Upstate Medical University
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HIV-1 has a sweet tooth: glucose metabolism drives the multistep process of HIV-1 latency reversalThe major barrier to a cure for HIV-1 is the establishment of latency in long-lived CD4+ T cells within lymphoid tissues which readily fuel viral rebound upon antiretroviral therapy (ART) interruption. Therapeutic approaches aimed at eliminating these HIV reservoirs with latency reversal agents (LRAs) have hitherto yielded underwhelming results in clinical trials owing to our incomplete understanding of the exact determinants of meaningful latency reversal in vivo. While previous studies have associated glycolysis with HIV productive replication and latency reversal, the exact role and mechanistic link of glycolysis to HIV latency reversal remains undefined. Furthermore, few studies have investigated HIV latency under physiologically relevant metabolic conditions found in the anatomical reservoirs of HIV in vivo. The studies in this thesis reveal that glycolysis is a metabolic determinant of HIV latency reversal, particularly during physiological hypoxia. We show that the capacity of LRAs to modulate glycolysis determines their efficacies over a physiological range of glucose and oxygen availabilities as found across tissues in vivo. Mechanistically, glycolysis fuels histone lactylation, a novel post-translational modification (PTM) which we show is a stronger predictor xviii of latency reversal than the canonically recognized acetylation marks, and promotes chromatin accessibility at the HIV LTR. Beyond histone PTM modulation, glycolysis also modulates HIV RNA splicing, a critical post-transcriptional step in HIV latency reversal. Specifically, multiple splicing of rev, an HIV regulatory gene, is significantly downmodulated by glycolytic restriction in a hypoxia-dependent fashion. Finally, we show that glucose and oxygen availability impact the phosphorylation and lactylation of splicing factor 3B subunit 1 (SF3B1), a core component of the U2 spliceosome complex and HIV dependency factor which provides preliminary mechanistic insight to how glycolysis and hypoxia modulate HIV RNA splicing. Collectively, our findings uncover glucose and oxygen availability as critical metabolic determinants of HIV-1 latency reversal and support the rationale that physiologically relevant experimental conditions should be utilized in studies aimed at identifying therapeutic agents that effectively target the latent reservoir in vivo.
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Racial and ethnic disparities in social isolation and 11-year dementia risk among older adults in the United States.Social isolation has been implicated in the development of cognitive impairment, but research on this association remains limited among racial-ethnic minoritized populations. Our study examined the interplay between social isolation, race-ethnicity and dementia.
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Kohlschütter-Tönz protein ROGDI is the homolog of yeast Rav2 and a novel Rabconnectin-3 subunitV-ATPases are rotary proton pumps that are extraordinarily well-conserved among eukaryotes. V-ATPases function primarily to acidify intracellular compartments, critical to maintaining cellular homeostasis. The V-ATPase-generated proton gradient provides the optimal environment for lysosomal catabolism and drives intracellular protein trafficking. V-ATPases serve important functions throughout the human body. For example, V-ATPase activity energizes the active transport of neurotransmitters into synaptic vesicles, regulates the acid/base balance in the kidney, and helps the immune system recognize invading pathogens. However, when V-ATPase activity is inappropriately increased or decreased, these processes are affected, and disease can result. V-ATPases are composed of peripheral V₁ and integral membrane V₀ subcomplexes; V₁ hydrolyzes ATP and transmits rotation to V₀, which moves protons across a membrane. V-ATPase activity is regulated in part through the reversible association of the V₁ subcomplex and V₁C subunit from V₀. Upon disassembly, both V₁ and V₀ are catalytically inactivated. In yeast, the RAVE complex catalyzes the efficient reassembly of V-ATPases. Rabconnectin-3 is the human homolog of the RAVE complex and functions similarly. Mutations in the Rabconnectin-3 complex can reduce V-ATPase activity through decreased assembly, which leads to disease. Both Rabconnectin-3 subunits share substantial homology with the RAVE subunit Rav1. We have identified the poorly characterized protein ROGDI as the mammalian homolog of the yeast RAVE subunit, Rav2. ROGDI shares strong functional and structural homology with yeast Rav2. Expression of ROGDI in a rav2Δ yeast strain partially rescues the growth phenotype characteristic of RAVE mutants. ROGDI binds to the structurally conserved N-terminal β-sheet rich domain. AlphaFold3 modeling predicts that ROGDI binds between the Rabconnectin-3 subunits. ROGDI coimmunoprecipitates with Rabconnectin-3 and V-ATPase subunits. Additionally, ROGDI is present alongside V-ATPase and Rabconnectin-3 subunits on lysosomal membranes. This indicates that, like RAVE and Rav2, Rabconnectin-3 and ROGDI localize intracellular regions rich in V-ATPases. Identifying ROGDI as a novel Rabconnectin-3 subunit is a substantial step forward in our understanding of Rabconnectin-3 and how it influences V-ATPase activity.
