Sister chromatid cohesion, which is mediated by the cohesin complex, is vital for faithful segregation of chromosomes in mitosis and meiosis (reviewed in). Cohesion is established during S phase, and this process requires the function of the acetyltransferase Eco1/Ctf7. The mechanism of the cohesion establishment is, however, still unclear. Here, we describe isolation and identification of genetic suppressors of budding yeast eco1-1 temperature-sensitive mutant. By using a recently described microarray-based method, we successfully mapped 11 intergenic suppressor mutations in two genes, wpl1 (also known as rad61) and pds5. Pds5 is a known accessory factor of cohesin complex, and we show that Wpl1/Rad61 protein forms a complex with Pds5 and colocalizes with cohesin on chromosomes, as its presumed human homolog Wapl. Impaired function of Wpl1-Pds5 complex makes Eco1 dispensable for cell survival. We also provide evidence that Wpl1 is required for efficient association of cohesin with G2 phase chromosomes and that Eco1 promotes dissociation of Wpl1-Pds5 from cohesin via acetylation of Smc3, a cohesin subunit. Taken together, the presented data suggest that Wpl1-Pds5 complex is inhibitory for cohesion establishment and that Eco1 establishes cohesion by hindering the function of Wpl1-Pds5 temporally in S phase.

GH3 (glycoside hydrolase family 3) BGLs (β-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 (Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N-glycan chains) in the dimer. The largest N-glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion.

For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulosedegrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on ␣-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His 6 ) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-␣-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley ␤-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and ␤-glucosidase 1 from Aspergillus aculeatus No.

Context. Although opioids and pregabalin are widely used for cancer-related neuropathic pain (CNP), no clinical trials exist to determine which medications are effective when an opioid-pregabalin combination therapy fails.Objectives. We investigated the efficacy of duloxetine for CNP nonresponsive or intolerant to opioid-pregabalin combination therapy.Methods. A multicenter, randomized, double-blind, placebo-controlled trial was performed at 12 specialized palliative care services in Japan. Patients with CNP average pain scores (Brief Pain Inventory [BPI]eItem 5) $ 4 in the previous 24 hours and nonresponsive or intolerant to opioid-pregabalin combination therapy were eligible. Patients with chemotherapy-induced peripheral neuropathies were excluded. Patients were administered duloxetine 20 mg/day titrated to 40 mg/day or placebo for 10 days. The primary endpoint was BPI-Item 5 on Day 10. Responder analysis measured proportions of patients with 30% and 50% pain decreases.Results. Seventy patients were enrolled. Complete case analysis revealed mean BPI-Item 5 on Day 10 of 4.03 for Group D vs. 4.88 for Group P (P ¼ 0.053). Baseline observation carried forward analysis revealed mean BPI-Item 5 on Day 10 of 4.06 and 4.91 for Groups D and P, respectively (P ¼ 0.048). Clinically meaningful pain improvement ($30%) was reported by 44.1% (n ¼ 15) of patients in Group D vs. 18.2% (n ¼ 6) in Group P (P ¼ 0.02); 32.4% (n ¼ 11) vs. 3.0% (n ¼ 1) of patients in Groups D and P, respectively, reported pain reduction $ 50% (P ¼ 0.002).Conclusion. Adding duloxetine to opioid-pregabalin therapy might have clinical benefit in alleviating refractory CNP. Further studies are needed to conclude the efficacy of adding duloxetine. J Pain Symptom

Filamentous fungi produce cellulolytic and hemicellulolytic enzymes in response to small inducer molecules liberated from cellulosic biomass. Enzyme production is mainly regulated at the level of transcription. The first transcription factor identified as being involved in cellulosic biomass degradation was XlnR, which mediates D-xylose-triggered induction of xylanolytic and cellulolytic genes in Aspergillus. XlnR has played the leading role for over a decade in studies aimed at clarification of gene regulation related to cellulosic biomass degradation. Very recently, several new transcription factors were identified, namely Clr-1/2 in Neurospora; ManR, McmA, and ClbR in Aspergillus; and BglR in Trichoderma, all of which participate in the regulation of cellulolytic and/or hemicellulolytic enzyme production. Furthermore, as well as the carbon sources available, other factors such as light signaling and anti-sense RNA accumulation have been shown to contribute to this regulation. Here, we review the recent advancements demonstrating that multiple factors coordinately regulate the expression of cellulosic biomass degrading enzyme genes.

The two types of intervention protocol well reflected the treatment intention and expected outcomes. Further, large-scale cohort studies are promising.

To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.

The registration number of this study in the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) is UMIN000008646.