Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

Background: How immune-tolerance is lost to pancreatic β-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin ER chaperone glucose-regulated protein (GRP) 94 leads to mishandling of proinsulin, ER stress and activation of the inducible proteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered β-cell MHC-I bound peptidome and inflammasome activation, sensitizing β-cells to immune attack. Methods: We used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20μM), or exposed to proinflammatory cytokines interleukin (IL)-1β or IFNγ (15 pg/ml and 10 ng/ml, respectively) for 24 hours. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse phase high performance liquid chromatography (RP-HPLC) followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). NALP1, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and (pro) IL-1β expression and secretion were investigated by Western blotting. Results: GRP94 KO increased RT1.A expression in β-cells as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptides diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NALP1 expression was upregulated in GRP94 deficient cells along with decreased (IκBα) content while proIL-1β cellular levels declined, coupled with an increased secretion of mature IL-1β. Our results suggest that limiting β-cell proinsulin chaperoning enhances RT1.A expression, alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress impeding proinsulin handling may sensitize β-cells to immune-attack.

Shared regulation and functional relevance of local gene co-expression revealed by single cell analysis

Most human genes are co-expressed with a nearby gene. Yet, previous studies only reported this extensive local gene co-expression using bulk RNA-seq. Here, we leverage single cell datasets in >85 individuals to identify gene co-expression across cells, unbiased by cell type heterogeneity and benefiting from the co-occurrence of transcription events in single cells. We discover thousands of co-expressed genes in two cell types and (i) compare single cell to bulk RNA-seq in identifying local gene co-expression, (ii) show that many co-expressed genes – but not the majority – are composed of functionally-related genes and (iii) provide evidence that these genes are transcribed synchronously and their co-expression is maintained up to the protein level. Finally, we identify gene-enhancer associations using multimodal single cell data, which reveal that >95% of co-expressed gene pairs share regulatory elements. Our in-depth view of local gene co-expression and regulatory element co-activity advances our understanding of the shared regulatory architecture between genes.

Distinct mechanisms involving diacylglycerol, ceramides, and inflammation underlie insulin resistance in oxidative and glycolytic muscles from high fat-fed rats

This study investigated whether oxidative and glycolytic rat skeletal muscles respond differently to a high-fat (HF) sucrose-enriched diet with respect to diacylglycerol (DAG) and ceramides accumulation, protein kinase C (PKC) activation, glucose metabolism, and the expression of inflammatory genes. HF diet (8 weeks) suppressed insulin-stimulated glycogen synthesis and glucose oxidation in soleus (Sol), extensor digitorum longus (EDL) and epitrochlearis (Epit) muscles. However, DAG and ceramides levels increased in Sol and EDL, but not in Epit muscles of HF-fed rats. Additionally, membrane-bound PKC-delta and PKC-theta increased in Sol and EDL, whereas in Epit muscles both PKC isoforms were reduced by HF diet. In Epit muscles, HF diet also increased the expression of tumor necrosis factor-α (TNF-α) receptors (CD40 and FAS), toll-like receptor 4 (TLR4), and nuclear factor kappa light polypeptide gene enhancer in B cells (NF-kB), whereas in Sol and EDL muscles the expression of these inflammatory genes remained unchanged upon HF feeding. In conclusion, HF diet caused DAG and ceramides accumulation, PKC activation, and the induction of inflammatory pathways in a fiber type-specific manner. These findings help explain why oxidative and glycolytic muscles similarly develop insulin resistance, despite major differences in their metabolic characteristics and responsiveness to dietary lipid abundance.

