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Plants produce a plethora of natural products, including many drugs. It has recently emerged that the genes encoding different natural product pathways may be organized as biosynthetic gene clusters in plant genomes, with >30 examples reported so far. Despite superficial similarities with microbes, these clusters have not arisen by horizontal gene transfer, but rather by gene duplication, neofunctionalization, and relocation via unknown mechanisms. Previously we reported that two Arabidopsis thaliana biosynthetic gene clusters are located in regions of the genome that are significantly enriched in transposable elements (TEs) . Other plant biosynthetic gene clusters also harbor abundant TEs. TEs can mediate genomic rearrangement by providing homologous sequences that enable illegitimate recombination and gene relocation. Thus, TE-mediated recombination may contribute to plant biosynthetic gene cluster formation. TEs may also facilitate establishment of regulons. However, a systematic analysis of the TEs associated with plant biosynthetic gene clusters has not been carried out. Here we investigate the TEs associated with clustered terpene biosynthetic genes in multiple plant genomes and find evidence to suggest a role for miniature inverted-repeat transposable elements in cluster formation in eudicots. Through investigation of the newly sequenced Amborella trichopoda, Aquilegia coerulea, and Kalanchoe fedtschenkoi genomes, we further show that the "block" mechanism of founding of biosynthetic gene clusters through duplication and diversification of pairs of terpene synthase and cytochrome P450 genes that is prevalent in the eudicots arose around 90-130 million years ago, after the appearance of the basal eudicots and before the emergence of the superrosid clade.

Significance Recently discovered biosynthetic gene clusters in plants are a striking example of the nonrandom complex structure of eukaryotic genomes. The mechanisms underpinning the formation of these clustered pathways are not understood. Here we carry out a systematic analysis of transposable elements associated with clustered terpene biosynthetic genes in plant genomes, and find evidence to suggest a role for miniature inverted-repeat transposable elements in cluster formation in eudicots. Our analyses provide insights into potential mechanisms of cluster assembly. They also shed light on the emergence of a "block" mechanism for the foundation of new terpene clusters in the eudicots in which microsyntenic blocks of terpene synthase and cytochrome P450 gene pairs duplicate, providing templates for the evolution of new pathways.

Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI) , a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60-70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNg expression by CD4+ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections.

Triose phosphate isomerase ( TPI) is a ubiquitous and highly conserved enzyme in intracellular glucose metabolism. Surprisingly, the human lymphatic filariai nematode parasite Brugia malayi, releases TPI into the extracellular environment, suggesting a role in helminth survival in the mammalian host. We first established that B. malayi-infected humans and rodents generate TPI-specific serum antibody responses, confirming presentation of this protein to the host immune system. However, immunisation of rodents with B. malayi TPI did not induce protection against infection. Furthermore, TPI from a related parasite, Litomosoides sigmodontis, did not induce protective immunity in mice. Notably, antibodies from infected hosts did not neutralise the enzymatic activity of TPI. We then generated twenty-three anti-TPI monoclonal antibodies, of which only two inhibited enzymatic activity. Transfer of neutralising antibody to mice prior to B. malayi infection effected a 69. 5% reduction in microfilarial levels in vivo and a 60% reduction in microfilariae produced by individual adult female parasites. Corresponding shifts in the host immune response included reduced Th1 cytokine production and enhanced macrophage numbers. Enzymatically active TPI therefore promotes production of the transmission stage of B. malayi filarial parasites and represents a rational target for new vaccine and drug development to protect against filarial infections.

Although the use of phylogenetic trees in epidemiological investigations has become commonplace, their epidemiological interpretation has not been systematically evaluated. Here, we use an HIV-1 within-host coalescent model to probabilistically evaluate transmission histories of two epidemiologically linked hosts. Previous critique of phylogenetic reconstruction has claimed that direction of transmission is difficult to infer, and that the existence of unsampled intermediary links or common sources can never be excluded. The phylogenetic relationship between the HIV populations of epidemiologically linked hosts can be classified into six types of trees, based on cladistic relationships and whether the reconstruction is consistent with the true transmission history or not. We show that the direction of transmission and whether unsampled intermediary links or common sources existed make very different predictions about expected phylogenetic relationships: (i) Direction of transmission can often be established when paraphyly exists, (ii) intermediary links can be excluded when multiple lineages were transmitted, and (iii) when the sampled individuals' HIV populations both are monophyletic a common source was likely the origin. Inconsistent results, suggesting the wrong transmission direction, were generally rare. In addition, the expected tree topology also depends on the number of transmitted lineages, the sample size, the time of the sample relative to transmission, and how fast the diversity increases after infection. Typically, 20 or more sequences per subject give robust results. We confirm our theoretical evaluations with analyses of real transmission histories and discuss how our findings should aid in interpreting phylogenetic results.

Significance Phylogenetic inference of who infected whom has great value in epidemiological investigations because it should provide an objective test of an explicit hypothesis about how transmission( s) occurred. Until now, however, there has not been a systematic evaluation of which phylogeny to expect from different transmission histories, and thus the interpretation of what an observed phylogeny actually means has remained somewhat elusive. Here, we show that certain types of phylogenies associate with different transmission histories, which may make it possible to exclude possible intermediary links or identify cases where a common source was likely but not sampled. Our systematic classification and evaluation of expected topologies should make future interpretation of phylogenetic results in epidemiological investigations more objective and informative.

A recent trend in drug development is to identify drug combinations or multi-target agents that effectively modify multiple nodes of disease-associated networks. Such polypharmacological effects may reduce the risk of emerging drug resistance by means of attacking the disease networks through synergistic and synthetic lethal interactions. However, due to the exponentially increasing number of potential drug and target combinations, systematic approaches are needed for prioritizing the most potent multi-target alternatives on a global network level. We took a functional systems pharmacology approach toward the identification of selective target combinations for specific cancer cells by combining large-scale screening data on drug treatment efficacies and drug-target binding affinities. Our model-based prediction approach, named TIMMA, takes advantage of the polypharmacological effects of drugs and infers combinatorial drug efficacies through system-level target inhibition networks. Case studies in MCF-7 and MDA-MB-231 breast cancer and BxPC-3 pancreatic cancer cells demonstrated how the target inhibition modeling allows systematic exploration of functional interactions between drugs and their targets to maximally inhibit multiple survival pathways in a given cancer type. The TIMMA prediction results were experimentally validated by means of systematic siRNA-mediated silencing of the selected targets and their pairwise combinations, showing increased ability to identify not only such druggable kinase targets that are essential for cancer survival either individually or in combination, but also synergistic interactions indicative of non-additive drug efficacies. These system-level analyses were enabled by a novel model construction method utilizing maximization and minimization rules, as well as a model selection algorithm based on sequential forward floating search. Compared with an existing computational solution, TIMMA showed both enhanced prediction accuracies in cross validation as well as significant reduction in computation times. Such cost-effective computational-experimental design strategies have the potential to greatly speed-up the drug testing efforts by prioritizing those interventions and interactions warranting further study in individual cancer cases.

Selective inhibition of specific panels of multiple protein targets provides an unprecedented potential for improving therapeutic efficacy of anticancer agents. We introduce a computational systems pharmacology strategy, which uses the concept of target inhibition networks to predict effective multi-target combinations for treating specific cancer types. The strategy is based on integration of two complementary information sources, drug treatment efficacies and drug-target binding affinities, which are readily available in drug screening labs. Compared to the cancer sequencing efforts, which often result in a huge number of non-targetable genetic alterations, the target combinations from our strategy are druggable, by definition, hence enabling more straightforward translation toward clinically actionable treatment strategies. The model predictions were experimentally validated using siRNA-mediated target silencing screens in three case studies involving MDA-MB-231 and MCF-7 breast cancer and BxPC-3 pancreatic cancer cells. In more general terms, the cancer cell-specific target inhibition networks provided additional insights into the drugs' mechanisms of action, for instance, how the cancer cell survival pathways can be targeted by synergistic and synthetic lethal interactions through multi-target perturbations. These results demonstrate that the principles introduced here offer the possibilities to move toward more systematic prediction and evaluation of the most effective drug target combinations.

Molecular ferroelectrics combine electromechanical coupling and electric polarizabilities, offering immense promise in stimuli-dependent metamaterials. Despite such promise, current physical realizations of mechanical metamaterials remain hindered by the lack of rapid-prototyping ferroelectric metamaterial structures. Here, we present a continuous rapid printing strategy for the volumetric deposition of water-soluble molecular ferroelectric metamaterials with precise spatial control in virtually any three-dimensional (3D) geometry by means of an electric-field-assisted additive manufacturing. We demonstrate a scaffold-supported ferroelectric crystalline lattice that enables self-healing and a reprogrammable stiffness for dynamic tuning of mechanical metamaterials with a long lifetime and sustainability. A molecular ferroelectric architecture with resonant inclusions then exhibits adaptive mitigation of incident vibroacoustic dynamic loads via an electrically tunable subwavelength-frequency band gap. The findings shown here pave the way for the versatile additive manufacturing of molecular ferroelectric metamaterials.

Significance Molecular ferroelectrics, which show the ability to switch the electromechanical activity by an external electric field, establish the basis for mechanical metamaterial technologies. Despite their theoretical promise, such mechanical metamaterials remain hindered by the lack of adaptive stimuli-responsive materials which can be effectively tuned "on demand" across time and length scales. Here, we unravel a printable mechanical metamaterial of imidazolium perchlorate with superior electromechanical coupling and reprogrammable stiffness. We propose a continuous rapid three-dimensional ( 3D) printing technique which can reduce the manufacturing time of ferroelectrics from hours down to minutes. The printed molecular ferroelectric metamaterial structure is then shown to enable a tunable-frequency vibration-isolating architecture. This study paves the way for rationally designed 3D-printable molecular ferroelectric metamaterials.

Selenocysteine (Sec) is known as the 21st amino acid, a cysteine analogue with selenium replacing sulphur. Sec is inserted co-translationally in a small fraction of proteins called selenoproteins. In selenoprotein genes, the Sec specific tRNA (tRNASec) drives the recoding of highly specific UGA codons from stop signals to Sec. Although found in organisms from the three domains of life, Sec is not universal. Many species are completely devoid of selenoprotein genes and lack the ability to synthesize Sec. Since tRNASec is a key component in selenoprotein biosynthesis, its efficient identification in genomes is instrumental to characterize the utilization of Sec across lineages. Available tRNA prediction methods fail to accurately predict tRNASec, due to its unusual structural fold. Here, we present Secmarker, a method based on manually curated covariance models capturing the specific tRNASec structure in archaea, bacteria and eukaryotes. We exploited the non-universality of Sec to build a proper benchmark set for tRNASec predictions, which is not possible for the predictions of other tRNAs. We show that Secmarker greatly improves the accuracy of previously existing methods constituting a valuable tool to identify tRNASec genes, and to efficiently determine whether a genome contains selenoproteins. We used Secmarker to analyze a large set of fully sequenced genomes, and the results revealed new insights in the biology of tRNASec, led to the discovery of a novel bacterial selenoprotein family, and shed additional light on the phylogenetic distribution of selenoprotein containing genomes. Secmarker is freely accessible for download, or online analysis through a web server at http: //secmarker. crg. cat.

Most proteins are made of twenty amino acids. However, there is a small group of proteins that incorporate a 21st amino acid, Selenocysteine ( Sec) . These proteins are called selenoproteins and are present in some, but not all, species from the three domains of life. Sec is inserted in selenoproteins in response to the UGA codon, normally a stop codon. A Sec specific tRNA ( tRNASec) , which only exists in the organisms that synthesize selenoproteins recognizes the UGA codon. tRNASec is not only indispensable for Sec incorporation into selenoproteins, but also for Sec synthesis, since Sec is synthesized on its own tRNA. The structure of tRNASec differs from that of canonical tRNAs, and general tRNA detection methods fail to accurately predict it. We developed Secmarker, a tRNASec specific identification tool based on the characteristic structural features of the tRNASec. Our benchmark shows that Secmarker produces nearly flawless tRNASec predictions. We used Secmarker to scan all currently available genome sequences. The analysis of the highly accurate predictions obtained revealed new insights into the biology of tRNASec.

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3) , as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.

Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, host cells up-regulate nitric oxide while/or parasites induce expression of host arginase, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter ( AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis as well as hypusination of eukaryotic translation Initiation Factor 5A. Here we report the discovery of a pathway whereby Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in the expression and activity of AAP3, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a mitogen-activated protein kinase 2 ( MPK2) -mediated signaling cascade in this response, and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. The arginine-sensing pathway might play an important role in Leishmania virulence and hence serve as target for drug development.

Background Chronic kidney disease (CKD) is associated with high morbidity and death, which increases as CKD progresses to end-stage kidney disease (ESKD). There has been increasing interest in developing innovative, effective and cost-efficient methods to engage with patient populations and improve health behaviours and outcomes. Worldwide there has been a tremendous increase in the use of technologies, with increasing interest in using eHealth interventions to improve patient access to relevant health information, enhance the quality of healthcare and encourage the adoption of healthy behaviours. Objectives This review aims to evaluate the benefits and harms of using eHealth interventions to change health behaviours in people with CKD. Search methods We searched the Cochrane Kidney and Transplant Register of Studies up to 14 January 2019 through contact with the Information Specialist using search terms relevant to this review. Studies in the Register are identified through searches of CENTRAL, MEDLINE, and EMBASE, conference proceedings, the International Clinical Trials Register (ICTRP) Search Portal and ClinicalTrials.gov. Selection criteria Randomised controlled trials (RCTs) and quasi-RCTs using an eHealth intervention to promote behaviour change in people with CKD were included. There were no restrictions on outcomes, language or publication type. Data collection and analysis Two authors independently assessed trial eligibility, extracted data and assessed the risk of bias. The certainty of the evidence was assessed using GRADE. Main results We included 43 studies with 6617 participants that evaluated the impact of an eHealth intervention in people with CKD. Included studies were heterogeneous in terms of eHealth modalities employed, type of intervention, CKD population studied and outcomes assessed. The majority of studies (39 studies) were conducted in an adult population, with 16 studies (37%) conducted in those on dialysis, 11 studies (26%) in the pre-dialysis population, 15 studies (35%) in transplant recipients and 1 studies (2%) in transplant candidates We identified six different eHealth modalities including: Telehealth; mobile or tablet application; text or email messages; electronic monitors; internet/websites; and video or DVD. Three studies used a combination of eHealth interventions. Interventions were categorised into six types: educational; reminder systems; self-monitoring; behavioural counselling; clinical decision-aid; and mixed intervention types. We identified 98 outcomes, which were categorised into nine domains: blood pressure (9 studies); biochemical parameters (6 studies); clinical end-points (16 studies); dietary intake (3 studies); quality of life (9 studies); medication adherence (10 studies); behaviour (7 studies); physical activity (1 study); and cost-effectiveness (7 studies). Only three outcomes could be meta-analysed as there was substantial heterogeneity with respect to study population and eHealth modalities utilised. There was found to be a reduction in interdialytic weight gain of 0.13kg (4 studies, 335 participants: MD -0.13, 95% CI -0.28 to 0.01; I 2 = 0%) and a reduction in dietary sodium intake of 197 mg/day (2 studies, 181 participants: MD -197, 95% CI -540.7 to 146.8; I 2 = 0%). Both dietary sodium and fluid management outcomes were graded as being of low evidence due to high or unclear risk of bias and indirectness (interdialytic weight gain) and high or unclear risk of bias and imprecision (dietary sodium intake). Three studies reported death (2799 participants, 146 events), with 45 deaths/1000 cases compared to standard care of 61 deaths/1000 cases (RR 0.74, CI 0.53 to 1.03; P = 0.08). We are uncertain whether using eHealth interventions, in addition to usual care, impact on the number of deaths as the certainty of this evidence was graded as low due to high or unclear risk of bias, indirectness and imprecision. Authors' conclusions eHealth interventions may improve the management of dietary sodium intake and fluid management. However, overall these data suggest that current evidence for the use of eHealth interventions in the CKD population is of low quality, with uncertain effects due to methodological limitations and heterogeneity of eHealth modalities and intervention types. Our review has highlighted the need for robust, high quality research that reports a core (minimum) data set to enable meaningful evaluation of the literature.

eHealth interventions for people with chronic kidney disease What is the issue? Chronic kidney disease (CKD) is a condition where kidneys have reduced function over a period of time. To remain well people with CKD need to follow complex diet, lifestyle and medication advice and often need to use several specialist medical services. Some people with advanced CKD may need dialysis or treatment with a kidney transplant. Enabling patients to manage this condition by themselves improves quality and length of life and reduces healthcare costs. Electronic health (eHealth) interventions may improve patients' ability to look after themselves and improve care provided by healthcare services. eHealth interventions refer to "health services and information delivered or enhanced through the Internet and related technologies". However, there is little research evaluating the impact of eHealth interventions in CKD. What did we do? We focused on randomised controlled trials (RCT), which enrolled people with CKD (including pre-dialysis, dialysis or kidney transplant), and compared eHealth interventions to usual care. What did we find? We found 43 studies involving 6617 people who had CKD that examined if eHealth interventions improve patient care and health outcomes. eHealth interventions used different modes of technology, such as Telehealth, electronic monitors, mobile or tablet applications, text message or emails, websites, and DVDs or videos. Interventions were classified by their intention: educational, reminder systems, self-monitoring, behavioural counselling, clinical decision-aids and mixed interventions. We categorised outcomes into nine domains: dietary intake, quality of life, blood pressure control, medication adherence, results of blood tests, cost-analysis, behaviour, physical activity and clinical end-points such as death. We found that it was uncertain whether using an eHealth interventions improved clinical and patient-centred outcomes compared with usual care. The quality of the included studies was low, meaning we could not be sure that future studies would find similar results. Conclusions We are uncertain whether using eHealth interventions improves outcomes for people with CKD. We need large and good quality research studies to help understand the impact of eHealth on the health of people with CKD.

