Stem Cell-Based Treatment Used to Prevent Blindness In Animal Models of Retinal Degeneration
Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly. With limited treatment options for AMD patients, scientists turned to regenerative medicine approaches, pursuing an induced pluripotent stem cell (iPSC) based therapy for AMD. IPSCs are cells taken from patient tissues and coaxed into a stem cell-like state where they can be become any cell type in the body. In a study led by Dr. Kapil Bharti, a Regenerative Medicine Program-funded investigator, dying retinal pigment epithelial (RPE) cells in pigs and rats with retinal deterioration were replaced with human iPSC-derived RPE cells. The replacement cells helped nurture photoreceptors, the light-sensing cells in the retina. Both healthy RPE and photoreceptor cells are necessary to maintain vision. With both cell types now in place, the investigators observed that the iPSC-based therapy successfully prevented blindness in the animals. The findings from this study will inform a first-in-human clinical trial to treat AMD.
Watch Dr. Bharti explain his research: https://youtu.be/k6DTj597Gv8
Image: This is a scanning electron microscope image of an RPE monolayer growing on top of a biodegradable scaffold (blue, left side of image). The polarity of the RPE cells is clearly visible (brown, bottom of cells in middle of image; green, top of cells on right side of image).
Clinical-grade stem cell–derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs. Sharma R, Khristov V, Rising A, Jha BS, Dejene R, Hotaling N, Li Y, Stoddard J, Stankewicz C, Wan Q, Zhang C, Campos MM, Miyagishima KJ, McGaughey D, Vilasmil R, Mattapallil M, Stanzel B, Qian H, Wong W, Chase L, Charles S, McGill T, Miller S, Maminishikis A, Amaral J, Bharti K. Science Translational Medicine. January 16, 2019 (Note: full text of the article may require institutional access)
In the news:
- NIH researchers rescue photoreceptors, prevent blindness in animal models of retinal degeneration-NEI Press Release
- Macular degeneration trial will be first human test of Nobel-winning stem cell technique-STAT
Honey, I Shrunk the Structures
Dr. Edward Boyden, a 2013 Pioneer Awardee, and his research team developed a technique that could be a giant breakthrough for science—ironically, by making structures very small. Boyden’s team invented a way to make nanoscale 3D objects of various shapes. The technique improves on conventional methods for creating nanostructures, as many are limited to two dimensions, rely on materials limited in function, or are prohibitively slow and challenging. The new method, called “Implosion Fabrication” was an innovative adaptation of a technique developed a few years ago for neuroscience. By placing small brain tissues in a polyacrylate gel (a substance commonly found in diapers), then expanding the gel, the brain tissues also expanded, making them easier to visualize for high resolution imaging. For Implosion Fabrication, Dr. Boyden’s team reversed the process. They created a structure inside of a gel, and then shrank the gel, reducing the structure inside to one-thousandth the volume of the original—creating an intact nano-sized 3D object.
How was this done?
First, Dr. Boyden’s team began with polyacrylate gel as scaffolding. The gel was bathed in a liquid, containing the light-reactive molecule fluorescein, which attached to the scaffolding. Afterwards, the fluorescein molecules were precisely targeted by laser light, binding them to specific locations within the gel. This was key, as the bound fluorescein molecules acted as a ‘sticky trap’ to bind other materials the researchers could add including metals, nanoparticles, or potentially even DNA. Because of the specificity in the technique, the attached materials could be arranged to form a 3D structure. Finally, the researchers added acid to shrink the gel, which also reduced the size the object, with over a 1000-fold reduction in volume.
How could it be useful?
Dr. Boyden’s team is exploring applications for Implosion Fabrication. Some of the earliest potential uses could be in optics, creating better lenses for cell phone cameras or microscopes. Because the technique uses equipment many labs already have access to, there is widespread potential for its use in many different topic areas. The accessibility of Implosion Fabrication could act as a field multiplier, laying the groundwork for future innovative nanoscale projects, including nanoscale electronics or robots.
Reference: 3D Nanofabrication by Volumetric Deposition and Controlled Shrinkage of Patterned Scaffolds. Oran, D., Rodriques, S. G., Gao, R., Asano, S., Skylar-Scott, M. A., Chen, F., Tillberg, P. W., Marblestone, A. H., & Boyden, E. S. Science, (2018). 362(6420), 1281-1285.
In the News:
- Incredible Shrinking Method Collapses Structures to the Nanoscale
- MIT Develops Incredible System to 'Shrink' Objects
- 'Implosion Fabrication' Allows to Shrink 3D Objects to the Nanoscale
Using Machine Learning to Identify a Signature of Bipolar Disorder
In collaboration with researchers from the Big Data to Knowledge (BD2K) ENIGMA Center, Abraham Nunes MD. ,M.B.A., and Tomas Hajek M.D., Ph.D., published a study evaluating machine-learning methods to identify diagnostic markers of bipolar disorder. "After assembling over 3,000 brain scans of bipolar disorder patients and controls from multiple sites, the researchers tested whether computers could train themselves to reliably distinguish patients from controls. The results, considered promising, provide a proof-of-concept for a brain-imaging signature of bipolar disorder to identify the illness in individuals," noted a news feature on the initiative, by the Brain & Behavior Research Foundation. The study, recently published in Molecular Psychiatry, reports the latest results from an international alliance to understand what brain features might consistently distinguish bipolar patients from healthy individuals.
Using structural MRI to identify bipolar disorders - 13 site machine learning study in 3020 individuals from the ENIGMA Bipolar Disorders Working Group. Abraham Nunes, Tomas Hajek, et al. Mol Psychiatry. 2018 Aug 31. doi: 10.1038/s41380-018-0228-9
ADHD or Adolescence?
