Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus

Sequences termed v-abl, which encode the protein-tyrosine kinase activity of Abelson murine leukemia virus, have been expressed in Escherichia coli as a fusion product (ptabl50 kinase). This fusion protein contains 80 amino acids of SV40 small t and the 403 amino acid protein kinase domain of v-abl. We report here the purification and characterization of this kinase. The purified material contains two proteins (Mr = 59,800 and 57,200), both of which possess sequences derived from v-abl. Overall purification was 3,750-fold, with a 31% yield, such that 117 micrograms of kinase could be obtained from 40 g of E. coli within 6-7 days. The specific kinase activity is over 170 mumol of phosphate min-1 mumol-1, comparable to the most active protein- serine kinases. Kinase activity is insensitive to K+, Na+, Ca2+, Ca2+- calmodulin, cAMP, or cAMP-dependent protein kinase inhibitor. The Km for ATP is dependent on the concentration of the second substrate. GTP can also be used as a phosphate donor. The enzyme can phosphorylate peptides consisting of as few as two amino acids and, at a very low rate, free tyrosine. Incubation of the kinase with [gamma-32P]ATP results in incorporation of 1.0 mol of phosphate/mol of protein. This reaction, however, cannot be blocked by prior incubation with unlabeled ATP. Incubation of 32P-labeled kinase with either ADP or ATP results in the synthesis of [32P]ATP. This suggests the phosphotyrosine residue on the Abelson kinase contains a high energy phosphate bond

New Classes of Alanine Racemase Inhibitors Identified by High-Throughput Screening Show Antimicrobial Activity against Mycobacterium tuberculosis

In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells.This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents

Development of selection indices for improvement of seed yield and lipid composition in bambara groundnut (Vigna subterranea (l.) verdc.)

The underutilised grain legume bambara groundnut (Vigna subterranea) has the potential to contribute significantly to nutritional security. However, the lack of commercial cultivars has hindered its wider adoption and utilisation as a food source. The development of competitive cultivars is impeded by (1) lack of systematic data describing variation in nutritional composition within the gene pool, and (2) a poor understanding of how concentrations of different nutritional components interact. In this study, we analysed seed lipid and protein concentration and lipid composition within a collection of 100 lines representing the global gene pool. Seed protein and lipid varied over twofold with a normal distribution, but no significant statistical correlation was detected between the two components. Seed lipid concentration (4.2–8.8 g/100 g) is primarily determined by the proportion of oleic acid (r2 = 0.45). Yield and composition data for a subset of 40 lines were then used to test selection parameters for high yielding, high lipid breeding lines. From five selection indices tested using 15 scenarios, an index based on the seed number, seed weight, and oleic acid yielded a >50% expected increase in each of the mean values of seed number, pod dry weight, seed dry weight, and seed size, as well as an expected 7% increase in seed lipid concentration

Comparison of Aerosol Classification From Airborne High Spectral Resolution Lidar and the CALIPSO Vertical Feature Mask

Knowledge of aerosol composition and vertical distribution is crucial for assessing the impact of aerosols on climate. In addition, aerosol classification is a key input to CALIOP aerosol retrievals, since CALIOP requires an inference of the lidar ratio in order to estimate the effects of aerosol extinction and backscattering. In contrast, the NASA airborne HSRL-1 directly measures both aerosol extinction and backscatter, and therefore the lidar ratio (extinction-to-backscatter ratio). Four aerosol intensive properties from HSRL-1 are combined to infer aerosol type. Aerosol classification results from HSRL-1 are used here to validate the CALIOP aerosol type inferences

Developmental Plasticity of the Major Alkyl Cannabinoid Chemotypes in a Diverse Cannabis Genetic Resource Collection

Cannabis is a chemically diverse domesticated plant genus which produces a unique class of biologically active secondary metabolites referred to as cannabinoids. The affinity and selectivity of cannabinoids to targets of the human endocannabinoid system depend on alkyl side chain length, and these structural-activity relationships can be utilized for the development of novel therapeutics. Accurate early screening of germplasm has the potential to accelerate selection of chemical phenotypes (chemotypes) for pharmacological exploitation. However, limited attempts have been made to characterize the plasticity of alkyl cannabinoid composition in different plant tissues and throughout development. A chemotypic diversity panel comprised of 99 individuals from 20 Cannabis populations sourced from the Ecofibre Global Germplasm Collection (ecofibre.com.au and anandahemp.com) was used to examine alkyl cannabinoid variation across vegetative, flowering and maturation stages. A wide range of di-/tri-cyclic as well as C3-/C5-alkyl cannabinoid composition was observed between plants. Chemotype at the vegetative and flowering stages was found to be predictive of chemotype at maturation, indicating a low level of plasticity in cannabinoid composition. Chemometric cluster analysis based on composition data from all three developmental stages categorized alkyl cannabinoid chemotypes into three classes. Our results suggest that more extensive chemical and genetic characterization of the Cannabis genepool could facilitate the metabolic engineering of alkyl cannabinoid chemotypes

Myocardial fibrosis as an early manifestation of hypertrophic cardiomyopathy

BACKGROUND: Myocardial fibrosis is a hallmark of hypertrophic cardiomyopathy and a proposed substrate for arrhythmias and heart failure. In animal models, profibrotic genetic pathways are activated early, before hypertrophic remodeling. Data showing early profibrotic responses to sarcomere-gene mutations in patients with hypertrophic cardiomyopathy are lacking. METHODS: We used echocardiography, cardiac magnetic resonance imaging (MRI), and serum biomarkers of collagen metabolism, hemodynamic stress, and myocardial injury to evaluate subjects with hypertrophic cardiomyopathy and a confirmed genotype. RESULTS: The study involved 38 subjects with pathogenic sarcomere mutations and overt hypertrophic cardiomyopathy, 39 subjects with mutations but no left ventricular hypertrophy, and 30 controls who did not have mutations. Levels of serum C-terminal propeptide of type I procollagen (PICP) were significantly higher in mutation carriers without left ventricular hypertrophy and in subjects with overt hypertrophic cardiomyopathy than in controls (31% and 69% higher, respectively; P<0.001). The ratio of PICP to C-terminal telopeptide of type I collagen was increased only in subjects with overt hypertrophic cardiomyopathy, suggesting that collagen synthesis exceeds degradation. Cardiac MRI studies showed late gadolinium enhancement, indicating myocardial fibrosis, in 71% of subjects with overt hypertrophic cardiomyopathy but in none of the mutation carriers without left ventricular hypertrophy. CONCLUSIONS: Elevated levels of serum PICP indicated increased myocardial collagen synthesis in sarcomere-mutation carriers without overt disease. This profibrotic state preceded the development of left ventricular hypertrophy or fibrosis visible on MRI. (Funded by the National Institutes of Health and others.

A method for comparing multiple imputation techniques: A case study on the U.S. national COVID cohort collaborative.

Healthcare datasets obtained from Electronic Health Records have proven to be extremely useful for assessing associations between patients’ predictors and outcomes of interest. However, these datasets often suffer from missing values in a high proportion of cases, whose removal may introduce severe bias. Several multiple imputation algorithms have been proposed to attempt to recover the missing information under an assumed missingness mechanism. Each algorithm presents strengths and weaknesses, and there is currently no consensus on which multiple imputation algorithm works best in a given scenario. Furthermore, the selection of each algorithm’s pa- rameters and data-related modeling choices are also both crucial and challenging

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts