Dear Editor:Clostridium acetobutylicum, a gram-positive, anaerobic, spore-forming bacterium, is capable of using a wide variety of carbon sources to produce acetone, butanol and ethanol. To improve solvent productivity of C. acetobutylicum, metabolic engineering is considered as a useful tool in developing strains with industrially desirable characteristics. However, to date, there are few useful methods for genetic manipulation of C. acetobutylicum, especially for gene disruption. To our knowledge, two types of vectors, including non-replicative and replicative integrative plasmids, have been developed for gene-inactivation in C. acetobutylicum. By using non-replicative integrative plasmids, buk and solR genes of C. acetobutylicum were inactivated [1,2]. However, due to their low frequencies of transformation and recombination, the non-replicative integrative plasmids are usually transformed at less than 1 integrative transformant per mg plasmid DNA. To obtain the integrative mutant, it may require higher transformation frequencies up to 10 5 , but the typical transformation frequencies were reported at 10 3 [3]. Harris et al. described the construction of a replicative integrative plasmid pETSPO and its application in the disruption of gene spo0A which could not be inactivated by using the non-replicative integrative plasmid [4]. With the functional replication origin in C. acetobutylicum, pETSPO increases opportunity for homologous recombination, but it is still time-consuming to screen for double crossover integration. Therefore, a more efficient tool for targeted gene inactivation in the C. acetobutylicum is much needed.Recently, a new strategy was developed to construct gene inactivation mutants by using group II intron-based Targetron technology. The mobile group II intron, originating from the Lactococcus lactis L1.LtrB intron, has been successfully used in a wide range of bacteria including Clostridium perfringens [5]. Without a proper replicon and/or promoter, the targetron plasmid pJIR750ai for C. perfringens from Sigma Aldrich was not applicable for gene disruption in the C. acetobutylicum directly (data not shown). Therefore, a modified targetron plasmid pSY6 was created by cloning the L1.LtrB group II intron fragment into the pIMP1-ptb, which was an E. coli-C. acetobutylicum shuttle vector containing a ptb (phosphotransbutyrylase) promoter [6].The gene buk, encoding the butyrate kinase, catalyzes the production of butyrate, and the gene solR located on the megaplasmid of the strain, encodes a putative repressor of solvent formation genes [7,8]. pSY6-buk and pSY6-solR vectors, constructed based on pSY6 ( Figure 1A and Supplementary information, Figure S1), were electroporated into C. acetobutylicum ATCC 824, respectively. Then, the cells were incubated overnight to induce the intron invasion (See Supplementary information, Materials and Methods). The overnight cultures were spread onto CGM medium (25 μg/ml erythromycin) and the transformants were analyzed

Chromatin modification is pivotal to the epithelial-mesenchymal transition (EMT), which confers potent metastatic potential to cancer cells. Here, we report a role for the chromatin remodeling factor lymphoid-specific helicase (LSH) in nasopharyngeal carcinoma (NPC), a prevalent cancer in China. LSH expression was increased in NPC, where it was controlled by the Epstein-Barr virus-encoded protein LMP1. In NPC cells in vitro and in vivo, LSH promoted cancer progression in part by regulating expression of fumarate hydratase (FH), a core component of the tricarboxylic acid cycle. LSH bound to the FH promoter, recruiting the epigenetic silencer factor G9a to repress FH transcription. Clinically, we found that the concentration of TCA intermediates in NPC patient sera was deregulated in the presence of LSH. RNAi-mediated silencing of FH mimicked LSH overexpression, establishing FH as downstream mediator of LSH effects. The TCA intermediates a-KG and citrate potentiated the malignant character of NPC cells, in part by altering IKKa-dependent EMT gene expression. In this manner, LSH furthered malignant progression of NPC by modifying cancer cell metabolism to support EMT.

Background: Inactivating mutations of the SLC12A3 gene are the most common cause of Gitelman’s syndrome (GS), a disorder inherited as an autosomal recessive trait. In a minority of cases, GS-like phenotypes are caused by mutations in the CLCNKB gene. Methods: We searched for SLC12A3 and CLCNKB gene mutations in 13 Chinese patients (9 males and 4 females, age 35 ± 14 years) from 8 unrelated families with the clinical and biochemical features of GS. All coding regions, including intron-exon boundaries, were analyzed using PCR followed by direct sequence analysis. Results: We identified 10 mutations distributed throughout the SLC12A3 gene. Seven are novel variants, including 4 missense mutations (Gly196Val, Cys430Gly, Gly439Val and Leu571Pro), 2 deletions (1384delG and 346–353delACTGATGG) and 1 in-frame insertion (997insCys). Three mutations were recurrent, including 2 missense mutations (Thr60Met and Asp486Asn) and 1 deletion (2883–2884delAG). The homozygous or heterozygous mutation Thr60Met was found in 8 of 13 patients. There were no mutations detected in the CLCNKB gene. Conclusions: Thr60Met may be the most common mutation in Chinese patients with GS. Possible specific genotype-phenotype correlations were difficult to identify.

We have identified 13 variants, including five novel variants in the SLC12A3 gene in 13 patients with Gitelman syndrome. T60M is the most frequent variant in our patients. There was no significant correlation between genotype and phenotype in our patients.