Attention deficit-hyperactivity disorder (ADHD) diagnoses have increased over the last two decades. Between 2007 and 2012, rates rose by a further 50%, which generated concerns from experts about an appropriate level of diagnosis. A potential contributing factor is the arbitrary age cutoff for public school entry in most U.S. states (i.e. children must be 5 years old by September 1st). As a result, students born in August, directly entering kindergarten, are almost a year younger than those born in September, who had to wait. This interacts with a unique aspect of ADHD diagnoses—reliance on teacher and parental assessments of a child compared to their same-grade peers. This is significant in kindergarten, as overly active behavior in younger students could be attributed to relative immaturity.
Understanding this issue, Anupam B. Jena, an Early Independence Awardee (2013), asked the question: Are the youngest children in a kindergarten class more often diagnosed with ADHD?
To address the query, Dr. Jena and his team scoured an insurance claims database with information on roughly 407,000 U.S. children, also providing information about ADHD and other diagnoses. They focused on students born between 2007-2009 and tracked them until 2015. They found younger “August-born” children, were diagnosed at rates approximately 34% higher than older “September-born” children (85.1 vs. 63.6 per 10,000 children, respectively). This finding also seemed to be specific to ADHD, as no differences were found in the rates of some other diseases including asthma, diabetes, or obesity. These data may suggest the children’s younger ages were playing a role in ADHD diagnosis.
While the results are informative, it is important to understand that this study does not prove students are being misdiagnosed. Another possible interpretation is that many students in September cutoff-states could be getting appropriate early diagnoses, with increased ADHD rates being caused by some currently unidentified factor. Additionally, relying only on insurance data makes it difficult to assess confounding factors, as other childhood issues appear as ADHD (e.g. medical conditions, overcrowded classrooms, or psychiatric issues). Even considering the caveats, the use of a large sample size and insurance records rather than subjective recall of subjects, lends credibility to the findings. The results may highlight shortcomings in how ADHD is identified, but more studies will be needed to understand what underlies this phenomenon.
Reference: Attention Deficit--Hyperactivity Disorder and Month of School Enrollment. Layton, T. J., Barnett, M. L., Hicks, T. R., & Jena, A. B. New England Journal of Medicine, (2018). 379(22), 2122-2130.
In the News:
Scientists Develop Molecular Tweezers with CRISPR-GO
One of the goals of the is to understand how organization of all the DNA in the nucleus – called the genome – affects cell function. To truly understand this relationship, scientists need to be able to manipulate genome organization to observe the effect on which genes are turned on or off. In a recent article in Cell, 4DN investigator Dr. Stanley Qi and his research team describe a new technique called CRISPR-genome organization (CRISPR-GO) to manipulate the organization of DNA in the cell nucleus. Described as “molecular tweezers,” CRISPR-GO allows scientists to grab a specific section of DNA and move it to a new targeted location in the nucleus. This tool works rapidly and is inducible and reversible, making it very useful for exploring the effects of genome reorganization on many genes. CRISPR-GO allowed the team to explore whether genes behave differently in different parts of the nucleus. They found that genes relocated to the periphery of the nucleus or to blobs of proteins and RNA in the nucleus called Cajal bodies were turned off, indicating a role for these regions in gene regulation. CRISPR-GO is a valuable tool for studying the relationship between genome organization and cell function that could improve our understanding of diseases associated with abnormal genome organization to help develop new treatments for these diseases.
Reference:. Wang H, Xu X, Nguyen CM, Liu Y, Gao Y, Lin X, Daley T, Kipniss NH, La Russa M, and Qi LS. November 2018. Cell 175: 1405-1417.
In the News:, Stanford Medicine News Center
Better Biomarkers for Muscular Dystrophy
Extracellular RNA Communication program researchers put urine biomarkers to the test
New and non-invasive methods to identify muscular dystrophy (MD) are greatly needed to replace painful and highly invasive muscle biopsy techniques. Currently, clinicians and scientists typically isolate an important biological molecule called RNA from muscle cells removed during a biopsy to look for RNA that has not been properly processed through a process called splicing. The mis-spliced RNA can serve as a diagnostic biomarker for MD. However, RNA doesn't have to be obtained only from cells taken from muscle biopsy. In some cases, RNA found outside of cells called extracellular RNA (exRNA), can be isolated from bodily fluids like urine and serve as biomarkers for some diseases. Researchers from the NIH Common Fund’s ExRNA Communication program examined if exRNA in urine could be used to detect the mis-spliced RNA from muscular dystrophy patients.
In this study, researchers isolated exRNA from the urine of patients with a certain type of Muscular Dystrophy (myotonic dystrophy type I (DM1)) and from healthy volunteers. They found several different RNA molecules that were not spliced properly in DM1 patients. When 10 of the mis-spliced RNA molecules were all present in one patient, the researchers could predict with 100% accuracy that the people had the disease. The presence of these 10 mis-spliced RNA molecules is the composite biomarker signature the researchers were hoping to identify. While more studies are needed, this approach is a promising alternative to invasive muscle biopsies. The exRNA approach also showed promise in detecting differences in RNA splicing in patients with DM1 who were being treated with drugs designed to target the RNA splicing process. This may also represent a painless and low-cost way to track disease activity or response to drug treatment.
Analysis of extracellular mRNA in human urine reveals splice variant biomarkers of muscular dystrophies. Antoury L, Hu N, Balaj L, Das S, Georghiou S, Darras B, Clark T, Breakefield XO, Wheeler TM. Nature Communications. 2018 Sep 25;9(1):3906.
In the News: Extracellular RNA in urine may provide useful biomarkers for muscular dystrophy. Mass General News Release. September 25, 2018.
Real-Time Virus Genetic Data Helped Nigeria Respond to a Lassa Fever Outbreak
Lassa fever is an infectious disease that can cause life threatening hemorrhaging (bleeding) throughout the body. The fever is caused by Lassa virus, which is normally transmitted to humans through contact with food or household items contaminated with urine or feces from infected rodents. Lassa fever is commonly found in parts of West Africa including Sierra Leone, Liberia, and Nigeria.
