This collection showcases journal articles, preprints, book chapters, and other publications and presentations produced by faculty, postdocs, and researchers at UMass Chan Medical School.


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Recently Published

  • Individual Development Plan for Career Development Professionals

    Ismael, Amber; Campbell, Brian; Chremos, Ioannis Vasileios; Fuhrmann, Cynthia N; Nordell, Shawn (2024-06-21)
    The Individual Development Plan for Career Development Professionals is an IDP worksheet to guide individuals who are pursuing careers in graduate/postdoctoral career development through the process of creating their own IDP. The worksheet is designed as an IDP tool for all career levels, including those preparing to enter this field, practitioners seeking to grow within their current positions, or those looking to advance or pursue a career transition. The tool includes prompts to guide one through exercises that help one assess their skills, values, interests, progress and growth; set goals and define an action plan; and consider the resources, mentorship, and community that they may need to help develop a fulfilling career. Though designed for professionals in the career development field, this tool may also be helpful for those in other academic administration or education roles. This tool was designed by members of the Professional Development Committee of the Graduate Career Consortium, an international member organization to help individuals advance the field of graduate and postdoctoral career and professional development.
  • Apheresis practice variation during the COVID-19 pandemic: Results of a survey

    Tanhehco, Yvette C; Alsammak, Mohamed; Chhibber, Vishesh; Ibeh, Nnaemeka; Li, Yanhua; Stephens, Laura D; Noland, Daniel K; Wu, Ding Wen; Zantek, Nicole D; DeChristopher, Phillip J; et al. (2024-06-01)
    Background: The COVID-19 pandemic affected healthcare delivery across all specialties including apheresis. To describe the changes in apheresis service practices that occurred during the pandemic, the American Society for Apheresis (ASFA) Apheresis Medicine Attending Physician Subcommittee conducted a survey study. Study design and methods: A 32-question survey was designed and distributed to 400 ASFA physician members on September 7, 2022. Attending physicians responded to questions about whether and how apheresis service practices changed during the COVID-19 pandemic compared with the time period prior to the pandemic in terms of: (1) procedure types and volumes, (2) patient consultation workflow, and (3) the use of telemedicine. Descriptive analyses were reported as number and frequency of responses. Results: The survey response rate was 13.8% (55/400). Of these respondents, 96.4% (53/55) were attending physicians. The majority of respondents (42/53, 79.2%) indicated that the types of procedures performed during COVID-19 compared to pre-pandemic did not change. Most frequently for apheresis procedure volume, respondents reported: no change in their monthly inpatient volume (21/47, 44.7%) and a decrease in their monthly outpatient volume (28/46, 60.9%). Prior to COVID-19, 75.0% (30/40) of respondents performed consultations at bedside for inpatients and 67.4% (29/43) performed consultations at bedside for outpatients. Bedside consultations decreased in both settings during the pandemic but were still most frequently performed by attending physicians. At the same time, the use of telemedicine increased for 15.4% of survey respondents during COVID-19. Conclusion: Some, but not all, respondents observed or made changes to their apheresis service during the COVID-19 pandemic. A subset of changes, such as increased utilization of telemedicine, may persist.
  • ADAM9 promotes type I interferon-mediated innate immunity during encephalomyocarditis virus infection

