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Welcome to the new eScholarship@UMassChan! eScholarship@UMassChan is a freely available digital repository offering worldwide access to the research and scholarly work of the UMass Chan Medical School community. We welcome submissions from our faculty, researchers, staff, and students. eScholarship@UMassChan is a service of the Lamar Soutter Library, Worcester, MA, USA.
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Adulting Shorts: The “TEA” on IEPs Part 2This info-comic is for high school students to help them understand what an Individualized Educational Plan or IEP is, what transition planning is, and the importance of the student being involved in them. This is Part 2 of the story. Find Part 1 here: https://www.umassmed.edu/TransitionsACR/publication/comic/2021/09/tea-on-ieps-part-1/
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Ultrasound in the Acute AbdomenThis presentation is part of the PEER Liberia Radiology Lecture Series. It provides an overview for clinicians of ultrasound in the acute abdomen.
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The Role of Non-Coding Regulatory Elements in Complex Traits and Immune-Mediated DiseaseThe completion of the Human Genome Project ushered in the age of genome-wide association studies (GWAS), which have associated thousands of single nucleotide polymorphisms (SNPs) and other sequence variants with complex traits and diseases. Despite this success, progress bridging these associations to pathophysiologic understanding and new therapeutic interventions has been limited. In large part, this owes to the fact that 90% of GWAS-identified variants are non-coding–they do not impact the structure or function of proteins. Unraveling the impacts of non-coding sequence variants is one of the most significant unsolved problems in biology. Non-coding GWAS variants are enriched within cis-regulatory elements (CREs), sequences of DNA which modulate the expression, rather than the function, of target genes. These include promoters, which are immediately adjacent to the gene they regulate; enhancers, which increase expression of distant genes; silencers, which reduce the expression of distant genes; and insulators, which divide chromatin into domains to regulate interactions between other CREs. The function of CREs is modulated in part by transcription factors (TFs), DNA binding proteins which recognize and bind short characteristic DNA sequences called motifs. TFs and CREs are tissue- and cell type-specific, frequently regulating gene expression in only a few of the thousands of distinct cell and tissue types comprising the human body. Here we present work leveraging deep sequencing data and evolutionary conservation to build comprehensive atlases of cis-regulatory elements and transcription factor binding sites in the human genome, along with work architecting visualization platforms to make these atlases more accessible to, and impactful for, the scientific community. We then illustrate a key role for the sites in our atlases, particularly those evolutionarily constrained throughout the mammalian lineage, in complex traits and diseases. We conclude by presenting two case studies utilizing these datasets: one to better understand the role of non-coding variants in primary sclerosing cholangitis, a rare immune-mediated liver disease, and a second to understand the sequence features underlying strong insulator elements in the human genome.
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Mucosal nanobody IgA as inhalable and affordable prophylactic and therapeutic treatment against SARS-CoV-2 and emerging variantsAnti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants.
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In vitro Methods to Better Evaluate Drug Responses in CancerEvaluating anti-cancer drugs in vitro is an important aspect of the drug development pipeline. When evaluating anti-cancer drugs, two different measurements are used: relative viability, which scores an amalgam of proliferative arrest and cell death, and fractional viability, which specifically scores the degree of cell killing. These two metrics are often used interchangeably despite measuring different aspects of a drug response. This study explored the relationship between drug-induced growth inhibition and cell death, and found that most drugs affect both proliferation and death, but in different proportions, and with different relative timing. This causes a non-uniform relationship between relative and fractional response measurements. To unify these measurements, I created a data visualization and analysis platform, called drug GRADE, which characterizes the degree to which death contributes to an observed drug response. GRADE captures drug- and genotype-specific responses, which are not captured using traditional pharmaco-metrics. Current in vitro anti-cancer drug evaluation practices measure drug responses with cancer cell lines in mono-culture. However, many cell types in the tumor microenvironment influence cancer’s drug response and disease progression. Therefore, current drug evaluation practices overlook complex cell-cell interactions that influence cancer’s drug response. In this study, I developed a high-throughput assay to study the effect of another cell type (cytotoxic T cells) on cancer viability in co-culture, in vitro. Further, I developed a reference framework to model the complex interaction between cancer cells and cytotoxic T cells, and to model how T cell-mediated cell death is modulated by anti-cancer drug treatment. Taken together, this study highlights two new methods which enable better in vitro evaluation of drug responses in cancer.
