razavi Bazaz, Mahsa Asadniaye Fardjahromi, Flyn McKinnirey, Brian Saputro, Balarka Banerjee, Graham Vesey, Majid Ebrahimi Warkiani in Bioresources and Bioprocessing
Researchers have developed a unique 3D printed system for harvesting stem cells from bioreactors, offering the potential for high quality, wide-scale production of stem cells in Australia at a lower cost.
Stem cells offer great promise in the treatment of many diseases and injuries, from arthritis and diabetes to cancer, due to their ability to replace damaged cells. However, current technology used to harvest stem cells is labour intensive, time consuming and expensive. Biomedical engineer Professor Majid Warkiani from the University of Technology Sydney led the translational research, in collaboration with industry partner Regeneus — an Australian biotechnology company developing stem cell therapies to treat inflammatory conditions and pain.
“Our cutting-edge technology, which uses 3D printing and microfluidics to integrate a number of production steps into one device can help make stem cell therapies more widely available to patients at a lower cost,” said Professor Warkiani.
“While this world-first system is currently at the prototype stage, we are working closely with biotechnology companies to commercialise the technology. Importantly, it is a closed system with no human intervention, which is necessary for current good manufacturing practices,” he said.
Microfluidics is the precise control of fluid at microscopic levels, which can be used to manipulate cells and particles. Advances in 3D printing have allowed for the direct construction of microfluidic equipment, and thus rapid prototyping and building of integrated systems. The new system was developed to process mesenchymal stem cells, a type of adult stem cell that can divide and differentiate into multiple tissue cells including bone, cartilage, muscle, fat, and connective tissue.
Mesenchymal stem cells are initially extracted from human bone marrow, fat tissue or blood. They are then transferred to a bioreactor in the lab and combined with microcarriers to allow the cells to proliferate. The new system combines four micromixers, one spiral microfluidic separator and one microfluidic concentrator to detach and separate the mesenchymal stem cells from microcarriers and concentrate them for downstream processing.
Professor Warkiani said other bioprocessing industrial challenges can also be addressed using the same technology and workflow, helping to reduce costs and increase the quality of a range of life-saving products including stem cells and CAR-T cells.
Clonal evolution and TCR specificity of the human T FH cell response to Plasmodium falciparum CSP
by Ilka Wahl, Anna S. Obraztsova, Julia Puchan, Rebecca Hundsdorfer, Sumana Chakravarty, B. Kim Lee Sim, Stephen L. Hoffman, Peter G. Kremsner, Benjamin Mordmüller, Hedda Wardemann in Science Immunology
Scientists from the German Cancer Research Center (DKFZ) studied the human immune response after immunization with the malaria pathogen Plasmodium falciparum. Their goal was to find out against which protein components the T helper cells induced in this way are directed. To the researchers’ surprise, the T helper cells reacted exclusively to the protein sequence of the vaccine strain and showed hardly any cross-reactivity with the naturally occurring pathogen variants. This could explain why natural infections, to which people in endemic areas are constantly exposed, offer little protection against new diseases with other strains, and why the effect of the vaccination available to date lasts only a short time.
Despite impressive successes in controlling malaria, more than 600,000 people worldwide still die from the tropical disease every year, according to the World Health Organization. The vast majority of fatal cases of malaria are caused by the pathogen Plasmodium falciparum. To date, there is only one approved vaccine against this single-celled organism, and its efficacy, which is already rather low, does not last long.
The vaccine is directed against CSP, the quantitatively dominant protein on the surface of the “sporozoites.” Sporozoites are the stage of the malaria pathogen which is transmitted with the bite of the mosquito and enters human blood. “To improve the vaccine, we need to understand which protective antibodies are induced by the immunization. But the production of such antibodies depends to a large extent on help from the so-called follicular T helper cells,” says Hedda Wardemann of the German Cancer Research Center. “They ensure that B cells transform into antibody-producing plasma cells and memory B cells.”
To study the T helper cell response against CSP in detail, the team led by DKFZ immunologist Wardemann examined the blood of volunteers infected with killed P. falciparum sporozoites from the vaccine strain. The volunteers were of European descent and had no prior contact with malaria pathogens. The researchers analyzed the induced Plasmodium-specific follicular T helper cells at the single cell level. In particular, they focused their investigation on which sequences of CSP are recognized by the receptors of the T helper cells.
