Bacteriocinogenic Potential of Bacillus amyloliquefaciens Isolated from Kimchi,a Traditional Korean Fermented Cabbage

Bacteriocin production is considered a favorable property for various beneficial cultures. In addition to their potential as biopreservatives, bacteriocins are also promising alternatives for the control of multidrug-resistant pathogens and the inhibition of some viruses and cancer cells. The objective of this study was to screen and characterize a bacteriocin-producing strain with the aim of its future application for control of Listeria monocytogenes, an important food-borne pathogen. A total of 22 potentially bacteriocinogenic strains active against L. monocytogenes ATCC15313 were isolated from locally produced kimchi through a three-level approach. Pure cultures were obtained according to good microbiological practices and differentiated through RAPD-PCR using the primers OPL01, OPL09, and OPL11. Altogether, 5 strains were selected for further study. Specific focus was given to strain ST05DL based on its specific inhibitory activity against L. monocytogenes ATCC15313, while not affecting different strains belonging to the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella, most of which are beneficial microorganisms. The strain ST05DL was identified as Bacillus amyloliquefaciens based on its sugar fermentation profile obtained through API50CHB analysis and 16S rRNA partial sequencing. The antimicrobial compound produced by B. amyloliquefaciens ST05DL was found to be sensitive to pepsin and α-chymotrypsin, evidence of its proteinaceous nature. The presence of skim milk, NaCl, Tween 80, glycerol, and SDS did not affect the antimicrobial activity. The addition of 20% cell-free supernatant (CFS) obtained from a 24-h culture of B. amyloliquefaciens ST05DL to an exponentially growing culture of L. monocytogenes ATCC15313 successfully inhibited the test microorganisms during the monitored 10-h incubation. Optimal bacteriocin production by B. amyloliquefaciens ST05DL was observed during the stationary phase at 12 h (800 AU/mL) and remained stable for the next 15 h. The ratio between live and dead cells during this period was 74.37% and 25.66%, respectively, as determined by flow cytometry. The presence of the virulence genes hblA, hblB, hblC, nheA, nheB, and nheC was not detected in the total DNA of B. amyloliquefaciens ST05DL, and the strain was resistant only to ampicillin out of 10 tested antibiotics. Future evaluation of expressed bacteriocin/s by B. amyloliquefaciens ST05DL (amino acid sequence, molecular mass, cytotoxicity, detailed mode of action, etc.), will be the next step in the characterization and its potential application as biopreservative and/or pharmaceutical product.

     

Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)

The presence of acetate exceeding 5 g/L is a major concern during E. coli fermentation due to its inhibitory effect on cell growth, thereby limiting high-density cell culture and recombinant protein production. Hence, engineered E. coli strains with enhanced acetate tolerance would be valuable for these bioprocesses. In this work, the acetate tolerance of E. coli was much improved by rewiring its global regulator cAMP receptor protein (CRP), which is reported to regulate 444 genes. Error-prone PCR method was employed to modify crp and the mutagenesis libraries (~3×10⁶) were subjected to M9 minimal medium supplemented with 5–10 g/L sodium acetate for selection. Mutant A2 (D138Y) was isolated and its growth rate in 15 g/L sodium acetate was found to be 0.083 h^-1, much higher than that of the control (0.016 h^-1). Real-time PCR analysis via OpenArray^® system revealed that over 400 CRP-regulated genes were differentially expressed in A2 with or without acetate stress, including those involved in the TCA cycle, phosphotransferase system, etc. Eight genes were chosen for overexpression and the overexpression of uxaB was found to lead to E. coli acetate sensitivity.     

