National Institute Of General Medical Sciences Research Articles

Abstract 7252: Characterization of targeting peptide sequences for peptide-mediated delivery of siRNA in HER2+ breast cancer

Abstract Introduction: This work presents the characterization of two novel HER2 targeting peptides to measure which has the most compatible properties to be used as a siRNA carrier to knock down the CD44 gene in HER2+ breast cancer. Procedures: Targeting peptides were electrostatically complexed with non-targeting siRNAs at different N:P ratios to determine the minimum binding ratios needed to form nanoparticles. Different ratios of peptide-siRNA complexes were tested to determine the minimum molar ratio at which the peptides would protect siRNA from degradation. Cytotoxicity assays were performed to determine cytotoxicity levels at different peptide concentrations. Dynamic light scattering assay was performed to determine peptide-siRNA nanoparticle size and zeta potential. Immunofluorescent microscopy was used to determine the targeting of the peptide to HER2+ cells compared to cells not expressing HER2. Summary of Results: Peptide-siRNA complexes for the P51 and P25 peptides formed at a minimum 20:1 N:P ratio. P25 and P51 peptides both successfully protects the siRNA at N:P ratios from 20:1 to 60:1 for both degradation agents. Cytotoxicity assays show that in the SKBR3 cell line, the P25 and P51 peptides both showed above 80% viability until the 70:1 ratio; however, in the BT474 cell line, the P25 showed above 80% viability to the 50:1 ratio, but the P51 was only viable in the 20:1 ratio. The results of these studies illustrate how both peptides protect siRNA but that the P51 peptide is toxic to some HER2+ breast cancer cell lines. Conclusions: Results show that both peptides target HER2+ breast cancer and are compatible carriers of siRNA, protecting the siRNA well within a cell-like environment. However, from MTS studies, P25 appears less cytotoxic to the cell lines than P51, which shows higher cytotoxicity at high molar ratios. We observed that the P25 protects siRNA from degradation, is cytocompatibility, and promotes efficient internalization into HER2+ cell lines. Future work will include the development of a tandem peptide, including a targeting peptide fused with a fusogenic peptide to increase cancer targeting and efficacy of this targeted treatment. Acknowledgments: This research was supported in part by the Dabo Swinney All-In Foundation as well as Materials Assembly and Design Excellence in South Carolina (MADE in SC) under the National Science Foundation EPSCoR Program under NSF Award #OIA-1655740. Data presented in this material was partially collected using SC BioCRAFT facilities supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number P30 GM131959. Citation Format: Audreanna Miserendino, Sevrina Tekle, James Kalogeros, Angela Alexander-Bryant. Characterization of targeting peptide sequences for peptide-mediated delivery of siRNA in HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7252.

Abstract 5390: Exploring signaling pathways of tumor-nerve interactions in prostate and breast cancer

Abstract A significant portion of prostate cancer (PCa) and breast cancer (BCa) patients develop bone metastatic disease. In some cases, cancer spreads through the nervous system, a process known as perineural invasion (PNI). Neurite outgrowth is believed to be a precursor to PNI. Snail is an important gene which regulates the epithelial-mesenchymal transition (EMT) process in which tumor cells at the invasive front undergo this transition to promote invasion, migration, and subsequent metastasis. We recently published that Snail promotes neurite outgrowth in PCa cells. We hypothesize that Snail expression will stimulate neurite outgrowth in both PCa and BCa cells through extracellular vesicles from cancer cells interacting with neurons. To test this hypothesis, we first collected conditioned media from PCa and BCa cells expressing Snail and isolated exosomes. Exosomal markers were detected using western blot to confirm the isolated exosomes. Additionally, we used Transmission Electron Microscopy (TEM) to observe the secretion of exosomes. Proteomics was performed to analyze proteins expressed within exosomes of LNCaP Snail overexpressing or C4-2 Snail knockdown PCa cells. Our results showed that Snail-expressing cells secrete exosomes containing Talin1, specifically, the proteolyzed C-terminal rod domain, while full-length Talin1 is found in whole cell lysates from Snail-expressing cells. Talin1 has previously been associated with neurite outgrowth. Neurite outgrowth assays were then performed using conditioned medium collected from C4-2 PCa cells with Snail knockdown and MCF-7 BCa cells with Snail overexpression. Increased neurite outgrowth is observed in PC-12 cells and NPC (Induced Pluripotent Stem Cells differentiated neuronal progenitor cells) when cultured with conditioned medium collected from PCa and BCa cells expressing high levels of Snail. AKT activation was observed in the neuronal cells in response to conditioned media from Snail-expressing cells. Furthermore, we found that mH4, a proprietary small molecule inhibitor of Talin1, reduces Snail-mediated neurite outgrowth and AKT activation (p-AKT Thr308 and Ser473). Overall, we have uncovered a pathway whereby Snail transcription factor promotes secretion of exosomes containing Talin1 which promotes AKT signaling in neuronal cells and neurite outgrowth. Talin1 small molecule inhibitor shows promise for therapeutic targeting of tumor-nerve interactions. Acknowledgements: The authors acknowledge the use of core facilities supported by the National Institute on Minority Health and Health Disparities through grant number 5U54MD013376 and 1U54GM128729-01-DaCCoTA Scholars Program (PI: Rezvani) National Institute of General Medical Sciences (NIGMS). Citation Format: Bor-Jang Hwang, Gabrielle J. Edwards, Cole Davis Knoblich, Khosrow Rezvani, Valerie Odero-Marah. Exploring signaling pathways of tumor-nerve interactions in prostate and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5390.