Confirmed Lassa fever cases in Nigeria spiked over 2017 to 2018. The Nigeria Centre for Disease and Control (NCDC) had little information to explain the increase in cases, which complicated mounting an effective response. There was concern that a particularly infectious strain of the virus or human-to-human transmission of the disease may have caused the spike in cases. This concern led a team of researchers, including members of the NIH Common Fund-supported Human Heredity and Health in Africa (H3Africa) program, to conduct a genetic analysis of viruses in samples collected from Lassa-infected patients. Their results showed that the increase in cases was not due to a single virus strain or to human-to-human transmission. Instead, the virus genetic make-up from the 2018 season was consistent with a diverse range of virus strains previously observed in the rodent population in Nigeria. This means that people were still getting Lassa virus by contact with rodent droppings. Additionally, genetic differences in the virus differed by geographic region, and indicated that natural barriers to rodent movement, like rivers, helped prevent spread of the disease. The researchers reported their findings to the NCDC and other public health officials in real time to help respond to the high number of Lassa fever cases in Nigeria.
Furthermore, this study is a great example of how modern research approaches can impact genomic research and public health in Africa. The findings from this study have the potential to improve the health of African populations.
Genomic Analysis of Lassa Virus during an Increase in Cases in Nigeria in 2018. Siddle KJ, Eromon P, Barnes KG, Mehta S, Oguzie JU, Odia I, Schaffner SF, Winnicki SM, Shah RR, Qu J, Wohl S, Brehio P, Iruolagbe C, Aiyepada J, Uyigue E, Akhilomen P, Okonofua G, Ye S, Kayode T, Ajogbasile F, Uwanibe J, Gaye A, Momoh M, Chak B, Kotliar D, Carter A, Gladden-Young A, Freije CA, Omoregie O, Osiemi B, Muoebonam EB, Airende M, Enigbe R, Ebo B, Nosamiefan I, Oluniyi P, Nekoui M, Ogbaini-Emovon E, Garry RF, Andersen KG, Park DJ, Yozwiak NL, Akpede G, Ihekweazu C, Tomori O, Okogbenin S, Folarin OA, Okokhere PO, MacInnis BL, Sabeti PC, Happi CT. N Engl J Med. 2018 Oct 17.
Photo: This transmission electron microscopic (TEM) image depicted numbers of Lassa virus virions adjacent to some cell debris by C. S. Goldsmith
In the News:
Genomic Analysis Offers Insight into 2018 Nigeria Lassa Fever Outbreak-NIAID News
Rapid genomic sequencing of Lassa virus in Nigeria enabled real-time response to 2018 outbreak-Medical Xpress
Pinpointing Plenty of Proteins - in a Single Cell
The cell is the fundamental unit of life, but until recently, it was very difficult for researchers to study the activities of an individual cell. Even cells that have the same job in our bodies can differ from each other in what genes are turned on or off and in what proteins they produce, and those differences can be important for understanding human health and disease. Recent advances made it possible to investigate a handful of proteins from a single cell, but Dr. Nikolai Slavov, a 2016 recipient of the NIH Director’s New Innovator Award, just changed the game. His approach now allows researchers to study 100 to 1000 times more proteins in individual cells.
Dr. Slavov and his team used a common research technique called mass spectrometry, which breaks down proteins and analyzes their building blocks, but they took steps to enhance the technique. Before mass spectrometry, Slavov’s team isolated individual mammalian cells, careful not to lose much of the cells’ protein content. Next, the team inventively applied another approach, tagging protein building blocks from the individual cells. The chemical tags were recognizable during mass spectrometry data analysis and were traced back to the individual cells submitted for analysis.
Combining these two steps in an innovative manner greatly increased the number of proteins identified and traced to a particular cell. Putting it into practice, Dr. Slavov showed that he could create protein profiles for individual stem cells allowed to develop randomly into many different cell types making many different proteins. Dr. Slavov’s method helps researchers move to a more concrete understanding of individual cells and the organisms they make up. In the future, this understanding may help patients by identifying biological markers for precision medicine and regeneration therapies.
Reference: SCoPE-MS: mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation. Budnik, B., Levy, E., Harmange, G., & Slavov, N. Genome biology. 2018 Oct 22; 19(1): 161
Initial Outcomes from the Undiagnosed Diseases Network Reveal Promising Number of Diagnoses
Members of the(UDN) published a summary of the progress that was made by the network during the first twenty months of accepting applicants in the New England Journal of Medicine. During that time, the network accepted 601 participants that remained undiagnosed by traditional medical practices. Of those people that competed their UDN evaluation in the first twenty months, 35% were given a diagnosis. Many of these diagnoses are rare genetic diseases including 31 previously unknown syndromes.
New coverage associated with this publication:
- Finding Answers for Patients with Rarest of Rare Diseases
October 10, 2018
- Doctor Detectives Help Diagnosis Mysterious Illnesses
October 11, 2018
- Disease Detectives Crack Cases of 130 Patients with Mysterious Illnesses
San Francisco Chronicle
October 10, 2018
Effect of Genetic Diagnosis on Patients with Previously Undiagnosed Disease. Splinter K, Adams DR, Bacino CA, Bellen HJ, Bernstein JA, Cheatle-Jarvela AM, Eng CM, Esteves C, Gahl WA, Hamid R, Jacob HJ, Kikani B, Koeller DM, Kohane IS, Lee BH, Loscalzo J, Luo X, McCray AT, Metz TO, Mulvihill JJ, Nelson SF, Palmer CGS, Phillips JA 3rd, Pick L, Postlethwait JH, Reuter C, Shashi V, Sweetser DA, Tifft CJ, Walley NM, Wangler MF, Westerfield M, Wheeler MT, Wise AL, Worthey EA, Yamamoto S, Ashley EA, Undiagnosed Diseases Network. N Engl J Med. 2018 Oct 10.
Cigarillos and Marijuana: It’s What’s on the Outside That Counts
Cigarillos, popularly repurposed mid-sized cigars, are often dumped of their tobacco to create “blunts” filled with marijuana. Under these conditions, smokers are still exposed to nicotine and other toxic agents through the cigarillo wrappings, which poses serious health risks. As Tobacco and Marijuana co-use rises, some people have asked: are cigarillos playing a role?
Concerned about this issue, Daniel Giovenco, a 2016 Early Independence awardee, used national sales data to study cigarillo products in three regions with legalized recreational marijuana. Some products were more “blunt maker-friendly” than others by being: inexpensive, easy to unwrap or flavored. As expected, Daniel’s team found “blunt maker-friendly” cigarillos were sold more than those used for smoking tobacco in Portland, Seattle, and Denver compared to the U.S. overall. One of the “blunt maker-friendly” products, Swisher Sweets, was so popular, it alone made up more than half of the sales in Portland and Seattle. Unexpectedly though, they found less overall cigarillo sales per capita (total dollar sales/population size) in the recreationally legal regions of Seattle (5.22), Denver (4.19), and Portland (3.02), compared to the U.S. average (7.98). However, this could be due to stronger tobacco control laws in these areas compared to other states.
The data used for the study did have some inherent limitations. Sales made at smoke shops and dispensaries were excluded. Additionally, the data do not allow the researchers to conclude how cigarillos are being used or if legalization specifically increases sales of “blunt maker-friendly” products. To build on the current analysis, follow up studies should look at sales “per person” as well as medical marijuana industry. As this is one of the first investigations to focus primarily on legalized recreational marijuana use in this context, Giovenco hopes that as legalization spreads to more states, research will continually be performed to question how recreationally legal marijuana is connected to tobacco use.
Reference: Cigarillo sales in legalized marijuana markets in the US.. Giovenco, D. P., Spillane, T. E., Mauro, C. M., & Martins, S. S. April 2018. Drug and alcohol dependence 185:347-350.
Sports Leagues Promote Unhealthy Food and Beverages to Children
Food and beverage companies spend millions of advertising dollars on professional sports organizations to reach young audiences. However, a question that often goes unasked is: what kind of messages about health are the advertisements sending to children? Thanks to the efforts of Early Independence Awardee, Marie Bragg, this overlooked aspect of marketing is being researched.
Dr. Bragg and her team analyzed Nielsen statistics for televised sports programs among 2-17 year-old viewers of 2015 televised events. The top 10 most-watched sports organizations advertised (on television, YouTube, and websites) food products that were “unhealthy” as judged by the Nutrient Profile Model (NPM), a respected nutritional value food rating system. Bragg’s team used a NPM based scoring index and found more than three-quarters of promoted products did not meet basic nutritional standards. On a 100-point scale, the average score for advertised foods such as potato chips and sugary cereals was around 38-39.
The researchers also examined the number of food and beverage sponsorship agreements (between 2006-2016) of the top 10 sports organizations with the most youth viewers which included: the National Football League (NFL), Major League Baseball (MLB), The National Hockey League (NHL), the National Basketball Association (NBA), the Fédération Internationale de Football Association (FIFA), and Little League Baseball. The NFL led the pack with 10 major “unhealthy” sponsorships, compared to the NHL and Little League baseball, which tied for second with 7 each.
Though extensive, the study has limitations including, a lack of sponsorship ad assessments within stadiums, and an inability to determine unique views from repeated views of YouTube ads. Even with these limits, Dr. Bragg suggests the overall message is clear: sports organizations promote unhealthy food and beverage products to children through their advertising partners.
The US is dealing with a child obesity epidemic. As part of efforts to address childhood obesity, Dr. Bragg recommends sports organizations create more health-conscious policies that limit partnerships with companies promoting unhealthy products. She also suggests that grassroots efforts through public involvement and media focus could generate attention required to shift current sponsorship practices.
Reference: Sports Sponsorships of Food and Nonalcoholic Beverages. Bragg, M. A., Miller, A. N., Roberto, C. A., Sam, R., Sarda, V., Harris, J. L., & Brownell, K. D. (2018). Pediatrics, 141(4), e20172822.
In the News:
Misfolded DNA Contributes to Neurodegenerative Disease
Over 1% of our DNA is made up of repeated sequences, which equals tens of thousands of short repeat tracts. Most repeat sequences are stable in length, but a small subset tend to expand or contract in length during processes such as DNA replication and repair. Over 30 human neurodegenerative disorders are associated with DNA repeat instability, including fragile X syndrome, Huntington’s disease, and Friedreich’s ataxia. For the disease-associated repeats, healthy individuals have a “normal” number of repeats, while those with the disease have repeats expanded beyond a threshold length. Researchers have long sought to understand why some DNA repeats are prone to expansion, while others are not.
The 3D arrangement of DNA impacts how information encoded by the DNA is “read” and used by the cell. To explore whether DNA folding plays a role in repeat instability, 4DN Program-funded researcher Dr. Jennifer Phillips-Cremins and her team analyzed the folding pattern of unstable regions of DNA repeats. They found that nearly all the repeat sequences associated with human disease are located at the boundaries between discreet 3D regions of the genome. To explore this in the context of human disease, they created genome folding maps around the FMR1 gene, the gene associated with fragile X syndrome, in samples from patients and healthy individuals. In samples from patients (which contained expanded repeats) they found misfolding at the expanded repeat tracts disrupted the boundary between domains, leading to the FMR1 gene being turned off. This study shows that regions of DNA repeats associated with human diseases can localize to genome domain boundaries and can disrupt 3D genome structure and gene function. Improved understanding of the link between DNA repeat instability and genome folding can aid in development of treatments for repeat expansion disorders.
Disease-Associated Short Tandem Repeats Co-Localize with Chromatin Domain Boundaries. Sun, JH, Zhou, L, Emerson, DJ, Phyo, SA, Titus, KR, Gong, W, Gilgenast, TG, Beagan, JA, Davidson, BL, Tassone, F, and Phillips-Cremins, JE. Cell 175, 38-40. September 2018.
Chromatin Domains Go on Repeat in Disease. Bruneau, BG and Nora, EP. Cell 175:1, 224-238. September 2018.
