Plecanatide and dolcanatide, novel guanylate cyclase-C agonists, ameliorate gastrointestinal inflammation in experimental models of murine colitis.

AIM: To evaluate the effect of orally administered plecanatide or dolcanatide, analogs of uroguanylin, on amelioration of colitis in murine models. METHODS: The cyclic guanosine monophosphate (cGMP) stimulatory potency of plecanatide and dolcanatide was measured using a human colon carcinoma T84 cell-based assay. For animal studies all test agents were formulated in phosphate buffered saline. Sulfasalazine or 5-amino salicylic acid (5-ASA) served as positive controls. Effect of oral treatment with test agents on amelioration of acute colitis induced either by dextran sulfate sodium (DSS) in drinking water or by rectal instillation of trinitrobenzene sulfonic (TNBS) acid, was examined in BALB/c and/or BDF1 mice. Additionally, the effect of orally administered plecanatide on the spontaneous colitis in T-cell receptor alpha knockout (TCRα(-/-)) mice was also examined. Amelioration of colitis was assessed by monitoring severity of colitis, disease activity index and by histopathology. Frozen colon tissues were used to measure myeloperoxidase activity. RESULTS: Plecanatide and dolcanatide are structurally related analogs of uroguanylin, which is an endogenous ligand of guanylate cyclase-C (GC-C). As expected from the agonists of GC-C, both plecanatide and dolcanatide exhibited potent cGMP-stimulatory activity in T84 cells. Once-daily treatment by oral gavage with either of these analogs (0.05-0.5 mg/kg) ameliorated colitis in both DSS and TNBS-induced models of acute colitis, as assessed by body weight, reduction in colitis severity (P < 0.05) and disease activity index (P < 0.05). Amelioration of colitis by either of the drug candidates was comparable to that achieved by orally administered sulfasalazine or 5-ASA. Plecanatide also effectively ameliorated colitis in TCRα(-/-) mice, a model of spontaneous colitis. As dolcanatide exhibited higher resistance to proteolysis in simulated gastric and intestinal juices, it was selected for further studies. CONCLUSION: This is the first-ever study reporting the therapeutic utility of GC-C agonists as a new class of orally delivered and mucosally active drug candidates for the treatment of inflammatory bowel diseases.

A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover.

BACKGROUND: Fungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes. Aspergillus nidulans, a well-studied saprophyte and close homologue to industrially important species such as A. niger and A. oryzae, was selected for this study. RESULTS: A. nidulans was grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14 days. Based on analysis of chitin content, A. nidulans grew to be 4-5% of the total biomass in the culture after 2 days and then maintained a steady state of 4% of the total biomass for the next 12 days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14 days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS) of the secretome of A. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance. CONCLUSIONS: Our results show that A. nidulans secretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin) by 1 day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes secreted is tailored to the specific substrates available. Our findings reveal that A. nidulans is capable of degrading the major polysaccharides in sorghum without any chemical pre-treatment.

Phanerochaete chrysosporium produces a diverse array of extracellular enzymes when grown on sorghum.

In an effort to understand how fungi degrade biomass, we grew Phanerochaete chrysosporium on sorghum stover and chronicled the growth of the fungus over the course of 14 days. The fungal mass grew steadily until the fifth day, reaching 0.06 mg of cells per milligram of dry mass, which fell by the seventh day and stayed at nearly the same level until day 14. After 1 day, hemicellulases, cellulases, and polygalacturonases were detected in the extracellular fluid at 1.06, 0.34, and 0.20 U/ml, respectively. Proteomic studies performed with the extracellular fluid using liquid chromatography­tandem mass spectrometry identified 57, 116, and 102 degradative enzymes targeting cellulose, hemicellulose, pectin, lignin, proteins, and lipids on days 1, 7, and 14, respectively. Significant concentrations of breakdown products of the sorghum polysaccharides were detected in the extracellular fluid indicating that the enzymes were breaking the polysaccharides, and after 14 days, almost 39% of the sorghum sugars had been used by the fungus. Our results suggest that P. chrysosporium produces a set of enzymes to degrade the components of lignocellulose from the beginning of its growth, but modifies the complement of enzymes it secretes over time to adapt to the particular substrate available.

Solid-state fermentation for production of griseofulvin on rice bran using Penicillium griseofulvum.

Griseofulvin is a secondary metabolite produced from fungal species that have morphology suitable for solid-state fermentation (SSF). Reports on production of griseofulvin by SSF are scarce. The present work investigates SSF for griseofulvin production, optimization of its process parameters vis-à-vis the conventional submerged fermentation and its downstream processing from the same. Rice bran adjusted to an initial moisture content (IMC) of 50% (v/w) inoculated with 1 mL of a suspension of 10(6) spores/mL under agitation at 250 rpm containing the modified Czapek-Dox medium and additional 0.1% choline chloride as a precursor gave a yield of griseofulvin in 9 days that was comparable to submerged fermentation after 28 days. The yield of griseofulvin (microg/g dry biomass) was comparable in SSF and submerged fermentation. The biomass was estimated by estimation of chitin. Discussions on the effect of each parameter in SSF have also been included.

Supercritical carbon dioxide extraction of griseofulvin from the solid matrix obtained after solid-state fermentation.

Globally there is an increasing concern to minimize the use of organic solvents, particularly the chlorinated ones because of their suspected human carcinogenicity. The use of ecofriendly carbon dioxide as an alternative to organic solvents would be appropriate in the perspective of green technology. Supercritical carbon dioxide (SC-CO(2)) extraction is suitable for extraction of nonpolar compound with molecular weights less than 400. Griseofulvin is an antifungal antibiotic having a molecular weight of 353, making it amenable to SC-CO(2) extraction. This work brings out the potential of supercritical carbon dioxide extraction (SCFE) for downstream processing of griseofulvin from the solid matrix obtained after solid-state fermentation (SSF). The optimized conditions for SCFE of griseofulvin from dried media after SSF were a flow rate of 0.4 L/min, temperature of 60 degrees C, and contact time of 90 min (30 min static + 60 min dynamic) at a pressure of 450-455 bar.