RTCpredictor: identification of read-through chimeric RNAs from RNA sequencing data.

Read-through chimeric RNAs are being recognized as a means to expand the functional transcriptome and contribute to cancer tumorigenesis when mis-regulated. However, current software tools often fail to predict them. We have developed RTCpredictor, utilizing a fast ripgrep tool to search for all possible exon-exon combinations of parental gene pairs. We also added exonic variants allowing searches containing common SNPs. To our knowledge, it is the first read-through chimeric RNA specific prediction method that also provides breakpoint coordinates. Compared with 10 other popular tools, RTCpredictor achieved high sensitivity on a simulated and three real datasets. In addition, RTCpredictor has less memory requirements and faster execution time, making it ideal for applying on large datasets.

Essential role for EGFR tyrosine kinase and ER stress in myocardial infarction in type 2 diabetes.

We previously reported that EGFR tyrosine kinase (EGFRtk) activity and endoplasmic reticulum (ER) stress are enhanced in type 2 diabetic (T2D) mice and cause vascular dysfunction. In the present study, we determined the in vivo contribution of EGFRtk and ER stress in acute myocardial infarction induced by acute ischemia (40 min)-reperfusion (24 h) (I/R) injury in T2D (db-/db-) mice. We treated db-/db- mice with EGFRtk inhibitor (AG1478, 10 mg/kg/day) for 2 weeks. Mice were then subjected to myocardial I/R injury. The db-/db- mice developed a significant infarct after I/R injury. The inhibition of EGFRtk significantly reduced the infarct size and ER stress induction. We also determined that the inhibition of ER stress (tauroursodeoxycholic acid, TUDCA, 150 mg/kg per day) in db-/db- significantly decrease the infarct size indicating that ER stress is a downstream mechanism to EGFRtk. Moreover, AG1478 and TUDCA reduced myocardium p38 and ERK1/2 MAP-kinases activity, and increased the activity of the pro-survival signaling cascade Akt. Additionally, the inhibition of EGFRtk and ER stress reduced cell apoptosis and the inflammation as indicated by the reduction in macrophages and neutrophil infiltration. We determined for the first time that the inhibition of EGFRtk protects T2D heart against I/R injury through ER stress-dependent mechanism. The cardioprotective effect of EGFRtk and ER stress inhibition involves the activation of survival pathway, and inhibition of apoptosis, and inflammation. Thus, targeting EGFRtk and ER stress has the potential for therapy to overcome myocardial infarction in T2D.

Essential Role of IL-12 in Angiogenesis in Type 2 Diabetes.

Recently, IL-12 emerged as a critical player in type 2 diabetes complications. We previously reported that ischemia-induced angiogenesis is compromised in type 2 diabetic mice. In this study, we determined that IL-12 disruption rescued angiogenesis and arteriogenesis in type 2 diabetic mice. To induce type 2 diabetes, wild-type (WT), p40IL-12-/- (p40-/-), and p35IL-12-/- (p35-/-) mice were fed a high-fat diet (HFD) for 12 weeks. Body weight, glucose test tolerance, and insulin test tolerance were assessed. After 12 weeks of an HFD, the femoral artery was ligated and blood flow recovery was measured every week for 4 weeks. WT, p40-/-, and p35-/- mice fed an HFD become obese after 12 weeks and exhibit glucose intolerance and insulin resistance. Blood flow recovery was fully restored in 2 to 3 weeks after femoral artery ligation in all groups of mice fed a normal diet. However, after 12 weeks of an HFD, blood flow recovery was compromised in WT mice, whereas it was fully recovered in p40-/- and p35-/- mice. The mechanism of blood flow recovery involves an increase in capillary/arteriole density, endothelial nitric oxide synthase/Akt/vascular endothelial growth factor receptor 2 signaling, and a reduction in oxidative stress and inflammation. The disruption of IL-12 promotes angiogenesis and increases blood flow recovery in obese type 2 diabetic mice by an endothelial nitric oxide synthase/Akt/vascular endothelial growth factor receptor 2/oxidative stress-inflammation-dependent mechanism.

