TL;DR
- Nuclear pore complexes (NPCs) are massive multi-protein complexes that act as passageways for the transport of molecules into and out of the nucleus. NPC defects are linked to many diseases such as viral infections, cancers and certain neurodegenerative diseases. Using rapid plunge freezing and cryo-EM (electron microscopy) with computational methods, researchers have produced a comprehensive model of the yeast NPC which reveals the interconnected architecture of its core scaffold. Findings provide an in-depth view of how the nucleus and cell body communicate.
- Researchers generated mice with a specific DNA sequence inverted to determine if orientation affects expression of a gene called H19. Expression can also be impacted if the surrounding DNA is altered by a process called methylation. Interestingly, methylation was only relevant when the inverted sequence was inherited from the father. When inherited from the mother, the inversion had the opposite effect on H19 expression, suggesting a more complex mechanism is at play.
- DNAzymes are precision biocatalysts that destroy unwanted RNA molecules. However, major obstacles to their use in medicine remain. Scientists have investigated with atomic resolution how DNAzymes work in real time.
- A new study shows how cell membranes curve to create the ‘mouths’ that allow the cells to consume things that surround them. Study solves a 40-year-old problem in cell biology.
- Researchers have developed a method that allows resting human immune cells to be genetically analyzed in detail for the first time.
- The secret to producing large batches of stem cells more efficiently may lie in the near-zero gravity conditions of space. Scientists have found that microgravity has the potential to contribute to life-saving advances on Earth by facilitating the rapid mass production of stem cells.
- A tiny region at the root tip has been found to be responsible for orchestrating the growth and development of the complex network of vascular tissues that transport sugars through plant roots.
- Plants, like other organisms, can be severely affected by heat stress. To increase their chances of survival, they activate the heat shock response, a molecular pathway also employed by human and animal cells for stress protection. Researchers have now discovered that plant steroid hormones can promote this response in plants.
- Genome sequencing is now much cheaper than it was, but still accounts for a large part of the costs in animal and plant breeding. One trick to reduce these costs is to sequence only a very small and randomly selected part of the genome and to complete the remaining gaps using mathematical and statistical techniques. Researchers have developed a new approach to do this.
- Prion diseases, such as Creutzfeldt-Jakob Disease (CJD), are fast-moving, fatal dementia syndromes associated with the formation of aggregates of the prion protein, PrP. How these aggregates form within and kill brain cells has never been fully understood, but a new study suggests that the aggregates kill neurons by damaging their axons, the narrow nerve fibers through which they send signals to other neurons.
- 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
Comprehensive structure and functional adaptations of the yeast nuclear pore complex
by Christopher W. Akey, Digvijay Singh, Christna Ouch, Ignacia Echeverria, Ilona Nudelman, Joseph M. Varberg, Zulin Yu, Fei Fang, Yi Shi, Junjie Wang, Daniel Salzberg, Kangkang Song, Chen Xu, James C. Gumbart, Sergey Suslov, Jay Unruh, Sue L. Jaspersen, Brian T. Chait, Andrej Sali, Javier Fernandez-Martinez, Steven J. Ludtke, Elizabeth Villa, Michael P. Rout in Cell
Nuclear pore complexes (NPCs) are massive multi-protein complexes that act as passageways for the transport of molecules into and out of the nucleus. Given their central role in gene expression, growth and development, it is not surprising that NPC defects are linked to many diseases such as viral infections, cancers and certain neurodegenerative diseases, and that nuclear transport is a target for possible therapeutics.
Using rapid plunge freezing and cryo-EM (electron microscopy) with computational methods, researchers from Boston University School of Medicine (BUSM) have produced a comprehensive model of the yeast NPC which reveals the interconnected architecture of its core scaffold. This work provides molecular models for two configurations: one that is easier to study in isolated samples to provide a more detailed overview of a radially-compact form and a second expanded form in the living yeast cell, albeit this “in situ” form is currently visualized with a lower level of detail.
“This research significantly extends our understanding of the architecture of the NPC from brewers’ yeast, a model organism that is used to study the biology of cells that contain a nucleus and thus provides new insights on multiple levels into the functions of this transport machine,” explains corresponding author Christopher W. Akey, PhD, professor of physiology and biophysics at BUSM.
According to researchers, this model will provide a better understanding of how these large mega-channels assemble and how they can flex and adapt to changes in transport by expansion of their central passageway.
