Lipid remodelling is a widespread strategy in marine heterotrophic bacteria upon phosphorus deficiency

Upon phosphorus (P) deficiency, marine phytoplankton reduce their requirements for P by replacing membrane phospholipids with alternative non-phosphorus lipids. It was very recently demonstrated that a SAR11 isolate also shares this capability when phosphate starved in culture. Yet, the extent to which this process occurs in other marine heterotrophic bacteria and in the natural environment is unknown. Here, we demonstrate that the substitution of membrane phospholipids for a variety of non-phosphorus lipids is a conserved response to P deficiency among phylogenetically diverse marine heterotrophic bacteria, including members of the Alphaproteobacteria and Flavobacteria. By deletion mutagenesis and complementation in the model marine bacterium Phaeobacter sp. MED193 and heterologous expression in recombinant Escherichia coli, we confirm the roles of a phospholipase C (PlcP) and a glycosyltransferase in lipid remodelling. Analyses of the Global Ocean Sampling and Tara Oceans metagenome data sets demonstrate that PlcP is particularly abundant in areas characterized by low phosphate concentrations. Furthermore, we show that lipid remodelling occurs seasonally and responds to changing nutrient conditions in natural microbial communities from the Mediterranean Sea. Together, our results point to the key role of lipid substitution as an adaptive strategy enabling heterotrophic bacteria to thrive in the vast P-depleted areas of the ocean.     

Effects of non-thermal plasma on mammalian cells

Thermal plasmas and lasers have been widely used in medicine to cut, ablate and cauterize tissues through heating; in contrast, non-thermal plasma produces no heat, so its effects can be selective. In order to exploit the potential for clinical applications, including wound healing, sterilization, blood coagulation, and cancer treatment, a mechanistic understanding of the interaction of non-thermal plasma with living tissues is required. Using mammalian cells in culture, it is shown here that non-thermal plasma created by dielectric barrier discharge (DBD) has dose-dependent effects that range from increasing cell proliferation to inducing apoptosis. It is also shown that these effects are primarily due to formation of intracellular reactive oxygen species (ROS). We have utilized γ-H2AX to detect DNA damage induced by non-thermal plasma and found that it is initiated by production of active neutral species that most likely induce formation of organic peroxides in cell medium. Phosphorylation of H2AX following non-thermal plasma treatment is ATR dependent and ATM independent, suggesting that plasma treatment may lead to replication arrest or formation of single-stranded DNA breaks; however, plasma does not lead to formation of bulky adducts/thymine dimers.     

Brain plasticity in the adult: modulation of function in amblyopia with rTMS

Amblyopia is a cortically based visual disorder caused by disruption of vision during a critical early developmental period. It is often thought to be a largely intractable problem in adult patients because of a lack of neuronal plasticity after this critical period [1]; however, recent advances have suggested that plasticity is still present in the adult amblyopic visual cortex [2-6]. Here, we present data showing that repetitive transcranial magnetic stimulation (rTMS) of the visual cortex can temporarily improve contrast sensitivity in the amblyopic visual cortex. The results indicate continued plasticity of the amblyopic visual system in adulthood and open the way for a potential new therapeutic approach to the treatment of amblyopia.     

Microbial community analysis of a coastal salt marsh affected by the Deepwater Horizon oil spill

Coastal salt marshes are highly sensitive wetland ecosystems that can sustain long-term impacts from anthropogenic events such as oil spills. In this study, we examined the microbial communities of a Gulf of Mexico coastal salt marsh during and after the influx of petroleum hydrocarbons following the Deepwater Horizon oil spill. Total hydrocarbon concentrations in salt marsh sediments were highest in June and July 2010 and decreased in September 2010. Coupled PhyloChip and GeoChip microarray analyses demonstrated that the microbial community structure and function of the extant salt marsh hydrocarbon-degrading microbial populations changed significantly during the study. The relative richness and abundance of phyla containing previously described hydrocarbon-degrading bacteria (Proteobacteria, Bacteroidetes, and Actinobacteria) increased in hydrocarbon-contaminated sediments and then decreased once hydrocarbons were below detection. Firmicutes, however, continued to increase in relative richness and abundance after hydrocarbon concentrations were below detection. Functional genes involved in hydrocarbon degradation were enriched in hydrocarbon-contaminated sediments then declined significantly (p<0.05) once hydrocarbon concentrations decreased. A greater decrease in hydrocarbon concentrations among marsh grass sediments compared to inlet sediments (lacking marsh grass) suggests that the marsh rhizosphere microbial communities could also be contributing to hydrocarbon degradation. The results of this study provide a comprehensive view of microbial community structural and functional dynamics within perturbed salt marsh ecosystems.     