The analyses revealed that the T-cell receptors mainly targeted amino acids 311 to 333 of the CSP. But another observation stunned the researchers: there was virtually no cross-reactivity between the individual T-cell clones. “The receptors highly specifically bind only the CSP epitopes of the vaccine strain used. Even deviations of only a single amino acid component were not tolerated in some cases,” Wardemann explains.
The immunologist points out that in the natural population of P. falciparum, sequence polymorphisms occur to a high degree in this region of the CSP. “The specificity of the T-cell clones prevents the constantly recurring natural infections with the pathogen from acting as a natural ‘booster.’ This could possibly explain why the protective effect of the malaria vaccine wears off so quickly,” Wardemann said. The researcher recommends that further development of the vaccine should test whether inducing a broader spectrum of T helper cells could generate longer-lasting immune protection.
Genetic epidemiology of blood type, disease and trait variants, and genome-wide genetic diversity in over 11,000 domestic cats
by Heidi Anderson, Stephen Davison, Katherine M. Lytle, Leena Honkanen, Jamie Freyer, Julia Mathlin, Kaisa Kyöstilä, Laura Inman, Annette Louviere, Rebecca Chodroff Foran, Oliver P. Forman, Hannes Lohi, Jonas Donner in PLOS Genetics
Thirteen genetic variants associated with disease in cats are present in more pedigreed breeds than previously thought, according to the largest ever DNA-based study of domestic cats, led by Heidi Anderson from Wisdom Panel in the United States and colleagues from the University of Helsinki in Finland. However, these variants are declining in frequency in breeds that are regularly screened for the genetic markers.
The researchers genotyped over 11,000 domestic cats, including 90 pedigreed breeds and breed types, and 617 non-pedigreed cats, for 87 genetic variants associated with disease, blood type or physical appearance. They found that there was more genetic diversity in the non-pedigreed cat population than in the pedigreed cat population, and three disease-associated variants were found solely in non-pedigreed cats. They also identified 13 disease-associated variants in 47 breeds for which the disease had not previously been documented. However, the results suggest that the frequency of some markers has declined since they were first identified. For example, PKD1, a variant associated with Polycystic Kidney Disease and previously reported to affect 40% of Persian cats, was identified in none of the 118 Persians in this study but was present in Maine Coons and Scottish Straights. Markers for certain coat colors and patterns, such as Colorpoints in Siamese cats, were also responsible for the same trait in other breeds, and the rarest color variant was the Amber coat color found in Norwegian Forest Cats, which was also detected in one non-pedigreed cat.
Genetic screening for known disease variants and the information on all breeds’ genetic diversity can inform breeders’ decisions, the authors say. In turn, these tools can also help to make well-balanced breeding plans that maintain genetic diversity and avoid breeding affected kittens.
Anderson adds, “This study demonstrates the clinical utility and importance of comprehensive genetic screening of feline variants in supporting domestic cat breeding programs, veterinary care and health research.”
Interpretable modeling of genotype–phenotype landscapes with state-of-the-art predictive power
by P.D. Tonner, A. Pressman, and D. Ross in PNAS
Researchers at the National Institute of Standards and Technology (NIST) have developed a new statistical tool that they have used to predict protein function. Not only could it help with the difficult job of altering proteins in practically useful ways, but it also works by methods that are fully interpretable — an advantage over the conventional artificial intelligence (AI) that has aided with protein engineering in the past.
The new tool, called LANTERN, could prove useful in work ranging from producing biofuels to improving crops to developing new disease treatments. Proteins, as building blocks of biology, are a key element in all these tasks. But while it is comparatively easy to make changes to the strand of DNA that serves as the blueprint for a given protein, it remains challenging to determine which specific base pairs — rungs on the DNA ladder — are the keys to producing a desired effect. Finding these keys has been the purview of AI built of deep neural networks (DNNs), which, though effective, are notoriously opaque to human understanding.
Described in a new paper, LANTERN shows the ability to predict the genetic edits needed to create useful differences in three different proteins. One is the spike-shaped protein from the surface of the SARS-CoV-2 virus that causes COVID-19; understanding how changes in the DNA can alter this spike protein might help epidemiologists predict the future of the pandemic. The other two are well-known lab workhorses: the LacI protein from the E. coli bacterium and the green fluorescent protein (GFP) used as a marker in biology experiments. Selecting these three subjects allowed the NIST team to show not only that their tool works, but also that its results are interpretable — an important characteristic for industry, which needs predictive methods that help with understanding of the underlying system.
