Anaerobic fungi habitat in the gastrointestinal tract of foregut fermenters or hindgut fermenters and degrade fibrous plant biomass through the hydrolysis reactions with a wide variety of cellulolytic enzymes and physical penetration through fiber matrix with their rhizoids. To date, seventeen genera have been described in family Neocallimasticaceae, class Neocallimastigomycetes, phylum Neocallimastigomycota and one genus has been described in phylum Neocallimastigomycota. In National Center for Biotechnology Information (NCBI) database (DB), 23,830 nucleotide sequences and 59,512 protein sequences have been deposited and most of them were originated from Piromyces, Neocallimastix and Anaeromyces. Most of protein sequences (44,025) were acquired with PacBio next generation sequencing system. The whole genome sequences of Anaeromyces robustus, Neocallimastix californiae, Pecoramyces ruminantium, Piromyces finnis and Piromyces sp. E2 are available in Joint Genome Institute (JGI) database. According to the results of protein prediction, average Isoelectric points (pIs) were ranged from 5.88 (Anaeromyces) to 6.57 (Piromyces) and average molecular weights were ranged from 38.7 kDa (Orpinomyces) to 56.6 kDa (Piromyces). In Carbohydrate-Active enZYmes (CAZY) database, glycoside hydrolases (36), carbohydrate binding module (11), carbohydrate esterases (8), glycosyltransferase (5) and polysaccharide lyases (3) from anaerobic fungi were registered. During four decades, 1,031 research articles about anaerobic fungi were published and 444 and 719 articles were available in PubMed (PM) and PubMed Central (PMC) DB.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively is able to increase apoptosis in cancer cells as agent with minimum toxicity to noncancerous cells. However, all cancer cells are not sensitive to TRAIL-induced apoptosis. In this study, we showed the sub-lethal concentrations of a lysosomotropic autophagy inhibitor, IITZ-01, sensitizes cancer cells (renal, lung, and breast carcinoma) to TRAIL-induced apoptosis through DR5 upregulation and survivin downregulation through ubiquitin-proteasome pathway. Knockdown of DR5 or overexpression of survivin inhibited combined treatment with IITZ-01 and TRAIL-induced apoptosis. IITZ-01 downregulated protein expression of Cbl, ubiquitin E3 ligase, and decreased expression level of Cbl markedly led to increase DR5 protein expression and TRAIL sensitivity. Moreover, IITZ-01 decreased expression level of survivin protein via downregulation of deubiquitinase ubiquitin-specific protease 9X (USP9X) expression. Taken together, these results provide the first evidence that IITZ-01 enhances TRAIL-mediated apoptosis through DR5 stabilization by downregulation of Cbl and USP9X-dependent survivin ubiquitination and degradation in renal carcinoma cells.

Objective Fasting may lead to changes in the microbiota and activity in the rumen. In the present study, the effects of fasting on rumen microbiota and the impact of fasting on in vitro rumen fermentation were evaluated using molecular culture-independent methods. Methods Three ruminally cannulated Holstein steers were fed rice straw and concentrates. The ruminal fluids were obtained from the same steers 2 h after the morning feeding (control) and 24 h after fasting (fasting). The ruminal fluid was filtrated through four layers of muslin, collected for a culture-independent microbial analysis, and used to determine the in vitro rumen fermentation characteristics. Total DNA was extracted from both control and fasting ruminal fluids. The rumen microbiota was assessed using denaturing gradient gel electrophoresis (DGGE) and quantitative polymerase chain reaction. Microbial activity was evaluated in control and fasting steers at various intervals using in vitro batch culture with rice straw and concentrate at a ratio of 60:40. Results Fasting for 24 h slightly affected the microbiota structure in the rumen as determined by DGGE. Additionally, several microorganisms, including Anaerovibrio lipolytica, Eubacterium ruminantium, Prevotella albensis, Prevotella ruminicola, and Ruminobacter amylophilus, decreased in number after fasting. In addition, using the ruminal fluid as the inoculum after 24 h of fasting, the fermentation characteristics differed from those obtained using non-fasted ruminal fluid. Compared with the control, the fasting showed higher total gas production, ammonia, and microbial protein production (p<0.05). No significant differences, however, was observed in pH and dry matter digestibility. Conclusion When in vitro techniques are used to evaluate feed, the use of the ruminal fluid from fasted animals should be used with caution.