    Bazzone, Lindsey E; Zhu, Junji; King, Michael; Liu, GuanQun; Guo, Zhiru; MacKay, Christopher R; Kyawe, Pyae P; Qaisar, Natasha; Rojas-Quintero, Joselyn; Owen, Caroline A; et al. (2024-05-16)
    Viral myocarditis, an inflammatory disease of the heart, causes significant morbidity and mortality. Type I interferon (IFN)-mediated antiviral responses protect against myocarditis, but the mechanisms are poorly understood. We previously identified A Disintegrin And Metalloproteinase domain 9 (ADAM9) as an important factor in viral pathogenesis. ADAM9 is implicated in a range of human diseases, including inflammatory diseases; however, its role in viral infection is unknown. Here, we demonstrate that mice lacking ADAM9 are more susceptible to encephalomyocarditis virus (EMCV)-induced death and fail to mount a characteristic type I IFN response. This defect in type I IFN induction is specific to positive-sense, single-stranded RNA (+ ssRNA) viruses and involves melanoma differentiation-associated protein 5 (MDA5)-a key receptor for +ssRNA viruses. Mechanistically, ADAM9 binds to MDA5 and promotes its oligomerization and thereby downstream mitochondrial antiviral-signaling protein (MAVS) activation in response to EMCV RNA stimulation. Our findings identify a role for ADAM9 in the innate antiviral response, specifically MDA5-mediated IFN production, which protects against virus-induced cardiac damage, and provide a potential therapeutic target for treatment of viral myocarditis.
  • Dominant negative mutations in yeast Hsp90 reveal triage decision mechanism targeting client proteins for degradation [preprint]

    Flynn, Julia M; Joyce, Margot E; Bolon, Daniel N A (2024-04-30)
    Most of the fundamental processes of cells are mediated by proteins. However, the biologically-relevant mechanism of most proteins are poorly understood. Dominant negative mutations have provided a valuable tool for investigating protein mechanisms but can be difficult to isolate because of their toxic effects. We used a mutational scanning approach to identify dominant negative mutations in yeast Hsp90. Hsp90 is a chaperone that forms dynamic complexes with many co-chaperones and client proteins. In vitro analyses have elucidated some key biochemical states and structures of Hsp90, co-chaperones, and clients; however, the biological mechanism of Hsp90 remains unclear. For example, high throughput studies have found that many E3 ubiquitin ligases bind to Hsp90, but it is unclear if these are primarily clients or acting to tag other clients for degradation. We introduced a library of all point mutations in the ATPase domain of Hsp90 into yeast and noticed that 176 were more than 10-fold depleted at the earliest point that we could analyze. There were two hot spot regions of the depleted mutations that were located at the hinges of a loop that closes over ATP. We quantified the dominant negative growth effects of mutations in the hinge regions using a library of mutations driven by an inducible promoter. We analyzed individual dominant negative mutations in detail and found that addition of the E33A mutation that prevents ATP hydrolysis by Hsp90 abrogated the dominant negative phenotype. Pull-down experiments did not reveal any stable binding partners, indicating that the dominant effects were mediated by dynamic complexes. DN Hsp90 decreased the expression level of two model Hsp90 clients, glucocorticoid receptor (GR) and v-src kinase. Using MG132, we found that GR was rapidly destabilized in a proteasome-dependent fashion. These findings provide evidence that the binding of E3 ligases to Hsp90 may serve a quality control function fundamental to eukaryotes.
  • Translation-dependent and -independent mRNA decay occur through mutually exclusive pathways defined by ribosome density during T cell activation

    Mercier, Blandine C; Labaronne, Emmanuel; Cluet, David; Guiguettaz, Laura; Fontrodona, Nicolas; Bicknell, Alicia; Corbin, Antoine; Wencker, Mélanie; Aube, Fabien; Modolo, Laurent; et al. (2024-04-25)
    mRNA translation and decay are tightly interconnected processes both in the context of mRNA quality-control pathways and for the degradation of functional mRNAs. Cotranslational mRNA degradation through codon usage, ribosome collisions, and the recruitment of specific proteins to ribosomes is an important determinant of mRNA turnover. However, the extent to which translation-dependent mRNA decay (TDD) and translation-independent mRNA decay (TID) pathways participate in the degradation of mRNAs has not been studied yet. Here we describe a comprehensive analysis of basal and signal-induced TDD and TID in mouse primary CD4+ T cells. Our results indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner. Our analysis further identifies the length of untranslated regions, the density of ribosomes, and GC3 content as important determinants of TDD magnitude. Consistently, all transcripts that undergo changes in ribosome density within their coding sequence upon T cell activation display a corresponding change in their TDD level. Moreover, we reveal a dynamic modulation in the relationship between GC3 content and TDD upon T cell activation, with a reversal in the impact of GC3- and AU3-rich codons. Altogether, our data show a strong and dynamic interconnection between mRNA translation and decay in mammalian primary cells.
  • CD20/MS4A1 is a mammalian olfactory receptor expressed in a subset of olfactory sensory neurons that mediates innate avoidance of predators

    Jiang, Hao-Ching; Park, Sung Jin; Wang, I-Hao; Bear, Daniel M; Nowlan, Alexandra; Greer, Paul L (2024-04-18)
    The mammalian olfactory system detects and discriminates between millions of odorants to elicit appropriate behavioral responses. While much has been learned about how olfactory sensory neurons detect odorants and signal their presence, how specific innate, unlearned behaviors are initiated in response to ethologically relevant odors remains poorly understood. Here, we show that the 4-transmembrane protein CD20, also known as MS4A1, is expressed in a previously uncharacterized subpopulation of olfactory sensory neurons in the main olfactory epithelium of the murine nasal cavity and functions as a mammalian olfactory receptor that recognizes compounds produced by mouse predators. While wildtype mice avoid these predator odorants, mice genetically deleted of CD20 do not appropriately respond. Together, this work reveals a CD20-mediated odor-sensing mechanism in the mammalian olfactory system that triggers innate behaviors critical for organismal survival.
  • Muscle-Specific Pyruvate Kinase Isoforms, Pkm1 and Pkm2, Regulate Mammalian SWI/SNF Proteins and Histone 3 Phosphorylation During Myoblast Differentiation [preprint]

    Olea-Flores, Monserrat; Sharma, Tapan; Verdejo-Torres, Odette; DiBartolomeo, Imaru; Thompson, Paul R; Padilla-Benavides, Teresita; Imbalzano, Anthony N. (2024-04-11)
    Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, Pkm1 and Pkm2, function in glycolysis, but Pkm2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of Pkm1 and Pkm2 during myoblast differentiation. RNA-seq analysis revealed that Pkm2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. Dpf2 and Baf250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for activation of myogenic gene expression during differentiation. Pkm2 also mediated the incorporation of Dpf2 and Baf250a into the regulatory sequences controlling myogenic gene expression. Pkm1 did not affect expression but was required for nuclear localization of Dpf2. Additionally, Pkm2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters, but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for Pkm2 and a novel function for Pkm1 in gene expression and chromatin regulation during myoblast differentiation.
  • Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer

    Mishra, Alok K; Ye, Tianyi; Banday, Shahid; Thakare, Ritesh P; Su, Chinh Tran-To; Pham, Ngoc N H; Ali, Amjad; Kulshreshtha, Ankur; Chowdhury, Shreya Roy; Simone, Tessa M; et al. (2024-04-03)
    CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.
  • Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis

    Min, Kyounghee; Yenilmez, Batuhan; Kelly, Mark; Echeverria, Dimas; Elleby, Michael; Lifshitz, Lawrence M; Raymond, Naideline; Tsagkaraki, Emmanouela; Harney, Shauna M; DiMarzio, Chloe; et al. (2024-04-02)
    Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline-deficient, high-fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.
  • Nucleic Acid Aptamers Protect Against Lead (Pb(II)) Toxicity [preprint]

    Anwar, Afreen; De Ayreflor Reyes, Solimar Ramis; John, Aijaz Ahmad; Breiling, Erik; O'Connor, Abigail M; Reis, Stephanie; Shim, Jae-Hyuck; Shah, Ali Asghar; Srinivasan, Jagan; Farny, Natalie G (2024-03-31)
    Lead (Pb(II)) is a pervasive heavy metal toxin with many well-established negative effects on human health. Lead toxicity arises from cumulative, repeated environmental exposures. Thus, prophylactic strategies to protect against the bioaccumulation of lead could reduce lead-associated human pathologies. Here we show that DNA and RNA aptamers protect C. elegans from toxic phenotypes caused by lead. Reproductive toxicity, as measured by brood size assays, is prevented by co-feeding of animals with DNA or RNA aptamers. Similarly, lead-induced behavioral anomalies are also normalized by aptamer feeding. Further, cultured human HEK293 and primary murine osteoblasts are protected from lead toxicity by transfection with DNA aptamers. The osteogenic development, which is decreased by lead exposure, is maintained by prior transfection of lead-binding DNA aptamers. Aptamers may be an effective strategy for the protection of human health in the face of increasing environmental toxicants.
  • Single-cell genomics and regulatory networks for 388 human brains [preprint]

