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Rationale: Methicillin resistant Staphylococcus aureus (MRSA) is prevalent and consequential in cystic fibrosis (CF). Whole genome sequencing (WGS) could reveal genomic differences in MRSA associated with poorer outcomes or detect MRSA transmission. Objectives: To identify MRSA genes associated with low lung function and identify potential MRSA transmission in CF. Methods: We collected 97 MRSA isolates from 74 individuals with CF from 2017 and performed short-read WGS. We determined sequence type (ST) and the phylogenetic relationship between isolates. We aligned accessory genes from 25 reference genomes to genome assemblies. We classified the MRSA by accessory gene content and correlated the accessory genome to clinical outcomes. Results: The most prevalent ST were ST5 (N=55), ST105 (N=14), and ST8 (N=14). Closely related MRSA strains were shared by family members with CF, but rarely between unrelated individuals. Three distinct clusters of MRSA were identified by accessory genome content. The first included ST5 and ST105 strains and was common among older patients with lower FEV. The second cluster included ST8, which was generally identified in younger patients. Sputum density of MRSA and Pseudomonas aeruginosa was higher in cultures from patients with ST5/ST105 compared to patients with ST8 at similar ages. Conclusions: In this CF cohort, we identified MRSA subtypes that predominate at different ages and differ by accessory gene content. ST5 and ST105 represented the most prevalent cluster of MRSA. ST8 MRSA was more common in younger patients and thus has the potential to rise in prevalence as these patients age.

Suitable experimental models of gallstone pancreatitis with systemic inflammation and mortality are limited. We developed a novel murine model of duct-ligation-induced acute pancreatitis associated with multiorgan dysfunction and severe mortality.Laparotomy was done on C57/BL6 mice followed by pancreatic duct (PD) ligation, bile duct (BD) ligation without PD ligation, or sham operation.Only mice with PD ligation developed acute pancreatitis and had 100% mortality. Pulmonary compliance was significantly reduced after PD ligation but not BD ligation. Bronchoalveolar lavage fluid neutrophil count and interleukin-1β concentration, and the plasma creatinine level, were significantly elevated with PD ligation but not BD ligation. Pancreatic nuclear factor κB (p65) and activator protein 1 (c-Jun) were activated within 1 h of PD ligation.PD-ligation-induced acute pancreatitis in mice is associated with systemic inflammation, acute lung injury, multiorgan dysfunction and death. The development of this novel model is an exciting and notable advance in the field.

Background Mitogen-activated protein (MAP) kinases and nuclear factor kappa B (NF-κB) are implicated in early stages of acute pancreatitis pathogenesis. We investigated the relationship between the p38 MAP kinase and NF-κB in isolated acinar cells. Methods Isolated rodent acinar cells were stimulated with agonists after infection with an adenovector containing a luciferase promoter driven only by NF-κB and an adenovector containing the dominant negative (DN) form of p38 (empty vector in controls). Results Initial immunoblots confirmed that the agonist stimulated p38 activation in acinar cells was substantially attenuated by DN p38 overexpression. Stimulation of native cholecystokinin (CCK)-A receptors or tumor necrosis factor-α (TNF-α) receptors promoted a significant increase in NF-κB-dependent gene transcription in cells infected with the empty vector, while overexpression of DN p38 significantly abrogated NF-κB-dependent luciferase activity. Conclusions These findings support our hypothesis that p38 is involved in the activation of proinflammatory nuclear transcription factors such as NF-κB in pancreatic exocrine cells.

Actin is a highly conserved cytoskeletal protein that is a key component of cells. Genes encoding actin occur in single copies in most green algae, in 2–3 copies in bryophytes, and in increasingly more complex gene families in ferns and seed plants. We use the well-resolved phylogenetic frameworks of the Streptophyta as a guide to reconstruct the patterns of actin gene duplication in early diverging land plants. Our working hypothesis is that the origin of novel tissues in the bryophytes (e.g. multicellular sporophyte) may be reflected in the functional diversification of duplicate actin genes in these taxa. Actin is used as a model cytoskeletal protein with the assumption that its evolutionary history represents those of other cytoskeletal elements and the coevolved binding proteins. Here we provide a phylogenetic perspective on the origin of green algal and land plant actin genes and use this information to speculate on the role of plant actin in early plant evolution.