The potential epidemiologic, clinical, and economic value of a universal coronavirus vaccine: a modelling study

With efforts underway to develop a universal coronavirus vaccine, otherwise known as a pan-coronavirus vaccine, this is the time to offer potential funders, researchers, and manufacturers guidance on the potential value of such a vaccine and how this value may change with differing vaccine and vaccination characteristics. Using a computational model representing the United States (U.S.) population, the spread of SARS-CoV-2 and the various clinical and economic outcomes of COVID-19 such as hospitalisations, deaths, quality-adjusted life years (QALYs) lost, productivity losses, direct medical costs, and total societal costs, we explored the impact of a universal vaccine under different circumstances. We developed and populated this model using data reported by the CDC as well as observational studies conducted during the COVID-19 pandemic. A pan-coronavirus vaccine would be cost saving in the U.S. as a standalone intervention as long as its vaccine efficacy is≥10% and vaccination coverage is≥10%. Every 1% increase in efficacy between 10% and 50% could avert an additional 395,000 infections and save $1.0 billion in total societal costs ($45.3 million in productivity losses, $1.1 billion in direct medical costs). It would remain cost saving even when a strain-specific coronavirus vaccine would be subsequently available, as long as it takes at least 2-3 months to develop, test, and bring that more specific vaccine to the market. Our results provide support for the development and stockpiling of a pan-coronavirus vaccine and help delineate the vaccine characteristics to aim for in development of such a vaccine. The National Science Foundation, the Agency for Healthcare Research and Quality, the National Institute ofGeneral Medical Sciences, the National Center for Advancing Translational Sciences, and the City University of New York.

A 10 Year Analysis of Gender Distribution in National Institutes of Health Funding for Non-Malignant Hematology

Introduction: Historically, there has been an underrepresentation of women in the field of hematology. Consequently, this makes them less likely to receive academic research grants. Here, we present a ten year retrospective analysis of the Research Project Grants (R01) awarded by the National Institutes of Health (NIH) through the fiscal years of 2012-2022. The grants were categorized by various NIH agencies. This temporal analysis examines gender disparities under the field of non-malignant hematology research. Methods:Data was extracted for the fiscal years 2012-2022 focusing exclusively on R01 grants awarded and categorized by the various NIH agencies. Data was leveraged using the NIH RePORTER tool, Tidyverse, and janitor packages in R. The dataset incorporated key grant parameters such as grant ID, agency code, activity code, abstract text, project title, fiscal year, activity status, award amount, organization, and principal investigator's(PI) name. In order to distinguish a PI's gender, PI's first names were processed using the gender package in R, which provided a gender-wise distribution of recipients. For statistical analyses, proportions of females with accepted R01 grants were compared between 2012 to 2022; this proportion analysis was also done among each NIH agency. Further, linear regression and associated statistical tests were used to identify whether there was significant change in grants received by either gender over this time period. Results: A total of 250,031 R01 grants were awarded by the NIH during the fiscal years of 2012 to 2022. Females (n=82,152: 32.9% (95% CI 0.41-0.43) received fewer grants than males (n=167,879: 67.1%, (95% CI 0.50-0.55). Across 2012 to 2022, there was no significant change in R01 grants awarded among men (16,221 to 15,601, p-value= 0.52). In contrast, there was a significant difference for females in the R01 grants awarded across 2012 to 2022 (6,865 to 9,339, p-value <0.001). In 2012, the National Institute of General Medical Sciences (NIGMS), National Institute of Neurological Disorders and Stroke (NINDS), and National Institute of Biomedical Imaging and Bioengineering (NIBIB) awarded less than 25% of the total grants to females. By 2022, of all the agencies, the NBIB remained the agency with the most extensive gender gap with females being awarded only 23% of grants. In contrast, the National Institute of Minority Health and Disparities (NIMHD) and the National Institute of Nursing Research (NINR) were the only agencies in 2012 to award more grants to females with approximately 52% and 74% of grants respectively. In 2022, the NINR and NIHMD continued to show more grants awarded to females versus males. In addition to those agencies, by 2022, the National Institute of Child Health and Human Development(NICHD) and National Center for Complementary and Integrative Health (NCCIH) joined the NINR and NIHMD in awarding more than 50% of grants awarded to females that year. Conclusion: This analysis highlights continued gender disparity as only 33% of total R01 NIH grants were awarded to females during the fiscal years 2012-2022 in non-malignant hematologic research. Although, it is noted that while some agencies have made strides towards gender parity, others continue to have a significant gap. While this remains a systemic and multifaceted issue, identifying these areas of gender disparity will enable targeted efforts to bridge this gap and advance gender equality.