Objective This study was conducted to evaluate the effect of homo or hetero fermentative inoculants on fermentation quality and aerobic stability of sweet potato vine (SPV) silage containing Italian ryegrass hay as moisture absorbent. Methods The SPV was harvested at 15% dry matter, mixed with Italian ryegrass hay at 1:1 ratio on a fresh weight basis, and chopped to 3 to 5 cm length. After then, the chopped forage mixture was ensiled into 20-L mini silos in quadruplicate for 7, 48, and 100 days after application of microbial inoculants at 1.2×105 colony forming units (cfu)/g of forage following: no inoculant (CON), Lactobacillus plantarum as a homo fermentative (LP), Lactobacillus buchneri as a hetero fermentative (LB), and mixture of LP and LB at 1:1 ratio as a combo fermentative (MIX). Results The LP and MIX silages had lowest pH (p<0.001) on 7 and 48 days, while MIX and CON silages had greatest lactate concentrations (p<0.05) on 7 and 48 days, respectively. Acetate concentrations were highest (p<0.01) in LB and MIX silages on 7 days, and in LB silage on 48 days, while lactate to acetate ratios were lowest (p<0.001) in LB silages. The chemical compositions and nutrient digestibility of silage ensiled for 100 days was not affected by inoculants. On 100 days of ensiling, LB silage had lowest (p<0.01) lactate concentration and lactate to acetate ratio, but highest acetate concentration. Aerobic stability was highest (p<0.001) in LB silage followed in MIX silage. On contrast, LB silage had lowest (p<0.05) lactic acid bacteria and mold. Conclusion The results indicated that application of LB solely had a better effect on aerobic stability than not only LP, but also MIX. However, LP application did not show beneficial effects from the viewpoints of fermentation quality and aerobic stability compared to CON.

Members of the Ras superfamily of small G-proteins serve as molecular switches of intracellular signaling pathways. Rac2 is a Rho subfamily GTPase switch that is specifically expressed in hematopoietic cells and regulates AKT activation in cell signaling. Ras activating protein-like 3 (RASAL3) is the recently identified Ras GTPase activating protein (GAP) that is also specifically expressed in hematopoietic cells and stimulates p21ras GTPase activity. The restricted expression of both Rac2 and RASAL3 suggests that they may serve critical roles in hematopoietic cell signaling. Here in the present study demonstrates that the catalytic domain of RASAL3 may also be able to interact with Rac2 and stimulate its GTPase activity in vitro. By contrast, p50 rhoGAP molecules did not markedly affect Rac2 GTPase activity, but did accelerate the activity of other Rho GTPases, including Rac1, RhoA and Cdc42. Collectively, the present results indicate, seemingly for the first time, that GAP activity for Rac2 is regulated by the RasGAP family protein, RASAL3.

Corosolic acid is one of the pentacyclic triterpenoids isolated from Lagerstroemia speciose and has been reported to exhibit anti-cancer and anti-proliferative activities in various cancer cells. In the present study, we investigated the molecular mechanisms of corosolic acid in cancer cell death. Corosolic acid induces a decrease of cell viability and an increase of cell cytotoxicity in human renal carcinoma Caki cells. Corosolic acid-induced cell death is not inhibited by apoptosis inhibitor (z-VAD-fmk, a pan-caspase inhibitor), necroptosis inhibitor (necrostatin-1), or ferroptosis inhibitors (ferrostatin-1 and deferoxamine (DFO)). Furthermore, corosolic acid significantly induces reactive oxygen species (ROS) levels, but antioxidants (N-acetyl-l-cysteine (NAC) and trolox) do not inhibit corosolic acid-induced cell death. Interestingly, corosolic acid induces lipid oxidation, and α-tocopherol markedly prevents corosolic acid-induced lipid peroxidation and cell death. Anti-chemotherapeutic effects of α-tocopherol are dependent on inhibition of lipid oxidation rather than inhibition of ROS production. In addition, corosolic acid induces non-apoptotic cell death in other renal cancer (ACHN and A498), breast cancer (MDA-MB231), and hepatocellular carcinoma (SK-Hep1 and Huh7) cells, and α-tocopherol markedly inhibits corosolic acid-induced cell death. Therefore, our results suggest that corosolic acid induces non-apoptotic cell death in cancer cells through the increase of lipid peroxidation.

Nitrogen metabolism in gut systems remains poorly studied in spite of its importance for microbial growth and its implications for the metabolism of the host. Prevotella spp. are the most predominant bacteria detected in the rumen, but their presence has also been related to health and disease states in the human gut and oral cavity. To explore the metabolic networks for nitrogen assimilation in this bacterium, changes in gene expression profiles in response to variations in the available nitrogen source and to different concentrations of ammonium were analyzed by microarray and reverse transcription quantitative PCR, and linked with function by further proteomic analysis. The observed patterns of transcript abundances for genes involved in ammonium assimilation differed from the classical "enteric paradigm" for nitrogen utilization. Expression of genes encoding high substrate affinity nitrogen assimilation enzymes (GS-GOGAT system) was similar in growth-limiting and non-limiting nitrogen concentrations in P. ruminicola 23, whereas E. coli and Salmonella spp. responses to excess nitrogen involve only low substrate affinity enzymes. This versatile behavior might be a key feature for ecological success in habitats such as the rumen and human colon where nitrogen is rarely limiting for growth, and might be linked to previously reported Prevotella spp. population imbalances relative to other bacterial species in gut systems.

