Project description:Comparison of transcriptome in adult control F344 kidneys versus Hsd11b2 knockout kidneys (model of syndrome of apparent mineralocorticoid excess; SAME)
Project description:SMARCD1, an auxiliary subunit of the SWI/SNF complex is essential for maintaining embryonic stem cell pluripotency and metabolic gene regulation. Here, we have tried to address the role of SMARCD1 in myeloid differentiation and leukemia. To address this, we transduced U937 cells with lentiviral particles expressing shcontrol and shSMARCD1. Next we subjected these cells to 30 nM vitamin D3 induction for 48 h. RNA-sequencing was conducted for shcontrol and shSMARCD1 in uninduced and vitamin D3 induced U937 cells. High quality RNA with RIN values above 9, was used to generate library using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760) following manufacturer’s instructions. Finally barcoded libraries were pooled, and a final concentration of 1.2 pM was loaded using NextSeq 500/550 High Output Kit v2 150 Cycles (15057931, Illumia) and paired-end sequenced in a Nextseq 550 (Illumina).
Project description:Description: SMARCD1, an auxiliary subunit of the SWI/SNF complex is essential for maintaining embryonic stem cell pluripotency and metabolic gene regulation. Here, we have tried to address the role of SMARCD1 in myeloid differentiation and leukemia. To address this, we transduced HL60 cells with lentiviral particles expressing shcontrol and shSMARCD1. Next we subjected these cells to 10 nM vitamin D3 induction for 48 h. RNA-sequencing was conducted for shcontrol and shSMARCD1 in uninduced and vitamin D3 induced HL-60 cells. High quality RNA with RIN values above 9, was used to generate library using TruSeq™ stranded mRNA kit (20020595, Illumina) following manufacturer’s instructions. Finally barcoded libraries were pooled, and a final concentration of 1.2 pM was loaded using NextSeq 500/550 High Output Kit v2 150 Cycles (15057931, Illumia) and paired-end sequenced in a Nextseq 550 (Illumina).
Project description:To identify candidates of interest that were more highly expressed in BAT than WAT, we conducted RNAseq in human primary brown and white adipocytes. Adipose tissue was obtained from the central compartment of the neck, superior to the clavicle and deep to the lateral thyroid lobe either adjacent to the longus colli muscle or to the oesophagus (brown adipose tissue) and more superficially from the subcutaneous neck tissue (white adipose tissue). The stromal vascular fraction was isolated and cultured as described (Ramage, Akyol et al. 2016 doi: 10.1016/j.cmet.2016.06.011). Following differentiation, cells were cultured in serum-stripped medium for 48 hours prior to RNA extraction and subsequent bulk RNA-seq.
Project description:RNAseq of enterocytes of the jejunum and Ileum from transgene C57BL/6 mice to assess the effects of a CKIα knock-out in combination with expression of a p53 mutant protein (R172H ) compared to a double knock-out of CKIα and p53. In addition, a group of mice exhibiting a CKIα knock-out and mutant p53 (R172H) was treated with gallic acid and compared to untreated mice (Jejunum only).
Project description:We reported previously that chronic treatment with the Cyclooxygenase-2 inhibitor, rofecoxib, increased acute mortality in rats exposed to ischemia/reperfusion injury (I/R). This manifestation of hidden cardiotoxicity was attributed to the proarrhythmic effect of the drug on the ischemic heart. However, rofecoxib also had beneficial effects on ischemic injury, manifesting as decreased infarct size. In the present study, we aimed to identify molecular changes caused by chronic rofecoxib treatment in the heart. Rats were treated with 5.12 mg/kg rofecoxib or its vehicle for four weeks. Messenger RNA (mRNA), microRNA (miRNA) deep sequencing data, and proteomic datasets of left ventricular tissue samples were used for an unbiased differential expression analysis followed by in silico molecular network analysis and experimental target validation. Using mass spectrometry and filtering criteria, 26 proteins were identified that exhibited pronounced changes in protein expression or phosphorylation due to chronic rofecoxib treatment. The transcriptomic analysis showed mild alterations in the heart´s mRNA- and miRNA expression. The posttranscriptional regulation of mRNAs by miRNAs did not result in differential protein expression. This is the first demonstration that chronic rofecoxib treatment affects posttranslational modification and expression of several proteins in the heart. These are potential off-target effects that could account for the hidden cardiotoxic and/or cardioprotective effects of rofecoxib.
