These new insights might lead to a new line of clinical laboratory testing, particularly if the results could guide the patient to microbiome-based repellents that would remain effective for months once applied
Researchers are beginning to identify what compounds make individuals more attractive to mosquitos. That is a first step in the development of a biomarker that could be developed into a clinical laboratory test. Question is: would there be enough consumers wanting to do a lab test to determine if they were highly attractive to mosquitos, thus making this a revenue-generating test for labs?
The SA article reported on their study published in the journal Cell titled, “Differential Mosquito Attraction to Humans Is Associated with Skin-Derived Carboxylic Acid Levels.” The researchers, according to SA, found that individual humans have “a unique scent profile made up of different chemical compounds” and that “mosquitoes were most drawn to people whose skin produces high levels of carboxylic acids.” The researchers also found that “attractiveness to mosquitoes remained steady over time, regardless of changes in diet or grooming habits.”
At a minimum, there would be widespread consumer interest to at least understand why some individuals get more mosquito bites than others. What may be of particular interest to microbiologists is the statement by molecular biologist Omar Akbari, PhD, of the University of California, San Diego, who told Scientific American that by “taking human-colonizing skin bacteria … and engineering them in such a way that they can either express a repellent compound or be able to degrade something that’s attractive,” a mosquito repellant could be developed that would last for months once applied.
“This study clearly shows that these acids are important,” neurogeneticist Matthew DeGennaro, PhD (above), told CNN. “… how the mosquitoes perceive these carboxylic acids is interesting because these particular chemicals … are hard to smell at a distance. It could be that these chemicals are being altered by … the skin microbiome … if we understand why mosquitoes find a host, we can design new repellents that will block the mosquitoes from sensing those chemicals, and this could be used to improve our current repellents.” Clinical laboratory testing will be needed to produce biomarkers for developing such improved repellents. (Photo copyright: Laboratory of Tropical Genetics.)
Clinical Laboratory Testing Needed to Identify Levels of Carboxylic Acids
To complete their study, the researchers had 64 participants wear nylon stockings for six hours on their arms to get their unique scent into the fabric. The scent on the stockings was not discernible to the human nose, but it was to the mosquitos.
Two pieces of the nylon were then placed in a closed container with Aedes aegypti mosquitoes. The researchers found that certain samples were more popular with the mosquitos than others. Upon further analysis the researchers found that the most popular samples came from subjects with higher levels of carboxylic acids, and the least popular had the lowest levels. The scientists ran the test with the same participants several times over three years and the results remained largely the same.
Carboxylic acid is an organic compound found in humans in sebum, the oily layer protecting our skin. The level at which humans release carboxylic acid varies from person to person. And there is no discernible way the human nose can determine whether a person has the level of carboxylic acid on the skin that mosquitos find desirable. The answer would need to be determined by a diagnostic test performed in a clinical laboratory.
Although the development of a test to determine someone’s susceptibility to mosquitos may be far away, there could be significant consumer interest in developing such a test.
“The question of why some people are more attractive to mosquitoes than others—that’s the question that everybody asks,” Leslie B. Vosshall, PhD, Chief Scientific Officer, Howard Hughes Medical Institute, who led the research team to find out why some people are more attractive to mosquitos than others, told Scientific American. “My mother, my sister, people in the street, my colleagues—everybody wants to know.” She credits their interest as the inspiration for embarking on the study.
“Understanding what makes someone a ‘mosquito magnet’ will suggest ways to rationally design interventions such as skin microbiota manipulation to make people less attractive to mosquitoes. We propose that the ability to predict which individuals in a community are high attractors would allow for more effective deployment of resources to combat the spread of mosquito-borne pathogens,” the researchers wrote in their Cell paper.
Preventing Spread of Deadly Diseases
Although mosquitos are an annoyance, they also can be dangerous vectors of disease.
“Every bite of these mosquitoes puts people into public health danger. Aedes aegypti mosquitoes are vectors for dengue, yellow fever, and Zika,” Vosshall told CNN. “Those people who are magnets are going to be much more likely to be infected with viruses.”
Further research into these early findings may help develop diagnostic tests to protect against the spread of these diseases and identify individuals who are more attractive to the mosquitos, and therefore, more likely to contract and spread disease.
Being able to identify which individuals are mosquito magnets could help keep individuals safe from dangerous diseases, and development of a better repellent could also make outdoor summer events more bearable for the (unfortunately) popular among the pests. Medical laboratory tests associated with determining an individual’s susceptibility to mosquito bites could give clinical laboratories a new way to add value to consumers and patients.
