Project description:A filtration method followed by microarray analyses allows PDE components to be identified in brain microvessels, and confirmed that PDE4D and PDE5A are the primary forms expressed in rat brain microvessels. Adult male F344 rats were sacrificed and blocks of the cerebral cortex and infratentorial areas were dissected. Microvessels were isolated using a filtration method, and total RNA was extracted. Two microarrays using four sample RNAs (microvessels from the cerebral cortex vs. microvessels from the infratentorial block) were included in the present study. Microarrays demonstrated that there were 16 PDE transcripts in the PDE superfamily, exhibiting quantifiable density in the microvessels.
Project description:The serine hydrolase (SH) superfamily is perhaps, one of the largest functional enzyme classes in all forms of life, and consists of proteases/peptidases, lipases, and carboxylesterases as representative members. Consistent with the name of this superfamily, all members, without any exception to date, use a nucleophilic serine residue in the enzyme active site to perform hydrolytic-type reactions via a two-step ping-pong mechanism involving a covalent enzyme intermediate. Given the highly conserved catalytic mechanism, this superfamily has served as a classical prototype in the development of several platforms of the chemical proteomics technique, activity-based protein profiling (ABPP), to globally interrogate the functions of its different members in various native, yet complex, biological settings. While ABPP-based proteome-wide activity atlas’ for SH activities are available in numerous organisms, including humans, to the best of our knowledge, such an analysis for this superfamily is lacking in any insect model. To address this, here, we first report a bioinformatics analysis towards the identification and classification of non-redundant SHs in Drosophila melanogaster. Following up on this in silico analysis, leveraging discovery chemoproteomics, we identify and globally map the activities of the SH superfamily during various developmental stages and in different adult tissues of Drosophila. Finally, as proof of concept of the utility of this activity atlas, we highlight sexual dimorphism in SH activities across different tissues in adult Drosophila melanogaster, and together, we prospect new research directions, resources and tools that this study can provide to the fly community.
Project description:A stable phylogenomic classification of Travunioidea (Arachnida, Opiliones, Laniatores) based on sequence capture of ultraconserved elements
Project description:Transporters mediate and control the flux of molecules across compartmental membranes. The human genome encodes 1500 genes with transport functions, of which the solute carriers (SLCs) form the largest superfamily with more than 450 members. Over 250 different SLCs are expressed in a typical human cell, many exhibiting overlapping expression patterns and substrate specificities. The collective role of these often seemingly redundant transporters in defining cellular outcomes, such as cell survival, remains unclear. Here, we performed pooled combinatorial KO screens to identify genetic interactions between 258 expressed SLCs, and between a subset of SLCs and selected metabolic enzymes under different growth conditions using both CRISPR-Cas12a and -Cas9 double knockout systems in the colorectal carcinoma cell line HCT116.
Project description:In mammals, members of the transforming growth factor-beta (TGF-beta) superfamily are known to have key roles in the regulation of follicular growth and development. The aim of the study was to evaluate the expression of TGF-beta superfamily growth factors, their receptors and downstream SMAD signalling molecules during early human folliculogenesis. Human preantral follicles were enzymatically isolated from surplus ovarian tissue obtained from women having ovarian cortical tissue frozen for fertility preservation. A total of 348 human preantral follicles, ranging from 40 to 200 um in diameter, were isolated from ovarian tissue obtained from 15 women, aged 24M-^V34 years. Isolated preantral follicles were grouped according to diameter in five size-matched populations spanning the primordial, primary and secondary stage follicles, and analyzed by whole-genome microarray analysis. Selected proteins/genes were analysed by immunocytochemistry and quantitative RT-PCR. TGF-beta superfamily genes with overall highest mRNA expressions levels included growth differentiation factors 9 (GDF9), bone morphogenic protein-15 (BMP15), BMP6, BMP-receptor-2 (BMPR2), anti-MM-|llerian hormone receptor 2 (AMHR2), TGFbetaR3, inhibin-alpha (INHA), and intracellular SMAD3 and SMAD4. Moreover, genes which were differentially expressed from the primordial to the late secondary stage follicles included GDF9 (p<0.01), BMP15 (p<0.001), AMH (p<0.0001), INHBB (p<0.05), TGFbetaR3 (p<0.05) and SMAD4 (p<0.05). Collectively, these data indicate that the active TGF-beta superfamily pathways in early human folliculogenesis consist of primarily GDF9 combined with synergistic effects of BMP15 through the BMPR2 and intracellular activation of SMAD3 and SMAD4, and that AMH and Inhibin B are engaged in intrafollicular events from the onset of follicular growth.
Project description:To identify more targets in soybean, particularly specific targets of Cd-stress-responsive miRNAs, high-throughput degradome sequencing was used. In total, we obtained 8913111 raw reads from the library which was constructed from a mixture of four samples (HX3-CK, HX3-Cd-treatment, ZH24-CK and ZH24-Cd-treatment). After removing the reads without the CAGAG adaptor, 5430126 unique raw-reads were obtained. The unique sequences were aligned to the G. max genome database, and 6516276 reads were mapped to the genome. The mapped reads from the libraries represented 51481 annotated G. max genes.