Circadian disruption dysregulates lung gene expression associated with inflammatory lung injury.

Rationale: Circadian systems drive the expression of multiple genes in nearly all cells and coordinate cellular-, tissue-, and system-level processes that are critical to innate immunity regulation. Objective: We examined the effects of circadian rhythm disorganization, produced by light shift exposure, on innate immunity-mediated inflammatory lung responses including vascular permeability and gene expression in a C57BL/6J murine model of inflammatory lung injury. Methods: A total of 32 C57BL/6J mice were assigned to circadian phase shifting (CPS) with intratracheal phosphate-buffered saline (PBS), CPS with intratracheal lipopolysaccharide (LPS), control (normal lighting) condition with intratracheal PBS, and control condition with intratracheal LPS. Bronchoalveolar lavage (BAL) protein, cell counts, tissue immunostaining, and differentially expressed genes (DEGs) were measured in lung tissues at 2 and 10 weeks. Measurements and results: In mice exposed to both CPS and intratracheal LPS, both BAL protein and cell counts were increased at both 2 and 10 weeks compared to mice exposed to LPS alone. Multiple DEGs were identified in CPS-LPS-exposed lung tissues compared to LPS alone and were involved in transcriptional pathways associated with circadian rhythm disruption, regulation of lung permeability, inflammation with Rap1 signaling, and regulation of actin cytoskeleton. The most dysregulated pathways included myosin light chain kinase, MAP kinase, profilin 2, fibroblast growth factor receptor, integrin b4, and p21-activated kinase. Conclusion: Circadian rhythm disruption results in exacerbated immune response and dysregulated expression of cytoskeletal genes involved in the regulation of epithelial and vascular barrier integrity-the mechanistic underpinnings of acute lung injury. Further studies need to explore circadian disorganization as a druggable target.

Interactive Effect of Combined Intermittent and Sustained Hypoxia and High-Fat Diet on the Colonic Mucosal Microbiome and Host Gene Expression in Mice.

Purpose: Gut dysbiosis can cause cardiometabolic disease. Gut dysbiosis can be independently caused by high-fat diet (HFD) and intermittent hypoxia (IH; characterizing obstructive sleep apnea), but the interactive effect of combined intermittent and sustained hypoxia (IH+SH) (characterizing obesity hypoventilation syndrome) and HFD on gut dysbiosis is unclear. We aimed to investigate the interactive effect of a combination of IH and SH and HFD on proximal colonic microbiota and colonic gene expression pattern. Methods: Male mice (n=16) were randomly received four different combinations of diet (normal versus HFD) and oxygen conditions (normoxia versus IH+SH) for 4 weeks. Bacterial DNA and mucosal epithelial cell RNA from proximal colon were collected for analysis of adherent microbiome and host's gene expression analysis. Results: HFD during IH+SH (22.6 ± 5.73; SD) led to greater Firmicutes: Bacteroidetes ratio than HFD during normoxia (5.89 ± 1.19; p=0.029). HFD significantly decreased microbial diversity as compared to normal diet, but the addition of IH+SH to HFD mildly reversed such effects. When compared to HFD during normoxia, HFD with combination of IH+SH resulted in changes to host mucosal gene expression for apical junctional complexes and adhesion molecules. Specifically, when compared to HFD during normoxia, HFD during IH+SH led to upregulation of Claudin 2 and Syk (tight junction dysfunction and increased mucosal permeability), while the barrier promoting claudin 4 was downregulated. Conclusion: HFD during combined IH and SH causes greater gut dysbiosis and potentially adverse changes in colonic epithelial transcriptome than HFD during normoxia. The latter changes are suggestive of impaired gut barrier function.

Hypoxia Suppresses High Fat Diet-Induced Steatosis And Development Of Hepatic Adenomas.

PURPOSE: Nonalcoholic fatty liver disease (NAFLD) is considered the most common form of silent liver disease in the United States and obesity is associated with increased risk of NAFLD. Obstructive sleep apnea (OSA) which is common in obese individuals is associated with a greater incidence of NAFLD, which in turn, increases the risk for hepatocellular carcinoma (HCC). It is unclear how obesity, OSA and NAFLD interrelate nor how they collectively contribute to an increased risk for developing HCC. PATIENTS AND METHODS: Male BALB/c mice were exposed to diethylnitrosamine and phenobarbital followed by 48 weeks of either standard chow diet (chow), chow with hypoxia, high-fat diet, or a combination of hypoxia and high-fat diet. We noninvasively monitored tumor development using micro-CT imaging. We tracked the total weight gained throughout the study. We evaluated liver histology, fat accumulation, carbonic anhydrase 9 (CA9) and hypoxia-inducible factor 1-alpha (HIF-1α) expression, as well as, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). RESULTS: A high-fat diet without hypoxia led to the development of obesity that induced hepatic steatosis and promoted tumorigenesis. Animals on a high-fat diet and that were also exposed to hypoxia had lower total weight gain, lower steatosis, lower serum AST and ALT levels, and fewer number of hepatic adenomas than a high-fat diet without hypoxia. CONCLUSION: These findings suggest that hypoxia abrogates obesity, hepatic steatosis, and hepatic tumorigenesis related to a high-fat diet.

Candida albicans Als proteins mediate aggregation with bacteria and yeasts.