Disparate natural killer T cell subsets exhibit opposing regulatory effects on the cytokine storm.

Abstract Inflammation is both a critical component of the immune system for effective clearance of microbial challenges and a mechanism by which some pathogens cause disease, e.g., tularemia, dengue fever, and COVID-19. The bacterium Francisella tularensis (Ft), the causative agent of tularemia, elicits a cytokine storm in which the immune system over-responds to the bacterium, releasing massive amounts of proinflammatory cytokines. Untreated, tularemia can reach a mortality rate as high as 30% in healthy adults with inhalation of as few as 10 colony forming units inducing respiratory tularemia. While Ftis spread from zoonotic sources in nature, the ease of aerosol spread and high mortality led to its weaponization and continued concern for bioterrorism applications. Natural Killer T (NKT) cells are important immune cells with the capability of modulating the inflammatory response. NKT cells are activated early following Ftinfection suggesting a role in the innate immune response in tularemia. NKT cells are categorized into two distinct subsets, type I and type II based on their antigen recognition, or not, of α-galactosylceramide. Literature suggests that these subsets have opposing regulatory phenotypes, however, a clear understanding of their roles in regulating inflammation remains unknown and an emerging frontier in NKT research. Our lab has observed opposing roles in suppressing the cytokine storm by NKT subtypes. Ongoing studies will elucidate the mechanism utilized to influence the cytokine storm. This project will yield a better understanding of NKT cell-mediated inflammation during Ftinfection and, more broadly, characterize their involvement and potential for therapeutic targeting for a number of NIH priority pathogens. Supported by grants from National Institute of General Medical Sciences (NIGMS) Research Initiative for Scientific Enhancement (RISE) grant R25 GM 06921-18

STAT1 gain of function mutation impairs immune response to viral infections

Abstract The coordinated effort of the immune system in host defense is dependent upon the proper release and interpretation of different cytokine signals. Several examples of monogenic diseases targeting the JAK-STAT pathway reveal there is still a critical need to understand basic principles of cytokine signaling and cytokine output. For example, patients with STAT1 gain-of-function (GOF) mutations exhibit a type 1/IFN-gamma bias that antagonizes a type 3/IL-17 immune response important for controlling fungal infection, yet patients also, paradoxically, exhibit chronic and sometimes lethal viral infections. Using a novel conditional knock in STAT1-GOF mouse model, we demonstrate that STAT1-GOF mice respond poorly to viral infections that are easily controlled by WT mice, such as MCMV and LCMV-Armstrong. High-dimensional flow cytometry and scRNA-seq reveal an impaired NK and CD8 T cell effector response in STAT1-GOF mice that is not simply due to an exhaustion phenotype. Instead, STAT1-GOF mice respond poorly to viral infections due to an unexpected defect in IFN-gamma production early during the innate immune response, leading to a cytokine storm, immunopathology, and an impaired adaptive immune response. scATAC/RNA-seq uncovers altered usage of different STAT complexes in STAT1-GOF mice, leading to a misinterpretation of early innate-stimulating cytokines. These results provide insight into why patients with interferonopathies or elevated interferons respond poorly to viral infections and highlight the importance of properly tuning STAT1 during an immune response. R.L.P. was supported by a Postdoctoral Research Associate (PRAT) fellowship from the National Institute of General Medical Sciences (NIGMS), award number 1Fi2GM137942-01

Telemedicine in Neurology: Lessons Learned During the COVID-19 Pandemic (P6-7.002)

