Project description:RNA-Sequencing performed on 177 honey bee whole-brains, divided into "soldier" and "forager" groups from Puerto Rican honey bee colonies.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Viral strains, age, and host factors including genetics and proteins are associated with variable immune responses against SARS-CoV-2 and disease severity. We hypothesized that unique proteins/pathways are associated with COVID-19 disease severity in Puerto Rican Hispanics. A total of 121 men and women aged 21-80 years-old were recruited in Puerto Rico. Plasma samples were collected from unvaccinated COVID-19 infected subjects during acute disease (n=39) and compared to COVID-19 negative individuals (n=56) during acute disease using proteomics and cytokine expression. Infected individuals were stratified based on symptomatology as follows: mild (n=18), moderate (n=13), and severe (n=8). Quantitative proteomics was performed in plasma samples using Tandem Mass Tag (TMT) labeling. Cytokines in plasma were quantified using a human cytokine array. Proteomics analyses revealed 56 differentially regulated proteins and the top 3 pathways that were predicted to be inhibited in severe patients including LXR/RXR signaling, Production of NO and ROS in macrophages, and Synaptogenesis signaling. Decreased cadherin-13 validated by ELISA, which participates in synaptogenesis, is a novel protein is a novel protein not previously reported in other studies of COVID-19 severity and validated by ELISA. Cytokine analyses showed that TNF⍺ levels decreased with disease severity. This study uncovers potential host predictors of COVID-19 severity and new avenues for treatment in Puerto Rican Hispanics.