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Role of Coronavirus Envelope Protein on Infectivity
No full textCoronaviruses infect a wide range of hosts and have caused recent human pandemics. While much has been learned, gaps remain in understanding viral assembly and egress, particularly the role of the envelope (E) protein in specific infectivity. To study this, recombinant MHV viruses were created with E gene deletions and HiBiT-tagged spike (S) proteins to monitor virion release. In mouse cells, MHV∆E S-HiBiT secreted less spike and fewer infectious particles, indicating reduced specific infectivity. Comparisons between human HeLa-CEACAM and mouse DBT cells showed species-specific support for virus production. TMED10, a host membrane protein, was tested as a potential E-interacting partner via CRISPR knockout, but results showed it was not required for E-mediated assembly. Additionally, E proteins increased IL-1β secretion, with variability among coronavirus strains. Overall, the study enhances understanding of E protein roles in coronavirus egress, infectivity, and host interactions
Investigating Whether Txnrd2 Is a Therapeutic Target Against Triple Negative Breast Cancer Using African American-Derived Models
No full textDespite similar breast cancer incidence rates, African American (AA) women experience a 40% higher mortality rate compared to Caucasian American (CA) women. This disparity is multifactorial and particularly pronounced in the Chicago area. AA women are twice as likely to be diagnosed with triple-negative breast cancer (TNBC), often at a younger age and with more aggressive disease. TNBC is a heterogeneous subtype lacking targeted therapies and is associated with the poorest prognosis among breast cancer types. Therefore, there is an urgent need for novel therapeutic strategies for TNBC, especially those tailored to AA patients. The thioredoxin pathway is essential for maintaining redox homeostasis and supports various cellular processes, including proliferation, cell death, and signaling. In cancer, the heightened reliance on this pathway presents a druggable vulnerability. In this study, we investigated the role of thioredoxin reductases (TXNRDs), with a particular focus on the mitochondrial isoform TXNRD2, in AA-derived TNBC cell models. We first observed that TXNRD2 levels are highest in breast tumors from AA patients. We then evaluated the therapeutic potential of targeting TXNRD2 using a new class of non-covalent TXNRD inhibitors, TXNRD(i)s, which bind an allosteric site rather than relying on traditional covalent inhibition. To distinguish the activity of TXNRD2 from its close homologue TXNRD1, we developed a dual-labeling live-cell microscopy approach. Silencing TXNRD2 using siRNA significantly impaired TNBC cell growth. While TXNRD1 also plays a role in supporting TNBC cell viability, dual silencing of TXNRD1 and TXNRD2 had a markedly stronger inhibitory effect, with minimal impact on non-tumorigenic MCF-10A control cells. Treatment of AA-derived TNBC cells with our TXNRD(i)s phenocopied this dual silencing, and our imaging data confirmed that these compounds act as pan-TXNRD1/2 inhibitors. Together, these findings support TXNRD2 as a viable therapeutic target in TNBC and suggest that simultaneous inhibition of both TXNRD1 and TXNRD2 may represent a more effective therapeutic strategy especially for treating AA-patients with lethal breast cancers
Investigating the Proviral and Immunogenic Effects of Sars-Cov-2 Accessory Protein 7A
Get PDFSARS-CoV-2 and related sarbecoviruses encode a set of accessory proteins (3a, 3b, 6, 7a, 7b, 8, 9b, and 10) that control host responses to infection and promote virus growth. Of these accessory proteins, 7a is set apart by its intracellular localization near coronavirus (CoV) budding sites and its incorporation into secreted virions. Initial studies utilizing a virus-like particle (VLP) system identified significant proviral effects mediated by 7a, but the system lacked the context of an actual CoV infection. To investigate 7a functions during CoV infections, we constructed recombinant Mouse Hepatitis Viruses (rMHV strain A59) that express 7a genes. Comparative infections revealed that 7a increased viral replication and viral output in immortalized murine cell cultures and in primary bone marrow-derived macrophages (BMDMs). This proviral effect was independent of a previously reported 7a-mediated interferon antagonizing activity. 7a is a type I transmembrane protein with a short cytoplasmic tail that operates in subcellular trafficking and signal transduction. To further elucidate tail functions, we generated a set of rA59 viruses expressing substitutions in the tail di-lysine motifs. Several substitutions reduced 7a proviral activities; notably the K119A change eliminated 7a support of virus yield. 7a expression was robustly proinflammatory in BMDMs, as measured cytokine arrays. Cytoplasmic tail substitutions tempered these proinflammatory responses, implying connections with proviral activities. SARS-CoV-2 infected macrophages have been implicated in inflammatory COVID-19 and these findings point to 7a cytoplasmic tails as potential contributors to cytokine-mediated disease. This study shows that SARS-CoV-2 accessory protein 7a promotes infection of a phylogenetically distinct embecovirus and in doing so elicits proinflammatory and potentially disease-relevant host responses. The findings localize and highlight a specific proviral component in a sarbecovirus accessory protein