<h3>Objective:</h3> Describe barriers and facilitators to telemedicine in neurology, and to examine the impact of telemedicine on neurologists’ job satisfaction. <h3>Background:</h3> Telemedicine facilitates patients access to specialized care and lowers patients physical and financial burden of traveling to appointments. But telemedicine is underutilized in neurology. Thus, little is known about neurologists’ experiences with telemedicine. Increased patients’ access via telemedicine may increase workload on neurologists, leading to increased stress and burnout. Exploring neurologists’ experiences with telemedicine will inform optimizing telemedicine in neurology. <h3>Design/Methods:</h3> A cross sectional study via a survey examined telemedicine related technological difficulties, need for technological support (IT), administrative support, neurologists’ motivation for work, change in work-life balance, and issues to improve telemedicine in neurology. Descriptive statistics were reported as percentages. <h3>Results:</h3> 110 neurologists participated. Specialties were stroke (21.82%), epilepsy (19.09%), movement disorders (14.55%), and headache (14.55%). (50.91%) of neurologists encounter technological issues in 1%–20% of telemedicine visits. And 94.55% of neurologists rated patient-related technological issues in setting up telemedicine visit is the most common difficulty encountered. Furthermore, 57.27% of neurologists required IT support in 1%–20% of telemedicine visits. 39.09% of neurologists rated improving administrative support, 37. 27% rated integration of video-telephone calls in EMR and 27.27% of neurologists rated facilitating ordering tests as the most important issues in telemedicine in neurology. On motivation for work, 48.18% of neurologists reported no change in their motivation, 30.91% of neurologists reported somewhat decreased motivation, and only 10.00% reported somewhat increased motivation. On work-life balance, 30.00% of neurologists stated it remained the same, 26.36% stated it somewhat decreased and 23.64% stated somewhat improved work-life balance. <h3>Conclusions:</h3> Technological difficulties in telemedicine are common. Improving administrative support and integration of virtual platforms in EMR are potential areas to optimize telemedicine visits. More studies are needed to examine the impact of telemedicine on neurologists’ burnout. <b>Disclosure:</b> Dr. Al-Faraj has nothing to disclose. Mr. Mohtar has nothing to disclose. Mr. Chen has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for ParaDocs Health. Mr. Jha has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for ParaDocs Health. Miss LeClair has received research support from The National Institute of General Medical Sciences (NIGMS) Interdisciplinary Training Grant for Biostatisticians (T32 GM74905).

Palmitic acid reprograms inflammatory responses to microbial ligands in macrophages, and mediates innate immune memory in vivo.

Abstract Dietary saturated fatty acids (SFAs) modulate circulating lipids in mice and humans, which can alter the inflammatory capacity of innate immune cells. The most common SFA in human serum, palmitic acid (PA), is enriched in Western diets, and while it is known to induce an inflammatory response in macrophages, the impact of PA on responses to pathogenic stimuli is unknown. We developed an in vitro model to determine the impact of PA on the macrophage response to lipopolysaccharide (LPS). We find that bone marrow-derived macrophages (BMDMs) exposed to PA for 12–24 hours exhibit significantly enhanced TNF, IL-6 and IL-1β secretion following LPS exposure. BMDMs treated simultaneously with PA and the unsaturated fatty acid oleic acid (OA) show a reversal of this PA-induced hyper-inflammatory response to LPS, suggesting that OA negatively regulates the memory-like feature of PA. While monocytes and splenocytes from mice fed a PA-enriched diet exhibit unaltered basal inflammation, ex vivo LPS treatment leads to significantly enhanced pro-inflammatory cytokine expression. Mice exposed to PA for 9 days, followed by 7 days without PA, exhibit increased LPS-induced endotoxemia severity and mortality, and enhanced mRNA expression of tnf, il-6, and il-1β in blood. Rag−/− mice exposed to PA for 12-hours showed significantly reduced kidney fungal burden after Candida albicans infection. We hypothesize that PA induces a long-lasting innate immune memory program in macrophages, and reprograms their response to subsequent inflammatory challenge via metabolic and epigenetic alterations. Our findings are the first to suggest dietary PA regulates innate immune memory and is detrimental in an acute septic response, yet beneficial for pathogen clearance. Supported by National Institute of General Medical Sciences (NIGMS) 525 grant 5R35GM133804-02 to B.A.N.

Transcription factor profiling reveals activation of Type II Natural Killer T cell effector functions in response to Francisella tularensis infection.