Background: Gitelman's syndrome (GS) is an autosomal recessive renal tubular disorder, which is caused by the mutations in SLC12A3. This study was designed to analyze the characteristics of the genotype and phenotype, and follow-up in the largest group of Chinese patients with GS. Methods: Sixty-seven patients with GS underwent SLCl2A3 analysis, and their clinical characteristics and biochemical findings as well as follow-up were reviewed, aiming to achieve a better description of GS. Additionally, the association of genotype and phenotype was explored. Results: Forty-one different mutations were identified within these 67 GS patients, including 11 novel mutations and 5 recurrent ones. Typical hypocalciuria and hypomagnesemia were not found in 6 (9%) and 8 (11.9%) patients, respectively. Male patients and those harboring severe mutations in both alleles had significant higher urinary fractional excretion (FE) of potassium, magnesium and chlorine. In addition, there were 2 patients who had chronic kidney disease (estimated glomerular filtration rate <60 ml/min/1.73 m²) and 32 patients with abnormal glucose metabolism. Conclusions: We identified 41 mutations related to GS, containing 11 novel variants and 5 high-frequency ones, which should facilitate earlier and more accurate diagnosis of GS. FE of electrolytes in urine may be more sensitive in the phenotype evaluation and differential diagnosis than corresponding serum electrolytes. Hypokalemia and hypomagnesemia in GS were difficult to correct; however, spironolactone might be helpful for hypokalemia to some degree. Compared with normal people, patients with GS were at higher risk of developing type 2 diabetes.

N-Acetyl-D: -neuraminic acid (Neu5Ac) can be produced from N-acetyl-D: -glucosamine (GlcNAc) and pyruvate by a chemoenzymatic process in which an alkaline-catalyzed epimerization transforms GlcNAc to N-acetyl-D: -manosamine (ManNAc). ManNAc is then condensed biocatalytically with pyruvate in the presence of N-acetyl-D: -neuraminic acid lyase (NAL) or by a two-step, fully enzymatic process involving bioconversions of GlcNAc to ManNAc and ManNAc to Neu5Ac using N-acetyl-D: -glucosamine 2-epimerase (AGE) and NAL. There are some drawbacks to this technique, such as lengthy reaction time, and the low conversion rate when the soluble forms of the enzymes are used in the two-step enzymatic process. In this study, the Escherichia coli-expressed AGE and NAL in the supernatant were purified by FP-based affinity chromatography and then immobilized on Amberzyme oxirane resin. These two immobilized enzymes, with a specific activity of 78.18 U/g for AGE and 69.30 U/g for NAL, were coupled to convert GlcNAc to Neu5Ac directly in one reactor. The conversion rate of the two-step reactions from GlcNAc to Neu5Ac was approximately 73% within 24 h. Furthermore, the immobilized AGE and NAL could both be used up to five reaction cycles without loss of activity or significant decrease of the conversion rate.

Cassava, due to its high starch content and low cost, is a promising candidate substrate for large-scale fermentation processes aimed at producing the solvents acetone, butanol and ethanol (ABE). However, the solvent yield from the fermentation of cassava reaches only 60% of that achieved by fermenting corn. We have found that the addition of ammonium acetate (CH(3)COONH(4)) to the cassava medium significantly promotes solvent production from cassava fermented by Clostridium acetobutylicum EA 2018, a mutant with a high butanol ratio. When cassava medium was supplemented with 30 mM ammonium acetate, the acetone, butanol and total solvent production reached 5.0, 13.0 and 19.4 g/l, respectively, after 48 h of fermentation. This level of solvent production is comparable to that obtained from corn medium. Both ammonium (NH(4) (+)) and acetate (CH(3)COO(-)) were required for increased solvent synthesis. We also demonstrated substantially increased acetic and butyric acid accumulation during the acidogenesis phase as well as greater acid re-assimilation during the solventogenesis period in ammonium acetate-supplemented cassava medium. Reverse transcription-polymerase chain reaction analysis indicated that the transcription of several genes encoding enzymes related to acidogenesis and solventogenesis in C. acetobutylicum EA 2018 were enhanced by the addition of ammonium acetate to the cassava medium.

Unimolecular dual agonists of the glucagon (GCG) receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) are a new class of drugs that are potentially superior to GLP-1R–specific agonists for the management of metabolic disease. The dual-agonist, peptide 15 (P15), is a glutamic acid 16 analog of GCG with GLP-1 peptide substitutions between amino acids 17 and 24 that has potency equivalent to those of the cognate peptide agonists at the GCGR and GLP-1R. Here, we have used cryo-EM to solve the structure of an active P15-GCGR-Gs complex and compared this structure to our recently published structure of the GCGR-Gs complex bound to GCG. This comparison revealed that P15 has a reduced interaction with the first extracellular loop (ECL1) and the top of transmembrane segment 1 (TM1) such that there is increased mobility of the GCGR extracellular domain and at the C terminus of the peptide compared with the GCG-bound receptor. We also observed a distinct conformation of ECL3 and could infer increased mobility of the far N-terminal His-1 residue in the P15-bound structure. These regions of conformational variance in the two peptide-bound GCGR structures were also regions that were distinct between GCGR structures and previously published peptide-bound structures of the GLP-1R, suggesting that greater conformational dynamics may contribute to the increased efficacy of P15 in activation of the GLP-1R compared with GCG. The variable domains in this receptor have previously been implicated in biased agonism at the GLP-1R and could result in altered signaling of P15 at the GCGR compared with GCG.