A budding yeast model for human disease mutations in the EXOSC2 cap subunit of the RNA exosome complex

RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies

Development and Validation of ML-DQA -- a Machine Learning Data Quality Assurance Framework for Healthcare

The approaches by which the machine learning and clinical research communities utilize real world data (RWD), including data captured in the electronic health record (EHR), vary dramatically. While clinical researchers cautiously use RWD for clinical investigations, ML for healthcare teams consume public datasets with minimal scrutiny to develop new algorithms. This study bridges this gap by developing and validating ML-DQA, a data quality assurance framework grounded in RWD best practices. The ML-DQA framework is applied to five ML projects across two geographies, different medical conditions, and different cohorts. A total of 2,999 quality checks and 24 quality reports were generated on RWD gathered on 247,536 patients across the five projects. Five generalizable practices emerge: all projects used a similar method to group redundant data element representations; all projects used automated utilities to build diagnosis and medication data elements; all projects used a common library of rules-based transformations; all projects used a unified approach to assign data quality checks to data elements; and all projects used a similar approach to clinical adjudication. An average of 5.8 individuals, including clinicians, data scientists, and trainees, were involved in implementing ML-DQA for each project and an average of 23.4 data elements per project were either transformed or removed in response to ML-DQA. This study demonstrates the importance role of ML-DQA in healthcare projects and provides teams a framework to conduct these essential activities.Comment: Presented at 2022 Machine Learning in Health Care Conferenc

Early Low-Titer Neutralizing Antibodies Impede HIV-1 Replication and Select for Virus Escape

Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC50) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1–V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one

The contribution of theory to the design, delivery, and evaluation of interprofessional curricula: BEME Guide No. 49

BACKGROUND: Interprofessional curricula have often lacked explicit reference to theory despite calls for a more theoretically informed field that illuminates curricular assumptions and justifies curricular practices. AIM: To review the contributions of theory to the design, delivery, and evaluation of interprofessional curricula. METHODS: Four databases were searched (1988-2015). Studies demonstrating explicit and a high-quality contribution of theory to the design, delivery or evaluation of interprofessional curricula were included. Data were extracted against a comprehensive framework of curricular activities and a narrative synthesis undertaken. RESULTS: Ninety-one studies met the inclusion criteria. The majority of studies (86%) originated from the UK, USA, and Canada. Theories most commonly underpinned "learning activities" (47%) and "evaluation" (54%). Theories of reflective learning, identity formation, and contact hypothesis dominated the field though there are many examples of innovative theoretical contributions. CONCLUSIONS: Theories contribute considerably to the interprofessional field, though many curricular elements remain under-theorized. The literature offers no "gold standard" theory for interprofessional curricula; rather theoretical selection is contingent upon the curricular component to which theory is to be applied. Theories contributed to interprofessional curricula by explaining, predicting, organizing or illuminating social processes embedded in interprofessional curricular assumptions. This review provides guidance how theory might be robustly and appropriately deployed in the design, delivery, and evaluation of interprofessional curricula

Microbiome Diversity and Differential Abundances Associated with Gastrointestinal Symptoms, BMI, Immune Markers, and Fecal Short Chain Volatile Fatty AcidProfiles

Objectives The gut microbiota and its metabolites – namely short chain volatile fatty acids (SCVFAs) – interact with the digestive, immune, and nervous systems. States of microbiome dysbiosis are highly associated with obesity and GI symptoms, and profiles of SCVFAs, which serve functions as fuel sources and signaling molecules, mimic this dysbiotic state. This study aimed to further our understanding of associations between bacterial diversity and GI symptoms, BMI, immune markers, and SCVFAs and to identify bacteria differentially abundant with changes to the previously mentioned variables. Methods Data (measures of GI distress, BMI, immunoglobulins, fecal proximate analysis, SCVFAs, and 16s RNA sequences) was extracted from a study containing non-celiac gluten-sensitive and control participants. QIIME2 was used to process 16s RNA data, analyze quantitative, qualitative, phylogenetic quantitative, and phylogenetic qualitative measures of alpha and beta diversity and to perform an analysis of composition of microbes (ANCOM) for differential abundances data. Results Many significant differences were seen, namely in multiple measures of alpha diversity for IgG4 (P \u3c 0.018), propionate (P \u3c 0.014), caproate (P \u3c 0.003), heartburn (P \u3c 0.004), urgent need to defecate (P \u3c 0.027), and feelings of incomplete evacuation (P \u3c 0.024). Statistical significance was seen in multiple measures of beta diversity between normal and overweight (P \u3c 0.01), normal and obese (P \u3c 0.005), and overweight and obese BMI (P \u3c 0.016), IgG4 (P \u3c 0.033), propionate (P \u3c 0.001), increased gas (P \u3c 0.024), and urgent need for defecation (P \u3c 0.026). The ANCOM identified multiple species of bacteria differentially abundant with changes to variables. Conclusions Findings suggest differences in both alpha and beta diversity with various GI symptoms, SCVFAs, and BMI. This research supports plans to apply analysis to larger sample sizes to train machine learning classifiers to identify important features of microbial profiles associated with certain SCVFA and markers of health. Funding Sources ETSU (CCRHS, Deans\u27s Research Enhancement Award and CPH, Health Sciences Funding, Honors College Summer Research Fellowship) and Shield Nutraceuticals, LLC