Childhood maltreatment (CM) comprises experiences of abuse and neglect during childhood. CM causes psychological as well as biological alterations in affected individuals. In humans, it is hardly explored whether these CM consequences can be transmitted directly on a biological level to the next generation. Here, we investigated the associations between maternal CM and mitochondrial bioenergetics (mitochondrial respiration and intracellular mitochondrial density) in immune cells of mothers and compared them with those of their newborns. In n = 102 healthy mother-newborn dyads, maternal peripheral blood mononuclear cells and neonatal umbilical cord blood mononuclear cells were collected and cryopreserved shortly after parturition to measure mitochondrial respiration and intracellular mitochondrial density with high-resolution respirometry and spectrophotometric analyses, respectively. Maternal CM was assessed with the Childhood Trauma Questionnaire. Maternal and neonatal mitochondrial bioenergetics were quantitatively comparable and positively correlated. Female newborns showed higher mitochondrial respiration compared to male newborns. Maternal CM load was significantly and positively associated with mitochondrial respiration and density in mothers, but not with mitochondrial respiration in newborns. Although maternal and neonatal mitochondrial bioenergetics were positively correlated, maternal CM only had a small effect on mitochondrial density in newborns, which was not significant in this study after adjustment for multiple comparisons. The biological relevance of our finding and its consequences for child development need further investigation in future larger studies. This study reports data on mitochondrial bioenergetics of healthy mother-newborn dyads with varying degrees of CM.

Significance Mitochondrial physiology affected by childhood maltreatment ( CM) might be a biological link for the intergenerational transmission of CM-related consequences. As mitochondria are intergenerationally transmitted from mother to child, alterations in mitochondrial bioenergetics might influence how maternal CM affects physical and mental development as well as functioning in their children. Our study reports data on mitochondrial bioenergetics in peripheral mononuclear immune cells of mother-newborn dyads, which were positively correlated. Higher maternal maltreatment load was associated with higher mitochondrial respiration and density in mothers, but not in their newborns. Thus, we found no evidence for an intergenerational effect of maternal CM on mitochondrial bioenergetics in immune cells of their newborns.

Bacterial cellulose nanofiber (BCNF) with high thermal stability produced by an ecofriendly process has emerged as a promising solution to realize safe and sustainable materials in the large-scale battery. However, an understanding of the actual thermal behavior of the BCNF in the full-cell battery has been lacking, and the yield is still limited for commercialization. Here, we report the entire process of BCNF production and battery manufacture. We systematically constructed a strain with the highest yield (31. 5%) by increasing metabolic flux and improved safety by introducing a Lewis base to overcome thermochemical degradation in the battery. This report will open ways of exploiting the BCNF as a "single-layer" separator, a good alternative to the existing chemical-derived one, and thus can greatly contribute to solving the environmental and safety issues.

Significance This report describes an opportunity for the bacterial cellulose nanofiber ( BCNF) membrane, which has recently been attracting much attention due to its sustainability and high thermal stability, as a "single-layer, " good alternative membrane to the existing chemical-derived one in batteries, and thus allows us to find possible solutions to the environmental and safety issues. We achieved highest BCNF yield ever reported so far and fabricated a cylindrical lithium ion battery ( LIB) by roll-to-roll process to realize remarkable cycle stability with 80% capacity retention even after 1, 000 cycles, which is comparable to those of commercialized batteries. Finally, we introduced a Lewis base to improve thermal safety of the battery to a level comparable to that of a commercial ceramic-coated separator.

Calmodulin (CaM) is a ubiquitous Ca2+ buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca2+-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD) . Induction of LTP and LTD require activation of Ca2+-CaM-dependent enzymes: Ca2+/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca2+ and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca2+ ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca2+ and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca2+ signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca2+-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca2+ channels, and to the microscopic injection rate of Ca2+ ions. We also demonstrate that Ca2+ saturation takes place via two different pathways depending on the Ca2+ injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca2+ sensors that can differentially transduce Ca2+ influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca2+-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.

Calmodulin is a versatile Ca2+ signal mediator and a buffer in a wide variety of body organs including the heart and brain. In the brain, calmodulin regulates intracellular molecular processes that change the strength of connectivity between neurons, thus contributing to various brain functions including memory formation. The exact molecular mechanism as to how calmodulin regulates these processes is not yet known. Interestingly, in other excitable tissues, including the heart, each of two lobes of calmodulin responds differentially toward Ca2+ influx and toward its target molecules ( e. g. , ion channels) . This way, calmodulin precisely controls the Ca2+ dynamics of the cell. We wish to test if a similar mechanism may be operational in neurons so that two lobes of calmodulin interact differentially with Ca2+ ions to activate different downstream molecules that control the strength of connections between neurons. We constructed a detailed simulation of calmodulin that allows us to keep track of its interactions with Ca2+ ions and target proteins at the single molecule level. The simulation predicts that two lobes of calmodulin respond differentially to Ca2+ influx both in space and in time. This work opens a door to future experimental testing of the lobe-specific control of neural function by calmodulin.

Although the effects of stereochemistry have been studied extensively for volatile insect pheromones, little is known about the effects of chirality in the nonvolatile methyl-branched hydrocarbons (MBCHs) used by many insects as contact pheromones. MBCHs generally contain one or more chiral centers and so two or more stereoisomeric forms are possible for each structure. However, it is not known whether insects biosynthesize these molecules in high stereoisomeric purity, nor is it known whether insects can distinguish the different stereoisomeric forms of MBCHs. This knowledge gap is due in part to the lack of methods for isolating individual MBCHs from the complex cuticular hydrocarbon (CHC) blends of insects, as well as the difficulty in determining the absolute configurations of the isolated MBCHs. To address these deficiencies, we report a straightforward method for the isolation of individual cuticular hydrocarbons from the complex CHC blend. The method was used to isolate 36 pure MBCHs from 20 species in nine insect orders. The absolute stereochemistries of the purified MBCHs then were determined by digital polarimetry. The absolute configurations of all of the isolated MBCHs were determined to be (R) by comparison with a library of synthesized, enantiomerically pure standards, suggesting that the biosynthetic pathways used to construct MBCHs are highly conserved within the Insecta. The development of a straightforward method for isolation of specific CHCs will enable determination of their functional roles by providing pure compounds for bioassays.

Significance Methyl-branched hydrocarbons ( MBCHs) are widely used for inter- and intraspecific communication by insects. MBCHs can exist as two or more stereoisomers, but it remains unknown which stereoisomer( s) insects produce, nor is it clear whether insects can distinguish MBCH stereoisomers. To address these questions, we developed a generic method for isolation of MBCHs ( and other lipids) from a range of insect species. Polarimetric analyses of 36 isolated MBCHs showed that their stereochemistry is conserved across nine orders of Insecta, with all compounds having the ( R) -configuration, regardless of methyl branch position or chain length. These results will enable further explorations of the role of stereochemistry in contact chemical communication, and the molecular mechanisms involved in recognition of hydrocarbons.

Background For people with limitations due to neurological conditions such as stroke, the routine practice of oral health care (OHC) may become a challenge. Evidence-based supported oral care intervention is essential for this patient group. Objectives To compare the effectiveness of staff-led OHC interventions with standard care for ensuring oral hygiene for individuals after a stroke. Search methods We searched the trials registers of the Cochrane Stroke Group (last searched April 2010) and Cochrane Oral Health Group (last searched May 2010), the Cochrane Central Register of Controlled Trials (CENTRAL) ( The Cochrane Library May 2010), MEDLINE (1966 to May 2010), CINAHL (1982 to May 2010), Research Findings Electronic Register (February 2006), National Research Register (Issue 1, 2006), ISI Science and Technology Proceedings (July 2010), Dissertation Abstracts and Conference Papers Index (August 2005), Zetoc (2000 to July 2010) and Proquest Dissertations and Theses (2000 to July 2010). We scanned reference lists from relevant papers and contacted authors and researchers in the field. Selection criteria Randomised controlled trials that evaluated one or more interventions designed to improve oral hygiene. We included trials with a mixed population provided we could extract the stroke-specific data. Data collection and analysis Two review authors independently classified trials according to the inclusion and exclusion criteria, assessed the trial quality and extracted data. We sought clarification from study authors when required. Main results We included three studies involving 470 participants. These trials were of limited comparability evaluating an OHC education training programme, a decontamination gel and a ventilator-associated pneumonia bundle of care augmented with an OHC component by comparing them to a deferred intervention, a placebo gel or standard care respectively. The OHC educational intervention demonstrated a significant reduction in denture plaque scores up to six months (P < 0.00001) after the intervention but not dental plaque. Staff knowledge (P = 0.0008) and attitudes (P = 0.0001) towards oral care also improved. The decontamination gel reduced the incidence of pneumonia amongst the intervention group (P = 0.03). Authors' conclusions Based on two trials involving a small number of stroke survivors, OHC interventions can improve staff knowledge and attitudes, the cleanliness of patients' dentures and reduce the incidence of pneumonia. Improvements in the cleanliness of patients teeth were not observed. Further evidence relating to staff-led oral care interventions is severely lacking.

Staff-led interventions for improving oral hygiene in patients following stroke A clean mouth not only feels good but the practice of oral hygiene (removing dental plaque and traces of food) is a crucial factor in maintaining the health of the mouth, teeth and gums. A clean and healthy mouth will also prevent pain or discomfort and allow people to eat a range of nutritious foods. Maintaining good oral hygiene may be difficult after a stroke and healthcare staff may have to assist in providing such care. This review of three studies involving 470 participants found little evidence of how this care is best delivered. Information on a small number of nursing home residents who had a stroke (67 participants from a larger trial) showed that training nursing staff improved their knowledge of oral care and resulted in improved oral hygiene in their patients. Another trial demonstrated the beneficial impact of a decontamination gel on the incidence of pneumonia amongst patients in a stroke ward. However, there was no other information on how best to provide oral hygiene and more studies are urgently needed.

Diverse efforts in protein engineering are beginning to produce novel kinds of symmetric self-assembling architectures, from protein cages to extended two-dimensional (2D) and three-dimensional (3D) crystalline arrays. Partial theoretical frameworks for creating symmetric protein materials have been introduced, but no complete system has been articulated. Only a minute fraction of the possible design space has been explored experimentally, in part because that space has not yet been described in theory. Here, in the form of a multiplication table, we lay out a complete rule set for materials that can be created by combining two chiral oligomeric components (e. g. , proteins) in precise configurations. A unified system is described for parameterizing and searching the construction space for all such symmetry-combination materials (SCMs) . In total, 124 distinct types of SCMs are identified, and then proven by computational construction. Mathematical properties, such as minimal ring or circuit size, are established for each case, enabling strategic predictions about potentially favorable design targets. The study lays out the theoretical landscape and detailed computational prescriptions for a rapidly growing area of protein-based nanotechnology, with numerous underlying connections to mathematical networks and chemical materials such as metal organic frameworks.

Significance Building nanoscale materials in a predictable way is a major goal in broad fields of molecular engineering. In pursuing that goal, protein molecules are gaining interest as a source of structurally defined, self-assembling building blocks, but methods and geometric rules for how they might be combined into novel materials have not been fully laid out yet. This work brings together ideas from structural biology, geometry, and group theory to articulate a complete rule set that describes all the kinds of protein-based materials that can be created by combining separate oligomeric components, thereby laying the foundation for an emerging area of science and biotechnology.

The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in C. elegans, the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5: UNC-40 heterodimer, inhibits growth cone protrusion. We have also shown that inhibition of growth cone protrusion downstream of the UNC-5: UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL (Molecule Interacting with CasL) also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases (FMO-1, -2, -3, -4, and 5) and another molecule, EHBP-1, similar to the non-FMO portion of MICAL. Here we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and UNC-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and unc-5 VD/DD axon guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and UNC-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance.

Mechanisms that guide axons to their targets in the developing nervous system have been elucidated, but how these pathways affect behavior of the growth cone of the axon during outgrowth remains poorly understood. We previously showed that the guidance cue UNC-6/Netrin and its receptors UNC-40/DCC and UNC-5 inhibit lamellipodial and filopodial growth cone protrusion to mediate repulsion from UNC-6/Netrin in C. elegans. Here we report a new mechanism downstream of UNC-6/Netrin involving flavin monooxygenase redox enzymes ( FMOs) . We show that FMOs are normally required for axon guidance and to inhibit growth cone protrusion. Furthermore, we show that they are required for the anti-protrusive effects of activated UNC-40 and UNC-5 receptors, and that they can partially compensate for loss of molecules in the pathway, indicating that they act downstream of UNC-6/Netrin signaling. Based on the function of the FMO-containing MICAL molecules in Drosophila and vertebrates, we speculate that the FMOs might directly oxidize actin, leading to filament disassembly and collapse, and/or lead to the phosphorylation of UNC-33/CRMP, which we show also genetically interacts with the FMOs downstream of UNC-6/Netrin. In conclusion, this is the first evidence that FMOs might act downstream of UNC-6/Netrin signaling in growth cone protrusion and axon repulsion.

Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK) -mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [^13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC) -fed male rats (control) , 2) high fat diet (HFD) -fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.

Significance Hepatic glycogen synthesis plays a critical role in maintaining normal glucose homeostasis; however, the rate-controlling step regulating this process is unknown. Applying metabolic control analysis in vivo, we show that the regulation of insulin-stimulated hepatic glycogen synthesis under both normal and pathophysiological conditions of fatty liver-associated hepatic insulin resistance is controlled at the glucokinase ( GCK) step through GCK translocation.

Influenza A virus (IAV) infection can be severe or even lethal in toddlers, the elderly and patients with certain medical conditions. Infection of apparently healthy individuals nonetheless accounts for many severe disease cases and deaths, suggesting that viruses with increased pathogenicity co-circulate with pandemic or epidemic viruses. Looking for potential virulence factors, we have identified a polymerase PA D529N mutation detected in a fatal IAV case, whose introduction into two different recombinant virus backbones, led to reduced defective viral genomes (DVGs) production. This mutation conferred low induction of antiviral response in infected cells and increased pathogenesis in mice. To analyze the association between low DVGs production and pathogenesis in humans, we performed a genomic analysis of viruses isolated from a cohort of previously healthy individuals who suffered highly severe IAV infection requiring admission to Intensive Care Unit and patients with fatal outcome who additionally showed underlying medical conditions. These viruses were compared with those isolated from a cohort of mild IAV patients. Viruses with fewer DVGs accumulation were observed in patients with highly severe/fatal outcome than in those with mild disease, suggesting that low DVGs abundance constitutes a new virulence pathogenic marker in humans.

Influenza A viruses are the causative agents of annual epidemics, sporadic zoonotic outbreaks and occasionally pandemics. Worldwide, acute respiratory infections caused by influenza A viruses continue to be one of the main causes of acute illness and death. The appearance in 2009 of a new H1N1 pandemic influenza strain reinforced the search to identify viral pathogenicity determinants for evaluation of the consequences of virus epidemics and potential pandemics for human health. Here we identify a new general virulence determinant found in a cohort of severe/fatal influenza virus-infected patients, a reduced accumulation of viral defective genomes. These molecules are incomplete viral genome segments that activate the innate immune response. This data will contribute to the prediction of influenza disease severity, to improved guidance of patient treatment and will enable the development of risk-based prevention strategies and policies.

Spontaneous canine head and neck squamous cell carcinoma (HNSCC) represents an excellent model of human HNSCC but is greatly understudied. To better understand and utilize this valuable resource, we performed a pilot study that represents its first genome-wide characterization by investigating 12 canine HNSCC cases, of which 9 are oral, via high density array comparative genomic hybridization and RNA-seq. The analyses reveal that these canine cancers recapitulate many molecular features of human HNSCC. These include analogous genomic copy number abnormality landscapes and sequence mutation patterns, recurrent alteration of known HNSCC genes and pathways (e. g. , cell cycle, PI3K/AKT signaling) , and comparably extensive heterogeneity. Amplification or overexpression of protein kinase genes, matrix metalloproteinase genes, and epithelial-mesenchymal transition genes TWIST1 and SNAI1 are also prominent in these canine tumors. This pilot study, along with a rapidly growing body of literature on canine cancer, reemphasizes the potential value of spontaneous canine cancers in HNSCC basic and translational research.

Head and neck squamous cell carcinoma ( HNSCC) represents the sixth leading cancer by incidence in humans; thus, developing effective therapeutic interventions is important. Although great advance has been made in our understanding of the biology of HNSCC over the past several decades, translating the research findings into clinical success has been frustratingly slow, and anticancer drug development remains a lengthy and expensive process. A significant challenge is that drug effects in current preclinical cancer models often do not predict clinical results, and there lacks translational models that can bridge the gap between preclinical research and human clinical trials. Here we report a pilot study that represents the first genome-wide characterization of spontaneously occurring HNSCCs in pet dogs. The study reveals a strong dog-human molecular homology at various levels, indicating the likelihood that spontaneous canine HNSCC molecularly represents its human counterpart. If conclusions of this pilot study are validated with a large sample size and more efforts are put into building better resource and infrastructure for canine cancer research, spontaneous canine HNSCCs could effectively serve as a much-needed translational model that bridges the gap between preclinical research and human trials.

