Bacteriophage Receptor Binding Protein Based Assays for the Simultaneous Detection of Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of foodborne gastroenteritis which is occasionally followed by a debilitating neuropathy known as Guillain-Barré syndrome. Rapid and specific detection of these pathogens is very important for effective control and quick treatment of infection. Most of the diagnostics available for these organisms are time consuming and require technical expertise with expensive instruments and reagents to perform. Bacteriophages bind to their host specifically through their receptor binding proteins (RBPs), which can be exploited for pathogen detection. We recently sequenced the genome of C. jejuni phage NCTC12673 and identified its putative host receptor binding protein, Gp047. In the current study, we localized the receptor binding domain to the C-terminal quarter of Gp047. CC-Gp047 could be produced recombinantly and was capable of agglutinating both C. jejuni and C. coli cells unlike the host range of the parent phage which is limited to a subset of C. jejuni isolates. The agglutination procedure could be performed within minutes on a glass slide at room temperature and was not hindered by the presence of buffers or nutrient media. This agglutination assay showed 100% specificity and the sensitivity was 95% for C. jejuni (n = 40) and 90% for C. coli (n = 19). CC-Gp047 was also expressed as a fusion with enhanced green fluorescent protein (EGFP). Chimeric EGFP_CC-Gp047 was able to specifically label C. jejuni and C. coli cells in mixed cultures allowing for the detection of these pathogens by fluorescent microscopy. This study describes a simple and rapid method for the detection of C. jejuni and C. coli using engineered phage RBPs and offers a promising new diagnostics platform for healthcare and surveillance laboratories.     

Impact of differential DNA methylation on transgene expression in cotton (Gossypium hirsutum L.) events generated by targeted sequence insertion

Targeted Genome Optimization (TGO) using site‐specific nucleases to introduce a DNA double‐strand break (DSB) at a specific target locus has broadened the options available to breeders for generation and combination of multiple traits. The use of targeted DNA cleavage in combination with homologous recombination (HR)‐mediated repair, enabled the precise targeted insertion of additional trait genes (2mepsps, hppd, axmi115) at a pre‐existing transgenic locus in cotton. Here we describe the expression and epigenome analyses of cotton Targeted Sequence Insertion (TSI) events over generations. In a subset of events, we observed variability in the level of transgene (hppd, axmi115) expression between independent but genetically identical TSI events. Transgene expression could also be differential within single events and variable over generations. This expression variability and silencing occurred independently of the transgene sequence and could be attributed to DNA methylation that was further linked to different DNA methylation mechanisms. The trigger(s) of transgene DNA methylation remains elusive but we hypothesize that targeted DSB induction and repair could be a potential trigger for DNA methylation.     

SIRT2 regulates adipocyte differentiation through FoxO1 acetylation/deacetylation

The family of mammalian Sirtuin proteins comprises seven members homologous to yeast Sir2. Here we show that SIRT2, a cytoplasmic sirtuin, is the most abundant sirtuin in adipocytes. Sirt2 expression is downregulated during preadipocyte differentiation in 3T3-L1 cells. Overexpression of SIRT2 inhibits differentiation, whereas reducing SIRT2 expression promotes adipogenesis. Both effects are accompanied by corresponding changes in the expression of PPARgamma, C/EBPalpha, and genes marking terminal adipocyte differentiation, including Glut4, aP2, and fatty acid synthase. The mechanism underlying the effects of reduced SIRT2 in 3T3-L1 adipocytes includes increased acetylation of FOXO1, with direct interaction between SIRT2 and FOXO1. This interaction enhances insulin-stimulated phosphorylation of FOXO1, which in turn regulates FOXO1 nuclear and cytosolic localization. Thus, Sirt2 acts as an important regulator of adipocyte differentiation through modulation of FOXO1 acetylation/phosphorylation and activity and may play a role in controlling adipose tissue mass and function.     

Biogeography of Southern Ocean prokaryotes: a comparison of the Indian and Pacific sectors

We investigated the Southern Ocean (SO) prokaryote community structure via zero-radius operational taxonomic unit (zOTU) libraries generated from 16S rRNA gene sequencing of 223 full water column profiles. Samples reveal the prokaryote diversity trend between discrete water masses across multiple depths and latitudes in Indian (71–99°E, summer) and Pacific (170–174°W, autumn-winter) sectors of the SO. At higher taxonomic levels (phylum-family) we observed water masses to harbour distinct communities across both sectors, but observed sectorial variations at lower taxonomic levels (genus-zOTU) and relative abundance shifts for key taxa such as Flavobacteria, SAR324/Marinimicrobia, Nitrosopumilus and Nitrosopelagicus at both epi- and bathy-abyssopelagic water masses. Common surface bacteria were abundant in several deep-water masses and vice-versa suggesting connectivity between surface and deep-water microbial assemblages. Bacteria from same-sector Antarctic Bottom Water samples showed patchy, high beta-diversity which did not correlate well with measured environmental parameters or geographical distance. Unconventional depth distribution patterns were observed for key archaeal groups: Crenarchaeota was found across all depths in the water column and persistent high relative abundances of common epipelagic archaeon Nitrosopelagicus was observed in deep-water masses. Our findings reveal substantial regional variability of SO prokaryote assemblages that we argue should be considered in wide-scale SO ecosystem microbial modelling.     

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.