Energy input and dissipation in a temperate lake during the spring transition

ADCP and temperature chain measurements have been used to estimate the rate of energy input by wind stress to the water surface in the south basin of Windermere. The energy input from the atmosphere was found to increase markedly as the lake stratified in spring. The efficiency of energy transfer (Eff), defined as the ratio of the rate of working in near-surface waters (RW) to that above the lake surface (P10), increased from ∼0.0013 in vertically homogenous conditions to ∼0.0064 in the first 40 days of the stratified regime. A maximum value of Eff∼0.01 was observed when, with increasing stratification, the first mode internal seiche period decreased to match the diurnal wind period of 24 h. The increase in energy input, following the onset of stratification was reflected in enhancement of the mean depth-varying kinetic energy without a corresponding increase in wind forcing. Parallel estimates of energy dissipation in the bottom boundary layer, based on determination of the structure function show that it accounts for ∼15% of RW in stratified conditions. The evolution of stratification in the lake conforms to a heating stirring model which indicates that mixing accounts for ∼21% of RW. Taken together, these estimates of key energetic parameters point the way to the development of full energy budgets for lakes and shallow seas

The evolution of methods for establishing evolutionary timescales

The fossil record is well known to be incomplete. Read literally, it provides a distorted view of the history of species divergence and extinction, because different species have different propensities to fossilize, the amount of rock fluctuates over geological timescales, as does the nature of the environments that it preserves. Even so, patterns in the fossil evidence allow us to assess the incompleteness of the fossil record. While the molecular clock can be used to extend the time estimates from fossil species to lineages not represented in the fossil record, fossils are the only source of information concerning absolute (geological) times in molecular dating analysis. We review different ways of incorporating fossil evidence in modern clock dating analyses, including node-calibrations where lineage divergence times are constrained using probability densities and tip-calibrations where fossil species at the tips of the tree are assigned dates from dated rock strata. While node-calibrations are often constructed by a crude assessment of the fossil evidence and thus involves arbitrariness, tip-calibrations may be too sensitive to the prior on divergence times or the branching process and influenced unduly affected by well-known problems of morphological character evolution, such as environmental influence on morphological phenotypes, correlation among traits, and convergent evolution in disparate species. We discuss the utility of time information from fossils in phylogeny estimation and the search for ancestors in the fossil record. This article is part of the themed issue ‘Dating species divergences using rocks and clocks’

Substrate Binding Mode and Its Implication on Drug Design for Botulinum Neurotoxin A

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC) has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25). An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide 197QRATKM202 and its variant 197RRATKM202 to 1.5 Å and 1.6 Å, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5′ sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion and replace the nucleophilic water. The P1′-Arg198, occupies the S1′ site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2′ subsite is formed by Arg363, Asn368 and Asp370, while S3′ subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4′-Lys201 makes hydrogen bond with Gln162. P5′-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin

Microbial ligand costimulation drives neutrophilic steroid-refractory asthma

Funding: The authors thank the Wellcome Trust (102705) and the Universities of Aberdeen and Cape Town for funding. This research was also supported, in part, by National Institutes of Health GM53522 and GM083016 to DLW. KF and BNL are funded by the Fonds Wetenschappelijk Onderzoek, BNL is the recipient of an European Research Commission consolidator grant and participates in the European Union FP7 programs EUBIOPRED and MedALL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Dynamics of Endoreplication during Drosophila Posterior Scutellar Macrochaete Development

Endoreplication is a variant type of DNA replication, consisting only of alternating G1 and S phases. Many types of Drosophila tissues undergo endoreplication. However, the timing and the extent to which a single endocycling macrochaete undergoes temporally programmed endoreplication during development are unclear. Here, we focused on the dynamics of endoreplication during posterior scutellar (pSC) macrochaete development. Quantitative analyses of C values in shaft cells and socket cells revealed a gradual rise from 8C and 4C at 8 hours after pupal formation (APF) to 72C and 24C at 29 hours APF, respectively. The validity of the values was further confirmed by the measurement of DNA content with a confocal laser microscope. BrdU incorporation assays demonstrated that shaft cells undergo four rounds of endoreplication from 18 to 29.5 hours APF. In contrast, socket cells undergo two rounds of endoreplication during the same period. Statistical analyses showed that the theoretical C values, based on BrdU assays, nearly coincide with the actually measured C values in socket cells, but not in shaft cells after 22 hours APF. These analyses suggest that socket cells undergo two rounds of endoreplication. However, the mechanism of endoreplication in the shaft cells may change from 22 hours APF, suggesting the possibility that shaft cells undergo two or four rounds of endoreplication during the periods. We also found that the timing of endoreplication differs, depending on the type of macrochaete. Moreover, endocycling in shaft cells of both the left and right sides of pSC bristle lineages occurs in the same pattern, indicating that the process is synchronized for specific types of macrochaete. Our findings suggest that endocycling in macrochaete cell lineages can be a model for understanding mechanisms of endoreplication at the single-cell level

The Reinforcing Therapist Performance (RTP) experiment: Study protocol for a cluster randomized trial

