Phylo SI: a new genome-wide approach for prokaryotic phylogeny

The evolutionary history of all life forms is usually represented as a vertical tree-like process. In prokaryotes, however, the vertical signal is partly obscured by the massive influence of horizontal gene transfer (HGT). The HGT creates widespread discordance between evolutionary histories of different genes as genomes become mosaics of gene histories. Thus, the Tree of Life (TOL) has been questioned as an appropriate representation of the evolution of prokaryotes. Nevertheless a common hypothesis is that prokaryotic evolution is primarily tree-like, and a routine effort is made to place new isolates in their appropriate location in the TOL. Moreover, it appears desirable to exploit non–tree-like evolutionary processes for the task of microbial classification. In this work, we present a novel technique that builds on the straightforward observation that gene order conservation (‘synteny’) decreases in time as a result of gene mobility. This is particularly true in prokaryotes, mainly due to HGT. Using a ‘synteny index’ (SI) that measures the average synteny between a pair of genomes, we developed the phylogenetic reconstruction tool ‘Phylo SI’. Phylo SI offers several attractive properties such as easy bootstrapping, high sensitivity in cases where phylogenetic signal is weak and computational efficiency. Phylo SI was tested both on simulated data and on two bacterial data sets and compared with two well-established phylogenetic methods. Phylo SI is particularly efficient on short evolutionary distances where synteny footprints remain detectable, whereas the nucleotide substitution signal is too weak for reliable sequence-based phylogenetic reconstruction. The method is publicly available at http://research.haifa.ac.il/ssagi/software/PhyloSI.zip.     

The complex methylome of the human gastric pathogen Helicobacter pylori

The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural transformation, the major driving force of genetic diversification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT®) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificity and the interaction of one MTase with two alternative specificity subunits resulting in different methylation patterns. The methylomes of these well-characterized H. pylori strains will provide a valuable resource for future studies investigating the role of H. pylori R-M systems in limiting transformation as well as in gene regulation and host interaction.     

Subtypes of cytotoxic lymphocytes and natural killer cells infiltrating cancer nests correlate with prognosis in patients with vulvar squamous cell carcinoma

Objective

Adaptive immune effectors do not influence prognosis in vulvar squamous cell carcinoma (vSCC). Therefore, we tried to clarify the prognostic role of innate immunity and granzyme B-dependent cytotoxicity as defined by intratumoral infiltrates of natural killer cells (CD56+) and lymphocytes expressing granzyme B (GrB+).

Methods

We analyzed 76 primary vSCCs and 35 lymph node metastases that were obtained from 76 patients with a full clinical history. The distribution and density of GrB+ and CD56+ cells within cancer tissues were evaluated by immunohistochemistry and correlated with clinicopathological features, commonly recognized prognostic factors and overall survival (OS).

Results

CD56+ cells were mostly detected within the cancer nests, while GrB+ cells were predominant in the tumor stroma. Intraepithelial (IE) CD56+ infiltrates at the primary site were correlated with depth of invasion (r = 0.339, p = 0.003) and recurrence (r = 0.295, p = 0.011), while IE GrB+ infiltrates were correlated with tumor grade (r = 0.304, p = 0.009) and age (r = 0.333, p = 0.004). The primary cancer nests of metastatic patients were infiltrated more by intraepithelial (IE) CD56+ cells than were those of the non-metastatic patients (p = 0.05). The median OS was 41.16 months (range 1.7–98.43). High IE GrB+ infiltrates predicted longer OS among patients without metastases (p = 0.028). High IE CD56+ infiltrates were correlated with longer OS in metastatic cases (p = 0.009).

Conclusion

The combined cytotoxicity of innate and adaptive immune effectors infiltrating cancer nests (IE GrB+) predicts an improved clinical outcome among non-metastatic vSCC patients. The functional status of prognostic IE CD56+ infiltrates in immune escaped (metastatic) tumors requires further investigation.     

Improved pregnancy and birth rates with routine application of nonsurgical embryo transfer

Nonsurgical embryo transfer (NSET) of blastocysts to pseudopregnant female recipients provides many benefits over surgical implantation with less distress for the mice, no anesthesia or analgesia required and a considerable reduction in implantation time per mouse. Although a disposable device to perform NSET is on the market since 2009, it is not generally used in transgenic facilities, most likely because surgical implantation is efficient and inexpensive. Here, we report that with several refinements to the original protocol, the NSET method becomes very attractive and outperforms the traditional surgical transfer on basis of pregnancy rate, birth rate and implantation-related discomfort. Furthermore, repeated use of the same NSET device on several recipient females reduces the costs to a reasonable level. The data presented covers all embryo transfers over the last 5 years at the transgenic facility of the Netherlands Cancer Institute, of which the last 2 years were performed exclusively with NSET.     

Genetics and epigenetics of aging and longevity

Evolutionary theories of aging predict the existence of certain genes that provide selective advantage early in life with adverse effect on lifespan later in life (antagonistic pleiotropy theory) or longevity insurance genes (disposable soma theory). Indeed, the study of human and animal genetics is gradually identifying new genes that increase lifespan when overexpressed or mutated: gerontogenes. Furthermore, genetic and epigenetic mechanisms are being identified that have a positive effect on longevity. The gerontogenes are classified as lifespan regulators, mediators, effectors, housekeeping genes, genes involved in mitochondrial function, and genes regulating cellular senescence and apoptosis. In this review we demonstrate that the majority of the genes as well as genetic and epigenetic mechanisms that are involved in regulation of longevity are highly interconnected and related to stress response.     

