Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection

Coulomb Interactions between Cytoplasmic Electric Fields and Phosphorylated Messenger Proteins Optimize Information Flow in Cells

Normal cell function requires timely and accurate transmission of information from receptors on the cell membrane (CM) to the nucleus. Movement of messenger proteins in the cytoplasm is thought to be dependent on random walk. However, Brownian motion will disperse messenger proteins throughout the cytosol resulting in slow and highly variable transit times. We propose that a critical component of information transfer is an intracellular electric field generated by distribution of charge on the nuclear membrane (NM). While the latter has been demonstrated experimentally for decades, the role of the consequent electric field has been assumed to be minimal due to a Debye length of about 1 nanometer that results from screening by intracellular Cl- and K+. We propose inclusion of these inorganic ions in the Debye-Huckel equation is incorrect because nuclear pores allow transit through the membrane at a rate far faster than the time to thermodynamic equilibrium. In our model, only the charged, mobile messenger proteins contribute to the Debye length.Using this revised model and published data, we estimate the NM possesses a Debye-Huckel length of a few microns and find this is consistent with recent measurement using intracellular nano-voltmeters. We demonstrate the field will accelerate isolated messenger proteins toward the nucleus through Coulomb interactions with negative charges added by phosphorylation. We calculate transit times as short as 0.01 sec. When large numbers of phosphorylated messenger proteins are generated by increasing concentrations of extracellular ligands, we demonstrate they generate a self-screening environment that regionally attenuates the cytoplasmic field, slowing movement but permitting greater cross talk among pathways. Preliminary experimental results with phosphorylated RAF are consistent with model predictions.This work demonstrates that previously unrecognized Coulomb interactions between phosphorylated messenger proteins and intracellular electric fields will optimize information transfer from the CM to the NM in cells

Less than 5 Netrin-1 molecules initiate attraction but 200 Sema3A molecules are necessary for repulsion

Guidance molecules, such as Sema3A or Netrin-1, induce growth cone (GC) repulsion or attraction. In order to determine the speed of action and efficiency of these guidance cues we developed an experimental procedure to deliver controlled amounts of these molecules. Lipid vesicles encapsulating 10-10 4 molecules of Sema3A or Netrin-1 were manipulated with high spatial and temporal resolution by optical tweezers and their photolysis triggered by laser pulses. Guidance molecules released from the vesicles diffused and reached the GC membrane in a few seconds. Following their arrival, GCs retracted or grew in 20-120 s. By determining the number of guidance molecules trapped inside vesicles and estimating the fraction of guidance molecules reaching the GC, we show that the arrival of less than 5 Netrin-1 molecules on the GC membrane is sufficient to induce growth. In contrast, the arrival of about 200 Sema3A molecules is necessary to induce filopodia repulsion

The Achene Mucilage Hydrated in Desert Dew Assists Seed Cells in Maintaining DNA Integrity: Adaptive Strategy of Desert Plant Artemisia sphaerocephala

Despite proposed ecological importance of mucilage in seed dispersal, germination and seedling establishment, little is known about the role of mucilage in seed pre-germination processes. Here we investigated the role of mucilage in assisting achene cells to repair DNA damage during dew deposition in the desert. Artemisia sphaerocephala achenes were first treated γ-irradiation to induce DNA damage, and then they were repaired in situ in the desert dew. Dew deposition duration can be as long as 421 min in early mornings. Intact achenes absorbed more water than demucilaged achenes during dew deposition and also carried water for longer time following sunrise. After 4-d dew treatment, DNA damage of irradiated intact and demucilaged achenes was reduced to 24.38% and 46.84%, respectively. The irradiated intact achenes exhibited much higher DNA repair ratio than irradiated demucilaged achenes. Irradiated intact achenes showed an improved germination and decreased nonviable achenes after dew treatment, and significant differences in viability between the two types of achenes were detected after 1020 min of dew treatment. Achene mucilage presumably plays an ecologically important role in the life cycle of A. sphaerocephala by aiding DNA repair of achene cells in genomic-stressful habitats

Substance use and dietary practices among students attending alternative high schools: results from a pilot study

