Structural basis of FliG–FliM interaction in Helicobacter pylori

FliG and FliM are switch proteins that regulate the rotation and switching of the flagellar motor. Several assembly models for FliG and FliM have recently been proposed; however, it remains unclear whether the assembly of the switch proteins is conserved among different bacterial species. We applied a combination of pull‐down, thermodynamic and structural analyses to characterize the FliM–FliG association from the mesophilic bacterium Helicobacter pylori. FliM binds to FliG with micromolar binding affinity, and their interaction is mediated through the middle domain of FliG (FliG_M), which contains the EHPQR motif. Crystal structures of the middle domain of H. pylori FliM (FliM_M) and FliG_M–FliM_M complex revealed that FliG binding triggered a conformational change of the FliM α3‐α1′ loop, especially Asp130 and Arg144. We furthermore showed that various highly conserved residues in this region are required for FliM–FliG complex formation. Although the FliM–FliG complex structure displayed a conserved binding mode when compared with Thermotoga maritima, variable residues were identified that may contribute to differential binding affinities across bacterial species. Comparison of the thermodynamic parameters of FliG–FliM interactions between H. pylori and Escherichia coli suggests that molecular basis and binding properties of FliM to FliG is likely different between these two species.     

Suicide and suicide attempts in children and adolescents in the child welfare system

Background:

Few population studies have examined the psychiatric outcomes of children and adolescents in the child welfare system, and no studies have compared outcomes before and after entry into care. Our objective was to assess the relative rate (RR) of suicide, attempted suicide, admission to hospital and visits to physicians’ offices among children and adolescents in care compared with those not in care. We also examined these outcomes within the child welfare population before and after entry into care.

Methods:

We used population-level data to identify children and adolescents 5 to 17 years of age who were in care in Manitoba for the first time between Apr. 1, 1997, and Mar. 31, 2006, and a comparison cohort not in care. We compared the two cohorts to obtain RRs for the specified outcomes. We also determined RRs within the child welfare population relative to the same population two years before entry into care.

Results:

We identified 8279 children and adolescents in care for the first time and a comparison cohort of 353 050 children and adolescents not in care. Outcome rates were higher among those in care than in the comparison cohort for suicide (adjusted RR 3.54, 95% confidence interval [CI] 2.11–5.95), attempted suicide (adjusted RR 2.11, 95% CI 1.84–2.43) and all other outcomes. However, adjusted RRs for attempted suicide (RR 0.27, 95% CI 0.21–0.34), admissions to hospital and physician visits decreased after entry into care.

Interpretation:

Children and adolescents in care were at greater risk of suicide and attempting suicide than those who were not in care. Rates of suicide attempts and hospital admissions within this population were highest before entry into care and decreased thereafter.In Canada, about 76 000 children and adolescents are under the care of provincial child and family services.¹ In Manitoba, more than 7000 children and adolescents were in the care of child and family services in 2008. Many of them had experienced abuse and neglect, or death or conflict in their families, along with disability or emotional problems.^2–13 Concerns have been raised that the Canadian child welfare system does not provide adequate resources and supports to mitigate the effects of abuse and neglect.¹⁴ Although the health outcomes of this population are a frequent topic of concern in the media, population-based research describing these outcomes is limited.To our knowledge, only two studies of a population cohort of children and adolescents in care have been published to date, both describing the psychiatric morbidity and mortality of children and adolescents in care in Sweden.^4,6 These studies found greater rates of suicide, suicide attempts and psychiatric hospital admissions among children in care than in the general population. However, these studies had substantial limitations. Although they used the general population as a comparison group, they did not analyze for the presence of psychiatric morbidity in the period before entry into care. This omission limits the ability to draw conclusions about whether the poor outcomes of these children were associated with disruptions in their lives and families related to involvement in the child welfare system or whether they were a consequence of their life, health and psychological characteristics before they entered the care system.The first objective of the current study was to assess the relative rates (RRs) of suicide and attempted suicide and the number of hospital admissions and visits to physicians’ offices among children and adolescents with a history of being in the care of child and family services in Manitoba, relative to the general population of children and adolescents not in care. The second objective was to assess the RR of attempting suicide and the number of hospital admissions and physician visits in the child welfare population before and after entry into care.     