    Emani, Prashant S; Liu, Jason J; Clarke, Declan; Jensen, Matthew; Warrell, Jonathan; Gupta, Chirag; Meng, Ran; Lee, Che Yu; Xu, Siwei; Dursun, Cagatay; et al. (2024-03-30)
    Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet, little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multi-omics datasets into a resource comprising >2.8M nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550K cell-type-specific regulatory elements and >1.4M single-cell expression-quantitative-trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized ~250 disease-risk genes and drug targets with associated cell types.
  • Vocal learning-associated convergent evolution in mammalian proteins and regulatory elements

    Wirthlin, Morgan E; Schmid, Tobias A; Elie, Julie E; Zhang, Xiaomeng; Kowalczyk, Amanda; Redlich, Ruby; Shvareva, Varvara A; Rakuljic, Ashley; Ji, Maria B; Bhat, Ninad S; et al. (2024-03-29)
    Vocal production learning ("vocal learning") is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat (Rousettus aegyptiacus) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution.
  • Casein kinase II promotes piRNA production through direct phosphorylation of USTC component TOFU-4

    Zhang, Gangming; Zheng, Chunwei; Ding, Yue-He; Mello, Craig (2024-03-28)
    Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function. We show that CK2 is required for the localization of PRG-1 and for the proper localization of several factors that comprise the 'upstream sequence transcription complex' (USTC), which is required for piRNA transcription. Loss of CK2 impairs piRNA levels suggesting that CK2 promotes USTC function. We identify the USTC component twenty-one-U fouled-up 4 (TOFU-4) as a direct substrate for CK2. Our findings suggest that phosphorylation of TOFU-4 by CK2 promotes the assembly of USTC and piRNA transcription. Notably, during the aging process, CK2 activity declines, resulting in the disassembly of USTC, decreased piRNA production, and defects in piRNA-mediated gene silencing, including transposons silencing. These findings highlight the significance of posttranslational modification in regulating piRNA biogenesis and its implications for the aging process. Overall, our study provides compelling evidence for the involvement of a posttranslational modification mechanism in the regulation of piRNA biogenesis.
  • Traumatic injury causes selective degeneration and TDP-43 mislocalization in human iPSC-derived -associated ALS/FTD motor neurons [preprint]

    Martin, Eric J; Santacruz, Citlally; Mitevska, Angela; Jones, Ian E; Krishnan, Gopinath; Gao, Fen-Biao; Finan, John D; Kiskinis, Evangelos (2024-03-26)
    A hexanucleotide repeat expansion (HRE) in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, patients with the HRE exhibit a wide disparity in clinical presentation and age of symptom onset suggesting an interplay between genetic background and environmental stressors. Neurotrauma as a result of traumatic brain or spinal cord injury has been shown to increase the risk of ALS/FTD in epidemiological studies. Here, we combine patient-specific induced pluripotent stem cells (iPSCs) with a custom-built device to deliver biofidelic stretch trauma to C9orf72 patient and isogenic control motor neurons (MNs) in vitro. We find that mutant but not control MNs exhibit selective degeneration after a single incident of severe trauma, which can be partially rescued by pretreatment with a C9orf72 antisense oligonucleotide. A single incident of mild trauma does not cause degeneration but leads to cytoplasmic accumulation of TDP-43 in C9orf72 MNs. This mislocalization, which only occurs briefly in isogenic controls, is eventually restored in C9orf72 MNs after 6 days. Lastly, repeated mild trauma ablates the ability of patient MNs to recover. These findings highlight alterations in TDP-43 dynamics in C9orf72 ALS/FTD patient MNs following traumatic injury and demonstrate that neurotrauma compounds neuropathology in C9orf72 ALS/FTD. More broadly, our work establishes an in vitro platform that can be used to interrogate the mechanistic interactions between ALS/FTD and neurotrauma.
  • Citizenship status and career self-efficacy: An intersectional study of biomedical trainees in the United States