Background Acute pancreatitis is potentially fatal but treatment options are limited as disease pathogenesis is poorly understood. IL-33, a novel IL-1 cytokine family member, plays a role in various inflammatory conditions but its role in acute pancreatitis is not well understood. Specifically, whether pancreatic acinar cells produce IL-33 when stressed or respond to IL-33 stimulation, and whether IL-33 exacerbates acute pancreatic inflammation is unknown. Methods/Results In duct ligation-induced acute pancreatitis in mice and rats, we found that (a) IL-33 concentration was increased in the pancreas; (b) mast cells, which secrete and also respond to IL-33, showed degranulation in the pancreas and lung; (c) plasma histamine and pancreatic substance P concentrations were increased; and (d) pancreatic and pulmonary proinflammatory cytokine concentrations were increased. In isolated mouse pancreatic acinar cells, TNF-α stimulation increased IL-33 release while IL-33 stimulation increased proinflammatory cytokine release, both involving the ERK MAP kinase pathway; the flavonoid luteolin inhibited IL-33-stimulated IL-6 and CCL2/MCP-1 release. In mice without duct ligation, exogenous IL-33 administration induced pancreatic inflammation without mast cell degranulation or jejunal inflammation; pancreatic changes included multifocal edema and perivascular infiltration by neutrophils and some macrophages. ERK MAP kinase (but not p38 or JNK) and NF-kB subunit p65 were activated in the pancreas of mice receiving exogenous IL-33, and acinar cells isolated from the pancreas of these mice showed increased spontaneous cytokine release (IL-6, CXCL2/MIP-2α). Also, IL-33 activated ERK in human pancreatic tissue. Significance As exogenous IL-33 does not induce jejunal inflammation in the same mice in which it induces pancreatic inflammation, we have discovered a potential role for an IL-33/acinar cell axis in the recruitment of neutrophils and macrophages and the exacerbation of acute pancreatic inflammation. Conclusion IL-33 is induced in acute pancreatitis, activates acinar cell proinflammatory pathways and exacerbates acute pancreatic inflammation.

The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20–50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor.

We have previously shown that distal pancreatic duct ligation-induced acute pancreatitis in mice is associated with substantial mortality. We examined the cause of death in duct ligation-induced acute pancreatitis in mice by serial examination of multiple parameters in three experimental groups: distal pancreatic duct ligation (PD), bile duct ligation alone (BD), and sham operation (S). BD and S had no mortality, while PD had 94% mortality with most deaths between days 2 and 4. Characteristics of mice with acute pancreatitis included (ANOVA; p

Neuronally enriched RGS4 plays a critical role attenuating G protein signaling in brain, although the mechanisms regulating RGS4 expression are unknown. Here we describe a novel mechanism for transcriptional activation of RGS4 in neuron-like PC6 cells, where RGS4 is markedly induced during confluence-induced growth arrest. Transcriptional activation of RGS4 in confluent PC6 cells was accompanied by impaired G(i/o)-dependent MAPK activation. In the human RGS4 gene promoter, we identified three phylogenetically conserved cis-elements: an inverted CCAAT box element (ICE), a cAMP response element, and a B-cell lymphoma 6 (Bcl6)-binding site. The ICE and the cAMP response element mediate activation, and the Bcl6 site mediates repression of RGS4 transcription. Activation of RGS4 transcription in confluent PC6 cells is accompanied by increases in NF-YA and C/EBPβ and decreases in Bcl6 levels in the nucleus. Increases in NF-YA and C/EBPβ lead to their increased binding to the RGS4 promoter in vivo, and dominant negative forms of these proteins repressed RGS4 promoter activity. Acetylation of NF-YA and Bcl6 were increased in postconfluent cells. Trichostatin A stimulation of RGS4 promoter activity, accompanied by increased binding of NF-YA and decreased binding of Bcl6 to the promoter, was abolished by mutation of the ICE and enhanced by mutation of the Bcl6 site. These findings demonstrate a dynamic and coordinated regulation of nuclear levels and acetylation status of trans-acting factors critical in determining the off/on state of the RGS4 promoter.

Background: The role of the p38 mitogen-activated protein (MAP) kinase in acute pancreatitis pathogenesis is controversial. We hypothesize that p38 plays a role in regulating NF-κB activation in exocrine pancreatic cells. Methods: AR42J cells incorporating an NF-κB-responsive luciferase reporter, with and without adenoviral transduction of DNp38, were stimulated with cholecystokinin (CCK) or tumor necrosis factor-α (TNF-α) prior to measuring NF-κB activation. Results: CCK-or TNF-α-stimulated NF-κB-dependent gene transcription (luciferase assay) was substantially subdued by DNp38 expression. These findings were confirmed by electrophoretic mobility shift assay. Nuclear translocation of the p65 NF-κB subunit following agonist stimulation was evident (supershift). Characterization studies showed excellent adenoviral infection efficiency and cell viability in our AR42J cell model. Agonist-stimulated dose- and time-dependent p38 activation, with inhibition by DNp38 expression, was also con- firmed. Conclusion: The p38 MAP kinase regulates NF-κB pathway activation in exocrine pancreatic cells, and thus potentially plays a role in the mechanism of acute pancreatitis pathogenesis.