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Research on Health Topics Communicated through TikTok: A Systematic Review of the LiteratureTikTok has more than 1.5 billion users globally. Health and wellness content on the application increased by more than 600% in 2021. This systematic review seeks to summarize which fields within medicine have embraced researching health communication on the TikTok platform and the most common measures reported within this literature. Research questions include what categories of health topics on TikTok are investigated in the literature, trends in topics by year, and types of outcomes reported. Embase, CINAHL, Scopus, and Ovid MEDLINE databases were searched in March 2024. Eligible studies met four criteria: (1) investigated human health topics on TikTok; (2) conducted in the United States; (3) published in English; and (4) published in a peer-reviewed journal. Of the 101 included studies, 50.5% (N = 51) discussed non-surgical specialties, 9.9% (N = 10) discussed topics within surgery, and 11.9% (N = 12) discussed COVID-19. The number of papers referencing non-surgical topics spiked in 2023, and no increase was seen in the number of COVID-19 papers over time. Most papers reported a number of interactions, and papers about mental health were least likely to report accuracy. Our findings highlight several health topics with a wide breadth of research dedicated to them, such as dermatology and COVID-19, and highlight areas for future research, such as the intersection of cancer and TikTok. Findings may be influential in the fields of medicine and healthcare research by informing health policy and targeted prevention efforts. This review reveals the need for future policies that focus on the role and expectations of the healthcare worker in health communication on social media. Implications for clinical practice include the need for providers to consider an individual's perception of health and illness, given the wide variety of information available on social media applications such as TikTok. This review was pre-registered on PROSPERO (CRD42024529182).
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Role of Cytoreductive Nephrectomy in Metastatic Clear Cell Renal cell Carcinoma in the Era of immunotherapy: An Analysis of the National Cancer DatabaseBackground: The effectiveness of the clinical outcome of CN (Cytoreductive Nephrectomy) in cases of mccRCC (Metastatic Clear Cell Renal cell Carcinoma) is still uncertain despite two trials, SURTIME and CARMENA. These trials, conducted with Sunitinib as the standard treatment, did not provide evidence supporting the use of CN. Methods: We queried the NCDB for stage IV mccRCC patients between the years of 2004 to 2020, who received (immunotherapy) IO with or without nephrectomy. Overall survival (OS) was calculated among three groups of IO alone, IO followed by CN (IOCN), CN followed by IO (CNIO). Cox models compared OS by treatment group after adjusting for sociodemographic, health, and facility variables. Results: From 1,549,101 renal cancer cases, 7983 clear and nonclear cell renal cell carcinoma cases were identified. After adjusting for sociodemographic and health covariates, patients who received IO followed by CN or CN followed by IO had a respective 64% (adjusted Hazard Ratio [aHR] = 0.36, 95% CI = 0.30-0.43, P = .006] and 47% (aHR = 0.53, 95% CI = 0.49-0.56, P = .001) mortality risk reduction respectively compared to patients who received IO alone. Compared to White adults, individuals who identified as Black exhibited 17% higher risk mortality (aHR = 1.17, 95% CI = 1.06-1.30, P = .002). Patients who received CN prior to IO had a 59% associated mortality risk compared to patients who received IO followed by CN who had a lower risk, 35.7% (P < .001). Conclusions: Patients receiving CN regardless of sequence with IO did better than IO alone in this national registry-based adjusted analysis for mccRCC. Presently available data indicates that the combination of CN and IO holds promise for enhancing clinical results in patients with mRCC.
