RSS 1.0Upstate Medical University
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Smoking History Intensity and Permanent Tooth Removal: Findings from a National United States SampleThe role of smoking in the development of periodontal disease has been well explored. However, this study aims to explore the relationship between intensity of smoking history and permanent tooth removal. We utilized the 2022 Behavioral Risk Factor Surveillance System (BRFSS), a nationally representative sample of 107,859 US adults, to explore this association. Smoking history intensity was a BRFSS-derived measure of pack-year smoking history. Permanent tooth removal was binarized as the presence or absence of a history of permanent tooth removal. A binary logistic regression was conducted to analyze this association after adjusting for a variety of sociodemographic, health, and substance-use covariates. There was a dose-dependent relationship in which increasing smoking history intensity was associated with increased odds for removal of one or more permanent teeth. For example, those who reported a pack-year history of 30 or more years had a 6.4 times significantly higher odds of reporting a history of permanent tooth removal when compared to those with a 0 pack-year history (adjusted odds ratio = 6.37, 95% CI = 3.80–10.69, p < 0.001). These findings can be used to promote smoking reduction or cessation as a means of decreasing risk of permanent tooth removal due to tooth decay or gum disease.
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The Bok-IP3R interaction and the impact it has on apoptosis, Bok stability, and IP3R-mediated calcium mobilizationBcl-2-related ovarian killer, Bok, is often considered a pro-apoptotic Bcl-2 family member due to its ability to induce mitochondrial outer membrane permeabilization (MOMP) when over-expressed. However, there are many conflicting reports regarding endogenous Bok's apoptotic function and stability. Some studies claim that endogenous Bok is constitutively a MOMP-mediator but is very unstable and kept at very low levels via ubiquitin proteasome pathway (UPP)-mediated degradation. On the other hand, many studies, including those from the Wojcikiewicz Lab, indicate that endogenous Bok is a stable protein constitutively bound to inositol 1,4,5-trisphosphate receptors (IP3Rs), a calcium (Ca2+) release channel in the endoplasmic reticulum (ER) membrane, where it would be unable to induce MOMP. In fact, Bok KO mice have no developmental of phenotypical abnormalities, further suggesting that Bok's "killer" characterization needs to be reconsidered. Since endogenous Bok is an ER protein, studying its MOMP-like properties seems futile. Therefore, focusing on the significance of the Bok-IP3R interaction has been the priority of the Wojcikiewicz Lab since we discovered the complex. Over the years, we found that Bok protects IP3Rs from proteolytic cleavage and that Bok stability is dependent on its ability to bind to IP3Rs. However, surprisingly, there is no clear evidence to date that Bok influences the Ca2+ mobilizing activity of IP3Rs. Here, I study in detail the Bok-IP3R interaction and the impact it has on apoptosis, Bok stability, and IP3R-mediated Ca2+ mobilization. I resolve the controversy surrounding the apoptotic function of exogenous and endogenous Bok and hypothesize why there is a discrepancy between the two. Also, I show that IP3R binding in the ER membrane is essential for Bok stability, and without them Bok is rapidly degraded by the UPP. This demonstrates that the Bok-IP3R interaction is critical to Bok function, likely including its newly emerged non-apoptotic roles, such as regulating mitochondrial dynamics and mitochondria associated-ER membranes. Finally, for the first time, I reveal that Bok has suppressive effects on IP3R-mediated Ca2+ mobilization and describe how these effects are reversed when Bok is phosphorylated at serine-8. Overall, this thesis demonstrates the importance of the Bok-IP3R interaction on both Bok and IP3R function.
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Diverging cognitive benefits from education between rural and urban middle-aged and older adults in the USA.Subjective cognitive decline (SCD) is defined as self-reported increase in confusion or memory loss. There is limited research on the interplay between rural-urban residence and education on SCD.
