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INVESTIGATING THE ROLES OF ZNFX-1 IN PROPAGATING EPIGENETIC STATES ACROSS GENERATIONS IN C. ELEGANS
No full textEpigenetic mechanisms of gene regulation allow organisms to transmit information to their progeny while retaining the same DNA sequence. One means by which epigenetic information is established and maintained is through germline Argonaute pathways. The Caenorhabditis elegans PIWI Argonaute PRG-1 engages thousands of endogenously encoded piRNAs within perinuclear nuage. Sufficient base-pairing between piRNAs and their mRNA targets can elicit a silencing response that is maintained transgenerationally by worm-specific Argonautes (WAGOs), even in the absence of continued PRG-1 activity. We previously described ZNFX-1 as a factor involved in the maintenance of piRNA-triggered inherited silencing. However, the functions of ZNFX-1 in balancing and maintaining inherited silencing and how it functions alongside other core components of germline small RNA pathways remain poorly understood. In this dissertation I explore how ZNFX-1 and PRG-1 work in parallel to promote properly directed silencing on endogenous genetic elements. I show that they function together to prevent thousands of endogenous mRNAs from becoming de-novo targets of small RNA templating. Surprisingly, I find that RNAi inheritance, while defective in znfx-1, is partially rescued in prg-1; znfx-1 double mutants. These data suggest that ZNFX-1 promotes but is not essential for the inheritance of silencing. I also report that znfx-1 mutants cause GLH-4—but not other nuage components analyzed to date—to become de-localized from the nuage. This includes not only null alleles but also catalytic point mutants, suggesting ZNFX-1 function contributes to GLH-4 localization. I hypothesize this mislocalization of GLH-4 contributes to some, but not all, of the small RNA phenotypes observed in znfx-1 mutants. Taken together, my findings shed light on how ZNFX-1 contributes to robust transmission of epigenetic information in C. elegans.Interdisciplinary Graduate Program1 year2025-12-1
The contextual interview: a cross-cutting patient-interviewing approach for social context
No full textPatient interviewing pedagogy in medical education has not evolved to comprehensively capture the biopsychosocial model of healthcare delivery. While gathering a patient's social history targets important aspects of social context it does not adequately capture and account for the real-time reassessment required to understand evolving factors that influence exposure to drivers of health inequities, social determinants of health, and access to supports that promote health. The authors offer a patient interviewing approach called the Contextual Interview (CI) that specifically targets dynamic and ever-changing social context information. To substantiate the use of the CI in medical education, the authors conducted a qualitative review of the Accreditation Council for Graduate Medical Education Milestones for primary care specialties (Family Medicine, Internal Medicine, and Pediatrics). Milestones were coded to the extent to which they reflected the learner's need to acknowledge, assess, synthesize and/or apply patient contextual data in real-time patient encounters. Approximately 1 in 5 milestones met the context-related and patient-facing criteria. This milestone review further highlights the need for more intentional training in eliciting meaningful social context data during patient interviewing. The CI as a cross-cutting, practical, time-conscious, and semi-structured patient interviewing approach that deliberately elicits information to improve the clinician's sense and understanding of a patient's social context. The authors reviewed future directions in researching adapted versions of the CI for undergraduate and graduate medical education
Lamar Soutter Library Annual Report Fiscal Year 2024
Get PDFAnnual report of the Lamar Soutter Library at UMass Chan Medical School, covering fiscal year July 1, 2023 - June 30, 2024.No embarg
Impact of preanalytical factors on liquid biopsy in the canine cancer model [preprint]