Investigating the Consequences of Conjugated and Secreted ISG15 on Coronavirus Pathogenesis
Get PDFCoronaviruses have evolved a breadth of mechanisms to antagonize host innate immune responses. One widely studied mechanism is the ability to deconjugate ISG15 from target proteins in a process called deISGylation, which is catalyzed by the papain-like protease (PLP) domain of viral non-structural protein (nsp) 3. Since PLP activity is required for viral replication, it has been challenging to generate an infectious virus with a PLP that maintains protease activity but has lost deISGylase activity. In this study, we generated a chimeric MHV-A59 with its PLP2 domain replaced with that of the human coronavirus HKU1, which has been documented to have minimal deISGylase activity in vitro. By using this virus, we determined that the deISGylase activity is critical for inhibiting the IFN-response in primary macrophages and promoting robust viral replication. Importantly, we also report that without deISGylase activity, the chimeric virus is highly attenuated in vivo and is more rapidly cleared from infected mice compared to MHV-A59. Accordingly, we found that at earlier stages of infection, MHV-HP2 induces a higher IFN response in the liver, providing a potential mechanism for the rapid clearance of the deISGylase-deficient coronavirus. These results document the critical role of viral deISGylase activity as a key contributor to innate immune evasion during coronavirus infection. We also investigated the ability of ISG15 to be secreted from the cell in response to coronavirus infection. The mechanism of ISG15 secretion was unknown. Here, we report that ISG15 is secreted from inflammasome-activated macrophages via gasdermin D (GSDMD) pores on the plasma membrane, mirroring the unconventional secretory pathway used by IL-1β and IL-18. Secretion of ISG15 is negated by deletion of GSDMD. It is also inhibited by treatment with the small-molecule pan-caspase inhibitor zVAD-fmk, the FDA-approved drug disulfiram, the caspase-1 inhibitor VX-765, and the NLRP3 inhibitor MCC-950, all of which block GSDMD pore formation. This study paves the way for investigating how extracellular ISG15 contributes to viral pathogenesis. Finally, we interrogated the effects of extracellular ISG15 on coronavirus replication and activation of the innate immune response through the addition of recombinant ISG15 (rISG15) to macrophages. By analyzing changes in gene expression and cytokine release in BMDMs treated with rISG15 and comparing them to IFN-driven responses, we determined that extracellular ISG15 has antiviral and proinflammatory effects during MHV-infection that is distinct from the IFN response. This study contributes to our understanding of how coronavirus infection drives inflammation within the infected host
Characterizing the Role of the FAK-HIF1a-CTGF Axis in Hepatocellular Carcinoma
No full textHepatocellular carcinoma (HCC) is a deadly cancer, and there is a pressing need to identify novel therapeutic targets. Our lab characterized focal adhesion kinase (FAK) as a driver of HCC, where FAK is required to induce HCC in mouse models. In HCC, 16% of patients harbor an amplification of the PTK2 gene, and these patients have decreased survival. Because of its role in tumor promotion, FAK has been investigated as a therapeutic target, however these therapies show little clinical efficacy. We hypothesize that selectively treating patients with high levels of FAK will significantly benefit from FAK-targeting therapeutics, however there are currently no known biomarkers for FAK. We used a proteolysis-targeting chimera (FP) to degrade FAK in vitro and showed significant growth inhibition following treatment. RNA-seq of FP-treated cells showed connective tissue growth factor (CTGF) was significantly downregulated. CTGF is secreted from cells and can be detected in serum, making it a suitable biomarker candidate. We confirmed the downregulation of CTGF following FP treatment at the mRNA, intracellular protein level, and secreted protein level. Overexpression of FAK led to an increase in intracellular and secreted CTGF protein levels. To assess CTGF alone in HCC, we used an shRNA knockdown and found that shCTGF significantly inhibited cell growth in vitro and in vivo. RNA-seq of shCTGF