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DevATLAS: A novel tool to monitor the sequence of neural circuit development and study neurodevelopmental disordersEarly postnatal brain development is the critical stage when the symptoms of many neurodevelopment disorders (NDDs) start manifesting. These functional deficits are often caused by abnormal neural circuit maturation without accompanying gross alteration to brain architecture, making it challenging to pinpoint disruptions in these NDD models. There is an urgent need for genetic tools to track the neural circuit maturation sequence on the whole brain level during the early postnatal period. One of the key driving factors of neural circuit maturation is neuronal activity. Our lab has developed DevATLAS, the Developmental Activation Timing-based Longitudinal Acquisition System, to overcome this challenge based on the immediate early gene Npas4 expression. Npas4 is selectively induced by neuronal activity, and its activation during development triggers activity-dependent synapse development, which is a critical step during the functional maturation of neural circuits. DevATLAS permanently labels neurons with tdTomato (tdT) as they are activated by neuronal activity to express Npas4. We demonstrate that DevATLAS captures the functional neural circuit maturation sequence across the whole brain during the early postnatal period. We also demonstrate that early environmental enrichment (EE) intervention can accelerate functional neural circuit development in the granule cells (GC) of the dentate gyrus (DG). Finally, we combine DevATLAS with the NDD model of Fragile-X Syndrome (FXS) and observe significant developmental perturbations in neural circuit maturation in multiple ASD-associated regions, including the dorsal striatum, primary motor cortex, medial prefrontal cortex, which can be associated with perturbed behaviors in juvenile FXS mice. Within the DG of FXS mice, we use DevATLAS to track perturbed neural circuit maturation with delayed emergence of contextual learning and memory with altered development of granule cell dendritic arbors and spines, as well as demonstrate how early EE can ameliorate. Our results indicate that DevATLAS can study neural circuit maturation in NDD models, enable researchers to gain an improved underlying NDD etiology, and ultimately help derive new therapeutic interventions to ameliorate NDD deficits.
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Longitudinal Engagement in Modifiable Lifestyle Behaviors and Racial–Ethnic Differences in Dementia RiskObjectives The objective was to examine racial–ethnic differences in longitudinal engagement for lifestyle behaviors and moderating role of race–ethnicity between lifestyle behaviors and dementia risk. Methods We analyzed 2011–2021 National Health and Aging Trends Study data, a nationally representative U.S. sample of 6155 White, Black, Hispanic, and Asian older adults aged 65+. Cox models regressed dementia on the interaction between lifestyle behaviors (physical activity, smoking, and social contacts) and race–ethnicity. Results Only smoking was associated with about a 45% higher dementia risk (aHR = 1.45, 95% CI = 1.11–1.89). On average, Black and Hispanic respondents exhibited less frequent physical activity and social contacts, along with more frequent smoking. There was one significant interaction; more social contacts were associated with lower dementia risk among Asian respondents (aHR = 0.16, 95% CI = 0.05–0.55). Discussion Racial–ethnic differences in lifestyle behaviors should be considered when addressing dementia disparities. Future research needs to explore the relationship between social contacts and lower dementia risk among Asian older adults.
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Mitochondria-induced Bioenergetic Independent Stress Signaling in the HeartMitochondria are well known for their function in providing energy supply to the cell. Aside from these "bioenergetic" functions, mitochondria perform many other essential processes. To accomplish these functions, mitochondria must import many proteins from the cytosol. This import process can sometimes become dysfunctional and induce a severe stress on the cell via the mistargeting of mitochondrial proteins to the cytosol. Our lab termed this specific type of cell stress as mitochondrial precursor overaccumulation stress (mPOS). Our work has focused on demonstrating that mPOS is able to occur in various models of disease, both in vivo and in vitro. In this thesis we demonstrate that mPOS can occur in the heart of mice and induce significant signaling and functional changes over the lifespan (chapter 2). Additionally, in a related work, we found that mitochondrial protein import clogging can induce mPOS in the central nervous system (CNS) and potentiate pathology in a mouse model of Parkinson's disease (appendix I). Most importantly, all the changes occurring in both animal models do not necessarily co-occur with bioenergetic deficiencies. The implication of this is that mPOS may be a bioenergetic independent mechanism liking mitochondrial dysfunction with tissue dysfunction. Speaking more generally, mPOS may occur in many clinically relevant conditions such as heart failure, normative ageing, muscle loss, and neurodegenerative diseases. This work and future work therefore aims to establish the basic mechanisms by which mPOS may occur within cells and how cells can in turn respond to this stress.