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Imbalanced specialty representation of USMLE and NBME test writersPurpose: The United States Medical Licensing Examination (USMLE) is an examination series required for allopathic physician licensure in the United States (US). USMLE content is created and maintained by the National Board of Medical Examinations (NBME). The specialty composition of the USMLE and NBME taskforce members involved in the creation of examination content is currently unknown. Methods: Using the 2021 USMLE and 2021 NBME Committees and Task Forces documents, we determined each member's board-certified primary specialty and involvement in test material development committees who we dubbed "test writers". Total active physicians by primary specialty were recorded from the 2020 Physician Specialty Data Report published by the Association of American Medical Colleges (AAMC). Descriptive statistics and chi-square analysis were used to analyze the cohorts. Results: The USMLE and NBME test writer primary specialty composition was found to be significantly different compared to the US active physician population (USMLE χ2 [32]=172, p<.001 and NBME χ2 [32]=200, p<.001). Only nineteen specialties were represented within USMLE test writers, with three specialties being proportionally represented. Two specialties were represented within NBME test writers. Obstetrics and Gynecology physicians were proportionally represented in USMLE but not within NBME test writers. Internal Medicine (IM) accounts for the largest percentage of all USMLE test writers (60/197, 30%) with an excess representation of 31 individuals. Conclusions: There is an imbalance in the specialty representation of USMLE and NBME test writers compared to the US active physician population. These findings may have implications for the unbiased and accurate portrayal of topics in such national examinations; thus, future investigation is warranted.
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Human cytomegalovirus (HCMV) exploits heat-shock transcription factor 1 (HSF1) to promote viral replication: a potential novel antiviral target to combat HCMV infectionHuman cytomegalovirus (HCMV) is a highly prevalent beta-Herpesviridae virus infecting almost 80-90 % of the world population. Though HCMV infection is typically asymptomatic, it can cause significant morbidity and mortality among immunocompromised individuals. Because of its obligate intracellular nature, HCMV modulates the cellular environment to promote infection. HCMV activates different cellular responses and signaling pathways to facilitate a favorable state for viral replication. During the lytic cycle of HCMV infection, viral entry, and replication inside the cell initiate stress response due to nutrient deficiency, energy depletion, hypoxia, and proteotoxic stress. Stress responses are designed to sense the damage, initiating a cascade of events to survive the stress. Several studies showed that HCMV usurps components of heat shock-stress response (HSR) to mitigate stress-associated damage and promote viral gene expression and replication. In this study, we found that HCMV infection in fibroblast cells induces a unique biphasic activation of heat shock transcription factor 1 (HSF1), a master transcription factor that is activated in response to heat-induced proteotoxic stress. HCMV binding to the integrin-ᵝ receptor activates HSF1 through Src- kinases. Importantly, HCMV infection drives the translocation of HSF1 into the infected cell nucleus. During canonical activation of HSF1, nuclear HSF1 binds to the specific sequence on the genome called heat shock element (HSE) and initiates transcription of a wide variety of stress-related genes. Interestingly, HCMV also utilizes this master transcription factor by harboring HSEs on major immediate early promoter (MIEP) to regulate viral immediate early (IE) gene expression. We found inhibition of HSF1 with a novel anti-HSF1 targeting drug SISU102 (Direct Targeted HSF1 InhiBitor) attenuated IE protein expression, indicating that the HSF1 regulates HCMV lytic replication. Additionally, inhibition of HSF1 reduced late (L) gene expression and subsequent viral progeny production. To explore HSF1 as a potential in vivo anti-HCMV target, we employed a murine model involving the subcutaneous transplantation of human skin into athymic nude mice. Treatment with SISU102 significantly diminishes viral replication in skin xenografts compared to the vehicle-treated group, indicating HSF1 as a possible cellular protein target for HCMV antiviral therapy. Overall, our data suggest that HCMV infection rapidly activates HSF1 during viral binding and entry, driving nuclear localization to promote lytic replication, which can be exploited as an antiviral strategy.
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Effectiveness of Artificial Intelligence Technologies in Cancer Treatment for Older Adults: A Systematic ReviewBackground: Aging is a multifaceted process that may lead to an increased risk of developing cancer. Artificial intelligence (AI) applications in clinical cancer research may optimize cancer treatments, improve patient care, and minimize risks, prompting AI to receive high levels of attention in clinical medicine. This systematic review aims to synthesize current articles about the effectiveness of artificial intelligence in cancer treatments for older adults. Methods: We conducted a systematic review by searching CINAHL, PsycINFO, and MEDLINE via EBSCO. We also conducted forward and backward hand searching for a comprehensive search. Eligible studies included a study population of older adults (60 and older) with cancer, used AI technology to treat cancer, and were published in a peer-reviewed journal in English. This study was registered on PROSPERO (CRD42024529270). Results: This systematic review identified seven articles focusing on lung, breast, and gastrointestinal cancers. They were predominantly conducted in the USA (42.9%), with others from India, China, and Germany. The measures of overall and progression-free survival, local control, and treatment plan concordance suggested that AI interventions were equally or less effective than standard care in treating older adult cancer patients. Conclusions: Despite promising initial findings, the utility of AI technologies in cancer treatment for older adults remains in its early stages, as further developments are necessary to enhance accuracy, consistency, and reliability for broader clinical use.