    Chatterjee, Deepshikha; Nogueira, Ana T; Wefes, Inge; Chalkley, Roger; Sturzenegger Varvayanis, Susi; Fuhrmann, Cynthia N; Varadarajan, Janani; Jacob, Gabrielle A; Gaines, Christiann H; Hubbard, Nisan M; et al. (2024-03-20)
    This study examines the intersectional role of citizenship and gender with career self-efficacy amongst 10,803 doctoral and postdoctoral trainees in US universities. These biomedical trainees completed surveys administered by 17 US institutions that participated in the National Institutes of Health Broadening Experiences in Scientific Training (NIH BEST) Programs. Findings indicate that career self-efficacy of non-citizen trainees is significantly lower than that of US citizen trainees. While lower career efficacy was observed in women compared with men, it was even lower for non-citizen female trainees. Results suggest that specific career interests may be related to career self-efficacy. Relative to US citizen trainees, both male and female non-citizen trainees showed higher interest in pursuing a career as an academic research investigator. In comparison with non-citizen female trainees and citizen trainees of all genders, non-citizen male trainees expressed the highest interest in research-intensive (and especially principal investigator) careers. The authors discuss potential causes for these results and offer recommendations for increasing trainee career self-efficacy which can be incorporated into graduate and postdoctoral training.
  • Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia

    Funes, Salome; Jung, Jonathan; Gadd, Del Hayden; Mosqueda, Michelle; Zhong, Jianjun; Shankaracharya; Unger, Matthew; Stallworth, Karly; Cameron, Debra; Rotunno, Melissa S; et al. (2024-03-20)
    Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism, autophagy dysregulation and deficient phagocytosis, a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P, a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways, as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders.
  • Vaping Cessation Support Recommendations from Adolescents Who Vape: A Qualitative Study [preprint]

    Pbert, Lori; Dube, Catherine E; Nagawa, Catherine S; Simone, Dante P; Wijesundara, Jessica G; Sadasivam, Rajani S (2024-03-20)
    Background: Youth vaping is an epidemic, being more prevalent than any other tobacco use. To inform cessation interventions, we explored what adolescents perceive to be their reasons for quitting and strategies to help in their quit efforts. Method: Semi-structured interviews were conducted with a convenience sample of 11 adolescents reporting vaping in the past 90 days and recruited from a high school in Massachusetts. Interviews were transcribed, and dual coded. Inductive thematic analysis was employed and thematic summaries were prepared. Results: Reasons adolescents reported for quitting included: cost; experiencing "nic-sick" from nicotine withdrawal or excess intake; negative impacts on mood, concentration, or health; and experiencing symptoms of nicotine dependence. Nearly all tried to quit multiple times. Barriers to quitting included: exposure to vaping; access to vape products; stress; and "cool" new products or flavors. Quit strategies included: avoiding others vaping; seeking social support to quit; addressing peer pressure to continue vaping; learning successful quit strategies from peers; and using distraction strategies or alternatives to vaping. Conclusion: Many adolescents who vape want to quit and most have tried multiple times. Interventions need to engage adolescents with varying reasons to quit, barriers, and quit strategy preferences. Clinical trial registration: This study is registered through ClinicalTrials.gov. The trial registration number is NCT05140915. The trial registration date is 11/18/2021.
  • Calcineurin promotes adaptation to chronic stress through two distinct mechanisms [preprint]

    Flynn, Mackenzie J; Harper, Nicholas W; Li, Rui; Zhu, Lihua Julie; Lee, Michael J; Benanti, Jennifer A (2024-03-20)
    Adaptation to environmental stress requires coordination between stress-defense programs and cell cycle progression. The immediate response to many stressors has been well characterized, but how cells survive in challenging environments long-term is unknown. Here, we investigate the role of the stress-activated phosphatase calcineurin (CN) in adaptation to chronic CaCl2 stress in Saccharomyces cerevisiae. We find that prolonged exposure to CaCl2 impairs mitochondrial function and demonstrate that cells respond to this stressor using two CN-dependent mechanisms - one that requires the downstream transcription factor Crz1 and another that is Crz1-independent. Our data indicate that CN maintains cellular fitness by promoting cell cycle progression and preventing CaCl2-induced cell death. When Crz1 is present, transient CN activation suppresses cell death and promotes adaptation despite high levels of mitochondrial loss. However, in the absence of Crz1, prolonged activation of CN prevents mitochondrial loss and further cell death by upregulating glutathione (GSH) biosynthesis genes thereby mitigating damage from reactive oxygen species. These findings illustrate how cells maintain long-term fitness during chronic stress and suggest that CN promotes adaptation in challenging environments by multiple mechanisms.
  • Multicenter integrated analysis of noncoding CRISPRi screens

    Yao, David; Tycko, Josh; Oh, Jin Woo; Bounds, Lexi R; Gosai, Sager J; Lataniotis, Lazaros; Mackay-Smith, Ava; Doughty, Benjamin R; Gabdank, Idan; Schmidt, Henri; et al. (2024-03-19)
    The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.
  • Radiation Therapy Quality Assurance Analysis of Alliance A021501: Preoperative mFOLFIRINOX or mFOLFIRINOX Plus Hypofractionated Radiation Therapy for Borderline Resectable Adenocarcinoma of the Pancreas

    Tchelebi, Leila T; Segovia, Diana; Smith, Koren; Shi, Qian; FitzGerald, Thomas J; Chuong, Michael D; Zemla, Tyler J; O'Reilly, Eileen M; Meyerhardt, Jeffrey A; Koay, Eugene J; et al. (2024-03-15)
    Purpose: Alliance A021501 is the first randomized trial to evaluate stereotactic body radiation therapy (SBRT) for borderline resectable pancreatic ductal adenocarcinoma (PDAC) after neoadjuvant chemotherapy. In this post hoc study, we reviewed the quality of radiation therapy (RT) delivered. Methods and materials: SBRT (6.6 Gy × 5) was intended but hypofractionated RT (5 Gy × 5) was permitted if SBRT specifications could not be met. Institutional credentialing through the National Cancer Institute-funded Imaging and Radiation Oncology Core (IROC) was required. Rigorous RT quality assurance (RT QA) was mandated, including pretreatment review by a radiation oncologist. Revisions were required for unacceptable deviations. Additionally, we performed a post hoc RT QA analysis in which contours and plans were reviewed by 3 radiation oncologists and assigned a score (1, 2, or 3) based on adequacy. A score of 1 indicated no deviation, 2 indicated minor deviation, and 3 indicated a major deviation that could be clinically significant. Clinical outcomes were compared by treatment modality and by case score. Results: Forty patients were registered to receive RT (1 planned but not treated) at 27 centers (18 academic and 9 community). Twenty-three centers were appropriately credentialed for moving lung/liver targets and 4 for static head and neck only. Thirty-two of 39 patients (82.1%) were treated with SBRT and 7 (17.9%) with hypofractionated RT. Five cases (13%) required revision before treatment. On post hoc review, 23 patients (59.0%) were noted to have suboptimal contours or plan coverage, 12 (30.8%) were scored a 2, and 11 (28.2%) were scored a 3. There were no apparent differences in failure patterns or surgical outcomes based on treatment technique or post hoc case score. Details related to on-treatment imaging were not recorded. Conclusions: Despite rigorous QA, we encountered variability in simulation, contouring, plan coverage, and dose on trial. Although clinical outcomes did not appear to have been affected, findings from this analysis serve to inform subsequent PDAC SBRT trial designs and QA requirements.

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