Project description:Cryptococcus neoformans is a fungal pathogen responsible for an increased mortality among immunocompromised individuals. Long antifungal therapies to treat cryptococcal infections have compounded the occurrence of resistant strains that threaten the efficacy of current treatments. In this senseDue to resistance mechanisms, discovery of compounds that inhibit virulence factors, rather than kill the fungus, have emerged as potential new strategies to combat infection and reduce the rate of resistance due to lower selective pressure. Invertebrates rely solely on an effective innate immune system to prevent infections, provide providing a potential one health approach for discovery of novel antifungal and antibacterial compounds. Here, we demonstrate a differentiated extraction of proteins from three mollusks (freshwater and terrestrial) and evaluate extract their effects against the growth and virulence factor production (thermotolerance, melanin, capsule, and biofilm) in C. neoformans. We show that clarified extracts of Planorbella pilsbryi have a fungicidal effect on cryptococcal cells in a comparable way to Fluconazolefluconazole. Similarly, crude and clarifiedall extracts of Cipangopaludina chinensis not only affects cryptococcal thermotolerance but also impairs biofilm and capsule production. In addition, incubation of C. neoformans with clarified extracts of Cepaea nemoralis extracts reduced capsule production. Using inhibitory activity of extracts against peptidases related with virulence factors and Quantitative quantitative proteomics arose distinct proteome signatures for each extract and proposed proteins driving the observed anti-virulence properties. Overall, this work demonstratesproves the potential of compounds derived from natural sources to inhibit virulence factor production in a clinically important fungal pathogen.
Project description:We investigate the molecular targets and pathways that the neem extracts and the associated compounds act through, to bring about tumor suppression by using a genome-wide functional pooled shRNA screen on head and neck squamous cell carcinoma cell line treated with crude neem leaf extracts. We analyzed differences in global clonal sizes of the shRNA-infected cells cultured under no treatment and treatment with neem leaf extract conditions, assayed using next-generation sequencing. We further analyzed differences in gene expression in HSC-4 cells, upon treatment with neem leaf extract in a time-dependent manner, followed by a time-dependent rescue. Our results indicate that neem extract simultaneously affects various important molecular signaling pathways in head and neck cancer cells, some (TGF-β/HSF-1) of which may be therapeutic targets for this devastating tumor.
Project description:To understand how GIPC3 exerts its effects on cuticular plate dimensions, we examined the GIPC3 protein-interaction network in hair cells. We immunoaffinity purified GIPC3 from crosslinked chicken inner ear extracts that were enriched for stereocilia, but still contain large amounts of hair-cell cytoplasmic proteins (Morgan et al., 2016). We carried out two separate experiments, each with ~1000 chicken ears, where we stabilized protein complexes using primary amine-reactive homo-bifunctional N-hydroxysuccimide ester crosslinkers that are thiol-cleavable and hence reversible (Mattson et al., 1993). In one experiment, we used dithiobis(succinimidyl propionate) (DSP), a membrane-permeable crosslinker that crosslinks extracellular and intracellular complexes; in the other experiment, we used 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), which is membrane impermeant and thus only stabilizes extracellular and transmembrane complexes. We prepared soluble extracts of crude, crosslinked stereocilia and used these fractions for identifying GIPC3-interacting proteins.
Project description:We perform RNA-Seq to analyze gene expression profiles in HeLa TAF7 (WT) cytoplasmic extracts, which are the inputs of RIP-Seq and PAR-CLIP.
Project description:Members of the Caryophyllaceae Juss. family possess anabolic, adaptogenic, radioprotective, antitumoral and hemorheological properties and have been used in medical practice for the treatment of various inflammatory disorders. Macrophages are key immune cells that coordinate the inflammatory reaction. To identify the effects of Silene sendtneri and Silene roemeri on macrophage activation, primary human macrophages were cultured with extracts of these plants .We used microarrays to determine the global expression pattern induced by the extracts in macrophages.
Project description:Background: RNA interference (RNAi) is an indispensable regulatory mechanism governing developmental processes and stress responses via sequence-specific control of target RNAs mediated by the action of small, 20–24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs. Biogenesis and sorting of miRNAs into ARGONAUTE (AGO) proteins are intensively investigated, however very few information is available about the presence and distribution of distinct sRNA pools in plant cells. Results: High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the canonical miRNAs were associated with high molecular weight RNA-induced silencing complexes co-migrating with AGO1 (HMW RISC). In contrast, the majority of 24-nt-long siRNAs were found in association with low molecular weight complexes co-migrating with AGO4 (LMW RISC). Intriguingly, we identified a large set of sRNAs in the cytoplasm, including mature miRNA sequences, in the low molecular size range corresponding to protein-unbound sRNAs. By comparing the RISC-loaded and protein-unbound pools of miRNAs, we identified miRNAs with highly different loading efficiencies. Investigation of some selected miRNAs in a transient expression system validated this finding. We also showed that the availability of RISCs is a limiting factor determining the loading efficiency of miRNAs. Conclusion: Our data reveal the existence of a regulatory checkpoint, likely controlled by information carried by the diverse miRNA precursors, determining the RISC-loading efficiencies of various miRNAs by sorting only a subset of the produced miRNAs into the biologically active RISCs.