Conserved and divergent DNA recognition specificities and functions of R2 retrotransposon N-terminal domains.

R2 non-long terminal repeat (non-LTR) retrotransposons are among the most extensively distributed mobile genetic elements in multicellular eukaryotes and show promise for applications in transgene supplementation of the human genome. They insert new gene copies into a conserved site in 28S ribosomal DNA with exquisite specificity. R2 clades are defined by the number of zinc fingers (ZFs) at the N terminus of the retrotransposon-encoded protein, postulated to additively confer DNA site specificity. Here, we illuminate general principles of DNA recognition by R2 N-terminal domains across and between clades, with extensive, specific recognition requiring only one or two compact domains. DNA-binding and protection assays demonstrate broadly shared as well as clade-specific DNA interactions. Gene insertion assays in cells identify the N-terminal domains sufficient for target-site insertion and reveal roles in second-strand cleavage or synthesis for clade-specific ZFs. Our results have implications for understanding evolutionary diversification of non-LTR retrotransposon insertion mechanisms and the design of retrotransposon-based gene therapies.

HMGB1 mediates microbiome-immune axis dysregulation underlying reduced neutralization capacity in obesity-related post-acute sequelae of SARS-CoV-2.

While obesity is a risk factor for post-acute sequelae of SARS-CoV-2 infection (PASC, "long-COVID"), the mechanism(s) underlying this phenomenon remains poorly understood. To address this gap in knowledge, we performed a 6-week longitudinal study to examine immune activity and gut microbiome dysbiosis in post-acute stage patients recovering from SARS-CoV-2 infection. Self-reported symptom frequencies and blood samples were collected weekly, with plasma assessed by ELISA and Luminex for multiple biomarkers and immune cell profiling. DNA from stool samples were collected at the early stage of recovery for baseline assessments of gut microbial composition and diversity using 16S-based metagenomic sequencing. Multiple regression analyses revealed obesity-related PASC linked to a sustained proinflammatory immune profile and reduced adaptive immunity, corresponding with reduced gut microbial diversity. In particular, enhanced signaling of the high mobility group box 1 (HMGB1) protein was found to associate with this dysregulation, with its upregulated levels in plasma associated with significantly impaired viral neutralization that was exacerbated with obesity. These findings implicate HMGB1 as a candidate biomarker of PASC, with potential applications for risk assessment and targeted therapies.

Sequencing the SARS-CoV-2 Genome from Stool Samples of Post-acute Cases Implicates a Novel Mutation Associated with Reduced Antibody Neutralization.

Whole-genome SARS-CoV-2 sequencing tools are crucial for tracking the COVID-19 pandemic. However, current techniques require sampling of actively infectious patients following COVID-19 testing to recover enough SARS-CoV-2 RNA from the nasopharyngeal passage, which rapidly clears during the first few weeks of infection. A prospective assessment of the viral genome sourced from recovered non-infectious patients would greatly facilitate epidemiological tracking. Thus, we developed a protocol to isolate and sequence the genome of SARS-CoV-2 from stool samples of post-acute SARS-CoV-2 patients, at timepoints ranging from 10-120 days after onset of symptoms. Stool samples were collected from patients at varying timepoints post-convalescence, and viral DNA was isolated and sequenced using the QIAamp Viral RNA Mini Kit (Qiagen Inc.) and Ion Ampliseq™ Library Kit Plus (Life Technologies Corporation). Capacity of neutralizing antibodies in patient plasma was tested using a Luminex panel (Coronavirus Ig Total Human 11-Plex ProcartaPlex™ Panel, ThermoFisher). Of 64 samples obtained from post-acute patients, 21 (32.8%) yielded sufficient material for whole-genome sequencing. This allowed us to identify widely divergent phylogenetic relativity of the SARS-CoV-2 genome from post-acute patients living in the same households and infected around the same time. Additionally, we observed that individuals who recovered from infection expressed varying degrees of antibodies against SARS-CoV-2 structural proteins that corresponded to distinct variants. Interestingly, we identified a novel point mutation in the viral genome where infected patients expressed antibodies with a significantly reduced capacity to neutralize the virus in vitro relative to that of those infected with the wild-type strain. Altogether, we demonstrate a protocol to successfully sequence the SARS-CoV-2 genome from stool samples from patients up to 4 months post-infection, which can be applied to studies that assess the relationship between variants and immune response post-hoc and safe monitoring of the SARS-CoV-2 genome during the pandemic.