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Francis Fontan (1929-2018): Pioneer pediatric cardiac surgeonUp until the mid-1900s, tricuspid atresia - a birth defect of the tricuspid valve, was once categorized as a "death sentence." The challenge of achieving positive health outcomes for affected patients was compounded by a hesitancy to operate on children. The main concern was safely administering anesthesia to young patients who were going through a strenuous operation that was often poorly tolerated. Despite these assumed limitations, Francis Fontan, a pediatric cardiothoracic surgeon at the Hospital of Tondu in Bordeaux, was able to redirect blood flow from the superior and inferior vena cava to the pulmonary arteries in 1971, which elucidated the process of advancing clinical practice in medicine. With the support of mentors and a firm belief in this new technique, Fontan pioneered his eponymous procedure and ultimately paved the way for modern cardiovascular surgical techniques that helped to prolong the life of those with single functioning ventricles. The aim of this study is to examine the genesis and the evolution of the Fontan procedure to elucidate the process of advancing clinical practice in medicine by utilizing personal interviews, Fontan's works, associated primary and secondary sources in the context of 20th century cardiothoracic surgery and innovations.
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Examining Pregnant Veterans' Acceptance and Beliefs Regarding the COVID-19 VaccineBackground: Pregnant persons have received mixed messages regarding whether or not to receive COVID-19 vaccines as limited data are available regarding vaccine safety for pregnant and lactating persons and breastfeeding infants. Objective: The aims of this study were to examine pregnant Veteran's acceptance of COVID-19 vaccines, along with perceptions and beliefs regarding vaccine safety and vaccine conspiracy beliefs. Design and participants: We conducted a cross-sectional survey of pregnant Veterans enrolled in VA care who were taking part in an ongoing cohort study at 15 VA medical centers between January and May 2021. Main measures: Pregnant Veterans were asked whether they had been offered the COVID-19 vaccine during pregnancy, and whether they chose to accept or refuse it. Additional questions focused on perceptions of COVID-19 vaccine safety and endorsements of vaccine knowledge and conspiracy beliefs. Logistic regression was utilized to examine predictors of acceptance of a vaccine during pregnancy. Key results: Overall, 72 pregnant Veterans were offered a COVID-19 vaccine during pregnancy; over two-thirds (69%) opted not to receive a vaccine. Reasons for not receiving a vaccine included potential effects on the baby (64%), side effects for oneself (30%), and immunity from a past COVID-19 infection (12%). Those who received a vaccine had significantly greater vaccine knowledge and less belief in vaccine conspiracy theories. Greater knowledge of vaccines in general (aOR: 1.78; 95% CI: 1.2-2.6) and lower beliefs in vaccine conspiracies (aOR: 0.76; 95% CI: 0.6-0.9) were the strongest predictors of acceptance of a COVID-19 vaccine during pregnancy. Conclusions: Our study provides important insights regarding pregnant Veterans' decisions to accept the COVID-19 vaccine, and reasons why they may choose not to accept the vaccine. Given the high endorsement of vaccine conspiracy beliefs, trusted healthcare providers should have ongoing, open discussions about vaccine conspiracy beliefs and provide additional information to dispel these beliefs.