Abstract The bacterium Francisella tularensis (Ft) is the causative agent of human tularemia. When untreated, tularemia can reach a mortality rate as high as 30% in healthy adults with inhalation of as few as 10 colony forming units (CFU) inducing respiratory tularemia. While Ft is spread from zoonotic sources in nature, the ease of aerosol spread and high mortality in respiratory form led to its weaponization and concern for bioterrorism. Exposure to Ft elicits the cytokine storm, in which the immune system over-responds to the presence of the bacterium releasing massive amounts of proinflammatory and regulatory cytokines. Natural Killer T (NKT) cells are important immune cells that have both activating and inhibitory effector mechanisms. NKT cells are activated very early following Ft infection suggesting a critical role in the innate immune response to the bacterium. NKT cells are categorized into two distinct subsets based on their antigen recognition of CD1d-tetramers loaded with α-galactosylceramide (αGalCer-tetramer), though their individual characteristics in regulating inflammation are ill-defined. Type I NKT cells, similar to CD4+ αβ T helper cells, have been shown to express varied transcription factor (TF) profiles, e.g., T-bet, GATA3, and RORγT, contributing to either Th1, Th2 or Th17 phenotypes. It is not currently known whether this same TF profile classification exists within type II NKTs. The purpose of this research project is to investigate the activation of the type II NKT cell subset and characterize their TF profiles in response to Ft infection. This data will lead to a better understanding of the role of type II NKT cells in Francisella-mediated immunity. Supported by National Institute of General Medical Sciences (NIGMS) Research Initiative for Scientific Enhancement (RISE) grant R25 GM 06921-18

Citrullination of a phage-displayed human peptidome library reveals the fine specificities of rheumatoid arthritis-associated autoantibodies.

BackgroundPost-translational modifications (PTMs) on proteins can be targeted by antibodies associated with autoimmunity. Despite a growing appreciation for their intrinsic role in disease, there is a lack of highly multiplexed serological assays to characterize the fine specificities of PTM-directed autoantibodies.MethodsIn this study, we used the programmable phage display technology, Phage ImmunoPrecipitation Sequencing (PhIP-Seq), to profile rheumatoid arthritis (RA) associated anti-citrullinated protein antibody (ACPA) reactivities.FindingsUsing both unmodified and peptidylarginine deiminase (PAD)-modified phage display libraries consisting of ~250,000 overlapping 90 amino acid peptide tiles spanning the human proteome, PTM PhIP-Seq robustly identified antibodies to citrulline-dependent epitopes.InterpretationPTM PhIP-Seq was used to quantify key differences among RA patients, including PAD isoform specific ACPA profiles, and thus represents a powerful tool for proteome-scale antibody-binding analyses.FundingThis research is based upon work supported in part by the Office of the Director of National Intelligence (ODNI), Intelligence Advanced Research Projects Activity (IARPA). The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of ODNI, IARPA, or the US Government. The US Government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright annotation therein. This study was made possible by a National Institute of General Medical Sciences (NIGMS) grant R01 GM136724 (HBL). MFK was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) grant T32AR048522. ED was supported by the Rheumatology Research Foundation.

An assessment tool for evaluating the National Institute of General Medical Sciences COBRE and INBRE mentoring programs

The National Institute of General Medical Sciences (NIGMS) mandates that its Centers of Biomedical Research Excellence (COBRE) and Institutional Development Award Networks of Biomedical Research Excellence (INBRE) institute formal mentoring programs to promote the core program objective of junior investigator development. Despite this NIGMS requirement, and the many career-related benefits associated with mentoring, few tools exist for purposes of rigorously evaluating COBRE and INBRE mentoring programs. The purpose of this project was to develop a mentoring assessment tool to aid in the evaluation of COBRE and INBRE mentoring programs. In study 1, a list of items comprising the tool was created via a multiphase item generation process based on input received from subject matter experts within the Cognitive and Neurobiological Approaches to Plasticity Center. In study 2, feedback about this tool was solicited from 78 grant directors, mentees, and mentors representing 21 unique COBRE programs and 8 unique INBRE programs from across the United States. The results provide initial evidence that this tool possesses suitable psychometric properties, is a flexible instrument with many potential uses, and represents a valuable resource for helping evaluate COBRE and INBRE mentoring programs. Having a tool for evaluating mentoring can help promote the grant success and career development of junior investigators in COBRE and INBRE programs and help program directors develop more sustainable research centers.

Phylogenetic Analysis and Molecular Evolution (PAME)

In the fall of 2009 and in the spring of 2012, supported by the National Institute of General Medical Sciences (NIGMS) in the National Institutes of Health (NIH), we designed a course “Phylogenetic Analysis and Molecular Evolution” (PAME), the first cross-listed course across three different colleges (College of Arts and Sciences, College of Engineering, and College of Agriculture). This course was the first at the author's university. The course will be made available to graduate students and advanced undergraduate students in biology and computer science, and undergraduate student in statistics and mathematics at regional universities and colleges. The graduate students will learn how to gather sequence data, use bioinformatics tools, and analyze biological data using mathematics and statistics throughout the project. In this paper, we propose an online course on PAME facilitated with the statistical software RStudio on Amazon Web Service (AWS).

Development of Privacy Features on Anecdata.org, a Free Citizen Science Platform for Collecting Datasets for Climate Change and Related Projects.

