Symbiodinium) inhabiting coral endodermal tissues are well known for their role as keystone symbiotic partners, providing corals with enormous amounts of energy acquired via photosynthesis and the absorption of dissolved nutrients. In the past few decades, corals reefs worldwide have been increasingly affected by coral bleaching , which carries important socio-economic implications. Consequently, the number of studies focusing on the molecular and cellular processes underlying this biological phenomenon has grown rapidly, and symbiosis is now widely recognized as a major topic in coral biology. However, obtaining a clear image of the interplay between the environment and this mutualistic symbiosis remains challenging. Here, we review the potential of recent technological advances in molecular biology and approaches using stable isotopes to fill critical knowledge gaps regarding coral symbiotic function. Finally, we emphasize that the largest opportunity to achieve the full potential in this field arises from the integration of these technological advances.The symbiotic dinoflagellates (genus  Reefs based on scleractinian corals are among the most productive and biologically diverse marine ecosystems on Earth . At the Since 1979, populations of scleractinian corals have been reported as increasingly affected by mass coral bleaching, which involves the breakdown of the symbiosis between the cnidarian host and the dinoflagellate symbionts . Given tSymbiodinium. This photosynthetic dysfunction results in the excessive production of reactive oxygen species (ROS) in the symbiont and promotes the degradation of host mitochondria, providing another potential site of harmful ROS production and oxidative stress  e stress . The cele stress . HoweverMontastraea faveolata to measure gene expression changes associated with thermal stress. Their results suggested that oxidative stress in thermal-stressed corals causes a disruption of Ca2+ homeostasis and the initiation of cell death via apoptosis and necrosis. In a following study, Acropora millepora and show that elevated temperature compromise some critical components of the coral immune defenses including a mannose-binding C-type lectin. More recently, microarrays studies demonstrated that coral host transcriptomic states are correlated with different Symbiodinium genotypes (S. pistillata may divert cellular energy into mechanisms such as the ER-unfolded protein response (UPR) and ER-associated degradation (ERAD) at the expense of growth and biomineralization processes in an effort to survive and subsequently recover from the stress. The emergence of next-generation sequencing technologies has further increased the speed of coverage and decreased the cost through massively parallel sequencing methods. As a result, large–scale transcriptomics are well established as part of the coral biologist’s “toolbox”  and repetitive DNA (S. minutum nuclear genome (Hydra magnipapillata have explored the use of the reverse-genetics technique RNA interference (RNAi), which consists of introducing a synthetic double strand of RNA into cells to selectively induce gene suppression. Unfortunately, this method has had limited success; the RNA delivery via electroporation often damages tissues and cells , most likely related to the instability or poor delivery of siRNA in vivo. Genetic modification is still in its infancy in symbiotic cnidarians, and efforts at using siRNA for gene silencing have often been hampered by the difficulty of effectively introducing it into cells of interest. As a result, no other attempts to use siRNA in corals have been published thus far.By allowing researchers to simultaneously investigate genes and their level of expression, genomics and transcriptomics approaches have greatly improved our understanding of coral bleaching. In the late 2000s, the first microarrays studies had a tremendous impact on revealing the cellular foundation of thermal stress-induced coral bleaching . In the enotypes  and thatenotypes . The fintoolbox”  and a totoolbox”  in databtive DNA . Howevertive DNA , 2011a,br genome . Despitend cells . In a piin vitro and in vivo and their uptake is easily carried out, the specific control of the cargo release is still a challenge. Some years ago, a new concept was presented in which hollow particles, typically on the micrometer scale , were used as a carrier system. The cavity of the capsules can be loaded with a large variety and high quantity of cargo  suffer less from photobleaching than conventional fluorophores. Thus, single-molecule based tracking of fluorescently labeled membrane-bound proteins received a great boost by moving from connectional fluorophores to fluorescent NPs . The smaof cargo . If the of cargo . This reynthesis . When leGiven the nutritional role of the symbionts, another intriguing question—and subsequent knowledge gap in coral symbiosis—arises as to whether bleaching may in part reflect a change in the nutritional status of the host–symbiont interaction. In this respect, recent studies have highlighted strong correlations between feeding, sustained photosynthetic activity and reduced bleaching . Coral sin situ environmental conditions . Shifting the balance of supply versus demand for nitrogen and carbon, for example due to an environmental perturbation, may well result in the disruption of the coral–symbiont relationship, which is referred to as bleaching. The development of research investigating environmental impacts on the metabolic function of coral reefs is particularly necessary given that (i) reefs are rapidly deteriorating worldwide and that (ii) most coral reef research does not consider biomass stoichiometry and metabolic function when studying bleaching. Providing redress for such an omission may be critical in understanding the issue of coral reef degradation.Ecological stoichiometry is an increasingly broad field of research that evaluates how the relative quantity of specific chemical elements (C:N:P ratio) constrains or facilitates the movement of nutrients through an ecosystem . Given tThe intertwined nature of the coral-dinoflagellate endosymbiosis has long hampered research on coral metabolic function, as studies often suffer from potential cross-contamination between coral host and dinoflagellate fractions . In this13CO3) combined with HPLC-MS to investigate the lipogenesis in symbiotic cnidarian. Interestingly, their results indicated that fatty acids derived from photosynthetically fixed carbon were not used directly in host lipogenesis, suggesting that additional sources of carbon, such as host respiration and heterotrophy may be especially important for the lipogenesis of fatty acids in the cnidarian host. Another technique that over- comes the use of stable isotopes to detect metabolically active microbes in their natural environment is nano-scale secondary ion mass spectrometry (NanoSIMS). With NanoSIMS, secondary ions are extracted from the surface of a sample under the impact of a primary ions beam and subsequently analyzed using mass spectrometry providing imaging and quantification of up to seven isotopes of elements simultaneously . Discovering the relationships between gene functions and the sum of all metabolic processes  occurring within symbiotic corals could be an especially important next step to better understand the biology and functioning of this symbiosis.When integrated with recent advances in molecular biology, including “omics” approaches and next generation siRNA delivery systems, approaches based on incubation with stable isotopes could also provide important opportunities for system biology (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this transcranial alternating current stimulation study, Randolph Helfrich, Andreas Engel and colleagues demonstrate that the selective modulation of synchronized neuronal activity in large-scale cortical networks impacts conscious perception. PLOS Biology, Helfrich and colleagues address this long-standing question by attempting to drive brain oscillations using transcranial electrical current stimulation. Remarkably, they were able to manipulate