Activated platelets rescue apoptotic cells via paracrine activation of EGFR and DNA-dependent protein kinase

Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK – an endogenous cytoprotective cascade.Platelets are small discoid anuclear cell fragments that are key players in haemostasis – a frontline physiological response to acute tissue injury. Under basal conditions, platelets circulate at 150–400 × 10⁹ per litre and sequester a diverse array of bioactive molecules within their intracellular granules.¹ Upon tissue injury, however, vascular disruption triggers localised platelet deposition, activation and release of their granular contents. These platelet-released molecules (PRMs) further recruit and activate platelets, resulting in a multi-cellular aggregate that restricts blood loss. In conjunction, the coagulation cascade becomes activated, leading to thrombin generation which consolidates the growing thrombus by promoting further platelet activation and by catalysing fibrin formation.In addition to this critical role in haemostasis, platelets also participate in numerous non-haemostatic processes, including inflammation,² tissue repair,³ angiogenesis and lymphatic development.⁴ Platelets also promote tumour cell proliferation and metastasis via peri-cellular adhesion and signalling.^{5, 6, 7} Such pleiotropy is attributed to the array of molecules that are released from activated platelets.The reparative influence of platelets also has important clinical utility. For decades, ‘platelet concentrates'' have been applied to sites of injury to expedite the recovery of organs such as bone,⁸ skin⁹ and tendon.¹⁰ The beneficial effects of platelet concentrates are thought to be due to the many growth factors that are released from activated platelets.³ This reasoning, however, may be considered speculative given the lack of direct experimental evidence. Furthermore, little-to-no information exists about the platelet-derived peri-cellular signals that facilitate tissue repair.Platelets are normally restricted to the intravascular compartment. However, extravascular platelet accumulation can also occur under certain pathological conditions. One notable example of this is neurotrauma. Here, we find that exceptionally high levels of platelet activation occur in the brain after neurotrauma, thereby providing in-principle support that platelet products may influence neuronal survival. We therefore examined the influence of PRMs on injured primary cortical neurons. As neurons are post-mitotic, this approach allowed the elucidation of effects that were unrelated to proliferation. We find that PRMs selectively and potently reduce neuronal apoptosis via paracrine activation of the epidermal growth factor receptor (EGFR) and downstream activation of DNA-dependent protein kinase (DNA-PK) – a ubiquitous DNA repair enzyme. Strikingly, the same platelet-dependent mechanism also protects several non-neuronal human cancer cell types from chemotherapy-induced apoptosis. Thus, activated platelets trigger a potent and broad-acting paracrine signal that attenuates apoptosis.Our findings highlight a beneficial action of platelets that likely operates within the injured brain where existing knowledge about platelets has been limited to its haemostatic role. Interestingly, we also find that this underappreciated cytoprotective action of platelets may have implications in cancer where the association between higher platelet count and poorer patient prognosis has been well established.^{11, 12, 13, 14} In addition, this newly identified anti-apoptotic mechanism should encourage rationally designed improvements to the clinical use of platelet concentrates.     

EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers

Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers. In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by shRNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation between EZH2 and DLC1 expression was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors.     

Cataloging and profiling genes expressed in Lentinula edodes fruiting body by massive cDNA pyrosequencing and LongSAGE

This study investigated the molecular mechanism of the fruiting body development and sporulation in the cap of the Shiitake mushroom, Lentinula edodes. Although there has been much research into L. edodes, there remain significant gaps in our knowledge of how the species reproduces. In order to provide molecular resources and to understand the molecular mechanism of the fruiting body development in basidiomycete comprehensively, we searched for the genes which are important for fruiting body development and sporulation in the cap of mature fruiting body of L. edodes by using the whole-genome approach. Massive cDNA pyrosequencing was used to generate >7000 sequence contigs from mature fruiting bodies. We used Gene Ontology to categorize the contigs to form the catalog of genes expressed at the stage of the mature fruiting body. We also assigned the contigs into the KEGG pathways. The catalog of expressed genes indicates that the mature fruiting bodies (1) sense the external environment, (2) transmit signals to express genes through regulatory systems, (3) produce many proteins, (4) degrade unwanted proteins, (5) perform extensive biosynthesis, (6) generate energy, (7) regulate the internal environment, (8) transport molecules, (9) carry out cell division, and (10) differentiate and develop. After establishing the catalog of expressed genes in L. edodes, we used the LongSAGE approach to analyze the expression levels of genes found in mature fruiting bodies before (FB) and after (FBS) spores appeared. Gene-expression patterns according to GO categories were similar in these two stages. We have also successfully identified genes differentially expressed in FB and FBS. Fold-changes in expression levels of selected genes based on LongSAGE tag counts were similar to those obtained by real-time RT-PCR. The consistency between real-time RT-PCR and LongSAGE results indicates reliability of the LongSAGE results. Overall, this study provides valuable information on the fruiting processes of L. edodes through a combination of massive cDNA pyrosequencing and LongSAGE sequencing, and the knowledge thereby obtained may provide insight into the improvement of the yield of commercially grown Shiitake mushrooms.     