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Cellular heterogeneity and gene regulatory network coordination during thymic epithelial cell developmentThymic epithelial cells, derived from the pharyngeal endoderm, perform essential functions for establishing a self-tolerant immune system. Unsurprisingly, dysfunction of thymic epithelial cells resulting from maldevelopment of the pharyngeal endoderm causes immunodeficiency or autoimmunity syndromes, some of which cannot be fully explained according to known genetic errors. Despite the functional significance and disease-relevance of pharyngeal endoderm with respect to thymic epithelial cells, we lack a comprehensive understanding of the gene regulatory networks driving pharyngeal endoderm differentiation. To close this gap, we applied transcriptome and chromatin accessibility single cell profiling to generate a multi-omic developmental resource covering pharyngeal differentiation toward organ-specific epithelia in the mouse embryo. We identified cell-type specific gene regulation of developing organ domains and characterized the role of an immunodeficiency-associated forkhead box transcription factor, Foxn1, during early thymus development. Furthermore, analyses of the pharyngeal endoderm multi-omics atlas led us to discover a novel gene associated with thymus development, namely Grainyhead-like3 (Grhl3). We assessed the expression pattern and the functional importance of Grhl3 in the prenatal and postnatal thymus, uncovering a putative role in a specialized medullary thymic subtype. In conclusion, this dissertation provides insight on the molecular basis of pharyngeal endoderm differentiation and subsequent development of the thymus.
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Regulated Gene Therapy Towards Glycosphingolipid Biosynthesis DeficienciesGlycosphingolipids (GSLs) are a group of amphipathic glycolipids essential for maintaining the normal ultrastructure and function of neural and oligodendrocyte cell membranes throughout the mammalian central nervous system (CNS). De novo GSL biosynthesis defects cause severe neurological diseases such as GM3 synthase deficiency (GM3SD) and hereditary sensory and autonomic neuropathy type 1A (HSAN1A), each lacking effective treatment. Here, we developed two distinct potential therapeutic approaches for these neurological diseases. For GM3SD that is caused by loss-of-function mutations in ST3GAL5, we employed a recombinant adeno-associated virus (rAAV)-mediated human ST3GAL5 gene replacement therapy. First, using ST3GAL5 mutant patient iPSC-derived neurons and St3gal5 knock-out mouse models, we have achieved ST3GAL5 gene normalization and restoration of the functional products, cerebral gangliosides. Importantly, we revealed the hepatic toxicity caused by ubiquitous expression of ST3GAL5 and optimized a CNS-restricted rAAV gene replacement therapy for the safe and efficacious rescue of the severe neurodevelopmental phenotypes and early lethality in disease mouse models, given by both intracerebroventricular and intravenous routes of administration. These results support for further clinical development of ST3GAL5 gene therapy. On the other hand, to target gain-of-function SPTLC1 mutation caused HSAN1A, we screened antisense oligonucleotides (ASOs) and achieved efficient reduction of mutant SPTLC1 transcripts and its toxic products in patient-fibroblasts. In summary, this thesis describes the potential of novel rAAV-mediated gene replacement therapy in GM3SD and allele-specific ASO silencing in HSAN1A, highlighting the significance of personalized gene therapy for monogenic neurological disorders.
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Investigating Proteolytic Processing of Ataxin 2, a Neurodegenerative Disease Associated ProteinAtaxin 2 (ATXN2) is a ubiquitously expressed mRNA binding protein involved in the development and progression of spinocerebellar ataxia 2 (SCA2) and amyotrophic lateral sclerosis (ALS). In the context of both neurodegenerative diseases, its N-terminal polyglutamine (polyQ) domain is mutated and expanded in length. Several other polyQ proteins, such as huntingtin (Htt), ataxin 3 (ATXN3), and ataxin 7 (ATXN7), undergo proteolytic processing that produces toxic fragments containing their polyQ domains. Investigating how ATXN2 is regulated by proteolysis is hindered by the lack of available molecular biological tools such as N-terminal ATXN2 antibodies to target and analyze the endogenous N-terminus of ATXN2. To circumvent this challenge, I developed a transient overexpression model of N-terminally tagged ATXN2 in HEK293E cells. Here, I demonstrate that both wild-type and mutant ATXN2 are targets of N-terminal proteolysis. I confirmed that ATXN2 produces an independent polyQ cleavage fragment like other polyQ proteins through basic molecular biology approaches such as Western blotting and immunoprecipitation. Additionally, I identified the specific region that is both necessary and sufficient for cleavage to occur via deletion mapping with multiple truncated ATXN2 mutants and reporter constructs. Further definition of ATXN2 as a target of proteolytic cleavage aligns it with other neurodegenerative polyQ proteins, and proteolysis is currently a less explored avenue of research for ATXN2-related disease development, progression, and therapeutic modalities. This work reveals a novel site that directs cleavage of ATXN2 and provides a potential avenue of investigation for how ATXN2 posttranslational modifications contribute to the progression of SCA2 and ALS.
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UMCCTS Newsletter, August 2022This is the August 2022 issue of the UMass Center for Clinical and Translational Science Newsletter containing news and events of interest.
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Journal of eScience Librarianship (JeSLIB) Publishing ProcessDescribes the roles and responsibilities, author guidelines, and peer review process for the Journal of eScience Librarianship.
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High bacillary burden and the ESX-1 type VII secretion system promote MHC class I presentation to CD8 T-cells during Mycobacterium tuberculosis infectionT-cell mediated immunity is required for optimal protection against Mycobacterium tuberculosis (Mtb) infection, but often fails to completely clear the pathogen. Mtb has evolved strategies to subvert host immunity so it can persist in host cells despite pressure from innate and adaptive immunity. While cytotoxic CD8 T-cells should recognize and clear infected host cells, eliminating Mtb’s intracellular niche, previous findings have demonstrated otherwise [1]. In fact, we have shown that CD8 T-cells specific to the immunodominant antigen TB10.4 poorly recognize Mtb infected macrophages in vitro. Here we extend our initial findings to show that class I MHC-restricted epitopes other than TB10.44-11 are inefficiently presented by Mtb-infected macrophages to CD8 T cells. The only exception we find is for heavily infected macrophages. During high burden infections, macrophages cross-present TB10.4 antigen to CD8 T-cells. These high burden infections result in considerable cell death, and we find that uninfected macrophages effectively scavenge dead cellular debris and cross-present this antigen to CD8 T cells. Furthermore, we find that cross-presentation by heavily infected cells is dependent on the ESX-1 type VII secretion system, suggesting that phagosomal membrane damage and host cell death are crucial for effective class I MHC cross-presentation during Mtb infection.
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International Controlled Study of Revascularization and Outcomes Following COVID-Positive Mechanical ThrombectomyBACKGROUND: Previous studies suggest that the mechanisms and outcomes in COVID-19-associated stroke differ from those with non-COVID-19 strokes, but there is limited comparative evidence focusing on these populations. Therefore, we aimed to determine if a significant association exists between COVID-19 status with revascularization and functional outcomes following thrombectomy for large vessel occlusion (LVO), after adjustment for potential confounding factors. METHODS: A cross-sectional, international multicenter retrospective study of consecutively admitted COVID-19 patients with concomitant acute LVO, compared to a control group without COVID-19. Data collected included age, gender, comorbidities, clinical characteristics, details of the involved vessels, procedural technique, and various outcomes. A multivariable adjusted analysis was conducted. RESULTS: In this cohort of 697 patients with acute LVO, 302 had COVID-19 while 395 patients did not. There was a significant difference (p < 0.001) in the mean age (in years) and gender of patients, with younger patients and more males in the COVID-19 group. In terms of favorable revascularization (mTICI 3), COVID-19 was associated with lower odds of complete revascularization [OR=0.33; 95% CI=0.23-0.48; p < 0.001], which persisted on multivariable modelling with adjustment for other predictors [aOR=0.30; 95% CI=0.12-0.77; p=0.012]. Moreover, endovascular complications, in-hospital mortality, and length of hospital stay were significantly higher among COVID-19 patients (p < 0.001). CONCLUSION: COVID-19 was an independent predictor of incomplete revascularization and poor functional outcome in patients with stroke due to LVO. Furthermore, COVID-19 patients with LVO were more often younger and suffered higher morbidity/mortality rates.
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The Role of Age-Associated B Cells (ABC) in Combating Influenza InfectionWith age, follicular helper T cell (TFH) dependent B cell responses erode, reducing B cell memory and long-term antibody responses. However, aged mice and humans develop an alternative B cell population, termed age-associated B cells (ABC), that may produce TFH independent antibodies. ABC lack CD21 and CD23 expression, and some express transcription factors and adhesion molecules indicative of antigen exposure. Some of these have been implicated in autoimmunity. We found a unique population of responding B cells following influenza A virus (IAV) infection, which was Fashi/GL7neg. We postulated that the CD21-CD23-ABC might be progenitors of these non-follicular B cells, that we called induced ABC (iABC). Using T-deficient RAG KO and TFH deficient SAP KO hosts, we found that the CD21-CD23-ABC can become iABC (FasHiGL7-) upon IAV infection. These iABC share the same phenotype of iABC found in infected aged mice and can become Ab-producing cells without T cell help. We showed that CD21-CD23-ABC can be separated into IgD+ (putative naïve B cells) vs. IgD- (memory-like B cells). We followed their ability to become iABC in SAP KO hosts. The IgD+ABC were most efficient at giving rise to iABC. Further transfer studies revealed that iABC generation from donor IgD+ABC requires extrinsic TLR signaling, and IgD+ABC can provide Ab-medicated protection. We concluded that upon T-independent stimulation, IgD+ABC (CD21-CD23-) become iABC (Fashi/GL7neg), some of which can secrete IAV-specific Ab and may provide protection against IAV.
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Data and Code from "Show me the data! Data sharing practices demonstrated in published research at the University of Massachusetts Chan Medical School"Data extracted from articles published by UMass Chan researchers to determine where and how data was being shared. Code from the analysis is also included.
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UMCCTS Newsletter, July 2022This is the July 2022 issue of the UMass Center for Clinical and Translational Science Newsletter containing news and events of interest.
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Novel Genetic Pathways in Functional Regulation of Hematopoietic Stem CellsHematopoietic stem cells (HSCs) are a rare population of bone marrow cells that have self-renewal and differentiating capabilities enabling them to produce all blood lineages during normal hematopoiesis. Many molecular pathways are involved in the regulation of HSCs, and the survival, maintenance and proliferation of these cells must be tightly controlled to avoid aberrant activities that can cause blood diseases, such as hematopoietic malignancies. Therefore, additional factors involved in the functional regulation of HSCs must be discovered to provide new therapeutic treatments for hematopoietic diseases. In chapter I, I briefly introduce the hematopoietic system and hierarchy through which HSCs can produce all mature blood cells in the lifespan. I also describe various methods to identify different hematopoietic cells, with a focus on using cell surface antigen markers. I additionally discuss an important method to study HSCs in mice by using bone marrow transplantation in which the donor cells are manipulated to examine the role of a gene of interest. I also briefly describe several signaling pathways important in HSCs, such as the Bmp, Wnt, Hedgehog and Notch signaling pathways. I provide known relevant information to my thesis work on c-Kit and Sca-1 receptors, as well as on LSK and LSK- cells. In chapter II, I describe my findings regarding a novel mechanism in which Ikzf3 plays a suppressive role in regulating HSC survival and maintenance. Ikzf3 suppresses the population of LSK (lineage-Sca-1+c-Kit+) cells that contains HSCs, and increases the LSK- (lineage-Sca-1+c-Kit-) population, which is highly apoptotic and derived from the LSK population. The DNA binding domain of Ikzf3 is required for its inhibition of HSCs, and Ikzf3 downregulates the expression of Bcl-2, Bcl-xL and c-Myc. Ikzf3 expression in HSCs is maintained at low levels by the c-Kit pathway. In chapter III, on