Schwann cells in the peripheral nervous systems extend their membranes to wrap axons concentrically and form the insulating sheath, called myelin. The spaces between layers of myelin are sealed by myelin junctions. This tight insulation enables rapid conduction of electric impulses (action potentials) through axons. Demyelination (stripping off the insulating sheath) has been widely regarded as one of the most important mechanisms altering the action potential propagation in many neurological diseases. However, the effective nerve conduction is also thought to require a proper myelin seal through myelin junctions such as tight junctions and adherens junctions. In the present study, we have demonstrated the disruption of myelin junctions in a mouse model (Pmp22+/-) of hereditary neuropathy with liability to pressure palsies (HNPP) with heterozygous deletion of Pmp22 gene. We observed a robust increase of F-actin in Pmp22+/- nerve regions where myelin junctions were disrupted, leading to increased myelin permeability. These abnormalities were present long before segmental demyelination at the late phase of Pmp22+/- mice. Moreover, the increase of F-actin levels correlated with an enhanced activity of p21-activated kinase (PAK1) , a molecule known to regulate actin polymerization. Pharmacological inhibition of PAK normalized levels of F-actin, and completely prevented the progression of the myelin junction disruption and nerve conduction failure in Pmp22+/- mice. Our findings explain how abnormal myelin permeability is caused in HNPP, leading to impaired action potential propagation in the absence of demyelination. We call it "functional demyelination", a novel mechanism upstream to the actual stripping of myelin that is relevant to many demyelinating diseases. This observation also provides a potential therapeutic approach for HNPP.

Demyelination has been widely regarded as one of the most important mechanisms impairing the nerve conduction of electrical impulses in many neurological diseases. Failure of the nerve conduction results in sensory and motor deficits. In the present study, we investigated a novel mechanism that also impairs nerve conduction without the removal of myelin, called functional demyelination. Deletion of one of two copies of the PMP22 gene causes hereditary neuropathy with liability to pressure palsies ( HNPP) . Our recent study reports that deficiency of PMP22 in HNPP disrupts myelin junctions of Schwann cells that seal the spaces between layers of myelin membrane. This disruption leads to "leaky" myelin that impairs the conduction of electrical impulses on the nerves. In the present study, using a HNPP mouse model ( Pmp22+/-) , we have identified a molecular pathway responsible for the disruption of myelin junctions. Our results showed an increase of actin polymerization in the areas where myelin junctions resided. This increase was associated with an up-regulation of PAK1 activity, a kinase function that is known to regulate actin polymerization. We thus treated the HNPP mice with PAK1 inhibitor. This treatment completely prevented the progression of nerve conduction failure and HNPP pathology. This work offers a promising therapeutic approach for HNPP. Moreover, myelin junction disruption takes place long before the actual stripping of myelin ( demyelination) in the late phase of HNPP. Therefore, our finding provides a mechanism upstream to segmental demyelination, a pathological process relevant to many demyelinating diseases.

Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-D7) . Both the mutant and the parental (rTGEV-wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-D7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-D7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2a) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-D7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c) , a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2a dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-D7 and rTGEV-wt viruses showed that rTGEV-D7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of gene 7 by the TGEV genome most likely has provided a selective advantage to the virus.

Innate immune response is the first line of antiviral defense. Viruses have developed diverse strategies to evade this deleterious response, ensuring their survival. Several CoV accessory genes play a central role in these pathways. Nevertheless, the molecular mechanisms by which they exert their function are still unknown. The generation of a rTGEV without gene 7 expression allowed us to study the role of protein 7 in the modulation of the antiviral response. The absence of protein 7 during TGEV infection caused an enhanced apoptosis and a translational shutoff, due to an increased cellular RNA degradation and eIF2a phosphorylation. We identified a protein phosphatase 1 ( PP1) binding motif in protein 7, and a TGEV protein 7-PP1 interaction was demonstrated. We propose a novel mechanism to counteract dsRNA-induced antiviral response by RNA viruses. In vitro results were in agreement with the enhanced virulence of the gene 7 deletion mutant virus in infected piglets. Our results demonstrated that protein 7 modifies TGEV virulence, reducing virus pathology and increasing the period of virus shedding. This effect also benefits the host decreasing clinical disease and extending its survival. These observations could justify the incorporation and maintenance of gene 7 to genus a1 CoVs during their evolution.

Thymine DNA glycosylase (TDG) is a pivotal enzyme with dual roles in both genome maintenance and epigenetic regulation. TDG is involved in cytosine demethylation at CpG sites in DNA. Here we have used molecular modeling to delineate the lesion search and DNA base interrogation mechanisms of TDG. First, we examined the capacity of TDG to interrogate not only DNA substrates with 5-carboxyl cytosine modifications but also G: T mismatches and nonmismatched (A: T) base pairs using classical and accelerated molecular dynamics. To determine the kinetics, we constructed Markov state models. Base interrogation was found to be highly stochastic and proceeded through insertion of an arginine-containing loop into the DNA minor groove to transiently disrupt Watson-Crick pairing. Next, we employed chain-of-replicas path-sampling methodologies to compute minimum free energy paths for TDG base extrusion. We identified the key intermediates imparting selectivity and determined effective free energy profiles for the lesion search and base extrusion into the TDG active site. Our results show that DNA sculpting, dynamic glycosylase interactions, and stabilizing contacts collectively provide a powerful mechanism for the detection and discrimination of modified bases and epigenetic marks in DNA.

Significance The most prominent epigenetic modification in mammalian genomes is cytosine methylation at position 5 on the pyrimidine ring. Thymine DNA glycosylase ( TDG) plays a central role in the pathways for 5-methyl cytosine removal and thus influences gene silencing, stem cell differentiation, and alterations in normal development. Additionally, methylation abnormalities in DNA are often observed in diseases, specifically cancer. Here we examine the mechanisms by which TDG detects, extrudes, and excises modified bases in DNA. Using path-sampling methodologies, we compute minimum free energy paths for TDG base extrusion. The computed paths reveal a unique mechanism underpinning TDG selectivity for DNA lesions or modified bases, which involves DNA sculpting, global protein dynamics, conformational gating, and specific protein-nucleic acid interactions.

Changes in the physical interaction between cis-regulatory DNA sequences and proteins drive the evolution of gene expression. However, it has proven difficult to accurately quantify evolutionary rates of such binding change or to estimate the relative effects of selection and drift in shaping the binding evolution. Here we examine the genome-wide binding of CTCF in four species of Drosophila separated by between ~2. 5 and 25 million years. CTCF is a highly conserved protein known to be associated with insulator sequences in the genomes of human and Drosophila. Although the binding preference for CTCF is highly conserved, we find that CTCF binding itself is highly evolutionarily dynamic and has adaptively evolved. Between species, binding divergence increased linearly with evolutionary distance, and CTCF binding profiles are diverging rapidly at the rate of 2. 22% per million years (Myr) . At least 89 new CTCF binding sites have originated in the Drosophila melanogaster genome since the most recent common ancestor with Drosophila simulans. Comparing these data to genome sequence data from 37 different strains of Drosophila melanogaster, we detected signatures of selection in both newly gained and evolutionarily conserved binding sites. Newly evolved CTCF binding sites show a significantly stronger signature for positive selection than older sites. Comparative gene expression profiling revealed that expression divergence of genes adjacent to CTCF binding site is significantly associated with the gain and loss of CTCF binding. Further, the birth of new genes is associated with the birth of new CTCF binding sites. Our data indicate that binding of Drosophila CTCF protein has evolved under natural selection, and CTCF binding evolution has shaped both the evolution of gene expression and genome evolution during the birth of new genes.

A large proportion of the diversity of living organisms results from differential regulation of gene transcription. Transcriptional regulation is thought to differ between species because of evolutionary changes in the physical interactions between regulatory DNA elements and DNA-binding proteins; these can generate variation in the spatial and temporal patterns of gene expression. The mechanisms by which these protein-DNA interactions evolve is therefore an important question in evolutionary biology. Does adaptive evolution play a role, or is the process dominated by neutral genetic drift? Insulator proteins are a special group of DNA-binding proteins--instead of directly serving to activate or repress genes, they can function to coordinate the interactions between other regulatory elements ( such as enhancers and promoters) . Additionally, insulator proteins can limit the spreading of chromatin condensation and help to demarcate the boundaries of regulatory domains in the genome. In spite of their critical role in genome regulation, little is known about the evolution of interactions between insulator proteins and DNA. Here, we use ChIP-seq to examine the distribution of binding sites for CTCF, a highly conserved insulator protein, in four closely related Drosophila species. We find that genome-wide binding profiles of CTCF are highly dynamic across evolutionary time, with frequent births of new CTCF-DNA interactions, and we demonstrate that this evolutionary process is driven by natural selection. By comparing these with RNA-seq data, we find that gain or loss of CTCF binding impacts the expression levels of nearby genes and correlates with structural evolution of the genome. Together these results suggest a potential mechanism of regulatory re-wiring through adaptive evolution of CTCF binding.

Nef is an accessory protein of primate lentiviruses that is essential for efficient replication and pathogenesis of HIV-1. A conserved feature of Nef proteins from different lentiviral lineages is the ability to modulate host protein trafficking and down-regulate a number of cell surface receptors to enhance replication and promote immune evasion. Notably, the inability of Nef to down-regulate CD3 from infected T cells distinguishes HIV-1 Nef and its direct simian precursors from other primate lentiviruses. Why HIV-1 does not employ this potential immune evasion strategy is not fully understood. Using chimeric HIV-1 constructs expressing lentiviral Nef proteins that differ in their ability to down-modulate CD3, we show that retaining CD3 on the surface of infected primary T cells results in increased viral replication and cell-to-cell spread. We identified increased expression of envelope (Env) trimers at the cell surface and increased Env incorporation into virions as the determinants for the Nef- and CD3-dependent enhancement of viral infectivity. Importantly, this was independent of Nef-mediated antagonism of the host restriction factor SERINC5. CD3 retention on the surface of infected primary T cells also correlated with increased T cell signaling, activation, and cell death during cell-to-cell spread. Taken together, our results show that loss of an otherwise conserved function of Nef has a positive effect on HIV-1 replication, allowing for more efficient replication while potentially contributing to HIV-1 pathogenesis by triggering T cell activation and cell death during viral spread.

Significance Lentiviruses encode accessory proteins to manipulate their host cells in order to efficiently replicate and evade antiviral defenses. Interestingly, most lentiviral Nefs down-regulate CD3 from the surface of infected T cells to perturb immune responses. However, for reasons that are incompletely understood, HIV-1 and its simian immunodeficiency virus ancestors lack this function. Here, we report that engineering HIV-1 for Nef-mediated down-regulation of CD3 reduces Env-dependent HIV-1 infectivity, resulting in less efficient cell-to-cell spread and replication. Our data suggest that HIV-1 may have evolved to lose the CD3 down-modulation function of Nef in order to allow T cell activation and to boost viral replication, possibly at the cost of less effective immune evasion and increased pathogenicity.

Effectors of the bacterial type III secretion system provide invaluable molecular probes to elucidate the molecular mechanisms of plant immunity and pathogen virulence. In this report, we focus on the AvrBs2 effector protein from the bacterial pathogen Xanthomonas euvesicatoria (Xe) , the causal agent of bacterial spot disease of tomato and pepper. Employing homology-based structural analysis, we generate a three-dimensional structural model for the AvrBs2 protein and identify catalytic sites in its putative glycerolphosphodiesterase domain (GDE) . We demonstrate that the identified catalytic region of AvrBs2 was able to functionally replace the GDE catalytic site of the bacterial glycerophosphodiesterase BhGlpQ cloned from Borrelia hermsii and is required for AvrBs2 virulence. Mutations in the GDE catalytic domain did not disrupt the recognition of AvrBs2 by the cognate plant resistance gene Bs2. In addition, AvrBs2 activation of Bs2 suppressed subsequent delivery of other Xanthomonas type III effectors into the host plant cells. Investigation of the mechanism underlying this modulation of the type III secretion system may offer new strategies to generate broad-spectrum resistance to bacterial pathogens.

The bacterial pathogen Xanthomonas euvesicatoria ( Xe) is the causal agent of bacterial leaf spot disease of pepper and tomato. This pathogen is capable of delivering more than 28 effector proteins to plant cells via the type three secretion and translocation system ( TTSS) . The AvrBs2 protein is a TTSS effector of Xe with a significant virulence contribution that depends on a conserved glycerolphosphodiesterase ( GDE) domain. Additionally, activation of the resistance protein Bs2 by AvrBs2 modulates the TTSS of Xe and suppresses the subsequent delivery of TTSS effectors.

MicroRNAs (miRNAs) , a large class of short noncoding RNAs found in many plants and animals, often act to post-transcriptionally inhibit gene expression. We report the generation of deletion mutations in 87 miRNA genes in Caenorhabditis elegans, expanding the number of mutated miRNA genes to 95, or 83% of known C. elegans miRNAs. We find that the majority of miRNAs are not essential for the viability or development of C. elegans, and mutations in most miRNA genes do not result in grossly abnormal phenotypes. These observations are consistent with the hypothesis that there is significant functional redundancy among miRNAs or among gene pathways regulated by miRNAs. This study represents the first comprehensive genetic analysis of miRNA function in any organism and provides a unique, permanent resource for the systematic study of miRNAs.

MicroRNAs ( miRNAs) are tiny endogenous RNAs that regulate gene expression in plants and animals. Individual miRNAs have important roles in development, immunity, and cancer. Although the investigation of miRNA function is of great importance, to date few miRNAs have been studied in the intact organism because of a lack of mutants in which specific miRNAs have been inactivated. Here we describe a collection of loss-of-function mutants representing the majority of all known miRNA genes in the nematode Caenorhabditis elegans. This study identifies a new role for miRNAs in C. elegans and also demonstrates that most miRNAs are not essential for viability or development. Our findings suggest that many miRNAs act redundantly with other miRNAs or other pathways. We expect that this collection of miRNA mutants will become a widely used resource to further our understanding of the biology of miRNAs.

The majority of cilia are formed and maintained by the highly conserved process of intraflagellar transport (IFT) . Mutations in IFT genes lead to ciliary structural defects and systemic disorders termed ciliopathies. Here we show that the severely truncated sensory cilia of hypomorphic IFT mutants in C. elegans transiently elongate during a discrete period of adult aging leading to markedly improved sensory behaviors. Age-dependent restoration of cilia morphology occurs in structurally diverse cilia types and requires IFT. We demonstrate that while DAF-16/FOXO is dispensable, the age-dependent suppression of cilia phenotypes in IFT mutants requires cell-autonomous functions of the HSF1 heat shock factor and the Hsp90 chaperone. Our results describe an unexpected role of early aging and protein quality control mechanisms in suppressing ciliary phenotypes of IFT mutants, and suggest possible strategies for targeting subsets of ciliopathies.

Cilia are 'antenna-like' structures that are present on nearly all cell types in animals. These structures are important for sensing and signaling external cues to the cell. Most cilia are formed by a protein transport process called 'intraflagellar transport' or IFT. Mutations in IFT genes result in severe cilia defects, and are causal to a large number of diverse human disorders called ciliopathies. Since the genes and processes by which cilia are formed are similar across species, studies in experimental models such as the nematode C. elegans can greatly inform our overall understanding of cilia formation and function. Here we report the surprising observation that the structures and functions of severely defective cilia in nematodes with disrupted IFT genes markedly improve upon aging. We find that protein quality control mechanisms that normally decline in aging are required for this age-dependent recovery of cilia structure. Our results raise the possibility that the effects of some mutations in IFT genes can be bypassed under specific conditions, thereby restoring cilia functions.

Spherical nucleic acids (SNAs) are nanostructures formed by chemically conjugating short linear strands of oligonucleotides to a nanoparticle template. When made with modified small interfering RNA (siRNA) duplexes, SNAs act as single-entity transfection and gene silencing agents and have been used as lead therapeutic constructs in several disease models. However, the manner in which modified siRNA duplex strands that comprise the SNA lead to gene silencing is not understood. Herein, a systematic analysis of siRNA biochemistry involving SNAs shows that Dicer cleaves the modified siRNA duplex from the surface of the nanoparticle, and the liberated siRNA subsequently functions in a way that is dependent on the canonical RNA interference mechanism. By leveraging this understanding, a class of SNAs was chemically designed which increases the siRNA content by an order of magnitude through covalent attachment of each strand of the duplex. As a consequence of increased nucleic acid content, this nanostructure architecture exhibits less cell cytotoxicity than conventional SNAs without a decrease in siRNA activity.

Significance Although small interfering RNAs ( siRNAs) are promising agents for treating many diseases, cellular delivery difficulties have prevented their widespread application. The use of spherical nucleic acids ( SNAs) as single-entity gene regulation agents could overcome this limitation, since SNAs naturally and rapidly enter over 50 cell types. Consequently, siRNA-SNAs have been extensively used for effecting knockdown in both research and clinical settings. However, the mechanism of gene regulation by siRNA-SNAs is not well understood. Herein, we study cytosolic processing of siRNA-SNAs and how it leads to gene knockdown. Informed by this mechanism, we designed an SNA architecture with an order-of-magnitude-higher siRNA density. Importantly, this higher density reduces the cellular toxicity of SNAs without loss in RNA interference performance.

