• Unraveling the Roles of C. elegans Vasa Homologs, GLH Proteins, in Argonaute Pathway Specificity and Transcriptome Surveillance

  Dai, Siyuan (2023-05-25)

  Epigenetic regulation of gene expression empowers organisms to alter phenotypic information without affecting DNA sequence. Germline Argonaute proteins, complexed with their cognate small-RNAs, are essential for transcriptome surveillance and maintenance of heritable gene silencing. In C. elegans, PIWI Argonaute PRG-1 employs piRNAs to screen thousands of germline transcripts through microRNA-like base pairing. Upon target recognition, RNA-directed RNA polymerases (RdRPs) are recruited to generate abundant antisense small RNAs (22G-RNAs), which are subsequently loaded onto worm-specific Argonautes (WAGOs) to establish long-term epigenetic memory. Both small RNA amplification and Argonaute surveillance are thought to occur within perinuclear liquid-like condensates called nuage or P granules. The precise mechanism by which the nearly one million different perinuclear Argonaute/guide complexes engage their targets to mediate gene regulation remain unclear. In this dissertation, we explore the functions of a family of VASA homologs, Germ Line Helicases (GLHs) in diverse germline small-RNA pathways. Our genetic and biochemical investigations reveal that these perinuclear-localized DEAD-box proteins engage germline transcripts and promote piRNA- and RNA interference-mediated transgenerational silencing in C. elegans. We provide evidence that GLH proteins scaffold multiple Argonautes responsible for epigenetic silencing, competing with paralogs for direct binding to target mRNAs. Additionally, GLHs enhance the specificity of Argonuate pathways by preventing WAGO/22G-RNA misrouting. Through examining mutants without severe P-granule disruptions, we sought to separate GLH-1 scaffolding and enzymatic functions. We found that the GLH-1 ATPase cycle promotes RNA duplex unwinding and the biogenesis of WAGO-bound 22G-RNAs while many P-granule components remain properly localized. Moreover, we show that GLH-1 N-terminal domains containing both FG repeats and zinc fingers are crucial for binding Argonautes and RNA substrates. Taken together, our findings support a model in which Argonautes recruit GLH proteins for target mRNA binding and retention, enabling GLHs to regulate pathway-specific small-RNA signals and transgenerational inheritance.
• Integrating evolution and genomics to investigate social development in wolf-dog hybrids

  Li, Xue (2023-05-05)

  Domesticated dogs separated from wolves around ~5000-7000 generations ago, with major differences in early social development that have enabled them to survive, and thrive, in close proximity to humans. Due to their unique evolutionary history and accessibility, canines serve as a natural model system to study the genetic factors underlying behavior adaptation within and between subspecies. The wolf/dog system can not only advance the understanding of evolutionary processes, but also help to better understand the neurogenetic pathways involved in human psychiatric disorders. Although wolves and dogs split relatively recently in evolutionary time, they are genetically distinct populations with numerous differences across their genome. This population structure makes it impossible to confidently associate particular genomic variants with domestication-related traits by simply comparing dogs and wolves. In this dissertation, I identified genes and pathways associated with domestication-related behavior using an unusual admixed population of wolf-dog hybrids housed in sanctuaries across the United States. I developed methods and approaches to map behavioral phenotypes in wolf-dog hybrids, and explored the overlap with dog social behaviors, and human psychiatric conditions. I first characterized the population history of wolf-dog hybrids using techniques including exploratory principal component analysis, ancestry calling, and population differentiation test. I defined the behavioral phenotypes by dimensional reduction analysis of coded video data, and identified associations between genes and regulatory elements with those phenotypes using admixture mapping and association test. Finally, I investigated the functional and biological mechanisms underlying the associated regions by gene-set analysis. I discovered that regions associated with domestication-related behavioral differences are enriched for brain expressed genes, especially those enriched in early infancy. To further investigate the candidate regions associated with canine domestication, I leveraged a powerful new data resource comparing the genomes of 240 mammalian species. Using data from massively parallel reporter assay experiments in human cells, I confirmed that this resource can distinguish which bases have regulatory function. Overall, variants in highly constrained positions are more likely to alter cellular function. In addition, I showed that dogs with ancestry from a single breed, which have shorter lifespans than outbred dogs, are also more likely to carry variants in constrained positions, suggesting they impact fitness. In the wolf-dog hybrids, I cataloged candidate causal variants that differed between dogs and wolves and were highly constrained across mammals. Overall, this thesis demonstrates how new genomic tools and data resources can be leveraged to investigate exceptional evolutionary adaptations in other species that may offer insight into human diseases. By utilizing the wolf-dog hybrid population, we can re-trace the ancient genetic changes of domestication that led to divergence of canine social and developmental behaviors, and potentially uncover genetic pathways that contribute to social behavioral disorders such as autism spectrum disorders.
• Phosphoregulation of Cell Cycle Transcription Factors by Cyclin-Dependent Kinase