The Anecdata website and its corresponding mobile app provide unique features to meet the needs of a wide variety of diverse citizen science projects from across the world. The platform has been developed with the help of continuous feedback from community partners, project leaders, and website users and currently hosts more than 200 projects. Over 8,000 registered users have contributed more than 30,000 images and over 50,000 observations since the platform became open to the public in 2014. From its inception, one of the core tenets of Anecdata’s mission has been to make data from citizen science projects freely accessible to project participants and the general public, and in the platform’s first few years, it followed a completely open data access model. As the platform has grown, hosting ever more projects, we have found that this model does not meet all project needs, especially where endangered species, property access rights, participant safety in the field, and personal privacy are concerned. We first introduced features for data and user privacy as part of “All About Arsenic,” a National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) Science Education Partnership Award (SEPA)-funded project at MDI Biological Laboratory, which engages middle and high school teachers and students from schools across Maine and New Hampshire in sampling their home well water for analysis of arsenic and other heavy metals. In order to host this project on Anecdata, we developed features for spatial privacy or “geoprivacy” to conceal the coordinates of samplers’ homes, partial data redaction tools we call “private fields” to withhold certain sample registration questions from public datasets, and “participant anonymity” to conceal which user account uploaded an observation. We describe the impetus for the creation of these features, challenges we encountered, and our technical approach. While these features were originally developed for the purposes of a public health and science literacy project, they are now available to all project leaders setting up projects on Anecdata.org and have been adopted by a number of projects, including Mass Audubon’s Eastern Meadowlark Survey, South Carolina Aquarium’s SeaRise, and Coastal Signs of the Seasons (SOS) Monitoring projects.

To train the qualified physician-scientists

As advances in medicine have progressed, the development of medical science faces the challenge of significant separation between basic medical research and medical clinical practice. As the bridge between medicine and science, physicians-scientists have a unique potential to combine clinical perspectives with scientific insights. They are defined as doctors with professional degrees who have training in clinical care and are engaged in independent biomedical research. Physician-scientists continue the great pace of recent biomedical research, translate scientific findings to clinical applications and play a vital role in promoting the development of medical science. Without supporting efforts of physician-scientists who have been systematically trained in medicine and science, pressing challenges cannot be met at the forefront of biomedicine. Given the importance of developing the physician-scientist workforce, well-known MD-PhD training programs started in the 1950s. These programs expanded significantly when the Medical Scientist Training Program (MSTP) was launched in 1964 by the National Institute of General Medical Sciences (NIGMS) at the National Institutes of Health. MSTP has supported over 10000 students who have successfully trained outstanding biomedical scientists and top talents for the United States. In China, the era of health promotion driven by innovation is upon us, and a large number of physician-scientists are needed to meet the development of medical science. Some practices provide preliminary exploration for training physician-scientists, such as MD-PhD programs supported by famous medical schools and the inception of a research hospital. However, physician-scientists have become an “endangered species”. It has been reported that physician-scientists are less likely to play a major role in biomedical research than they were in the past in the United States. This article examines the barriers of physician-scientists with regard to participation in clinical research, such as physician-, research subject-, and institution- and research hospital-related barriers. The future viability and vitality of the physician-scientist workforce pose unique challenges. To encourage physician-scientist participation, strategies are proposed to provide a new path for cultivating excellent physician-scientists. These strategies include proper understanding of the role of physician-scientists in medical innovation, scientific training systems at critical stages of career development, adequate research support from institutions and hospitals, reasonable evaluation and incentive systems, encouraging innovative academic culture, and personal active participation and effort. The great potential of translating scientific achievements into clinical practice and the complex challenges facing the development of medical science suggest the importance and necessity of physician-scientists. A variety of potential approaches should be taken to ensure physicians-scientists’ participation in clinical trials and basic research as well as the sustainable development of the physician-scientist team.

SARS-CoV-2 Variants of Concern Have Acquired Mutations Associated With an Increased Spike Cleavage

