One-step production of lactate from cellulose as the sole carbon source without any other organic nutrient by recombinant cellulolytic Bacillus subtilis

Although intensive efforts have been made to create recombinant cellulolytic microorganisms, real recombinant cellulose-utilizing microorganisms that can produce sufficient secretory active cellulase, hydrolyze cellulose, and utilize released soluble sugars for supporting both cell growth and cellulase synthesis without any other organic nutrient (e.g., yeast extract, peptone, amino acids), are not available. Here we demonstrated that over-expression of Bacillus subtilis endoglucanase BsCel5 enabled B. subtilis to grow on solid cellulosic materials as the sole carbon source for the first time. Furthermore, two-round directed evolution was conducted to increase specific activity of BsCel5 on regenerated amorphous cellulose (RAC) and enhance its expression/secretion level in B. subtilis. To increase lactate yield, the alpha-acetolactate synthase gene (alsS) in the 2,3-butanediol pathway was knocked out. In the chemically defined minimal M9/RAC medium, B. subtilis XZ7(pBscel5-MT2C) strain (ΔalsS), which expressed a BsCel5 mutant MT2C, was able to hydrolyze RAC with cellulose digestibility of 74% and produced about 3.1g/L lactate with a yield of 60% of the theoretical maximum. When 0.1% (w/v) yeast extract was added in the M9/RAC medium, cellulose digestibility and lactate yield were enhanced to 92% and 63% of the theoretical maximum, respectively. The recombinant industrially safe cellulolytic B. subtilis would be a promising consolidated bioprocessing platform for low-cost production of biocommodities from cellulosic materials.     

Investigation on Mycobacterium tuberculosis diversity in China and the origin of the Beijing clade

Background

Investigation of the genetic diversity of Mycobacterium tuberculosis in China has shown that Beijing genotype strains play a dominant role in the tuberculosis (TB) epidemic. In order to examine the strain diversity in the whole country, and to study the evolutionary development of Beijing strains, we sought to genotype a large collection of isolates using different methods.

Methodology/Principal Findings

We applied a 15-loci VNTR typing analysis on 1,586 isolates from the Beijing municipality and 12 Chinese provinces or autonomous regions. The data was compared to that of 900 isolates from various other worldwide geographic regions outside of China. A total of 1,162/1,586 (73.2%) of the isolates, distributed into 472 VNTR types, were found to belong to the Beijing genotype family and this represented 56 to 94% of the isolates in each of the localizations. VNTR typing revealed that the majority of the non-Beijing isolates fall into two genotype families, which represented 17% of the total number of isolates, and seem largely restricted to China. A small number of East African Indian genotype strains was also observed in this collection. Ancient Beijing strains with an intact region of difference (RD) 181, as well as strains presumably resembling ancestors of the whole Beijing genotype family, were mainly found in the Guangxi autonomous region.

Conclusions/Significance

This is the largest M. tuberculosis VNTR-based genotyping study performed in China to date. The high percentage of Beijing isolates in the whole country and the presence in the South of strains representing early branching points may be an indication that the Beijing lineage originated from China, probably in the Guangxi region. Two modern lineages are shown here to represent the majority of non-Beijing Chinese isolates. The observed geographic distribution of the different lineages within China suggests that natural frontiers are major factors in their diffusion.     

Discovery of novel microRNAs in rat kidney using next generation sequencing and microarray validation

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. The latest version of the miRBase database (Release 18) includes 1,157 mouse and 680 rat mature miRNAs. Only one new rat mature miRNA was added to the rat miRNA database from version 16 to version 18 of miRBase, suggesting that many rat miRNAs remain to be discovered. Given the importance of rat as a model organism, discovery of the completed set of rat miRNAs is necessary for understanding rat miRNA regulation. In this study, next generation sequencing (NGS), microarray analysis and bioinformatics technologies were applied to discover novel miRNAs in rat kidneys. MiRanalyzer was utilized to analyze the sequences of the small RNAs generated from NGS analysis of rat kidney samples. Hundreds of novel miRNA candidates were examined according to the mappings of their reads to the rat genome, presence of sequences that can form a miRNA hairpin structure around the mapped locations, Dicer cleavage patterns, and the levels of their expression determined by both NGS and microarray analyses. Nine novel rat hairpin precursor miRNAs (pre-miRNA) were discovered with high confidence. Five of the novel pre-miRNAs are also reported in other species while four of them are rat specific. In summary, 9 novel pre-miRNAs (14 novel mature miRNAs) were identified via combination of NGS, microarray and bioinformatics high-throughput technologies.     