  Conti, Michelle (2023-04-27)

  To prevent the development of cancer, cells must regulate the cell division cycle. Cell cycle events are coordinated by an oscillatory gene expression program, established by a conserved transcription factor (TF) network. Most TFs in the network are phosphorylated by cyclin-dependent kinases (CDKs), which regulate their activity. However, the physiological consequences of disrupting TF phosphorylation remain poorly understood. The budding yeast repressive TFs Yhp1 and Yox1 are degraded following multisite phosphorylation by CDK. Surprisingly, I discovered that blocking phosphorylation of Yhp1 and Yox1 increased fitness compared to wild type cells, despite decreased expression of several essential cell cycle genes. We found that cells expressing non-phosphorylatable Yhp1 and Yox1 accelerated the G1/S transition and delayed mitotic exit. This suggests that by lengthening mitosis mutant cells have more time to correct chromosome segregation errors, which confers a fitness advantage to cells. Although hundreds of CDK targets have been identified, it is challenging to determine which phosphosites within a domain are required to regulate protein function. The conserved S-phase TF Hcm1 is activated by CDK-dependent phosphorylation of eight sites in its transactivation domain (TAD). Like Yox1 and Yhp1, disruption of Hcm1 TAD phosphorylation impacts cellular fitness. I leveraged these fitness phenotypes to develop a high-throughput approach, Phosphosite Scanning, that determines the importance of each phosphosite within a multisite phosphorylated domain. I identified multiple combinations of phosphosites that can activate Hcm1 and found that specific phosphorylations are required for phosphorylation throughout the TAD. These results highlight the importance of precise TF phosphoregulation and demonstrate that disruption of phosphoregulatory networks can have unexpected consequences on cellular physiology.
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  Rigid Plate Fixation for Reconstruction of Symptomatic Sternal Nonunion: A Retrospective Review

  Sjoquist, Jan; Joo, Alex; Bello, Ricardo; Dunn, Raymond (2023-04-26)