<p>Abstract</p> <p>Background</p> <p>Rewarding provider performance has been recommended by the Institute of Medicine as an approach to improve the quality of treatment, yet little empirical research currently exists that has examined the effectiveness and cost-effectiveness of such approaches. The aim of this study is to test the effectiveness and cost-effectiveness of providing monetary incentives directly to therapists as a method to improve substance abuse treatment service delivery and subsequent client treatment outcomes.</p> <p>Design</p> <p>Using a cluster randomized design, substance abuse treatment therapists from across 29 sites were assigned by site to either an implementation as usual (IAU) or pay-for-performance (P4P) condition.</p> <p>Participants</p> <p>Substance abuse treatment therapists participating in a large dissemination and implementation initiative funded by the Center for Substance Abuse Treatment.</p> <p>Intervention</p> <p>Therapists in both conditions received comprehensive training and ongoing monitoring, coaching, and feedback. However, those in the P4P condition also were given the opportunity to earn monetary incentives for achieving two sets of measurable behaviors related to quality implementation of the treatment.</p> <p>Outcomes</p> <p>Effectiveness outcomes will focus on the impact of the monetary incentives to increase the proportion of adolescents who receive a targeted threshold level of treatment, months that therapists demonstrate monthly competency, and adolescents who are in recovery following treatment. Similarly, cost-effectiveness outcomes will focus on cost per adolescent receiving targeted threshold level of treatment, cost per month of demonstrated competence, and cost per adolescent in recovery.</p> <p>Trial Registration</p> <p>Trial Registration Number: NCT01016704</p

Horizontal DNA transfer mechanisms of bacteria as weapons of intragenomic conflict

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated

The effect of statin therapy on heart failure events: a collaborative meta-analysis of unpublished data from major randomized trials

The effect of statins on risk of heart failure (HF) hospitalization and HF death remains uncertain. We aimed to establish whether statins reduce major HF events.We searched Medline, EMBASE, and the Cochrane Central Register of Controlled Trials for randomized controlled endpoint statin trials from 1994 to 2014. Collaborating trialists provided unpublished data from adverse event reports. We included primary- and secondary-prevention statin trials with >1000 participants followed for >1 year. Outcomes consisted of first non-fatal HF hospitalization, HF death and a composite of first non-fatal HF hospitalization or HF death. HF events occurring <30 days after within-trial myocardial infarction (MI) were excluded. We calculated risk ratios (RR) with fixed-effects meta-analyses. In up to 17 trials with 132 538 participants conducted over 4.3 [weighted standard deviation (SD) 1.4] years, statin therapy reduced LDL-cholesterol by 0.97 mmol/L (weighted SD 0.38 mmol/L). Statins reduced the numbers of patients experiencing non-fatal HF hospitalization (1344/66 238 vs. 1498/66 330; RR 0.90, 95% confidence interval, CI 0.84-0.97) and the composite HF outcome (1234/57 734 vs. 1344/57 836; RR 0.92, 95% CI 0.85-0.99) but not HF death (213/57 734 vs. 220/57 836; RR 0.97, 95% CI 0.80-1.17). The effect of statins on first non-fatal HF hospitalization was similar whether this was preceded by MI (RR 0.87, 95% CI 0.68-1.11) or not (RR 0.91, 95% CI 0.84-0.98).In primary- and secondary-prevention trials, statins modestly reduced the risks of non-fatal HF hospitalization and a composite of non-fatal HF hospitalization and HF death with no demonstrable difference in risk reduction between those who suffered an MI or not

Identification of novel functional sequence variants in the gene for peptidase inhibitor 3

BACKGROUND: Peptidase inhibitor 3 (PI3) inhibits neutrophil elastase and proteinase-3, and has a potential role in skin and lung diseases as well as in cancer. Genome-wide expression profiling of chorioamniotic membranes revealed decreased expression of PI3 in women with preterm premature rupture of membranes. To elucidate the molecular mechanisms contributing to the decreased expression in amniotic membranes, the PI3 gene was searched for sequence variations and the functional significance of the identified promoter variants was studied. METHODS: Single nucleotide polymorphisms (SNPs) were identified by direct sequencing of PCR products spanning a region from 1,173 bp upstream to 1,266 bp downstream of the translation start site. Fourteen SNPs were genotyped from 112 and nine SNPs from 24 unrelated individuals. Putative transcription factor binding sites as detected by in silico search were verified by electrophoretic mobility shift assay (EMSA) using nuclear extract from Hela and amnion cell nuclear extract. Deviation from Hardy-Weinberg equilibrium (HWE) was tested by χ(2 )goodness-of-fit test. Haplotypes were estimated using expectation maximization (EM) algorithm. RESULTS: Twenty-three sequence variations were identified by direct sequencing of polymerase chain reaction (PCR) products covering 2,439 nt of the PI3 gene (-1,173 nt of promoter sequences and all three exons). Analysis of 112 unrelated individuals showed that 20 variants had minor allele frequencies (MAF) ranging from 0.02 to 0.46 representing "true polymorphisms", while three had MAF ≤ 0.01. Eleven variants were in the promoter region; several putative transcription factor binding sites were found at these sites by database searches. Differential binding of transcription factors was demonstrated at two polymorphic sites by electrophoretic mobility shift assays, both in amniotic and HeLa cell nuclear extracts. Differential binding of the transcription factor GATA1 at -689C>G site was confirmed by a supershift. CONCLUSION: The promoter sequences of PI3 have a high degree of variability. Functional promoter variants provide a possible mechanism for explaining the differences in PI3 mRNA expression levels in the chorioamniotic membranes, and are also likely to be useful in elucidating the role of PI3 in other diseases

Evidence for a Role of srGAP3 in the Positioning of Commissural Axons within the Ventrolateral Funiculus of the Mouse Spinal Cord

Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus