Microdiversity and temporal dynamics of marine bacterial dimethylsulfoniopropionate genes

Dimethylsulfoniopropionate (DMSP) is an abundant organic sulfur metabolite produced by many phytoplankton species and degraded by bacteria via two distinct pathways with climate-relevant implications. We assessed the diversity and abundance of bacteria possessing these pathways in the context of phytoplankton community composition over a 3-week time period spanning September–October, 2014 in Monterey Bay, CA. The dmdA gene from the DMSP demethylation pathway dominated the DMSP gene pool and was harboured mostly by members of the alphaproteobacterial SAR11 clade and secondarily by the Roseobacter group, particularly during the second half of the study. Novel members of the DMSP-degrading community emerged from dmdA sequences recovered from metagenome assemblies and single-cell sequencing, including largely uncharacterized gammaproteobacteria and alphaproteobacteria taxa. In the DMSP cleavage pathway, the SAR11 gene dddK was the most abundant early in the study, but was supplanted by dddP over time. SAR11 members, especially those harbouring genes for both DMSP degradation pathways, had a strong positive relationship with the abundance of dinoflagellates, and DMSP-degrading gammaproteobacteria co-occurred with haptophytes. This in situ study of the drivers of DMSP fate in a coastal ecosystem demonstrates for the first time correlations between specific groups of bacterial DMSP degraders and phytoplankton taxa.     

Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibody during fed-batch culture

In this study we have analyzed the dynamic covariation of the mammalian cell proteome with respect to functional phenotype during fed-batch culture of NS0 murine myeloma cells producing a recombinant IgG(4) monoclonal antibody. GS-NS0 cells were cultured in duplicate 10 L bioreactors (36.5 degrees C, 15% DOT, pH 7.0) for 335 h and supplemented with a continuous feed stream after 120 h. Cell-specific growth rate declined continuously after 72 h of culture. Cell-specific recombinant monoclonal antibody production rate (qP) varied sixfold through culture. Whilst qP correlated with relative recombinant heavy chain mRNA abundance up to 216 h, qP subsequently declined, independent of recombinant heavy chain or light chain mRNA abundance. GS-NS0 cultures were sampled at 48 h intervals between 24 and 264 h of culture for proteomic analyses. Total protein abundance and nascent polypeptide synthesis was determined by 2D PAGE of unlabeled proteins visualized by SYPRO Ruby and autoradiography of (35)S-labeled polypeptides, respectively. Covariation of nascent polypeptide synthesis and abundance with biomass-specific cell growth, glucose and glutamate consumption, lactate and Mab production rates were then examined using two partial least squares regression models. Most changes in polypeptide synthesis or abundance for proteins previously identified by mass spectrometry were positively correlated with biomass-specific growth rate. We conclude that the substantial transitions in cell physiology and qP that occur during culture utilize a relatively constant complement of the most abundant host cell machines that vary primarily with respect to induced changes in cell growth rate.     

Parent overweight predicts daughters' increase in BMI and disinhibited overeating from 5 to 13 years

Objective: To assess whether parental overweight status and disinhibited overeating are predictive of daughters’ accelerated weight gain and disinhibited overeating. Research Methods and Procedures: Participants were part of a longitudinal study of girls (N = 197) and their parents. Measured height and weight were used to calculate BMI [weight (kilograms)/height (meters)²]. Parents’ disinhibited eating behavior was assessed using the Eating Inventory. Girls’ disinhibited eating was assessed using a behavioral protocol to measure eating in the absence of hunger. Girls were classified based on parental overweight at study entry into four groups: neither, mother only, father only, or both parents overweight. Results: Girls with both parents overweight had the most rapid increases in BMI from 5 to 13 years of age; BMI increased most slowly among the neither parent overweight group, with intermediate increases in BMI among mother only and father only overweight groups. Daughters with both parents overweight at study entry were eight times more likely to be overweight at age 13, controlling for daughters’ weight at age 5. Girls with both parents overweight had higher levels of disinhibited eating across all ages than all other groups. Although girls in all parental weight status groups showed increases in disinhibited eating over time, girls with both parents overweight had larger increases in disinhibited eating over time compared with all other groups. Discussion: Girls growing up in families differing in parental overweight had divergent developmental trajectories for BMI and disinhibited overeating. Findings reveal the need to focus prevention efforts on overweight parents of young children.     

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.     

Chloride-sensitive fluorescent indicators

Three fluorescent halide-sensitive quinolinium dyes have been produced by the reaction of the 6-methylquinoline heterocyclic nitrogen base with methyl bromide, methyl iodide, and 3-bromo-1-propanol. The quaternary salts, unlike the precursor molecule, are readily water soluble and the fluorescence intensity of these salts is reduced in the presence of aqueous chloride, bromide, and iodide ions, allowing halide solution concentrations to be determined using well-known Stern-Volmer kinetics. One of the dyes, dye 1, has a chloride Stern-Volmer constant of 255 mol(-1) dm(3) which is more than twice that of SPQ [6-methoxy-N-(3-sulfopropyl)quinolinium] used in recent physiological measurements to measure intracellular chloride levels. The dyes have been characterized using steady-state fluorescence spectroscopy and are compared to three similar dyes based on the 6-methoxyquinoline nucleus, reported earlier by the authors, and also to dyes reported by Krapf et al. (Anal. Biochem. 169, 142-150, 1988). The interference of aqueous anions and the potential for using these dyes in biological halide-sensing applications are discussed.     

Identification of glucosinolates on the leaf surface of plants from the Cruciferae and other closely related species

Leaf-surface extracts prepared from 18 non-cultivated (wild) plant species, derived from the Capparidaceae, Cruciferae, Resedaceae and Tropaeolaceae were ranked for their ability to stimulate oviposition by the cabbage root fly, and analysed for glucosinolates. A total of 28 different glucosinolates were identified. A clear relationship was detected between the indolyl-, benzyl- and the total glucosinolate composition on the leaf surface and oviposition preference by cabbage root fly females. However, as the results are not fully explained by differences in leaf surface glucosinolates, other important oviposition deterrents and stimuli on the leaf surface of these wild crucifers must also be present.     

A cysteine protease gene is expressed early in resistant potato interactions with Phytophthora infestans

A potato cysteine protease (cyp) cDNA expressed at an early stage of an incompatible interaction with Phytophthora infestans was isolated. Both the nucleotide and deduced amino acid sequences are highly homologous to those of a tomato cysteine protease, CYP1. Striking protein similarity to all known cathepsins in animals, particularly cathepsin K, was also observed. However, unlike cathepsins, a granulin binding domain is located near the carboxyl terminus of the putative CYP protein. In animals, granulins bind to receptors in the plasma membrane and signal cell growth and division. A ribonuclease protection assay demonstrated that the cyp gene is tightly regulated and is induced 15 h post inoculation with P. infestans in potato leaves either with high field resistance or in which a resistance (R) gene is activated. We conclude that a common signaling pathway is activated in each form of resistance.