visual perception by forcing brain oscillations of the left and right visual hemispheres into synchrony using oscillatory currents over both hemispheres. Under this condition, human observers more often perceived an inherently ambiguous visual stimulus in one of its perceptual instantiations. These findings shed light on the mechanisms underlying neuronal computation. They show that it is the neuronal oscillations that drive the visual experience, not the experience driving the oscillations. And they indicate that synchronized oscillatory activity groups brain areas into functional networks. This points to new ways for controlled experimental and possibly also clinical interventions for the study and modulation of brain oscillations and associated functions.Do neuronal oscillations play a causal role in brain function? In a study in this issue of  How does the brain work? How does it code, transfer, and store information? How are conscious experiences generated? These, among others, are long-standing questions neuroscientists try to answer. One way to approach this is to study how the brain orchestrates behaviour, for instance, by measuring brain activity and relating it to behaviour. Yet, studying the brain–behaviour relationship raises another series of questions: What type of brain activity should one look at? Do we need to record directly from single neurons? Or can we make inferences also by recording from larger pools of neurons? And importantly, do these measures of brain activity provide mechanistic accounts of how the brain implements function, or are they just inevitable side-products, with limited explanatory power for the neural mechanisms underlying our experiences, thoughts, or actions?PLOS Biology.Certainly, one would have a good argument for brain activity causally underlying brain function if (i) this brain activity not only relates to sensory experiences or behavioural performance measures , but (ii) interventions into this brain activity would also modulate our experiences or performance . Recent developments allow addressing these central points for oscillatory brain activity, which is what Helfrich et al. At the basis of Helfrich et al.'s study are two lines of research, one of which is concerned with the interpretation of a special type of brain activity, namely, brain oscillations. This type of brain activity represents voltage fluctuations of neuronal elements and was initially observed from one scalp electrode by Hans Berger Brain oscillations in the gamma frequency band (gamma-oscillations): This is a class of brain oscillations cycling at rapid frequencies (35–100 Hz). Gamma-oscillations are prominent in visual cortex (among other areas) and become evident also in scalp recordings when participants view specific types of visual stimuli.Alpha-band brain oscillations cycle at 8–12 Hz. Alpha-oscillations can co-occur with gamma-oscillations in visual areas, where these two classes of oscillations show an inverse relationship in terms of amplitude.Transcranial direct current stimulation (tDCS) and transcranial alternating current stimulation (tACS) use electrical currents applied through two or more scalp electrodes for transient, non-invasive brain stimulation, whereas transcranial magnetic stimulation (TMS) uses the principle of electromagnetic induction. In tACS, the currents are modulated in an oscillatory  pattern, and can therefore be frequency-tuned to underlying brain oscillations. Likewise, TMS in its rhythmic form (rhythmic TMS) allows for periodic brain stimulation at frequencies of brain oscillations.The other line of research that is at the heart of Helfrich et al.'s study is concerned with interventions into brain activity by non-invasive brain stimulation techniques; this to probe the brain–behaviour relationship along a more causal dimension Capitalizing on the above, Helfrich et al. convincingly address in healthy human volunteers the long-standing issue of whether oscillatory brain activity indeed coordinates functional brain architecture, as opposed to representing a mere by-product, and thereby bridge a gap between recordings and interventional studies into brain oscillations see , in whicThe findings of Helfrich et al. make an important contribution. They more firmly link the dynamics of oscillatory brain activity to the formation of functional networks, as well as the orchestration of brain function  and this along a causal dimension. This corroborates and extends a growing number of studies showing that brain oscillations can serve as targets for controlled interventions into brain activity and function, by non-invasive brain stimulation in periodic patterns 2) and require a “return” electrode which excites widespread areas. To render stimulation more focal, special electrode montages have been proposed These findings are exciting and indicate that it is promising to study brain oscillations, even at a macroscopic scale (such as measured with EEG/MEG), to answer some of the long-standing questions of how the brain works. They also take the emerging new approach of using periodic transcranial stimulation to interact with brain oscillations and function beyond the proof-of-principle stage. However, the usefulness of this approach will depend on the extent to which its specificity can be improved  and its mechanisms of actions understood. One unresolved point is the spatial extent of stimulation. With tACS, the conventional stimulation electrodes are large (several cm


AtTZF1, AtTZF2, and AtTZF3 homolog gene regulation by salt stress in evolutionary distant plant species”, D'Orso et al.  proteins comprise a small subfamily of zinc finger proteins that is characterized by presence of a unique tandem zinc finger domain and an arginine rich (RR) motif and hence referred as arginine rich tandem zinc finger (RR-TZF) protein Hall, . Till daArabidopsis and rice protein as abiotic stress modulator have already been established using various wet-lab and in silico techniques. Major objective of this work was to gain proper insight regarding the orthologous origin and functional conservation of durum wheat RR-TZF protein, which was previously identified and have been reported as a cold responsive protein, along with other already reported RR-TZF proteins present in plant belonging to different evolutionary scale including Arabidopsis. Authors have initially cloned the full length genomic and cDNA sequences of durum wheat (TdTZF1-A and TdTZF1-B). Based on sequence similarity, gene structure and expression analysis, they have successfully established the orthologous origin of Arabidopsis and durum wheat RR-TZF proteins. They used several computational methods freely available on the internet to identify the RR-TZF proteins present in the genome of 54 different plant species ranging from green algae to angiosperm using the BLASTP program with protein sequence of Arabidopsis tandem zinc finger proteins as the query sequence. They also constructed a phylogenetic tree with 9 different plant species belonging to different evolutionary scale in order to reveal the evolutionary relationship among the RR-TZF proteins. Along with this in silico analysis, they also deduced the expression level analysis of RR-TZF gene in durum wheat, Arabidopsis and P. patens, when exposed to different abiotic stresses during different developmental stages, and also constructed an overexpressing and attenuated Arabidopsis line for AtTZF3 gene. To our knowledge this is the first study of this kind dealing with comprehensive analysis of RR-TZF proteins belonging to different plant genera. It makes a significant contribution in understanding the evolutionary conservation of this group of genes during the evolutionary development of both non seed and seed bearing plants and how their functions are conserved and maintained through structural integrity of conserved motifs irrespective of its phylogenetic status.D'Orso et al.  have invHowever, the lacunae lie in the fact that their mode of action is still not completely elucidated. That they play an important role in regulating seed germination and abiotic stress response is clear