Get PDFThis article is a preprint. Preprints are preliminary reports of work that have not been certified by peer review.While liquid biopsy has potential to transform cancer diagnostics through minimally-invasive detection and monitoring of tumors, the impact of preanalytical factors such as the timing and anatomical location of blood draw is not well understood. To address this gap, we leveraged pet dogs with spontaneous cancer as a model system, as their compressed disease timeline facilitates rapid diagnostic benchmarking. Key liquid biopsy metrics from dogs were consistent with existing reports from human patients. The tumor content of samples was higher from venipuncture sites closer to the tumor and from a central vein. Metrics also differed between lymphoma and non-hematopoietic cancers, urging cancer-type-specific interpretation. Liquid biopsy was highly sensitive to disease status, with changes identified soon after post chemotherapy administration, and trends of increased tumor fraction and other metrics observed prior to clinical relapse in dogs with lymphoma or osteosarcoma. These data support the utility of pet dogs with cancer as a relevant system for advancing liquid biopsy platforms
Multicenter integrated analysis of noncoding CRISPRi screens
Get PDFThe 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
Enhancing Safety and Efficacy of Genome Editing In Vivo with Compact Cas9 and Guide Chemical Modification
Get PDFClustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) have revolutionized science and medicine. However, applying the CRISPR/Cas system for in vivo therapeutic genome editing remains challenging due in part to delivery obstacles. Adeno-associated virus (AAV) vectors remain one of the most promising vehicles for gene therapies, particularly in extrahepatic tissues. Nevertheless, employing AAV for in vivo CRISPR/Cas genome editing presents difficulties, including limited packaging capacity, prolonged expression, redosing complexities, modest multiplexing capability, dose-related toxicity, and immunogenicity. This thesis addresses AAV limitations in delivering CRISPR/Cas in vivo in two ways. First, I focus on developing a novel delivery modality in which the CRISPR RNA (crRNA) is delivered in the form of a naked oligonucleotide, separate from AAV-expressed effector and trans-activating crRNA (tracrRNA). I find that a short, fully stabilized oligonucleotide (a ‘protecting oligo’) can significantly enhance the potency, stability, and uptake of a heavily chemically modified crRNA. The establishment of AAV/crRNA co-delivery modality offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation. Second, I use a compact Nme2Cas9 adenine base editor (Nme2-ABE) to engineer a single AAV delivery system for base editing in vivo. The capability to deliver a base editor via a single AAV holds the potential to enhance safety by minimizing immune responses, mitigating dose-related toxicity, and reducing manufacturing complexity.Interdisciplinary Graduate Progra
CAPES Newsletter January 2024
Get PDFThis is the January 2024 issue of the CAPES Newsletter containing relevant news and center updates
Volumetric microscopy of cerebral arteries with a miniaturized optical coherence tomography imaging probe
No full textEndovascular interventions are increasingly becoming the preferred approach for treating strokes and cerebral artery diseases. These procedures rely on sophisticated angiographical imaging guidance, which encounters challenges because of limited contrast and spatial resolution. Achieving a more precise visualization of the underlying arterial pathology and neurovascular implants is crucial for accurate procedural decision-making. In a human study involving 32 patients, we introduced the clinical application of a miniaturized endovascular neuro optical coherence tomography (nOCT) imaging probe. This technology was designed to navigate the tortuous paths of the cerebrovascular circulation and to offer high-resolution imaging in situ. The nOCT probe is compatible with standard neurovascular microcatheters, integrating with the procedural workflow used in clinical routine. Equipped with a miniaturized optical fiber and a distal lens, the probe illuminates the tissue and collects the backscattered, near-infrared light. While rotating the fiber and the lens at high speed, the probe is rapidly retracted, creating a spiral-shaped light pattern to comprehensively capture the arterial wall and implanted devices. Using nOCT, we demonstrated volumetric microscopy of cerebral arteries in patients undergoing endovascular procedures. We imaged the anterior and posterior circulation of the brain, including distal segments of the internal carotid and middle-cerebral arteries, as well as the vertebral, basilar, and posterior cerebral arteries. We captured a broad spectrum of neurovascular pathologies, such as brain aneurysms, ischemic stroke, arterial stenoses, dissections, and intracranial atherosclerotic disease. nOCT offered artifact-free, high-resolution visualizations of intracranial artery pathology and neurovascular devices
Inflammasomes in neurological disorders - mechanisms and therapeutic potential