Pentahydroxy flavonoid isolated from Madhuca indica ameliorated adjuvant-induced arthritis via modulation of inflammatory pathways

Rheumatoid arthritis (RA) is an autoimmune disease associated with advanced joint dysfunction. Madhuca indica J. F. Gmel, from the family Sapotaceae, is an Indian medicinal plant reported to have an array of pharmacological properties. The aim of present investigation was to determine the anti-arthritic potential of an isolated phytoconstituent from methanolic leaf extract of Madhuca indica (MI-ALC) against FCA-induced experimental arthritis. Polyarthritis was induced in female rats (strain: Wistar) via an intradermal injection of FCA (0.1 mL) into the tail. Polyarthritis developed after 32 days of FCA administration. Then rats were treated orally with an isolated phytoconstituent from MI-ALC at doses of 5, 10, and 20 mg/kg. Findings suggested that High-Performance Thin-Layer Chromatography, Fourier-Transform Infrared Spectroscopy, and Liquid Chromatography-Mass Spectrometry spectral analyses of the phytoconstituent isolated from MI-ALC confirmed the structure as 3,5,7,3′,4′-Pentahydroxy flavone (i.e., QTN). Treatment with QTN (10 and 20 mg/kg) showed significant (p < 0.05) inhibition of increased joint diameter, paw volume, paw withdrawal threshold, and latency. The elevated synovial oxidative stress (Superoxide dismutase, reduced glutathione, and malondialdehyde) and protein levels of Tumor necrosis factor-α (TNF-α) and Interleukin (ILs) were markedly (p < 0.05) reduced by QTN. It also effectively (p < 0.05) ameliorated cyclooxygenase-2 (COX-2), Nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-kβ) and its inhibitor-α (Ikβα), and ATP-activated P2 purinergic receptors (P2X7) protein expressions as determined by western blot analysis. In conclusion, QTN ameliorates FCA-induced hyperalgesia through modulation of elevated inflammatory release (NF-kβ, Ikβα, P2X7, and COX-2), oxido-nitrosative stress, and pro-inflammatory cytokines (ILs and TNF-α) in experimental rats.

MIREyA: a computational approach to detect miRNA-directed gene activation

Emerging studies demonstrate the ability of microRNAs (miRNAs) to activate genes via different mechanisms. Specifically, miRNAs may trigger an enhancer promoting chromatin remodelling in the enhancer region, thus activating the enhancer and its target genes. Here we present MIREyA, a pipeline developed to predict such miRNA-gene-enhancer trios based on an expression dataset which obviates the need to write custom scripts. We applied our pipeline to primary murine macrophages infected by Mycobacterium tuberculosis (HN878 strain) and detected Mir22, Mir221, Mir222, Mir155 and Mir1956, which could up-regulate genes related to immune responses. We believe that MIREyA is a useful tool for detecting putative miRNA-directed gene activation cases. MIREyA is available from: https://github.com/veania/MIREyA

A Reassessment of Copy Number Variations in Congenital Heart Defects: Picturing the Whole Genome

Copy number variations (CNVs) can modulate phenotypes by affecting protein-coding sequences directly or through interference of gene expression. Recent studies in cancer and limb defects pinpointed the relevance of non-coding gene regulatory elements such as long non-coding RNAs (lncRNAs) and topologically associated domain (TAD)-related gene-enhancer interactions. The contribution of such non-coding elements is largely unexplored in congenital heart defects (CHD). We performed a retrospective analysis of CNVs reported in a cohort of 270 CHD patients. We reviewed the diagnostic yield of pathogenic CNVs, and performed a comprehensive reassessment of 138 CNVs of unknown significance (CNV-US), evaluating protein-coding genes, lncRNA genes, and potential interferences with TAD-related gene-enhancer interactions. Fifty-two of the 138 CNV-US may relate to CHD, revealing three candidate CHD regions, 19 candidate CHD genes, 80 lncRNA genes of interest, and six potentially CHD-related TAD interferences. Our study thus indicates a potential relevance of non-coding gene regulatory elements in CNV-related CHD pathogenesis. Shortcomings in our current knowledge on genomic variation call for continuous reporting of CNV-US in international databases, careful patient counseling, and additional functional studies to confirm these preliminary findings.