Examining the immunoepigenetic-gut microbiome axis in the context of self-esteem among Native Hawaiians and other Pacific Islanders.

Introduction: Native Hawaiian and other Pacific Islander (NHPI) populations experience higher rates of immunometabolic diseases compared to other racial-ethnic groups in Hawaii. As annual NHPI mortality rates for suicide and type 2 diabetes mellitus (T2DM) exceed those of the state as a whole, understanding the social and biological mechanisms underlying these disparities are urgently needed to enable preventive strategies. Methods: A community-based approach was used to investigate the immunoepigenetic-gut microbiome axis in an NHPI-enriched cohort of Oahu residents (N = 68). Self-esteem (SE) data was collected using a modified Rosenberg self-esteem (SE) assessment as a proxy measure for mental wellbeing in consideration for cultural competency. T2DM status was evaluated using point-of-care A1c (%) tests. Stool samples were collected for 16s-based metagenomic sequencing analyses. Plasma from blood samples were isolated by density-gradient centrifugation. Peripheral blood mononuclear cells (PBMCs) were collected from the same samples and enriched for monocytes using negative selection techniques. Flow-cytometry was used for immunoprofiling assays. Monocyte DNA was extracted for Illumina EPIC array-based methylation analysis. Results: Compared to individuals with normal SE (NSE), those with low SE (LSE) exhibited significantly higher plasma concentrations (pg/ml) of proinflammatory cytokines IL-8 (p = 0.051) and TNF-α (p = 0.011). Metagenomic analysis revealed that the relative abundance (%) of specific gut bacteria significantly differed between SE groups - some of which directly correlated with SE scores. Gene ontology analysis revealed that 104 significantly differentially methylated loci (DML) between SE groups were preferentially located at genes involved in immunometabolic processes. Horvath clock analyses indicated epigenetic age (Epi-Age) deceleration in individuals with LSE and acceleration in individuals with NSE (p = 0.042), yet was not reproduced by other clocks. Discussion: These data reveal novel differences in the immunoepigenetic-gut microbiome axis with respect to SE, warranting further investigation into its relationship to brain activity and mental health in NHPI. Unexpected results from Epi-Age analyses warrant further investigation into the relationship between biological age and disparate health outcomes among the NHPI population. The modifiable component of epigenetic processes and the gut microbiome makes this axis an attractive target for potential therapeutics, biomarker discovery, and novel prevention strategies.

Exploration of Cardiology Patient Hospital Presentations, Health Care Utilisation and Cardiovascular Risk Factors During the COVID-19 Pandemic.

OBJECTIVES: COVID-19 and the lockdowns have affected health care provision internationally, including medical procedures and methods of consultation. We aimed to assess the impact of COVID-19 at two Australian hospitals, focussing on cardiovascular hospital admissions, the use of community resources and cardiovascular risk factor control through a mixed methods approach. METHODS: Admissions data from the quaternary referral hospital were analysed, and 299 patients were interviewed from July 2020 to December 2021. With the admissions data, the number, complexity and mortality of cardiology hospital admissions, prior to the first COVID-19 lockdown (T0=February 2018-July 2019) were compared to after the introduction of COVID-19 lockdowns (T1=February 2020-July 2021). During interviews, we asked patients about hospital and community health resource use, and their control of cardiovascular risk factors from the first lockdown. RESULTS: Admission data showed a reduction in hospital presentations (T0=138,099 vs T1=128,030) and cardiology admissions after the lockdown period began (T0=4,951 vs T1=4,390). After the COVID-19-related lockdowns began, there was an increased complexity of cardiology admissions (T0=18.7%, 95% CI 17.7%-19.9% vs T1=20.3%, 95% CI 19.1%-21.5%, chi-square test: 4,158.658, p<0.001) and in-hospital mortality (T0=2.3% of total cardiology admissions 95% CI 1.9%-2.8% vs T1=2.8%, 95% CI 2.3%-3.3%, chi-square test: 4,060.217, p<0.001). In addition, 27% of patients delayed presentation due to fears of COVID-19 while several patients reported reducing their general practitioner or pathology/imaging appointments (27% and 11% respectively). Overall, 19% reported more difficulty accessing medical care during the lockdown periods. Patients described changes in their cardiovascular risk factors, including 25% reporting reductions in physical activity. CONCLUSION: We found a decrease in hospital presentations but with increased complexity after the introduction of COVID-19 lockdowns. Patients reported being fearful about presenting to hospital and experiencing difficulty in accessing community health services.