“Moreover, we have observed multiple types of NPC in the same cell for the first time, which reflects the lego-like ability of this assembly to use interchangeable parts to modify its architecture on the nuclear side. This adaptability may play a role in tailoring the functions of these machines for different local environments at the periphery of the nucleus,” says Akey.
The researchers believe that these findings now set the table for studies of how viruses may usurp this important pathway to infect cells and alter their physiology to cause disease.
Orientation of mouse H19 ICR affects imprinted H19 gene expression through promoter methylation-dependent and -independent mechanisms
by Hitomi Matsuzaki, Yu Miyajima, Akiyoshi Fukamizu, Keiji Tanimoto in Communications Biology
Mammalian offspring inherit two versions, or alleles, of each gene with one allele from each biological parent. However, gene expression is tightly regulated and certain genes undergo the phenomenon of “genomic imprinting,” which is where only the allele received by the male or the female parent is expressed. Imprinted genes play diverse roles in development and disruption of their mono-allelic expression can cause diseases, thus understanding the mechanisms behind their regulation is critical. In a recent article, a team led by researchers at the University of Tsukuba examined genomic imprinting of a specific genetic locus in mice. Their experiments helped reveal the molecular details of how this mechanism governs expression levels of these genes.
The team focused on the H19 gene locus, which was previously shown to be controlled by the H19 imprinted control region (ICR) via genomic imprinting. The paternal H19 ICR is modified via DNA methylation while the maternal H19 ICR allele isn’t methylated. Methylation of the H19 ICR is in part responsible for repressing the expression of H19. However, H19 itself can also be methylated, and the effects of this had yet to be clarified.
“While the general imprinting mechanism for the mouse H19 locus is well established, it is less clear how expression of H19 is affected by its own methylation status,” explains Assistant Professor Hitomi Matsuzaki, lead author of the study. “Our previous finding suggested that the methylation state of the H19 ICR is transferred directionally downstream to H19 in the fertilized embryo post-implantationwhich makes it difficult to study the two in isolation.”
Then, the team hypothesized that by inverting the H19 ICR, thus reversing its direction, they could reduce H19 methylation and they created mutant mice to test this. Interestingly, with paternally inherited inverted ICR, H19 had decreased levels of methylation and as a result was derepressed. However, when the same experiments were conducted for the maternally inherited inverted ICR, H19 expression levels were lower compared with the un-inverted ICR allele, despite having low methylation.
“Our findings involving the maternally inherited allele were quite unexpected, especially given the paternal data,” describes Assistant Professor Matsuzaki. “We did observe slightly more ICR methylation in the inverted allele compared with the wild type one.”
Further work did not provide evidence that ICR methylation status was responsible for the differential H19 expression in the maternally inherited alleles. Collectively, these data suggest that, for maternal inheritance, H19 expression is in fact affected by the ICR orientation, but it is independent of DNA methylation.
Overall, Assistant Professor Matsuzaki and colleagues provided compelling insights into the complex nature of genomic imprinting in mice. The methylation status and direction of certain DNA sequences can affect genes found at the locus in different manners, and the effects also vary based on which parent the allele was inherited from. These results shed new light on the current knowledge and raise intriguing questions to be addressed by further studies.
Time-resolved structural analysis of an RNA-cleaving DNA catalyst
by Jan Borggräfe, Julian Victor, Hannah Rosenbach, Aldino Viegas, Christoph G. W. Gertzen, Christine Wuebben, Helena Kovacs, Mohanraj Gopalswamy, Detlev Riesner, Gerhard Steger, Olav Schiemann, Holger Gohlke, Ingrid Span, Manuel Etzkorn in Nature
DNAzymes are precision biocatalysts that destroy unwanted RNA molecules. However, major obstacles to their use in medicine remain. Together with Jülich Research Centre (FZJ) and the University of Bonn, a research team from Heinrich Heine University Düsseldorf (HHU) has investigated with atomic resolution how DNAzymes work in real time.
DNAzymes — a word made up of DNA and enzyme — are catalytically active DNA sequences. They comprise a catalytic core comprising around 15 nucleic acids flanked by short binding arms on the right- and left-hand sides, each with around ten nucleic acids. While the sequence of the core is fixed, the binding arms can be modified specifically match virtually any RNA target sequence.
The aim is to target unwanted RNA molecules of viruses, cancer or damaged nerve cells, using DNAzymes to attack and destroy them. This is achieved via binding sequences that match a sequence of nucleotides on the targeted RNA molecule. The DNAzyme docks precisely to the matching position and the core cleaves the RNA molecule, the fragments of which are then quickly degraded in the cell. The binding arms can be exchanged quickly and easily.
