Search

Cronobacter sakazakii is an emerging opportunistic foodborne pathogen causing rare but severe infections in neonates. Furthermore, the formation of biofilm allows C. sakazakii to persist in different environments. We have demonstrated that the mutator phenotype ascribed to deficiency of the pmrA gene results in more biomass in the first 24 h but less during the post maturation stage (7–14 d) compared with BAA 894. The present study aimed to investigate the regulatory mechanism modulating biofilm formation due to pmrA mutation. The transcriptomic analyses of BAA 894 and s-3 were performed by RNA-sequencing on planktonic and biofilm cells collected at different time points. According to the results, when comparing biofilm to planktonic cells, expression of genes encoding outer membrane proteins, lysozyme, etc. were up-regulated, with LysR family transcriptional regulators, periplasmic proteins, etc. down-regulated. During biofilm formation, cellulose synthase operon genes, flagella-related genes, etc. played essential roles in different stages. Remarkably, pmrA varies the expression of a number of genes related to motility, biofilm formation, and antimicrobial resistance, including srfB, virK, mviM encoding virulence factor, flgF, fliN, etc. encoding flagellar assembly, and marA, ramA, etc. encoding AraC family transcriptional regulators in C. sakazakii. This study provides valuable insights into transcriptional regulation of C. sakazakii pmrA mutant during biofilm formation.

The CRISPR-Cas12a system has been demonstrated as an attractive tool for bacterial genome engineering. In particular, FnCas12a recognizes protospacer-adjacent motif (PAM) sites with medium or low GC content, which complements the Cas9-based systems. Here we explored Francisella novicida Cas12a (FnCas12a) for genome editing in Pseudomonas aeruginosa. By using a two-plasmid system expressing the constitutive FnCas12a nuclease, the inducible λRed recombinase, a CRISPR RNA (crRNA), we achieved gene deletion, insertion and replacement with high efficiency (in most cases > 75%), including the deletion of large DNA fragments up to 15 kb and the serial deletion of duplicate gene clusters. This work should provide a useful and complementary addition to the genome engineering toolbox for the study of P. aeruginosa biology and physiology.

This study investigated the effect of the β2 receptor agonist terbutaline on the single channel activity of BKCa channel. The effects of racemate and two isomers of terbutaline were all assessed. β2 adrenoceptors were stably overexpressed on HEK293 cells by lentiviral transduction method and chicken BKCa channels were transiently expressed on normal HEK293 cell line or HEK293 cells overexpressing β2 receptors. Data showed that terbutaline significantly increased the single channel open probability of BKCa channel within 10min. The channel activating effects of terbutaline are stereoselective and mainly stay with the R-enantiomers. The opening probability of BKCa channel at 10min after drug application normalized to that just before drug application (Po10/Po0s) for R- and S-terbutaline were 7.85±3.20 and 1.06±0.45 respectively at 1μM concentration, corresponding to 28.37±9.96 and 2.68±1.09 at the higher concentration of 10μM. ICI 118551 blocked the effect of R- but not S-terbutaline (10μM), whereas atropine blocked the channel activating effects of S-terbutaline of higher concentration. In addition, the muscarinic receptor agonist carbachol increased the BKCa channel activity in an atropine-sensitive manner as an positive control experiment, which indicate the involvement of M receptor in the channel activating effect of S-terbutaline.

Background Existing treatments are inadequate for patients at high risk of coronary heart disease caused by elevated levels of plasma low-density lipoprotein cholesterol (LDL-C). Bambuterol is a prodrug of β2-agonist commonly used for the treatment of asthma and chronic obstructive pulmonary disease (COPD) with the advantage of once daily dosing and favorable side effect profile. The potential lipid-lowering effects of bambuterol were unclear, possibly due to the racemic bambuterol (rac-bambuterol) that was used in previous studies. Methods The lipid-lowering effects of R-bambuterol were examined in a randomized phase I trial in 48 healthy Chinese volunteers aged 18–45 years. Participants were randomly assigned to five groups to receive a single dose (2.5 mg, 5 mg or 10 mg) or multiple doses (5 mg) of oral medications of R-bambuterol, or a single dose of rac-bambuterol (10 mg). Plasma lipid levels were measured at baseline, time to peak concentration (Tmax) and 24 h after the treatment. Findings Administration of a single-dose of R-bambuterol resulted in dose-dependent reductions in the levels of plasma LDL-C, high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), apolipoprotein B (ApoB) and apolipoprotein A1 (ApoA1) at Tmax. Levels of LDL-C exhibited the most reductions, which were statistically significant in all three single-dose R-bambuterol groups (all P values, Highlights • Oral administration of R-bambuterol, a long-acting bronchodilator, can significantly lower LDL cholesterol in healthy Chinese volunteers. • R-bambuterol was more potent than rac-bambuterol in lowering LDL-C and raising the ratio of ApoA1/ApoB. • It may provide an alternative for treating high cholesterol, especially for those also suffering from COPD.