Deconstructing Ableism in Health Care Settings Through Case-Based Learning.

Introduction: Patients with disabilities face health disparities, and providers with disabilities confront professional roadblocks and institutional bias. Yet their experiences are often excluded from medical education, and few case studies address culturally humble care for those with disabilities. Methods: We created two 1-hour case-based modules on disability and ableism from patient and provider perspectives. Modules were piloted in June 2020 and presented at two conferences in April 2021. Modules included a prereading, introductory disability presentation, and facilitated case discussions. Sessions were evaluated with pre- and/or postsession surveys. Modules were rated on 5-point Likert scales for educational value, professional growth contribution, and interactive/engaging design. Results: Participants rated the patient and provider modules 4.5, 4.4, and 4.4 and 4.5, 4.4, and 4.5 for the three categories, respectively. Participants noted that the sessions were insightful and validating and improved their understanding of ableism and the importance of disability curricula. There were significant improvements in participants' perceptions of ability to discuss ableism's impacts, recognize barriers, identify resource/support gaps for trainees, and advocate. A total of 171 participants completed our modules, with survey response rates of 38% (60 out of 160) and 48% (77 out of 160) for one conference and a postsurvey response rate of 64% (seven out of 11) for the second. Discussion: Designed for health care trainees, providers, administration, and staff, our sessions introduced concepts of ableism, accommodation, and health care barriers. Our results suggest the modules can contribute to professional growth, understandings of ableism, and participants' disability advocacy tool kit.

Gut microbial indicators of metabolic health underlie age-related differences in obesity and diabetes risk among Native Hawaiians and Pacific Islanders.

Native Hawaiians and Pacific Islanders (NHPIs) suffer from higher prevalence of and mortality to type 2 diabetes mellitus (T2DM) than any other major race/ethnic group in Hawaii. Health inequities in this indigenous population was further exacerbated by the SARS-CoV-2 pandemic. T2DM progression and medical complications exacerbated by COVID-19 are partially regulated by the gut microbiome. However, there is limited understanding of the role of gut bacteria in the context of inflammation-related diseases of health disparities including T2DM and obesity. To address these gaps, we used a community-based research approach from a cohort enriched with NHPI residents on the island of Oahu, Hawaii (N=138). Gut microbiome profiling was achieved via 16s rDNA metagenomic sequencing analysis from stool DNA. Gut bacterial capacity for butyrate-kinase (BUK)-mediated fiber metabolism was assessed using quantitative PCR to measure the abundance of BUK DNA and RNA relative to total bacterial load per stool sample. In our cohort, age positively correlated with hemoglobin A1c (%; R=0.39; P<0.001) and body mass index (BMI; R=0.28; P<0.001). The relative abundance of major gut bacterial phyla significantly varied across age groups, including Bacteroidetes (P<0.001), Actinobacteria (P=0.007), and Proteobacteria (P=0.008). A1c was negatively correlated with the relative levels of BUK DNA copy number (R=-0.17; P=0.071) and gene expression (R=-0.33; P=0.003). Interestingly, we identified specific genera of gut bacteria potentially mediating the effects of diet on metabolic health in this cohort. Additionally, α-diversity among gut bacterial genera significantly varied across T2DM and BMI categories. Together, these results provide insight into age-related differences in gut bacteria that may influence T2DM and obesity in NHPIs. Furthermore, we observed overlapping patterns between gut bacteria and T2DM risk factors, indicating more nuanced, interdependent interactions among these factors as partial determinants of health outcomes. This study adds to the paucity of NHPI-specific data to further elucidate the biological characteristics associated with pre-existing health inequities in this racial/ethnic group that is significantly underrepresented in biomedical research.