the basis of data from microarray analysis previously performed to compare gene expression profiles between Hif1a knockout HSCs and wild type HSCs, the effects of both Hif1a and Notch1 deletion on HSC regulation are examined. Loss of both Hif1a and Notch1 induces the development of myelodysplastic/myeloproliferative diseases, which are clonal hematopoietic stem cell neoplasms characterized by abnormal regulation of the myeloid pathways for cellular proliferation, maturation and survival. Loss of both Hif1a and Notch1 also leads to a loss of HSC function. These findings indicate a mechanism through which the hypoxia pathway acts in coordination with the Notch pathway in HSCs. In chapter IV, I summarize the findings from chapter II and III and discuss the importance of these results in the field. I also provide the future directions that can answer more questions to expand our knowledge on these pathways. Together, the findings reveal two novel pathways involved in functional regulation of HSCs: (i) the Ikzf3 pathway, which involves c-Kit, Icsbp, Bcl-2, Bcl-xL and c-Myc, and suppresses normal HSCs to maintain homeostasis and (ii) the synergy of Hif1a and Notch1 in regulating HSCs. The loss of both genes can cause myelodysplastic/myeloproliferative-like diseases.
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COVID-19: a gray swan's impact on the adoption of novel medical technologiesThe COVID-19 pandemic offers a unique context and opportunity to investigate changes in healthcare professional perceptions towards the adoption of novel medical technologies, such as point-of-care technologies (POCTs). POCTs are a nascent technology that has experienced rapid growth as a result of COVID-19 due to their ability to increase healthcare accessibility via near-patient delivery, including at-home. We surveyed healthcare professionals before and during COVID-19 to explore whether the pandemic altered their perceptions about the usefulness of POCTs. Our network analysis method provided a structure for understanding this changing phenomenon. We uncovered that POCTs are not only useful for diagnosing COVID-19, but healthcare professionals also perceive them as increasingly important for diagnosing other diseases, such as cardiovascular, endocrine, respiratory, and metabolic diseases. Healthcare professionals also viewed POCTs as facilitating the humanization of epidemiology by improving disease management/monitoring and strengthening the clinician-patient relationship. As the accuracy and integration of these technologies into mainstream healthcare delivery improves, hurdles to their adoption dissipate, thereby encouraging healthcare professionals to rely upon them more frequently to diagnose, manage, and monitor diseases. The technological advances made in POCTs during COVID-19, combined with shifting positive perceptions of their utility by healthcare professionals, may better prepare us for the next pandemic.
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Gliotransmission Orchestrates Neuronal Type-specific Axon RegenerationWhy closely related neuronal types differ in their axon regenerative abilities remains elusive. Here, I demonstrate gliotransmission determines such a difference in Drosophila larval sensory neurons. Axotomy activates ensheathing glia, which signal to regenerative neurons through the gliotransmitter adenosine, to mount regenerative programs including neuronal activity and Ras. Surprisingly, ensheathing glia do not signal to non-regenerative neurons. Such neuronal type-specific responses to gliotransmission result from specific expression of adenosine receptors in regenerative neurons. Disrupting gliotransmission impedes regeneration of regenerative neurons. Strikingly, reconstitution of gliotransmission in non-regenerative neurons enables them to regenerate. Furthermore, activation of an adenosine receptor in adult mice promotes both regeneration and survival of retinal ganglion cells, uncovering a conserved pro-regenerative role of adenosine receptors. My studies demonstrate gliotransmission as a novel mechanism by which glia instruct axon regeneration, with neuronal type-specificity, and suggest targeting purinergic signaling as a new strategy for mammalian central nervous system repair.