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Engineering Generalized Protein-Based Biosensors for Molecular Detection and Clinical ApplicationsProtein-based conformational switches serve as powerful tools for the construction of biosensors and for the control of cellular processes. These proteins feature a binding domain that recognizes a specific analyte and is coupled to an output domain in such a way that the binding event causes the output domain to provide an observable signal. These signals can either be turn-on of fluorescence, luminescence, or enzymatic activity or consist of the sensor changing its color. A challenge in constructing these protein switches is finding binding domains capable of relaying a ligand binding event to the conformational change of an output domain. Generalized binding domains can address these challenges by providing a scaffold that can easily be modified to detect a different ligand. These generalized binding domains are small proteins with modifiable residues that can be selected to bind a ligand of choice, usually through phage display and similar selection techniques. Here, we present two approaches to make generalized protein switches. In the first approach, antibody mimetics nanobodies and monobodies are inserted in fluorescent proteins such that binding of their ligand causes an increase in fluorescence. This technique, named adaptable turn-on maturation (ATOM), was used to develop biosensors for WD-40 repeat protein 5 (WDR5), c-Abl src homology 2 (SH2) domain, hRas, postsynaptic density protein 95 (PSD95), gephyrin, HOMER1, and mCherry for use in mammalian cells. ATOM is, therefore, compatible with a variety of ligands due to its input domain being a generalized binding domain. Additionally, the ATOM mechanism can be used to convert many fluorescent proteins into biosensors. For demonstration, we made biosensors from Clover, mTurqoise, mTagRFP-t, mStayGold, mBaoJin, and GCaMP6s. In the second approach, we develop a luminescent protein switch from the enzyme nanoluciferase (nLucAFF) that switches color from green to blue upon DNA binding. We show that DNA-based devices can then be used to detect various ligands and relay that event to nLucAFF, which provides an output easily quantifiable by a cell phone. The nLucAFF protein was used to detect DNA sequences amplified from cytomegalovirus (CMV), dengue, and nCoV. Additionally, aptamers binding to serotonin and aptamers were used to detect these molecules by directing the nLucAFF color change. The initial version of nLucAFF was slow, dim, and had low sensitivity. These drawbacks were resolved in the next version, nLucAFF2, to achieve turn-on within 5 minutes and detect ligands down to 40 pM with a cell phone camera. The last chapter combines two ligand-binding domains to activate a small cytotoxic RNase, barnase, and paves the way for the development of multi-input protein switches that can potentially be generalized ligand-binding domains.
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Nanobody development for therapeutically targeting Vacuolar H+-ATPasesThe vacuolar H+-ATPase (V-ATPase, V1Vo) is a dedicated proton pump that is highly conserved amongst eukaryotes, and is necessary for pH homeostasis within subcellular compartments. The V-ATPase consists of two subcomplexes: the soluble V1 responsible for hydrolyzing ATP, and the membrane integral Vo responsible for proton translocation across membranes. V1 and Vo are each comprised of multiple subunits, A3B3CDE3FG3 and ac8c'c"def Voa1 respectively. Many basic cellular functions depend on the differential pH gradient across cellular membranes to operate properly, making regulation of V-ATPases through "reversible disassembly" immensely important. Global loss of V-ATPase activity is lethal to all mammalian cell types, while aberrant activity and incorrectly localized V-ATPase results in various disease states. Current therapeutics struggle to target specific V-ATPase populations, and as a potential solution to this problem we generated 94 nanobody clones against the yeast nanodisc reconstituted Vo (VoND). Nanobodies (Nbs) are the small 15 kDa VHH domain isolated from heavy-chain only antibodies that are known for their high specificity. In this dissertation we describe the characterization of three α-yeast VoND Nbs, N27, N125, and N2149. Using an ATPase assay, we found that N27, but not N125 or N21149, fully inhibited the activity of assembled V-ATPase. Contrastingly, N2149, but not N27 or N125, was found to inhibit the assembly of the two subcomplexes. BLI was used to identify the binding affinity of each Nb, with affinities being observed in the nM-pM range. High-resolution structures obtained from cryoEM revealed the subunit specificity of each Nb, with N27 and N125 found to bind the c-ring in different stoichiometries, and N2149 found to bind the d subunit. Furthermore, we determined that N125 has cross affinity for the human enzyme. Overall, this study provides evidence that novel nanobody mediated inhibition of assembly or activity of V-ATPases is an effective technique with broader implications of nanobody development into therapeutics.