Several SARS-CoV-2 lineages have emerged leading to the divergence of more transmissible variants termed Variants of Concern (VOCs). The natural selection of mutations in the spike protein can impact viral cell entry, transmission, and pathogenesis. Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to demonstrate that the substitution S:655Y, included in the highly prevalent Gamma variant, enhances viral replication and spike protein cleavage. Moreover, viral competition experiments demonstrate that the S:655Y transmits more efficiently than the ancestor 655H in the hamster model. Finally, we analyze a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibit an increased spike cleavage and fusogenic capacity. This study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-CoV-2 VOCs show a convergent evolution that promotes the spike protein processing.Funding Information: This research was partly funded by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), a NIAID funded Center of Excellence for Influenza Research and Response (CEIRR, contract #75N93021C00014) (AGS), NCI SeroNet grant U54CA260560 (AGS), NIAID grants U19AI135972 and U19AI142733 (AGS), DARPA grant HR0011-19-2-0020 (AGS), JPB Foundation (AGS), Open Philanthropy Project (research grant 2020-215611 (5384) (AGS), anonymous donors to AGS, NBAF Transition Funds from the State of Kansas (JAR), NIAID Centers of Excellence for Influenza Research and Surveillance under contract number HHSN 272201400006C (JAR), AMP Core of the Center for Emerging and Zoonotic Infectious Diseases of the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number P20GM130448 (JAR) and Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases under grant number HSHQDC 16-A-B0006 (JAR). AGR is funded by Marion Alban MSCIC Scholars Award and the 2020 Robin Chemers Neustein Postdoctoral fellowship. ML is funded by a fellowship of the Belgian American Education FoundationDeclaration of Interests: The A.G.-S. laboratory has received research support from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, N-fold LLC, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer and Merck, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories and Pfizer, outside of the reported work. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections, owned by the Icahn School of Medicine at Mount Sinai, New York. The Icahn School of Medicine at Mount Sinai has filed a patent application relating to SARS-CoV-2 serological assays, which lists Viviana Simon as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. All other authors have nothing to declare. Ethics Approval Statement: Human SARS-CoV-2: Nasopharyngeal swab specimens were collected as part of the routine SARS-CoV-2 surveillance conducted by the Mount Sinai Pathogen Surveillance program (IRB approved, HS#13-00981).All hamster animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Icahn School of Medicine at Mount Sinai (ISMMS).The Institutional Animal Care and Use Committee (IACUC) of the Icahn School of Medicine at Mount Sinai (ISMMS) reviewed and approved the mink model of COVID-19.

Remdesivir is a Delayed Translocation Inhibitor of SARS CoV-2 Replication

Remdesivir is a nucleoside analog approved by the FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryoEM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of three copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of three bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugsFunding: This work was supported in part by Welch Foundation grantsF-1604 (to K.A.J.) and F-1938 (to D.W.T.), National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) R01AI110577 (to K.A.J) and National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) R01114223 (to K.A.J.) and R35GM138348 (to D.W.T.), Army Research Office Grant W911NF-15-1-0120(to D.W.T.), and a Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation Medical Research Award (to D.W.T.). D.W.T is a CPRIT Scholar supported by the Cancer Prevention andResearch Institute of Texas (RR160088) and an Army Young Investigator supported by the Army Research Office (W911NF-19-1-0021).Conflict of Interest: All authors declare no competing interests.

Developing a culture of safety in biomedical research training.

The National Institute of General Medical Sciences (NIGMS) at the U.S. National Institutes of Health (NIH) is committed to supporting the safety of the nation’s biomedical research and training environments. Institutional training grants affect many trainees and can have a broad influence across their parent institutions, making them good starting points for our initial efforts to promote the development and maintenance of robust cultures of safety at U.S. academic institutions. In this Perspective, we focus on laboratory safety, although many of the strategies we describe for improving laboratory safety are also applicable to other forms of safety including the prevention of harassment, intimidation, and discrimination. We frame the problem of laboratory safety using a number of recent examples of tragic accidents, highlight some of the lessons that have been learned from these and other events, discuss what NIGMS is doing to address problems related to laboratory safety, and outline steps that institutions can take to improve their safety cultures.

Assessing the Global Impact of an Adenylate Kinase Isoenzyme on AMPK and mTOR Signaling Pathways Through Metabolic Profiling in both Brain and Lung Cancer Cell Lines

In a variety of human cancers, it is well observed that specific altered bioenergetic programs must be sustained in order to maintain the cancer cell phenotype. Well‐described genes play a role in sustaining energy flux for the cancer cell and recent studies add the regulation of AK4 expression, an adenylate kinase localized to the mitochondrial matrix, to the list. Our results demonstrate that in response to altered AK4 expression in cancer cells, intracellular ATP levels drastically increase. Secondly, we demonstrate that in response to perturbed AK4 expression in cancer cells, two critical homeostatic signaling pathways, the 5′ AMP‐activated protein kinase (AMPK) and a mammalian target of rapamycin (mTOR) pathways, are significantly altered. Both mTOR and AMPK proteins govern a metabolic pathway in which they sense cellular energy levels and nutrient status in response to regulate cancer cell growth and/or proliferation. Lastly, AK4 also has an impact on other bioenergetic pathways. For instance, AK4 can catalyze the reaction GTP + AMP ↔ GDP + ADP24 and sustain critical energy flux of nucleotides in the cell. Because AK4 expression appears to regulate cellular metabolic responses at multiple levels, further analyses by obtaining a metabolomic profile is necessary to highlight the global impact AK4 exerts on all the metabolites in the cancer cell. To do this, cell lines will be engineered without the AK4 gene in glioblastomas, lung adenocarcinomas, and HeLa cells then analyzed to identify all known metabolites in the system. It is hypothesized that without the expression of an AK4 protein there will be indirect or direct disruptions in the global nucleotide pools by either increasing nucleotide biosynthesis or decreasing nucleotide consumption. The outcome of this study will add to the small library of research on AK4 as well as provide a comprehensive understanding on the enzymatic activity and interactions where AK4 exerts its biological effects on cancer cell metabolism.Support or Funding InformationFunded by National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) Grant 1R15GM123382‐01.