cells showed an increase in SKP2 and a decrease in p27. Furthermore, we showed that HIF1 mediates the regulation of CTGF by FAK. Using immunohistochemistry of HCC patient tumor samples, we saw a strong correlation between FAK and CTGF protein. Using the DEN/CCl4 murine model, FAK knockout attenuated tumor formation, and in wild type mice, an increase in serum CTGF levels was seen as tumors progressed. Finally, serum collected from patients at high risk for developing HCC suggest CTGF can be used as a marker for tumor formation for tumors with high FAK expression. Overall, FAK promotes the expression and secretion of CTGF, which promotes HCC. FAK is a promising therapeutic target in HCC, and identification of CTGF as a downstream target of FAK suggests CTGF as a treatment-indicating biomarker for FAK in HCC patients
ML Model to Better Identify Instances of Bullying Faced by Members of the LGBTQ+ Community
Get PDFCyberbullying poses a significant threat to online communities, with the LGBTQ+ community facing disproportionately higher rates of harassment. While existing cyberbullying detection systems have made progress in identifying general instances of online harassment, they often fail to capture the nuanced and context-dependent nature of LGBTQ+-targeted bullying. This thesis presents a novel approach to this challenge by developing SpectrumNet, an LGBTQ+-centric transformer-based model for cyberbullying detection. Our research was conducted in two phases. In Phase 1, we evaluated the effectiveness of pre-trained transformer models (RoBERTa, BERT, and GPT-2) in identifying LGBTQ+-related cyberbullying. Building on these findings, Phase 2 introduced SpectrumNet which integrates identity-aware attention mechanisms with hierarchical attention networks to understand the contextual nuances of LGBTQ+-targeted harassment better. The model was trained and evaluated on Instagram comments, demonstrating SpectrumNet\u27s superior performance in detecting LGBTQ+-specific harassment, with notable improvements in identifying subtle forms of bullying that traditional models often miss. This work contributes to the field of cyberbullying detection by introducing specialized architectural components designed specifically for identifying LGBTQ+-targeted harassment, potentially offering new directions for creating safer online spaces for marginalized communities
Early Social Experience and Biased Attention to Threat-Related Emotion in Infancy
Get PDFBy 7 months of age, infants typically develop a bias toward threatening facial expressions, such as fear and anger (Leppänen & Nelson, 2009; Leppänen et al., 2018; Peltola et al., 2009; 2013). However, individual differences in threat-related attention biases in adulthood are related to socioemotional functioning across a broad range of outcomes, including racial biases and psychological well-being. Links between attention and socioemotional development may begin in infancy, with prior evidence demonstrating that both maternal well-being and race may bias visual processing of emotional expressions (Morales et al., 2017; Vogel et al., 2017; Quinn et al., 2018; 2020). Thus, the current study aimed to examine the impact of two potential modulators of infant threat bias, maternal anxiety and racial and ethnic identity, on attention to emotional expressions. In pursuit of these aims, 40 mother-infant dyads participated in this study asynchronously online. Infants completed a preferential looking paradigm viewing pairs of own- and other-race faces expressing happy, angry, fearful, and sad emotion. Mothers completed measures of community and caregiving network diversity, depression, and anxiety. Results showed that infants were biased toward fearful expressions regardless of in- or out-group racial categorization, while race simultaneously biased attention to other negatively-valenced expressions (i.e., sad, angry). Maternal anxiety was uncorrelated with attention biases in our sample of 7-month-olds; however, experience-dependent shaping of emotion perception was observed through infant exposure to racial and ethnic diversity. These findings provide evidence of distinct responses to affect by in- and out-group racial categorizations by 7-months-of-age and has implications for the role of early experience in shaping visual attention biases for emotion
Discerning the Role of Microtubule Acetylation During HIV-1 Infection