Past studies have revealed major difficulties in applications of xylanase in the pulp and paper industry as enzymes isolated from many different species could not tolerate high temperatures or highly alkaline conditions. The thermostable xylanase A from Bacillus halodurans C-125 (C-125 xylanase A) was successfully cloned and expressed in Pichia pastoris with a yield as high as 3361 U/mL in a 2 L reactor. Its thermophilic and basophilic properties (optimal activity at 70 °C and pH 9.0), together with the fact it is cellulase-free, render this enzyme attractive for compatible applications in the pulp and paper industry. The pretreatment of wheat straw pulp with C-125 xylanase A at pH 9.0 and 70 °C for 90 min induced the release of both chromophores (Ab(237), Ab(254), Ab(280)) and hydrophobic compounds (Ab(465)) into the filtrate as well as sugar degradation. Moreover, the addition of 10 U xylanase to 1 g wheat straw pulp (dry weight) as pretreatment improved brightness by 5.2% ISO and decreased the kappa number by 5.0% when followed by hydrogen peroxide bleaching. In addition, compared with two commercial enzymes, Pulpzyme HC and AU-PE89, which are normally incorporated in ECF bleaching of wheat straw pulp, C-125 xylanase A proved to be more effective in enhancing brightness as well as preserving paper strength properties. When evaluating the physical properties of pulp samples, such as tensile index, tearing index, bursting index, and post-color (PC) number, the enzymes involved in pretreating pulps exhibited better or the same performances as chemical treatment. Compared with chemical bleaching, chlorine consumption can be significantly reduced by 10% for xylanase-pretreated wheat straw pulp while maintaining the brightness together with the kappa number at the same level. Scanning electron microscopy revealed significant surface modification of enzyme-pretreated pulp fibers with no marked fiber disruptions.

Conventional lipase screening methods are mostly based on hydrolytic activity, which may not always be the best method to assess the enzyme activity, especially for evaluating synthetic activity. Here we developed a high throughput and visual method to screen clones with high synthetic activity and used it to assess lipases thermostability. All mutants’ lipase synthetic activity were identified through esterification of caprylic acid and ethanol with methyl red as the pH indicator adding in the substrates on according to the color change halo around the colony on culture plates since synthetic reaction was often accompanied with a rise in pH. After two rounds operation with the pH indicator screening method, we obtained a double mutant Asn120Lys/Lys131Phe from the Rhizomucor miehei lipase saturation mutated library based on amino acid residue B factors. The mutant's initial synthetic activity was a little higher than wild type and its thermostability in synthetic reaction was enhanced, which remained 63.1% residual activity after being heated at 70 °C for 5 h comparing to 51.0% of wild type. The double mutant with the two residue replacements balanced well between stability and activity. Yeast surface display technology and the pH indicator method, combined with colony screening were shown to facilitate high-throughput screening for lipase synthetic activity.

Trehalose and glycerol have been implicated as potential stress protectants that accumulate in yeasts during various stress conditions. We investigated the levels of glycerol and trehalose and the expression profiles of genes involved in their metabolism to determine their involvement in the response of Saccharomyces cerevisiae XQ1 to thermal, sorbitol and ethanol stresses. The results showed that the genes involved in the synthesis and degradation of trehalose and glycerol were stress induced, and that trehalose and glycerol were synthesized simultaneously during the initial stages (a sensitive response period) of diverse stress treatments. Trehalose accumulated markedly under heat treatment, but not under sorbitol or ethanol stress, whereas glycerol accumulated strikingly under sorbitol stress conditions. Interestingly, extracellular trehalose seemed to be involved in protecting cells from damage under unfavorable conditions. Moreover, our results suggest that the stress-activated futile ATP cycles of trehalose and glycerol turnover are of general importance during cellular stress adaptation.

Extensive alteration of gene expression and metabolic remodeling enable the budding yeast Saccharomyces cerevisiae to ensure cellular homeostasis and adaptation to heat shock. The response logic of the cells to heat shock is still not entirely clear. In this study, we combined the expression profiles with metabolic pathways to investigate the logical relations between heat shock response metabolic pathways. The results showed that the heat-stressed S. cerevisiae cell accumulated trehalose and glycogen, which protect cellular proteins against denaturation, and modulate its phospholipid structure to sustain stability of the cell wall. The TCA cycle was enhanced, and the heat shock-induced turnover of amino acids and nucleotides served to meet the extra energy requirement due to heat-induced protein metabolism and modification. The enhanced respiration led to oxidative stress, and subsequently induced the aldehyde detoxification system. These results indicated that new insight into the response logic of S. cerevisiae to heat shock can be gained by integrating expression profiles and the logical relations between heat shock response metabolic pathways.

Measuring the viability of the cells after chemical or physical stresses is a routine method in the determination of phenotypes in yeast. Numerous analytical techniques have been applied to the measurement of yeast cell viability, but few of them are suitable for real-time monitoring. Here we propose a microplate-based method for real-time monitoring of Saccharomyces cerevisiae cell viability. This method is also suitable for high-throughput measurement.