Regulation of gene expression via specific cis-regulatory promoter elements has evolved in cellular organisms as a major adaptive mechanism to respond to environmental change. Assuming a simple model of transcriptional regulation, genes that are differentially expressed in response to a large number of different external stimuli should harbor more distinct regulatory elements in their upstream regions than do genes that only respond to few environmental challenges. We tested this hypothesis in Arabidopsis thaliana using the compendium of gene expression profiling data available in AtGenExpress and known cis-element motifs mapped to upstream gene promoter regions and studied the relation of the observed breadth of differential gene expression response with several fundamental genome architectural properties. We observed highly significant positive correlations between the density of cis-elements in upstream regions and the number of conditions in which a gene was differentially regulated. The correlation was most pronounced in regions immediately upstream of the transcription start sites. Multistimuli response genes were observed to be associated with significantly longer upstream intergenic regions, retain more paralogs in the Arabidopsis genome, are shorter, have fewer introns, and are more likely to contain TATA-box motifs in their promoters. In abiotic stress time series data, multistimuli response genes were found to be overrepresented among early-responding genes. Genes involved in the regulation of transcription, stress response, and signaling processes were observed to possess the greatest regulatory capacity. Our results suggest that greater gene expression regulatory complexity appears to be encoded by an increased density of cis-regulatory elements and provide further evidence for an evolutionary adaptation of the regulatory code at the genomic layout level. Larger intergenic spaces preceding multistimuli response genes may have evolved to allow greater regulatory gene expression potential.

The induction or repression of specific genes has evolved in living organisms as a mechanism to respond to environmental changes. At the molecular level, this process is mediated via molecular switches, so-called regulatory elements, generally located in the genomic region adjacent to the gene they control, the gene promoter. Upon environmental change, specific proteins bind to such regulatory elements, thereby turning on or off the associated genes. As this molecular response is often specific to the external signal, genes that respond to a large number of different external stimuli should harbor more distinct regulatory elements in their promoter regions than should genes responding only to few environmental challenges. In analyzing data for the plant Arabidopsis thaliana, we observed that indeed an increased number of regulatory elements is associated with a broader range of responses. Several other genome structural properties, such as gene size, the occurrence of similar genes in the Arabidopsis genome, and the distance between genes, were also observed to be correlated with a broader breadth of response. The results suggest that greater regulatory complexity appears encoded by an increased density of regulatory elements and provide further evidence for an evolutionary adaptation of the regulatory code at the genomic architectural level.

Each year influenza virus infections cause hundreds of thousands of deaths worldwide and a significant level of morbidity with major economic burden. At the present time, vaccination with inactivated virus vaccine produced from embryonated chicken eggs is the most prevalent method to prevent the infections. However, current influenza vaccines are only effective against closely matched circulating strains and must be updated and administered yearly. Therefore, generating a vaccine that can provide broad protection is greatly needed for influenza vaccine development. We have previously shown that vaccination of the major surface glycoprotein hemagglutinin (HA) of influenza virus with a single N-acetylglucosamine at each of the N-glycosylation sites [monoglycosylated HA (HAmg) ] can elicit better cross-protection compared with the fully glycosylated HA (HAfg) . In the current study, we produced monoglycosylated inactivated split H1N1 virus vaccine from chicken eggs by the N-glycosylation process inhibitor kifunensine and the endoglycosidase Endo H, and intramuscularly immunized mice to examine its efficacy. Compared with vaccination of the traditional influenza vaccine with complex glycosylations from eggs, the monoglycosylated split virus vaccine provided better cross-strain protection against a lethal dose of virus challenge in mice. The enhanced antibody responses induced by the monoglycosylated vaccine immunization include higher neutralization activity, higher hemagglutination inhibition, and more HA stem selectivity, as well as, interestingly, higher antibody-dependent cellular cytotoxicity. This study provides a simple and practical procedure to enhance the cross-strain protection of influenza vaccine by removing the outer part of glycans from the virus surface through modifications of the current egg-based process.

Significance Glycosylation is important for the proper folding and function of glycoproteins. However, glycosylation on viral surface protein may mask the conserved epitopes and causes poor host immune responses and protection against viral infection. To expose more antigenic sites shielded by glycans, we developed a strategy to remove the glycans on the viral surface by modification of the traditional egg-based influenza vaccine production procedure. By means of immunization with the monoglycosylated split virus vaccine, mice were induced with broader immune responses, and thus better protection against cross-strain H1N1 virus infections. The results showed that the monoglycosylated split virus vaccine is a broadly protective vaccine and can be produced through simple modifications of the current vaccine manufacturing process.

DNA nanotechnology has established approaches for designing programmable and precisely controlled nanoscale architectures through specific Watson-Crick base-pairing, molecular plasticity, and intermolecular connectivity. In particular, superior control over DNA origami structures could be beneficial for biomedical applications, including biosensing, in vivo imaging, and drug and gene delivery. However, protecting DNA origami structures in complex biological fluids while preserving their structural characteristics remains a major challenge for enabling these applications. Here, we developed a class of structurally well-defined peptoids to protect DNA origamis in ionic and bioactive conditions and systematically explored the effects of peptoid architecture and sequence dependency on DNA origami stability. The applicability of this approach for drug delivery, bioimaging, and cell targeting was also demonstrated. A series of peptoids (PE1-9) with two types of architectures, termed as "brush" and "block, " were built from positively charged monomers and neutral oligo-ethyleneoxy monomers, where certain designs were found to greatly enhance the stability of DNA origami. Through experimental and molecular dynamics studies, we demonstrated the role of sequence-dependent electrostatic interactions of peptoids with the DNA backbone. We showed that octahedral DNA origamis coated with peptoid (PE2) can be used as carriers for anticancer drug and protein, where the peptoid modulated the rate of drug release and prolonged protein stability against proteolytic hydrolysis. Finally, we synthesized two alkyne-modified peptoids (PE8 and PE9) , conjugated with fluorophore and antibody, to make stable DNA origamis with imaging and cell-targeting capabilities. Our results demonstrate an approach toward functional and physiologically stable DNA origami for biomedical applications.

Significance DNA nanotechnology provides a structural toolkit for the fabrication of programmable DNA nano-constructs; however, their use in biomedical applications is challenging due the limited structural integrity in complex biological fluids. Here, we report a class of tailorable molecular coatings, peptoids, which can efficiently stabilize three-dimensional wireframed DNA constructs under a variety of biomedically relevant conditions, including magnesium-ion depletion and presence of degrading nuclease. Furthermore, we show that peptoid-coated DNA constructs offer a controllable anticancer drug release and an ability to display functional biomolecules on the DNA surfaces. Our study demonstrates an approach for building multifunctional and environmentally robust DNA-based molecular structures for nanomedicine and biosensing.

Identifying the structure and dynamics of synaptic interactions between neurons is the first step to understanding neural network dynamics. The presence of synaptic connections is traditionally inferred through the use of targeted stimulation and paired recordings or by post-hoc histology. More recently, causal network inference algorithms have been proposed to deduce connectivity directly from electrophysiological signals, such as extracellularly recorded spiking activity. Usually, these algorithms have not been validated on a neurophysiological data set for which the actual circuitry is known. Recent work has shown that traditional network inference algorithms based on linear models typically fail to identify the correct coupling of a small central pattern generating circuit in the stomatogastric ganglion of the crab Cancer borealis. In this work, we show that point process models of observed spike trains can guide inference of relative connectivity estimates that match the known physiological connectivity of the central pattern generator up to a choice of threshold. We elucidate the necessary steps to derive faithful connectivity estimates from a model that incorporates the spike train nature of the data. We then apply the model to measure changes in the effective connectivity pattern in response to two pharmacological interventions, which affect both intrinsic neural dynamics and synaptic transmission. Our results provide the first successful application of a network inference algorithm to a circuit for which the actual physiological synapses between neurons are known. The point process methodology presented here generalizes well to larger networks and can describe the statistics of neural populations. In general we show that advanced statistical models allow for the characterization of effective network structure, deciphering underlying network dynamics and estimating information-processing capabilities.

To appreciate how neural circuits control behaviors, we must understand two things. First, how the neurons comprising the circuit are connected, and second, how neurons and their connections change after learning or in response to neuromodulators. Neuronal connectivity is difficult to determine experimentally, whereas neuronal activity can often be readily measured. We describe a statistical model to estimate circuit connectivity directly from measured activity patterns. We use the timing relationships between observed spikes to predict synaptic interactions between simultaneously observed neurons. The model estimate provides each predicted connection with a curve that represents how strongly, and at which temporal delays, one circuit element effectively influences another. These curves are analogous to synaptic interactions of the level of the membrane potential of biological neurons and share some of their features such as being inhibitory or excitatory. We test our method on recordings from the pyloric circuit in the crab stomatogastric ganglion, a small circuit whose connectivity is completely known beforehand, and find that the predicted circuit matches the biological one -- a result other techniques failed to achieve. In addition, we show that drug manipulations impacting the circuit are revealed by this technique. These results illustrate the utility of our analysis approach for inferring connections from neural spiking activity.

Background Symptomatic peripheral arterial disease may be treated by a number of options including exercise therapy, angioplasty, stenting and bypass surgery. Atherectomy is an alternative technique where atheroma is excised by a rotating cutting blade. Objectives The objective of this review was to analyse randomised controlled trials comparing atherectomy against any established treatment for peripheral arterial disease in order to evaluate the effectiveness of atherectomy. Search methods The Cochrane Peripheral Vascular Diseases Group Trials Search Co-ordinator searched the Specialised Register (last searched November 2013) and CENTRAL (2013, Issue 10). Trials databases were searched for details of ongoing or unpublished studies. Selection criteria Randomised controlled trials (RCTs) comparing atherectomy and other established treatments were selected for inclusion. All participants had symptomatic peripheral arterial disease with either claudication or critical limb ischaemia and evidence of lower limb arterial disease. Data collection and analysis Two review authors (GA and CT) screened studies for inclusion, extracted data and assessed the quality of the trials. Any disagreements were resolved through discussion. Main results Four trials were included with a total of 220 participants (118 treated with atherectomy, 102 treated with balloon angioplasty) and 259 treated vessels (129 treated with atherectomy, 130 treated with balloon angioplasty). All studies compared atherectomy with angioplasty. No study was properly powered or assessors blinded to the procedures and there was a high risk of selection, attrition, detection and reporting biases. The estimated risk of success was similar between the treatment modalities although the confidence interval (CI) was compatible with small benefits of either treatment for the initial procedural success rate (Mantel-Haenszel risk ratio (RR) 0.92, 95% CI 0.44 to 1.91, P = 0.82), patency at six months (Mantel-Haenszel RR 0.92, 95% CI 0.51 to 1.66, P = 0.79) and patency at 12 months (Mantel-Haenszel RR 1.17, 95% CI 0.72 to 1.90, P = 0.53) following the procedure. The reduction in all-cause mortality with atherectomy was most likely due to an unexpectedly high mortality in the balloon angioplasty group in one of the two trials that reported mortality (Mantel-Haenszel RR 0.24, 95% CI 0.06 to 0.91, P = 0.04). Cardiovascular events were not reported in any study. There was a reduction in the rate of bailout stenting following atherectomy (Mantel-Haenszel RR 0.45, 95% CI 0.24 to 0.84, P = 0.01), and balloon inflation pressures were lower following atherectomy (mean difference -2.73 mmHg, 95% CI -3.48 to -1.98, P < 0.00001). Complications such as embolisation and vessel dissection were reported in two trials indicating more embolisations in the atherectomy group and more vessel dissections in the angioplasty group, but the data could not be pooled. From the limited data available, there was no clear evidence of different rates of adverse events between the atherectomy and balloon angioplasty groups for target vessel revascularisation and above-knee amputation. Quality of life and clinical and symptomatic outcomes such as walking distance or symptom relief were not reported in the studies. Authors' conclusions This review has identified poor quality evidence to support atherectomy as an alternative to balloon angioplasty in maintaining primary patency at any time interval. There was no evidence for superiority of atherectomy over angioplasty on any outcome, and distal embolisation was not reported in all trials of atherectomy. Properly powered trials are recommended.

Atherectomy for peripheral arterial disease A person with diseased arteries in the legs can experience pain on walking (also known as intermittent claudication), pain at rest (especially at night), or ulcers due to poor blood flow. Established treatments include surgery, where a bypass is inserted to carry blood from an artery above the diseased (blocked or narrowed) section to below the diseased section, and balloon angioplasty, where a deflated balloon is inserted into the vessel and then blown up to stretch the artery thus opening up the narrow or blocked section. Stents may be inserted during angioplasty. In addition to these two established treatments, a less commonly used technique is to core out the artery, cutting or grinding away the disease which is causing the vessel to narrow or block. This is known as atherectomy. In this review, we compared atherectomy to the more established treatments such as balloon angioplasty and bypass surgery. We identified four studies with a total of 220 participants. All studies compared atherectomy with balloon angioplasty. The studies were of low quality as there was no blinding of the procedures, the studies were not properly powered to show an effect, not all study outcomes were reported and a large number of the initial study populations did not complete the studies. Although the results of the meta-analyses were imprecise, the average effect of the two treatments was similar in terms of initial success and unobstructed arteries (patency) at six months or 12 months following the procedure. There was a lower risk of death with atherectomy, most likely due to an unexpectedly high number of deaths in the balloon angioplasty group in one of the two trials reporting deaths. Cardiovascular events were not reported in any of the included studies. There was a reduction in the rate of emergency stenting procedures following atherectomy, and balloon inflation pressures were lower following atherectomy. Complications such as formation of clots (embolisation) and tears along the vessels (vessel dissection) were reported in two trials indicating more embolisations in the atherectomy group and more vessel dissections in the angioplasty group but the data could not be combined. The limited data available indicated that there was no clear evidence of a difference between the atherectomy and balloon angioplasty groups for adverse events such as the need for re-intervention due to obstruction of the treated vessel and above-knee amputation. Quality of life and clinical and symptomatic outcomes such as walking distance or symptom relief were not reported in the studies. We showed that the limited evidence available does not support a significant advantage of atherectomy over conventional balloon angioplasty.

Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O2^*-) by catalyzing its conversion to dioxygen (O2) and hydrogen peroxide (H2O2) . Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major aspect of the antioxidant function of Sod1 is to integrate O2 availability to promote NADPH production. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the glycolytic enzyme, GAPDH, which in turn reroutes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is dependent on and rate-limited by Sod1. Thus, Sod1 senses O2 via O2^*- to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O2^*- toxicity, which only requires 2O2 is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.

Significance Cu/Zn superoxide dismutase ( Sod1) is a key antioxidant enzyme, and its importance is underscored by the fact that its ablation in cell and animal models results in oxidative stress; metabolic defects; and reductions in cell proliferation, viability, and lifespan. Curiously, Sod1 detoxifies superoxide radicals ( O2^*-) in a manner that produces an oxidant as byproduct, hydrogen peroxide ( H2O2) . While much is known about the necessity of scavenging O2^*-, it is less clear what the physiological roles of Sod1-derived H2O2 are. We discovered that Sod1-derived H2O2 plays an important role in antioxidant defense by stimulating the production of NADPH, a vital cellular reductant required for reactive oxygen species scavenging enzymes, as well as redox regulating a large network of enzymes.

Over the last decades, researchers have characterized a set of "clock genes" that drive daily rhythms in physiology and behavior. This arduous work has yielded results with far-reaching consequences in metabolic, psychiatric, and neoplastic disorders. Recent attempts to expand our understanding of circadian regulation have moved beyond the mutagenesis screens that identified the first clock components, employing higher throughput genomic and proteomic techniques. In order to further accelerate clock gene discovery, we utilized a computer-assisted approach to identify and prioritize candidate clock components. We used a simple form of probabilistic machine learning to integrate biologically relevant, genome-scale data and ranked genes on their similarity to known clock components. We then used a secondary experimental screen to characterize the top candidates. We found that several physically interact with known clock components in a mammalian two-hybrid screen and modulate in vitro cellular rhythms in an immortalized mouse fibroblast line (NIH 3T3) . One candidate, Gene Model 129, interacts with BMAL1 and functionally represses the key driver of molecular rhythms, the BMAL1/CLOCK transcriptional complex. Given these results, we have renamed the gene CHRONO (computationally highlighted repressor of the network oscillator) . Bi-molecular fluorescence complementation and co-immunoprecipitation demonstrate that CHRONO represses by abrogating the binding of BMAL1 to its transcriptional co-activator CBP. Most importantly, CHRONO knockout mice display a prolonged free-running circadian period similar to, or more drastic than, six other clock components. We conclude that CHRONO is a functional clock component providing a new layer of control on circadian molecular dynamics.

Daily rhythms are ever-present in the living world, driving the sleep-wake cycle and many other physiological changes. In the last two decades, several labs have identified "clock genes" that interact to generate underlying molecular oscillations. However, many aspects of circadian molecular physiology remain unexplained. Here, we used a simple "machine learning" approach to identify new clock genes by searching the genome for candidate genes that share clock-like features such as cycling, broad-based tissue RNA expression, in vitro circadian activity, genetic interactions, and homology across species. Genes were ranked by their similarity to known clock components and the candidates were screened and validated for evidence of clock function in vitro. One candidate, which we renamed CHRONO ( Gm129) , interacted with the master regulator of the clock, BMAL1, disrupting its transcriptional activity. We found that Chrono knockout mice had prolonged locomotor activity rhythms, getting up progressively later each day. Our experiments demonstrated that CHRONO interferes with the ability of BMAL1 to recruit CBP, a bona fide histone acetylase and key transcriptional coactivator of the circadian clock.