In the News:
Shocking Nerves to Treat Gut Disorders
The gut, or gastrointestinal tract, is an organ system responsible for the digestion of food. Several human diseases and conditions, including gastroparesis, obesity, and reflux disease, are associated with impaired regulation of gut function. Along with pharmaceutical, surgical, and dietary interventions, which are not always successful in treating these disorders, researchers have begun to explore electrical stimulation of the vagus nerve to precisely control gastrointestinal function. For this treatment strategy to work, scientists must understand the function of vagal nerve signals carried between the central nervous system and the gut.
A SPARC program-funded research team at Purdue University is studying the effect of vagus nerve stimulation on emptying the stomach as food moves into the small intestine (gastric emptying) in rats. They observe the process of gastric emptying using a technique called Magnetic Resonance Imaging (MRI). They found that selective electrical stimulation of the vagus nerve significantly increased gastric emptying through relaxation of the pyloric sphincter, a valve-like band of muscle that separates the stomach from the small intestine, which allows movement of food out of the stomach. These findings suggest that electrical stimulation of the vagus nerve could be used to improve gut function to treat gastric emptying disorders like gastroparesis. In addition, their MRI protocol describes a new non-invasive method to assess the effectiveness of many different types of therapies on gastrointestinal function.
Vagus nerve stimulation promotes gastric emptying by increasing pyloric opening measured with magnetic resonance imaging. Lu KH, Cao J, Oleson S, Ward MP, Phillips RJ, Powley TL, and Liu Z. September 2018. Wiley Publishers: Neurogastroenterology & Motility. e13380.
Making the Nucleus Measure Up
The dynamic 3D organization of DNA in the tiny nucleus of a cell plays a critical role in determining which genes are turned on in a cell (gene expression), which affects cell function. Determining the 3D organization of DNA in the nucleus of live cells has been extremely difficult. To overcome this obstacle, the NIH Common Fund 4D Nucleome ( ) program is developing new tools to explore nuclear organization in relation to cell function and human health. 4DN-funded investigator Dr. Andrew Belmont and a team of researchers have developed a new technique called “TSA-seq” that can measure the positions of genes in the nucleus relative to nuclear landmarks such as the nuclear lamina (surrounds the nucleus) or nuclear speckles (found in the center of the nucleus). The technique works by targeting an enzyme called “horseradish peroxidase” to a specific nuclear structure. The enzyme generates reactive molecules that label the surrounding DNA, with DNA closer to the enzyme being more heavily labeled. The DNA is isolated and sequenced, and the amount of labeling at each gene can then be used to calculate how close each gene is to the tagged nuclear structure. This information can be used to build a genome-wide 3D picture of nuclear organization. Combining TSA-seq with measurements of gene expression showed that nuclear speckles tend to be “hot zones” of gene activity, with more of the genes close to the nuclear speckles being active. This finding suggests that even small changes in the position of a gene, that move the gene closer to or farther from a nuclear speckle, could have significant consequences on gene expression and cell function.
In the News:, Science Daily
Turning on Genes is No Lonely Job
At any given time, only some of the in our DNA are turned on, or “active,” while others are turned off. Making sure only the correct genes are turned on in the correct cells is critical for our health. Scientists aim to understand how in cells coordinate to turn a gene on, which could help identify strategies for treating improper regulation of gene activity. Two -funded studies recently published in the journal Science explore the protein interactions that help turn a gene on. Both studies found that proteins cluster together at regions in the DNA called enhancers and interact to trigger gene activation.
In one study, Dr. Ibrahim Cisse and a team of researchers used live-cell super-resolution microscopy to view single molecules of proteins in mouse cells. They were able to view the interactions of a protein complex called Mediator, which helps kick-start, and RNA polymerase II, the protein that carries out transcription by copying DNA into . They found that both Mediator and RNA polymerase II group into stable clusters forming liquid-like droplets, a process known as phase-separation. Protein interactions were found to be brief, with proteins able to move in and out of the droplets, and droplets able to fuse together. They propose that Mediator droplets cluster at enhancers and fuse with RNA polymerase II droplets, allowing interactions between Mediator and RNA polymerase II that spur transcription to turn on genes.
In another study, led by Dr. Robert Tijan and Dr. Xavier Darzacq, the research team used live-cell single-molecule imaging to explore how proteins called transcription factors bind to the DNA enhancer and interact to initiate gene activation. They found that transcription factors also form high-concentration clusters that localize at the enhancer to stabilize DNA binding, recruit RNA polymerase II, and activate transcription. The interactions between transcription factors and RNA Polymerase II were rapid, reversible, and selective, making them a potential class of drug targets for regulating the process of gene activation.
. Cho, WK, Spille, JH, Hecht, M, Lee, C, Li, C, Grube, V, and Cisse, II. Science 361, 412-415. 2018 July 27.
. Chong, S, Dugast-Darzacq, C, Liu, Z, Dong, P, Dailey, GM, Cattoglio, C, Heckert, A, Banala, S, Lavis, L, Darzacq, X, and Tijan, R. Science 361 (6400). 2018 July 27.
In the News:
It may take a village (of proteins) to turn on genes, Science News
Classification of Career Pathways for Biomedical Trainees
A national movement is building to provide transparent information on career paths of biomedical graduates and postdoctoral alumni. One problem standing in the way is that institutions do not yet have an intuitive, complete, and replicable career classification (or “taxonomy”) that concisely and decidedly describes alumni career outcomes. To address this problem, several members of the NIH BEST consortium, Association of American Medical Colleges Graduate Research Education and Training (GREAT) group, and Rescuing Biomedical Research (RBR), collaborated to propose a three-tier career taxonomy. The first tier includes five career sectors (e.g. Government or For-Profit), the second tier includes five career types (e.g. primarily research or primarily teaching), and the third tier defines 24 job functions (e.g. administration or regulatory affairs). The developed classification is also suitable for use in other academic disciplines beyond the biomedical research fields.