  Background: Median sternotomy, the most common approach to open cardiac surgery, is performed in over 500,000 patients annually in the United States. This approach involves an incision from the manubrium to xyphoid and vertical division of the bony sternum. Wire cerclage remains the standard technique for sternal closure after median sternotomy. Complications following median sternotomy include infection, hematoma, seroma, and sternal nonunion or dehiscence. Sternal nonunion occurs when either significant bony motion, fracture, or separation occurs with the two sternal halves. It is clinically defined as greater than 6 months of pain, clicking, or sternal instability. This represents a failure of primary cerclage “fixation.” Risk factors for nonunion after sternotomy include obesity, bilateral internal mammary artery harvesting, diabetes, and off-midline sternotomy. While sternal nonunion has an incidence of less than 1%, this complication can serve as a nidus for life-threatening infection and can cause significant discomfort for the patient. There is currently no standard-of-care treatment for sternal nonunion after median sternotomy. In fact, sternal nonunion most commonly goes untreated, leaving patients continuously symptomatic. Rigid plate fixation (RPF) has been employed in certain cases for primary sternal closure in patients at higher risk for sternal healing complications. RPF has been shown to significantly reduce the incidence of complications and mortality after median sternotomy in high-risk patients when compared to wire cerclage. We have recently employed traditional orthopedic techniques of bony debridement, anatomic bony reduction, and have extended the use of RPF to patients with symptomatic sternal nonunion. Objectives: The goal of this retrospective review is to investigate and describe long term clinical outcomes in patients at our institution who have undergone RPF for sternal nonunion after median sternotomy. Our objectives are to investigate long term outcomes and complications in patients at our institution who have undergone rigid plate fixation for sternal nonunion after median sternotomy. Methods: All patients who underwent sternal reconstruction for sterile sternal nonunion between 2017 and 2023 were reviewed. Patients were excluded if they underwent prophylactic RPF during primary sternotomy or if they did not meet the clinical definition of sternal nonunion. Data regarding demographics, risk factors, initial sternotomy procedure, nonunion presentation, reconstructive procedure, and clinical and radiographic follow up. For sternal reconstruction, all patients underwent debridement of nonviable sternal tissue, rigid fixation with locking plates and screws with or without pectoralis muscle advancement flaps, layered closure, and incisional negative pressure wound therapy (NPWT). A total of 18 eligible patients, 14 male and 4 female, were identified. Average age was 63 years. Preoperative risk factors included obesity (n = 14), smoking (n = 9), diabetes (n = 8), and LIMA harvest (n = 16). Indication for median sternotomy included coronary artery bypass grafting (n = 16) and aortic valve replacement (n = 2). Patients presented with symptoms of sternal nonunion an average of 4.5 months after initial sternotomy. The most common presenting symptoms were pain (n = 17) and sternal clicking (n = 14). 8 patients (44%) showed evidence of fractured sternal wires. Results: Average time from symptom presentation to sternal reconstruction was 3.2 months. Average time from initial sternotomy to reconstruction was 7.7 months. Regarding the sternal reconstruction procedure, 100% of patients underwent debridement of sternal edges and rigid plate fixation using locking plates and screws. Bilateral pectoralis advancement flaps were performed in 17 patients (94%). The average clinical follow-up period was 3 years, ranging from 39 days to 4.9 years. 100% of patients had sternal nonunion confirmed by CT scan and demonstrated clinical evidence of sternal healing. Complications following RPF included seroma (n=3), hematoma (n = 2), and wound infections (n = 2). One patient presented with osteomyelitis/mediastinitis one month post sternal reconstruction, with full resolution after receiving IV and oral antibiotics. Another patient had all hardware removed after presenting with cellulitis and CT evidence of perihardware infection 3 months post reconstruction. Sternal union was noted at time of hardware removal. Conclusion: Rigid plate fixation is a reliable method of treatment for symptomatic sternal nonunion and should be offered to all patients demonstrating signs and symptoms of sternal nonunion after median sternotomy.
• The Molecular Mechanism of RIPK1-Induced Cell Death And Its Impact On The Immune Response

  Park, Christa (2023-04-25)

  Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is a critical adapter protein with pleiotropic functions that regulate cell survival and death. RIPK1 is essential for immune homeostasis and thus is closely controlled during development and inflammation. RIPK1 overexpression has been implicated in multiple inflammatory disorders such as multiple sclerosis, atherosclerosis, cardiovascular disease, obesity, psoriasis, and tumor growth. To study the effects of the overactivation of RIPK1, a system was established that drives its overexpression in mouse fibroblasts. Remarkably, the overexpression of RIPK1 resulted in the induction of both apoptosis and necroptosis. While apoptosis is known to be immunologically silent, necroptosis is highly inflammatory. Additional assays using chemical inhibitors and genetic knockout mice established that RIPK1 kinase activity promotes both types of cell death. Furthermore, RIPK1-induced apoptosis and necroptosis require caspase 8 and MLKL, respectively, and the absence of both caspase 8 and MLKL inhibits RIPK1-induced cell death. RIPK1 induction activates NF-κB/MAPK and increases cytokine/chemokine production driven by cell death. This system was further explored to elucidate the effects of RIPK1-induced cell death on immune effector cells, revealing that RIPK1-induced apoptosis and necroptosis can promote DC activation. Lastly, to study the role of RIPK1 in DCs and its contribution to intestinal homeostasis and injury, mice lacking RIPK1 in the DC population were characterized. Importantly, RIPK1 functions in DCs to support colon homeostasis, but also plays a detrimental role during DSS-induced colitis. Collectively, these data further provide novel insights into the multifaceted functions of RIPK1 in cell death and inflammation, highlighting its critical contributions to the immune response.
• Artificial Intelligence for the Diagnosis of Pediatric Appendicitis: A Systematic Review