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored gut and non-gut environments. Additionally many studies are now known to have used primers that provide incomplete coverage of the Neocallimastigomycota. For ecological studies the internal transcribed spacer 1 region (ITS1) has been the taxonomic marker of choice, but due to various limitations the large subunit rRNA (LSU) is now being increasingly used. How the continued expansion of our knowledge regarding anaerobic fungal diversity will impact on our understanding of their biology and ecological role remains unclear; particularly as it is becoming apparent that anaerobic fungi display niche differentiation. As a consequence, there is a need to move beyond the broad generalization of anaerobic fungi as fiber-degraders, and explore the fundamental differences that underpin their ability to exist in distinct ecological niches. Application of genomics, transcriptomics, proteomics and metabolomics to their study in pure/mixed cultures and environmental samples will be invaluable in this process. To date the genomes and transcriptomes of several characterized anaerobic fungal isolates have been successfully generated. In contrast, the application of proteomics and metabolomics to anaerobic fungal analysis is still in its infancy. A central problem for all analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit.

Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine α-helixes and 12 β-strands. The enzyme expressed in E.coli had the highest activity at 40°C and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at 40°C, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.

Silibinin, an effective anti-cancer and chemopreventive agent, has been shown to exert multiple effects on cancer cells, including inhibition of both cell proliferation and migration. However, the molecular mechanisms responsible for these effects are not fully understood. We observed that silibinin significantly induced the expression of the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in both p53 wild-type and p53-null cancer cell lines, suggesting that silibinin-induced NAG-1 up-regulation is p53-independent manner. Silibinin up-regulates early growth response-1 (EGR-1) expression. The ectopic expression of EGR-1 significantly increased NAG-1 promoter activity and NAG-1 protein expression in a dose-dependent manner. Furthermore, down-regulation of EGR-1 expression using siRNA markedly reduced silibinin-mediated NAG-1 expression, suggesting that the expression of EGR-1 is critical for silibinin-induced NAG-1 expression. We also observed that reactive oxygen species (ROS) are generated by silibinin; however, ROS did not affect silibinin-induced NAG-1 expression and apoptosis. In addition, we demonstrated that the mitogen-activated protein kinase (MAP kinase) signal transduction pathway is involved in silibinin-induced NAG-1 expression. Inhibitors of p38 MAP kinase (SB203580) attenuated silibinin-induced NAG-1 expression. Furthermore, we found that siRNA-mediated knockdown of NAG-1 attenuated silibinin-induced apoptosis. Collectively, the results of this study demonstrate for the first time that up-regulation of NAG-1 contributes to silibinin-induced apoptosis in cancer cells.

In this study, protein domains with cellulase activity in goat rumen microbes were investigated using metagenomic and bioinformatic analyses. After the complete genome of goat rumen microbes was obtained using a shotgun sequencing method, 217,892,109 pair reads were filtered, including only those with 70% identity, 100-bp matches, and thresholds below E-10 using METAIDBA. These filtered contigs were assembled and annotated using blastN against the NCBI nucleotide database. As a result, a microbial community structure with 1431 species was analyzed, among which Prevotella ruminicola 23 bacteria and Butyrivibrio proteoclasticus B316 were the dominant groups. In parallel, 201 sequences related with cellulase activities (EC.3.2.1.4) were obtained through blast searches using the enzyme.dat file provided by the NCBI database. After translating the nucleotide sequence into a protein sequence using Interproscan, 28 protein domains with cellulase activity were identified using the HMMER package with threshold E values below 10(-5). Cellulase activity protein domain profiling showed that the major protein domains such as lipase GDSL, cellulase, and Glyco hydro 10 were present in bacterial species with strong cellulase activities. Furthermore, correlation plots clearly displayed the strong positive correlation between some protein domain groups, which was indicative of microbial adaption in the goat rumen based on feeding habits. This is the first metagenomic analysis of cellulase activity protein domains using bioinformatics from the goat rumen.

