ResultsRecipient Abx pretreatment attenuates, but adjunctive fecal microbiota transfer recreates, hepatic IRI in mouse allogeneic OLT. We first aimed to determine the influence of Abx treatment on IRI severity in a clinically relevant allogeneic mouse OLT model (BALB/c> C57BL/6) with ex vivo cold storage (4°C for 18 hours), which mimics marginal human liver grafts. Mouse OLT recipients pretreated for 10 days (14, 15) with oral Abx (amoxicillin, 50 mg/mL) showed decreased serum aspartate aminotransferase (sAST) levels (OLT = 7047 ± 1332 vs. OLT + Abx = 3609 ± 447 IU/L, P = 0.0317; Figure 1A); attenuated sinusoidal congestion, edema/vacuolization and hepatocellular necrosis ( Figure 1B); decreased Suzuki's histological grading of IRI (OLT = 6.8 ± 0.6 vs. OLT + Abx = 3.6 ± 0.5, P

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3 to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the L-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.Maintenance of cell polarity and tissue architecture is vital to the normal growth and differentiation program of epithelial cells (1). Disruption of cell polarity and tissue architecture, which is associated with uncontrolled cell proliferation and differentiation, is a feature of neoplastic transformation (2). A complex network of cell-cell and cell-extracellular matrix interactions mediated in part by adhesion molecules play an important role in epithelial tissue shape and function. Not surprisingly, many cancers have altered expression of various cell adhesion molecules. One of them is CEACAM1, 4 whose aberrant expression has been linked to a variety of carcinomas (3, 4).CEACAM1 is a glycosylated transmembrane protein and is a member of the CEA immunoglobulin superfamily (5). In humans, CEACAM1 pre-mRNA undergoes alternative splicing, giving rise to 11 splice variants (6). All splice variants include a highly conserved N-terminal ectodomain that defines the CEA gene family and is responsible for the homotypic adhesion function of these gene products (5, 7). CEACAM1 is expressed as a type I transmembrane protein in human tissues with 1, 3, or 4 extracellular Ig-like domains and either short or long cytoplasmic domains that engage distinct signal transduction pathways based on their different amino acid sequences (5). The two cytoplasmic domain splice varia...

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ikaros-SIRT1 signaling axis regulates hepatic inflammation and pyroptosis in ischemia-stressed mouse and human liver,

Background: Despite tremendous progress in understanding the mechanisms of constitutive and alternative splicing, an important and widespread step along the gene expression pathway, our ability to deliberately regulate gene expression at this step remains rudimentary. The present study was performed to investigate whether a theophylline-dependent "splice switch" that sequesters the branchpoint sequence (BPS) within RNA-theophylline complex can regulate alternative splicing.

Fibromyalgia syndrome (FMS) is a chronic musculoskeletal pain disorder affecting 2% to 5% of the general population. Both genetic and environmental factors may be involved. To ascertain in an unbiased manner which genes play a role in the disorder, we performed complete exome sequencing on a subset of FMS patients. Out of 150 nuclear families (trios) DNA from 19 probands was subjected to complete exome sequencing. Since >80,000 SNPs were found per proband, the data were further filtered, including analysis of those with stop codons, a rare frequency (<2.5%) in the 1000 Genomes database, and presence in at least 2/19 probands sequenced. Two nonsense mutations, W32X in C11orf40 and Q100X in ZNF77 among 150 FMS trios had a significantly elevated frequency of transmission to affected probands (p = 0.026 and p = 0.032, respectively) and were present in a subset of 13% and 11% of FMS patients, respectively. Among 9 patients bearing more than one of the variants we have described, 4 had onset of symptoms between the ages of 10 and 18. The subset with the C11orf40 mutation had elevated plasma levels of the inflammatory cytokines, MCP-1 and IP-10, compared with unaffected controls or FMS patients with the wild-type allele. Similarly, patients with the ZNF77 mutation have elevated levels of the inflammatory cytokine, IL-12, compared with controls or patients with the wild type allele. Our results strongly implicate an inflammatory basis for FMS, as well as specific cytokine dysregulation, in at least 35% of our FMS cohort.

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