  Chekmeyan, Mariam (2023-04-21)

  BACKGROUND: While acute appendicitis is the most frequent surgical emergency in children, its diagnosis remains complex. Artificial intelligence (AI) and machine learning (ML) tools have been employed to improve the accuracy of various diagnoses, including appendicitis. The purpose of this study was to systematically review the current body of evidence regarding the efficacy of AL and ML approaches for the diagnosis of acute pediatric appendicitis. METHODS: This systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to identify articles from Pubmed, Scopus, and iEEE Xplore. Eligible articles included full text, English-language articles assessing the use of AI technologies for the diagnosis of acute pediatric appendicitis. Study quality of reporting was appraised using The Transparent Reporting of a multivariable prediction model of Individual Prognosis Or Diagnosis (TRIPOD) statement. RESULTS: A total of fourteen studies were included in the final analysis of which ten were published after 2019. Two studies originated in the United States while half were carried out in Europe. Artificial Neural Network and Random Forest AI methods were the most commonly used modeling approaches. Commonly used predictors were pain and laboratory blood findings. The average area under the curve that was reported among the fourteen studies was greater than 80%. CONCLUSIONS: AI and ML technologies have the potential to improve the accuracy of acute appendicitis diagnosis in pediatric patients. Further investigation is needed to identify barriers to adoption of these technologies and to assess their efficacy in real world usage once integrated into clinical workflows.
• A pilot study of robotic surgery case videos for first-year medical student anatomy

  Palleiko, Benjamin A; Maxfield, Mark W; Czerniach, Donald R; Cherng, Nicole B; Giannaris, Eustathia Lela (2023-04-17)

  There has been a recent shift in medical student anatomy education with greater incorporation of virtual resources. Multiple approaches to virtual anatomy resources have been described, but few involve video or images from surgical procedures. In this pilot study, a series of surgical case videos was created using robotic surgery video footage for a first-year medical student anatomy course. Five operations were included that covered thoracic, abdominal, and pelvic anatomy. Students were surveyed at the end of the course regarding their experience with the videos and their perceptions towards a surgical career. Overall, participants agreed that the videos were an effective learning tool, were useful regardless of career interest, and that in the future it would be useful to incorporate additional surgical case videos. Respondents highlighted the importance of audio narration with future videos and provided suggestions for future operations that they would like to see included. In summary, this pilot study describes the creation and implementation of a surgical video anatomy curriculum and student survey results suggest this may be an effective approach to video-based anatomy education for further curricular development.
• The HHV-6B U20 Glycoprotein Inhibits NK Cell Responses By Binding ULBP1 And Blocking NKG2D Activation

  Weaver, Grant (2023-04-06)

  Many viruses impede host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Hosts will often counter this through NK cells that sense the absence of MHC-I and kill the infected cell. Human Herpesvirus 6B (HHV-6B) has also been shown to downregulate MHC-I but this does not result in NK-mediated elimination of the virus. Previous work has shown that HHV-6B downregulates three NK-activating stress ligands: MICB, ULBP1, and ULBP3. More recently, the U20 glycoprotein of HHV-6A was implicated in the downregulation of ULBP1 but the precise mechanism remains undetermined. We set out to better understand the role of HHV-6B U20 in modulating NK cell activity. We performed structural modeling studies that suggest that U20 is likely a viral nonclassical MHC (vMHC). These vMHCs are common tools used by herpesviruses to modulate host immune responses. Through in vitro studies with recombinant protein, we demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity (225nM). Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels (at the surface or in toto). Soluble U20 has the same effect and can also inhibit activation of ULBP1-stimulated primary NK cells. When taken together, these data suggest that U20 helps to downregulate NK cell activity by binding ULBP1, masking it from recognition by NKG2D at the surface of infected cells, resulting in a decrease in antiviral NK cell activation and killing.
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  Improved Performance of ChatGPT-4 on the OKAP Exam: A Comparative Study with ChatGPT-3.5 [preprint]