Candida albicans occupies a microniche on mucosal surfaces where diverse microbial populations interact within a biofilm. Because C. albicans is intimately involved with other microbes in this environment we studied the interactions of C. albicans with other fungi and bacteria that form mixed microbial aggregates. Once aggregation is initiated, aggregates form rapidly and incorporate fungal as well as bacterial cells. The fungus formed mixed microbial aggregates with homotypic cells (i.e., self to self, e.g., C. albicans or Als1p-expressing yeast cells aggregating with cells bearing Als1p); with heterotypic cells (i.e., self to non-self, e.g., C. albicans or Alsp-expressing yeast cells aggregating with other Candida species); and with xenotypic cells (e.g., C. albicans or Alsp-expressing yeast cells forming aggregates with bacteria). When either of the C. albicans adhesins Als1p or Als5p was displayed on the surface of non-adherent Saccharomyces cerevisiae cells, the S. cerevisiae also mediated these mixed microbial interactions. Thus the Als adhesins are potentially important for the co-adhesion of mixed microbial communities in biofilms and on mucus surfaces.

Threonine-rich repeats increase fibronectin binding in the Candida albicans adhesin Als5p.

Commensal and pathogenic states of Candida albicans depend on cell surface-expressed adhesins, including those of the Als family. Mature Als proteins consist of a 300-residue N-terminal region predicted to have an immunoglobulin (Ig)-like fold, a 104-residue conserved Thr-rich region (T), a central domain of a variable number of tandem repeats (TR) of a 36-residue Thr-rich sequence, and a heavily glycosylated C-terminal Ser/Thr-rich stalk region, also of variable length (N. K. Gaur and S. A. Klotz, Infect. Immun. 65: 5289-5294, 1997). Domain deletions in ALS5 were expressed in Saccharomyces cerevisiae to excrete soluble protein and for surface display. Far UV circular dichroism indicated that soluble Ig-T showed a single negative peak at 212 nm, consistent with previous data indicating that this region has high beta-sheet content with very little alpha-helix. A truncation of Als5p with six tandem repeats (Ig-T-TR(6)) gave spectra with additional negative ellipticity at 200 nm and, at 227 to 240 nm, spectra characteristic of a structure with a similar fraction of beta-sheet but with additional structural elements as well. Soluble Als5p Ig-T and Ig-T-TR(6) fragments bound to fibronectin in vitro, but the inclusion of the TR region substantially increased affinity. Cellular adhesion assays with S. cerevisiae showed that the Ig-T domain mediated adherence to fibronectin and that TR repeats greatly increased cell-to-cell aggregation. Thus, the TR region of Als5p modulated the structure of the Ig-T region, augmented cell adhesion activity through increased binding to mammalian ligands, and simultaneously promoted fungal cell-cell interactions.

Evidence for the presence of a guanine quadruplex forming region within a polypurine tract of the hypoxia inducible factor 1alpha promoter.

The promoter of the hypoxia inducible factor 1 alpha (HIF-1alpha) gene has a polypurine/polypyrimidine tract (-65 to -85) overlapping or adjacent to several putative transcription factor binding sites, and we found that mutagenesis of this region diminished basal HIF-1alpha expression. Oligonucleotides representing this region of the HIF-1alpha promoter were analyzed by electrophoretic mobility shift, chemical probing, circular dichroism, and DNA polymerase arrest assays. The guanine-rich strand was found to form a parallel, unimolecular quadruplex in the presence of potassium that was further stabilized by two known quadruplex binding compounds, the cationic porphyrin TmPyP4 and the natural product telomestatin, while TmPyP2, a positional isomer of TmPyP4, did not stabilize quadruplex formation. These data suggest that a quadruplex structure may form in a region of the HIF-1alpha promoter that regulates basal HIF-1alpha expression.

Multisite inhibition by phenylacetate of PC-3 cell growth.

Phenylacetate (PA) is a reversible inhibitor of tumor cell growth and an inhibitor of mevalonate pyrophosphate decarboxylase (MPD). We hypothesized that MPD inhibition should lower rates of protein accumulation and accretion of cell number in all cell lines regardless of tumorigenic status or origin of the cell lines. PA treatment inhibited growth of MCF-7, NIH-3T3, Detroit 551, UT-2, NCTC-929, COS-1 and PC-3 cell lines. NCTC-929 cells lack cadherins and Cos-1 cells are deficient in PPARalpha and PPARgamma, proteins suggested to be central to the action of PA. Oxidative metabolism was not impeded by PA treatment. One-dimensional and two-dimensional FACS analysis of BrdU incorporation failed to demonstrate a redistribution of nuclei in the cell cycle or that the rate of cells entering S phase had changed. Time-lapse photo-microscopy studies reveal a process that left condensed nuclei with little or no cytoplasm. However, negative TUNEL assay results and failure to block cell loss with z-VAD-fmk suggest this type of cell death is not typical apoptosis, but cell death is responsible for the lower rates of cell and protein accumulation. Supplementation studies with mevalonate pathway intermediates during inhibition of the mevalonate pathway of cholesterol biosynthesis by lovastatin confirmed MPD as a site of PA inhibition of growth, but in the presence of lovastatin with or without farnesyl pyrophosphate plus geranylgeranyl pyrophosphate, additive inhibition by PA revealed additional site(s). The existence of site(s) in addition to MPD suggests effective PA-based agents might be developed that would not inhibit MPD.