HER2 Phosphorylates and Destabilizes Pro-Apoptotic PUMA,Leading to Antagonized Apoptosis in Cancer Cells

HER2 is overexpressed in 15–20% of breast cancers. HER2 overexpression is known to reduce apoptosis but the underlying mechanisms for this association remain unclear. To elucidate the mechanisms for HER2-mediated survival, we investigated the relationship between HER2 and p53 upregulated modulator of apoptosis (PUMA), a potent apoptosis inducer. Our results showed that HER2 interacts with PUMA, which was independent of HER2 activation. In addition, we observed that HER2 interacted with PUMA in both mitochondrial and non-mitochondrial compartments. We next examined whether HER2 phosphorylates PUMA. Notably, PUMA tyrosine phosphorylation has never been reported. Using an intracellular assay, we found PUMA to be phosphorylated in breast cancer cells with activated HER2. Via cell-free HER2 kinase assay, we observed that PUMA was directly phosphorylated by HER2. Activation of HER2 decreased PUMA protein half-life. To identify which of the three tyrosines within PUMA are targeted by HER2, we generated three PUMA non-phosphorylation mutants each with a single Tyr→Phe substitution. Results indicated that each PUMA single mutant had lost some, but not all phosphorylation by HER2 indicating that HER2 targets all three tyrosines. Consequently, we created an additional PUMA mutant with all three tyrosines mutated (TM-PUMA) that could not be phosphorylated by HER2. Importantly, TM-PUMA was found to have a longer half-life than PUMA. An inverse association was observed between HER2 and PUMA in 93 invasive breast carcinoma samples. We further found that TM-PUMA suppressed growth of breast cancer cells to a greater degree than PUMA. Also, TM-PUMA had a stronger propensity to induce apoptosis than PUMA. Together, our results demonstrate, for the first time, that PUMA can be tyrosine phosphorylated and that HER2-mediated phosphorylation destabilizes PUMA protein. The HER2-PUMA interplay represents a novel mechanism by which PUMA is regulated and a new molecular basis for HER2-mediated growth and survival of cancer cells.     

A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14⁺ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.     

Impacts of natural history and exhibit factors on carnivore welfare

To improve the welfare of nonhuman animals under professional care, zoological institutions are continuously utilizing new methods to identify factors that lead to optimal welfare. Comparative methods have historically been used in the field of evolutionary biology but are increasingly being applied in the field of animal welfare. In the current study, data were obtained from direct behavioral observation and institutional records representing 80 individual animals from 34 different species of the order Carnivora. Data were examined to determine if a variety of natural history and animal management factors impacted the welfare of animals in zoological institutions. Output variables indicating welfare status included behavioral diversity, pacing, offspring production, and infant mortality. Results suggested that generalist species have higher behavioral diversity and offspring production in zoos compared with their specialist counterparts. In addition, increased minimum distance from the public decreased pacing and increased offspring production, while increased maximum distance from the public and large enclosure size decreased infant mortality. These results have implications for future exhibit design or renovation, as well as management practices and priorities for future research.     

Attenuation of insulin resistance by chronic beta2-adrenergic agonist treatment possible muscle specific contributions

A possible mechanism by which chronic clenbuterol treatment causes multiple physiological changes in skeletal muscle that leads to reduced insulin resistance in the obese Zucker rat (falfa) was investigated. Animals were gavaged with clenbuterol (CB) (0.8 mg x kg(-1) day(-1)), terbutaline (TB) (1.0 mg x kg(-1)day(-1)), or control (CT) vehicle for six weeks. Oral glucose tolerance and insulin responses were markedly improved in CB rats and impaired in TB rats. CB treatment caused a 24-34% gain in muscle mass in all muscle fiber types, and increases in 3-O-methyglucose transport (2-fold) and GLUT4 concentration (57%) in fast twitch glycolytic (FG) muscle. Oxidative capacity was reduced in both FG (47%) and fast twitch oxidative (FO) muscle (30%), but not in slow twitch oxidative (SO) muscle. Null model analysis for receptor occlusion demonstrated that most functional beta-adrenoceptors were lost in FO (82%) and FG (89%) fibers, but not in SO fibers. We propose that hypertrophy is the result of continuous direct activation of beta-adrenoceptors while loss in oxidative capacity may be the result of receptor down regulation. Improvements in insulin resistance may have been due, in part, to both increases in lean body mass and specific adaptations in FG muscle.