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Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127.
No full textIncreasing use of covalent and noncovalent inhibitors of Bruton\u27s tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves \u3e80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients
The origin of genetic diversity of indigenous cockfighting chickens of Pakistan by analyzing the mtDNA.
Get PDFIn Pakistan, the origin of the indigenous cockfighting chicken (ICC) or gamecock population is unknown. However, it is speculated that this might have been associated with domestication, an event linked to recreational, entertainment (cockfighting), religious or ornamental activities. This study aims to understand the origin and genetic diversity of the ICC population in Pakistan. A total of 185 ICC population and 10 captive Indian red junglefowl (Gallus gallus murghi) were analyzed for genetic diversity indices and phylogenetic reconstruction using a 397 bp of mtDNA D-loop region. It is reported that a total of 43 haplotypes from 38 polymorphic nucleotide sites. The haplotype and nucleotide diversity are also estimated in the range of 0.643–0.909, and 0.00585–0.01575, respectively. The total genetic diversity within the population was 91.52%. Four mitochondrial haplogroups A, B, C and D were identified by median-joining network analysis, two of them have high percentages, haplogroup D (81.6%) and A (15.1%). Phylogenetic analysis showed that the ICC population of Pakistan and Gallus gallus murghi shared haplogroup D. The results of this study showed that sub-haplogroup D17a05, has significantly high haplotype diversity and percentage as compared to previously published studies, this indicated that Pakistan might be one of the centres of domestication for chicken, as it is considered that Southeast Asia is the centre of domestication. Frequencies of Haplogroup A also indicate South-North indices. This research work showed that the indigenous cockfighting chicken population of Pakistan is genetically introgressed from Gallus gallus murghi, and significant variations could be attributed to the underlying differences in the geographics, selection pressures, introgression, and regional practices; and multiple origins of cockfighting chickens’ populations around the world which reflected the past trading routes between human communities and civilizations
Improved DNA Extraction and Amplification Strategy for 16S rRNA Gene Amplicon-Based Microbiome Studies.
Get PDFNext-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent (\u27Rapid\u27 protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel \u27Rapid\u27 method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the \u27Rapid\u27 protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel \u27Rapid\u27 DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel \u27Rapid\u27 DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing
Alterations in Adipose Tissue Distribution, Cell Morphology, and Function Mark Primary Insulin Hypersecretion in Youth With Obesity.
No full textExcessive insulin secretion independent of insulin resistance, defined as primary hypersecretion, is associated with obesity and an unfavorable metabolic phenotype. We examined the characteristics of adipose tissue of youth with primary insulin hypersecretion and the longitudinal metabolic alterations influenced by the complex adipo-insular interplay. In a multiethnic cohort of adolescents with obesity but without diabetes, primary insulin hypersecretors had enhanced model-derived β-cell glucose sensitivity and rate sensitivity but worse glucose tolerance, despite similar demographics, adiposity, and insulin resistance measured by both oral glucose tolerance test and euglycemic-hyperinsulinemic clamp. Hypersecretors had greater intrahepatic and visceral fat depots at abdominal MRI, hypertrophic abdominal subcutaneous adipocytes, higher free fatty acid and leptin serum levels per fat mass, and faster in vivo lipid turnover assessed by a long-term 2H2O labeling protocol. At 2-year follow-up, hypersecretors had greater fat accrual and a threefold higher risk for abnormal glucose tolerance, while individuals with hypertrophic adipocytes or higher leptin levels showed enhanced β-cell glucose sensitivity. Primary insulin hypersecretion is associated with marked alterations in adipose tissue distribution, cellularity, and lipid dynamics, independent of whole-body adiposity and insulin resistance. Pathogenetic insight into the metabolic crosstalk between β-cell and adipocyte may help to identify individuals at risk for chronic hyperinsulinemia, body weight gain, and glucose intolerance
Unraveling the genetics of arsenic toxicity with cellular morphology QTL.
Get PDFThe health risks that arise from environmental exposures vary widely within and across human populations, and these differences are largely determined by genetic variation and gene-by-environment (gene-environment) interactions. However, risk assessment in laboratory mice typically involves isogenic strains and therefore, does not account for these known genetic effects. In this context, genetically heterogenous cell lines from laboratory mice are promising tools for population-based screening because they provide a way to introduce genetic variation in risk assessment without increasing animal use. Cell lines from genetic reference populations of laboratory mice offer genetic diversity, power for genetic mapping, and potentially, predictive value for in vivo experimentation in genetically matched individuals. To explore this further, we derived a panel of fibroblast lines from a genetic reference population of laboratory mice (the Diversity Outbred, DO). We then used high-content imaging to capture hundreds of cell morphology traits in cells exposed to the oxidative stress-inducing arsenic metabolite monomethylarsonous acid (MMAIII). We employed dose-response modeling to capture latent parameters of response and we then used these parameters to identify several hundred cell morphology quantitative trait loci (cmQTL). Response cmQTL encompass genes with established associations with cellular responses to arsenic exposure, including Abcc4 and Txnrd1, as well as novel gene candidates like Xrcc2. Moreover, baseline trait cmQTL highlight the influence of natural variation on fundamental aspects of nuclear morphology. We show that the natural variants influencing response include both coding and non-coding variation, and that cmQTL haplotypes can be used to predict response in orthogonal cell lines. Our study sheds light on the major molecular initiating events of oxidative stress that are under genetic regulation, including the NRF2-mediated antioxidant response, cellular detoxification pathways, DNA damage repair response, and cell death trajectories
Lethal phenotypes in Mendelian disorders.