Co-expression and Synergism Analysis of Vip3Aa29 and Cyt2Aa3 Insecticidal Proteins from Bacillus thuringiensis

Vegetative insecticidal protein (Vip3) from Bacillus thuringiensis shows high activity against lepidopteran insects. Cytolytic δ-endotoxin (Cyt) also has high toxicity to dipteran larvae and synergism with other crystal proteins (Cry), but synergism between Cyt and Vip3 proteins has not been tested. We analyzed for synergism between Cyt2Aa3 and Vip3Aa29. Both cyt2Aa3 and vip3Aa29 genes were co-expressed in Escherichia coli strain BL21 carried on vector pCOLADuet-1. Vip3Aa29 showed insecticidal activity against Chilo suppressalis and Spodoptera exigua, with 50% lethal concentration (LC(50)) at 24.0 and 36.6 μg ml(-1), respectively. It could also inhibit Helicoverpa armigera growth, with 50% inhibition concentration at 22.6 μg ml(-1). While Cyt2Aa3 was toxic to Culex quinquefasciatus (LC(50): 0.53 μg ml(-1)) and Chironomus tepperi (LC(50): 36 μg ml(-1)), it did not inhibit C. suppressalis, S. exigua, and H. armigera. However, the co-expression of Cyt2Aa3 and Vip3Aa29 showed synergistic effect on C. suppressalis and S. exigua, and the individual activities were strengthened 3.35- and 4.34-fold, respectively. The co-expression had no synergism against C. tepperi and H. armigera, but exerted some antagonistic effect on Cx. quinquefasciatus. The synergism between Cyt2Aa and Vip3Aa was thus discovered for the first time, which confirmed that Cyt toxin can enhance the toxicity of other toxins against some non-target insects. By synergism analysis, the effectiveness of microbial insecticides can be verified.     

Efficacy and safety of metformin and sitagliptin based triple antihyperglycemic therapy (STRATEGY): a multicenter,randomized, controlled,non-inferiority clinical trial

Despite the current guideline’s recommendation of a timely stepwise intensification therapy, the “clinical inertia”, termed as the delayed treatment intensification, commonly exists in the real world, which may be partly due to the relatively little substantial evidence and no clear consensus regarding the efficacy and safety of triple oral agents in patients inadequately controlled with dual therapy. In this clinical trial performed in 237 centers in China, 5,535 type 2 diabetic patients inadequately controlled by previous therapies were treated with a stable metformin/sitagliptin dual therapy for 20 weeks. The patients who did not reach the glycated hemoglobin A1c (HbA1c) goal were then further randomized into glimepiride, gliclazide, repaglinide, or acarbose group for an additional 24-week triple therapy. A mean HbA1c reduction of 0.85% was observed when sitagliptin was added to the patients inadequately controlled with metformin in 16 weeks. Further HbA1c reductions in the 24-week triple therapy stage were 0.65% in glimepiride group, 0.70% in gliclazide group, 0.61% in repaglinide group, and 0.45% in acarbose group. The non-inferiority criterion for primary hypotheses was met for gliclazide and repaglinide, but not for acarbose, compared with glimepiride, when added to metformin/sitagliptin dual therapy. The incidences of adverse events (AEs) were 29.2% in the dual therapy stage and 30.3% in the triple therapy stage. Metformin/sitagliptin as baseline therapy, with the addition of a third oral antihyperglycemic agent, including glimepiride, gliclazide, repaglinide, or acarbose, was effective, safe and well-tolerated for achieving an HbA1c <7.0% goal in type 2 diabetic patients inadequately controlled with previous therapies. The timely augmentation of up to three oral antihyperglycemic agents is valid and of important clinical benefit to prevent patients from exposure to unnecessarily prolonged hyperglycemia.     