Background Severe aortic valve stenosis (AS) is a major cause of morbidity and mortality worldwide. The definitive management for severe AS is aortic valve replacement (AVR). The choice of transcatheter approach versus open-heart surgery for AVR in people with severe AS and low surgical risk remains a matter of debate. Objectives To assess the benefits and harms of transcatheter aortic valve implantation (TAVI) compared to surgical aortic valve replacement (SAVR) in people with severe AS and low surgical risk. Search methods We searched the following databases for randomised controlled trials (RCTs) on 29 April 2019: Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, and Web of Science Core Collection. We also searched ClinicalTrials.gov and the World Health Organization International Clinical Trials Registry Platform. We searched all databases from inception to present and imposed no restriction on language or date of publication. Selection criteria We included RCTs that compared TAVI and SAVR in adults (18 years of age or older) with severe AS and low surgical risk. Data collection and analysis We used the standard methodological procedures expected by Cochrane. Two authors independently screened titles and abstracts for inclusion, performed data extraction, and assessed risk of bias in the studies included. We analysed dichotomous data using the risk ratio (RR) and continuous data using the mean difference (MD), with respective 95% confidence intervals (CI). We assessed the certainty of evidence for each outcome using the GRADE approach. Our outcomes of interest were assessed in the short term (i.e. during hospitalisation and up to 30 days of follow-up). Primary outcomes were all-cause mortality, stroke, and rehospitalisation. Secondary outcomes were myocardial infarction (MI), cardiac death, length of hospital stay (LOS), permanent pacemaker (PPM) implantation, new-onset atrial fibrillation, acute kidney injury (AKI), and any bleeding. Main results We identified four studies (13 reports), with 2818 participants, and one ongoing study. Overall certainty of evidence ranged from high to very low. There is probably little or no difference between TAVI and SAVR for the following short-term outcomes: all-cause mortality (RR 0.69, 95% CI 0.33 to 1.44; SAVR 11 deaths per 1000, TAVI 8 deaths per 1000 (95% CI 4 to 16); 2818 participants; 4 studies; moderate-certainty evidence); stroke (RR 0.73, 95% CI 0.42 to 1.25; SAVR 21 strokes per 1000, TAVI 16 strokes per 1000 (95% CI 9 to 27); 2818 participants; 4 studies; moderate-certainty evidence); MI (RR 0.82, 95% CI 0.42 to 1.58; SAVR 14 MI per 1000, TAVI 11 MI per 1000 (95% CI 6 to 21); 2748 participants; 3 studies; moderate-certainty evidence); and cardiac death (RR 0.71, 95% CI 0.32 to 1.56; SAVR 10 cardiac deaths per 1000, TAVI 7 cardiac deaths per 1000 (95% CI 3 to 16); 2818 participants; 4 studies; moderate-certainty evidence). TAVI may reduce the risk of short-term rehospitalisation, although the confidence interval also includes the possibility of no difference in risk between groups (RR 0.64, 95% CI 0.39 to 1.06; SAVR 30 cases per 1000, TAVI 19 cases per 1000 (95% CI 12 to 32); 2468 participants; 2 studies; low-certainty evidence). TAVI, compared with SAVR, probably increases the risk of PPM implantation (RR 3.65, 95% CI 1.50 to 8.87; SAVR 47 per 1000, TAVI 170 cases per 1000 (95% CI 70 to 413); number needed to treat for an additional harmful outcome (NNTH) = 7; 2683 participants; 3 studies; moderate-certainty evidence). We are uncertain whether TAVI, compared with SAVR, affects the LOS in days, although it appears to be associated with shorter LOS. TAVI, compared with SAVR, reduces the risk of atrial fibrillation (RR 0.21, 95% CI 0.15 to 0.30; 2683 participants; 3 studies), AKI (RR 0.30, 95% CI 0.16 to 0.58; 2753 participants; 4 studies), and bleeding (RR 0.31, 95% CI 0.16 to 0.62; 2753 participants; 4 studies) (all high-certainty evidence). Authors' conclusions Our meta-analysis indicates that, in the short term, TAVI probably has little or no mortality difference compared to SAVR for severe AS in individuals with low surgical risk. Similarly, there is probably little or no difference in risk of stroke, MI, and cardiac death between the two approaches. TAVI may reduce the risk of rehospitalisation, but we are uncertain about the effects on LOS. TAVI reduces the risk of atrial fibrillation, AKI, and bleeding. However, this benefit is offset by the increased risk of PPM implantation. Long-term follow-up data are needed to further assess and validate these outcomes, especially durability, in the low surgical risk population.

Transcatheter aortic valve implantation versus surgical aortic valve replacement in people with severe aortic stenosis and low surgical risk Review question Does transcatheter approach, compared to open-heart surgery, for aortic valve replacement improve outcomes in people with severe aortic stenosis and low surgical risk? Background Aortic stenosis (AS) is the narrowing of the exit of the left ventricle of the heart (where the aorta begins). It typically gets worse over time. Its severity can be divided into mild, moderate, severe, and very severe; and it is distinguishable by ultrasound scan of the heart and other clinical features. Once it has become severe, treatment primarily involves surgery to replace the valve. An alternative approach is to use transcatheter aortic valve implantation (TAVI). This approach improves outcomes in individuals who are inoperable or at a high to intermediate risk for surgery. However, it remains unclear if TAVI is beneficial to those who have severe AS and are at a low surgical risk. Search Date The evidence is current to April 2019. We searched the literature and found a total of 3092 citations that were potentially relevant. After reviewing each of these, we found 13 published articles describing four clinical trials that could help us answer our question. Study characteristics The four clinical trials included 2818 participants who were randomly allocated to undergo either TAVI or surgical aortic valve replacement (SAVR). The trials were multicentre and took place in Australia, Canada, France, Japan, the Netherlands, New Zealand, the USA, Denmark, and Sweden. Key Results Moderate-certainty evidence from clinical trials shows that, in the short term (i.e. during hospitalisation and up to 30 days of follow-up), there is probably little or no difference between TAVI compared with SAVR in risk of death due to any cause, stroke (insult to the brain), myocardial infarction (injury or death of heart muscle), or death due to cardiac causes (e.g. myocardial infarction or failure of the heart muscle pump). Low-certainty evidence shows that TAVI may reduce the risk of rehospitalisation compared with SAVR. We are uncertain whether TAVI, compared with SAVR, affects the length of hospital stay, although it appears to be associated with shorter duration of hospitalisation. High-certainty evidence shows that fewer people had atrial fibrillation (a type of irregular heart rhythm), acute kidney injury (insult to the kidney), and bleeding when they underwent TAVI, compared with SAVR. However, moderate-certainty evidence shows that TAVI probably increases the risk of permanent pacemaker implantation (a device that is placed to artificially set the heart rhythm), compared with SAVR. Quality of the evidence We consider the overall quality of evidence to be moderate for most relevant outcomes (death, stroke, myocardial infarction, cardiac death, and risk of permanent pacemaker implantation), with the exception of rehospitalisation (low-quality evidence) and length of hospital stay (very low quality evidence). The evidence for atrial fibrillation, acute kidney injury, and bleeding was of high quality.

Development is often strongly regulated by interactions among close relatives, but the underlying molecular mechanisms are largely unknown. In eusocial insects, interactions between caregiving worker nurses and larvae regulate larval development and resultant adult phenotypes. Here, we begin to characterize the social interactome regulating ant larval development by collecting and sequencing the transcriptomes of interacting nurses and larvae across time. We find that the majority of nurse and larval transcriptomes exhibit parallel expression dynamics across larval development. We leverage this widespread nurse-larva gene co-expression to infer putative social gene regulatory networks acting between nurses and larvae. Genes with the strongest inferred social effects tend to be peripheral elements of within-tissue regulatory networks and are often known to encode secreted proteins. This includes interesting candidates such as the nurse-expressed giant-lens, which may influence larval epidermal growth factor signaling, a pathway known to influence various aspects of insect development. Finally, we find that genes with the strongest signatures of social regulation tend to experience relaxed selective constraint and are evolutionarily young. Overall, our study provides a first glimpse into the molecular and evolutionary features of the social mechanisms that regulate all aspects of social life.

Social interactions are fundamental to all forms of life, from single-celled bacteria to complex plants and animals. Despite their obvious importance, little is known about the molecular causes and consequences of social interactions. In this paper, we study the molecular basis of nurse-larva social interactions that regulate larval development in the pharaoh ant Monomorium pharaonis. We infer the effects of social interactions on gene expression from samples of nurses and larvae collected in the act of interaction across a developmental time series. Gene expression appears to be closely tied to these interactions, such that we can identify genes expressed in nurses with putative regulatory effects on larval gene expression. Genes which we infer to have strong social regulatory effects tend to have weak regulatory effects within individuals, and highly social genes tend to experience relatively weaker natural selection in comparison to fewer social genes. This study represents a novel approach and foundation upon which future studies at the intersection of genetics, behavior, and evolution can build.

Electron-transfer theories predict that an increase in the quantum-mechanical mixing (HDA) of electron donor and acceptor wavefunctions at the instant of electron transfer drives equilibrium constants toward unity. Kinetic and equilibrium studies of four acceptor-bridge-donor (A-B-D) compounds reported herein provide experimental validation of this prediction. The compounds have two redox-active groups that differ only by the orientation of the aromatic bridge: a phenyl-thiophene bridge (p) that supports strong electronic coupling of HDA > 1, 000 cm^-1; and a xylyl-thiophene bridge (x) that prevents planarization and decreases HDA without a significant change in distance. Pulsed-light excitation allowed kinetic determination of the equilibrium constant, Keq. In agreement with theory, Keq(p) were closer to unity compared to Keq(x) . A van't Hoff analysis provided clear evidence of an adiabatic electron-transfer pathway for p-series and a nonadiabatic pathway for x-series. Collectively, the data show that the absolute magnitude of the thermodynamic driving force for electron transfers are decreased when adiabatic pathways are operative, a finding that should be taken into account in the design of hybrid materials for solar energy conversion.

Significance Nature's use of electronic coupling ( HDA) and free-energy ( DG^o) gradients to vectorially control electron transport provides inspiration for artificial photosynthesis. Theoretical predictions indicate that HDA and DG^o are not independent parameters, and are instead linked. Reported here is a broadly applicable kinetic approach that was utilized to demonstrate such behavior for four acceptor-bridge-donor compounds. When the electronic coupling was large and electron transfer was adiabatic, the free energy of the reaction |DG^o| was less than that for nonadiabatic transfer. This finding should be taken into account in the design of hybrid materials for solar energy conversion and has broad implications to the many classes of electron-transfer reactions in biology and chemistry.

Obesity and the metabolic syndrome have evolved to be major health issues throughout the world. Whether loss of genome integrity contributes to this epidemic is an open question. DNA polymerase e (pol e) , encoded by the xeroderma pigmentosum (XP-V) gene, plays an essential role in preventing cutaneous cancer caused by UV radiation-induced DNA damage. Herein, we demonstrate that pol e deficiency in mice (pol e^-/-) causes obesity with visceral fat accumulation, hepatic steatosis, hyperleptinemia, hyperinsulinemia, and glucose intolerance. In comparison to WT mice, adipose tissue from pol e^-/- mice exhibits increased DNA damage and a greater DNA damage response, indicated by up-regulation and/or phosphorylation of ataxia telangiectasia mutated (ATM) , phosphorylated H2AX (gH2AX) , and poly[ADP-ribose] polymerase 1 (PARP-1) . Concomitantly, increased cellular senescence in the adipose tissue from pol e^-/- mice was observed and measured by up-regulation of senescence markers, including p53, p16^Ink4a, p21, senescence-associated (SA) b-gal activity, and SA secretion of proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor a (TNF-a) as early as 4 wk of age. Treatment of pol e^-/- mice with a p53 inhibitor, pifithrin-a, reduced adipocyte senescence and attenuated the metabolic abnormalities. Furthermore, elevation of adipocyte DNA damage with a high-fat diet or sodium arsenite exacerbated adipocyte senescence and metabolic abnormalities in pol e^-/- mice. In contrast, reduction of adipose DNA damage with N-acetylcysteine or metformin ameliorated cellular senescence and metabolic abnormalities. These studies indicate that elevated DNA damage is a root cause of adipocyte senescence, which plays a determining role in the development of obesity and insulin resistance.

Significance The metabolic syndrome has evolved to be a major health issue globally. The association between genome integrity and metabolic abnormalities is not well understood. Our results indicate that increased DNA damage and persistent activation of the DNA damage response induce adipocyte senescence in DNA polymerase e knockout ( pole^-/-) mice. Suppression of adipocyte senescence with a p53 inhibitor, pifithrin-a, alleviated metabolic abnormalities in pol e^-/- mice. An increase or decrease in DNA damage affected the senescence status of adipocytes accordingly, which was also in concordance with the severity of metabolic abnormalities in the pol e^-/- mice. Our current results indicate that reduced genome integrity plays a causative role in provoking adipocyte senescence that leads to development of obesity and insulin resistance.

Using a computational model, we simulated mitochondrial deoxynucleotide metabolism and mitochondrial DNA replication. Our results indicate that the output from the mitochondrial salvage enzymes alone is inadequate to support a mitochondrial DNA replication duration of as long as 10 hours. We find that an external source of deoxyribonucleoside diphosphates or triphosphates (dNTPs) , in addition to those supplied by mitochondrial salvage, is essential for the replication of mitochondrial DNA to complete in the experimentally observed duration of approximately 1 to 2 hours. For meeting a relatively fast replication target of 2 hours, almost two-thirds of the dNTP requirements had to be externally supplied as either deoxyribonucleoside di- or triphosphates, at about equal rates for all four dNTPs. Added monophosphates did not suffice. However, for a replication target of 10 hours, mitochondrial salvage was able to provide for most, but not all, of the total substrate requirements. Still, additional dGTPs and dATPs had to be supplied. Our analysis of the enzyme kinetics also revealed that the majority of enzymes of this pathway prefer substrates that are not precursors (canonical deoxyribonucleosides and deoxyribonucleotides) for mitochondrial DNA replication, such as phosphorylated ribonucleotides, instead of the corresponding deoxyribonucleotides. The kinetic constants for reactions between mitochondrial salvage enzymes and deoxyribonucleotide substrates are physiologically unreasonable for achieving efficient catalysis with the expected in situ concentrations of deoxyribonucleotides.

The powerhouses of human cells, mitochondria, contain DNA that is distinct from the primary genome, the DNA in the nucleus of cells. The mitochondrial genome needs to be replicated often to ensure continued generation of ATP ( adenosine triphosphate) which is the energy currency of the cell. Problems with maintenance of mitochondrial DNA, arising from genetic mutations as well as from antiviral drugs, can lead to debilitating diseases that are often fatal in early life and childhood, or reduced compliance to therapy from patients suffering drug toxicity. It is therefore important to understand the processes that contribute to the upkeep of mitochondrial DNA. The activities of a set of enzymes, which together generate the chemical building blocks of mitochondrial DNA, are important in this regard. We used computational methods to analyze the properties of these enzymes. Results from our approach of treating these enzymes as a system rather than studying them one at a time suggest that in most conditions, the activities of the enzymes are not sufficient for completing replication of mitochondrial DNA in the observed duration of around 2 hours. We propose that a source of building blocks in addition to this set of enzymes appears to be essential.

The slow spontaneous inactivation of potassium channels exhibits classic signatures of transmembrane allostery. A variety of data support a model in which the loss of K^+ ions from the selectivity filter is a major factor in promoting inactivation, which defeats transmission, and is allosterically coupled to protonation of key channel activation residues, more than 30 A from the K^+ ion binding site. We show that proton binding at the intracellular pH sensor perturbs the potassium affinity at the extracellular selectivity filter by more than three orders of magnitude for the full-length wild-type KcsA, a pH-gated bacterial channel, in membrane bilayers. Studies of F103 in the hinge of the inner helix suggest an important role for its bulky sidechain in the allosteric mechanism; we show that the energetic strength of coupling of the gates is strongly altered when this residue is mutated to alanine. These results provide quantitative site-specific measurements of allostery in a bilayer environment, and highlight the power of describing ion channel gating through the lens of allosteric coupling.

Significance Although C-type inactivation in potassium channels is crucial in many biological signaling processes, its mechanism is debated. We address a hypothesis that K^+ affinity reduction and ion loss due to allosterically coupled activation is a major factor in promoting inactivation. We show that in KcsA, activation by proton binding at the intracellular pH sensor can significantly reduce K^+ affinity at the extracellular selectivity filter, which would be expected to contribute to current cessation ( inactivation) . This allosteric coupling is substantially reduced by mutating a residue that connects the inner transmembrane helix hinge ( involved in activation) to the base of the selectivity filter, thus providing clear evidence for the allosteric coupling network and explaining why this mutant is inactivationless.

Proinflammatory signaling pathways are commonly up-regulated in breast cancer. In estrogen receptor-negative (ER^-) and triple-negative breast cancer (TNBC) , nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) have been described as independent predictors of disease outcome. We further explore these findings by investigating the impact of their coexpression on breast cancer survival. Elevated coexpression of NOS2/COX2 proteins is a strong predictor of poor survival among ER^- patients (hazard ratio: 21) . Furthermore, we found that the key products of NOS2 and COX2, NO and prostaglandin E2 (PGE2) , respectively, promote feed-forward NOS2/COX2 crosstalk in both MDA-MB-468 (basal-like) and MDA-MB-231 (mesenchymal-like) TNBC cell lines in which NO induced COX2 and PGE2 induced NOS2 proteins. COX2 induction by NO involved TRAF2 activation that occurred in a TNFa-dependent manner in MDA-MB-468 cells. In contrast, NO-mediated TRAF2 activation in the more aggressive MDA-MB-231 cells was TNFa independent but involved the endoplasmic reticulum stress response. Inhibition of NOS2 and COX2 using amino-guanidine and aspirin/indomethacin yielded an additive reduction in the growth of MDA-MB-231 tumor xenografts. These findings support a role of NOS2/COX2 crosstalk during disease progression of aggressive cancer phenotypes and offer insight into therapeutic applications for better survival of patients with ER^- and TNBC disease.