New colchicine analogs have been synthesized with the aim of developing stronger potential anticancer activities. Among the analogs, CT20126 has been previously reported to show immunosuppressive activities. Here, we report that CT20126 also shows potential anticancer effects via an unusual mechanism: the modulation of microtubule integrity and cell cycle arrest at the G2/M phase before apoptosis. When we treated COS-7 cells with CT20126 (5 μ M ), the normal thread-like microtubules were disrupted into tubulin dimers within 10 min and thereafter repolymerized into short, thick filaments. In contrast, cells treated with the same concentration of colchicine exhibited microtubule depolymerization after 20 min and never underwent repolymerization. Furthermore, optical density (OD) analysis (350 nm) with purified tubulin showed that CT20126 had a higher repolymerizing activity than that of Taxol, a potent microtubule-polymerizing agent. These results suggest that the effects of CT20126 on microtubule integrity differ from those of colchicine: the analog first destabilizes microtubules and then stabilizes the disrupted tubulins into short, thick polymers. Furthermore, CT20126 induced a greater level of apoptotic activity in Jurkat T cells than colchicine (assessed by G2/M arrest, caspase-3 activation and cell sorting). At 20 n M , CT20126 induced 47% apoptosis among Jurkat T cells, whereas colchicine induced only 33% apoptosis. Our results suggest that the colchicine analog CT20126 can potently induce apoptosis by disrupting microtubule integrity in a manner that differs from that of colchicine or Taxol.

Although there have been many studies of native Korean cattle, Hanwoo, there have been no selective sweep studies in these animals. This study was performed to characterize genetic variation and identify selective signatures. We sequenced the genomes of 12 cattle, and identified 15125420 SNPs, 1768114 INDELs, and 3445 CNVs. The SNPs, INDELs, and CNVs were similarly distributed throughout the genome, and highly variable regions were shown to contain the BoLA family and GPR180, which are related to adaptive immunity. We also identified the domestication footprints of the Hanwoo population by searching for selective sweep signatures, which revealed the RCN2 gene related to BPV resistance. The results of this study may contribute to genetic improvement of the Hanwoo population in Korea.

Phospholipase C-γl (PLC-γl) expression is associated with cellular transformation. Notably, PLC-gamma is up-regulated in colorectal cancer tissue and breast carcinoma. Because exotoxins released by Clostridium botulinum have been shown to induce apoptosis and promote growth arrest in various cancer cell lines, we examined here the potential of Clostridium difficile toxin A to selectively induce apoptosis in cells transformed by PLC-γl overexpression. We found that PLC-γl-transformed cells, but not vectortransformed (control) cells, were highly sensitive to C. difficile toxin A-induced apoptosis and mitotic inhibition. Moreover, expression of the proapoptotic Bcl2 family member, Bim, and activation of caspase-3 were significantly up-regulated by toxin A in PLC-γl-transformed cells. Toxin A-induced cell rounding and paxillin dephosphorylation were also significantly higher in PLC-γl-transformed cells than in control cells. These findings suggest that C. difficile toxin A may have potential as an anticancer agent against colorectal cancers and breast carcinomas in which PLC-γl is highly up-regulated.

We report a systematic study of gene expression during myogenesis and transdifferentiation in four bovine muscle tissues and of adipogenesis in three bovine fat tissues using DNA microarray analysis. One hundred hybridizations were performed and 7245 genes of known and unknown function were identified as being differentially expressed. Supervised hierarchical cluster analysis of gene expression patterns revealed the tissue specificity of genes. A close relationship in global gene expression observed for adipocyte-like cells derived from muscle and adipocytes derived from intramuscular fat suggests a common origin for these cells. The role of transthyretin in myogenesis is a novel finding. Different genes were highly induced during the transdifferentiation of myogenic satellite cells and in the adipogenesis of preadipocytes, indicating the involvement of different molecular mechanisms in these processes. Induction of CD36 and FABP4 expression in adipocyte-like cells and adipocytes may share a common pathway.

Clostridium difficile toxin A glucosylates Rho family proteins, resulting in actin filament disaggregation and cell rounding in cultured colonocytes. Given that the cellular toxicity of toxin A is dependent on its receptor binding and subsequent entry into the cell, we herein sought to identify additional colonocyte proteins that might bind to toxin A following its internalization. Our results revealed that toxin A interacted with ERK1 and ERK2 in two human colonocyte cell lines (NCM460 and HT29). A GST-pulldown assay also showed that toxin A can directly bind to ERK1 and ERK2. In NCM460 cells exposed to PMA (an ERK1/2 activator), the phosphorylation of ERK1/2 did not affect the interaction between toxin A and ERK1/2. However, an in vitro kinase assay showed that the direct binding of toxin A to ERK1 or ERK2 inhibited their kinase activities. These results suggest a new molecular mechanism for the cellular toxicity seen in cells exposed to toxin A.