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Microbiota Colonization Dynamics Dictate Systemic IgAEvolution of the mammalian gut is intimately linked with the microbes that inhabit this space. Immunological development of gastrointestinal and systemic tissues is fundamentally dependent on stimulation by symbiotic microorganisms. In some cases, the same species that are critical for host immunity display pathogenic qualities when homeostasis is disrupted. Bacteroides fragilis is one such species with numerous symbiotic and pathogenic characteristics. This thesis explores the generation of B. fragilis-specific systemic IgA and the role of this response in protecting the host from B. fragilis pathogenicity. Induction of systemic IgA specific to B. fragilis requires exposure of this bacterium to small intestinal Peyer's patches and results in migration of newly generated IgA plasma cells to systemic tissues. Colonization dynamics of B. fragilis in mouse models with endogenous gut microbiota revealed that the magnitude of systemic IgA responses occurs in a dose-dependent fashion. Finally, a framework for establishing B. fragilis colonization and subsequent immune modulation within a highly diverse intestinal ecosystem was developed.
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Investigating the role of formin FHOD3 during myofibrillogenesis in embryonic chick cardiomyocytesFormins are major actin polymerizing proteins which act via the FH2 domain to promote actin nucleation and polymerization, as well as the FH1 domain to accelerate FH2 mediated actin elongation. FHOD3 is a formin that has been shown to be expressed predominantly in the heart and is critical for myofibril maturation during development in mice. FHOD3 has been shown to localize where actin filaments overlap myosin filaments within the sarcomeres of mice, rat, and human induced pluripotent stem-cell derived cardiomyocytes, flanking both sides of the M-line in the sarcomere. However, the role of FHOD3 in the myofibrillogenesis and the timing of FHOD3's activity in myofibrils has yet to be determined. Using RT-PCR, I successfully identified expression of at least two different isoforms of FHOD3 within heart tissue, matching to predicted isoforms X5 and X6. I also identified two chemically conserved regions within the FHOD3 amino acid sequence that are related to the cardiac FHOD3 isoform's localization to myofibrils. Using immunofluorescence microscopy and western blotting I found that FHOD3 is present within embryonic chick cardiomyocytes and that the localization of FHOD3 matches prior reports. FHOD3 was determined to be transiently expressed at significantly higher rates on Days 3 and 4 of culture in cardiomyocyte myofibrils. 90% of measured sarcomeres containing FHOD3 had a Z-line to Z-line length ranging from 1.4-1.9 µm, suggesting not only a length-dependent role of FHOD3, but a myofibril maturity dependent localization of FHOD3. These observations illustrate that FHOD3 likely does not have a function in the initiation of myofibrillogenesis but may instead have a role in the maturation and elongation of sarcomeres. The transient nature observed also suggests that FHOD3 may be localized within the sarcomere only as needed. Knockdowns of FHOD3 performed with shRNAs showed no indication of knockdown causing myofibrillar disruption. Knockdowns of FHOD3 using DsiRNAs were statistically inconclusive for knockdown occurring but did have an upwards nonsignificant trend in the percentage of myofibril disruption in cardiomyocytes.
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HSV-1 targets a novel antiviral response of the STING pathwayIn order to establish a successful infection, herpes simplex virus-1 (HSV-1), a ubiquitous virus with high seropositivity in the human population, must undermine a multitude of host innate and intrinsic immune defense mechanisms, including key players of the stimulator of interferon genes (STING) pathway. Recently it was discovered that not only de novo produced intracellular 2'-3'cGAMP, but also extracellular 2'-3'cGAMP can activate the STING pathway by being transported across the cell membrane via the folate transporter, SLC19A1, the first identified extracellular antiporter of this critical signaling molecule in cancer cells. We hypothesized that the import of exogenous 2'-3'cGAMP would function to establish an antiviral state similar to that seen with the paracrine antiviral activities of interferon. Further, to establish a successful infection, viruses, such as HSV-1, must undermine this induction of the STING pathway by inhibiting the biological functions of SLC19A1. Herein, we report that treatment of the monocytic cell line, THP-1 cells and SH-SY5Y neuronal cell line with exogenous 2'-3'cGAMP induces interferon production and establishes an antiviral state. Using either pharmaceutical inhibition or genetic knockout of SLC19A1 blocks the 2'-3'cGAMP-induced inhibition of viral replication. Additionally, HSV-1 infection results in the reduction of SLC19A1 transcription, translation, and importantly, the rapid removal of SLC19A1 from the cell surface of infected cells. Our data indicate SLC19A1 functions as a newly identified antiviral mediator for extracellular 2'-3'cGAMP which is undermined by HSV-1 protein ICP27. This work presents novel and important findings about how HSV-1 manipulates the host's immune environment for viral replication and discovers details about an antiviral mechanism which information could aid in the development of better antiviral drugs in the future.
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Structural insights of the histone H3 tail and its role in the mechanism of histone H3 lysine-4 methylationStructural insights of the histone H3 tail and its role in the mechanism of H3 lysine-4 methylation Gene expression relies on the proper chromatin structure to provide the necessary access to the DNA for the large transcription complexes to carry out their tasks. If the chromatin is tightly condensed, transcription is unable to occur. To regulate and initiate access to the DNA, an elaborate network of histone modifying enzymes, chromatin remodeling complexes, and other supporting proteins must coordinate the writing, reading, and erasing of histone post-translational modifications (PTMs). One such PTM, methylation of histone H3 on the lysine-4 (H3K4) residue, is critically important for maintenance of gene expression states. This is done in a spatiotemporal manner, which is influenced by the number of methyl groups that are present. However, an understanding of how the degree of H3K4 methylation is regulated remains elusive. In this dissertation, we demonstrate the remarkable conservation of length and composition in the flexible N-terminal tails of histone proteins across evolution. Recent structural studies indicate several methyltransferase complexes bind to the nucleosome core, often leaving the N-terminal tails unbound. Research from our lab has also demonstrated that non-processive buildup of lysine-4 methyl groups takes place at multiple active sites. Based on these observations, we propose a hypothesis whereby the histone H3 tail acts as a swinging arm substrate, delivering residue side chains to different active sites to facilitate the progressive establishment of these epigenetic states. To investigate this hypothesis, we employed the CRISPR/Cas9 system in Saccharomyces cerevisiae to systematically modify the length of the H3 tail. We monitored histone H3 lysine 4 (H3K4) methylation, mediated by SET1, the primary H3K4 methyltransferase in budding yeast. Our findings demonstrate that altering the length of the H3 tail has varying effects on the extent of H3K4 methylation, in accordance with the swinging arm model. We also demonstrate that three proline residues are responsible for providing a segmented, tripartite structure with hinge-like joints that likely influence the tail's range of motion. Furthermore, the results support the proposed multiple active-site model, where mono-, di-, and trimethylation occur at distinct active sites within the COMPASS or MLL Core Complexes.
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Healthcare staff perceptions of an electronic hand hygiene monitoring system within a large university systemObjective: The acceptability of an electronic HH monitoring system (EHHMS) was evaluated among hospital staff members. Design: An electronic HH monitoring system was implemented in June 2020 at a large, academic medical center. An interdisciplinary team developed a cross-sectional survey to gather staff perceptions of the EHHMS. Setting: The survey was conducted at a public, tertiary acute care hospital. Participants: The survey included current employees and staff. 1,273 participants responded. The mean age was 44.9 years (SD = 13.5). Most of the samples were female (71%) and non-Hispanic white (83%). Methods: A survey was conducted between June and July 2021. Responses were analyzed using Stata statistical software. Multiple logistic regression models were constructed to examine factors associated with negative perceptions of the EHHMS and its radiofrequency identification (RFID) badge. Supporting qualitative analyses were performed using Atlas.ti version 9. Results: Three-quarters (75%) of respondents reported neutral to negative perceptions of the EHHMS and its associated badge. Respondents reported limited influence on HH practices. Age, campus location, length of employment, job role, and opinion on data sharing were associated with negative perceptions of the EHHMS and RFID badge. Position in a direct patient care role was associated with negative perceptions of the RFID badge. Conclusions: Perceptions of the EHHMS aligned with previous research. Identified associations provide opportunities for targeted education, outreach, and intervention to increase acceptability and uptake. Lack of acceptance is explained by poorly perceived ease of use and usefulness, as well as challenges in implementation.