<p>Abstract</p> <p>Background</p> <p>Substance use and poor dietary practices are prevalent among adolescents. The purpose of this study was to examine frequency of substance use and associations between cigarette, alcohol and marijuana use and selected dietary practices, such as sugar-sweetened beverages, high-fat foods, fruits and vegetables, and frequency of fast food restaurant use among alternative high school students. Associations between multi-substance use and the same dietary practices were also examined.</p> <p>Methods</p> <p>A convenience sample of adolescents (n = 145; 61% minority, 52% male) attending six alternative high schools in the St Paul/Minneapolis metropolitan area completed baseline surveys. Students were participants in the Team COOL (Controlling Overweight and Obesity for Life) pilot study, a group randomized obesity prevention pilot trial. Mixed model multivariate analyses procedures were used to assess associations of interest.</p> <p>Results</p> <p>Daily cigarette smoking was reported by 36% of students. Cigarette smoking was positively associated with consumption of regular soda (p = 0.019), high-fat foods (p = 0.037), and fast food restaurant use (p = 0.002). Alcohol (p = 0.005) and marijuana use (p = 0.035) were positively associated with high-fat food intake. With increasing numbers of substances, a positive trend was observed in high-fat food intake (p = 0.0003). There were no significant associations between substance use and fruit and vegetable intake.</p> <p>Conclusions</p> <p>Alternative high school students who use individual substances as well as multiple substances may be at high risk of unhealthful dietary practices. Comprehensive health interventions in alternative high schools have the potential of reducing health-compromising behaviors that are prevalent among this group of students. This study adds to the limited research examining substance use and diet among at-risk youth.</p> <p>Trial registration number</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01315743">NCT01315743</a></p

Genomic Sequence around Butterfly Wing Development Genes: Annotation and Comparative Analysis

, where a whole-genome BAC library allows targeted access to large genomic regions. genes. Comparative analysis with orthologous regions of the lepidopteran reference genome allowed assessment of conservation of fine-scale synteny (with detection of new inversions and translocations) and of DNA sequence (with detection of high levels of conservation of non-coding regions around some, but not all, developmental genes)., both involved in multiple developmental processes including wing pattern formation

Power analysis of single-cell RNA-sequencing experiments

Single-cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, thereby revealing new cell types and providing insights into developmental processes and transcriptional stochasticity. A key question is how the variety of available protocols compare in terms of their ability to detect and accurately quantify gene expression. Here, we assessed the protocol sensitivity and accuracy of many published data sets, on the basis of spike-in standards and uniform data processing. For our workflow, we developed a flexible tool for counting the number of unique molecular identifiers (https://github.com/vals/umis/). We compared 15 protocols computationally and 4 protocols experimentally for batch-matched cell populations, in addition to investigating the effects of spike-in molecular degradation. Our analysis provides an integrated framework for comparing scRNA-seq protocols.The study was supported by Cancer Research UK grant number C45041/A14953 to A Cvejic and C Labalette, European Research Council project 677501-ZF_Blood to A Cvejic and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute. The ERC grant ThSWITCH to SA Teichmann (grant no. 260507) and a Lister Institute Research Prize to SA Teichmann. KN Natarajan was supported by the Wellcome Trust Strategic Award “Single cell ge nomics of mouse gastrulation”

Sequencing and Comparative Genome Analysis of Two Pathogenic Streptococcus gallolyticus Subspecies: Genome Plasticity, Adaptation and Virulence

Streptococcus gallolyticus infections in humans are often associated with bacteremia, infective endocarditis and colon cancers. The disease manifestations are different depending on the subspecies of S. gallolyticus causing the infection. Here, we present the complete genomes of S. gallolyticus ATCC 43143 (biotype I) and S. pasteurianus ATCC 43144 (biotype II.2). The genomic differences between the two biotypes were characterized with comparative genomic analyses. The chromosome of ATCC 43143 and ATCC 43144 are 2,36 and 2,10 Mb in length and encode 2246 and 1869 CDS respectively. The organization and genomic contents of both genomes were most similar to the recently published S. gallolyticus UCN34, where 2073 (92%) and 1607 (86%) of the ATCC 43143 and ATCC 43144 CDS were conserved in UCN34 respectively. There are around 600 CDS conserved in all Streptococcus genomes, indicating the Streptococcus genus has a small core-genome (constitute around 30% of total CDS) and substantial evolutionary plasticity. We identified eight and five regions of genome plasticity in ATCC 43143 and ATCC 43144 respectively. Within these regions, several proteins were recognized to contribute to the fitness and virulence of each of the two subspecies. We have also predicted putative cell-surface associated proteins that could play a role in adherence to host tissues, leading to persistent infections causing sub-acute and chronic diseases in humans. This study showed evidence that the S. gallolyticus still possesses genes making it suitable in a rumen environment, whereas the ability for S. pasteurianus to live in rumen is reduced. The genome heterogeneity and genetic diversity among the two biotypes, especially membrane and lipoproteins, most likely contribute to the differences in the pathogenesis of the two S. gallolyticus biotypes and the type of disease an infected patient eventually develops