Capsaicin enhances the pro‐apoptotic activity of Camptothecin in human small cell lung cancer

Small cell lung cancer (SCLC) is characterized by early dissemination, aggressive clinical course and a dismal survival rate. The first line therapy for SCLC patients is cisplatin‐based combination chemotherapy along with radiation. Initially, cisplatin shows excellent results causing remission in 60–80% of the SCLC patients but within a year the tumor inevitably relapses and this relapsed tumor is resistant to cisplatin. A subset of SCLC tumors are unresponsive to cisplatin from the outset. These patients are said to have platinum‐refractory SCLC tumors. Patients with platinum resistant or refractory disease have very limited options, as the only standard chemotherapy with an FDA‐approved drug, camptothecin (and its derivations), has an objective response rate of approximately 3% and little or no survival benefit. Such sobering statistics define the arena where there is an urgent need for drugs which enhance the pro‐apoptotic activity of camptothecin in human SCLC. The present study tests the hypothesis that capsaicin can sensitize human SCLC cells to the apoptotic effects of camptothecin. Caspase‐3 activity assays reveal that the combination of camptothecin and capsaicin displayed greater apoptotic activity than the drugs used alone. We verified these results using a second apoptosis assay, namely the Cell Death ELISA and obtained similar results. Chou‐Talalay isobologram analysis showed the interaction between camptothecin and capsaicin is synergistic. Chicken chorioallantoic membrane (CAM) assays confirmed the combinatorial apoptotic effects of capsaicin and camptothecin in human SCLC. The co‐operative effects of capsaicin and camptothecin were also confirmed in human SCLCs xenografted on athymic mice. The results of our experiments pave the way to novel combination therapies for human small cell lung cancer.Support or Funding InformationSupport or Funding Information Funding for our study was supported by a NIH R15‐AREA Grant (2R15CA161491‐02). Furthermore, this study was supported in part by a Center for Natural Products pilot grant from Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH).

A Realistic View of Proteins : Solution Scattering at the Life Sciences X‐ray Scattering Beamline (LIX)

Small angle X‐ray scattering is an important tool that reveals the behavior of proteins in their native environment. The scattering pattern produced from a SAXS experiment represents an average of all conformations present in the solution. Since most proteins in our body function in a solution environment, SAXS data can reveal additional biologically relevant states of a molecule or molecular complex. Furthermore, SAXS provides data about the shape and size of the molecule of interest and is a complementary technique to other structural methods. Combining SAXS data with X‐ray crystallography, NMR or Cryo‐EM can lead to a more robust understanding of the species being studied. High quality SAXS data can be used to build ab initio low resolution bead models, which provide a 3D envelope of your molecule. Here at the life sciences X‐ray scattering beamline (LiX), we collect data by flowing sample through the X‐ray path in a 3 channel flow cell. As the sample enters the X‐ray path, the simultaneous collection of SAXS and wide angle X‐ray scattering (WAXS) is obtained via a 3 detector setup. The detector setup has a q‐range of 0.006Å−1 to 3.2 Å−1, primarily to obtain scattering data from the water peak to normalize buffer subtraction. Automated sample handling at LiX allows for collection of up to 360 samples, packaging of data and processing. One challenge in obtaining high quality SAXS data is preparation of pure, monodisperse samples. For these complicated samples that have multiple species, our in‐line HPLC system seamlessly integrates with our beamline for SEC‐SAXS (size‐exclusion chromatography‐SAXS), and increases the chances of obtaining a more pure, monodisperse sample immediately before data collection.Support or Funding InformationLiX beamline is part of the Life Science Biomedical Technology Research resource, co‐funded by the National Institute of General Medical Sciences (NIGMS) under grant P41 GM111244 and by the DOE Office of Biological and Environmental Research under grant KP1605010, with additional support from NIH under grant S10 OD012331. The operation of NSLS2 is supported by US Department of Energy, Office of Basic Energy Sciences, under contract No. DE‐SC0012704.