Clinical laboratories involved in genetic testing may find this welcomed news, after a pair of studies conducted in 2019 raised concerns about CRISPR base editing. The researchers of those studies observed that it “causes a high number of unpredictable mutations in mouse embryos and rice,” Chemical and Engineering News (C&EN) reported, adding, “Other groups have raised concerns about off-target mutations caused when the traditional CRISPR-Cas9 form of gene editing cuts DNA at a location that it wasn’t supposed to touch. The results of the new studies are surprising, however, because scientists have lauded base editors as one of the most precise forms of gene editing yet.”
Nevertheless, UC Berkeley’s latest breakthrough is expected to drive development of new and more accurate CRISPR-Cas genome-editing tools, which consist of base editors as well as nucleases, transposases, recombinases, and prime editors.
Understanding CRISPR Base Editors At a ‘Deeper Level’
Harvard University Chemistry and Chemical Biology Professor David Liu, PhD, who co-authored the study, explained the significance of this latest discovery.
“While base editors are now widely used to introduce precise changes in organisms ranging from bacteria to plants to primates, no one has previously observed the three-dimensional molecular structure of a base editor,” he said in a UC Berkeley news release. “This collaborative project reveals the beautiful molecular structure of a state-of-the-art highly-active base editor—ABE8e—caught in the act of engaging a target DNA site.”
UC Berkeley Professor Jennifer Doudna, PhD (above), who served as senior author of the study, says scientists may now have the information necessary to refine base editors and improve their precision and genome-targeting ability. “This structure helps us understand base editors at a much deeper level,” she said in the UC Berkeley statement. “Now that we can see what we’re working with, we can develop informed strategies to improve the system.” (Photo copyright: UC Berkeley.)
Jennifer Doudna, PhD, UC Berkeley Professor, Howard Hughes Medical Institute Investigator, and senior author of the study, has been a leading figure in the development of CRISPR-Cas9 gene editing. In 2012, Doudna and Emmanuelle Charpentier, PhD, Founding, Scientific and Managing Director at Max Planck Unit for the Science of Pathogens in Berlin, led a team of researchers who “determined how a bacterial immune system known as CRISPR-Cas9 is able to cut DNA, and then engineered CRISPR-Cas9 to be used as a powerful gene editing technology.” In a 2017 news release, UC Berkeley noted that the work has been described as the “scientific breakthrough of the century.”
Viewing the Base Editor’s 3D Shape
CRISPR-Cas9 gene editing allows scientists to permanently edit the genetic information of any organism, including human cells, and has been used in agriculture as well as medicine. A base editor is a tool that manipulates a gene by binding to DNA and replacing one nucleotide with another.
According to the recent UC Berkeley news release, the research team used a “high-powered imaging technique called cryo-electron microscopy” to reveal the base editor’s 3D shape.
Genetic Engineering and Biotechnology News notes that, “The high-resolution structure is of ABE8e bound to DNA, in which the target adenine is replaced with an analog designed to trap the catalytic conformation. The structure, together with kinetic data comparing ABE8e to earlier ABEs [adenine base editors], explains how ABE8e edits DNA bases and could inform future base-editor design.”
The graphic above, taken from the UC Berkeley news release, shows the “3D structure of a base editor, comprised of the Cas9 protein (white and gray), which binds to a DNA target (teal and blue helix) complementary to the RNA guide (purple), and the deaminase proteins (red and pink), which switch out one nucleotide for another.” (Image and caption copyright: UC Berkeley.)
Knowing the Cas9 fusion protein’s 3D structure may help eliminate unintended off-target effects on RNA, extending beyond the targeted DNA. However, until now, scientists have been hampered by their inability to understand the base editor’s structure.
“If you really want to design truly specific fusion protein, you have to find a way to make the catalytic domain more a part of Cas9, so that it would sense when Cas9 is on the correct target and only then get activated, instead of being active all the time,” study co-first author Audrone Lapinaite, PhD, said in the news release. At the time of the study, Lapinaite was a postdoctoral fellow at UC Berkeley. She is now an assistant professor at Arizona State University.
“As a structural biologist, I really want to look at a molecule and think about ways to rationally improve it. This structure and accompanying biochemistry really give us that power,” added UC Berkeley postdoctoral fellow Gavin Knott, PhD, another study co-author. “We can now make rational predications for how this system will behave in a cell, because we can see it and predict how it’s going to break or predict ways to make it better.”