Integration of structural MRI and epigenetic analyses hint at linked cellular defects of the subventricular zone and insular cortex in autism: Findings from a case study.

Introduction: Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social interaction, communication and repetitive, restrictive behaviors, features supported by cortical activity. Given the importance of the subventricular zone (SVZ) of the lateral ventrical to cortical development, we compared molecular, cellular, and structural differences in the SVZ and linked cortical regions in specimens of ASD cases and sex and age-matched unaffected brain. Methods: We used magnetic resonance imaging (MRI) and diffusion tractography on ex vivo postmortem brain samples, which we further analyzed by Whole Genome Bisulfite Sequencing (WGBS), Flow Cytometry, and RT qPCR. Results: Through MRI, we observed decreased tractography pathways from the dorsal SVZ, increased pathways from the posterior ventral SVZ to the insular cortex, and variable cortical thickness within the insular cortex in ASD diagnosed case relative to unaffected controls. Long-range tractography pathways from and to the insula were also reduced in the ASD case. FACS-based cell sorting revealed an increased population of proliferating cells in the SVZ of ASD case relative to the unaffected control. Targeted qPCR assays of SVZ tissue demonstrated significantly reduced expression levels of genes involved in differentiation and migration of neurons in ASD relative to the control counterpart. Finally, using genome-wide DNA methylation analyses, we identified 19 genes relevant to neurological development, function, and disease, 7 of which have not previously been described in ASD, that were significantly differentially methylated in autistic SVZ and insula specimens. Conclusion: These findings suggest a hypothesis that epigenetic changes during neurodevelopment alter the trajectory of proliferation, migration, and differentiation in the SVZ, impacting cortical structure and function and resulting in ASD phenotypes.

Office of Medical Education: Opportunities for Trainees to Engage and Lead in Curricular Innovation and Reform.

Introduction: The AAMC prioritizes promoting a diverse and culturally competent workforce which is thought to have a positive impact on the health of people living in the US. There is a lack of diversity in the current landscape of academic medicine and strategies are needed to effect change. This module introduced undergraduate and graduate medical trainees to leadership skills and opportunities in curriculum innovation and reform by learning about and interacting with the office of medical education (OME) at their institutions. Methods: We implemented a workshop using small-group case discussions and didactics to help medical students and residents learn how to: (1) describe the structure and functions of an OME, (2) describe leadership competencies associated with various roles within the OME, and (3) identify opportunities for trainees to engage with the OME on curricular innovation and reform, especially advancing diversity and inclusion. Results: Across three sites, 45 learners completed partial or full workshop evaluations. Of learners, 22 (49%) were not knowledgeable and 13 (29%) were somewhat knowledgeable in identifying leadership opportunities for trainees to become engaged through the OME. There was a statistically significant increase in confidence after the workshop in "discussing an interdisciplinary approach to the creation of a medical education innovation," and, "assessing the need for curricula change." Over 90% of attendees agreed learning objectives were met. Discussion: This workshop succeeded in promoting awareness of the structure and function of OMEs and confidence in seeking opportunities to become engaged in medical education, especially in advancing diversity and inclusion.

How Should Medical School Admissions Drive Health Care Workforce Diversity?

Over the past decade, holistic review has been implemented to motivate schools' compliance with state and federal laws about how to regard race in admissions processes and decisions. From clinical, ethical, and public health standpoints, physician workforce diversification is widely regarded as foundational to medicine's capacity as a profession to respond justly to the health care needs of a pluralistic nation. In response to a case, this commentary considers merits and limitations of holistic review's roles in advancing health professional workforce diversity and health equity.

KIAA0319 Genotype Predicts the Number of Past Concussions in a Division I Football Team: A Pilot Study.

This candidate gene study evaluated the relationship of a past history of concussion with single nucleotide polymorphisms (SNPs) in nine genes in a small cohort (N = 87) of a nationally ranked Division I football team. Genes and SNPs studied were selected based on their published connection to brain injury and brain development, as well as impulsivity. We used multinomial logistic regression analysis (MLRA) to quantify how well genotype predicted the number of previously diagnosed concussions (three categories: none, one, two or more), while covarying race and number of years participating in football. The rs4504469 SNP for KIAA0319 was the only locus that significantly predicted number of previously diagnosed concussions (p = 0.005, meeting Bonferroni correction for multiple comparisons). The KIAA0319 results raise the hypothesis that having the CT or TT genotype of KIAA0319 may be predictive of a lower incidence of previously diagnosed concussion. This finding raises a number of hypotheses for future pre-clinical research, particularly whether alterations in neural organization related to KIAA0319 rs4504469 lead to reduced susceptibility for lasting head trauma, or greater resilience in the face of repeated subconcussive injury.