The therapeutic benefits are obvious: Unwanted RNA can be destroyed precisely, while other, useful RNA strands in a cell remain untouched. In some viruses like SARS-CoV2 and Ebola, the genetic material is coded on an RNA molecule. Like healthy cells, cancer cells use so-called messenger RNA (mRNA) to copy the blueprints for proteins from their DNA and transfer them to the molecule factories. The mRNA sequence in cancer cells is often slightly different to that of healthy cells or present in different amounts, meaning that DNAzymes can specifically attack cancer cells while sparing others.
“What sounds outstanding in theory and was already proposed 20 years ago, unfortunately doesn’t work like that in medical practice,” says Dr Manuel Etzkorn, working group leader at the HHU Institute of Physical Biology and last author of the study, which has now been published in Nature. “In a test tube, the DNAzymes are highly effective at destroying the RNA molecules, but this rarely happens in a cell. There must be a competing process that blocks the DNAzymes. However, without a fundamental understanding of how they function, it is very difficult to develop improved DNAzyme variants that can accomplish their work in cells. Our insights have now brought movement into this deadlocked situation.”
In their study, the authors from HHU and a team from Jülich Research Centre (FZJ), the University of Bonn and a Swiss company sought to understand how the system as a whole functions dynamically, what steps occur in the binding and cleaving process and what cofactors support the reaction.
The researchers observed the processes at atomic resolution and in part in real time using high-resolution nuclear magnetic resonance (NMR) spectroscopy. This enabled them to depict the three-dimensional atomic arrangement assumed by the DNAzyme to bind to and cleave the RNA: The core wraps around the RNA strand in a highly effective way, cleaving it into two pieces in several intermediate steps. After cleaving, the DNAzyme releases the fragments and can bind again elsewhere.
Professor Dr Holger Gohlke from the HHU Chair of Pharmaceutical and Medicinal Chemistry and the Institute of Bio- and Geosciences at FZJ, whose team conducted molecular dynamics simulations on the DNAzyme/RNA complex, adds: “In the best sense of integrative modelling, we were able to put forward a plausible RNA cleaving mechanism at atomic level and supply information on RNA base preference at the cleavage site.”
Jan Borggräfe, doctoral researcher in Etzkorn’s working group and lead author of the study, explains why the DNAzymes do not work well in cells: “We established that magnesium, as a key cofactor, plays various essential roles in the mechanism, but that it binds relatively poorly and only briefly to the DNAzyme. There are other components in the cell with a greater affinity for magnesium that “steal” the magnesium from the DNAzyme so to speak.”
The next step is to conduct structural investigations into cell cultures and organoids. The goal for therapeutic applications is to improve the magnesium affinity of the DNAzymes through targeted modifications in order to increase their activity in biological tissue.
Biomanufacturing in low Earth orbit for regenerative medicine
Arun Sharma, Rachel A. Clemens, Orquidea Garcia, D. Lansing Taylor, Nicole L. Wagner, Kelly A. Shepard, Anjali Gupta, Siobhan Malany, Alan J. Grodzinsky, Mary Kearns-Jonker, Devin B. Mair, Deok-Ho Kim, Michael S. Roberts, Jeanne F. Loring, Jianying Hu, Lara E. Warren, Sven Eenmaa, Joe Bozada, Eric Paljug, Mark Roth, Donald P. Taylor, Gary Rodrigue, Patrick Cantini, Amelia W. Smith, Marc A. Giulianotti, William R. Wagner in Stem Cell Reports
The secret to producing large batches of stem cells more efficiently may lie in the near-zero gravity conditions of space. Scientists at Cedars-Sinai have found that microgravity has the potential to contribute to life-saving advances on Earth by facilitating the rapid mass production of stem cells.
A new paper, led by Cedars Sinai, highlights key opportunities discussed during the 2020 Biomanufacturing in Space Symposium to expand the manufacture of stem cells in space.
Biomanufacturing — a type of stem cell production that uses biological materials such as microbes to produce substances and biomaterials suitable for use in preclinical, clinical, and therapeutic applications — can be more productive in microgravity conditions.
“We are finding that spaceflight and microgravity is a desirable place for biomanufacturing because it confers a number of very special properties to biological tissues and biological processes that can help mass produce cells or other products in a way that you wouldn’t be able to do on Earth,” said stem cell biologist Arun Sharma, PhD, research scientist and head of a new research laboratory in the Cedars-Sinai Board of Governors Regenerative Medicine Institute, Smidt Heart Institute and Department of Biomedical Sciences.
