Sequence and function of LuxO, a negative regulator of luminescence in Vibrio harveyi

Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) In the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode a function required for the density-dependent response. Transposon Tn5 Insertions in the recombinant clone were isolated, and the mutations were transferred to the genome of V. harveyi for examination of mutant phenotypes. Expression of luminescence in V. harveyi strains with transposon insertions in one locus, luxO, was independent of the density of the culture and was similar in intensity to the maximal level observed in wild-type bacteria. Sequence analysis of luxO revealed one open reading frame that encoded a protein, LuxO, similar in amino acid sequence to the response regulator domain of the family of two-component, signal transduction proteins. The constitutive phenotype of LuxO∼ mutants indicates that LuxO acts negatively to control expression of luminescence, and relief of repression by LuxO in the wild type could result from interactions with other components in the Lux signalling system.     

Effects of cyclic mechanical stimulation of the cellular components of the heart: In vitro

Summary The response of the cellular components of the heart to cyclic mechanical stimulation is of particular importance because these cells are continually subjected to mechanical forces as a result of changes in blood volume and pressure. To directly investigate how mechanical tension affects these cellular components of the heart, an in vitro system that exposes the particular cell type (cardiac myocytes, endothelial cells, or fibroblasts) to a calibrated increase in cyclical linear stretch was developed. Cells were grown on silastic membranes coated with laminin and subjected to a 10% cyclical distention 10 times a minute for 72 h. Within 24 h of being exposed to the mechanical stretch, the cells became elongated and oriented perpendicular to the direction of the stretch. These results indicate that cyclical mechanical stimulation directly influences the cellular organization of the heart cells in vitro. This work was supported in part by grants HL 33656 and HL 24935 from the National Institutes of Health, Bethesda, MD.     

Characterization of the Fecal Microbiota of Pigs before and after Inoculation with “Brachyspira hampsonii”

“Brachyspira hampsonii” causes disease indistinguishable from swine dysentery, and the structure of the intestinal microbiome likely plays a role in determining susceptibility of individual pigs to infection and development of clinical disease. The objectives of the current study were to determine if the pre-inoculation fecal microbiota differed between inoculated pigs that did (INOC MH) or did not (INOC non-MH) develop mucohaemorrhagic diarrhea following challenge with “B. hampsonii”, and to quantify changes in the structure of the microbiome following development of clinical disease. Fecal microbiota profiles were generated based on amplification and sequencing of the cpn60 universal target sequence from 89 samples from 18 pigs collected at −8, −5, −3 and 0 days post-inoculation, and at termination. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of INOC MH and INOC non-MH pigs. However, the development of bloody diarrhea in inoculated pigs was associated with perturbation of the microbiota relative to INOC non-MH or sham-inoculated control pigs. Specifically, the fecal microbiota of INOC MH pigs was less dense (fewer total 16S rRNA copies per gram of feces), and had a lower Bacteroidetes:Firmicutes ratio. Further investigation of the potential long-term effects of Brachyspira disease on intestinal health and performance is warranted.     

Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway

Density-dependent expression of luminescence in Vibrio harveyl is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. One signal-response system is encoded by the luxL,M,N locus. The luxL and luxM genes are required for the production of an autoinducer (probably β-hydroxybutryl homoserine lactone), and the luxN gene is required for the response to that autoinducer. Analysis of the phenotypes of LuxL,M and N mutants indicated that an additional signal-response system also controls density sensing. We report here the identification, cloning and analysis of luxP and luxQ, which encode functions required for a second density-sensing system. Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance. LuxQ, like LuxN, is similar to members of the family of two-component, signal transduction proteins and contains both a histidine protein kinase and a response regulator domain. Analysis of signalling mutant phenotypes indicates that there are at least two separate signal-response pathways which converge to regulate expression of luminescence in V. harveyl.     

