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Anapc5 and Anapc7 as genetic modifiers of KIF18A function in fertility and mitotic progression.
Full text linkThe kinesin family member 18 A (KIF18A) is an essential regulator of microtubule dynamics and chromosome alignment during mitosis. Functional dependency on KIF18A varies by cell type and genetic context but the heritable factors that influence this dependency remain unknown. To address this, we took advantage of the variable penetrance observed in different mouse strain backgrounds to screen for loci that modulate germ cell depletion in the absence of KIF18A. We found a significant association at a Chr5 locus where anaphase promoting complex subunits 5 (Anapc5) and 7 (Anapc7) were the top candidate genes. We found that both genes were differentially expressed in a sensitive strain background when compared to resistant strain background at key timepoints in gonadal development. We also identified a novel retroviral insertion in Anapc7 that may in part explain the observed expression differences. In cell line models, we found that depletion of KIF18A induced mitotic arrest, which was partially rescued by co-depletion of ANAPC7 (APC7) and exacerbated by co-depletion of ANAPC5 (APC5). These findings suggest that differential expression and activity of Anapc5 and Anapc7 may influence sensitivity to KIF18A depletion in germ cells and CIN cells, with potential implications for optimizing antineoplastic therapies
Increasing the efficiency of transfecting vectors encoding KZFP genes into mouse embryonic stem cells
No full textFuture downstream studies to understand the regulatory genes responsible for cleft lip palate (CLP) in the A/WySn mouse model require a large supply of mouse embryonic stem cells (mESCs) transfected with large candidate regulatory KRAB zinc finger protein (KZFP) genes. However, current mESC transfection protocols yield on average 2-5%. This originates from their cohesive colonial nature, the 2i standard media they culture in, the conventional method of transfecting cells through adhesive cultures, and the large DNA cargo being transfected. Previous chemical transfection protocols could only yield 2-5% transfected mESCs, and yet we require a greater yield for large scale research into Clf1 and Clf2 roles in CLP development. Our project will experiment on factors that could impact yield rates, such as liposomal and non-liposomal transfection reagents, vector sizes, reagents supplemented through adhesive and/or suspensions, and modifications to the 2i standard ESC culture media. We concluded an optimal liposomal- based transfection protocol utilizing Lipofectamine 2000 in a trypsinized non-antibiotic cellular suspension under a 5-minute incubation sequence offers an average of 10-30% transfection yield. This improved transfection efficiency method will prove vital for future downstream studies of early embryonic development of CLP in A/WySn mouse strains
Evaluating the impact of mutant alleles on venetoclax and cytarabine sensitivity in acute myeloid leukemia (AML) cell lines
No full textAcute Myeloid Leukemia (AML) is a common form of blood cancer that has a poor prognosis in older adults due to limited low-toxicity treatment options [1]. AML is a heterogenous disease that can be driven by several types of somatic mutations. Two common drugs used in in the treatment of AML are venetoclax and cytarabine. While these drugs have been shown to cause apoptosis of AML cells [2,3], it is unknown whether different genetic subtypes of AML respond differently to venetoclax and cytarabine. I hypothesize that OCI-AML2 and OCI-AML3 will have a different sensitivity to venetoclax and cytarabine because of their different mutational makeups. After 3-day and 5-day treatments of cytarabine, we saw that as concentrations of drug treatment increased this lead to lower cell viability in both cell lines. However, after a 3-day and 5-day venetoclax treatment, OCI-AML2 was sensitive to venetoclax while OCI-AML3 showed to resistance to the treatment. A combined treatment of both drugs was tested on the cell lines to see if using both drugs in combination would further reduce AML cell viability. While a combined treatment of cytarabine and venetoclax showed additive effect for OCI-AML2, there was no significant additive effect in OCI-AML3. Together, these data suggest that different in genetic mutations in AML may affect sensitivity to drug treatments
Characterizing the Molecular and Mitochondrial Changes in Neuronal Pathology of CMT2D
No full textCharcot–Marie–Tooth disease type 2D (CMT2D) is a rare, inherited peripheral neuropathy characterized by a spectrum of sensory and motor deficits, including tremor, numbness, pain, and muscle weakness. In CMT2D, reduced translation through chronic activation of the Integrated Stress Response (ISR) is thought to be the primary driver of disease. However, the changes at the molecular level in the diseased state are not well understood. In the present study, we report mitochondrial abnormalities in motor neuron cell bodies, including reduced cristae density and reduced mitochondria count. We also explore the expression of candidate proteins responsible for driving pathology in mitochondria and motor axons. Additionally, we assessed the therapeutic efficacy of 2Bact, an ISR inhibitor drug, which significantly improved neuromuscular junction innervation in Gars mutant mice. Collectively, these findings elucidate the molecular and mitochondrial changes in neuronal pathology of CMT2D
Systematic evaluation of how Hand2 dimerization partners regulate DNA binding specificity and biological activity
No full textOur study aims to characterize how different dimerization partners alter Hand2 biological activity and binding site specificity during mandible development. To address these questions, we applied complementary experimental and computational approaches to assess Hand2 function as either an obligate homo- and heterodimer and modeled its 3D structure with different interactors. Twist1, Tcf12, and Tcf4 were identified using mass spectrometry as candidate Hand2 dimerization partners that modulate E-box binding specificity in the mandible. In this study, we used transient transfections in NIH-3T3 cells to verify nuclear localization and confirm protein production of synthetic Hand2-dimers. Thus far, our work has validated all obligate dimers can be imported into the nucleus and protein for monomeric Hand2 and dimeric Hand2-Hand2 can be detected via Western blot. Currently, studies are underway to perform Western blot for the other dimer pairs. We have shown successful transfection and expression in transfected NIH3T3 cell lines for Hand2-Hand2-V5 and Hand2-V5 constructs. Colocalization of Hand2-Hand2-V5 and Hand2-V5 were also verified with Hoechst stain, indicating nuclear colocalization of constructs
Cutaneous dysbiosis characterizes the post-allogeneic hematopoietic stem cell transplantation period.