Significance Nitric oxide synthase-2 ( NOS2) and cyclooxygenase-2 ( COX2) are inflammation-associated enzymes with oncogenic function in breast cancer. We show that crosstalk between NOS2/COX2 promotes aggressive phenotypes and that elevated coexpression of NOS2/COX2 in tumors predict significantly reduced patient survival ( 33%) when compared with 95% survival of estrogen receptor-negative patients with low NOS2/COX2 tumor expression. In addition, we have identified a tumor subtype-specific mechanism showing involvement of TNFa and/or endoplasmic reticulum stress as key players in this autocrine loop. Importantly, the simultaneous inhibition of NOS2/COX2 significantly reduced tumor growth in a xenograft murine model, suggesting that targeted inhibition of NOS2/COX2 may be therapeutically beneficial.

Background Superior oblique palsy is a common cause of vertical strabismus in adults and children. Patients may be symptomatic from binocular vertical diplopia or compensatory head tilt required to maintain single vision. Most patients who are symptomatic elect to undergo strabismus surgery, but the optimal surgical treatment for vertical strabismus in people with superior oblique palsy is unknown. Objectives To assess the relative effects of surgical treatments compared with another surgical intervention, non-surgical intervention, or observation for vertical strabismus in people with superior oblique palsy. Search methods We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (which contains the Cochrane Eyes and Vision Trials Register) (2016, Issue 12), MEDLINE Ovid (1946 to 13 December 2016), Embase Ovid (1947 to 13 December 2016), Latin American and Caribbean Health Sciences Literature Database (LILACS) (1982 to 13 December 2016), the ISRCTN registry ( www.isrctn.com/editAdvancedSearch ); searched 13 December 2016, ClinicalTrials.gov ( www.clinicaltrials.gov ); searched 13 December 2016, and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) ( www.who.int/ictrp/search/en ); searched 13 December 2016. We did not use any date or language restrictions in the electronic searches for trials. Selection criteria We included randomized trials that compared at least one type of surgical intervention to another surgical or non-surgical intervention or observation. Data collection and analysis Two review authors independently completed eligibility screening, data abstraction, 'Risk of bias' assessment, and grading of the evidence. Main results We identified two randomized trials comparing four different surgical treatments for this condition, two methods in each trial. The studies included a total of 45 children and adults. The surgical treatments were all procedures to weaken the ipsilateral inferior oblique muscle. One study compared inferior oblique myectomy to recession of 10 mm; the other study compared inferior oblique disinsertion to anterior transposition (2 mm anterior to the temporal border of the inferior rectus insertion). We judged both studies to be at unclear risk of bias due to incomplete reporting of methods and other methodological deficiencies. Neither study reported data on the primary outcome of this review, which was the proportion of participants with postoperative surgical success, defined as hypertropia less than 3 prism diopters (PD) in primary gaze. However, both studies reported the average reduction in hypertropia in primary gaze. One study found that at 12 months' postoperatively the average decrease in hypertropia was higher in participants who underwent inferior oblique myectomy than in those who underwent recession, however data were not available for statistical comparison. The other trial found that after at least six months of follow-up, the mean decrease in primary position hypertropia was lower in participants who underwent inferior oblique disinsertion than in those who underwent anterior transposition (mean difference (MD) -5.20 PD, 95% confidence interval (CI) -7.76 to -2.64; moderate-quality evidence). Both trials also reported the average postoperative reduction in vertical deviation in adduction. One study reported that the average reduction in hypertropia in adduction was greater in participants who underwent inferior oblique myectomy than in those who underwent recession, but data were not available for statistical comparison. The other study found a lower decrease in hypertropia in contralateral gaze in participants who underwent inferior oblique disinsertion than in those who underwent anterior transposition (MD -7.10 PD, 95% CI -13.85 to -0.35; moderate-quality evidence). Secondary outcomes with sufficient data for analysis included proportion of participants with preoperative head tilt that resolved postoperatively and proportion of participants who underwent a second surgery. These outcomes were assessed in the trial comparing inferior oblique anterior transposition to disinsertion; both outcomes favored anterior transposition (risk ratio 7.00, 95% CI 0.40 to 121.39 for both outcomes; very low-quality evidence). None of the participants who underwent inferior oblique anterior transposition or disinsertion developed postoperative hypotropia or reversal of the vertical deviation. All participants who underwent inferior oblique anterior transposition developed elevation deficiency, which the authors deemed to be clinically insignificant in all cases, whereas no participants who underwent inferior oblique disinsertion experienced this complication. Additionally, the trial comparing inferior oblique myectomy to recession reported that no participant in either group required another strabismus surgery during the postoperative period. Authors' conclusions The two trials included in this review evaluated four inferior oblique weakening procedures for surgical treatment of superior oblique palsy. We found no trials comparing other types of surgical procedures for this disorder. Both studies had enrolled a small number of participants and provided low-quality evidence due to limitations in completeness and applicability. We therefore found no high-quality evidence to support recommendations for optimal surgical treatment of superior oblique palsy. Rigorously designed, conducted, and reported randomized trials are needed to identify the optimal surgical treatment for vertical strabismus in this disorder.

Surgical treatments for vertical eye misalignment (strabismus) in superior oblique palsy Review aim The aim of this Cochrane Review was to determine whether surgery for vertical strabismus in people with superior oblique palsy works better than other surgical or non-surgical interventions. We searched for all relevant studies and identified two clinical trials. Key messages There is no high-quality evidence regarding the effects of surgery on vertical strabismus in people with superior oblique palsy. Consequently, we were unable to determine the best surgery for this disorder. Carefully designed studies are needed to enable treatment recommendations for this common problem. What did we study in this review? We compared different types of surgery to reduce vertical strabismus in children and adults with a diagnosis of superior oblique palsy. Superior oblique palsy occurs when there is weakness of one of the muscles (superior oblique) involved in eye movement, causing a characteristic pattern of strabismus, or misalignment of the eyes, that usually varies with head positioning. Superior oblique palsy is a common cause of vertical strabismus, and can lead to double vision or abnormal head positioning in order to maintain single vision. Main results Each of the two included trials compared two different surgical procedures to weaken the inferior oblique muscle, and thus balance the weakness in the superior oblique muscle. A total of four different inferior oblique muscle-weakening surgeries were studied: myectomy (removing part of the muscle), recession (moving the muscle to a position where it exerts less force), anterior transposition (moving the muscle to a position where the direction of force is altered), and disinsertion (detaching the muscle from the sclera). Neither of the trials examined the main outcome we wished to study, that is the proportion of participants deemed to have successful eye realignment after surgery. Additionally, we judged the quality of the data in both studies to be low. How up-to-date is this review? We searched for trials with outcome data published by 13 December 2016. The included trials were published between 2001 and 2003.

To study VSV entry and the fate of incoming matrix (M) protein during virus uncoating we used recombinant viruses encoding M proteins with a C-terminal tetracysteine tag that could be fluorescently labeled using biarsenical (Lumio) compounds. We found that uncoating occurs early in the endocytic pathway and is inhibited by expression of dominant-negative (DN) Rab5, but is not inhibited by DN-Rab7 or DN-Rab11. Uncoating, as defined by the separation of nucleocapsids from M protein, occurred between 15 and 20 minutes post-entry and did not require microtubules or an intact actin cytoskeleton. Unexpectedly, the bulk of M protein remained associated with endosomal membranes after uncoating and was eventually trafficked to recycling endosomes. Another small, but significant fraction of M distributed to nuclear pore complexes, which was also not dependent on microtubules or polymerized actin. Quantification of fluorescence from high-resolution confocal micrographs indicated that after membrane fusion, M protein diffuses across the endosomal membrane with a concomitant increase in fluorescence from the Lumio label which occurred soon after the release of RNPs into the cytoplasm. These data support a new model for VSV uncoating in which RNPs are released from M which remains bound to the endosomal membrane rather than the dissociation of M protein from RNPs after release of the complex into the cytoplasm following membrane fusion.

Vesicular stomatitis virus ( VSV) is a prototypic enveloped virus that enters cells following endocytosis and a low pH-dependent membrane fusion event between the viral and endosomal membrane. To initiate a productive infection the viral nucleocapsid must dissociate from the matrix ( M) protein, which underlies the viral membrane, in a process known as uncoating. The requirements for VSV uncoating are poorly understood. Here we used a virus containing fluorescent M protein to follow VSV uncoating in live cells. This analysis resulted in three new findings which provide for the first time a description of matrix and nucleocapsid trafficking during VSV uncoating. We found that most of the M protein remains bound to the endosomal membrane after virus-endosome fusion and that the nucleocapsid is released into the cytoplasm where replication occurs. While most of M remains membrane-bound, a small but detectable fraction is released during uncoating and is trafficked to nuclear pores. This has not been previously observed and may aid in shutting down host responses to infection. Collectively we provide the first spatio-temporal description of VSV uncoating by visualizing the uncoating process in live cells.

Background Carbonic anhydrase inhibitors such as acetazolamide cause a mild metabolic acidosis and may stimulate breathing. Some patients with severe chronic obstructive pulmonary disease (COPD) develop chronic hypercapnic ventilatory failure. In theory, they may benefit from use of these drugs with a fall in arterial carbon dioxide level (PCO2) and a rise in arterial oxygen (PO2). Objectives To determine the effectiveness and safety of acetazolamide in the treatment of hypercapnic ventilatory failure due to COPD. Search methods The Cochrane Airways Group Specialised Register was searched with predefined search terms. Searches were current as of October 2010. Selection criteria Trials were included in the review provided they were placebo controlled, carried out in patients with stable chronic ventilatory failure due to COPD. Data collection and analysis Data were extracted and analysed by two reviewers (PJ and MG) and agreement was reached by consensus. Where data could be aggregated they were analysed using a fixed effects model and reported as a weighted mean difference (MD) and its associated 95% confidence interval (95% CI). Main results Four trials were included in the review. Of these, two were randomised parallel studies, one was a crossover study and the other had a sequential design. A total of 84 patients were involved. Study quality was mixed and the studies were short (typically two weeks). All studies showed a similar direction and size of effect. In the randomised parallel studies, acetazolamide caused a metabolic acidosis and produced a non-significant fall in PCO2 (MD -0.41 kPa; 95% CI -0.91, 0.09; N=2) and a significant rise in PO2 (MD 1.54 kPa; 95% CI 0.97, 2.11; N=2). One study reported an improvement in sleep but there were no data concerning outcomes such as health status, symptoms, exacerbation rate, hospital admissions or deaths. Side effects were reported infrequently. An update search conducted in October 2005 did not identify any further studies. Authors' conclusions Acetazolamide can produce a small increase in arterial PO2 and fall in PCO2. These conclusions are drawn from a few small short studies that were not all of high quality. It is not known whether this physiological improvement is associated with clinical benefit.

Carbonic anhydrase inhibitors for hypercapnic ventilatory failure in chronic obstructive pulmonary disease Some people with advanced chronic lung disease (COPD - chronic bronchitis or emphysema) can experience breathing failure. This involves chemical changes which in turn can lower the drive to breathe. The drug acetazolamide is used for mountain sickness, and it can stimulate breathing in some circumstances. The review of trials found that a few days of using acetazolamide can improve the level of oxygen in the blood of people with COPD. It is not clear if this leads to better outcomes, so more research is needed. Not enough data were reported on the safety of the drug.

Germ-line specification is essential for sexual reproduction. In the ovules of most flowering plants, only a single hypodermal cell enlarges and differentiates into a megaspore mother cell (MMC) , the founder cell of the female germ-line lineage. The molecular mechanisms restricting MMC specification to a single cell remain elusive. We show that the Arabidopsis transcription factor WRKY28 is exclusively expressed in hypodermal somatic cells surrounding the MMC and is required to repress these cells from acquiring MMC-like cell identity. In this process, the SWR1 chromatin remodeling complex mediates the incorporation of the histone variant H2A. Z at the WRKY28 locus. Moreover, the cytochrome P450 gene KLU, expressed in inner integument primordia, non-cell-autonomously promotes WRKY28 expression through H2A. Z deposition at WRKY28. Taken together, our findings show how somatic cells in ovule primordia cooperatively use chromatin remodeling to restrict germ-line cell specification to a single cell.

Significance In flowering plants, the female germ line begins as a single cell known as the megaspore mother cell ( MMC) in each ovule. The mechanisms that restrict MMC fate to a single cell remain largely unknown. We show that the Arabidopsis cytochrome P450 gene KLU acts through the chromatin remodeling complex SWR1 to promote WRKY28 expression in ovule primordia. We show that WRKY28 is expressed in a few somatic cells surrounding the MMC and is required to inhibit these cells from acquiring the MMC-like cell fate. Consistent with non-cell-autonomous KLU activity, KLU-expressing cells and WRKY28-expressing cells are neither identical nor adjacently positioned. Our study demonstrates that cell-cell interactions involving only somatic cells in ovule primordia ensure the specification of a single MMC.

Soil microorganisms determine the fate of soil organic matter (SOM) , and their activities compose a major component of the global carbon (C) cycle. We employed a multisubstrate, DNA-stable isotope probing experiment to track bacterial assimilation of C derived from distinct sources that varied in bioavailability. This approach allowed us to measure microbial contributions to SOM processing by measuring the C assimilation dynamics of diverse microorganisms as they interacted within soil. We identified and tracked 1, 286 bacterial taxa that assimilated ^13C in an agricultural soil over a period of 48 d. Overall ^13C-assimilation dynamics of bacterial taxa, defined by the source and timing of the ^13C they assimilated, exhibited low phylogenetic conservation. We identified bacterial guilds composed of taxa that had similar ^13C assimilation dynamics. We show that C-source bioavailability explained significant variation in both C mineralization dynamics and guild structure, and that the growth dynamics of bacterial guilds differed significantly in response to C addition. We also demonstrate that the guild structure explains significant variation in the biogeographical distribution of bacteria at continental and global scales. These results suggest that an understanding of in situ growth dynamics is essential for understanding microbial contributions to soil C cycling. We interpret these findings in the context of bacterial life history strategies and their relationship to terrestrial C cycling.

Significance Soil organic matter contains a large fraction of global C whose fate is largely governed by soil microbial communities. Global C-cycle models can be improved using information about microbial processes in soil, but the inner workings of soil communities remain largely hidden from view. We used stable isotopes to characterize the guild structure of soil bacteria based on how they assimilated C from different sources. Guild structure corresponded with growth dynamics and rrn copy number. We also show that this guild structure explains microbial biogeographical patterns at continental and global scales. These results suggest that microbial contributions to soil C cycling are largely governed by an anabolic process that governs energy allocation to growth, competition, and survival.

Neddylation, an important type of post-translational modification, has been implicated in innate and adapted immunity. But the role of neddylation in innate immune response against RNA viruses remains elusive. Here we report that neddylation promotes RNA virus-induced type I IFN production, especially IFN-a. More importantly, myeloid deficiency of UBA3 or NEDD8 renders mice less resistant to RNA virus infection. Neddylation is essential for RNA virus-triggered activation of Ifna gene promoters. Further exploration has revealed that mammalian IRF7undergoes neddylation, which is enhanced after RNA virus infection. Even though neddylation blockade does not hinder RNA virus-triggered IRF7 expression, IRF7 mutant defective in neddylation exhibits reduced ability to activate Ifna gene promoters. Neddylation blockade impedes RNA virus-induced IRF7 nuclear translocation without hindering its phosphorylation and dimerization with IRF3. By contrast, IRF7 mutant defective in neddylation shows enhanced dimerization with IRF5, an Ifna repressor when interacting with IRF7. In conclusion, our data demonstrate that myeloid neddylation contributes to host anti-viral innate immunity through targeting IRF7 and promoting its transcriptional activity.

With the features of high mutation rates and fast propagation, RNA viruses remain a great challenge for the control and prevention of epidemic. Better understanding of the molecular mechanisms involved in host innate immunity against RNA viruses will facilitate the development of anti-viral drugs and vaccines. Neddylation has been implicated in innate and adapted immunity. But the role of neddylation in RNA virus-triggered type I IFN production remains elusive. Here, using mouse models with myeloid deficiency of UBA3 or NEDD8, we report for the first time that neddylation contributes to innate immunity against RNA viruses in mammals. Neddylation is indispensable for RNA virus-induced IFN-a production although its role in IFN-b production is much blunted in macrophages. In mechanism, neddylation directly targets IRF7 and enhances its transcriptional activity through, at least partially, promoting its nuclear translocation and preventing its dimerization with IRF5, an Ifna repressor when interacting with IRF7. Our study provides insight into the regulation of IRF7 and innate immune signaling.