Clinical laboratory leaders and pathologists will want to monitor these new advances in CRISPR technology. Each breakthrough has the power to fuel development of cost-effective, rapid point-of-care diagnostics.
The scientist also employed machine learning “to gauge how easily accessible genes are for transcription” in research that could lead to new clinical laboratory diagnostic tests
Anatomic pathologists and clinical laboratories are of course familiar with the biological science of genomics, which, among other things, has been used to map the human genome. But did you know that a three-dimensional (3D) map of a genome has been created and that it is helping scientists understand how DNA regulates its organization—and why?
The achievement took place at St. Jude Children’s Research Hospital (St. Jude) in Memphis, Tenn. Scientists there created “the first 3D map of a mouse genome” to study “the way cells organize their genomes during development,” a St. Jude news release noted.
Some experts predict that this new approach to understanding how changes happen in a genome could eventually provide new insights that anatomic pathologists and clinical laboratory scientists could find useful when working with physicians to diagnose patients and using the test results to identify the most appropriate therapy for those patients.
In addition to 3D modeling, the researchers applied machine learning to data from multiple sources to see how the organization of the genome changed at different times during development. “The changes are not random, but part of the developmental program of cells,” Dyer said in the news release.
The St. Jude study focused on the rod cells in a mouse retina. That may seem like a narrow scope, but there are more than 8,000 genes involved in retinal development in mice, during which those genes are either turned on or off.
To see what was happening among the cells, the researchers used HI-C analysis, an aspect of ultra-deep chromosome conformation capture, in situ. They found that the loops in the DNA bring together regions of the genome, allowing them to interact in specific ways.
Until this study, how those interactions took place was a
mystery.
“Understanding the way cells organize their genomes during development will help us to understand their ability to respond to stress, injury and disease,”Michael Dyer, PhD (above), Chair of St. Jude’s Developmental Neurobiology Department, co-leader of the Developmental Biology and Solid Tumor Program, and Investigator at Howard Hughes Medical Institute (HHMI), said in the news release. (Photo copyright: St. Jude Children’s Research Hospital.)
The scientists also discovered there were DNA promoters, which encourage gene expression, and also DNA enhancers that increase the likelihood gene expression will occur.
“The research also included the first report of a powerful regulator of gene expression, a super enhancer, that worked in a specific cell at a specific stage of development,” the news release states. “The finding is important because the super enhancers can be hijacked in developmental cancers of the brain and other organs.”
St. Jude goes on to state, “In this study, the scientists determined that when a core regulatory circuit super-enhancer for the VSX2 gene was deleted, an entire class of neurons (bipolar neurons) was eliminated. No other defects were identified. Deletion of the VSX2 gene causes many more defects in retinal development, so the super-enhancer is highly specific to bipolar neurons.”
The St. Jude researchers developed a genetic mouse model of
the defect that scientists are using to study neural circuits in the retina,
the news release states.
Research Technologist Victoria Honnell (left); Developmental Neurobiologist Jackie Norrie, PhD (center); and Postdoctoral Researcher Marybeth Lupo, PhD (right), work in the St. Jude clinical laboratory of Michael Dyer, PhD, using 3D genomic mapping to study gene regulation during development and disease. (Photo copyright: St. Jude Children’s Research Hospital.)
DNA Loops May Matter to Pathology Sooner Rather than
Later
Previous researcher studies primarily used genomic sequencing technology to locate and investigate alterations in genes that lead to disease. In the St. Jude study, the researchers examined how DNA is packaged. If the DNA of a single cell could be stretched out, it would be more than six feet long. To fit into the nucleus of a cell, DNA is looped and bundled into a microscopic package. The St. Jude scientists determined that how these loops are organized regulates how the cell functions and develops.
Scientists around the world will continue studying how the loops in DNA impact gene regulation and how that affects the gene’s response to disease. At St. Jude Children’s Research Hospital, Dyer and his colleagues “used the same approach to create a 3D genomic map of the mouse cerebellum, a brain structure where medulloblastoma can develop. Medulloblastoma is the most common malignant pediatric brain tumor,” noted the St. Jude’s news release.