Selection of Motor Programs for Suppressing Food Intake and Inducing Locomotion in the Drosophila Brain

Central mechanisms by which specific motor programs are selected to achieve meaningful behaviors are not well understood. Using electrophysiological recordings from pharyngeal nerves upon central activation of neurotransmitter-expressing cells, we show that distinct neuronal ensembles can regulate different feeding motor programs. In behavioral and electrophysiological experiments, activation of 20 neurons in the brain expressing the neuropeptide hugin, a homolog of mammalian neuromedin U, simultaneously suppressed the motor program for food intake while inducing the motor program for locomotion. Decreasing hugin neuropeptide levels in the neurons by RNAi prevented this action. Reducing the level of hugin neuronal activity alone did not have any effect on feeding or locomotion motor programs. Furthermore, use of promoter-specific constructs that labeled subsets of hugin neurons demonstrated that initiation of locomotion can be separated from modulation of its motor pattern. These results provide insights into a neural mechanism of how opposing motor programs can be selected in order to coordinate feeding and locomotive behaviors.     

The Effectiveness of Community Action in Reducing Risky Alcohol Consumption and Harm: A Cluster Randomised Controlled Trial

Background

The World Health Organization, governments, and communities agree that community action is likely to reduce risky alcohol consumption and harm. Despite this agreement, there is little rigorous evidence that community action is effective: of the six randomised trials of community action published to date, all were US-based and focused on young people (rather than the whole community), and their outcomes were limited to self-report or alcohol purchase attempts. The objective of this study was to conduct the first non-US randomised controlled trial (RCT) of community action to quantify the effectiveness of this approach in reducing risky alcohol consumption and harms measured using both self-report and routinely collected data.

Methods and Findings

We conducted a cluster RCT comprising 20 communities in Australia that had populations of 5,000–20,000, were at least 100 km from an urban centre (population ≥ 100,000), and were not involved in another community alcohol project. Communities were pair-matched, and one member of each pair was randomly allocated to the experimental group. Thirteen interventions were implemented in the experimental communities from 2005 to 2009: community engagement; general practitioner training in alcohol screening and brief intervention (SBI); feedback to key stakeholders; media campaign; workplace policies/practices training; school-based intervention; general practitioner feedback on their prescribing of alcohol medications; community pharmacy-based SBI; web-based SBI; Aboriginal Community Controlled Health Services support for SBI; Good Sports program for sports clubs; identifying and targeting high-risk weekends; and hospital emergency department–based SBI. Primary outcomes based on routinely collected data were alcohol-related crime, traffic crashes, and hospital inpatient admissions. Routinely collected data for the entire study period (2001–2009) were obtained in 2010. Secondary outcomes based on pre- and post-intervention surveys (n = 2,977 and 2,255, respectively) were the following: long-term risky drinking, short-term high-risk drinking, short-term risky drinking, weekly consumption, hazardous/harmful alcohol use, and experience of alcohol harm. At the 5% level of statistical significance, there was insufficient evidence to conclude that the interventions were effective in the experimental, relative to control, communities for alcohol-related crime, traffic crashes, and hospital inpatient admissions, and for rates of risky alcohol consumption and hazardous/harmful alcohol use. Although respondents in the experimental communities reported statistically significantly lower average weekly consumption (1.90 fewer standard drinks per week, 95% CI = −3.37 to −0.43, p = 0.01) and less alcohol-related verbal abuse (odds ratio = 0.58, 95% CI = 0.35 to 0.96, p = 0.04) post-intervention, the low survey response rates (40% and 24% for the pre- and post-intervention surveys, respectively) require conservative interpretation. The main limitations of this study are as follows: (1) that the study may have been under-powered to detect differences in routinely collected data outcomes as statistically significant, and (2) the low survey response rates.

Conclusions

This RCT provides little evidence that community action significantly reduces risky alcohol consumption and alcohol-related harms, other than potential reductions in self-reported average weekly consumption and experience of alcohol-related verbal abuse. Complementary legislative action may be required to more effectively reduce alcohol harms.

Trial registration

Australian New Zealand Clinical Trials Registry ACTRN12607000123448 Please see later in the article for the Editors'' Summary     

Heterogeneity and Convergence of Olfactory First-Order Neurons Account for the High Speed and Sensitivity of Second-Order Neurons

In the olfactory system of male moths, a specialized subset of neurons detects and processes the main component of the sex pheromone emitted by females. It is composed of several thousand first-order olfactory receptor neurons (ORNs), all expressing the same pheromone receptor, that contact synaptically a few tens of second-order projection neurons (PNs) within a single restricted brain area. The functional simplicity of this system makes it a favorable model for studying the factors that contribute to its exquisite sensitivity and speed. Sensory information—primarily the identity and intensity of the stimulus—is encoded as the firing rate of the action potentials, and possibly as the latency of the neuron response. We found that over all their dynamic range, PNs respond with a shorter latency and a higher firing rate than most ORNs. Modelling showed that the increased sensitivity of PNs can be explained by the ORN-to-PN convergent architecture alone, whereas their faster response also requires cell-to-cell heterogeneity of the ORN population. So, far from being detrimental to signal detection, the ORN heterogeneity is exploited by PNs, and results in two different schemes of population coding based either on the response of a few extreme neurons (latency) or on the average response of many (firing rate). Moreover, ORN-to-PN transformations are linear for latency and nonlinear for firing rate, suggesting that latency could be involved in concentration-invariant coding of the pheromone blend and that sensitivity at low concentrations is achieved at the expense of precise encoding at high concentrations.