from all the above evidences, but exactly “how” is yet to be determined. The future work should focus on decoding this particular “how” which will enhance our knowledge and enable us to draw a complete picture.A. thaliana, Durum wheat and P. patens RR-TZF genes increase considerably with increase in environmental salt concentration, indicating their role in stress alleviation. But their primary target genes or the pathways which they regulate are yet to be discovered. Deciphering their interaction with various stress regulatory pathways such as antioxidative enzyme regulation, osmolyte biosynthesis, ion homeostasis maintenance, hormone regulations and polyamine metabolism could be an interesting avenue of further studies. Another very interesting outcome of this particular work is the role of RR-TZF encoding genes in regulating germination process both in arabidopsis and durum wheat. This is congruent with earlier studies on arabidopsis carried out by different worker (Lopez-Molina et al., Various studies with mammalian TZF proteins reveal their close association with cytoplasmic mRNP complexes known as processing-bodies and stress granules, which contribute essentially in post transcriptional and epigenetic regulation of gene expression by triggering mRNA degradation and post translational repression by binding with them (Anderson and Kedersha, Future research prospect include identification of putative target genes for wheat RR-TZF proteins, their mode of regulation and their evolution. It would also be fascinating to know whether the target genes too, like the RR-TZF proteins themselves, are evolutionarily conserved across the plant kingdom. Another very promising field of research includes detailed analysis of the crosstalk that interlinks RR-TZF proteins with both developmental and stress adaptation pathways so that the target of these proteins could be easily identified and can be used in further scientific studies Figure .All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Computational methods have been developed to reconstruct evolutionary lineages from tumors using single-cell genomic data. The resulting tumor trees have important applications in cancer research and clinical oncology.http://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0929-9 and http://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0936-x.Please see related Research articles:  Tumors evolve from single cells. As they evolve, clonal lineages begin to diverge, resulting in distinct subpopulations and intratumor heterogeneity. This genomic diversity fuels tumor growth and enables the population of cells to survive various selective pressures in the tumor microenvironment . Intratumor heterogeneity is generally considered to be ‘bad news’ from a clinical standpoint because it complicates the diagnosis and therapeutic treatment of cancer patients. However, from the perspective of an evolutionary biologist, intratumor heterogeneity provides a permanent record of the mutations that occurred during tumor growth, providing a window into time. In the same way that evolutionary biologists can infer the ancestral lineages of species that are living on our planet today, so too can cancer biologists infer the evolutionary history of a tumor by comparing clones from a single patient sample. SCITE (single cell inference of tumor evolution) and OncoNEM (oncogenetic nested effects model) , 2 are tTumor lineages are typically represented as tree structures, of which there are three major classes: 1) phylogenetic trees, 2) clonal lineages, and 3) mutational trees . SCS provides the highest possible resolution for resolving clonal subpopulations and can report the precise combination of mutations that occur in any given clone or cell. However, SCS datasets are limited by the total number of cells that can be sampled at a reasonable cost and extensive technical errors that arise during whole-genome amplification (WGA). This process is necessary in order to amplify a sufficient DNA quantity from a single cell to perform next-generation sequencing (NGS). The most common WGA errors include: allelic dropout (ADO) errors, false-positive errors (FPs), coverage non-uniformity, and low-coverage sites Fig. . These eSCITE and OncoNEM are computational tools that were specifically designed to address technical errors in SCS data during phylogenetic inference , 2. SCITTumor phylogenies have several important applications in cancer research and clinical oncology. In addition to delineating the clonal substructure of a tumor, these methods provide an inferred chronological order of mutations that occurred during tumor growth, which can be traced to understand which mutations are involved in early tumorigenic processes  compared with late cancer processes . Tumor trees can also address questions of whether tumors evolve from a single normal initiating cell, or alternatively from multiple initiating cells, as would be predicted from a mutagenic field effect. Tumor trees are also very useful for resolving clonal dynamics to improve our understanding of whether mutations occur in early punctuated bursts , or alteAn important limitation of both SCITE and OncoNEM is that they make the ‘infinite sites assumption’, which implies that mutations occur only once at a specific nucleotide site and are never reversed. This assumption is of course false in human tumors, where genome-wide aneuploidy is common and often results in loss of heterozygosity (LOH) and hemizygous deletions that can eliminate mutations and reverse the genotype. Another violation of this assumption is convergent evolution, in which activating driver mutations can occur at the same nucleotide site in independent clones or lineages owing to strong positive selection. An additional criticism of both studies is that there is no straightforward way to know whether the clonal lineages or mutational trees generated from the human tumors truly reflect the biological lineages in any way. This is admittedly a very difficult problem to address and would require a ‘gold-standard’ tumor lineage to which different methods can be benchmarked to determine their accuracy. These experiments would be difficult to perform on a human tumor sample (owing to limited tissue), but might be feasible in artificial lineages that are generated in cell culture systems that are passaged for many generations, which are intentionally diverged and documented.SCITE and OncoNEM are innovative statistical methods that address a crucial problem in the construction of phylogenetic lineages from SCS data by using error models. These methods were shown to improve the accuracy of clonal lineages and mutational trees, as demonstrated in simulated datasets and human tumor samples. New technologies are on the horizon that will soon increase the throughput of DNA SCS to thousands of single cells, which will improve the accuracy of tumor lineages. OncoNEM and SCITE are well prepared to analyze these large-scale datasets and greatly facilitate the interpretation of the resulting lineages. Future versions of OncoNEM and SCITE could be improved in several ways. Removing the infinite sites assumption will be an important next step, which has already been addressed in classical phylogenetic methods such as maximum parsimony. Another important step will be to incorporate single-cell copy number and LOH into the tumor lineages, which can lead to missing mutations in the genotype matrix and violate the perfect phylogenies. Another avenue of progress will be to improve the probabilistic error models. Both methods currently assume a fixed probability of error at every site, but could instead assume higher error probabilities for lower-confidence mutation calls. However, even without these improvements, SCITE and OncoNEM are a major step forward in the analysis of SCS data and will have immediate applications in both cancer research and medicine.LOH, loss of heterozygosity; MCMC, Markov chain Monte Carlo; OncoNEM, oncogenetic nested effects model; SCITE, single cell inference of tumor evolution; SCS, single-cell DNA sequencing; WGA, whole-genome amplification.