Protein kinase C and NF-κB-dependent CD4 downregulation in macrophages induced by T cell-derived soluble factors: consequences for HIV-1 infection

Upon activation, CD4(+) T cells release cytokines, chemokines, and other soluble factors that influence the kinetics of HIV-1 replication in macrophages (M). In this article, we show that activation of human primary T cells suppresses the early stages of HIV-1 replication in human primary Mφ by downregulating the main cellular receptor for the virus CD4. The secreted factors responsible for this effect have a molecular mass greater than conventional cytokines, are independent of Th1 or Th2 polarization, and are not IFN-γ, IL-16, RANTES, or macrophage inhibitory factor, as revealed by cytokine array analysis and neutralization assays. CD4 downregulation is entirely posttranslational and involves serine phosphorylation of CD4 and its targeting to an intracellular compartment destined for acidification and degradation. CD4 downregulation is dependent on the activities of both protein kinase C and NF-κB as well as the proteasomes. Using high-resolution liquid chromatography-tandem mass spectrometry analysis in conjugation with label-free protein quantitation software, we found that proteins that promote Mφ adherence and spreading, such as attractin, fibronectin, and galectin-3-binding protein, were significantly overrepresented in the activated T cell supernatant fractions. These results reveal the existence of previously unreported anti-HIV-1 proteins, released by activated T cells that downregulate CD4 expression, and are of fundamental importance to understand the kinetics of HIV infection in vivo.     

Rates and intensity of re-infection with human helminths after treatment and the influence of individual, household, and environmental factors in a Brazilian community

This study quantifies the rate and intensity of re-infection with human hookworm and Schistosoma mansoni infection 12 months following successful treatment, and investigates the influence of socio-economic, geographical and environmental factors. A longitudinal study of 642 individuals aged over 5 years was conducted in Minas Gerais State, Brazil from June 2004 to March 2006. Risk factors were assessed using interval censored regression for the rate and negative binomial regression for intensity. The crude rate and intensity of hookworm re-infection was 0·21 per year (95% confidence interval (CI) 0·15-0·29) and 70·9 epg (95% CI 47·2-106·6). For S. mansoni the rate was 0·06 per year (95% CI 0·03-0·10) and intensity 6·51 epg (95% CI 3·82-11·11). Rate and intensity of re-infection with hookworm were highest among males and positively associated with previous infection status, absence of a toilet and house structure. Rate and intensity of S. mansoni re-infection were associated with previous infection status as well as geographical, environmental and socio-economic factors. The implications of findings for the design of anti-helminth vaccine trials are discussed.     

Decision-making under great uncertainty: environmental management in an era of global change

Global change issues are complex and the consequences of decisions are often highly uncertain. The large spatial and temporal scales and stakes involved make it important to take account of present and potential consequences in decision-making. Standard approaches to decision-making under uncertainty require information about the likelihood of alternative states, how states and actions combine to form outcomes and the net benefits of different outcomes. For global change issues, however, the set of potential states is often unknown, much less the probabilities, effect of actions or their net benefits. Decision theory, thresholds, scenarios and resilience thinking can expand awareness of the potential states and outcomes, as well as of the probabilities and consequences of outcomes under alternative decisions.     

Bioturbation: impact on the marine nitrogen cycle

Sediments play a key role in the marine nitrogen cycle and can act either as a source or a sink of biologically available (fixed) nitrogen. This cycling is driven by a number of microbial remineralization reactions, many of which occur across the oxic/anoxic interface near the sediment surface. The presence and activity of large burrowing macrofauna (bioturbators) in the sediment can significantly affect these microbial processes by altering the physicochemical properties of the sediment. For example, the building and irrigation of burrows by bioturbators introduces fresh oxygenated water into deeper sediment layers and allows the exchange of solutes between the sediment and water column. Burrows can effectively extend the oxic/anoxic interface into deeper sediment layers, thus providing a unique environment for nitrogen-cycling microbial communities. Recent studies have shown that the abundance and diversity of micro-organisms can be far greater in burrow wall sediment than in the surrounding surface or subsurface sediment; meanwhile, bioturbated sediment supports higher rates of coupled nitrification-denitrification reactions and increased fluxes of ammonium to the water column. In the present paper we discuss the potential for bioturbation to significantly affect marine nitrogen cycling, as well as the molecular techniques used to study microbial nitrogen cycling communities and directions for future study.     

Sparse estimation for structural variability

Background  

Proteins are dynamic molecules that exhibit a wide range of motions; often these conformational changes are important for protein function. Determining biologically relevant conformational changes, or true variability, efficiently is challenging due to the noise present in structure data.