Essential Role of Smooth Muscle STIM1 in Hypertension and Cardiovascular Dysfunction.

OBJECTIVES: Chronic hypertension is the most critical risk factor for cardiovascular disease, heart failure, and stroke. APPROACH AND RESULTS: Here we show that wild-type mice infused with angiotensin II develop hypertension, cardiac hypertrophy, perivascular fibrosis, and endothelial dysfunction with enhanced stromal interaction molecule 1 (STIM1) expression in heart and vessels. All these pathologies were significantly blunted in mice lacking STIM1 specifically in smooth muscle (Stim1(SMC-/-)). Mechanistically, STIM1 upregulation during angiotensin II-induced hypertension was associated with enhanced endoplasmic reticulum stress, and smooth muscle STIM1 was required for endoplasmic reticulum stress-induced vascular dysfunction through transforming growth factor-ß and nicotinamide adenine dinucleotide phosphate oxidase-dependent pathways. Accordingly, knockout mice for the endoplasmic reticulum stress proapoptotic transcriptional factor, CCAAT-enhancer-binding protein homologous protein (CHOP(-/-)), were resistant to hypertension-induced cardiovascular pathologies. Wild-type mice infused with angiotensin II, but not Stim1(SMC-/-) or CHOP(-/-) mice showed elevated vascular nicotinamide adenine dinucleotide phosphate oxidase activity and reduced phosphorylated endothelial nitric oxide synthase, cGMP, and nitrite levels. CONCLUSIONS: Thus, smooth muscle STIM1 plays a crucial role in the development of hypertension and associated cardiovascular pathologies and represents a promising target for cardiovascular therapy.

Landscape of Chimeric RNAs in Non-Cancerous Cells.

Gene fusions and their products (RNA and protein) have been traditionally recognized as unique features of cancer cells and are used as ideal biomarkers and drug targets for multiple cancer types. However, recent studies have demonstrated that chimeric RNAs generated by intergenic alternative splicing can also be found in normal cells and tissues. In this study, we aim to identify chimeric RNAs in different non-neoplastic cell lines and investigate the landscape and expression of these novel candidate chimeric RNAs. To do so, we used HEK-293T, HUVEC, and LO2 cell lines as models, performed paired-end RNA sequencing, and conducted analyses for chimeric RNA profiles. Several filtering criteria were applied, and the landscape of chimeric RNAs was characterized at multiple levels and from various angles. Further, we experimentally validated 17 chimeric RNAs from different classifications. Finally, we examined a number of validated chimeric RNAs in different cancer and non-cancer cells, including blood from healthy donors, and demonstrated their ubiquitous expression pattern.

Systems genetics in diversity outbred mice inform BMD GWAS and identify determinants of bone strength.

Genome-wide association studies (GWASs) for osteoporotic traits have identified over 1000 associations; however, their impact has been limited by the difficulties of causal gene identification and a strict focus on bone mineral density (BMD). Here, we use Diversity Outbred (DO) mice to directly address these limitations by performing a systems genetics analysis of 55 complex skeletal phenotypes. We apply a network approach to cortical bone RNA-seq data to discover 66 genes likely to be causal for human BMD GWAS associations, including the genes SERTAD4 and GLT8D2. We also perform GWAS in the DO for a wide-range of bone traits and identify Qsox1 as a gene influencing cortical bone accrual and bone strength. In this work, we advance our understanding of the genetics of osteoporosis and highlight the ability of the mouse to inform human genetics.

Targeted detection and quantitation of histone modifications from 1,000 cells.