Successful collective action often depends on the presence of leaders, who bear greater responsibility than other group members for the logistics of coordination, monitoring of effort, and reward and punishment. Leaders may be expected to shoulder more risk, are vulnerable to retaliation from sanctioned group members, and suffer greater reputational damage from failed collective action. What then motivates individuals to be leaders? From an evolutionary perspective, the answer is not straightforward since most of human history occurred in societies lacking significant disparities in material wealth and institutions that grant leaders coercive power. One possibility is that group members share costs by distributing leadership roles over iterations of collective action. However, this is uncommon where inter-individual differences in leadership ability have an impact on collective action. Whether in small-scale egalitarian societies or large-scale stratified societies, group members typically prefer leaders who are superlative in traits such as physical size, knowledge, and prosociality  not contributing to collective action, (2) not rewarding leaders, and (3) not punishing group members who fail to reward leaders. This is where the service-for-prestige theory makes unique predictions relative to other theories of leadership: followers will experience punitive sentiment toward other group members who fail to reward effective leaders . Price and van Vugt  present direct benefits from collective action. Examples of the latter include collective actions that produce goods more beneficial to leaders and their kin , followers' contributions to leaders are a product of group selection, or leaders recoup their costs by receiving greater As Price and van Vugt  suggest,The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In westernised countries, obesity is the most commonly diagnosed nutritional disorder in dogs and cats, with an estimated prevalence of between 22% and 44% in investigated populations. In humans, it is well established that obesity predisposes to lifestyle-related diseases such as type 2 diabetes mellitus and atherosclerosis. Dogs and cats seem less prone to developing atherosclerosis compared with humans, while diabetes is a relatively common endocrine disease in both species. The high prevalence of obesity in pet dogs and cats is believed to be multifactorial, often relating to lifestyle and the fact that pets have moved from being working animals to becoming close family members. Influencing factors include neutering, inactivity, genetic predisposition as well as factors such as interaction with owners through feeding/treats and the owners lacking recognition of their pets being overweight. As in humans, obesity causes insulin resistance and impaired glucose metabolism in cats and dogs. In cats, this may progress into feline diabetes because long-term elevated insulin demands together with obesity-related metabolic alterations result in destruction of the beta cells – through mechanisms similar to development of human type 2 diabetes mellitus. In dogs, the pancreas seems to better cope with increasing demands for insulin production and type 2-like diabetes seldom develops. Obesity may however, still predispose for diabetes in dogs. A possible explanation could be that obese dogs are at risk of developing pancreatitis as a frequent consequence of pancreatitis in dogs is inflammation-mediated destruction of the beta cells.


Substantial attention and resources have been directed to improving outcomes of patients with critical illnesses, in particular sepsis, but all recent clinical trials testing various interventions or strategies have failed to detect a robust benefit on mortality. Acute heart failure is also a critical illness, and although the underlying etiologies differ, acute heart failure and sepsis are critical care illnesses that have a high mortality in which clinical trials have been difficult to conduct and have not yielded effective treatments. Both conditions represent a syndrome that is often difficult to define with a wide variation in patient characteristics, presentation, and standard management across institutions. Referring to past experiences and lessons learned in acute heart failure may be informative and help frame research in the area of sepsis. Academic heart failure investigators and industry have worked closely with regulators for many years to transition acute heart failure trials away from relying on dyspnea assessments and all-cause mortality as the primary measures of efficacy, and recent trials have been designed to assess novel clinical composite endpoints assessing organ dysfunction and mortality while still assessing all-cause mortality as a separate measure of safety. Applying the lessons learned in acute heart failure trials to severe sepsis and septic shock trials might be useful to advance the field. Novel endpoints beyond all-cause mortality should be considered for future sepsis trials.The online version of this article (doi:10.1186/s40560-016-0151-6) contains supplementary material, which is available to authorized users. Sepsis, defined as "life-threatening organ dysfunction due to a dysregulated host response to infection" , is a maSepsis mortality has declined over the last decade from ~40 to ~20 % . ImproveEffective therapies are needed to better manage sepsis patients . TherapeOther critical care conditions  have faced similar challenges with efforts to prolong survival in clinical trials. Acute heart failure and sepsis are both critical care illnesses with high mortality. Both conditions represent syndromes with wide variation in patient characteristics, presentation, and standard management. Further, the underlying pathophysiology in both conditions is related to many processes, but pharmacologic interventions generally target single pathways and have not translated into survival benefits. All-cause mortality is usually the primary endpoint chosen for phase 3 pivotal trials in sepsis and acute heart failure, but no treatments to date have effectively reduced the high mortality associated with either of these conditions Fig. . In a suThe European Drug Development Hub brought together experts in critical care/sepsis and acute heart failure with the objective of sharing the collective clinical research experience in these critical care illnesses. The ultimate goal was to discuss better approaches to conducting clinical trials in critical care illnesses with high mortality  to promote advances in the care of these patients . This paper summarizes the key developments from the meeting, focusing on clinical trial designs and endpoints that should be considered for use in future sepsis trials.An overview of the results from a selection of recent large, rigorously designed and conducted sepsis clinical trials reveals a consistent theme Table . All triMortality rates due to sepsis are declining but remain high . StatistOver- or under-estimating treatment effects should be avoided when designing clinical trials . ResearcSepsis is a complex syndrome characterized by the interplay of many pathways and systems. Sepsis therapies must either (1) control several pathways with several interventions or (2) hit “upstream” nodes that control a number of pathways. The treatment approach for sepsis has ranged from inhibiting the uncontrolled, inflammatory host response to enhancing the host immune response . These sWhether sepsis treatments targeting a single aspect of this complex syndrome could be reasonably expected to reduce all-cause mortality is uncertain. All-cause mortality is a robust endpoint because it reflects the net benefit of an intervention . A benefIn sepsis trials, significant patient heterogeneity exists in time to presentation and diagnosis, organisms(s), type and source of infection, organ involvement, degree of organ impairment, severity of illness, location of enrollment , pre-existing conditions, and differences in standard of care across institutions or geographical regions . Although the European Medicines Agency guideline still specifies all-cause mortality as the preferred primary endpoint in acute heart failure trials, it states that symptomatic improvement might be acceptable as a primary endpoint for short-term trials provided mortality is not adversely affected . RegulatRegulatory agencies might consider a similar clinical composite endpoint adapted for sepsis trials, where endpoints describing end-organ function, need for mechanical support, or need for other interventions are combined with short-term mortality  as a primary endpoint, with longer-term all-cause mortality assessed for safety. This approach also has the advantage of reflecting relevant factors other than survival that are important to patients. Rigorous definitions for such endpoints are keys to ensure consistency and to reduce bias in the results and