Single chamber microbial fuel cell with spiral anode for dairy wastewater treatment

The circulating population of peripheral T lymphocytes obtained from a blood sample can provide a large amount of information about an individual's medical status and history. Recent evidence indicates that the detection and functional characterization of antigen-specific T cell subsets within the circulating population may provide a diagnostic indicator of disease and has the potential to predict an individual's response to therapy. In this report, a microarray detection platform that combines grating-coupled surface plasmon resonance imaging (GCSPRI) and grating-coupled surface plasmon coupled emission (SPCE) fluorescence detection modalities were used to detect and characterize CD4(+) T cells. The microspot regions of interest (ROIs) printed on the array consisted of immobilized antibodies or peptide loaded MHC monomers (p/MHC) as T cell capture ligands mixed with additional antibodies as cytokine capture ligands covalently bound to the surface of a corrugated gold sensor chip. Using optimized parameters, an unlabeled influenza peptide reactive T cell clone could be detected at a frequency of 0.1% in a mixed T cell sample using GCSPRI. Additionally, after cell binding was quantified, differential TH1 cytokine secretion patterns from a T cell clone cultured under TH1 or TH2 inducing conditions was detected using an SPCE fluorescence based assay. Differences in the secretion patterns of 3 cytokines, characteristic of the inducing conditions, indicated that differences were a consequence of the functional status of the captured cells. A dual mode GCSPRI/SPCE assay can provide a rapid, high content T cell screening/characterization tool that is useful for diagnosing disease, evaluating vaccination efficacy, or assessing responses to immunotherapeutics.     

Lrp5 is not required for the proliferative response of osteoblasts to strain but regulates proliferation and apoptosis in a cell autonomous manner

Although Lrp5 is known to be an important contributor to the mechanisms regulating bone mass, its precise role remains unclear. The aim of this study was to establish whether mutations in Lrp5 are associated with differences in the growth and/or apoptosis of osteoblast-like cells and their proliferative response to mechanical strain in vitro. Primary osteoblast-like cells were derived from cortical bone of adult mice lacking functional Lrp5 (Lrp5(-/-)), those heterozygous for the human G171V High Bone Mass (HBM) mutation (LRP5(G171V)) and their WT littermates (WT(Lrp5), WT(HBM)). Osteoblast proliferation over time was significantly higher in cultures of cells from LRP5(G171V) mice compared to their WT(HBM) littermates, and lower in Lrp5(-/-) cells. Cells from female LRP5(G171V) mice grew more rapidly than those from males, whereas cells from female Lrp5(-/-) mice grew more slowly than those from males. Apoptosis induced by serum withdrawal was significantly higher in cultures from Lrp5(-/-) mice than in those from WT(HBM) or LRP5(G171V) mice. Exposure to a single short period of dynamic mechanical strain was associated with a significant increase in cell number but this response was unaffected by genotype which also did not change the 'threshold' at which cells responded to strain. In conclusion, the data presented here suggest that Lrp5 loss and gain of function mutations result in cell-autonomous alterations in osteoblast proliferation and apoptosis but do not alter the proliferative response of osteoblasts to mechanical strain in vitro.     

Diacylglycerol and ceramide formation induced by dopamine D2S receptors via Gbeta gamma -subunits in Balb/c-3T3 cells

Diacylglycerol (DAG)and ceramide are important second messengers affecting cell growth,differentiation, and apoptosis. Balb/c-3T3 fibroblast cellsexpressing dopamine-D2S (short) receptors (Balb-D2S cells) provide amodel of G protein-mediated cell growth and transformation. In Balb-D2Scells, apomorphine (EC₅₀ = 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicatingG_i/G_o proteins. To address which G proteins areinvolved, Balb-D2S clones expressing individual PTX-insensitiveG_i proteins were treated with PTX and tested forapomorphine-induced responses. Neither PTX-insensitive G_i2 nor G_i3 rescued D2S-induced DAG orceramide formation. Both D2S-induced DAG and ceramide signals requiredG-subunits and were blocked by inhibitors of phospholipaseC[1-(6-[([17]-3-methoxyestra-1,2,3[10]-trien- 17yl)amino]hexyl)-1H-pyrrole-2,5-dione(U-73122) and partially by D609]. The similar G protein specificity ofD2S-induced calcium mobilization, DAG, and ceramide formation indicatesa common G-dependent phospholipase C-mediated pathway. Both D2agonists and ceramide specifically induced mitogen-activated proteinkinase (ERK1/2), suggesting that ceramide mediates a novel pathway ofD2S-induced ERK1/2 activation, leading to cell growth.

     

A multiwell format assay for heparanase

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.