Background Chronic obstructive pulmonary disease (COPD) is characterised by progressive airflow obstruction, worsening exercise performance and health deterioration. It is associated with significant morbidity, mortality and health system burden. Objectives To evaluate the effectiveness of outreach respiratory health care worker programmes for COPD patients in terms of improving lung function, exercise tolerance and health related quality of life (HRQL) of patient and carer, and reducing mortality and medical service utilisation. Search methods The Cochrane Airways Group Specialised Register of Trials was searched (November 2011). Study references were hand-searched for additional studies we contacted study authors to identify other unpublished studies. Selection criteria We included only randomised controlled trials of COPD patients. We included interventions involving an outreach nurse visiting patients in their homes, providing support, education, monitoring health and liaising with physicians. Studies in which the therapeutic intervention under test was physical training were not included. Data collection and analysis Two reviewers independently assessed trial quality and extracted data. We contacted study authors for additional information. Main results We pooled mortality data from eight studies and found a non-significant reduction in mortality at 12 months (OR 0.72, 95% CI 0.45 to, 1.15). We pooled four studies that assessed disease-specific heath-related quality of life (HRQL) and found a statistically significant improvement in HRQL (mean difference -2.61, 95% CI -4.82 to -0.40). Hospitalisations were reported in five studies. Although there was no statistically significant difference in the number of hospitalisations (OR 1.01, 95% CI 0.71 to 1.44), there was significant heterogeneity. Although this heterogeneity appeared to be caused by one outlying study with a statistically significant decrease in hospitalisations in patients receiving home care, whereas the other studies showed a non-significant increase in hospitalisations, we could not draw firm conclusions about why this heterogeneity exists. Data on GP visits and emergency department presentations were available, however no consistent effect in these was observed with the intervention. The intervention also incurred higher health care costs than standard care as reported in a single study. Very few studies provided data on lung function or exercise performance, so there was insufficient evidence to assess impact on these outcomes. Authors' conclusions Outreach nursing programmes for COPD improved disease-specific HRQL. However the effect on hospitalisations was heterogeneous, reducing admissions in one study, but increasing them in others, therefore we could not draw firm conclusions for this outcome.

Does delivery of home care by outreach nurses improve outcomes for people with chronic obstructive pulmonary disease? Home visits from nurses for people with chronic lung disease (chronic obstructive pulmonary disease, COPD - a combinations of emphysema and chronic bronchitis) aim to help people maintain their health and reduce the need for hospital stays. The nurses delivering this care aim to help people use their treatments well, provide education about coping strategies, and monitor the lung disease. However, this review of nine randomised controlled trial found that home care resulted in an improvement in people's quality of life, but has an unpredictable effect on the risk of being admitted to hospital. We could only find information on the cost of care from one study, but this indicated that home care was an expensive form of care. More research is needed to confirm the usefulness of home visits for people with COPD.

b-Barrel pore-forming toxins (bPFTs) form an obligatory oligomeric prepore intermediate before the formation of the b-barrel pore. The molecular components that control the critical prepore-to-pore transition remain unknown for bPFTs. Using the archetype bPFT perfringolysin O, we show that E183 of each monomer within the prepore complex forms an intermolecular electrostatic interaction with K336 of the adjacent monomer on completion of the prepore complex. The signal generated throughout the prepore complex by this interaction irrevocably commits it to the formation of the membrane-inserted giant b-barrel pore. This interaction supplies the free energy to overcome the energy barrier (determined here to be ~19 kcal/mol) to the prepore-to-pore transition by the coordinated disruption of a critical interface within each monomer. These studies provide the first insight to our knowledge into the molecular mechanism that controls the prepore-to-pore transition for a bPFT.

Significance Bacterial pathogens produce pore-forming toxins that damage eukaryotic membranes, whereas the pore-forming immune defense proteins produced by vertebrates can damage bacterial membranes. Despite the opposite functions of these proteins in pathogenesis or protection, many use a common pore-forming mechanism whereby membrane-bound monomers oligomerize into a circular structure, termed the prepore, which then assembles a b-barrel structure that punches a hole in the membrane. Here we show that once the prepore is assembled, an intermolecular electrostatic interaction is established that drives the formation of the pore. This mechanism is likely to be used by toxins and other pore-forming proteins that span the biological domains of life.

A central debate in cognitive science concerns the nativist hypothesis, the proposal that universal features of behavior reflect a biologically determined cognitive substrate: For example, linguistic nativism proposes a domain-specific faculty of language that strongly constrains which languages can be learned. An evolutionary stance appears to provide support for linguistic nativism, because coordinated constraints on variation may facilitate communication and therefore be adaptive. However, language, like many other human behaviors, is underpinned by social learning and cultural transmission alongside biological evolution. We set out two models of these interactions, which show how culture can facilitate rapid biological adaptation yet rule out strong nativization. The amplifying effects of culture can allow weak cognitive biases to have significant population-level consequences, radically increasing the evolvability of weak, defeasible inductive biases; however, the emergence of a strong cultural universal does not imply, nor lead to, nor require, strong innate constraints. From this we must conclude, on evolutionary grounds, that the strong nativist hypothesis for language is false. More generally, because such reciprocal interactions between cultural and biological evolution are not limited to language, nativist explanations for many behaviors should be reconsidered: Evolutionary reasoning shows how we can have cognitively driven behavioral universals and yet extreme plasticity at the level of the individual--if, and only if, we account for the human capacity to transmit knowledge culturally. Wherever culture is involved, weak cognitive biases rather than strong innate constraints should be the default assumption.

Significance A central debate in cognitive science concerns the nativist hypothesis: the proposal that universal human behaviors are underpinned by strong, domain-specific, innate constraints on cognition. We use a general model of the processes that shape human behavior--learning, culture, and biological evolution--to test the evolutionary plausibility of this hypothesis. A series of analyses shows that culture radically alters the relationship between natural selection and cognition. Culture facilitates rapid biological adaptation yet rules out nativism: Behavioral universals arise that are underpinned by weak biases rather than strong innate constraints. We therefore expect culture to have dramatically shaped the evolution of the human mind, giving us innate predispositions that only weakly constrain our behavior.

Feline infectious peritonitis virus (FIPV) is an alphacoronavirus that causes a nearly 100% mortality rate without effective treatment. Here we report a 3. 3-A cryoelectron microscopy (cryo-EM) structure of the serotype I FIPV spike (S) protein, which is responsible for host recognition and viral entry. Mass spectrometry provided site-specific compositions of densely distributed high-mannose and complex-type N-glycans that account for 1/4 of the total molecular mass; most of the N-glycans could be visualized by cryo-EM. Specifically, the N-glycans that wedge between 2 galectin-like domains within the S1 subunit of FIPV S protein result in a unique propeller-like conformation, underscoring the importance of glycosylation in maintaining protein structures. The cleavage site within the S2 subunit responsible for activation also showed distinct structural features and glycosylation. These structural insights provide a blueprint for a better molecular understanding of the pathogenesis of FIP.

Significance We report here a 3. 3-A cryo-EM structure of feline infectious peritonitis virus ( FIPV) S protein derived from the serotype I FIPV UU4 strain. The near-atomic EM map enabled ab initio modeling of 27 out of the 33 experimentally verified high-mannose and complex-type N-glycans that mask most of the protein surface. We demonstrated the feasibility to directly visualize the core fucose of a complex-type glycan, which was independently cross-validated by glycopeptide mass spectrometry analyses. There exist 3 N-glycans that wedge between 2 galectin-like domains within the S1 subunit of FIPV-UU4 S protein, resulting in a propeller-like conformation unique to all reported CoV S proteins. The results highlight a structural role of glycosylation in maintaining complex protein structures.

Mechanical patterns control a variety of biological processes in plants. The microviscosity of cellular structures effects the diffusion rate of molecules and organelles, thereby affecting processes such as metabolism and signaling. Spatial variations in local viscosity are also generated during fundamental events in the cell life cycle. While crucial to a complete understanding of plant mechanobiology, resolving subcellular microviscosity patterns in plants has remained an unsolved challenge. We present an imaging microviscosimetry toolbox of molecular rotors that yield complete microviscosity maps of cells and tissues, specifically targeting the cytosol, vacuole, plasma membrane, and wall of plant cells. These boron-dipyrromethene (BODIPY) -based molecular rotors are rigidochromic by means of coupling the rate of an intramolecular rotation, which depends on the mechanics of their direct surroundings, with their fluorescence lifetime. This enables the optical mapping of fluidity and porosity patterns in targeted cellular compartments. We show how apparent viscosity relates to cell function in the root, how the growth of cellular protrusions induces local tension, and how the cell wall is adapted to perform actuation surrounding leaf pores. These results pave the way to the noninvasive micromechanical mapping of complex tissues.

Significance Spatial variations in microviscosity are triggered throughout plant cells, and these provide insight into local mechanobiological processes. However, it has so far been challenging to visualize such variations in living plant cells. Here we report an imaging microviscosity toolbox of chemically modified molecular rotors that yield complete microviscosity maps of several key plant cell structures. This toolbox opens up new ways to understand the role of mechanical stress in the regulation of biological processes.

Determining how proteins interact to form stable complexes is of crucial importance, for example in the development of novel therapeutics. Computational methods to determine the thermodynamically stable conformation of complexes from the structure of the binding partners, such as RosettaDock, might potentially emerge to become a promising alternative to traditional structure determination methods. However, while models virtually identical to the correct experimental structure can in some cases be generated, the main difficulty remains to discriminate correct or approximately correct models from decoys. This is due to the ruggedness of the free-energy landscape, the approximations intrinsic in the scoring functions, and the intrinsic flexibility of proteins. Here we show that molecular dynamics simulations performed starting from a number top-scoring models can not only discriminate decoys and identify the correct structure, but may also provide information on an initial map of the free energy landscape that elucidates the binding mechanism.

Determining how proteins fold and form complexes is of crucial importance, for example in the development of novel therapeutics. Experimental determination of structures is costly and lengthy. Computational methods to determine the thermodynamically stable conformation of complexes from the structure of the binding partners are available and constantly improving. Such methods generate a large number of diverse conformations and rank them for their likelihood to be correct. Even a model very similar to the correct structure is rarely the top-scoring one, but, as in the examples presented here, only within the top ~10-100 ( the exact number depends on the complexity of the structure, and could be much higher) . Here we show through atomistic simulation that good models are kinetically stable and bad models most often are not. More surprisingly, we also see that some bad models spontaneously find the correct ( i. e. , experimentally determined) conformation. This is remarkable, and could become an additional tool to contribute to structure determination of protein complexes. Such a result can also be expected, because evolution sculpted the free energy landscape in a way that the biologically active state is not only the one of lowest free energy ( i. e. , the most likely state) but also robustly reachable and kinetically stable ( i. e. , at the bottom of a funnel on the free energy landscape) .

During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4+ and CD8+ T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naive T cells. Indeed, dendritic cells were required for optimal CD4+ and CD8+ T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naive mice potently induced CD4+ and CD8+ T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4+ and CD8+ T cell responses.

CD4+ and CD8+ T cells are critical for controlling many infections. To generate a T cell response during infection, T cells must encounter the microbial peptides that they recognize bound to MHC molecules on the surfaces of other cells, such as dendritic cells. It is currently unclear how dendritic cells acquire the antigens they present to T cells during infection with many intracellular pathogens. It is possible that these antigens are phagocytosed and processed by dendritic cells, or antigens may be presented by cells that are infected by pathogens such as Toxoplasma gondii, which invades host cells independently of phagocytosis. To differentiate these pathways, we developed a novel technique to track the fate of T. gondii in vivo that distinguishes actively infected cells from those that phagocytosed parasites. This technique was used to examine each of these cell populations. We also used pharmacological inhibitors of parasite invasion, and the transfer of sort-purified infected or uninfected dendritic cells and macrophages to determine what roles phagocytosis and active invasion have in the initiation of T cell responses. Our results demonstrate that phagocytosis of parasites is not sufficient to induce CD4+ or CD8+ T cell responses, whereas infected cells are critical for this process.

During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1. 1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A) -binding protein C1 (PABPC1) , which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX) protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A) tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.

Almost all eukaryotic messenger RNAs ( mRNAs) have a string of 150-200 adenylates at the 3' end. This poly( A) tail has been implicated as important for regulating mRNA translation, stability and export. During the lytic phase of infection of Kaposi's Sarcoma-Associated Herpesvirus ( KSHV) , a noncoding viral RNA is synthesized that resembles an mRNA in that it is transcribed by RNA polymerase II, is methyl-G capped at the 5' end, and is polyadenylated at the 3' end; yet this RNA is never exported to the cytoplasm for translation. Rather, it builds up in the nucleus to exceedingly high levels. We present evidence that the function of this abundant, polyadenylated nuclear ( PAN) RNA is to bind poly( A) binding protein, which normally binds poly( A) tails of mRNAs in the cytoplasm but is re-localized into the nucleus during lytic KSHV infection. The interaction between PAN RNA and re-localized poly( A) binding protein is important for formation of new virus, in particular for the synthesis of proteins made late in infection. Our study provides new insight into the function of this noncoding RNA during KSHV infection and expands recent discoveries regarding re-localization of poly( A) binding protein during many viral infections.

Mycobacterium tuberculosis (M. tb) , the cause of tuberculosis (TB) , is estimated to infect a new host every second. While analyses of genetic data from natural populations of M. tb have emphasized the role of genetic drift in shaping patterns of diversity, the influence of natural selection on this successful pathogen is less well understood. We investigated the effects of natural selection on patterns of diversity in 63 globally extant genomes of M. tb and related pathogenic mycobacteria. We found evidence of strong purifying selection, with an estimated genome-wide selection coefficient equal to -9. 5x10-4 (95% CI -1. 1x10-3 to -6. 8x10-4) ; this is several orders of magnitude higher than recent estimates for eukaryotic and prokaryotic organisms. We also identified different patterns of variation across categories of gene function. Genes involved in transport and metabolism of inorganic ions exhibited very low levels of non-synonymous polymorphism, equivalent to categories under strong purifying selection (essential and translation-associated genes) . The highest levels of non-synonymous variation were seen in a group of transporter genes, likely due to either diversifying selection or local selective sweeps. In addition to selection, we identified other important influences on M. tb genetic diversity, such as a 25-fold expansion of global M. tb populations coincident with explosive growth in human populations (estimated timing 1684 C. E. , 95% CI 1620-1713 C. E. ) . These results emphasize the parallel demographic histories of this obligate pathogen and its human host, and suggest that the dominant effect of selection on M. tb is removal of novel variants, with exceptions in an interesting group of genes involved in transportation and defense. We speculate that the hostile environment within a host imposes strict demands on M. tb physiology, and thus a substantial fitness cost for most new mutations. In this respect, obligate bacterial pathogens may differ from other host-associated microbes such as symbionts.

Mycobacterium tuberculosis ( M. tb) , the etiologic agent of tuberculosis ( TB) , is a highly prevalent pathogen of humans, estimated to infect one-third of the world's population. Previous investigations of M. tb evolution have emphasized the influence of chance events on populations of these bacteria. To understand why this organism is so well adapted to its niche, we sought to characterize the influence of natural selection on M. tb. DNA sequence data from M. tb populations appeared to be strongly influenced by genome-wide selection against deleterious mutations. This type of selection was particularly evident in three functional categories: genes essential for infection ( identified in an animal model of TB) , genes involved in protein translation, and genes involved in trafficking and metabolism of inorganic ions. By contrast, a fourth category ( 'defense' genes) exhibited high levels of diversity, consistent with selection for advantageous mutations. In addition to effects of selection on M. tb genomic data, we identified the influence of a pronounced recent expansion in M. tb populations, coincident with explosive growth of human populations around the world. Our results suggest that growth of M. tb populations parallels that of its human host population, and that complex influences lead to emergence and maintenance of adaptive traits in M. tb.

In mice, Quaking (Qk) is required for myelin formation; in humans, it has been associated with psychiatric disease. QK regulates the stability, subcellular localization, and alternative splicing of several myelin-related transcripts, yet little is known about how QK governs these activities. Here, we show that QK enhances Hnrnpa1 mRNA stability by binding a conserved 3' UTR sequence with high affinity and specificity. A single nucleotide mutation in the binding site eliminates QK-dependent regulation, as does reduction of QK by RNAi. Analysis of exon expression across the transcriptome reveals that QK and hnRNP A1 regulate an overlapping subset of transcripts. Thus, a simple interpretation is that QK regulates a large set of oligodendrocyte precursor genes indirectly by increasing the intracellular concentration of hnRNP A1. Together, the data show that hnRNP A1 is an important QK target that contributes to its control of myelin gene expression.

Myelin is a lipid-rich structure that insulates neuronal axons, facilitating electrical conductance and protecting neurons from degeneration. Myelin comprises multiple compact layers of phospholipid bilayer and specific myelin proteins that occupy distinct positions within the structure. In the central nervous system, an RNA-binding protein termed Quaking is required for formation of compact myelin. Quaking regulates the production of several myelin-related proteins by binding to their mRNAs. Quaking controls the overall levels of these proteins and controls the relative amount of sequence variants of the proteins generated through alternative splicing. Here, we identify a new Quaking mRNA target, the Hnrnpa1 transcript. We show that Quaking regulates the overall level of hnRNP A1. Because hnRNP A1 is itself an RNA regulatory factor and has been implicated in the control of alternative splicing, regulation of hnRNP A1 by Quaking may have consequences for the expression of multiple additional targets. We show that hnRNP A1 and Quaking regulate an overlapping set of transcripts and exons in myelin-forming cells of the central nervous system.

We examined the application and review materials of three calls (n = 2, 823) of a prestigious grant for personal research funding in a national full population of early career scientists awarded by the Netherlands Organization for Scientific Research (NWO) . Results showed evidence of gender bias in application evaluations and success rates, as well as in language use in instructions and evaluation sheets. Male applicants received significantly more competitive "quality of researcher" evaluations (but not "quality of proposal" evaluations) and had significantly higher application success rates than female applicants. Gender disparities were most prevalent in scientific disciplines with the highest number of applications and with equal gender distribution among the applicants (i. e. , life sciences and social sciences) . Moreover, content analyses of the instructional and evaluation materials revealed the use of gendered language favoring male applicants. Overall, our data reveal a 4% "loss" of women during the grant review procedure, and illustrate the perpetuation of the funding gap, which contributes to the underrepresentation of women in academia.