to ensure that the endpoint can be translated into a metric that is important to patients.Total or ICU length of stay has been considered as an endpoint for sepsis trials. It is relevant because ICU stays are costly, but it is dependent on external factors that are unrelated to drug therapy . These same limitations have been recognized in acute heart failure trials . Thus, tOrgan dysfunction is a relevant endpoint for sepsis trials. Multiple organs are impaired in sepsis , but allpreventing organ dysfunction (in patients who do not have organ dysfunction) and/or preventing progression of organ dysfunction . An adequate organ dysfunction scoring system must capture both of these possibilities.Days alive and free from mechanical ventilation, renal replacement therapy, or vasopressors  has also been proposed. These endpoints are clinically meaningful, and widespread use of the Surviving Sepsis Campaign guidelines has led to more consistent timing and application of life support interventions. Nonetheless, the decision to institute supportive therapies is often subjective and can be influenced by external factors ), which introduces increased variability  in the study and possibly bias if the study is not blinded. Other complex issues also warrant consideration, including whether patients value more event-free days equally regardless of when they occur , handling inclusion of multiple organs , and methodology to account for pre-existing organ dysfunction. Interventions can be effective in In general, there are no accepted surrogates for safety , althougAdaptive designs or seamless phase II/III designs have the potential to improve the efficiency of clinical trials. Adaptive designs can be particularly useful in fields in which data are limited to inform trial planning assumptions in the areas of expected event rates, anticipated effect sizes, heterogeneity of treatment effect, variance, safety, or drop-outs , 62. In Realistic trial simulation is the key tool to address these challenges and advance the field. Trial simulation of traditional and adaptive trial designs furthers understanding of strengths and weaknesses of proposed trial designs and will illustrate vulnerabilities from minor deviations to study design assumptions .Ideally, trial design should be a multi-step, collaborative, and interactive multidisciplinary process between scientific, clinical, and statistical domain experts to increase the quality and chance of success. This concept applies to all types of trials, but it is particularly important for adaptive design. Early interaction with regulators is highly recommended when using adaptive designs in the later stages of a drug development program , 62.Sepsis is a major burden with high mortality, and the lack of progress in identifying effective treatments is discouraging for researchers and industry. The clinical research challenges that have been encountered in sepsis trials closely resemble those experienced by investigators in acute heart failure trials. After decades of research, it has become clear in the acute heart failure community that the substantial patient heterogeneity contributes to the difficulties in identifying effective therapies for the condition. The recent consensus definitions for sepsis and septic shock are important advances in this regard , 37.  Ad


Recent investigations have established that significant accumulation of nanoparticles (NPs) occurs in aquatic biofilms. These studies point to the emerging roles of biofilms for influencing partitioning and possibly transformations of NPs in both natural and engineered systems. While attached biofilms are efficient “sponges” for NPs, efforts to elucidate the fundamental mechanisms guiding interactions between NPs and biofilms have just begun. In this mini review, special attention is focused on NP–biofilm interactions within the aquatic environment. We highlight key physical, chemical, and biological processes that affect interactions and accumulation of NPs by bacterial biofilms. We posit that these biofilm processes present the likely possibility for unique biological and chemical transformations of NPs. Ultimately, the environmental fate of NPs is influenced by biofilms, and therefore requires a more in-depth understanding of their fundamental properties.Bacteria are essential components of all natural and many engineered systems. The most active fractions of bacteria are now recognized to occur as  Microbial biofilms are an omnipresent component in many environments supporting life. Hence, the multifaceted roles of biofilms in the sequestration, accumulation, transformation, and trophic transfer of environmental contaminants have been a subject of much study and controversy. A biofilm is, in its simplest form, a collection of surface-attached microbial cells that are surrounded by a matrix of extracellular polymers. The inherent properties and physical structure of biofilms resemble that of a sorptive sponge capable of capturing various chemical and biological components in their vicinity. Natural and engineered systems that are significantly impacted by biofilms include soil mineral surfaces, microbial mats, wastewater treatment, and biofouling of ships and pipes. These will not be addressed further here. Rather, this review focuses on the mechanistic interactions of bacterial biofilm with natural and synthetic nanoparticles (NPs); an emerging concern in both the environment and health. Such NP–biofilm interactions within the aquatic environment are highlighted.2 NPs  attachment to the biofilm surface; and (3) migration within the biofilm   transporm Figure . Similarm Figure . For exaEngineered and naturally forming NPs can vary widely in their physicochemical characteristics such as shape, size, and charge . As discPseudomonas aeruginosa PAO1 biofilms than QDs having surface carboxyl (–COOH) groups . Dawson and colleagues introduced the concept of the “protein corona” as an important entity for NPs interacting with the external environment . The cor) groups . In anotexposure , suggest4–) groups on NPs resulted in greater sorption onto Alteromonas macleodii biofilms compared to NPs functionalized with amine (–NH) or carboxyl groups (P. fluorescens biofilms with their self-diffusion coefficients decreasing with increasing size and negative charge (Lactococcus lactis) and high  EPS contents , most of the water is not bound to EPS but rather is localized in pore spaces between adjacent polymer chains . These p2O3) NPs (2 NPs onto synthetic biofilms (60) NPs onto surfaces coated with EPS extracted from Escherichia coli  NPs . Differebiofilms  and for hia coli . On the hia coli . While thia coli .Once a NP binds to EPS, it can subsequently migrate deeper into the EPS matrix. NP penetration into and movement within the biofilm is considered to be driven primarily by diffusion . In thisWhile accumulation of NPs within biofilms results from initial attachment and migration of NPs, these two processes need not always occur sequentially. It is likely that more NPs are depositing onto the outermost surface simultaneously as other NPs are penetrating into the biofilm matrix and vice versa. Another possible mechanism that contributes to NP accumulation within the matrix is the active outgrowth of the biofilm, forming new layers above the surface-deposited NPs. In such cases, penetration of NPs into the biofilm matrix is not necessary to occur for accumulation to take place. However, differentiation between these different processes would be a difficult task in practice. All three are likely to occur in the complex and dynamic environments where biofilms naturally occur. Most recent studies on NP–biofilm interactions have examined either the combined effects of initial surface NP deposition and penetration into the biofilm  or all tAccumulation of NPs within biofilms has been previously documented . Is the E. coli biofilms were shown to aggregate to a larger degree than in planktonic cultures, possibly due to differences in ionic strength used in the experiments. On the other hand, organic ligands, including some that are found in EPS, typically stabilize NPs against aggregation, suggesting that NPs that enter the biofilm matrix as monomers may indeed stay as such within the biofilm. Another potential outcome of NP accumulation is NP dissolution as the NPs may be surrounded by enzymes and other organic matter that speed up those processes. This may especially be an important outcome for metallic NPs. Carbon-based NPs may undergo different changes in biofilms compared to metallic NPs. For example, microbial transformation of carbon-based NPs has been documented (One question we could ask is whether the NPs stay nano-sized and as particles. Given the right conditions, NPs will easily aggregate to form micro-size agglomerates. As shown by cumented , which mThe EPS matrix is the primary emergent- and adaptive-property of the microbial cells forming a biofilm . TherefoThe potential of biofilms to act as a factory for NP production has been increasingly recognized in both natural and engineered systems. Natural biofilms play a critical role in the biogeochemical cycling of elements which can lead to NP formation. For example, microbes precipitate metals in the form of NPs as a detoxification mechanism. There has been significant progress in understanding the micro- and nano-scale complexity within biofilms. Despite their complexity, it is now possible to experimentally examine the nature of NP interactions and penetration into biofilms. The ability to understand these fundamental and decisive processes can be approached using carefully controlled laboratory manipulations of EPS and NPs. Recent development of surface-sensitive techniques, high-resolution microscopies, and synchrotron-based spectroscopies provide powerful tools to promote an integrated approach to understanding NP–biofilm interactions. Multi-scale computational modeling efforts will be useful in complementing empirical data and enhancing the predictability of NP behavior within the biofilm matrix. Ultimately, improving our mechanistic understanding of NP–biofilm interactions will enable better risk assessment of nanotechnology as well as sustainable design of NPs.