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Pulse Pressure as a Hemodynamic Parameter in Preeclampsia with Severe Features Accompanied by Fetal Growth RestrictionBackground: Modern management of preeclampsia can be optimized by tailoring the targeted treatment of hypertension to an individual's hemodynamic profile. Growing evidence suggests different phenotypes of preeclampsia, including those with a hyperdynamic profile and those complicated by uteroplacental insufficiency. Fetal growth restriction (FGR) is believed to be a result of uteroplacental insufficiency. There is a paucity of research examining the characteristics of patients with severe preeclampsia who do and who do not develop FGR.We aimed to elucidate which hemodynamic parameters differed between these two groups. Methods: All patients admitted to a single referral center with severe preeclampsia were identified. Patients were included if they had a live birth at 23 weeks of gestation or higher. Multiple gestations and pregnancies complicated by fetal congenital anomalies and/or HELLP syndrome were excluded. FGR was defined as a sonographic estimation of fetal weight (EFW) < 10th percentile or abdominal circumference (AC) < 10th percentile. Results: There were 76% significantly lower odds of overall pulse pressure upon admission for those with severe preeclampsia comorbid with FGR (aOR = 0.24, 95% CI = 0.07-0.83). Advanced gestational age on admission was associated with lower odds of severely abnormal labs and severely elevated diastolic blood pressure in preeclampsia also complicated by FGR. Conclusions: Subtypes of preeclampsia with and without FGR may be hemodynamically evaluated by assessing pulse pressure on admission.
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Correlates of Loneliness and Social Isolation among Older Adults during the COVID-19 Outbreak: A Comprehensive Assessment from a National United States SampleThis study examined the correlates of loneliness and social isolation among older adults in the United States (U.S.) during the COVID-19 outbreak. We analyzed data from the 2020 National Health and Aging Trends Study, a nationally representative sample of 3257 U.S. older adults aged 65 years and older. We analyzed and identified the sociodemographic, health, social support, and community correlates of loneliness, higher loneliness during versus before the COVID-19 outbreak, and social isolation using weighted multiple logistic regression models. About 35.2% of U.S. older adults reported loneliness during the COVID-19 outbreak, 21.9% reported higher loneliness compared to before the COVID-19 outbreak, and 32.8% were socially isolated during the outbreak. Correlates for increased odds of loneliness included female gender, higher education, physical activity, depression, anxiety, functional limitations, and virtual communication access (only for higher loneliness during COVID-19 outbreak). Correlates for increased odds of social isolation included higher age, non- Hispanic Black, Hispanic, higher number of household children, and metropolitan residence. Our findings provide insights into evidence-based approaches to address social disconnection among U.S. older adults. The wide range of sociodemographic, health, social support, and community correlates identified in this study warrants multifaceted interventions that traverse individual, community, and societal levels to address the loneliness and social isolation epidemic.
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Role of TLDc Proteins Oxr1 and Rtc5 in Yeast V-ATPase Reversible DisassemblyThe vacuolar H+-ATPase (V-ATPase; V1Vo-ATPase) is a highly conserved, ATP hydrolysis-driven dedicated proton pump found on the membranes of intracellular organelles in virtually all eukaryotic cells and on the plasma membrane of specialized cell types. Regulation of V-ATPase activity is key to maintaining normal physiological functions, as aberrations in its activity are associated with several pathophysiological conditions. V-ATPase activity is mainly regulated by a mechanism called reversible disassembly, in which the assembly state - and hence the activity - of the enzyme is controlled by nutrient availability and extracellular cues. During the process, V-ATPase activity becomes either turned off by dissociation of the V1-ATPase from the Vo proton channel, or turned on by reassembling the two subcomplexes into an active enzyme. While the process is well-characterized at the cellular level, the molecular mechanism at the level of the enzyme remains elusive. Here, we show that two TLDc proteins, Oxr1p and Rtc5p, control the assembly state of yeast V-ATPase, with the former promoting disassembly, and the latter (re)assembly of the enzyme. Based on cryoEM analysis and in vitro and in vivo approaches, we discovered that Oxr1p is a V-ATPase disassembly factor. Oxr1p binding to V-ATPase results in autoinhibited V1 in two steps - first producing a disassembly intermediate, which, upon ATP hydrolysis, gets converted into autoinhibited V1. From in vitro experiments, we find that the second TLDc protein, Rtc5p, primes autoinhibited V1 for (re)assembly with Vo. CryoEM structures of Rtc5p bound V1 show Rtc5p's C-terminal ⍺ helix inserted into the catalytic core of the enzyme, thereby opening a second catalytic site, a conformational change that may facilitate (re)assembly of V1Vo. In vivo experiments, however, suggest that Rtc5p is not essential for V-ATPase reassembly in the cell, suggesting redundancy and/or alternative pathways. Overall, this study enhances our understanding of the molecular basis for the regulation of V-ATPase activity by reversible disassembly.