NASA Lunabotics

The goal of this project is to build a semi-autonomous lunar mining robot for the 2022 NASA Lunabotics Competition. As NASA carries out the Artemis program, the Lunabotics robots act as prototypes for autonomous sample collection rovers for lunar exploration. The robot is designed to autonomously navigate rough terrain, mine icy regolith simulant, and deposit regolith into a collection sieve. At the conclusion of the project, the majority of the performance metrics were met by the robot. The social implication of this project allows for a growing number of students and future engineers to learn and apply systems engineering skills through the Lunabotics challenge, which prepares them for industries that rely on these principles

The HIV-1 Antisense Protein (ASP) induces CD8 T cell responses during chronic infection

International audienceBackground : CD8+ T cells recognize HIV-1 epitopes translated from a gene’s primary reading frame (F1) and any one of its five alternative reading frames (ARFs) in the forward (F2, F3) or reverse (R1-3) directions. The 3’ end of HIV-1’s proviral coding strand contains a conserved sequence that is directly overlapping but antiparallel to the env gene (ARF R2) and encodes for a putative antisense HIV-1 protein called ASP. ASP expression has been demonstrated in vitro using HIV-transfected cell lines or infected cells. Although antibodies to ASP were previously detected in patient sera, T cell recognition of ASP-derived epitopes has not been evaluated. We therefore investigated the ex vivo and in vitro induction of ASP-specific T cell responses as a measure of immune recognition and protein expression during HIV-1 infection.Results : A panel of overlapping peptides was initially designed from the full-length ASP sequence to perform a global assessment of T cell responses. Recognition of ASP-derived antigens was evaluated in an IFN-γELISpot assay using PBMCs from HIV-1 seropositive and seronegative individuals. Eight of 25 patients had positive responses to ASP antigens and none of the seronegative donors responded. As a complimentary approach, a second set of antigens was designed using HLA-I binding motifs and affinities. Two ASP-derived peptides with high predicted binding affinities for HLA-A*02 (ASP-YL9) and HLA-B*07 (ASP-TL10) were tested using PBMCs from HIV-1 seropositive and seronegative individuals who expressed the matching HLA-I-restricting allele. We found that HLA-I-restricted ASP peptides were only recognized by CD8+ T cells from patients with the relevant HLA-I and did not induce responses in any of the seronegative donors or patients who do not express the restrictive HLA alleles. Further, ASP-YL9-specific CD8+ T cells had functional profiles that were similar to a previously described HLA-A*02-restricted epitope (Gag-SL9). Specific recognition of ASP-YL9 by CD8+ T cells was also demonstrated by tetramer staining using cells from an HLA-A*02 HIV-infected patient.Conclusion : Our results provide the first description of CD8+ T cell-mediated immune responses to ASP in HIV-1-infected patients, demonstrating that ASP is expressed during infection. Our identification of epitopes within ASP has implications for designing HIV vaccines

HLA-E-restricted HIV-1-specific CD8+ T cell responses in natural infection.

CD8+ T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E-restricted CD8+ T cell responses in HIV infection, however, remains unknown. In this study, CD8+ T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E-restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8+ T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8+ T cells. Importantly, bulk CD8+ T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E-restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E-restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8+ T cell responses in HIV infection

HLA-E-restricted HIV-1-specific CD8+ T cell responses in natural infection.

CD8+ T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E-restricted CD8+ T cell responses in HIV infection, however, remains unknown. In this study, CD8+ T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E-restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8+ T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8+ T cells. Importantly, bulk CD8+ T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E-restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E-restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8+ T cell responses in HIV infection