  Teebagy, Sean; Colwell, Lauren; Wood, Emma; Yaghy, Antonio; Faustina, Misha (2023-04-03)

  This study aims to evaluate the performance of ChatGPT-4, an advanced Artificial Intelligence (AI) language model, on the Ophthalmology Knowledge Assessment Program (OKAP) examination compared to its predecessor, ChatGPT-3.5. Both models were tested on 180 OKAP practice questions covering various ophthalmology subject categories. Results showed that ChatGPT-4 significantly outperformed ChatGPT-3.5 (81% vs. 57%; p<0.001), indicating improvements in medical knowledge assessment. The superior performance of ChatGPT-4 suggests potential applicability in ophthalmologic education and clinical decision support systems. Future research should focus on refining AI models, ensuring a balanced representation of fundamental and specialized knowledge, and determining the optimal method of integrating AI into medical education and practice.
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  The EN-TEx resource of multi-tissue personal epigenomes & variant-impact models

  Rozowsky, Joel; Gao, Jiahao; Borsari, Beatrice; Yang, Yucheng T; Galeev, Timur; Gürsoy, Gamze; Epstein, Charles B; Xiong, Kun; Xu, Jinrui; Li, Tianxiao; et al. (2023-03-30)

  Understanding how genetic variants impact molecular phenotypes is a key goal of functional genomics, currently hindered by reliance on a single haploid reference genome. Here, we present the EN-TEx resource of 1,635 open-access datasets from four donors (∼30 tissues × ∼15 assays). The datasets are mapped to matched, diploid genomes with long-read phasing and structural variants, instantiating a catalog of >1 million allele-specific loci. These loci exhibit coordinated activity along haplotypes and are less conserved than corresponding, non-allele-specific ones. Surprisingly, a deep-learning transformer model can predict the allele-specific activity based only on local nucleotide-sequence context, highlighting the importance of transcription-factor-binding motifs particularly sensitive to variants. Furthermore, combining EN-TEx with existing genome annotations reveals strong associations between allele-specific and GWAS loci. It also enables models for transferring known eQTLs to difficult-to-profile tissues (e.g., from skin to heart). Overall, EN-TEx provides rich data and generalizable models for more accurate personal functional genomics.
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  Mechanism of Sliding Clamp Loading During DNA Replication and Repair in Atomic Detail

  Liu, Xingchen (2023-03-30)

  DNA replication is a fundamental process that is essential for all forms of life, and it is made efficient by ring-shaped sliding clamp proteins. One such protein is the eukaryotic sliding clamp, Proliferating Cellular Nuclear Antigen (PCNA), which not only facilitates replication but also coordinates multiple cellular pathways, such as DNA repair, cell cycle regulation, and apoptosis. The proper function of PCNA is critical for maintaining genome stability, making it a crucial factor in human health. The clamp loader complex is the primary regulator of sliding clamp activity. Replication Factor C (RFC), the eukaryotic clamp loader, is responsible for opening the closed PCNA and loading it onto DNA. However, the mechanism by which RFC accomplishes this task has been elusive for years. Our research has contributed to this field by revealing multiple cryo-electron microscopy (cryo-EM) structures of the RFC:PCNA complex that describe the steps involved in the clamp loading reaction. Specifically, we found that RFC opens PCNA with a 'crab-claw' motion, allowing it to preferentially bind to PCNA before DNA. Additionally, during replication, primer-template DNA, which is RFC's primary DNA substrate, directly binds to the central chamber of the complex. Our study also sheds light on the mechanism by which RFC performs its role in loading PCNA during DNA repair. When RFC binds to gapped or nicked DNA during DNA repair, it uses an additional DNA binding site, and both sites work together to melt the DNA strands with their 'separation pins.' This discovery provides the first structural insight into how RFC accomplishes its crucial functions in DNA replication and repair. Overall, our findings provide detailed atomic-level insights into how RFC efficiently loads PCNA onto different DNA substrates, advancing our understanding of this essential biological process.
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  Activation of the NLRP1 inflammasome in human keratinocytes by the dsDNA mimetic poly(dA:dT)

