Two New Dihydrosphingosine Analogs Against Mycobacterium tuberculosis Affect gltA1, lprQ, and rpsO Expression

The emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis strains threaten the control of tuberculosis. New antitubercular dihydrosphingosine analogs, named UCIs, have been evaluated in preclinical studies but their cellular and molecular mechanisms of action against M. tuberculosis are still unknown. The aim of this study was to evaluate the effect of UCI exposure on gene expression of drug-sensitive H37Rv and MDR CIBIN:UMF:15:99 clones of M. tuberculosis which were isolated, phenotypically, and genetically characterized, cultured to log phase and treated with UCI compounds; followed by total RNA isolation, reverse transcription and hybridization assays on Affymetrix genomic microarrays. Data were validated with RT-qPCR assays. As results, UCI-05 and UCI-14 exposure increased gltA1 expression in drug-sensitive H37Rv clones. Furthermore, UCI-05 increased lprQ expression in MDR CIBIN:UMF:15:99 M. tuberculosis clones while UCI-14 reduced the expression of this gene in drug-sensitive H37Rv clones. In addition, UCI-05 reduced rpsO expression in drug-sensitive H37Rv clones. We found gene expression alterations that suggest these molecules may alter carbon and lipid metabolism as well as interfere in the protein-producing machinery in M. tuberculosis.

PSXII-23 Sirtuins expression and activity in bovine granulosa cells are regulated by hormones that influence ovarian steroidogenesis

Sirtuins (SIRTs) are a family of seven NAD+-dependent histone deacetylases that regulate several biological reactions. How SIRTs regulate ovarian steroidogenesis in cattle remains to be fully unveiled. We hypothesize that SIRTs expression and activity are regulated by hormones that influence steroidogenesis in bovine granulosa cells (GC). Bovine ovaries were collected at an USDA-inspected commercial slaughterhouse and GC were isolated from small antral follicles (1–5 mm on surface diameter). Cells were treated with hormones that regulate ovarian folliculogenesis: FSH, IGF1, fibroblast growth factor (FGF) 2, FGF9, and their combinations. Cells were cultured for 24h for total RNA isolation (n = 6 pools) with miRNeasy microkit (Qiagen) or for 48h for isolation of nuclear and cytoplasmic extracts (n = 3 pools) with EpiQuik Nuclear Extraction Kit (Epigentek) according to the manufacturers’ instructions. Relative mRNA abundance was quantified via qPCR and expressed as 2-ΔΔCt using the relative comparative threshold cycle (Ct) whereas SIRTs activity in nuclear (SIRTs 1, 6, and 7) and cytoplasmic (SIRTs 2, 4, and 5) extracts was analyzed with the Epigenase Universal SIRT Activity/Inhibition assay kit (Epigentek) following the manufacturer’s instructions. Data were analyzed via ANOVA with GLM procedures of SAS for Windows. In terms of mRNA relative abundance, FSH+IGF1+IGF9 increased mRNA relative expression of SIRTs 2 to 7 in comparison to negative control and of SIRTs 2, 3, 4, 6, and 7 in comparison to FSH+IGF1; FSH+IGF1+IGF2 increased mRNA relative abundance of SIRTs 2 and 6 in comparison to FSH+IGF1; FGF2 alone increased SIRT1 in comparison to negative control (P < 0.05). In term of SIRTs activity, FGF2 alone increased nuclear SIRTs activity in comparison to FSH, IGF1, FSH+IGF1, and FGF9 alone; FSH+IGF1+IGF2 increased cytoplasmic SIRTs activity in comparison to all treatments (P < 0.05). Taken together, our data demonstrate that SIRTs expression and activity in bovine GC are regulated by hormones that influence steroidogenesis.

Development of new total RNA isolation method for tissues with rich phenolic compounds

BackgroundRNAs to be used in transcriptome analysis must be of high quality and pure in order to ensure maximum representation of the expressed genes. RNA isolation is difficult in hazelnut tissues containing large amounts of secondary metabolite, phenolic compounds and the cell wall structure. Commonly used protocols for RNA isolation are those that require a lot of labor and time and also do not allow sufficient RNA isolation when applied to tissues rich in phenolic compounds. This study was aimed to develop an efficient method for isolation of total RNAs from bud of hazelnut to be used in RNA sequencing.Materials and methodsAn optimized new method was successfully applied on three different hazelnuts genotypes (Çakıldak, Palaz, Tombul) and about 25 times higher amount of total RNAs per mg fresh tissues were obtained compared to classical CTAB method. Different methods have been tried for the isolation of RNA from hazelnut tissues and the determination of the quality of the obtained RNAs.ResultsThe quality and quantity of isolalated total RNAs were determined by spectrophotometer, electrophoresis and PCR. This success has been caught without any compromise of purity since A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were >2.0 in all purified RNAs.ConclusionThe total RNAs isolated with new protocol was found to be suitable for RNA sequencing and other molecular applications.