Using a uniform classification across institutions in reporting outcomes of alumni has many advantages. It is valuable for comparing outcomes between institutions and providing data to potential applicants to help select institutions that may more closely match their career ambitions. Furthermore, being aware of the outcomes of trainees should help the training and mentoring community comprehensively aggregate, analyze, and disseminate information about career outcomes to guide professional development programs, teaching curricula, and possibly influence faculty opinions.
Importantly, many BEST institutions have already reported their data publicly for several years on websites and in publications using a similar taxonomy. Many schools outside the BEST consortium are doing this too (e.g. Stanford, University of Pennsylvania), and it is likely to become a standard in the field soon. Several NIH BEST consortium institutions are piloting the newly developed taxonomy for their institution’s doctoral alumni career outcomes and are contributing these data to populate a developing aggregated database. Combined data could be analyzed and reported to funding agencies, the public, science policymakers, and trainees to better understand and appreciate the full range of careers that Ph.D. trained scientists follow.
Evolution of a functional taxonomy of career pathways for biomedical trainees. Mathur, A., Brandt, P., Chalkley, R., Daniel, L., Labosky, P., Stayart, C., & Meyers, F. (2018) Journal of Clinical and Translational Science, 2(2), 63-65. doi:10.1017/cts.2018.22
Viewing the Moment a Gene Turns On
Genes are segments of our DNA that code for that determine our traits. Over 90% of our DNA does not encode genes and was long considered “junk” DNA that had no known purpose. We now know that much of this DNA does have a purpose, for example, some of this non-coding DNA contains enhancers- regions of DNA that help “turn on” genes to ultimately produce proteins. The timing of turning a gene on is very important for normal development and issues with timing can lead to development of disease. Enhancers are typically located far away from the gene they turn on, and how the enhancers find their target genes within the of the cell and how they interact with gene- coding regions to result in protein production is not well understood. In a recent study by -funded investigator Dr. Thomas Gregor and his research team, a live imaging approach was used to track the position of an enhancer and its target gene in developing fly embryos, while also monitoring gene activity. Using this technique, they were able to observe the moment when a gene was turned on. The results showed that close proximity between the enhancer and target gene was required not only to turn the gene on, but also to keep the gene active. When the enhancer disconnected from the target gene, the gene turned off. They also found that when the gene was turned on, the structure formed by the enhancer and target gene became more compact, and the results suggest changes in the 3D DNA arrangement improve the stability of this structure, allowing the gene to remain active. The results of this study improve our understanding of how gene activity is regulated and may help provide insight into how improper regulation of gene activity leads to developmental defects and disease.
Reference:. Chen, H, Levo, M, Barinov, L, Fujioka, M, Jaynes, JB, and Gregor, T. Nature Genetics. 2018.
In the news:
, Princeton University
, Genetic Engineering & Biotechnology News
Accelerating a Paradigm Shift
The behavior and function of individual cells in the body can vary greatly, even between cells that are very close together. These differences can play a role in determining health, disease, and therapy outcomes, making the ability to study single cells crucial. The NIH Common Fundlaunched in 2012 to speed up the discovery, development, and translation of approaches to analyze single cells in humans. A variety of new tools and technologies for single cell analysis were developed through the program. SCAP funding ended in 2017. During the period of SCAP funding, there was a significant increase in interest and funding for “single cell analysis” studies, indicating that the program accelerated research in the field. The technological advances made possible by SCAP will undoubtedly have a broad impact on health and disease research.
Variation in cells in human tissues can play a role in determining health, disease, and therapeutic outcomes, making the ability to analyze single cells critical. The NIH Common Fundlaunched in 2012 to speed up the discovery, development, and translation of approaches to analyze single cells in humans. SCAP particularly focused on technology development and a variety of single cell technologies were developed to analyze DNA sequence, DNA methylation, chromosome conformation, and chromatin state. Technology development evolved around three broad themes: droplet-based sequencing approaches, enhanced spatial resolution via fluorescent-based techniques, and barcoding techniques to multiplex microscopic approaches. In 2014, SCAP instituted a grand challenge, called “ ” to stimulate development of new tools for analyzing changes in individual cells over time. The winning project demonstrated a new nanopipette technology that can be used to repeatedly and non-destructively monitor the molecular properties of single cells over time. In addition, the program established the SCAP Transcriptome Consortium project, which developed a containing phenotypic information and whole transcriptome data from 56 human subjects. SCAP-developed resources have been used by the and the , among others. SCAP funding ended in 2017. The technological advances made possible by SCAP, particularly in single cell RNA-seq and multiplexed imaging combined with computational methods, will undoubtedly have a broad impact on health and disease research.
Reference: Accelerating a Paradigm Shift- the Common Fund Single Cell Analysis Program. Roy AL, Conroy R, Smith J, Yao Y, Beckel-Mitchener AC, Anderson JM, and Wilder EL. Science Advances. August 2018.
Exploring Unexplained Bowel Pain
Understanding the causes of inflammatory bowel disease is difficult because the associated abdominal pain can occur without any obvious changes to the structure of the colon or signs of inflammation. Because pain sensations can be carried by sensory neurons, one factor in the generation of abdominal pain could be abnormal sensory neuron activity, which can be influenced by secretions from cells called “epithelial cells” that line the interior of the colon. Understanding interactions between colon epithelial cells and sensory neurons could help us understand and treat abdominal pain. To study the interactions between the types of cells, a SPARC-funded team led by investigators Dr. Brian Davis and Dr. Kathryn Albers used genetically modified mice that contain blue light-activated colon epithelial cells to examine signaling between colon epithelial cells and neurons. These mouse colon epithelial cells could be specifically stimulated by blue light without any physical or chemical stimulation, allowing the study of their effects apart from other factors. Stimulating the epithelial cells caused activity in the pain-sensing neurons and behavioral responses similar to those that result from pain-inducing physical stimulation of the colon. Further study found that firing of neurons was likely triggered by release of a molecule called ATP from the colon epithelial cells. The results indicate that the activity of the colon epithelial cells alone, without any physical or chemical stimulation, could lead to abdominal pain through activation of the pain-sensing neurons. This study advances the understanding of how colon sensory neuron activity is regulated and could aid in development of new treatment strategies for inflammatory bowel disease.