“The last two decades have seen remarkable advances in regenerative medicine and exponential advancement in space technologies enabling new opportunities to access and commercialize space,” he said.
Attendees at the virtual space symposium in December identified more than 50 potential commercial opportunities for conducting biomanufacturing work in space, according to the Cedars-Sinai paper. The most promising fell into three categories: disease modeling, biofabrication, and stem-cell-derived products.
The first, disease modeling, is used by scientists to study diseases and possible treatments by replicating full-function structures — whether using stem cells, organoids (miniature 3D structures grown from human stem cells that resemble human tissue), or other tissues.
Investigators have found that once the body is exposed to low-gravity conditions for extended periods of time, it experiences accelerated bone loss and aging. By developing disease models based on this accelerated aging process, research scientists can better understand the mechanisms of the aging process and disease progression.
“Not only can this work help astronauts, but it can also lead to us manufacturing bone constructs or skeletal muscle constructs that could be applied to diseases like osteoporosis and other forms of accelerated bone aging and muscle wasting that people experience on Earth,” said Sharma, who is the corresponding author of the paper.
Another highly discussed topic at the symposium was biofabrication, which uses manufacturing processes to produce materials like tissues and organs. 3D printing is one of the core biofabrication technologies.
A major issue with producing these materials on Earth involves gravity-induced density, which makes it hard for cells to expand and grow. With the absence of gravity and density in space, scientists are hopeful that they can use 3D printing to print unique shapes and products, like organoids or cardiac tissues, in a way that can’t be replicated on Earth.
The third category has to do with the production of stem cells and understanding how some of their fundamental properties are influenced by microgravity. Some of these properties include potency, or the ability of a stem cell to renew itself, and differentiation, the ability for stem cells to turn into other cell types.
Understanding some of the effects of spaceflight on stem cells can potentially lead to better ways to manufacture large numbers of cells in the absence of gravity. Scientists from Cedars-Sinai will be sending stem cells into space early next year, in conjunction with NASA and a private contractor, Space Tango, to test whether it is possible to produce large batches in a low gravity environment.
“While we are still in the exploratory phase of some of this research, this is no longer in the realm of science fiction,” Sharma said. “Within the next five years we may see a scenario where we find cells or tissues that can be made in a way that is simply not possible here on Earth. And I think that’s extremely exciting.”
Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization
by Pawel Roszak, Jung-ok Heo, Bernhard Blob, Koichi Toyokura, Yuki Sugiyama, Maria Angels de Luis Balaguer, Winnie W. Y. Lau, Fiona Hamey, Jacopo Cirrone, Ewelina Madej, Alida M. Bouatta, Xin Wang, Marjorie Guichard, Robertas Ursache, Hugo Tavares, Kevin Verstaen, Jos Wendrich, Charles W. Melnyk, Yoshihisa Oda, Dennis Shasha, Sebastian E. Ahnert, Yvan Saeys, Bert De Rybel, Renze Heidstra, Ben Scheres, Guido Grossmann, Ari Pekka Mähönen, Philipp Denninger, Berthold Göttgens, Rosangela Sozzani, Kenneth D. Birnbaum, Yrjö Helariutta in Science
A tiny region at the root tip has been found to be responsible for orchestrating the growth and development of the complex network of vascular tissues that transport sugars through plant roots.
In a paper, an international team of scientists present a detailed blueprint of how plants construct phloem cells — the tissue responsible for transporting and accumulating sugars and starch in the parts of the plant that we harvest (seeds, fruits and storage tubers) to feed much of the world. This pivotal research reveals how global signals in root meristems coordinate distinct maturation phases of the phloem tissue.
Phloem is a highly specialised vascular tissue that forms an interconnected network of continuous strands throughout a plant’s body. It transports sugars, nutrients and a range of signalling molecules between leaves, roots, flowers and fruits. As a result, phloem is central to plant function. Understanding how the phloem network is initiated and develops is important for future applications in agriculture, forestry and biotechnology as it could reveal how to better transport this sugar energy to where it is needed.
Plant roots continue to grow throughout a plant’s life. This phenomenon, known as indeterminate growth, means roots continually elongate as they add new tissues to the tip of the root — like constructing a never-ending highway. A continuous file of specialised phloem cells running the length of roots (analogous to a lane on a highway) delivers the primary nutrient, sucrose, to the parts of the plant where it is needed for growth. To fulfil this vital role, phloem tissue must develop and mature rapidly so it can supply sugars to surrounding tissues — akin to building a service lane that needs to be completed in the first stage of constructing a multi-lane highway.