Full text linkGut dysbiosis is linked to mortality and the development of graft-versus-host disease after hematopoietic stem cell transplantation (HSCT), but the impact of cutaneous dysbiosis remains unexplored. We performed a pilot observational study, obtained retroauricular and forearm skin swabs from 12 adult patients before conditioning chemotherapy/radiation and at 1 week, 1 month, and 3 months after allogeneic HSCT, and performed shotgun metagenomic sequencing. The cutaneous microbiome among HSCT patients was enriched for gram-negative bacteria such as Escherichia coli and Pseudomonas, fungi, and viruses. Enrichment with bacteriophages and Polyomavirus species was observed among patients who died within 1 year. We observed longitudinal stability of the cutaneous microbiome at the 3-month time point among those who survived beyond 1 year after HSCT, although these may simply be a reflection of the overall medical status of the patients. There was no association with fungal abundance and any of the outcomes observed. The cutaneous microbiome may be a reservoir of pathobionts among allogeneic HSCT patients. Our findings suggest that cutaneous dysbiosis exists after HSCT, but the ultimate implication of this to patient outcomes remains to be seen through larger studies
A Framework for Strengthening The Quantitative Skills/Reasoning Support Ecosystem at Small Liberal Arts Colleges
Full text linkWe developed a framework for characterizing an institution’s quantitative skills/reasoning support ecosystem to consider how various activities contribute to student success in areas connected to students’ quantitative preparation. Through discussions with faculty and staff stakeholders at eight selective small liberal arts colleges, we established that the quantitative skills/reasoning support ecosystem at these institutions consists of four domains: bridge programs with a quantitative component, assessment of readiness, curricular on-ramps, and supplementary support for courses that require quantitative skills/reasoning. The framework includes questions about each domain that can be used by stakeholders in different institutional positions to reflect on existing efforts to support student success in quantitative disciplines and identify opportunities to align or change their institutional quantitative skills/quantitative reasoning support systems to better meet student needs
Rare disease gene association discovery in the 100,000 Genomes Project.
Full text linkUp to 80% of rare disease patients remain undiagnosed after genomic sequencin
Genomic reanalysis of a pan-European rare-disease resource yields new diagnoses.
Full text linkGenetic diagnosis of rare diseases requires accurate identification and interpretation of genomic variants. Clinical and molecular scientists from 37 expert centers across Europe created the Solve-Rare Diseases Consortium (Solve-RD) resource, encompassing clinical, pedigree and genomic rare-disease data (94.5% exomes, 5.5% genomes), and performed systematic reanalysis for 6,447 individuals (3,592 male, 2,855 female) with previously undiagnosed rare diseases from 6,004 families. We established a collaborative, two-level expert review infrastructure that allowed a genetic diagnosis in 506 (8.4%) families. Of 552 disease-causing variants identified, 464 (84.1%) were single-nucleotide variants or short insertions/deletions. These variants were either located in recently published novel disease genes (n = 67), recently reclassified in ClinVar (n = 187) or reclassified by consensus expert decision within Solve-RD (n = 210). Bespoke bioinformatics analyses identified the remaining 15.9% of causative variants (n = 88). Ad hoc expert review, parallel to the systematic reanalysis, diagnosed 249 (4.1%) additional families for an overall diagnostic yield of 12.6%. The infrastructure and collaborative networks set up by Solve-RD can serve as a blueprint for future further scalable international efforts. The resource is open to the global rare-disease community, allowing phenotype, variant and gene queries, as well as genome-wide discoveries
The Unified Phenotype Ontology : a framework for cross-species integrative phenomics.
Full text linkPhenotypic data are critical for understanding biological mechanisms and consequences of genomic variation, and are pivotal for clinical use cases such as disease diagnostics and treatment development. For over a century, vast quantities of phenotype data have been collected in many different contexts covering a variety of organisms. The emerging field of phenomics focuses on integrating and interpreting these data to inform biological hypotheses. A major impediment in phenomics is the wide range of distinct and disconnected approaches to recording the observable characteristics of an organism. Phenotype data are collected and curated using free text, single terms or combinations of terms, using multiple vocabularies, terminologies, or ontologies. Integrating these heterogeneous and often siloed data enables the application of biological knowledge both within and across species. Existing integration efforts are typically limited to mappings between pairs of terminologies; a generic knowledge representation that captures the full range of cross-species phenomics data is much needed. We have developed the Unified Phenotype Ontology (uPheno) framework, a community effort to provide an integration layer over domain-specific phenotype ontologies, as a single, unified, logical representation. uPheno comprises (1) a system for consistent computational definition of phenotype terms using ontology design patterns, maintained as a community library; (2) a hierarchical vocabulary of species-neutral phenotype terms under which their species-specific counterparts are grouped; and (3) mapping tables between species-specific ontologies. This harmonized representation supports use cases such as cross-species integration of genotype-phenotype associations from different organisms and cross-species informed variant prioritization