Identification of quantitative trait loci for resistance to rice black-streaked dwarf virus disease and small brown planthopper in rice

Small brown planthopper (SBPH) and its transmitted rice black-streaked dwarf virus disease (RBSDVD) cause serious damage to rice (Oryza sativa L.) production. Though breeding of resistant cultivars is believed to be one of the most important strategies for RBSDVD management, few high-resistance lines have been found to date. In the present study, we identified an indica variety, 9194, that is highly resistant to RBSDVD and analyzed the quantitative trait loci (QTLs) underlying this resistance . In total, four QTLs for RBSDVD resistance, viz. qRBSDV3, qRBSDV6, qRBSDV9, and qRBSDV11, were identified. Among them, qRBSDV6, qRBSDV9, and qRBSDV11 with LOD (logarithm [base 10] of odds) scores of 4.42–4.48, 2.11–7.26, and 5.01–7.16 were repeatedly detected in 2 years, accounting for 10.3–16.7%, 8.3–35.5%, and 20.0–31.1% of the total phenotypic variation, respectively. Further, introgression of single- or multiple-resistance QTLs into a susceptible rice variety by marker-assisted selection (MAS) indicated that stacking the QTLs could progressively enhance RBSDVD resistance, suggesting that these QTLs act additively. The same population was also used for QTL mapping of SBPH resistance. Four QTLs, viz. qSBPH1, qSBPH5, qSBPH8, and qSBPH9, with LOD scores of 2.72, 2.78, 2.15, and 2.85 were detected, explaining 13.7%, 11.0%, 12.0%, and 21.0% of the phenotypic variation, respectively. The identification of RBSDVD and SBPH resistance QTLs, and the development of single and multiple genes with pyramided lines, in this study provides innovative resources for molecular breeding of resistant rice cultivars.     

Evidence for the involvement of nitric oxide and reactive oxygen species in osmotic stress tolerance of wheat seedlings: Inverse correlation between leaf abscisic acid accumulation and leaf water loss

Nitric oxide (NO) and reactive oxygen species (ROS) play important roles in both abscisic acid (ABA) signaling and stress-induced ABA accumulation. However, little is known about their physiological roles in the whole plant. In this study, the effects of NO and ROS on leaf water control and the roles of ABA were determined using wheat (Triticum aestivum L.) seedlings. As compared with the control, osmotic stress reduced leaf water loss (LWL) while it increased leaf ABA content. The effects of osmotic stress on LWL and ABA contents were partially reversed by NO scavengers or NO synthase (NOS) inhibitors. Furthermore, sodium nitroprusside (SNP) at concentrations between 0.01 and 10 mM all reduced LWL efficiently and induced ABA accumulation in a dose-dependent manner. When ABA synthesis was inhibited by fluridone or actidione, the effects of SNP on LWL were partially reversed. These results suggest that NO is involved in leaf water maintenance of wheat seedlings under osmotic stress, and one of the possible mechanisms is by stimulating ABA synthesis. The ROS scavengers used in our experiments had no effects on either LWL or ABA accumulation induced by osmotic stress. However, all ROS induced LWL reduction and ABA accumulation significantly. Hydrogen peroxide had the same effects as SNP on LWL and induced ABA accumulation in a dose-dependent manner but had a maximal effect at 1 mM. Fluridone reversed the effects of H₂O₂ on both LWL reduction and ABA accumulation, while actidione had no effect. These results suggest that ROS are also involved in leaf water maintenance of wheat seedlings by stimulating ABA biosynthesis, but with a different mechanism to that of NO. The ABA-independent mechanism in NO/ROS regulation of leaf water balance is discussed, in relation to our results.     

Characterization of the C-terminal domain of a potassium channel from Streptomyces lividans (KcsA)

KcsA, a potassium channel from Streptomyces lividans, is a good model for probing the general working mechanism of potassium channels. To date, the physiological activator of KcsA is still unknown, but in vitro studies showed that it could be opened by lowering the pH of the cytoplasmic compartment to 4. The C-terminal domain (CTD, residues 112-160) was proposed to be the modulator for this pH-responsive event. Here, we support this proposal by examining the pH profiles of: (a) thermal stability of KcsA with and without its CTD and (b) aggregation properties of a recombinant fragment of CTD. We found that the presence of the CTD weakened and enhanced the stability of KcsA at acidic and basic pH values, respectively. In addition, the CTD fragment oligomerized at basic pH values with a transition profile close to that of channel opening. Our results are consistent with the CTD being a pH modulator. We propose herein a mechanism on how this domain may contribute to the pH-dependent opening of KcsA.     

Systems Properties and Spatiotemporal Regulation of Cell Position Variability during Embryogenesis

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