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Exploring the role of single nucleotide polymorphisms in varicella zoster virus vaccine attenuation in skinVaricella zoster virus (VZV) is a disease that can be detrimental to the health of children in its primary form, chicken pox, and later in the elderly as its reactivated form, shingles. Before the advent of the vaccine, Varivax, VZV was endemic in the United States as it is highly contagious and can be spread through both direct contact and aerosol particles. Varivax, or vOka, is a live attenuated vaccine, and while effective, has side effects ranging from rashes to possible VZV reactivation. While the vaccine has reduced the incidence and severity of VZV, there is still little known about the mechanism of its attenuation in skin. vOka is genetically heterogeneous with hundreds of single nucleotide polymorphisms (SNPs) that are a mixture of wild-type and vOka nucleotides. Previous studies have demonstrated the key to attenuation may be through five SNPs in the open reading frame (ORF) 62 region found to be fixed and stable across different licensed vOka preparations around the world. ORF62 contains the gene for IE62, a transactivator protein responsible for regulating the expression of viral genes and the host gene for KRT15, a cytokeratin protein. This project focused on if two SNPs, located in the loci positions 106262 and 107252, that are found to be almost 100% conserved across all variations of vOka are responsible for the attenuation in human skin and induction of KRT15. We evaluated four mutant viruses with SNPs found in vOka and discovered that a double SNP mutation stunted virus growth in HFF cells. In addition, we found no significant difference in the growth of our viruses in skin but variability in successful infection. Furthermore, in infected skin, we found that VZV-ORF57-Luc and single mutant virus, 68-958, upregulate KRT15 expression with VZV infection while single mutant virus, 68-62S-A, may downregulate KRT15 expression with VZV infection. This project is important because it may reveal the molecular basis of attenuation of the licensed varicella vaccine. This information could be used to make a vaccine that contains only the attenuated genotype.
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From bud scars to molecular insights: investigating V-ATPase function and assembly in yeast replicative agingAging is a complex process that involves the progressive decline of physiological functions over time. As the global population continues to age, understanding the mechanisms underlying aging has become an important area of study. Lysosomes play a crucial role in maintaining protein quality control and degrading unneeded or damaged proteins through proteolysis. Therefore, lysosomes play a prominent role in theories of aging due to their significant role in cellular homeostasis. Interestingly, many of the hallmarks of aging are conserved between yeast and humans, highlighting the relevance of yeast as a model organism to study aging processes. One key enzyme responsible for acidifying lysosomes and lysosome-like vacuoles in yeast is the vacuolar-type H+-ATPase (V-ATPase). Despite evidence showing that lysosomes alkalinize with age, compromising their proteolytic function, little is known about the regulation of V-ATPase in aging cells. Yeast cells divide asymmetrically with each division leaving a "bud scar" that can be stained to determine replicative age. In comparing cells of distinct replicative age, we find significant decreases in V-ATPase assembly, accompanied by poor vacuolar acidification, in older cells. Remarkably, partial disassembly of the V-ATPase occurs at a relatively early age, indicating its potential as a phenotypic driver in the aging process. Reversible disassembly is controlled in part by the activity of two opposing and conserved factors, the RAVE complex and Oxr1. The RAVE complex promotes V-ATPase assembly and a rav1∆ mutant has a significantly shorter lifespan than wild-type cells; Oxr1 promotes disassembly and an oxr1∆ mutation significantly extends lifespan. These data indicate that reduced V-ATPase assembly may drive the loss of lysosome acidification with age and place the balance of V-ATPase assembly factors at the center of this process. Caloric restriction, defined as reduced calories with adequate nutrition, has been shown to extend lifespan in multiple organisms including yeast. We find that caloric restriction reverses the age-related decreases in V-ATPase assembly and vacuolar acidification in yeast as well as restoring balance of assembly factors. We investigated three conserved metabolic signaling pathways that have been linked to acidification, caloric restriction, and aging: PKA, mTORC1/S6K (TORC1/Sch9 in yeast), and AMPK (Snf1 in yeast). By utilizing non-essential nutrient mutations in these signaling pathways, we determined the impact on V-ATPase assembly during replicative aging. Mutations compromising TORC1 function were known to extend lifespan and preserved V-ATPase assembly even in older cells. In contrast, a mutation that prevents recruitment of Snf1/AMPK to vacuoles prevented V-ATPase assembly even in young cells and shortened lifespan. This study provides novel insights into the importance of V-ATPase assembly and function in the aging process and suggests novel interventions to promote health aging.