The problem that has long puzzled plant scientists is how a single instructive gradient of proteins are able to stage the construction phases across all the different specialised cell files (highway lanes) that are present in roots. How does one cell type read the same gradient as its neighbours, but interprets it differently to stage its own specialised development is a question that plant scientists have been working to resolve.
Over the past 15 years, researchers in Yrjö Helariutta’s teams at the University of Cambridge and University of Helsinkihave uncovered the central role of cell-to-cell communication and complex feedback-mechanisms involved in vascular patterning. This new research, undertaken with collaborators at New York University and North Carolina State University, reveals how this single lane of phloem cells is constructed independently of surrounding cells.
The Sainsbury/Helsinki group dissected each step in the construction of the phloem cell file (the sugar transport lane) in the model plant Arabidopsis thaliana using single-cell RNA-seq and live imaging. Their work showed how the proteins that control the broad maturation gradient of the root interact with the genetic machinery that specifically controls phloem development.
This is one mechanism that appears to help the phloem cell file to fast-track maturation using its own machinery to interpret the maturation cues. Dr Pawel Roszak, co-first-author of the study and researcher at the Sainsbury Laboratory Cambridge University (SLCU), explains: “We have shown how global signals in the root meristem interact with the cell type specific factors to determine distinct phases of phloem development at the cellular resolution. Using cell sorting followed by deep, high-resolution single-cell sequencing of the underlying gene regulatory network revealed a “seesaw” mechanism of reciprocal genetic repression that triggers rapid developmental transitions.”
The group also showed how phloem development is staged over time, with early genetic programs inhibiting late genetic programs and vice versa — just as the road asphalt-laying work crews’ hand over construction to lane painters in the latter stages of highway construction. In addition, they showed how early phloem regulators instructed specific genes to split the phloem cells into two different subtypes — like the construction of a fork in the road leading to two separate destinations.
Co-leader of the work, Professor Yrjö Helariutta, said his teams’ reconstruction of the steps from birth to terminal differentiation of protophloem in the Arabidopsis root exposed the steps.
Helariutta said: “Broad maturation gradients interfacing with cell-type specific transcriptional regulators to stage cellular differentiation is required for phloem development.”
“By combining single-cell transcriptomics with live imaging, here we have mapped the cellular events from the birth of the phloem cell to its terminal differentiation into phloem sieve element cells. This allowed us to uncover genetic mechanisms that coordinate cellular maturation and connect the timing of the genetic cascade to broadly expressed master regulators of meristem maturation. The precise timing of developmental mechanisms was critical for proper phloem development, with apparent “fail safe” mechanisms to ensure transitions.”
The researchers plan to further explore the evolution of these mechanisms and whether these steps are replicated in other regions of plants and other plant species.
Transcription factor BES1 interacts with HSFA1 to promote heat stress resistance of plants
by Pablo Albertos, Gönül Dündar, Philipp Schenk, Sergio Carrera, Philipp Cavelius, Tobias Sieberer, Brigitte Poppenberger in The EMBO Journal
It may be hard to remember in winter, but July 2021 was the hottest month ever documented. In the USA, the mean temperature was higher than the average for July by 2,6 degrees Fahrenheit, and many southern European countries saw temperatures above 45 degrees Celsius including an all-time high temperature of 48,8 degrees Celsius recorded on the eastern coast of Sicily in Italy.
The past few decades have seen an increased incidence of heat waves with record highs around the globe, and this is seen as a result of climate change. Heat waves have been occurring more frequently, have been hotter, and have been lasting longer with severe consequences not only for humans and animals but also for plants. “Heat stress can negatively affect plants in their natural habitats and destabilize ecosystems while also drastically reducing crop harvests, thereby threatening our food security,” says Brigitte Poppenberger, Professor for Biotechnology of Horticultural Crops.
To survive short periods of heat stress, plants activate a molecular pathway called the heat-shock response. This heat-shock response (common to all organisms) protects cells from damage inflicted by proteotoxic stress, which damages proteins. Such stress is not only caused by heat but can also result from exposure to certain toxins, UV light, or soil salinity.
The heat shock response protects cells in various ways, one of them being production of so-called heat-shock proteins, which serve as molecular shields that protect proteins by preventing misfolding.