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Nanotherapeutics for Immune Modulation in SepsisDue to its complexity and heterogeneity, managing immune dysregulation in sepsis poses a significant clinical challenge. Thus, there is great demand to both improve our understanding of mediators of immune dysregulation in sepsis and develop nuanced therapeutic approaches to provide precise immune modulation for sepsis treatment. This thesis first investigates the novel phenomenon of cytokine charge disparity as a potential regulator of cytokine function. Then, two novel telodendrimer immune modulation approaches are presented as a personalized medicine strategy for sepsis. Through extensive database and literature review, we have established cytokine charge disparity as a potential mechanism for immune regulation. Using our versatile telodendrimers (TDs), we then optimized and validated our TD nanotrap approach for effective and selective targeting of plasma cytokines. Our lead selective TD nanotraps displayed charge selective cytokine targeting and our lead pan-affinitive TD nanotrap demonstrated superior cytokine removal efficacy compared to commercial resin control. Additionally, pan-affinitive TD nanotrap maintained efficacy across a wide range patient immune status, indicating promising therapeutic potential to reduce mortality risk associated with overwhelming cytokine profiles. To further expand our immune modulation tool set for sepsis treatment, we optimized our TD nanodrug for delivery of dimethyl itaconate (ITA) to control both hyperinflammation and pyroptosis. Encapsulating ITA into TD nanoparticles (ITA:TDNPs) resulted in a sustained-release profile and improved biocompatibility compared to free ITA. ITA:TDNPs more effectively inhibited both LPS- and LTA-induced inflammation and pyroptosis in macrophages compared to ITA or TDNP alone. Finally, ITA:TDNPs demonstrated superior therapeutic efficacy in both an IV LPS and polymicrobial cecal slurry sepsis model compared to individual therapies. Collectively, we have uncovered a novel phenomenon of cytokine charge disparity and validated it as a potential mechanism to regulate cytokine activity, as well as established it as targeting mechanism for effective immune modulation via charge selective TD nanotrap. We further developed an immune modulating TD nanodrug for ITA delivery to control both hyperinflammation and immune cell pyroptosis in sepsis. Through precise targeting of immune dysregulation in sepsis using a systematic multimodal TD therapeutic approach for personalized medicine, we may successfully improve patient outcomes in this devastating disease.
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Mechanism of Mitochondria-Induced Muscle Atrophy During AgingMitochondrial dysfunction is strongly associated with aging-related degenerative diseases including muscle atrophy. However, whether bioenergetic defects are the sole drivers of mitochondria-induced muscle atrophy remains unknown. The Chen lab discovered that various forms of mitochondrial damage can disrupt protein import, leading to the toxic accumulation of unimported mitochondrial precursors in the cytosol. This causes a stress termed mitochondrial Precursor Over-accumulation Stress (mPOS). A mouse model of mPOS was developed in which the mitochondrial inner membrane protein, ANT1, was overexpressed to saturate the protein import machinery. Ant1Tg/+ mice were found to have a severe muscle wasting phenotype. The overarching goal of this dissertation is to investigate the mechanism by which mPOS drives muscle wasting and its implications for normative muscle aging. The findings presented in this thesis led to three conclusions. First, we identified a novel mitochondria-to-lysosomal proteostatic axis through which mPOS induces lysosomal damage. Lysosomal damage subsequently causes the release proteolytic enzymes, which leads to excessive protein degradation and subsequent progressive muscle atrophy. Importantly, we found that this pathway operates independently of mitochondrial respiratory complex activity and reactive oxygen species (ROS) production. Second, we demonstrated the presence of mPOS in physiologically aged muscle. Sarcopenic muscle exhibited phenotypes similar to those found in Ant1Tg/+ mice, evidenced by overlapping transcriptional and proteomic profiles, and lysosomal damage. These findings indicate that mitochondria-induced changes to autophagic activity may play a central role in the pathogenesis of sarcopenia. However, considering the overall protein content of muscle is elevated during aging, we propose that reduced protein quality, rather than absolute protein content, drives sarcopenia. We therefore termed this phenomenon Muscle Atrophy Independent of Protein Content (MAIPC). Finally, we explored additional cellular factors that affect proteostasis and muscle mass maintenance. We found that the GCN2 kinase, a well-established activator of the Integrated Stress Response (ISR), plays a role in protecting myofibers from mPOS-induced stress and muscle wasting in Ant1Tg/+ muscle. Interestingly, we found that this effect is ISR-independent. The data presented in this dissertation provide valuable insights into the mechanistic role of mitochondrial dysfunction in both normative aging and chronic muscle wasting conditions. Our findings conclude that mitochondria-induced muscle atrophy is induced by mechanisms that extend beyond bioenergetic defects. By characterizing these alternative pathways, this work opens new avenues for therapeutic strategies targeting mitochondrial stress in chronic muscle wasting conditions.