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Kcnh2 Expression Profile and Continuous EEG/ECG Monitoring in a Rabbit Model of Long QT Syndrome Type 2Long QT Syndrome (LQTS) is a classically studied cardiac condition characterized by a prolonged ventricular excitation-repolarization interval (QT interval) on an electrocardiogram (ECG). LQTS is associated with an increased risk of arrhythmias and sudden cardiac death. People with LQTS, particularly those with Long QT Syndrome Type 2 (LQT2), are also at an increased risk of seizures/epilepsy. LQT2 is caused by loss of function variants in the KCNH2 gene. The dual neuro-cardiac phenotype of LQT2 can likely be explained by expression of KCNH2 in both the brain and heart. Using a rabbit model that harbors an endogenous knock-in mutation in one allele of the pore domain of the Kcnh2 gene, I characterized the molecular expression of WT vs. mutant Kcnh2 and developed a protocol for long-term subcutaneous EEG/ECG implantation. To better understand the molecular profile of WT vs. mutant rabbits, the relative expression of WT vs mutant Kcnh2 transcripts was evaluated using quantitative PCR (qPCR) with verification via Oxford Nanopore Technology (ONT) sequencing. Additionally, 44 RNA sequencing libraries were prepared and sequenced for further analysis of the molecular profile of WT vs mutant rabbits. Micro-C and high molecular weight DNA libraries were also prepared for the construction of a more thorough rabbit genome. In mutant rabbits, total Kcnh2 expression is roughly half that of WT rabbits. In mutant rabbits, the mutant Kcnh2 RNA represents 11% of the total Kcnh2. These data suggest that most mutant Kcnh2 RNA is degraded shortly after generation. To continuously monitor the rabbits’ cardiac and neuronal electrical function in vivo, a method of constant EEG/ECG recording was designed and implemented. It involves the surgical placement of subdermal electrodes and the design and manufacturing of a wiring system. The surgical placement of electrodes has been optimized to minimize time and number of incisions and improve outcomes. The wiring system enables the rabbits to have free range of motion within the housing cage and keeps all wires protected and out of reach of the rabbits. This system is functional and generates high quality continuous EEG/ECG recordings.
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The murine absolute visual threshold: behavior & retinal pathwaysConnexin 36 (Cx36) gap junctions are important for governing the sensitivity of the dark-adapted retina. Despite its importance for physiological sensitivity, the degree to which retinal Cx36 governs the psychophysical absolute threshold is not known. The purpose of this work is to study to what extent inner- and outer-retinal Cx36 governs the absolute visual threshold. In Chapter 2 we developed a one-alternative forced choice (1AFC) task for measuring murine absolute visual thresholds to full-field flash stimuli. We found that our 1AFC task, in conjunction with the theory of signal detection, gave response bias-independent absolute visual threshold estimated. Using this assay, we found that decision criteria are related to response times. In Chapter 3 we used the 1AFC task and the power of transgenic mice to assess the relative contributions of inner and outer retinal Cx36 to the absolute visual threshold. We concluded that inner, not outer, retinal Cx36 is most responsible for governing the absolute visual threshold. In parallel, by testing mice with disrupted rod vision, we determined that rod OFF pathways, and not cones, set the absolute visual threshold in the absence of Cx36. Finally, we studied the impact of Cx36 on temporal summation at absolute threshold, by obtaining thresholds for a range of flash durations. Threshold-vs-duration data was then fit with a model of temporal summation that allowed us to determine whether Cx36 influences the temporal filtering properties of scotopic vision. Our model fits suggest that photoreceptor Cx36 may play a role in temporal processing at absolute visual threshold. Overall, this work sheds new light on the behavioral dynamics and neural underpinnings of rod mediated vision.