In addition to providing an understanding of how genes
function, these 3D studies are providing valuable insight into how some
diseases develop and mature. While nascent research such as this may not impact
pathologists and clinical laboratories at the moment, it’s not a stretch to
think that this work may lead to greater understanding of the pathology of
diseases in the near future.
CRISPR-Cas9 connection to cancer prompts research to investigate different approaches to gene editing
Dark Daily has covered CRISPR-Cas9 many times in previous e-briefings. Since its discovery, CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, has been at the root of astonishing breakthroughs in genetic research. It appears to fulfill precision medicine goals for patients with conditions caused by genetic mutations and has anatomic pathologists, along with the entire scientific world, abuzz with the possibilities such a tool could bring to diagnostic medicine.
All of this research has contributed to a deeper understanding of how cells function. However, as is often the case with new technologies, unforeseen and problematic questions also have arisen.
“Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors, and a human differentiated cell line,” wrote the authors in their introduction.
Another study, this one conducted by biomedical researches at Cambridge, Mass., and published in Nature, describes possible toxicity caused by Cas9.
“Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR-Cas9 for genome engineering and screening in hPSCs [human pluripotent stem cells]. Moreover, as hPSCs can acquire P53 mutations, cell replacement therapies using CRISPR-Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.”
Essentially what both groups of researchers found is that CRISPR-Cas9 cuts through the double helix of DNA, which the cell responds to as it would any injury. A gene called p53 then directs a cellular “first-aid kit” to the “injury” site that either initiates self-destruction of the cell or repairs the DNA.
Therefore, in some instances, CRISPR-Cas9 is inefficient because the repaired cells continue to function. And, the repair process involves the p53 gene. P53 mutations have been implicated in ovarian, colorectal, lung, pancreatic, stomach, liver, and breast cancers.
Though important, some experts are downplaying the significance of the findings.
Erik Sontheimer, PhD (above), Professor, RNA Therapeutics Institute, at the University of Massachusetts Medical School, told Scientific American that the two studies are important, but not show-stoppers. “This is something that bears paying attention to, but I don’t think it’s a deal-breaker,” he said. (Photo copyright: University of Massachusetts.)
“It’s something we need to pay attention to, especially as CRISPR expands to more diseases. We need to do the work and make sure edited cells returned to patients don’t become cancerous,” Sam Kulkarni, PhD, CEO of CRISPR Therapeutics, told Scientific American.
Both studies are preliminary. The implications, however, is in how genes that have become corrupted are used.
A team from the Salk Institute may have found a solution. They are investigating a different enzyme—Cas13d—which, in conjunction with CRISPR would target RNA rather than DNA. “DNA is constant, but what’s always changing are the RNA messages that are copied from the DNA. Being able to modulate those messages by directly controlling the RNA has important implications for influencing a cell’s fate,” Silvana Konermann, PhD, a Howard Hughes Medical Institute (HHMI) Hanna Gray Fellow and member of the research team at Salk, said in a news release.
The Salk team published their findings in the journal Cell. The paper describes how “scientists from the Salk Institute are reporting for the first time the detailed molecular structure of CRISPR-Cas13d, a promising enzyme for emerging RNA-editing technology. They were able to visualize the enzyme thanks to cryo-electron microscopy (cryo-EM), a cutting-edge technology that enables researchers to capture the structure of complex molecules in unprecedented detail.”
The researchers think that CRISPR-Cas13d may be a way to make the process of gene editing more effective and allow for new strategies to emerge. Much like how CRISPR-Cas9 led to research into recording a cell’s history and to tools like SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing), a new diagnostic tool that works with CRISPR and changed clinical laboratory diagnostics in a foundational way.
Each discovery will lead to more branches of inquiry and, hopefully, someday it will be possible to cure conditions like sickle cell anemia, dementia, and cystic fibrosis. Given the high expectations that CRISPR and related technologies can eventually be used to treat patients, pathologists and medical laboratory professionals will want to stay informed about future developments.
Even in its early stages the Human Cell Atlas project is impacting the direction of research and development of RNA sequencing and other genetic tests
No one knows exactly how many cell types exist in the human body. Though traditional texts place numbers in the hundreds, recent studies have found ranges from thousands to tens of thousands. Anatomic pathologists and clinical laboratory scientists know that the discovery of new types of human cells could lead to the creation of new medical laboratory tests.
So, it’s an important development that leaders of the Human Cell Atlas Consortium, a project comparable to the Human Genome Project, have set out to determine the exact numbers of cell types. And their findings could open up an entirely new field of diagnostic testing for clinical laboratories and anatomic pathology and lead to advances in precision medicine.