The Contribution of Malodour in Quality of Life of Patients With Hidradenitis Suppurativa.

BACKGROUND: Malodourous discharge in patients with hidradenitis suppurativa (HS) has a strong psychosocial impact and is often reported as a source of embarrassment, low self-esteem, social stigma, and barriers to interpersonal relationships. Malodour is a maker of bacterial colonization, and its role in HS is understudied. OBJECTIVES: The aim of this study is to assess the relationship between severity of malodourous discharge and quality-of-life impairment in patients with HS. METHODS: This is a cross-sectional study of 51 patients recruited from the Women's College Hospital and the York Dermatology Centre. Quality of life was assessed using both the Dermatology Life Quality Index (DLQI) and the Skindex-29 instruments. RESULTS: Severity of odour significantly predicts the total Skindex score after controlling for disease severity as measured by the Hurley stage and Sartorius score ( R2 = 0.39, F = 8.11, P < .0001). However, odour severity is not a predictor of DLQI ( R2 = 0.17, F = 2.63, P = .064). There was no difference in mean DLQI scores for the low- vs high-odour groups, but patients with high odour had a greater quality of life impairment as measured by the Skindex tool ( t = -4.19, df = 43, P < .0001, mean difference = -18.87). CONCLUSION: Malodour is a common symptom that significantly impairs quality of life in patients with HS. The fact that this effect is captured in Skindex and not the DLQI may be attributed to the nonspecificity of the DLQI in terms of unique disease characteristics. It is important to address odour in the management of patients with HS.

Four-Station Group Observed Structured Clinical Encounter for Formative Assessment of Communication Skills for Internal Medicine Clerks.

INTRODUCTION: Communication with patients and among colleagues is critical to effective clinical care. A group observed structured clinical encounter (GOSCE) is an effective and resource-saving tool for teaching communication skills to medical students. While objective structured clinical exams (OSCEs) are a well-established assessment tool for communication skills, a GOSCE allows for formal observation of communication skills while also providing an opportunity for peer observation and feedback. Additionally, a GOSCE costs less and requires fewer faculty per learner than a traditional OSCE. METHODS: This is a four-station GOSCE to teach advanced communication skills to medical students. The stations are smoking cessation, difficult doctor-patient encounter, shared decision making, and delivering bad news. A group is made up of four to six students and one faculty member. At each station, one student takes the lead in the patient interview, followed by a group interview and ending with feedback by all participants. RESULTS: In the pilot phase, a total of 44 students were administered the GOSCE and were surveyed about their experience. Students felt the GOSCE was an enjoyable and educational experience. The GOSCE has subsequently been administered to more than 600 students, and 25 internal medicine faculty have participated. DISCUSSION: Our work demonstrates that the GOSCE is a feasible curricular enhancement for formative assessment of communication skills during the internal medicine clerkship. It is easy to implement and has been well received by all participants, with minimal impact on limited medical school and faculty resources.

[High conjugated linoleic acid (CLA) content in milk and dairy products using a dietary supplementation of sunflower seed in cows. Thrombogenic/atherogenic risk issues]. / Alto contenido de ácido linoleico conjugado (CLA) en leche y productos derivados al incorporar semillas de girasol a la dieta vacuna. Implicaciones sobre el riesgo trombo/aterogénico.

