Monitoring growth phase-related changes in phosphatidylcholine-specific phospholipase C production,adhesion properties and physiology of <Emphasis Type="Italic">Bacillus cereus</Emphasis> vegetative cells

The physiological status and metabolic heterogeneity of Bacillus cereus cells within a culture during an 8-h batch fermentation process was measured using flow cytometry (FCM). Concurrently, production of the toxin, PC-PLC, and the extent of cell adhesion of live and dead cells were monitored using novel fluorescent assays. Flow cytometry analysis detected growth phase-related changes in the physiological profiles of cells over the course of the fermentation, with variation in the percentage of cells displaying membrane damage and intracellular esterase and redox activities. As the exponential phase proceeded, populations became more uniform in terms of protein content as measured using FCM in tandem with a cell tracking dye, with the majority of cells becoming membrane intact, esterase positive and redox active. PC-PLC activity appeared strongly related to cell density. Permeabilisation of cells was accompanied by a loss in adherent properties, while 25–100% of cells with intracellular esterase activity possessed adhesion properties. Cells in late exponential phase appeared to have reduced adherence properties compared to cells in early exponential or lag phase. As well as demonstrating the utility of FCM for measuring heterogeneity in terms of cell physiological status throughout the course of batch cultures, the methods utilised in this study could be used to relate processes such as toxin production or cell adhesion to cell physiological state.     

Antibiotic amoxicillin induces DNA lesions in mammalian cells possibly via the reactive oxygen species

Amoxicillin is a commonly prescribed drug for anti- bacterial infection. In this study, we are interested in the effect of the drug on the cellular DNA integrity. Amoxicillin was added to the human or hamster cells in culture, and the DNA lesions induced by the drug were assessed by a comet assay with nuclear extract incubation (Wang et al., 2005 Anal Biochem 337: 70-75). Amoxicillin at 5mM rapidly induced DNA lesions in human AGS cells. The level of DNA lesions attained a maximum at about 1h, and then declined steadily and reached almost the basal level at 6h following the drug treatment. Similar induction pattern of DNA lesions was found with amoxicillin-related antibiotics such as ampicillin but not with the unrelated antibiotics such as kanamycin. For studying the repair kinetics, the cells were treated with amoxicillin for only 1h and continued culture in the absence of the drug for a certain period of time before subsequent analysis. Repair of the amoxicillin-induced DNA lesions was essentially completed within 4h. Such repair may not involve nucleotide excision repair (NER) pathway because the repair was completed with similar kinetics in both NER proficient Chinese hamster CHO-K1 cells and its isogenic NER deficient UV24 cells. Instead, the repair may involve base excision repair (BER) pathway because immunodepletion of OGG1/2, glycosylases involved in BER rendered the nuclear extract unable to excise DNA lesions induced by amoxicillin in the modified comet assay. Furthermore, amoxicillin induced intracellular reactive oxygen species (ROS) at the tempo similar to that of DNA lesions induction. Thus, we hypothesize that amoxicillin causes oxidative DNA damage in mammalian cells via ROS.     

Isolation and characterization of enteroaggregative,enterotoxigenic, diffusely adherent <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella</Emphasis> Worthington from human diarrhoeic faecal samples in Kashmir and Secunderabad,India

Seven hundred and thirty-five diarrhoeic faecal samples from children were investigated for presence of enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), diffusely adherent E. coli (DAEC) and Salmonella spp. by polymerase chain reaction (PCR) and bacterial culture. Out of 675 samples from Kashmir, 55 isolates were obtained, which carried at least one virulence gene studied. Out of the 55 isolates, 36 (65.45%) were EAEC, 18 (32.72%) were ETEC while only one isolate (1.81%) was DAEC. All the EAEC isolates were found to be typical as they possessed aggR gene. Six (16.66%) EAEC isolates carried the astA gene. Out of the 18 ETEC isolates, 13 carried the elt gene alone, four possessed both the elt and est genes and the remaining one harboured the est gene alone. Five ETEC isolates also possessed astA gene. Nineteen EAEC isolates belonged to 10 different serogroups. Serogroup O153 was most frequent. The ETEC isolates also belonged to 10 different serogroups of which O159 was most predominant. Out of 224 E. coli isolates from 60 samples of Secunderabad, 27 isolates carried at least one virulence gene. Out of 27 isolates 22 (81.48%) were typical EAEC, three (11.11%) were ETEC and two (7.4%) were DAEC. Fifteen EAEC isolates belonged to seven different serogroups with O86 as most frequent. Four EAEC isolates also possessed the astA gene. All the three ETEC isolates harboured elt gene only and belonged to three different serogroups. Two isolates of Salmonella Worthington were obtained from only two samples in Kashmir.     

SNP-based pool genotyping and haplotype analysis accelerate fine-mapping of the wheat genomic region containing stripe rust resistance gene <Emphasis Type="Italic">Yr26</Emphasis>

Key message

NGS-assisted super pooling emerging as powerful tool to accelerate gene mapping and haplotype association analysis within target region uncovering specific linkage SNPs or alleles for marker-assisted gene pyramiding.