High-resolution characterization of gene function using single-cell CRISPR tiling screen

Identification of novel functional domains and characterization of detailed regulatory mechanisms in cancer-driving genes is critical for advanced cancer therapy. To date, CRISPR gene editing has primarily been applied to defining the role of individual genes. Recently, high-density mutagenesis via CRISPR tiling of gene-coding exons has been demonstrated to identify functional regions in genes. Furthermore, breakthroughs in combining CRISPR library screens with single-cell droplet RNA sequencing (sc-RNAseq) platforms have revealed the capacity to monitor gene expression changes upon genetic perturbations at single-cell resolution. Here, we present “sc-Tiling,” which integrates a CRISPR gene-tiling screen with single-cell transcriptomic and protein structural analyses. Distinct from other reported single-cell CRISPR screens focused on observing gene function and gene-to-gene/enhancer-to-gene regulation, sc-Tiling enables the capacity to identify regulatory mechanisms within a gene-coding region that dictate gene activity and therapeutic response.

Involvement of ABA Responsive SVB Genes in the Regulation of Trichome Formation in Arabidopsis

Trichome formation in Arabidopsis is regulated by several key regulators, and plants hormones such as gibberellin, salicylic acid, jasmonic acid and cytokinins have been shown to regulate trichome formation by affecting the transcription or activities of the key regulators. We report here the identification of two abscisic acid (ABA) responsive genes, SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB) and SVB2 as trichome formation regulator genes in Arabidopsis. The expression levels of SVB and SVB2 were increased in response to ABA treatment, their expression levels were reduced in the ABA biosynthesis mutant aba1-5, and they have similar expression pattern. In addition to the trichome defects reported previously for the svb single mutant, we found that even though the trichome numbers were largely unaffected in both the svb and svb2 single mutants generate by using CRISPR/Cas9 gene editing, the trichome numbers were greatly reduced in the svb svb2 double mutants. On the other hand, trichome numbers were increased in SVB or SVB2 overexpression plants. RT-PCR results show that the expression of the trichome formation key regulator gene ENHANCER OF GLABRA3 (EGL3) was affected in the svb svb2 double mutants. Our results suggest that SVB and SVB2 are ABA responsive genes, and SVB and SVB2 function redundantly to regulate trichome formation in Arabidopsis.

Distinct mechanisms involving diacylglycerol, ceramides, and inflammation underlie insulin resistance in oxidative and glycolytic muscles from high fat-fed rats

This study investigated whether oxidative and glycolytic skeletal rat muscles respond differently to a high-fat (HF) sucrose-enriched diet with respect to diacylglycerol (DAG) and ceramides accumulation, protein kinase C (PKC) activation, glucose metabolism, and the expression of inflammatory genes. HF diet (8 weeks) suppressed insulin-stimulated glycogen synthesis and glucose oxidation in soleus (Sol), extensor digitorum longus (EDL) and epitrochlearis (Epit) muscles. However, DAG and ceramides levels increased in Sol and EDL, but not in Epit muscles of HF-fed rats. Additionally, membrane-bound PKC-delta and PKC-theta increased in Sol and EDL, whereas in Epit muscles both PKC isoforms were reduced by HF diet. In Epit muscles, HF diet also increased the expression of tumor necrosis factor-α (TNF-α) receptors (CD40 and FAS), toll-like receptor 4 (TLR4), and nuclear factor kappa light polypeptide gene enhancer in B cells (NF-kB), whereas in Sol and EDL muscles the expression of these inflammatory genes remained unchanged upon HF feeding. In conclusion, HF diet caused DAG and ceramides accumulation, PKC activation, and the induction of inflammatory pathways in a fiber type-specific manner. These findings help explain why oxidative and glycolytic muscles similarly develop insulin resistance, despite major differences in their metabolic characteristics and responsiveness to dietary lipid abundance.