Get PDFThe host-pathogen interactions underlying Human Immunodeficiency Virus type 1 (HIV-1) infection are complex and involve the exploitation of host cellular machinery. This thesis investigates the roles of dynein adaptors, microtubule acetylation, and TRIM69-mediated restriction in HIV-1 infection. Through a series of detailed experiments, we explore how HIV-1 utilizes multiple dynein adaptors, such as BICDR1 and Hook3, for intracellular transport, and reveal that these adaptors are critical for efficient viral trafficking. Conversely, the knockdown of Ninein surprisingly enhances HIV-1 infectivity, suggesting a nuanced regulatory role for this adaptor in viral transport. Further, this thesis challenges the prevailing paradigm that microtubule acetylation is essential for HIV-1 infection. Using CRISPR-Cas9 technology to knock out the αTAT1 enzyme responsible for microtubule acetylation, and overexpressing an αTAT1-mCherry construct, we demonstrate that HIV-1 can replicate effectively without microtubule acetylation. This finding prompts a re-evaluation of the role of microtubule modifications in HIV-1 pathogenesis. Additionally, we investigate the antiviral mechanisms of TRIM69, a member of the tripartite motif (TRIM) family of proteins. Our results show that TRIM69 restricts HIV-1 independently of microtubule acetylation, indicating alternative pathways and mechanisms at play. By using a doxycycline-inducible TRIM69 expression system in conjunction with CRISPR-Cas9 knockout of αTAT1, we confirm that TRIM69 retains its antiviral function even in the absence of acetylated microtubules. Moreover, TRIM69 demonstrates broad-spectrum antiviral activity, restricting viruses such as chikungunya virus (CHIKV), Coxsackie virus B3 (CVB3), and Zika virus (ZIKV). This comprehensive examination provides significant insights into the adaptability and complexity of HIV-1 in hijacking host cellular machinery, the dispensability of microtubule acetylation in HIV-1 infection, and the multifaceted antiviral mechanisms of TRIM69. These findings not only advance our understanding of HIV-1 biology but also highlight potential targets for novel therapeutic interventions aimed at disrupting the virus\u27s interaction with the host cell. Future research directions include expanding the range of dynein adaptors studied, investigating other post-translational modifications of microtubules, and further elucidating the broad antiviral mechanisms of TRIM69
Tribalism, Science Denial, and its Violation of Stephen J. Gould\u27s NOMA Model
No full textThis presentation explores how tribalism fuels science denial by breaching Stephen J. Gould’s Non-Overlapping Magisteria model, which distinguishes empirical science from value systems. Tribalism leads to group polarization, imposing ideological beliefs on the realm of science and dismissing evidence that threatens group identity. Case studies, like the leaded gasoline controversy of the 20th century, demonstrate how loyalty to identity distorts scientific discourse. Psychological factors, such as confirmation bias, exacerbate this issue. By recognizing these violations, the presentation highlights the importance of interdisciplinary strategies to restore trust in science while respecting Gould’s framework, offering hope in combating science denial
Plastic Mediated Photolysis of Emerging Contaminants in Aqueous Solutions
Get PDFPlastic pollution is ubiquitous. As of 2021, global plastic production was estimated to be 390 million metric tons and of the total plastic waste generated so far, only about 9% has been recycled. Plastic pollution may cause ecological damage in aquatic and terrestrial environments in several ways. Plastic particles can adsorb different compounds and transfer them to animals and then to humans farther up the food chain. Plastic debris in aquatic ecosystems may also transform water pollutants into different, potentially more toxic compounds. My research is concerned with how different types of plastics affect the photolysis of emerging contaminants in freshwater environments. Photolysis studies of triclosan (TCS, 5-chloro-2-(2,4-dichlorophenoxy)phenol and methyl triclosan (2,4,4’-trichloro-2’-methoxydiphenyl ether) have shown that this compound undergoes photolysis twenty to twenty-five times faster when adsorbed on polyethylene (PE) in aqueous solutions than in aqueous solutions alone. TCS is known to undergo photolysis in surface waters to 2,8-DCDD. We are using the light induced unimolecular transformation of TCS to 2,8-dichlorodibenzodioxin (2,8-DCDD) as a probe of the ability of different types of plastics to catalyze the photolysis process. We have found that plastics composed of primarily aliphatic polymers will accelerate the conversion of TCS to 2,8-DCDD while the rate of photolysis of TCS adsorbed onto aromatic polymers (polystyrene and polycarbonate) is much slower. Fluorescence studies of TCS adsorbed onto different plastics suggest that the transfer of adsorbed energy from TCS to the aromatic polymers competes with the fragmentation process, thus slowing the overall rate of photolysis. The surface area of the plastic is also an important factor affecting the rate of photolysis as the rate of photolysis of TCS is found to increase with increasing surface area. Photolysis studies of the flame retardants tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) are consistent with the TCS studies as well