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Sociodemographic Factors and Vulnerability to Multiple Extreme Weather Events: A National Study in the U.S.Background Millions of U.S. residents experience increasingly more prevalent weather events due to climate change, however, there is limited research exploring the vulnerability to multiple extreme weather events using a national U.S. sample. Aims Identify patterns in exposures to climate events, and examine sociodemographic factors associated with increased climate event vulnerability. Method Data was retrieved from the May 2022 American Trends Panel, a nationally representative sample of 10,282 United States adults. We performed a latent class analysis, a statistical method used to identify unobserved subgroups (latent classes) within a population, to group respondents by patterns in five climate event experiences (heatwave, intense storm, wildfire, drought, and sea level rise), and analyzed variables associated with vulnerability to climate events using weighted multinomial logistic regression, a statistical method that models the probability of membership in one of several outcome categories (climate vulnerability groups) relative to a reference category, while accounting for survey weights to ensure generalizability to the U.S. population. Results Respondents were categorized into four latent classes, which are unobserved subgroups identified through patterns in exposures to five climate events (heatwave, intense storm, wildfire, drought, and sea level rise). These subgroups were based on exposures to heatwave (42.5 %), intense storm (43.2 %), wildfire (21.3 %), drought (30.8 %) and sea level rise (15.8 %): high (9.8 %), heat-storm (22.2 %), heat-drought (13.4 %), and low (54.6 %) climate event vulnerability. Relative risk for high climate event vulnerability refers to the likelihood of belonging to the “high vulnerability” group compared to the “low vulnerability” group. It is assessed using the relative risk ratio (RRR), which is a measure of the association between a particular sociodemographic factor (e.g., age, gender, region) and the likelihood of being in a specific vulnerability group relative to the reference group. For instance, an RRR <1 indicates a reduced risk, while an RRR >1 indicates an increased risk compared to the reference category. Relative risk for high climate event vulnerability was lower for older adults (RRR = 0.39, p < 0.001), potentially reflecting a greater capacity to cope with certain climate events, such as access to stable housing or resources. However, this finding should not be interpreted as older adults being universally less vulnerable. Numerous studies have shown that older adults are at significantly higher risk during heatwaves due to physiological and social factors, which our analysis may not fully capture. Relative risk for high vulnerability was higher for females (RRR = 1.42, p = 0.01) and residents in the South (RRR = 2.05, p = 0.003) and West (RRR = 9.31, p < 0.001) geographic regions. Relative risk for heat-drought was higher for Hispanic adults (RRR = 1.51, p = 0.03), but lower for high school graduates (RRR = 0.40, p = 0.01) compared to those who did not complete high school. Conclusions We identified several underlying climate event exposure subpopulations, ranging from low to high vulnerability. As climate-related events become more frequent, our results provide critical insights for stakeholders to identify high-risk individuals and prioritize resources for disaster management.
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Non-neutralizing Antibodies in the Complex Dance of Dengue Clearance and Immune AvoidanceDengue virus (DENV) is endemic in over 100 countries causing widespread morbidity and mortality. Approximately 400 million people are infected annually with one of the four immunologically and genetically distinct serotypes of DENV, resulting in 100 million symptomatic cases and at least 40,000 deaths. While the mechanisms behind the pathophysiology of severe DENV infection are complex and incompletely understood, it has been previously suggested that antibodies directed against the DENV envelope (E) protein can facilitate antibody dependent enhancement (ADE) of the virus during secondary DENV infections, increasing the number of infected cells and the clinical severity of infection in an exposed individual. However, there are other functional roles for antibodies outside neutralization of the virion. In this thesis, we describe the roles of non-neutralizing antibodies during DENV-infection. We show that IgG and IgA non-structural protein 1 (NS1)- and E-reactive antibodies are capable of mediating monocytic phagocytosis of DENV-infected cells. We show that secreted NS1 (sNS1) acts as immunological chaff and abrogates NS1-reactive antibody-mediated phagocytosis. We also begin to investigate the potential of phagocytosis of DENV-infected cells to lead to lead to infection of phagocytic monocytes. The findings described in these studies have implications in therapeutics and vaccinations targeting both NS1 and E protein.