Plants respond to heat stress by activating heat shock factors and also other molecular players. In particular, hormones as chemical messengers are involved. Among the hormones that plants produce are the brassinosteroids, which primarily regulate their growth and developments. But, in addition to their growth-promoting properties, brassinosteroids have other interesting abilities, one of them being their ability to increase the heat stress resistance of plants, and researchers at TUM have recently discovered what contributes to this protective ability.
Using the model plant Arabidopsis thaliana, a research group led by Prof. Brigitte Poppenberger has been able to elucidate how a specific transcription factor — a special protein responsible for switching certain sections of the DNA on or off — is regulated by brassinosteroids. This transcription factor, called BES1, can interact with heat shock factors thereby allowing genetic information to be targeted towards increased synthesis of heat shock proteins. When BES1 activity is increased, plants become more resistant to heat stress, and when it is decreased, they become more sensitive to it. Furthermore, the group has demonstrated that BES1 is activated by heat stress and that this activation is stimulated by brassinosteroids.
“These results are not only of interest to biologists trying to expand our understanding of the heat shock response but also have potential for practical application in agriculture and horticulture,” says Prof. Poppenberger.
Bio-stimulants containing brassinosteroids are available and can be tested for their ability to increase heat stress resistance in plants. Such substances are natural products that are approved for organic farming and thus could be used without problems. Alternatively, BES1 may be an interesting target for breeding approaches. This could be used to create varieties that are more resistant to heat stress and thus provide more stable yields in the event of future heat waves.
Increasing calling accuracy, coverage, and read-depth in sequence data by the use of haplotype blocks
by Torsten Pook, Adnane Nemri, Eric Gerardo Gonzalez Segovia, Daniel Valle Torres, Henner Simianer, Chris-Carolin Schoen in PLOS Genetics
The use of genetic information is now indispensable for modern plant breeding. Even though DNA sequencing has become much cheaper since the human genome was decoded for the very first time in 2003, collecting the full genetic information still accounts for a large part of the costs in animal and plant breeding. One trick to reduce these costs is to sequence only a very small and randomly selected part of the genome and to complete the remaining gaps using mathematical and statistical techniques. An interdisciplinary research team from the University of Göttingen has developed a new methodological approach for this.
“The core idea of the method is to recognise ‘haplotype blocks’, by which we mean longer sections in the genome that are very similar in different plants due to inheritance, and to use this mosaic structure for compiling the rest of the information,” says Dr Torsten Pook from the Center for Integrated Breeding Research at Göttingen University. “In breeding populations, the sequences completed using this new method have quality comparable to collecting a hundred times as much information from the DNA strand.”
The researchers’ goal is to breed maize plants with low susceptibility to frost and drought damage as part of the MAZE project. KWS Saat SE, a partner in the project, is already using the method in breeding programmes because of its cost efficiency. “Another advantage is that the method not only allows us to detect differences in individual nucleotides in the DNA strand, but also to recognise structural differences that have so far been practically unusable for breeding purposes,” says Pook.
As things stand, however, the method can currently only be used efficiently for inbred lines in plant breeding. A follow-up study to extend the method to organisms with a regular double set of chromosomes is already planned. This would mean their new method could be used for most vertebrates, including humans.
Endosomal sorting drives the formation of axonal prion protein endoggresomes
by Romain Chassefeyre, Tai Chaiamarit, Adriaan Verhelle, Sammy Weiser Novak, Leonardo R. Andrade, André D. G. Leitão, Uri Manor, Sandra E. Encalada in Science Advances
Prion diseases, such as Creutzfeldt-Jakob Disease (CJD), are fast-moving, fatal dementia syndromes associated with the formation of aggregates of the prion protein, PrP. How these aggregates form within and kill brain cells has never been fully understood, but a new study from scientists at Scripps Research suggests that the aggregates kill neurons by damaging their axons, the narrow nerve fibers through which they send signals to other neurons.
The accumulation of protein aggregates in axons, along with axonal swellings and other signs of dysfunction, are also early features of other neurodegenerative disorders including Alzheimer’s and Parkinson’s diseases. The discovery of how these prion aggregates form in axons and how to inhibit them, may ultimately have a significance that goes far beyond prion diseases.
“We’re hopeful that these findings will lead to a better understanding of prion and other neurodegenerative diseases, as well as new strategies for treating them,” says study senior author Sandra Encalada, PhD, Arlene and Arnold Goldstein Associate Professor in the Department of Molecular Medicine at Scripps Research.