Get PDFPURPOSE: Existing resources that characterize the essentiality status of genes are based on either proliferation assessment in human cell lines, viability evaluation in mouse knockouts, or constraint metrics derived from human population sequencing studies. Several repositories document phenotypic annotations for rare disorders; however, there is a lack of comprehensive reporting on lethal phenotypes.
METHODS: We queried Online Mendelian Inheritance in Man for terms related to lethality and classified all Mendelian genes according to the earliest age of death recorded for the associated disorders, from prenatal death to no reports of premature death. We characterized the genes across these lethality categories, examined the evidence on viability from mouse models and explored how this information could be used for novel gene discovery.
RESULTS: We developed the Lethal Phenotypes Portal to showcase this curated catalog of human essential genes. Differences in the mode of inheritance, physiological systems affected, and disease class were found for genes in different lethality categories, as well as discrepancies between the lethal phenotypes observed in mouse and human.
CONCLUSION: We anticipate that this resource will aid clinicians in the diagnosis of early lethal conditions and assist researchers in investigating the properties that make these genes essential for human development
An interactive web application for exploring systemic lupus erythematosus blood transcriptomic diversity.
Get PDFIn the field of complex autoimmune diseases such as systemic lupus erythematosus (SLE), systems immunology approaches have proven invaluable in translational research settings. Large-scale datasets of transcriptome profiling have been collected and made available to the research community in public repositories, but remain poorly accessible and usable by mainstream researchers. Enabling tools and technologies facilitating investigators\u27 interaction with large-scale datasets such as user-friendly web applications could promote data reuse and foster knowledge discovery. Microarray blood transcriptomic data from the LUPUCE cohort (publicly available on Gene Expression Omnibus, GSE49454), which comprised 157 samples from 62 adult SLE patients, were analyzed with the third-generation (BloodGen3) module repertoire framework, which comprises modules and module aggregates. These well-characterized samples corresponded to different levels of disease activity, different types of flares (including biopsy-proven lupus nephritis), different auto-antibody profiles and different levels of interferon signatures. A web application was deployed to present the aggregate-level, module-level and gene-level analysis results from LUPUCE dataset. Users can explore the similarities and heterogeneity of SLE samples, navigate through different levels of analysis, test hypotheses and generate custom fingerprint grids and heatmaps, which may be used in reports or manuscripts. This resource is available via this link: https://immunology-research.shinyapps.io/LUPUCE/. This web application can be employed as a stand-alone resource to explore changes in blood transcript profiles in SLE, and their relation to clinical and immunological parameters, to generate new research hypotheses
Active Molecular Network Discovery Links Lifestyle Variables to Breast Cancer in the Long Island Breast Cancer Study Project.
Get PDFA healthy lifestyle has been associated with decreased risk of developing breast cancer. Using untargeted metabolomics profiling, which provides unbiased information regarding lifestyle choices such as diet and exercise, we aim to identify the molecular mechanisms connecting lifestyle and breast cancer through network analysis. A total of 100 postmenopausal women, 50 with breast cancer and 50 cancer-free controls, were selected from the Long Island Breast Cancer Study Project (LIBCSP). We measured untargeted plasma metabolomics using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Using the enet package, we retained highly correlated metabolites representing active molecular network (AMN) clusters for analysis. LASSO was used to examine associations between cancer status and AMN metabolites and covariates such as BMI, age, and reproductive factors. LASSO was then repeated to examine associations between AMN metabolites and 10 lifestyle-related variables including smoking, physical activity, alcohol consumption, meat consumption, fruit and vegetable consumption, and supplemental vitamin use. Results were displayed as a network to uncover biological pathways linking lifestyle factors to breast cancer status. After filtering, 851 active metabolites out of 1797 metabolomics were retained in 197 correlation AMN clusters. Using LASSO, breast cancer status was associated with 71 active metabolites. Several of these metabolites were associated with lifestyle variables including meat consumption, alcohol consumption, and supplemental β-carotene, B12, and folate use. Those metabolites could potentially serve as molecular-level biological intermediaries connecting healthy lifestyle factors to breast cancer, even though direct associations between breast cancer and the investigated lifestyles at the phenotype level are not evident. In particular, DiHODE, a metabolite linked with inflammation, was associated with breast cancer status and connected to β-carotene supplement usage through an AMN. We found several plasma metabolites associated with lifestyle factors and breast cancer status. Future studies investigating the mechanistic role of inflammation in linking supplement usage to breast cancer status are warranted
Microglia depletion leads to increased susceptibility to ocular hypertension-dependent glaucoma
Get PDFIn recent years, microglia have been highlighted for playing integral roles in neurodegenerative diseases, like glaucoma. To better understand the role of microglia during chronic ocular hypertension, we depleted microglia from aged (9–12 months old) DBA/2 J (D2) mice, which exhibit age-related increases in intraocular pressure, using a dietary CSF1R antagonist, PLX5622. Retinal ganglion cell (RGC) somas were counted, and optic nerve cross-sections stained and assessed for glaucomatous damage. Sustained administration of dietary PLX5622 significantly reduced the numbers of retinal microglia. Dietary PLX5622 did not lead to changes in intraocular pressure in D2 or normotensive DBA/2 J-Gpnmb+ (D2-Gpnmb+) control mice. While PLX5622-treated D2-Gpnmb+ did not develop optic nerve damage, PLX5622-treated D2 mice showed a significant increase in moderate-to-severe optic nerve damage compared to D2 mice fed a control diet. In conclusion, global reduction of microglia exacerbated glaucomatous neurodegeneration in D2 mice suggesting microglia play an overall beneficial role in protecting from ocular hypertension associated RGC loss