Abstract

Conventional gene mapping methods to identify genes associated with important agronomic traits require significant amounts of financial support and time. Here, a single nucleotide polymorphism (SNP)-based mapping approach, RNA-Seq and SNP array assisted super pooling analysis, was used for rapid mining of a candidate genomic region for stripe rust resistance gene Yr26 that has been widely used in wheat breeding programs in China. Large DNA and RNA super-pools were genotyped by Wheat SNP Array and sequenced by Illumina HiSeq, respectively. Hundreds of thousands of SNPs were identified and then filtered by multiple filtering criteria. Among selected SNPs, over 900 were found within an overlapping interval of less than 30 Mb as the Yr26 candidate genomic region in the centromeric region of chromosome arm 1BL. The 235 chromosome-specific SNPs were converted into KASP assays to validate the Yr26 interval in different genetic populations. Using a high-resolution mapping population (>?30,000 gametes), we confined Yr26 to a 0.003-cM interval. The Yr26 target region was anchored to the common wheat IWGSC RefSeq v1.0 and wild emmer WEWSeq v.1.0 sequences, from which 488 and 454 kb fragments were obtained. Several candidate genes were identified in the target genomic region, but there was no typical resistance gene in either genome region. Haplotype analysis identified specific SNPs linked to Yr26 and developed robust and breeder-friendly KASP markers. This integration strategy can be applied to accelerate generating many markers closely linked to target genes/QTL for a trait of interest in wheat and other polyploid species.

     

Characterization of the tunicamycin gene cluster unveiling unique steps involved in its biosynthesis

Tunicamycin, a potent reversible translocase I inhibitor, is produced by several Actinomycetes species. The tunicamycin structure is highly unusual, and contains an 11-carbon dialdose sugar and an α, β-1″,11′-glycosidic linkage. Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression (HHE) strategy combined with a bioassay. Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains, demonstrating the role of the genes for the biosynthesis of tunicamycins. Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes (tunA– tunL). Amongst these is a putative radical SAM enzyme (Tun B) with a potentially unique role in biosynthetic carbon-carbon bond formation. Hence, a seven-step novel pathway is proposed for tunicamycin biosynthesis. Moreover, two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827. These data provide clarification of the novel mechanisms for tunicamycin biosynthesis, and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.     

Synthesis of Monomethoxypolyethyleneglycol—Cholesteryl Ester and Effect of its Incorporation in Liposomes

The objective of the present study was to synthesize monomethoxypolyethyleneglycol-5000 cholesteryl ester [PEG–CH] as a cost-effective substitute for polyethyleneglycol–phosphatidylethanolamine and to evaluate the influence of its incorporation in liposomal bilayers for surface modification. PEG–CH was synthesized and characterized by infrared (IR), proton nuclear magnetic resonance spectroscopy (¹H NMR), and differential scanning calorimetry (DSC) studies. Influence of incorporation of PEG–CH in liposomes was evaluated on various parameters such as zeta potential, DSC, and encapsulation efficiency of a hydrophilic drug pentoxyfylline. Conventional and PEG–CH containing pentoxyfylline liposomes were formulated and their stability was evaluated at 4°C for 3 months. PEG–CH could be successfully synthesized with good yields and the structure was confirmed by IR, DSC, and ¹H NMR. The incorporation of PEG–CH in liposomes resulted in reduction of the zeta potential and broadening of the DSC endotherm. Furthermore, incorporation of PEG–CH in liposomes decreased the encapsulation efficiency of pentoxifylline in liposomes when compared to conventional liposomes. Conventional and PEG–CH containing pentoxyfylline liposomes did not show any signs of pentoxyfylline degradation when stored at 4°C for 3 months.     

A new species of <Emphasis Type="Italic">Alcantarea</Emphasis> (Bromeliaceae,Tillandsioideae) from Serra dos Órgãos,Rio de Janeiro,Brazil

Alcantarea martinellii is described and illustrated as a new species endemic to Serra dos Órgãos, Petrópolis, Rio de Janeiro State, Brazil. The presence of a simple and erect inflorescence concomitant with a conspicuous water impounding rosette is an unreported combination of characters for Alcantarea. This new lithophytic taxon is unique, with no obvious close relative, but it is similar to A. benzingii, A. farneyi, and A. hatschbachii, which also have unbranched inflorescences.     

High CO2 levels impair alveolar epithelial function independently of pH

Background

In patients with acute respiratory failure, gas exchange is impaired due to the accumulation of fluid in the lung airspaces. This life-threatening syndrome is treated with mechanical ventilation, which is adjusted to maintain gas exchange, but can be associated with the accumulation of carbon dioxide in the lung. Carbon dioxide (CO₂) is a by-product of cellular energy utilization and its elimination is affected via alveolar epithelial cells. Signaling pathways sensitive to changes in CO₂ levels were described in plants and neuronal mammalian cells. However, it has not been fully elucidated whether non-neuronal cells sense and respond to CO₂. The Na,K-ATPase consumes ∼40% of the cellular metabolism to maintain cell homeostasis. Our study examines the effects of increased pCO₂ on the epithelial Na,K-ATPase a major contributor to alveolar fluid reabsorption which is a marker of alveolar epithelial function.

Principal Findings

We found that short-term increases in pCO₂ impaired alveolar fluid reabsorption in rats. Also, we provide evidence that non-excitable, alveolar epithelial cells sense and respond to high levels of CO₂, independently of extracellular and intracellular pH, by inhibiting Na,K-ATPase function, via activation of PKCζ which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into intracellular pools.

Conclusions

Our data suggest that alveolar epithelial cells, through which CO₂ is eliminated in mammals, are highly sensitive to hypercapnia. Elevated CO₂ levels impair alveolar epithelial function, independently of pH, which is relevant in patients with lung diseases and altered alveolar gas exchange.