Project description:We reported previously that chronic treatment with the Cyclooxygenase-2 inhibitor, rofecoxib, increased acute mortality in rats exposed to ischemia/reperfusion injury (I/R). This manifestation of hidden cardiotoxicity was attributed to the proarrhythmic effect of the drug on the ischemic heart. However, rofecoxib also had beneficial effects on ischemic injury, manifesting as decreased infarct size. In the present study, we aimed to identify molecular changes caused by chronic rofecoxib treatment in the heart. Rats were treated with 5.12 mg/kg rofecoxib or its vehicle for four weeks. Messenger RNA (mRNA), microRNA (miRNA) deep sequencing data, and proteomic datasets of left ventricular tissue samples were used for an unbiased differential expression analysis followed by in silico molecular network analysis and experimental target validation. Using mass spectrometry and filtering criteria, 26 proteins were identified that exhibited pronounced changes in protein expression or phosphorylation due to chronic rofecoxib treatment. The transcriptomic analysis showed mild alterations in the heart´s mRNA- and miRNA expression. The posttranscriptional regulation of mRNAs by miRNAs did not result in differential protein expression. This is the first demonstration that chronic rofecoxib treatment affects posttranslational modification and expression of several proteins in the heart. These are potential off-target effects that could account for the hidden cardiotoxic and/or cardioprotective effects of rofecoxib.
Project description:Cornelia de Lange syndrome (CdLS) is a rare genetic disease associated with cohesinopathy. A novel iPSC line was generated from the CdLS patient carrying a heterozygous missense point-mutation of the NIPBL gene. iPSC lines prepared from the healthy parents and the mutation-corrected isogenic cell lines are used as the respective controls. Upon differentiation into hepatocytes, the patient-derived iPSC demonstrate the capacity to express hepatocyte-specific marker transcripts and the respective proteins, however, the efficiency of differentiation is significantly inferior to those of the respective controls, demonstrated by single-cell RNA sequencing and immune-fluorescent analyses. Global change of transcriptome in the patient-derived iPSC relative to the control cells is associated with altered chromatin accessibility, assessed by RNAseq combined with ATACseq analysis. Differentially down-regulated genes in the patient-derived iPSC, in particular, are those coding for proteins related to neural differentiation and transcription factors, while up-regulated genes contain some of antisense RNA coding genes, pseudo genes and long non-coding RNAs. Some of the differentially regulated genes are consistent with the altered chromatin accessibility and this observation is consistent during hepatocyte differentiation. Thus, the mutation in the NIPBL gene of the CdLS patient could be responsible for defects of differentiation primarily due to alteration of chromatin accessibility.
Project description:This study addresses this gap by conducting a direct comparison of eight platforms, representing both affinity-based and diverse mass spectrometry approaches, and covering over 13,000 proteins. By applying these platforms to the same cohort, we systematically assess their performance, identifying key differences and complementary strengths. Our findings offer valuable insights for researchers, highlighting trade-offs in coverage and their implications for biomarker discovery and clinical applications. This study serves as an essential resource, offering both technical evaluation and biological insights to support the development of novel diagnostics and therapeutics through plasma proteomics.
Project description:This study addresses this gap by conducting a direct comparison of eight platforms, representing both affinity-based and diverse mass spectrometry approaches, and covering over 13,000 proteins. By applying these platforms to the same cohort, we systematically assess their performance, identifying key differences and complementary strengths. Our findings offer valuable insights for researchers, highlighting trade-offs in coverage and their implications for biomarker discovery and clinical applications. This study serves as an essential resource, offering both technical evaluation and biological insights to support the development of novel diagnostics and therapeutics through plasma proteomics.