This study was undertaken to determine the effect of dietary supplementation of sunflower seed in cows on the chemical composition of milk and dairy products. Cream, butter and butter oil were prepared from milk produced by cows fed a control diet (control products) or diet supplemented with 11.2% sunflour seed (CLA-rich products). Milk samples collected were determined for lactose. A sample of CLA-rich or control product was determined for fatty acid profile as well as fat, protein and ash contents. The index of atherogenicity (IA) and the index of thrombogenicity (IT) were also calculated. Results revealed that there was no effect of the inclusion of sunflower seed in the diet on the lactose content in milk and total fat, protein and ash contents in the dairy products. Average contents of conjugated linoleic acid (CLA) and transvaccenic acid (TVA), expressed as g/100g total fatty acid were 0.54 and 1.6, respectively in the control products, and 2 and 6.4, respectively in the CLA-rich products. The content of either CLA or TVA was approximately four fold higher in the latter products. Moreover, CLA-rich products showed considerably low IA and IT, which were, respectively, 38.4 and 25.0% less than those from control products. Fatty acid profiles were unaffected during processing, which demonstrates that CLA is a stable component in the dairy products analyzed. It was concluded that dietary supplementation of sunflower seed in cows increases the CLA and TVA contents in milk, which may contribute to the reduction of the risk of cardiovascular diseases in humans.

Alto contenido de ácido linoleico conjugado (CLA) en leche y productos derivados al incorporar semillas de girasol a la dieta vacuna: implicaciones sobre el riesgo trombo/aterogénico / High conjugated linoleic acid (CLA) content in milk and dairy products using a dietary supplementation of sunflower seed in cows: thrombogenic/atherogenic risk issues

Con la finalidad de determinar el perfil de ácidos grasos y la composición química de productos lácteos enriquecidos con ácido linoleico conjugado (CLA) de manera natural, se elaboraron crema, mantequilla y grasa butírica con leche obtenida de vacas que recibieron una dieta control o suplementación con semilla de girasol en un 11.2%. El análisis químico incluyó el perfil de ácidos grasos,materia grasa, proteína y cenizas; en la leche se determinó además el contenido de lactosa. Se calcularon los índices de aterogenicidad (IA) y trombogenicidad (IT) en la leche y productos elaborados. Los resultados indicaron que los contenidos de grasa, proteína, lactosa y ceniza no fueron afectados por la incorporación de semilla de girasol en la dieta de los animales. El contenido promedio de CLA y ácidotrans vaccénico (TVA) expresados en g/100 g de lípidos totales fue, para los productos control, 0.54 y 1.6; mientras que para los productos ricos en CLA fueron 2 y 6.4, lo cual representa un incremento de cuatro veces. Además, en los productos ricos en CLA los IA e IT disminuyeron considerablemente (38.4 y 25% menos, respectivamente). Se observó que los perfiles de ácidos grasos no se modificaron durante el procesamiento, indicando que el CLA es un componente estable en los productos lácteos analizados. El uso de semilla de girasol en la dieta de las vacas, incrementa el contenido de CLA y TVA en los productos lácteos y disminuye el riesgo de enfermedades cardiovasculares en humanos sin afectar la proporción de los componentes mayoritarios.

High conjugated linoleic acid (CLA) content in milk and dairy products using a dietary supplementation of sunflower seed in cows. Thrombogenic/atherogenic risk issues. This studywas undertaken to determine the effect of dietary supplementation of sunflower seed in cows on the chemical composition of milk and dairy products. Cream, butter and butter oil were prepared from milk produced by cows fed a control diet (control products) or diet supplemented with 11.2% sunflour seed (CLA-rich products). Milk samples collected were determined for lactose. A sample of CLArich or control product was determined for fatty acid profile as well as fat, protein and ash contents. The index of atherogenicity (IA) and the index of thrombogenicity (IT) were also calculated. Results revealed that there was no effect of the inclusion of sunflower seed in the diet on the lactose content in milk and total fat, protein and ash contents in the dairy products. Average contents of conjugated linoleic acid (CLA) and transvaccenic acid (TVA), expressed as g/ 100g total fatty acid were 0.54 and 1.6, respectively in the control products, and 2 and 6.4, respectively in the CLA-rich products. The content of either CLA or TVA was approximately four fold higher in the latter products. Moreover, CLA-rich products showed considerably low IA and IT, which were, respectively, 38.4 and 25.0% less than those from control products. Fatty acid profiles were unaffected during processing, which demonstrates that CLA is a stable component in the dairy products analyzed. It was concluded that dietary supplementation of sunflower seed in cows increases the CLA and TVA contents in milk, which may contribute to the reduction of the risk of cardiovascular diseases in humans.