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Unveiling cell-type-specific transcriptome and genetic regulation in postmortem brains of schizophrenia patientsSchizophrenia is a complex psychiatric disorder with a poorly understood etiology. This dissertation addresses three critical questions in schizophrenia research: identifying involved cell types, characterizing their transcriptomic changes, and elucidating how these changes mediate genetic risk. After rigorous evaluation, we conducted a comprehensive analysis of cell-type-specific gene expression in postmortem brains of schizophrenia patients and controls with single-cell RNA sequencing and cell deconvolution methods. Our findings provide compelling evidence for the involvement of upper-layer neurons and multiple non-neuronal cell types in schizophrenia. We observed significant alterations in synaptic function, neurodevelopment, immune response, and vascular transport within their respective cell types. Notably, we demonstrate that genetic risk for schizophrenia is predominantly enriched in neurons, particularly upper-layer neurons, with partial enrichment in oligodendrocyte precursor cells and vascular cells. This cell-type-specific approach offers novel insights into the molecular underpinnings of schizophrenia, potentially bridging the gap between genetic risk factors and clinical manifestations. By highlighting key genes and pathways, our study establishes a robust foundation for future research and opens avenues for innovative preventive and therapeutic approaches.
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Sleep disturbances and racial-ethnic disparities in 10-year dementia risk among a national sample of older adults in the USARace/ethnicity and sleep disturbances are associated with dementia risk.
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Mucosal Innate Immunity of Human Surfactant Protein A Genetic Variants against SARS-CoV-2 InfectionMore than 7 million people have died of the coronavirus disease-2019 (COVID-19) since first reported in December of 2019. Infection in some patients manifests as life-threatening ALI/ARDS, multi-organ dysfunctions, and/or death characterized by active viral replication and profound inflammatory cell influx into tissues/organs. SARS coronavirus-2 (SARS-CoV-2) infects human angiotensin-converting enzyme 2 (hACE2)-expressing cells through its spike protein (S protein). The S protein is highly glycosylated and could be a target for lectins. Surfactant protein A (SP-A) is a collectin, expressed by lung alveolar type II cells and other mucosal epithelial cells; it plays a crucial role in innate immunity and inflammatory regulation. SP-A modulates pathogenic infection and disease severity by binding to microbial and host glycoproteins to alter infectivity and regulate host inflammation. The human SP-A gene is located on chromosome 10q22-23, which contains two functional genes SP-A1 and SP-A2 (gene names: SFTPA1 and SFTPA2), and a pseudogene. SP-A1 and SP-A2 are highly polymorphic and consist of several genetic variants, such as SP-A1 (variants 6A2, 6A4) and SP-A2 (variants 1A0, 1A3). It has been demonstrated that these variants have differential antiviral and immunoregulatory capacities in response to various viral infections. The goal of this study was to investigate the mechanistic role of human SP-A variants in response to SARS-CoV-2 infection and COVID-19 susceptibility and severity. The results from this study showed that native human SP-A can bind SARS-CoV-2 S protein, receptor-binding domain (RBD), and hACE2 in a dose-dependent manner. A decrease in S protein and RBD binding was observed in the presence of EDTA and sugars, indicating that the SP-A carbohydrate-recognition domain (CRD) mediates S protein binding in a calcium-dependent manner. We further showed that human SP-A can attenuate viral infectivity in susceptible host cells, evidenced by the dose-dependent reduction in viral load in infected cells. These results suggest that human SP-A can bind SARS-CoV-2 S protein, RBD, and hACE2 to attenuate SARS-CoV-2 infectivity in susceptible host cells. Next, we examined the variations in antiviral and immunoregulatory roles of human SP-A variants in response to SARS-CoV-2 infection. The binding studies showed that in vitro-expressed SP-A variants differentially interact with S protein. Moreover, cells inoculated with SARS-CoV-2 pretreated with the 1A0 variant had a more reduced virus titer than those pretreated with the 6A2 variant, indicative of their differential antiviral capacities. These findings from in vitro studies demonstrated that human SP-A and their genetic variants directly interact with viral S protein to differentially modulate SARS-CoV-2 infectivity. To perform in vivo study, six genetically modified double-hTG mouse lines, expressing both hACE2 and the respective SP-A variants: (hACE2/6A2 (6A2), hACE2/6A4 (6A4), hACE2/1A0 (1A0), and