Critical role for NLRP3 in necrotic death triggered by Mycobacterium tuberculosis

Induction of necrotic death in macrophages is a primary virulence determinant of Mycobacterium tuberculosis. The ESX-1 secretion system and its substrate ESAT-6 are required for M. tuberculosis to induce necrosis, but host factors that mediate the ESAT-6-promoted necrosis remain unknown. Here we report that ESAT-6-promoted necrotic death in THP-1 human macrophages is dependent on the NLRP3 inflammasome, as shown by RNA interference and pharmacological inhibitions. Phagosomes containing ESAT-6-expressing M. tuberculosis recruit markers previously associated with damaged phagosomal membrane, such as galectin-3 and ubiquitinated protein aggregates. In addition, ESAT-6 promoted lysosomal permeabilization by M. tuberculosis. ESAT-6 mutants defective for ubiquitination were unable to trigger NLRP3 activation and necrotic death. Furthermore, Syk tyrosine kinase, recently implicated in NLRP3 activation during fungal and malarial infections, was necessary for mediating the ESAT-6-promoted necrosis and NLRP3 activation. Our results thus link phagosomal damage and Syk activity to NLRP3-mediated necrotic death triggered by M. tuberculosis ESAT-6 during infection.     

Risk factors for severe outcomes following 2009 influenza A (H1N1) infection: a global pooled analysis

Background

Since the start of the 2009 influenza A pandemic (H1N1pdm), the World Health Organization and its member states have gathered information to characterize the clinical severity of H1N1pdm infection and to assist policy makers to determine risk groups for targeted control measures.

Methods and Findings

Data were collected on approximately 70,000 laboratory-confirmed hospitalized H1N1pdm patients, 9,700 patients admitted to intensive care units (ICUs), and 2,500 deaths reported between 1 April 2009 and 1 January 2010 from 19 countries or administrative regions—Argentina, Australia, Canada, Chile, China, France, Germany, Hong Kong SAR, Japan, Madagascar, Mexico, the Netherlands, New Zealand, Singapore, South Africa, Spain, Thailand, the United States, and the United Kingdom—to characterize and compare the distribution of risk factors among H1N1pdm patients at three levels of severity: hospitalizations, ICU admissions, and deaths. The median age of patients increased with severity of disease. The highest per capita risk of hospitalization was among patients <5 y and 5–14 y (relative risk [RR] = 3.3 and 3.2, respectively, compared to the general population), whereas the highest risk of death per capita was in the age groups 50–64 y and ≥65 y (RR = 1.5 and 1.6, respectively, compared to the general population). Similarly, the ratio of H1N1pdm deaths to hospitalizations increased with age and was the highest in the ≥65-y-old age group, indicating that while infection rates have been observed to be very low in the oldest age group, risk of death in those over the age of 64 y who became infected was higher than in younger groups. The proportion of H1N1pdm patients with one or more reported chronic conditions increased with severity (median = 31.1%, 52.3%, and 61.8% of hospitalized, ICU-admitted, and fatal H1N1pdm cases, respectively). With the exception of the risk factors asthma, pregnancy, and obesity, the proportion of patients with each risk factor increased with severity level. For all levels of severity, pregnant women in their third trimester consistently accounted for the majority of the total of pregnant women. Our findings suggest that morbid obesity might be a risk factor for ICU admission and fatal outcome (RR = 36.3).

Conclusions

Our results demonstrate that risk factors for severe H1N1pdm infection are similar to those for seasonal influenza, with some notable differences, such as younger age groups and obesity, and reinforce the need to identify and protect groups at highest risk of severe outcomes. Please see later in the article for the Editors'' Summary     

The impact of LED illumination on antioxidant properties of sprouted seeds

The objective of this study was to evaluate the effect of the light emitting diode (LED) spectra on the antioxidant properties of sprouted wheat (Triticum aestivum L.), radish (Raphanus sativus L.), and lentil (Lens esculenta Moenh.) seeds. Lighting experiments were performed under controlled conditions (PPFD - 100 μmol m⁻² s⁻¹; 12 h photoperiod; 27°C). The LED conditions used were: L1 - 638 nm; L2 - 455 nm, 638 nm, 669 nm, 731 nm (basal components); L3 - basal + 385 nm; L4 - basal + 510 nm and L5 - basal + 595 nm. Wheat and lentil sprouts were shown to accumulate less phenolic compounds and were more sensitive to light spectral differences when compared to radish sprouts. The antioxidant properties and contents of antioxidant compounds in seeds germinated in the dark were significantly lower than LED treated seeds. The higher content of total phenols and significant increase in alpha-tocopherol and vitamin C concentration resulted in altered DPPH free-radical scavenging capacity. Therefore we conclude that the LED spectra, based on basal components supplemented with green (510 nm) light can improve the antioxidant properties of sprouted seeds of lentil and wheat. The highest antioxidant properties of radish seeds were caused by radiation with supplemental amber (595 nm) light.