No full textInflammasomes are molecular scaffolds that are activated by damage-associated and pathogen-associated molecular patterns and form a key element of innate immune responses. Consequently, the involvement of inflammasomes in several diseases that are characterized by inflammatory processes, such as multiple sclerosis, is widely appreciated. However, many other neurological conditions, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, stroke, epilepsy, traumatic brain injury, sepsis-associated encephalopathy and neurological sequelae of COVID-19, all involve persistent inflammation in the brain, and increasing evidence suggests that inflammasome activation contributes to disease progression in these conditions. Understanding the biology and mechanisms of inflammasome activation is, therefore, crucial for the development of inflammasome-targeted therapies for neurological conditions. In this Review, we present the current evidence for and understanding of inflammasome activation in neurological diseases and discuss current and potential interventional strategies that target inflammasome activation to mitigate its pathological consequences
Safety and efficacy of losmapimod in facioscapulohumeral muscular dystrophy (ReDUX4): a randomised, double-blind, placebo-controlled phase 2b trial
No full textBackground: Facioscapulohumeral muscular dystrophy is a hereditary progressive myopathy caused by aberrant expression of the transcription factor DUX4 in skeletal muscle. No approved disease-modifying treatments are available for this disorder. We aimed to assess the safety and efficacy of losmapimod (a small molecule that inhibits p38α MAPK, a regulator of DUX4 expression, and p38β MAPK) for the treatment of facioscapulohumeral muscular dystrophy.
Methods: We did a randomised, double-blind, placebo-controlled phase 2b trial at 17 neurology centres in Canada, France, Spain, and the USA. We included adults aged 18-65 years with type 1 facioscapulohumeral muscular dystrophy (ie, with loss of repression of DUX4 expression, as ascertained by genotyping), a Ricci clinical severity score of 2-4, and at least one skeletal muscle judged using MRI to be suitable for biopsy. Participants were randomly allocated (1:1) to either oral losmapimod (15 mg twice a day) or matching placebo for 48 weeks, via an interactive response technology system. The investigator, study staff, participants, sponsor, primary outcome assessors, and study monitor were masked to the treatment allocation until study closure. The primary endpoint was change from baseline to either week 16 or 36 in DUX4-driven gene expression in skeletal muscle biopsy samples, as measured by quantitative RT-PCR. The primary efficacy analysis was done in all participants who were randomly assigned and who had available data for assessment, according to the modified intention-to-treat principle. Safety and tolerability were assessed as secondary endpoints. This study is registered at ClinicalTrials.gov, number NCT04003974. The phase 2b trial is complete; an open-label extension is ongoing.
Findings: Between Aug 27, 2019, and Feb 27, 2020, 80 people were enrolled. 40 were randomly allocated to losmapimod and 40 to placebo. 54 (68%) participants were male and 26 (33%) were female, 70 (88%) were White, and mean age was 45·7 (SD 12·5) years. Least squares mean changes from baseline in DUX4-driven gene expression did not differ significantly between the losmapimod (0·83 [SE 0·61]) and placebo (0·40 [0·65]) groups (difference 0·43 [SE 0·56; 95% CI -1·04 to 1·89]; p=0·56). Losmapimod was well tolerated. 29 treatment-emergent adverse events (nine drug-related) were reported in the losmapimod group compared with 23 (two drug-related) in the placebo group. Two participants in the losmapimod group had serious adverse events that were deemed unrelated to losmapimod by the investigators (alcohol poisoning and suicide attempt; postoperative wound infection) compared with none in the placebo group. No treatment discontinuations due to adverse events occurred and no participants died during the study.
Interpretation: Although losmapimod did not significantly change DUX4-driven gene expression, it was associated with potential improvements in prespecified structural outcomes (muscle fat infiltration), functional outcomes (reachable workspace, a measure of shoulder girdle function), and patient-reported global impression of change compared with placebo. These findings have informed the design and choice of efficacy endpoints for a phase 3 study of losmapimod in adults with facioscapulohumeral muscular dystrophy.
Funding: Fulcrum Therapeutics