“We have an approach that is fully interpretable and that also has no loss in predictive power,” said Peter Tonner, a statistician and computational biologist at NIST and LANTERN’s main developer. “There’s a widespread assumption that if you want one of those things you can’t have the other. We’ve shown that sometimes, you can have both.”
The problem the NIST team is tackling might be imagined as interacting with a complex machine that sports a vast control panel filled with thousands of unlabeled switches: The device is a gene, a strand of DNA that encodes a protein; the switches are base pairs on the strand. The switches all affect the device’s output somehow. If your job is to make the machine work differently in a specific way, which switches should you flip? Because the answer might require changes to multiple base pairs, scientists have to flip some combination of them, measure the result, then choose a new combination and measure again. The number of permutations is daunting.
“The number of potential combinations can be greater than the number of atoms in the universe,” Tonner said. “You could never measure all the possibilities. It’s a ridiculously large number.”
Because of the sheer quantity of data involved, DNNs have been tasked with sorting through a sampling of data and predicting which base pairs need to be flipped. At this, they have proved successful — as long as you don’t ask for an explanation of how they get their answers. They are often described as “black boxes” because their inner workings are inscrutable.
“It is really difficult to understand how DNNs make their predictions,” said NIST physicist David Ross, one of the paper’s co-authors. “And that’s a big problem if you want to use those predictions to engineer something new.”
LANTERN, on the other hand, is explicitly designed to be understandable. Part of its explainability stems from its use of interpretable parameters to represent the data it analyzes. Rather than allowing the number of these parameters to grow extraordinarily large and often inscrutable, as is the case with DNNs, each parameter in LANTERN’s calculations has a purpose that is meant to be intuitive, helping users understand what these parameters mean and how they influence LANTERN’s predictions.
The LANTERN model represents protein mutations using vectors, widely used mathematical tools often portrayed visually as arrows. Each arrow has two properties: Its direction implies the effect of the mutation, while its length represents how strong that effect is. When two proteins have vectors that point in the same direction, LANTERN indicates that the proteins have similar function. These vectors’ directions often map onto biological mechanisms. For example, LANTERN learned a direction associated with protein folding in all three of the datasets the team studied. (Folding plays a critical role in how a protein functions, so identifying this factor across datasets was an indication that the model functions as intended.) When making predictions, LANTERN just adds these vectors together — a method that users can trace when examining its predictions.
Other labs had already used DNNs to make predictions about what switch-flips would make useful changes to the three subject proteins, so the NIST team decided to pit LANTERN against the DNNs’ results. The new approach was not merely good enough; according to the team, it achieves a new state of the art in predictive accuracy for this type of problem.
“LANTERN equaled or outperformed nearly all alternative approaches with respect to prediction accuracy,” Tonner said. “It outperforms all other approaches in predicting changes to LacI, and it has comparable predictive accuracy for GFP for all except one. For SARS-CoV-2, it has higher predictive accuracy than all alternatives other than one type of DNN, which matched LANTERN’s accuracy but didn’t beat it.”
LANTERN figures out which sets of switches have the largest effect on a given attribute of the protein — its folding stability, for example — and summarizes how the user can tweak that attribute to achieve a desired effect. In a way, LANTERN transmutes the many switches on our machine’s panel into a few simple dials.
“It reduces thousands of switches to maybe five little dials you can turn,” Ross said. “It tells you the first dial will have a big effect, the second will have a different effect but smaller, the third even smaller, and so on. So as an engineer it tells me I can focus on the first and second dial to get the outcome I need. LANTERN lays all this out for me, and it’s incredibly helpful.”
Rajmonda Caceres, a scientist at MIT’s Lincoln Laboratory who is familiar with the method behind LANTERN, said she values the tool’s interpretability.
“There are not a lot of AI methods applied to biology applications where they explicitly design for interpretability,” said Caceres, who is not affiliated with the NIST study. “When biologists see the results, they can see what mutation is contributing to the change in the protein. This level of interpretation allows for more interdisciplinary research, because biologists can understand how the algorithm is learning and they can generate further insights about the biological system under study.”
Tonner said that while he is pleased with the results, LANTERN is not a panacea for AI’s explainability problem. Exploring alternatives to DNNs more widely would benefit the entire effort to create explainable, trustworthy AI, he said.
“In the context of predicting genetic effects on protein function, LANTERN is the first example of something that rivals DNNs in predictive power while still being fully interpretable,” Tonner said. “It provides a specific solution to a specific problem. We hope that it might apply to others, and that this work inspires the development of new interpretable approaches. We don’t want predictive AI to remain a black box.”
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