Significance Women remain underrepresented in academia as they continue to face a leadership gap, salary gap, and funding gap. Closing the funding gap is of particular importance, because this may directly retain women in academia and foster the closing of other gaps. In this study, we examined the grant funding rates of a national full population of early career scientists. Our results reveal gender bias favoring male applicants over female applicants in the prioritization of their "quality of researcher" ( but not "quality of proposal") evaluations and success rates, as well as in the language use in instructional and evaluation materials. This work illuminates how and when the funding gap and the subsequent underrepresentation of women in academia are perpetuated.

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb) . However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS) , thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2) -Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

Significance RNA polymerase II ( Pol II) is stalled at the transcription starting site for a large number of genes in higher eukaryotes. We previously found that JMJD5/7 proteins act as both endopeptidases and exopeptidases to cleave arginine methylated histone tails including those of H2A, H3, and H4, thus generating relatively "tailless nucleosomes", which can be overcome by Pol II. This report shows that knockout of JMJD5 in mice leads to accumulation of nucleosomes at +1 and loss of expression of a large number of genes. We find that the N-terminal domain of JMJD5 binds to CTD of Pol II with a unique combination of phosphorylation generated by CDK9. We propose that JMJD5 couples with CDK9 to release the paused Pol II.

The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called "van Gogh bundles") of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.

Some problems can be solved only when individuals act together. This applies to bacteria in the same way that it applies to humans. Here we study how bacteria overcome the environmental challenge of migration over a solid surface by bundling their forces. Migration can be a significant environmental challenge for bacteria, especially when food sources are distributed far apart and have to be reached by movement along a solid surface, where swimming motility does not work. We show that Bacillus subtilis--a common inhabitant of the soil--migrates over a solid surface by forming multicellular structures. Migration depends on the synergistic interaction of two cell types: surfactin-producing and matrix-producing cells. Surfactin-producing cells facilitate migration by reducing the friction between cells and their substrate, thereby allowing matrix-producing cells to organize themselves into bundles that form filamentous loops at the colony edge. Using time-course microscopy, we observe that the filamentous loops drive migration by pushing themselves away from the colony. A mathematical model further shows that the folding properties of these loops are critical for the rate of colony expansion. Thus, not only do cells act together to overcome the challenge of migration, they also divide labor, in that different cell types specialize on distinct tasks.

DNA-dependent protein kinase (DNA-PK) is a large protein complex central to the nonhomologous end joining (NHEJ) DNA-repair pathway. It comprises the DNA-PK catalytic subunit (DNA-PKcs) and the heterodimer of DNA-binding proteins Ku70 and Ku80. Here, we report the cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs at 4. 4-A resolution and the DNA-PK holoenzyme at 5. 8-A resolution. The DNA-PKcs structure contains three distinct segments: the N-terminal region with an arm and a bridge, the circular cradle, and the head that includes the kinase domain. Two perpendicular apertures exist in the structure, which are sufficiently large for the passage of dsDNA. The DNA-PK holoenzyme cryo-EM map reveals density for the C-terminal globular domain of Ku80 that interacts with the arm of DNA-PKcs. The Ku80-binding site is adjacent to the previously identified density for the DNA-binding region of the Ku70/Ku80 complex, suggesting concerted DNA interaction by DNA-PKcs and the Ku complex.

Significance Double-stranded DNA breaks pose a serious threat to the survival of cells. Nonhomologous end joining ( NHEJ) is a crucial DNA repair pathway in which the DNA-dependent protein kinase ( DNA-PK) complex, a key holoenzyme consisting of the Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, senses DNA breaks and initiates the NHEJ repair pathway. Here, we present cryo-EM structures of the DNA-PK complex. Together with previous structural and biochemical studies, our structures lead to a partial model for the interactions between DNA-PKcs and the Ku70/80 complex, and reveal how the DNA-PK complex may bring free ends of damaged DNA for repair.

Background People with cancer with febrile neutropenia are at risk of severe infections and mortality and are thus treated empirically with broad-spectrum antibiotic therapy. However, the recommended duration of antibiotic therapy differs across guidelines. Objectives To assess the safety of protocol-guided discontinuation of antibiotics regardless of neutrophil count, compared to continuation of antibiotics until neutropenia resolution in people with cancer with fever and neutropenia, in terms of mortality and morbidity. To assess the emergence of resistant bacteria in people with cancer treated with short courses of antibiotic therapy compared with people with cancer treated until resolution of neutropenia. Search methods We searched the Cochrane Central Register of Controlled Trials (CENTRAL; 2018, Issue 10) in the Cochrane Library, MEDLINE, Embase, and LILACS up to 1 October 2018. We searched the metaRegister of Controlled Trials and the US National Institutes of Health Ongoing Trials Register ClinicalTrials.gov for ongoing and unpublished trials. We reviewed the references of all identified studies for additional trials and handsearched conference proceedings of international infectious diseases and oncology and haematology conferences. Selection criteria We included randomised controlled trials (RCTs) that compared a short antibiotic therapy course in which discontinuation of antibiotics was guided by protocols regardless of the neutrophil count to a long course in which antibiotics were continued until neutropenia resolution in people with cancer with febrile neutropenia. The primary outcome was 30-day or end of follow-up all-cause mortality. Data collection and analysis Two review authors independently reviewed all studies for eligibility, extracted data, and assessed risk of bias for all included trials. We calculated risk ratios (RRs) with 95% confidence intervals (CIs) whenever possible. For dichotomous outcomes with zero events in both arms of the trials, we conducted meta-analysis of risk differences (RDs) as well. For continuous outcomes, we extracted means with standard deviations (SD) from the studies and computed mean difference (MD) and 95% CI. If no substantial clinical heterogeneity was found, trials were pooled using the Mantel-Haenszel fixed-effect model. Main results We included eight RCTs comprising a total of 662 distinct febrile neutropenia episodes. The studies included adults and children, and had variable design and criteria for discontinuation of antibiotics in both study arms. All included studies but two were performed before the year 2000. All studies included people with cancer with fever of unknown origin and excluded people with microbiological documented infections. We found no significant difference between the short-antibiotic therapy arm and the long-antibiotic therapy arm for all-cause mortality (RR 1.38, 95% CI 0.73 to 2.62; RD 0.02, 95% CI -0.02 to 0.05; low-certainty evidence). We downgraded the certainty of the evidence to low due to imprecision and high risk of selection bias. The number of fever days was significantly lower for people in the short-antibiotic treatment arm compared to the long-antibiotic treatment arm (mean difference -0.64, 95% CI -0.96 to -0.32; I2 = 30%). In all studies, total antibiotic days were fewer in the intervention arm by three to seven days compared to the long antibiotic therapy. We found no significant differences in the rates of clinical failure (RR 1.23, 95% CI 0.85 to 1.77; very low-certainty evidence). We downgraded the certainty of the evidence for clinical failure due to variable and inconsistent definitions of clinical failure across studies, possible selection bias, and wide confidence intervals. There was no significant difference in the incidence of bacteraemia occurring after randomisation (RR 1.56, 95% CI 0.91 to 2.66; very low-certainty evidence), while the incidence of any documented infections was significantly higher in the short-antibiotic therapy arm (RR 1.67, 95% CI 1.08 to 2.57). There was no significant difference in the incidence of invasive fungal infections (RR 0.86, 95% CI 0.32 to 2.31) and development of antibiotic resistance (RR 1.49, 95% CI 0.62 to 3.61). The data on hospital stay were too sparse to permit any meaningful conclusions. Authors' conclusions We could make no strong conclusions on the safety of antibiotic discontinuation before neutropenia resolution among people with cancer with febrile neutropenia based on the existing evidence and its low certainty. Results of microbiological outcomes favouring long antibiotic therapy may be misleading due to lower culture positivity rates under antibiotic therapy and not true differences in infection rates. Well-designed, adequately powered RCTs are required that address this issue in the era of rising antibiotic resistance.

Stopping antibiotic therapy early versus continuing until normal neutrophil count in people with cancer with fever and low neutrophil counts Review question People with cancer who are treated with chemotherapy may have a low number of white blood cells, a condition known as neutropenia. White blood cells are crucial to defending the immune system against infection. In people with neutropenia who develop fever, it is unknown whether it is safe to stop giving antibiotics before the white blood cell count returns to normal or whether it is better to continue antibiotics until the white blood cell count recovers (usually to a number higher than 500 cells per microlitre). Background Neutrophils are a type of white blood cell crucial to the defence of the immune system against many infective pathogens including bacteria. People with cancer receiving chemotherapy suffer from decreased production of neutrophils, which means that they are exposed to severe, life-threatening infections. When a cancer patient with neutropenia develops fever, it is crucial to start broad-range antibacterial treatment as soon as possible in order to lower the risk of death and serious complications. To date, the best duration of course of antibiotics to give is unclear. The continuation of antibiotics beyond the required duration might result in the development of side effects and resistant bacteria. Search date The evidence is current to 1 October 2018. Study characteristics We included eight studies involving people with neutropenia and fever and comparing short antibiotic therapy to long antibiotic therapy until normalisation of neutrophils. A total of 662 episodes of fever in people with neutropenia were randomly assigned to a treatment group (314 to short antibiotic treatment and 348 to long antibiotic treatment). All trials excluded people who had bacteria growing in any culture before the time of randomisation. All studies except two excluded people with infection in a specific organ. Study funding sources Three trials did not report funding sources; three were funded by academic sponsors; one had academic sponsorship, but the antibiotics and placebos were provided by pharmaceutical companies; and one was sponsored by government funding. Key results There was no difference in mortality between the short- and long-antibiotic therapy arm. There was no difference in the number of people with severe infections presenting as bacteria in blood. There were more cases of infections with positive cultures in people treated with short antibiotic courses compared to long antibiotic courses, but there was no difference in the rate of unfavourable outcome such as recurrence of fever, need for rehospitalization, and change or restart of antibiotics. We found no differences in the rate of fungal infections and development of antibiotic resistance, with few studies reporting the latter outcome. The number of days with fever was lower for people treated with short antibiotic courses compared to those treated with long antibiotic courses. In all trials the number of antibiotic treatment days was fewer in the short-antibiotic therapy arm by three to seven days compared to the long-antibiotic therapy arm. Data on hospital length of stay were insufficient to permit any meaningful conclusions. Certainty of the evidence The overall certainty of evidence was low or very low, permitting little confidence in the results presented. Most of the included studies were old and not adequately designed. There were also many differences between the studies in terms of design and inclusion criteria. We assessed the certainty of the evidence for the primary outcome of all-cause mortality as low and for the outcomes of clinical failure and bacteraemia occurring after randomisation as very low.

Extreme events and the related anomalous statistics are ubiquitously observed in many natural systems, and the development of efficient methods to understand and accurately predict such representative features remains a grand challenge. Here, we investigate the skill of deep learning strategies in the prediction of extreme events in complex turbulent dynamical systems. Deep neural networks have been successfully applied to many imaging processing problems involving big data, and have recently shown potential for the study of dynamical systems. We propose to use a densely connected mixed-scale network model to capture the extreme events appearing in a truncated Korteweg-de Vries (tKdV) statistical framework, which creates anomalous skewed distributions consistent with recent laboratory experiments for shallow water waves across an abrupt depth change, where a remarkable statistical phase transition is generated by varying the inverse temperature parameter in the corresponding Gibbs invariant measures. The neural network is trained using data without knowing the explicit model dynamics, and the training data are only drawn from the near-Gaussian regime of the tKdV model solutions without the occurrence of large extreme values. A relative entropy loss function, together with empirical partition functions, is proposed for measuring the accuracy of the network output where the dominant structures in the turbulent field are emphasized. The optimized network is shown to gain uniformly high skill in accurately predicting the solutions in a wide variety of statistical regimes, including highly skewed extreme events. The technique is promising to be further applied to other complicated high-dimensional systems.

Significance Understanding and predicting extreme events as well as the related anomalous statistics is a grand challenge in complex natural systems. Deep convolutional neural networks provide a useful tool to learn the essential model dynamics directly from data. A deep learning strategy is proposed to predict the extreme events that appear in turbulent dynamical systems. A truncated KdV model displaying distinctive statistics from near-Gaussian to highly skewed distributions is used as the test model. The neural network is trained using data only from the near-Gaussian regime without the occurrence of large extreme values. The optimized network demonstrates uniformly high skill in successfully capturing the solution structures in a wide variety of statistical regimes, including the highly skewed extreme events.

The phenols 4-methylphenol, 4-methoxyphenol, and N-acetyl-tyrosine form hydrogen-bonded adducts with N-methyl-4, 4'-bipyridinium cation (MQ^+) in aqueous solution as evidenced by the appearance of low-energy, low-absorptivity features in UV-visible spectra. They are assigned to the known examples of optically induced, concerted electron-proton transfer, photoEPT. The results of ultrafast transient absorption measurements on the assembly MeOPhO-H---MQ^+ are consistent with concerted EPT by the instantaneous appearance of spectral features for MeOPhO*---H-MQ^+ in the transient spectra at the first observation time of 0. 1 ps. The transient decays to MeOPhO-H---MQ^+ in 2. 5 ps, accompanied by the appearance of oscillations in the decay traces with a period of ~1 ps, consistent with a vibrational coherence and relaxation from a higher u(N-H) vibrational level or levels on the timescale for back EPT.

Significance Concerted proton-coupled electron transfer ( EPT) reactions in which both electrons and protons transfer in tandem are at the heart of many chemical and biological conversions including photosystem II. We report here the direct observation of absorption bands arising from photoEPT transitions, in this case, in H-bonded complexes between N-methyl-4, 4'-bipyridinium cation and biologically relevant donors including tyrosine. The importance of these observations follows from the earlier experimental observations by Taube and coworkers on intervalence transfer in mixed-valence complexes. The observation of these photoEPT transitions and the appearance of reactive radical products also points to a possible, if inefficient, role in DNA photodamage and, possibly, in the formation of reactive oxygen intermediates.

Fossil-fuel emissions may impact phytoplankton primary productivity and carbon cycling by supplying bioavailable Fe to remote areas of the ocean via atmospheric aerosols. However, this pathway has not been confirmed by field observations of anthropogenic Fe in seawater. Here we present high-resolution trace-metal concentrations across the North Pacific Ocean (158degW from 25degto 42degN) . A dissolved Fe maximum was observed around 35degN, coincident with high dissolved Pb and Pb isotope ratios matching Asian industrial sources and confirming recent aerosol deposition. Iron-stable isotopes reveal in situ evidence of anthropogenic Fe in seawater, with low d^56Fe (-0. 23%0 > d^56Fe > -0. 65%0) observed in the region that is most influenced by aerosol deposition. An isotope mass balance suggests that anthropogenic Fe contributes 21-59% of dissolved Fe measured between 35deg and 40degN. Thus, anthropogenic aerosol Fe is likely to be an important Fe source to the North Pacific Ocean.

Significance Mineral dust has long been considered an important source of Fe to the surface ocean, especially in remote areas away from continental margins. However, anthropogenic Fe derived from fossil-fuel burning may also contribute Fe to surface waters. Here we show in situ evidence of anthropogenic Fe in seawater, based on the concentrations and isotopic composition of Fe measured during a springtime cruise which transected the North Pacific Ocean. Our results suggest that anthropogenic Fe could play a globally important role in marine productivity and carbon cycling.

Artificial neural networks, taking inspiration from biological neurons, have become an invaluable tool for machine learning applications. Recent studies have developed techniques to effectively tune the connectivity of sparsely-connected artificial neural networks, which have the potential to be more computationally efficient than their fully-connected counterparts and more closely resemble the architectures of biological systems. We here present a normalisation, based on the biophysical behaviour of neuronal dendrites receiving distributed synaptic inputs, that divides the weight of an artificial neuron's afferent contacts by their number. We apply this dendritic normalisation to various sparsely-connected feedforward network architectures, as well as simple recurrent and self-organised networks with spatially extended units. The learning performance is significantly increased, providing an improvement over other widely-used normalisations in sparse networks. The results are two-fold, being both a practical advance in machine learning and an insight into how the structure of neuronal dendritic arbours may contribute to computation.

Neurons receive contacts from other cells on extensively branched processes known as dendrites. When a contact is formed, activity in one cell is communicated to another by altering the conductance of the receiving cell's membrane and allowing an ionic current to flow. A neuron with longer dendrites is intrinsically less excitable as these currents can more easily dissipate both across the larger cell membrane and along the dendrites themselves. We have recently shown that, in real neurons, this effect is precisely cancelled by the increased number of contacts allowed by longer dendrites. This in turn implies that the ability of a single synapse to influence a neuron is likely to be inversely proportional to the total number of synapses that that neuron receives. Here we study the computational implications of this effect using the well-established framework of artificial neural networks. Sparsely-connected artificial neural networks adapt their connectivity to solve defined computational tasks such as classifying inputs and are at the forefront of modern machine learning. We apply the normalisation implied by dendritic structure to such networks: artificial neurons receiving more contacts require larger dendrites and so each individual contact will have proportionately less influence. Our normalisation allows networks to learn desired tasks faster and more consistently. Our result is both a practical advance in machine learning and a previously unappreciated way in which intrinsic properties of neurons may contribute to their computational function.