  Zhou, Jeffrey Y. (2023-03-29)

  The accrual of cytosolic DNA leads to transcription of type I IFNs, proteolytic maturation of the IL-1 family of cytokines and pyroptotic cell death. Caspase-1 cleaves pro-IL-1b to generate mature bioactive cytokine and gasdermin D which facilitates IL-1 release and pyroptotic cell death. Absent in melanoma-2 (AIM2) is a sensor of dsDNA leading to caspase-1 activation, although in human monocytes, cGAS-STING acting upstream of NLRP3 mediate the dsDNA activated inflammasome response. In healthy human keratinocytes, AIM2 is not expressed yet caspase-1 is activated by the synthetic dsDNA mimetic poly(dA:dT). Here, we show that this response is not mediated by either AIM2 or the cGAS-STING-NLRP3 pathway and is instead dependent on NLRP1. Poly(dA:dT) is unique in its ability to activate NLRP1, as conventional linear dsDNAs fail to elicit NLRP1 activation. DsRNA was recently shown to activate NLRP1 and prior work has shown that poly(dA:dT) is transcribed into an RNA intermediate that stimulates the RNA sensor RIG-I. However, poly(dA:dT) dependent RNA-intermediates are insufficient to activate NLRP1. Instead, poly(dA:dT) results in oxidative nucleic acid damage and cellular stress, events which activate MAP3 kinases including ZAKa that converge on p38 to activate NLRP1. Collectively, this work defines a new activator of NLRP1, broadening our understanding of sensors that recognize poly(dA:dT), and advances understanding of the immunostimulatory potential of this potent adjuvant.
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  Mechanistic Role of the Sliding Clamp in Genome Stability and Disease

  Magrino, Joseph (2023-03-27)

  DNA replication is an essential task to all life. To ensure precise genome duplication, cells utilize a network of factors that copy, surveil, and repair DNA. The coordination of all these factors heavily relies on the homotrimeric sliding clamp protein, Proliferating Cell Nuclear Antigen (PCNA). Like other sliding clamps, PCNA slides along DNA and acts as a molecular tether that increases the processivity of various DNA-acting enzymes. In addition, PCNA plays a much more multifaceted role in coordinating the concerted efforts of dozens of proteins involved in DNA replication, DNA repair, chromatin remodeling, cell cycle, and apoptosis. Proper interactions with PCNA are necessary for these enzymes to perform their functions. Yet, how PCNA controls its vast network remains unclear. How does partner binding affinity, PCNA levels, and lifetime on DNA influence PCNA’s ability to coordinate various DNA metabolic events? To understand the biophysical principles behind these questions, I investigated how two disease-associated substitutions in PCNA impact partner binding, stability, and its loading onto DNA. Furthermore, I made C. elegans strains that harbored these substitutions to understand their effects at the organismal level. Finally, I identified an electrostatic patch within PCNA that plays a role in partner binding affinity. My work collectively identifies residues that are critical for PCNA function. More broadly, my work provides insight into the evolution of PCNA and how substitutions impact both genome stability and human health.
• The Interplay Between Stress and the Drosophila piRNA Pathway