The researchers in their study closely observed mutant, disease-causing copies of the prion-disease protein PrP forming large aggregates in the axons of neurons, but not in the neurons’ main cell bodies. The formation of these aggregates was followed by signs of axon dysfunction and ultimately neuronal death. The scientists found evidence that neurons’ waste-disposal processes normally are able to cope with such aggregates when they are within or close to neurons’ main cell bodies, but are much less able to do so when the aggregates accumulate far out within axons.
The researchers also identified a complex of key proteins as being responsible for steering PrP into axons and causing aggregation associated with large axonal swellings. They demonstrated that by silencing any one of these proteins they could inhibit the aggregates from forming and protect the neurons from damage and death.
CJD is the most common human prion disease, occurring at the rate of about one case per million people per year worldwide. Most cases are thought to arise spontaneously when PrP somehow is altered in the brain and starts aggregating. Because these aggregates grow by a chain-reaction process that draws in healthy copies of PrP, they can transmit CJD in rare cases — for example, during corneal transplant surgery — from one person to another. About 15 percent of cases are hereditary, caused by mutations that make PrP more likely to aggregate. Prion disorders occur in other mammals and are thought to be due to similar toxic aggregations of different species’ PrP proteins.
In the study, Encalada’s team used mouse brain cells containing mutant PrP, along with microscopic motion-picture techniques, to study the initial accumulation of PrP aggregates in axons. A neuron’s axon is often very long in relation to its main body — the soma — and has been found to be uniquely vulnerable to disruptions of its delicate systems for transporting essential molecules and getting rid of waste.
PrP’s ordinary function in neurons has never been clear, but the protein appears to be normally secreted, via sac-like containers called vesicles, from the soma and the axon, where it sometimes returns to be recycled or degraded as waste. The researchers found in their experiments that mutant PrP produced in the soma is also largely encapsulated in vesicles that get moved into the axon along railways called microtubules.
This movement involves a somewhat complex vesicle trafficking system, and the researchers observed that this system shunts much of the PrP far out into axons, where PrP-containing vesicles gather and merge. Mutant PrP in this situation forms large aggregates — Encalada calls them endoggresomes — that axons can’t get rid of. The aggregates lead to axonal swellings, and other signs of dysfunction including reduced neuronal calcium signaling, and ultimately a much faster neuronal death rate compared to neurons with normal PrP.
The researchers also found a way of countering endoggresomes formation. They identified four proteins, Arl8, kinesin-1, Vps41, and SKIP, that are responsible for directing PrP-containing vesicles into axons, carrying them far out into the soma, and merging them with other PrP-containing vesicles to trigger aggregate formation. When they silenced any of these proteins, far fewer PrP-containing vesicles entered axons, the axons showed few or no signs of aggregation, and the neurons functioned normally or almost normally and survived just as well as normal brain cells.
The results point to the tantalizing possibility that prion diseases, and perhaps many other protein-aggregate diseases of the brain, can be prevented or treated by interrupting at least transiently the trafficking process that brings vesicle-encapsulated, aggregate-prone proteins out into axons.
“We’re very enthusiastic about discovering molecules that can inhibit this aggregate-forming pathway and studying the effects of such inhibitors in animal models of prion and other neurodegenerative diseases,” Encalada says.
De novo endocytic clathrin coats develop curvature at early stages of their formation
by Nathan M. Willy, Joshua P. Ferguson, Ata Akatay, Scott Huber, Umidahan Djakbarova, Salih Silahli, Cemal Cakez, Farah Hasan, Henry C. Chang, Alex Travesset, Siyu Li, Roya Zandi, Dong Li, Eric Betzig, Emanuele Cocucci, Comert Kural in Developmental Cell
A new study shows how cell membranes curve to create the “mouths” that allow the cells to consume things that surround them.
“Just like our eating habits basically shape anything in our body, the way cells ‘eat’ matters for the health of the cells,” said Comert Kural, associate professor of physics at The Ohio State University and lead author of the study. “And scientists did not, until now, understand the mechanics of how that happened.”
The study found that the intercellular machinery of a cell assembles into a highly curved basket-like structure that eventually grows into a closed cage. Scientists had previously believed that structure began as a flat lattice. Membrane curvature is important, Kural said: It controls the formation of the pockets that carry substances into and out of a cell.
The pockets capture substances around the cell, forming around the extracellular substances, before turning into vesicles — small sacs one-one millionth the size of a red blood cell. Vesicles carry important things for a cell’s health — proteins, for example — into the cell. But they can also be hijacked by pathogens that can infect cells. But the question of how those pockets formed from membranes that were previously believed to be flat had stymied researchers for nearly 40 years.