Reference: Optogenetic Activation of Colon Epithelium of the Mouse Produces High-Frequency Bursting in Extrinsic Colon Afferents and Engages Visceromotor Responses. Makadia, PA, Naijar, SA, Saloman, JL, Adelman, P, Feng, B, Margiotta, J, Albers, KM, Davis, BM. June 2018. J Neurosci. 38(25): 5788-5798.
New Technique for 3D Genome Mapping
The 3D organization of DNA in the nucleus of a cell plays an important role in determining which genes are turned on in that cell. This has important implications for human health, as problems with DNA organization are linked to human diseases such as cancer and early aging. Understanding how the DNA is organized in healthy cells is a critical step in identifying targets and developing treatments for abnormal nuclear organization. A current method for mapping 3D genome organization uses a technique called “proximity ligation” in which regions of DNA that are very close together, or “touching,” are linked together and then sequenced to determine where these DNA “touches” occur. This technique mostly identifies interactions of DNA regions within the same chromosome. However, imaging of the genome using microscopy techniques has shown that there are interactions between chromosomes and that these interactions tend to occur at discrete regions of the nucleus known as nuclear bodies. This indicates limitations of proximity ligation techniques in identifying interactions between chromosomes that occur over longer-range distances. In addition, both proximity ligation and microscopy techniques are limited to measuring simultaneous contacts between a small number of DNA regions, making it difficult to develop a comprehensive model of global genome organization.
A recent study led by NIH Common Fund 4D Nucleome Program-funded investigator Dr. Mitchell Guttman, developed a new technique for detecting simultaneous genome-wide interactions within the nucleus, called Split-Pool Recognition of Interactions by Tag Extension (SPRITE). SPRITE works by linking interacting DNA, RNA, and proteins in cells, isolating the nuclei, fragmenting the chromatin, “barcoding” interacting molecules within a complex, and sequencing and matching the areas with identical “barcodes” to identify interacting regions. Unlike proximity ligation and microscopy techniques, SPRITE is not limited in the number of simultaneous DNA interactions that it can identify. Using SPRITE, they were able to detect interactions that occur across larger distances than those found by proximity ligation techniques. They found two “hubs” of interactions between chromosomes, both associated with nuclear bodies: an inactive gene-poor hub that organizes around the nucleolus and an active gene-rich hub that organizes around regions called “nuclear speckles.” Using the SPRITE results, they created a global model of 3D genome organization, in which nuclear bodies act as inter-chromosomal hubs that shape the 3D packaging of DNA in the nucleus.
Reference: Higher-order inter-chromosomal hubs shape three-dimensional genome organization in the nucleus. Quinodoz, SA, Ollikainen, N, Tabak, B, Palla, A, Schmidt, JM, Detmar, E, Lai, MM, Shishkin, AA, Bhat, P, Takei, Y, Trinh, V, Aznauryan, E, Russell, P, Cheng, C, Jovanovic, M, Chow, A, Cai, L, McDonel, P, Garber, M, and Guttman, M. Cell. 2018.
Using Sugars to Find Cancer Cells
Our cells are covered with a unique coating of various types of sugar molecules collectively called glycans. Glycans on the surface of diseased cells, such as cancer cells, are distinct from those of healthy cells. A glycan called sialyl-T is present in low levels in some normal cells but is present in higher levels on the surface of many types of cancer cells where it is attached to proteins on the cell surface, forming glycoproteins. The presence of sialyl-T on cell surfaces is believed to be correlated with tumor development and progression. This makes sialyl-T an important molecule for the recognition of cancer cells (a biomarker) and a potential target for treatment. However, sialyl-T has a complex structure and occurs at relatively low levels, even on cancer cells. The current two-step biochemical labeling methods for detecting sialyl-T are not sensitive or specific enough for detecting the presence of sialyl-T and analyzing the role of this glycan in disease.
In a recent study led by NIH Common Fund Glycoscience program awardee Dr. Peng George Wang, a new method was developed for labeling sialyl-T for visualization, quantification, and analysis. This simple method requires only one step and is more sensitive, specific, and rapid than existing methods of sialyl-T labeling. The new method uses an enzyme called ST6GalNAc-IV, which specifically recognizes sialyl-T and can attach another molecule to sialyl-T and its bound protein. ST6GalNAc-IV transfers a compound containing the molecule biotin onto the sialyl-T bound protein. Biotin can be used as a tag allowing sialyl-T glycoproteins to be visualized or captured and analyzed to identify unknown sialyl-T-glycoproteins. The authors were able to successfully identify 78 sialyl-T-linked proteins on the surface of human breast cancer cells and 43 sialyl-T-linked proteins on the surface of human colon cancer cells using cells grown under laboratory conditions. This method could speed up the study of sialyl-T in a variety of biological processes such as cancer progression. The next step of this research will be to test the labeling method in living animals.
Reference: A One-Step Chemoenzymatic Labeling Strategy for Probing Sialylated Thomsen-Friedenreich Antigen. Wen, L, Liu, D, Zheng, Y, Huang, K, Cao, X, Song, J, and Wang, PG. ACS Central Science. Feb 23, 2018. DOI: 10.1021/acscentsci.7b00573.
In the News: Detecting the Sweet Biomarker on Cancer Cells, American Chemical Society First Reactions
No Llama? No Problem!