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is compelling evidence that more diverse ecosystems deliver greater benefits to people, and these ecosystem services have become a key argument for biodiversity conservation. However, it is unclear how much biodiversity is needed to deliver ecosystem services in a cost-effective way. Here we show that, while the contribution of wild bees to crop production is significant, service delivery is restricted to a limited subset of all known bee species. Across crops, years and biogeographical regions, crop-visiting wild bee communities are dominated by a small number of common species, and threatened species are rarely observed on crops. Dominant crop pollinators persist under agricultural expansion and many are easily enhanced by simple conservation measures, suggesting that cost-effective management strategies to promote crop pollination should target a different set of species than management strategies to promote threatened bees. Conserving the biological diversity of bees therefore requires more than just ecosystem-service-based arguments. One argument for conserving biological diversity is that it delivers beneficial ecosystem services. However, Kleijn et al. show that the economic benefits of crop pollination are delivered by only a small subset of relatively common species, arguing that threatened species should be considered separately. Worldwide, biodiversity is declining at unprecedented rates, threatening species persistence as well as the benefits humans gain from ecosystems124567The use of ecosystem services arguments for justifying biodiversity conservation is, however, not without risk or controversy. Many experimental studies show that biodiversity increases the magnitude and/or stability of ecosystem functioning (of which ecosystem services are the subset that benefit people), and that most species contribute to ecosystem functioning in some way10111215181920−1 to the production of insect-pollinated crops. However, a limited subset of all known bee species provides the majority of pollination services because, across different crops, years and large biogeographical regions, crop-visiting bee communities are dominated by a small number of common species and rarely contain regionally threatened species. Dominant crop pollinators are furthermore able to persist under agricultural expansion and many are relatively easily enhanced by simple conservation measures. Focusing conservation on the services delivered by pollinators may therefore lead to management strategies that predominantly benefit the limited set of species currently providing the majority of crop pollination. Consequently, conservation of the biological diversity of bees should be motivated not only by immediate benefits from ecosystem services but also by the full richness of arguments for conservation.Here we test these hypotheses using data from 90 studies and 1,394 crop fields on crop-visiting bee communities from five continents. Pollination is an important ecosystem service. The economic contribution of pollinators to crop production is significant−1 to production of the examined crops , about the same as the contribution of managed honey bees . Individual wild bee species contribute substantially to crop production value with contributions up to $963 per crop ha−1 per species  of the individual bees observed on crops. Second, we determined whether the dominant crop-visiting bee species are common in agricultural landscapes generally, using an independent data set of bee communities in 264 sites in agricultural landscapes in Europe and North America (see Methods section). These studies compared bee communities in agricultural habitats such as arable fields , grasslands, old fields and hedgerows with bee communities in nearby sites that are actively managed for biodiversity enhancement  , 24. We The dominant crop-visiting bee species dominate bee communities in agricultural landscapes generally, constituting 75.4±6.9% of individuals in these habitats in Europe and 59.2±10.5% in North America. This suggests that the species that are the dominant crop pollinators are the most widespread and abundant species in agricultural landscapes in general. Furthermore, the proportion of all bees on crops that belong to the dominant crop-visiting species was inversely related to the proportion of semi-natural habitats around study sites , and decTo test whether dominant crop-visiting species can easily be enhanced (hypothesis 3), we compared their abundance on sites with biodiversity-enhancing management with that in ‘background' agricultural habitats (as defined above). Across all studies, biodiversity management raised the abundance of dominant crop-visiting bees by a factor of 3.2. Organic farming, planting wildflowers and establishing grass margin strips significantly enhanced dominant crop-visiting bees in arable landscapes . On grasHere we show that wild bee pollinators provide important pollination services to crops around the globe , with thOur data sets supported all three of our hypotheses about the disconnect between the ecosystem services approach to conservation and the protection of biodiversity at large. First, few species are needed to provide ecosystem services, with almost 80% of the crop pollination provided by only 2% of bee species. Second, the species currently contributing most to pollination service delivery are generally regionally common species, whereas threatened species contribute little, particularly in the most agriculturally productive areas. Thus, a strictly ecosystem-service-based approach to conservation would not necessitate the conservation of threatened species. Third, the most important ecosystem-service-providing species are relatively robust to agricultural intensification, and furthermore can be readily enhanced in those systems by simple management actions. This suggests that the rarer species, which are already absent from such systems, would benefit less from ecosystem-service-based actions than they would from traditional biodiversity conservation that targets threatened species in the areas where they are found.The first two points have been raised before in opinion and perspective papers as arguments for why the usefulness of ecosystem service provision as an argument to conserve biodiversity may be limited181967At first sight, our findings contrast with results of earlier studies, several of which were part of this study26272826One benefit of biodiversity to ecosystem services is that it may provide insurance effects that stabilize services over time or space34Many previous studies have found that species richness of bee communities in agricultural landscapes declines with decreasing proportion of semi-natural habitats3638Measures to mitigate loss of pollination services are most cost effective in relatively intensively farmed landscapes because here measures have the highest impact39Highlighting the economic benefits people might obtain from biodiversity can be an effective instrument to motivate people or institutions to support biodiversity conservation. However, too much focus on the services delivered by pollinators may lead to adoption of practices that will not benefit species that could potentially contribute under changing agricultural conditions nor species that will never contribute to crop pollination. Benefits of biodiversity should therefore not be used as the sole rationale for biodiversity conservation as, for example, is currently done in the new strategy of the Convention on Biological DiversityOur data