“It was a controversy in cellular studies,” Kural said. “And we were able to use super-resolution fluorescence imaging to actually watch these pockets form within live cells, and so we could answer that question of how they are created.
“Simply put, in contrast to the previous studies, we made high-resolution movies of cells instead of taking snapshots,” Kural said. “Our experiments revealed that protein scaffolds start deforming the underlying membrane as soon as they are recruited to the sites of vesicle formation.”
That contrasts with previous hypotheses that the protein scaffolds of a cell had to go through an energy-intensive reorganization in order for the membrane to curve.
The way cells consume and expel vesicles plays a key role for living organisms. The process helps clear bad cholesterol from blood; it also transmits neural signals. The process is known to break down in several diseases, including cancer and Alzheimer’s disease.
“Understanding the origin and dynamics of membrane-bound vesicles is important — they can be utilized for delivering drugs for medicinal purposes but, at the same time, hijacked by pathogens such as viruses to enter and infect cells,” Kural said. “Our results matter, not only for our understanding of the fundamentals of life, but also for developing better therapeutic strategies.”
Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4 T cells allows complex functional analyses
by Manuel Albanese, Adrian Ruhle, Jennifer Mittermaier, Ernesto Mejías-Pérez, Madeleine Gapp, Andreas Linder, Niklas A. Schmacke, Katharina Hofmann, Alexandru A. Hennrich, David N. Levy, Andreas Humpe, Karl-Klaus Conzelmann, Veit Hornung, Oliver T. Fackler, Oliver T. Keppler in Nature Methods
LMU researchers have developed a method that allows resting human immune cells to be genetically analyzed in detail for the first time.
CD4+ T cells are important parts of the immune system and play a key role in defending the body against pathogens. As they possess a great variety of defense mechanisms against HIV in their resting state, they are infected only very rarely — but these few infected cells form a latent reservoir for HIV in the body that currently cannot be reached by antiviral drugs. Consequently, the virus can spread again from there after activation of the CD4+ T cells. Understanding how HIV interacts with resting CD4+ T cells is essential for finding new therapeutic approaches.
Scientists led by Prof. Oliver T. Keppler from the Max von Pettenkofer Institute at LMU have now developed a method that for the first time allows these specific immune cells to be genetically manipulated under physiological conditions in an efficient and uncomplicated manner.
Resting CD4+ T cells had been scarcely amenable to genetic manipulations, because the available methods generally presuppose dividing cells, as Keppler explains. “And resting cells do not divide by definition.” As the first step in the development of the new method, the team of scientists optimized the cultivation conditions. As a result, the researchers were able to keep these cells alive in the laboratory after extracting them from the blood of healthy donors not just for 3–4 days as before, but for up to six weeks. The decisive progress came with an advance in nucleofection, a special method that allows reagents to be delivered into the nucleus of a cell. Using this technique, the researchers introduced the genetic scissors CRISPR-Cas into resting CD4+ T cells, enabling them to make targeted modifications to the genome of the host cells — for example, by eliminating genes by means of so-called knockouts.
“This combination worked very efficiently, and we were able to reach and genetically manipulate around 98 percent of the cells. Moreover, we did this without activating the CD4+ T cells,” says Keppler. “What was particularly exciting was that we were able to eliminate up to six genes simultaneously with high efficiency by means of a single nucleofection. Nobody had managed to do that in primary cells before — and we did it with cells isolated from an intact organ.”
In the future, the researchers will thus be able to eliminate individual genes and whole signaling pathways and analyze their functions. By knocking out the corresponding genes, they have already managed to clarify whether four previously controversial cellular factors play a role in infection with HIV or not.
On top of this, they pursued a second “knock-in” approach, whereby additional or slightly modified genes are inserted, such as a gene for green fluorescent protein (GFP). With the help of this protein, researchers can analyze how the activity of a target gene changes under certain conditions, or they can mark specific proteins.
“All these things together give us the opportunity for the first time to investigate the interaction of HIV with human resting CD4+ T cells under physiological conditions,” explains Adrian Ruhle, co-lead author of the study. “But we can also investigate these cells better in their general role as immune cells beyond HIV.”
In the long term, the researchers hope that having a better understanding of the biology of these cells will lead to new approaches for the total elimination of HIV from the bodies of patients, as there are still around 37 million people worldwide infected with the virus.
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