The importance of llamas, alpacas, camels, and other camelids in protein research is little-known, but their antibodies play a key role in solving protein structures. They also serve as a major obstacle and bottleneck to researchers where access to camelids is limited, and generating the desired antibody is time-consuming (often taking six months), expensive, and frequently doesn’t work. Now, a team of researchers including Andrew Kruse (2015), Aashish Manglik (2016), and Aaron Ring (2016), have created a synthetic library that uses yeast cells instead of camelids to create the essential antibodies. Camelids produce a unique class of antibodies called nanobodies that can bind to key proteins because of their much smaller size than regular antibodies. Nanobodies can lock a protein into a particular conformation, which is necessary in determining a protein’s structure. Instead of laboriously generating several milligrams of a target protein to use to inoculate a llama and hoping the llama’s immune system creates the desired antibodies, Kruse and Manglik’s team created a library of 500 million camelid antibodies using yeast cells. Each tube is like a miniature llama immune system. Researchers can label their protein of interest with a fluorescent tag and add it to the yeast library. Yeast with nanobodies that recognize and bind to the protein will glow and can be separated out using fluorescence-activated cell sorting. The yeast cells can then be sequenced to learn what the nanobodies are and E. coli bacteria used to grow more nanobodies. The entire process takes three to six weeks. The system was tested on the beta-2 adrenergic receptor and adenosine receptor and successfully and robustly bound nanobodies to their target receptors. Kruse and Manglik are offering the yeast nanobody library free of charge to any interested nonprofit labs and hope their platform will accelerate discoveries and eliminate the need of llamas for protein research.
Reference: Yeast Surface Display Platform For Rapid Discovery of Conformationally Selective Nanobodies. McMahon C, Baier AS, Pascolutti R, Wegrecki M, Zheng S, Ong JX, Erlandson SC, Hilger D, Rasmussen SGF, Ring AM, Manglik A, Kruse AC. Nature Structural & Molecular Biology. 2018 Feb 12. doi: 10.1038/s41594-018-0028-6.
In the News:
- Camels and Alpacas Have Special Antibodies. Now Researchers Can Make Them With Yeast
- Researchers Produce Alpaca Antibodies Using Yeast
- No Llamas Required
Speedy Sugar Binding Analysis
Glycans are types of sugars that play important roles in the function of our bodies. They modify proteins and lipids to form more complex molecules, and they can be recognized and bound specifically by glycan-binding proteins and antibodies. These protein-glycan interactions play critical roles in many cellular functions such as cell signaling, initiation of viral infection, the development of cancer, and initiation of the immune response. It has been difficult for scientists to study glycans because of the limited tools available to detect and quantify them. Methods that do exist can be technically challenging, are limited in the number of glycans that can be analyzed, have long turn-around times, and can be expensive. This lack of appropriate analysis tools has hindered progress on the study of glycans and glycan-binding proteins for biomedical research, clinical diagnoses, and therapies. There is therefore an urgent need for improved tools and methods for rapidly analyzing large numbers of glycans and glycan-binding proteins.
In a study led by Common Fund Glycoscience program investigator Dr. Jin-Xiong She, a new tool was developed for the speedy analysis of interactions between glycans and proteins that bind to them. This new, highly sensitive and specific high-throughput platform is called “multiplex glycan bead array” (MGBA) and uses microbeads, with a specific glycan attached to each individual uniquely tagged bead. Scientists can read the array to determine which beads and their attached glycans bind to which proteins. Because there are so many beads on an array, this allows analysis of a protein’s ability to bind to hundreds of individual glycans and can be used to greatly speed up studies of glycan-binding proteins. This tool may be adapted for use in clinical tests, with the potential to identify diagnostic biomarkers for diseases such as cancer. The utility of this platform was illustrated by identifying a glycan-binding antibody protein that predicts the survival of patients with ovarian cancer. This method is inexpensive, simple enough to be carried out by non-specialists, and relatively quick (~4 hours), making it suitable for clinical use by those not extensively trained as glycan scientists.
Reference: Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins. Purohit, S, Tiehai, L, Guan, W, Song, X, Song, J, Tian, Y, Li, L, Sharma, A, Dun, B, Mysona, D, Ghamande, S, Rungruang, B, Cummings, RD, Wang, PG, and She, JX. Nature Communications. 2018 Jan 17. 9(1):258.
Packaging Chromosomes for Cell Division
As a cell moves through the cell cycle, the shape of the DNA in the nucleus changes dramatically, from a ball of DNA during normal cell activities to distinct X-shaped chromosomes as the cell prepares to divide. This change in shape is important for the orderly passage of one copy of the DNA to each new cell. Before the cells begin to divide, the DNA compacts into the dense X-shaped chromosomes that are made up of consecutive DNA loops. How the compaction of loops occurs is not well understood. One of the goals of the 4D Nucleome program is to determine how the structure of DNA in the nucleus of a cell changes over time.
In a recent study in Science, a team led by 4DN-funded researchers Dr. Leonid Mirny and Dr. Job Dekker combined techniques that are used to determine where regions of the genome “touch” with imaging and modeling techniques at one-minute time intervals to investigate how DNA rearrangement occurs before cell division. Based on their results, they propose a model in which cells use a protein called “condensin” to drive the compaction of DNA. Condensin proteins create DNA loops by pushing DNA through their ring-like structures. In this model, condensin I creates wide loops in the DNA that are then split into smaller loops by condensin II. The loops twist around a condesin scaffold in a structure resembling a spiral staircase, creating a condensed helix of consecutive DNA loops that makes up the X-structure of the chromosomes and results in formation of compact units of DNA that can be easily divided between new cells. Understanding how the organization of DNA changes throughout the cell cycle is critical for determining how problems with cell cycle-associated DNA rearrangement (such as chromosome breakage) lead to human diseases such as cancer.
Reference: A pathway for mitotic chromosome formation. Gibcus, JH, Samejima, K, Goloborodko, A, Samejima, I, Naumova, N, Nuebler, J, Kanemaki, MT, Xie, L, Paulson, JR, Earnshaw, WC, Mirny, LA, Dekker, J. Science. 2018 Jan 18. doi: 10.1126/science.aao6135.
In the news: Packing a Genome, Step-by-Step, Howard Hughes Medical Institute
This page last reviewed on January 17, 2019