With the ability to identify cell types and sub-types associated with human disease and health conditions, medical labs could have a useful new way to help physicians make diagnoses and select appropriate therapies.
Begun in 2016, the group’s mission according to the Human Cell Atlas website is “To create comprehensive reference maps of all human cells—the fundamental units of life—as a basis for both understanding human health and diagnosing, monitoring, and treating disease.”
The ambitious project aims to catalog every cell type in the human body and “account for and better understand every cell type and sub-type, and how they interact.”
Striving for Deeper Understanding of the Basics
Cells are the basic building blocks of life, but scientists don’t know exactly how many different types of cells there are.
In an NPR interview, Aviv Regev, PhD, Professor of Biology and a core member at the Broad Institute of MIT and Harvard, investigator at the Howard Hughes Medical Institute, and co-leader of the Human Cell Atlas Consortium, said, “No one really knows how many [cells types] there will be,” adding, “People guess anything from the thousands to the tens of thousands. I’m not guessing. I would rather actually get the measurements done and have a precise answer.”
In an innovative move, Regev and her team improved the method they were already using to sort cells—single-cell RNA sequencing. “All of sudden we moved from something that was very laborious—and we could do maybe a few dozen or a few hundred—to something where we could do many, many thousands in a 15- to 20-minute experiment,” she told NPR.
But the project is massive. A typical human body contains about 37.2 trillion cells. So, the Human Cell Atlas scientists decided to complete preliminary pilot projects to identify the most efficient and effective strategies for sampling and analyzing the various cells to create the full atlas.
“It’s kind of like we’re trying to find out what are all the different colors of Lego building blocks that we have in our bodies,” Sarah Teichmann, PhD, Head of Cellular Genetics and Senior Group Leader at Wellcome Sanger Institute in the UK, and co-leader of the Human Cell Atlas Consortium, told NPR. “We’re trying to find out how those building blocks—how those Lego parts—fit together in three dimensions within each tissue.”
Sarah Teichmann, PhD (left), and Aviv Regev, PhD (right), are co-leaders of the Human Cell Atlas Consortium, an ambitious project of MIT/Harvard Broad Institute that seeks to “create comprehensive reference maps of all human cells—the fundamental units of life—as a basis for both understanding human health and diagnosing, monitoring, and treating disease.” Such an advance could lead to significant advances in clinical laboratory and pathology testing and move healthcare closer to true precision medicine. (Photo copyrights: University of Cambridge and MIT/Broad Institute.
Some of the early pilot projects include a partnership with the Immunological Genome Project (ImmGen) to study and map the cells in the immune system. According to the Human Cell Atlas website, the partnership “will combine:
“deep knowledge of immunological lineages;
“clinical expertise and infrastructure needed to procure and process diverse samples;
“genomic and computational expertise to resolve the hundreds of finely differentiated cell types that compose all facets of the immune system; and,
the genomic signatures that define them.”
Other areas the pilot projects will address include:
the Human Developmental Cell Atlas (HDCS), which will investigate the highly specialized cells involved in human development.
Progress So Far
In the two short years since the Human Cell Atlas project began much work has already been accomplished, according to a news release. In addition to organizing the consortium and obtaining funding, the collaborators have published a white paper describing their goals and a framework for reaching them, as well as launching the pilot projects.
Such an ambitious project, however, is not without barriers and challenges. Regev and Teichmann, along with other collaborators, outlined some of those challenges in an article published in Nature.
The complexity of the human body combined with rapidly changing technology make simply agreeing on the scope of the project challenging. In order to meet that particular challenge, the collaborators plan to work in phases and drafts, which will allow for some flexibility and increasing focus on specifics as they go.
Other challenges include:
keeping the entire project open and fair;
procuring samples with consent and in an appropriate manner; and,
organizing in an efficient and effective manner.
The collaborators have developed and detailed strategies for meeting each of these challenges.
The Human Cell Atlas could impact treatments for every disease that affects humans and bring healthcare closer to accomplishing precision medicine goals. By knowing what cells exist in what parts of the human body—and how they typically behave at their most basic levels—the MIT/Harvard/Broad Institute scientists hope to understand what’s happening when those cells “misbehave” in expected ways. The knowledge garnered from the Human Cell Atlas is likely to be invaluable to anatomic pathologists and clinical laboratories.