  Ho, Samantha (2023-03-22)

  piRNAs are required to silence transposons in the germline to maintain genome stability and transmission of an intact genome to the next generation. In Drosophila, biogenesis of these small RNAs occurs in three distinct compartments: the nucleus, perinuclear nuage, and the outer mitochondria membrane. My thesis focuses on how genomic instability via transposon-initiated DNA damage and heat stress impact piRNA pathway organization and function. We show that activation of Chk2, a Checkpoint kinase required for DNA damage signaling, disrupts nuage composition and this complicates piRNA mutant phenotypes. Stripping away DNA damage signaling in piRNA mutants provided new insight into how the nuclear piRNA proteins organize the cytoplasmic nuage. Additionally, we found that localization of key components to nuage is dispensable for piRNA production and transposon silencing. piRNA pathway proteins are not only susceptible to genomic instability, but also sensitive to heat shock. Rhino is a core component of the nuclear piRNA pathway and displays drastic localization changes upon heat shock that recovers with time. piRNAs have been proposed to help localize Rhino and heat stress provided a unique platform to test this model. We show that Rhino recovery after heat shock does not require Piwi, the sole nuclear protein bound to piRNAs, and this points to piRNAs having a less significant role in Rhino localization. Taken together, we show how different types of stress can modulate the piRNA pathway in unexpected manners.
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  Canonical Wnt Signaling Maintains Human Mesenchymal Progenitor Cell Multipotency During Adult Adipose Tissue Development

  Yang Loureiro, Zinger (2023-03-21)

  Human adipose tissue development and repair requires mesenchymal progenitor cells capable of self-renewal and differentiation into adipocytes. These progenitors are within adipose tissue throughout life, and how a progenitor pool is sustained is not well understood. As adipocytes have exceedingly slow turnover rate in vivo, we used a human adult primary adipose tissue-derived progenitor model to study adipogenesis at scale. Initially, single-cell transcriptome profiling of progenitors during early adipogenic induction identified common-origin developmental trajectories toward two fates. One fate is toward adipocyte terminal differentiation, and the other is toward a non-differentiated state characterized by up-regulation of extracellular matrix and canonical Wnt target genes. This latter cell population, herein referred to as Structural Wnt Adipose Tissue resident (SWAT) cells, retains features of progenitors, including the capability to proliferate and to differentiate towards multiple mesenchymal fates, but SWAT cells have a distinct transcriptional profile compared to progenitor cells. We conducted an in-depth investigation of Wnt signaling in SWAT cells. Canonical Wnt signaling reporter assay revealed Wnt activity was rapidly and transiently up-regulated upon adipogenic induction, and perturbation of Wnt activity alters the proportion of differentiated adipocytes. In additional to experimental validations, this research includes meta-analyses of published human adult adipose tissue single-nuclei transcriptome profiles, providing in vivo clinical evidence for expression of Wnt target genes and the transcriptomic signature of the maintained adipose progenitors. Our study identifies canonical Wnt signaling as a critical mechanism for mesenchymal progenitor maintenance and demonstrates its essential role in human adult adipose tissue homeostasis.
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  Structure and Assembly of a Thermophilic Bacteriophage

  Agnello, Emily (2023-03-20)

  Bacteriophages (phages) are ubiquitously abundant bacterial viruses and the most numerous biological entities on Earth. In order to fully understand the phage life cycle, we must understand how phage particles self-assemble. Further, an understanding of phage structure and assembly permits the application of phages to human health. Phage assembly is largely governed by two parallel pathways: capsid assembly and tail assembly. The capsid houses the genome, while the tail is the channel through which the genome travels to infect its host, making both components essential for successful infections. In this dissertation, I investigate the assembly pathways of the hyperthermophilic phage P74-26 as a model for understanding long-tailed phages (~85% of all phages). Primarily, I combine cryoEM structures of the P74-26 tail tube with an in vitro system for studying assembly kinetics to propose the first molecular model for the assembly of long-tailed phage tail tubes. Additionally, the tail is attached to a Tail Tip Complex (TTC) which recognizes the surface of the host. I present a cryo-EM structure of the P74-26 TTC, identifying the protein components for the first time. Finally, I explore the self-assembly of the capsid protein with the potential for establishing an in vitro system for studying capsid assembly and present proof-of-concept studies for using the particles as functional nanoparticle therapeutics. Together, this work explores principles of phage assembly and thermostability in tailed bacteriophages and how we can take advantage of these principles for future development of therapeutic delivery tools and nanoparticle applications.
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  Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses

  Zvornicanin, Sarah N; Shaqra, Ala M; Huang, Qiuyu J; Ornelas, Elizabeth; Moghe, Mallika; Knapp, Mark; Moquin, Stephanie; Dovala, Dustin; Schiffer, Celia A; Kurt Yilmaz, Nese (2023-03-18)

  With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (Mpro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 Mpro, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 Mpro. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors.
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  Cryo-EM structure of the human Sirtuin 6-nucleosome complex [preprint]

  Chio, Un Seng; Rechiche, Othman; Bryll, Alysia R; Zhu, Jiang; Feldman, Jessica L; Peterson, Craig L; Tan, Song; Armache, Jean-Paul (2023-03-18)

  Sirtuin 6 (SIRT6) is a multifaceted protein deacetylase/deacylase and a major target for small-molecule modulators of longevity and cancer. In the context of chromatin, SIRT6 removes acetyl groups from histone H3 in nucleosomes, but the molecular basis for its nucleosomal substrate preference is unknown. Our cryo-electron microscopy structure of human SIRT6 in complex with the nucleosome shows that the catalytic domain of SIRT6 pries DNA from the nucleosomal entry-exit site and exposes the histone H3 N-terminal helix, while the SIRT6 zinc-binding domain binds to the histone acidic patch using an arginine anchor. In addition, SIRT6 forms an inhibitory interaction with the C-terminal tail of histone H2A. The structure provides insights into how SIRT6 can deacetylate both H3 K9 and H3 K56. Teaser: The structure of the SIRT6 deacetylase/nucleosome complex suggests how the enzyme acts on both histone H3 K9 and K56 residues.
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  Leveraging Base Pair Mammalian Constraint to Understand Genetic Variation and Human Disease [preprint]

  Sullivan, Patrick F; Meadows, Jennifer R S; Gazal, Steven; Phan, BaDoi N; Li, Xue; Genereux, Diane P; Dong, Michael X; Bianchi, Matteo; Andrews, Gregory; Sakthikumar, Sharadha; et al. (2023-03-10)

  Although thousands of genomic regions have been associated with heritable human diseases, attempts to elucidate biological mechanisms are impeded by a general inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function that is agnostic to cell type or disease mechanism. Here, single base phyloP scores from the whole genome alignment of 240 placental mammals identified 3.5% of the human genome as significantly constrained, and likely functional. We compared these scores to large-scale genome annotation, genome-wide association studies (GWAS), copy number variation, clinical genetics findings, and cancer data sets. Evolutionarily constrained positions are enriched for variants explaining common disease heritability (more than any other functional annotation). Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.
• Molecular Mechanisms of Amino Acid Sensing Upstream of mTORC1

  Egri, Shawn B. (2023-03-10)

  In order to initiate cellular growth, a cell must weigh its nutrient availability against its anabolic needs. A critical pathway responsible for this process is the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 is a serine threonine protein kinase complex that will phosphorylate its downstream substrates in order to promote anabolic reactions. mTORC1 activity is dependent upon the Rag GTPase heterodimer. In the presence of amino acids, the Rags will activate mTORC1 by promoting its translocation to the lysosomal surface. In contrast, amino acid withdrawal results in mTORC1 inactivation. In order to ensure faithful mTORC1 signaling, the nucleotide loading state of the Rag GTPase is tightly regulated. In this thesis, we combine biochemical and biophysical approaches to investigate the molecular mechanism of how the nucleotide loading state of the Rag GTPase subunits are regulated. First, we characterized a conserved interdomain hydrogen bond within the Rag GTPases that is responsible for maintaining the GDP-bound state of the subunits. Elimination of this hydrogen bond abolishes the ability of the Rag GTPase to maintain its functional state, resulting in dysregulated mTORC1 signaling. Second, we utilize cryo-EM to describe the molecular mechanism of how GATOR1, a potent negative regulator of the mTORC1 pathway, modulates the nucleotide loading state of the Rag subunits in response to nutrient deprivation. These results reveal the molecular details of how the Rag GTPases are regulated in response to changes in amino acid availability, and furthermore how disruptions to those mechanisms can lead to dysregulation of the mTORC1 signaling pathway.

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