Protective effect of ß-glucan on Poly(I:C)-induced acute lung injury/inflammation: Therapeutic implications of viral infections in the respiratory system.

AIMS: Acute lung inflammation, particularly acute respiratory distress syndrome (ARDS), is caused by a variety of pathogens including bacteria and viruses. ß-Glucans have been reported to possess both anti-inflammatory and immunomodulatory properties. The current study evaluated the therapeutic effect of ß-glucans on polyinosinic:polycytidylic acid (Poly(I:C)) induced lung inflammation in both hamster and mice models. MAIN METHODS: Poly(I:C)-induced ALI/inflammation models were developed in hamsters (2.5 mg/kg) and mice (2 mg/kg) by delivering the Poly(I:C) intratracheally, and followed with and without ß-glucan administration. After treatment, lung mechanics were assessed and lung tissues were isolated and analyzed for mRNA/protein expression, and histopathological examinations. KEY FINDINGS: Poly(I:C) administration, caused a significant elevation of inflammatory marker's expression in lung tissues and showed abnormal lung mechanics in mice and hamsters. Interestingly, treatment with ß-glucan significantly (p < 0.001) reversed the Poly(I:C)-induced inflammatory events and inflammatory markers expression in both mRNA (IL-6, IL-1ß, TNF-α, CCL2 and CCL7) and protein levels (TNF-α, CD68, myeloperoxidase, neutrophil elastase, MUC-5Ac and iNOS). Lung functional assays revealed that ß-glucan treatment significantly improved lung mechanics. Histopathological analysis showed that ß-glucan treatment significantly attenuated the Poly(I:C) induced inflammatory cell infiltration, injury and goblet cell population in lung tissues. Consistent with these findings, ß-glucan treatment markedly reduced the number of neutrophils and macrophages in lung tissues. Our findings further demonstrated that ß-glucan could reduce inflammation by suppressing the MAPK pathway. SIGNIFICANCE: These results suggested that ß-glucan may attenuate the pathogenic effects of Poly(I:C)-induced ALI/ARDS via modulating the MAPK pathway, indicating ß-glucan as a possible therapeutic agent for the treatment of viral-pulmonary inflammation/injury.

Transcription-dependent enrichment of the yeast FACT complex influences nucleosome dynamics on the RNA polymerase III-transcribed genes.

The FACT (FAcilitates Chromatin Transactions) complex influences transcription initiation and enables passage of RNA polymerase (pol) II through gene body nucleosomes during elongation. In the budding yeast, ~280 non-coding RNA genes highly transcribed in vivo by pol III are found in the nucleosome-free regions bordered by positioned nucleosomes. The downstream nucleosome dynamics was found to regulate transcription via controlling the gene terminator accessibility and hence, terminator-dependent pol III recycling. As opposed to the enrichment at the 5'-ends of pol II-transcribed genes, our genome-wide mapping found transcription-dependent enrichment of the FACT subunit Spt16 near the 3'-end of all pol III-transcribed genes. Spt16 physically associates with the pol III transcription complex and shows gene-specific occupancy levels on the individual genes. On the non-tRNA pol III-transcribed genes, Spt16 facilitates transcription by reducing the nucleosome occupany on the gene body. On the tRNA genes, it maintains the position of the nucleosome at the 3' gene-end and affects transcription in gene-specific manner. Under nutritional stress, Spt16 enrichment is abolished in the gene downstream region of all pol III-transcribed genes and reciprocally changed on the induced or repressed pol II-transcribed ESR genes. Under the heat and replicative stress, its occupancy on the pol III-transcribed genes increases significantly. Our results show that Spt16 elicits a differential, gene-specific and stress-responsive dynamics, which provides a novel stress-sensor mechanism of regulating transcription against external stress. By primarily influencing the nucleosomal organization, FACT links the downstream nucleosome dynamics to transcription and environmental stress on the pol III-transcribed genes.

A Novel Method to Generate MNase Ladders Reveal Rapid Chromatin Remodeling upon Gametogenesis and Mating in Chlamydomonas.

To circumvent nuclei isolation for nucleosomal mapping of wild-type (cell walled) algal cells, we developed a quick and versatile methodology, by abrasion of whole cells (Chlamydomonas, Scenedesmus and yeast), allowing Micrococcal Nuclease (MNase) direct access to nuclear chromatin, in situ. Varying parameters such as bead abrasion, vortex and incubation conditions, we optimized capture of an 'early digest' which may probe chromatin differentially, based on nucleosome accessibility. A comparison of such ladders across vegetative cells, gametes and zygotes revealed an increase in the average nucleosomal repeat length (+17-34nt) upon gametogenesis, indicating a trend of chromatin compaction. Using PCR, we compared promoter enrichment in increasing orders of fractionated nucleosomal repeats (mono-, di-, up to penta-), each differing in cleavability based on chromatin accessibility. Concordant with higher gene expression (mating locus), promoters revealed an enrichment in mono-nucleosomal fractions. Interestingly, the zygote specific gene, MT0828 displayed rapid remodelling from penta-nucleosomal enrichment when completely repressed (vegetative), to intermediate states during gametogenesis (24hrs), which finally shifted to being largely mono-nucleosomal, when induced (1h zygotes). Summarizing three candidate genes from the mating locus, we conclude that the MNase based 'Chromatin Accessibility Assay' can track a range of large-scale rapid chromatin remodelling transitions within the binaries of gene expression.