hACE2/1A3 (1A3), one SP-A knockout (hACE2/SP-A KO (KO) and one hACE2/mouse SP-A (K18) mice, were generated and challenged intranasally with 103 PFU SARS-CoV-2 (Delta) or saline (Sham). We observed that these infected mice had differential COVID-19 severity. Infected KO and 1A0 mice had more mortality and lung injury compared to other mouse lines, and disease severity correlated with enhanced upregulations of inflammatory genes that play vital roles in host immunity such as MyD88 and Stat3 in the lungs of KO and 1A0 mice. Furthermore, pathway analysis identified several important signaling pathways involved in lung defense, including pathogen-induced cytokine storm, NOD1/2, toll-like receptor, neuroinflammation, and Trem1 signaling pathways. Consistent with the transcriptomic data, expressions of inflammatory mediators such as G-CSF, IL-6, and IL-1β were comparatively higher in the lungs and sera of KO and 1A0 mice with the highest mortality rate. We further examined other organ injuries (kidney, intestine, and brain) in the infected mice; we found a more severe acute kidney injury (AKI) and intestinal damage in KO and 6A4 mice compared to other double-hTG mice. Viral titers were generally lower in the kidneys and brains of infected double-hTG mice relative to KO mice. Inflammatory mediators like TNF-α, IL-6, IL-1β, and MCP-1 were comparatively higher in KO and 6A4 mice with the most severe AKI. High virus presence and inflammatory markers were also observed in the brain and hippocampus of all infected mice. The results from in vivo studies suggest that SP-A variants differentially protect against severe COVID-19. Furthermore, the human COVID-19 patient studies revealed increased SP-A levels in the saliva of COVID-19 patients compared to healthy controls and highlighted the potential use of SP-A levels as a biomarker for COVID-19 severity. Collectively, these findings underscore the importance of host innate immune collectins and contribute to our understanding of the roles of host genetic variations in the observed population-level differences in COVID-19 susceptibility and severity.
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Deciphering cellular dynamics and crosstalk of trabecular meshwork and Schlemm's canal cells in a bioengineered 3D extracellular matrix hydrogel microenvironmentIn the conventional outflow pathway, Schlemm's canal (SC) inner wall endothelium interfaces with the trabecular meshwork (TM). Biomechanical changes in this microenvironment contribute to increased resistance to aqueous outflow, a characteristic of ocular hypertensive glaucoma. Notably, TM undergoes fibrotic-like remodeling and stiffening. Existing in vitro TM/SC models fail to accurately replicate native cell-cell and cell-extracellular matrix (ECM) interactions, limiting their use for studying glaucomatous outflow pathobiology. In this dissertation, we utilized a biomimetic ECM hydrogel system made from natural polymers resembling native tissue proteins. This ECM hydrogel can be (i) used to encapsulate donor-derived primary human TM cells or (ii) employed as a substrate for culturing donor-derived primary human SC cells on top. As ECM hydrogels gradually emerge as a preferred model in diverse research laboratories, a standardized fabrication method is essential to improve accessibility and consistency across experimental protocols. Thus, a detailed methodology for producing these ECM hydrogels is provided in Chapter 2. In Chapter 3, using the 3D TM hydrogel system, we demonstrated that simvastatin-mediated inactivation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) attenuates pathological changes in TM cells. YAP/TAZ are key mechanotransducers involved in glaucoma pathogenesis and are shown to be regulated by the mevalonate pathway. By inhibiting this pathway, we hypothesized that statins could potentially improve TM cell pathobiology by modulating YAP/TAZ activity. Thus, targeting the mevalonate pathway with statins may offer therapeutic potential for glaucoma. Despite significant progress in understanding TM and SC cells individually, the dynamic interactions between them and their role in glaucoma pathogenesis remain poorly understood. These interactions are crucial in the pathogenesis of glaucoma, yet no effective model exists to study them. Therefore, in Chapter 4, we developed a novel co-culture hydrogel system to explore TM-SC interactions and assess how glaucomatous TM cells affect SC behavior. Our findings show that glaucomatous TM cells alone can induce pathological changes in SC cells, underscoring the critical role of cell-cell and cell-ECM interactions in glaucoma progression. Collectively, these biomimetic ECM hydrogels provide a unique platform for investigating glaucomatous outflow mechanisms and offering insights into disease pathogenesis.
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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).