Histone post-translational modifications (PTMs) create a powerful regulatory mechanism for maintaining chromosomal integrity in cells. Histone acetylation and methylation, the most widely studied histone PTMs, act in concert with chromatin-associated proteins to control access to genetic information during transcription. Alterations in cellular histone PTMs have been linked to disease states and have crucial biomarker and therapeutic potential. Traditional bottom-up mass spectrometry of histones requires large numbers of cells, typically one million or more. However, for some cell subtype-specific studies, it is difficult or impossible to obtain such large numbers of cells and quantification of rare histone PTMs is often unachievable. An established targeted LC-MS/MS method was used to quantify the abundance of histone PTMs from cell lines and primary human specimens. Sample preparation was modified by omitting nuclear isolation and reducing the rounds of histone derivatization to improve detection of histone peptides down to 1,000 cells. In the current study, we developed and validated a quantitative LC-MS/MS approach tailored for a targeted histone assay of 75 histone peptides with as few as 10,000 cells. Furthermore, we were able to detect and quantify 61 histone peptides from just 1,000 primary human stem cells. Detection of 37 histone peptides was possible from 1,000 acute myeloid leukemia patient cells. We anticipate that this revised method can be used in many applications where achieving large cell numbers is challenging, including rare human cell populations.

Treg cells depletion is a mechanism that drives microvascular dysfunction in mice with established hypertension.

BACKGROUND: Microvascular dysfunction is a major complication in hypertensive patients. We previously reported that CD4+CD25+ T regulatory cells (Treg) play an important preventive role in hypertension-induced vascular dysfunction. However, whether Treg cells therapy and autophagy inhibition could rescue Treg cells survival and microvascular function in established hypertension is an important question that remained unanswered. METHODS & RESULTS: Here we showed that Treg cells from mice model of established hypertension displayed an enhanced apoptotic rate, which was rescued with Treg cells transfer and autophagy inhibition. We also showed increased autophagy in mesenteric resistance artery (MRA) in mice with established hypertension. Importantly, the inhibition of autophagy or one single transfer of Treg cells into mice with established hypertension improved the microvascular function independently of high blood pressure. The protection involves the modulation of interleukin-10 (IL-10), inflammation, endoplasmic reticulum (ER) stress, oxidative stress, Akt, and eNOS. CONCLUSIONS: The present study suggests that Treg cells survival is regulated by autophagy. Also, Treg cells as a cellular therapy aimed at rescuing the microvascular function through an autophagy-dependent mechanism and independently of arterial blood pressure lowering effects. Because our mouse model of established hypertension mimics the clinical situation, our results have the potential for new therapeutic approaches that involve the manipulation of Treg cells and autophagy to overcome established hypertension-induced cardiovascular complications.

Essential role for smooth muscle cell stromal interaction molecule-1 in myocardial infarction.

OBJECTIVES: Stromal interacting molecule-1 (STIM1) plays a role in coordinating calcium signaling in different cell types. The increase or deletion of STIM1 expression in cardiomyocyte causes cardiac complication. Moreover, the deletion of STIM1 in endothelial cell causes vascular endothelial dysfunction. However, the disruption of STIM1 in smooth muscle cells (SMC) has no effect on endothelial function but protects vascular function when mice are infused with angiotensin-II. Nevertheless, the role of SMC-STIM1 in acute and chronic myocardial infarction (MI) induced by acute ischemia-reperfusion injury and permanent coronary artery occlusion is unknown. METHODS AND RESULTS: Stim1 were generated and crossed into the SM22α-Cre backgrounds. SM22α-Cre causes deletion of STIM1 floxed genes in adult SMC (Stim1). Control and Stim1 mice were subjected to acute ischemia-reperfusion injury. Hearts were then harvested and incubated with triphenyltetrazolium chloride to determine the infarct size. In control mice which are subjected to ischemia-reperfusion, the heart developed a significant infarct associated with an increase in STIM1 expression. Interestingly, the infarct size was substantially reduced in Stim1 mice. The protection in Stim1 mice against ischemia-reperfusion injury involves the modulation of endoplasmic reticulum stress, apoptosis, oxidative stress, protein kinase B, and mitogen-activated protein (MAP) kinase (ERK1/2 and p38) signaling, and inflammation. Furthermore, in another model of chronic MI induced by permanent coronary artery occlusion, SMC-STIM1 disruption significantly reduced myocardial infarct size and improved cardiac function. CONCLUSION: Our results provide new evidence that SMC-STIM1 disruption is a novel mechanism that protects the heart from MI through reduction of endoplasmic reticulum stress, oxidative stress, MAP-Kinase, apoptosis, and inflammation.