sets record the relative visitation rate of bees to crop flowers, which is a good proxy for the relative contribution to pollination service delivery (see next section). We used data from 90 studies and 1,394 crop fields around the world that used standardized protocols to examine the abundance and identity of wild bees visiting flowers of 20 different crops that depend on bee pollinators for maximum yield . We deteCitrullus lanatus, Helianthus annuus and Phaseolus coccineus). We therefore additionally analyse the relationship between visitation frequency , per-visit pollen deposition (measured as the number of conspecific pollen grains deposited during a single visit4546Xylocopa virginica) was included in several of our data sets. This would tend to increase the importance of per-visit deposition, and decrease the importance of visitation, in driving total pollination, which is a bias against the assumption tested here.Pollination is a function of both pollinator visitation frequency to flowers and per-visit pollen deposition (or efficiency)r between visitation and total pollination were high , was only considered to be non-managed in South Africa because here the species is native and wild populations still exist . In Indonesia, the Asian honey bee (A. cerana) is occasionally kept by local people and so was considered to be a managed pollinator. In all other countries, honey bees were considered to be managed pollinators and therefore irrelevant for ecosystem service provisioning. However, honey bee abundance was incorporated in the calculations of the contribution of bees to crop production value. On average, western honey bees had similar flower visitation rates as wild bees , although this varied among crops , P is the price ($ tonne−1), Y is the yield (tonne ha-1), D is the proportional reduction in crop yield without pollinationρ is the proportion of the visits to crop flowers made by a particular bee species (including honey bees).For 53 studies, the data allowed us to calculate the economic contribution of wild bees to crop production using the production value methodBee species were characterized as being dominant within a study when their relative abundance on crop flowers was 5% or higher. This threshold corresponds to the cumulative set of species that collectively provide 80% of the crop flower visits . SensitiConservation policy objectives are often formulated at national or even continental levels. We therefore also explored how the number of bee species encountered in our studies compared with the total number of unique bee species existing in the political territories in which the studies were performed . We used a database compiled from published and unpublished sources by J.S.A. of all described bee species currently known to exist in each country, state or province . We obtained these data for the German federal states of HessenTo examine what proportion of the regional bee species pool visited crop flowers, and what proportion of them was dominant in at least one study, we similarly generated species accumulation curves for (dominant) crop-visiting bee species. Using the full data set of all observed bee species on crop flowers in our data set, we computed the nonparametric, asymptotic true species richness estimator Chao1 with log-linear 95% confidence intervalsTo examine what proportion of the bee communities observed on crops had a recognized threat status, we used Red Data Books. Red Data Books were only available for four of the countries from which we had data of crop-visiting bee species: GermanyTo address the hypotheses that dominant crop-visiting bee species are generally common species and that these species can be easily enhanced by simple management actions, we used data from a number of European and North American studies examining the effects of measures to promote biodiversity in agricultural areas. These studies used paired designs and standardized protocols to compare bee community composition on sites with biodiversity-enhancing management with that on control sites (sites that were as similar as possible to the treatment sites but were not exposed to biodiversity management). Full details of the study locations and methodologies of the European studies collected in the EU-funded EASY project are given in refs In the United States, unpublished 2012 data were used from two studies in CA, one in NJ and one in Michigan (MI). Biodiversity-enhancing management involved establishment of hedgerows of native perennial plants (study CA1) or establishment of wildflower plantings . In contrast to the European studies, experimental sites in the United States were generally located adjacent to agricultural fields on pre-existing field edges or old fields. For the CA1 study, 20 field edges were selected containing native plant restorations , which were paired with 20 non-restored control sites. Restorations were ∼350 m long and 3–6 m wide and contained a mix of native perennial shrubs and treesn=7) were used, whereas in the United States the average per study was used. For the studies MI, NJ and CA2, sample size was six, whereas for CA1 sample size was nine, since land cover data (see below) for all 20 site pairs were not available. To explain differences in the proportional contribution of dominant species between studies, this variable was tested against a number of variables known to affect bee species community composition: the percentage of semi-natural habitat in the vicinity of sampling sites, latitude and continent48http://www.nj.gov/dep/gis/lulc07cshp.html). In MI, land cover was manually digitized from 2012 National Agriculture Imagery Program orthoimagery at the 1:2,000 scale . The other two US studies used the National Agricultural Statistics Service crop data file (http://nassgeodata.gmu.edu/CropScape/).To examine whether dominant crop-visiting bee species are common species in agricultural landscapes, generally (hypothesis 2) only data from the control sites were used because they were situated in agricultural habitats such as arable fields , grasslands, old fields and hedgerows. The proportion of the bee communities consisting of individuals from bee species dominating crop vistitation rates  were theWe used standard multiple linear regression models to relate the proportion of individuals from dominant crop-visiting species in bee communities to the proportion of semi-natural habitat, thereby correcting for latitude and continent. Plotting residuals versus fitted values confirmed that model assumptions were met satisfactorily. The often used arcsine transformation of proportional data or binomial regression increased heteroscedasticity, and we therefore present the results of untransformed data. To subsequently explain the patterns in the proportional data, we calculated standardized abundances of dominant crop-visiting bees and, separately, for all other bees for each of the European study regions by dividing the per region bee abundance by the mean abundance across all 15 regions. Since the study in each region had used exactly the same survey protocol, a standardized bee abundance >1 indicates above-average bee abundance compared with the cross-study mean, and a value <1 indicates a below-average bee abundance. We similarly calculated standardized abundances of dominant crop-visiting bees and, separately, all other bees for the three US studies that used the same survey protocol . The same approach was used to calculate per study standardized species richness. This allowed us to use the European and US data sets in a joint analysis. We used log-linear models assuming a Poisson distribution with standardized abundance or species richness as response variables, and the proportion semi-natural habitat, bee type  and their interaction as main explanatory variables of interest. A significant interaction would indicate that dominant crop-visiting bees and all other bees are differently related to semi-natural habitat. Latitude was again included as a correcting variable. Continent was not included because we had standardized the response variables between the studies on each continent.F8,267=3.94, P<0.001). We therefore chose to perform separate analyses for each study with treatment pair as a random factor and mitigation measure as a fixed factor. We chose not to correct for multiple testing, as correction reduces type I error, but tends to inflate type II errorWe used site-level count data as the statistical unit and used generalized linear mixed models assuming Poisson error distribution and using a log-link functionAll models were fitted using standard facilities in GenstatHow to cite this article: Kleijn, D. et al. Delivery of crop pollination services is an insufficient argument for wild pollinator conservation. Nat. Commun. 6:7414 doi: 10.1038/ncomms8414 (2015).Supplementary Figures 1-4, Supplementary Table 1-4, and Supplementary References.


There is a rising medical need for novel therapeutic targets of physical activity. Physical activity spans from spontaneous, low intensity movements to voluntary, high-intensity exercise. Regulation of spontaneous and voluntary movement is distributed over many brain areas and neural substrates, but the specific cellular and molecular mechanisms responsible for mediating overall activity levels are not well understood. The hypothalamus plays a central role in the control of physical activity, which is executed through coordination of multiple signaling systems, including the orexin neuropeptides. Orexin producing neurons integrate physiological and metabolic information to coordinate multiple behavioral states and modulate physical activity in response to the environment. This review is organized around three questions: (1) How do orexin peptides modulate physical activity? (2) What are the effects of aging and lifestyle choices on physical activity? (3) What are the effects of aging on hypothalamic function and the orexin peptides? Discussion of these questions will provide a summary of the current state of knowledge regarding hypothalamic orexin regulation of physical activity during aging and provide a platform on which to develop improved clinical outcomes in age-associated obesity and metabolic syndromes. Physical activity can improve overall health. For example, it can prevent obesity and reduce age-associated cognitive decline. There is wide variation between individuals in their drive to be physically active. The drive for physical activity is operationally defined as spontaneous physical activity (SPA). In humans, SPA includes time spent standing and ambulating, but not voluntary exercise. The energy expended by SPA is termed “nonexercise activity thermogenesis” or NEAT. Exercise is a necessary part of a healthy lifestyle but many people cannot or do not exercise. New treatments to target exercise-independent aspects of achieving and maintaining a healthy weight are greatly needed. Spontaneous physical activity is an excellent candidate, but our understanding of the brain mechanisms driving SPA is incomplete. Therapies that enhance SPA will contribute to better clinical outcomes for obesity and metabolic syndrome, diseases of high prevalence in the developed world. This review describes recent advances in our understanding of neuronal processes that regulate SPA, with a specific focus on changes that occur in the orexin neuropeptide system during normal and pathological aging.The orexin (hypocretin) neurons are a group of hypothalamic neurons defined by expression of the orexin peptides. The orexin signaling system regulates a variety of complex behaviors, including sleep/arousal, reward, food intake and SPA, with an overall effect of increasing energy expenditure. Orexin neuron activity is affected by multiple environmental and physiological variables like fasting and circadian rhythms. Function of the orexin system varies with lifestyle and age  and two G-protein coupled receptors  , perifornical area, and dorsomedial hypothalamus  and global (across the brain) network involved in the control of behavior and energy balance , which are critically linked to obesity and often become dysregulated with age  and physical activity (energy expenditure). Orexin neurons alter their activity in response to metabolic signals from the periphery, including leptin, glucose, and insulin (Håkansson et al., While much has been done in animal models and in humans to show that SPA significantly impacts body weight, metabolic and cognitive health, more work is needed to fully understand the neurocircuitry and molecular mechanisms which regulate SPA, in particular, what happens to this network during aging. Given our current knowledge, therapies should be developed that aim at producing behavioral and lifestyle changes that prevent or ameliorate age-associated declines in physical activity. There is a clear need for multifaceted approaches to altering SPA that include targeted manipulations of the neural systems that drive SPA. Knowing that aging is associated with an altered metabolic and hormonal milieu, an important future research direction is to understand how these molecular changes directly impact orexin signaling and SPA.In summary, hypothalamic orexin activity fluctuates over the lifespan to impact physical activity and body weight throughout the aging process Figure . Aged anThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stem cells can be valuable model systems for drug discovery and modelling human diseases as well as to investigate cellular interactions and molecular events in the earlystages of development. Controlling the differentiation of stem cells into specific germ layers provides a potential source of highly specialized cells for therapeutic applications. Inrecent years, finding individual properties of stem cells such as their ultimate self-renewalcapacity and the generation of particular cell lines by differentiation under specific culture conditions underpins the development of regenerative therapies. These futures makestem cells a leading candidate to treat a wide range of diseases. Nevertheless, as with allnovel treatments, safety issues are one of the barriers that should be overcome to guarantee the quality of a patient’s life after stem cell therapy. Many studies have pointed toa large gap in our knowledge about the therapeutic applications of these cells. This gapclearly shows the importance of biosafety concerns for the current status of cell-basedtherapies, even more than their therapeutic efficacy. Currently, scientists report that tumorigenicity and immunogenicity are the two most important associated cell-based therapyrisks. In principle, intrinsic factors such as cell characteristics and extrinsic elementsintroduced by manufacturing of stem cells can result in tumor formation and immunological reactions after stem cell transplantation. Therapeutic research shows there are manybiological questions regarding safety issues of stem cell clinical applications. Stem celltherapy is a rapidly advancing field that needs to focus more on finding a comprehensivetechnology for assessing risk. A variety of risk factors (from intrinsic to extrinsic) should beconsidered for safe clinical stem cell therapies.  Stem cells have the strong potential to differentiate into all three germ layers  and subsequently generate a broad range of cells such as cardiomyocytes, neurons, and hepatocytes . AccordiThere are two types of stem cells which can be used or have the potential to be used in stemcell-based therapies, multipotent [adult stem cells (ASCs)]  and plurDespite rapid progress, stem cell therapy is surrounded by major difficulties that affect transplantation efficacy. Allogeneic transplantation can raise sharp ethical issues about human identity, especially when the original stem cells have been isolated from an embryo for further culture and manipulation. In addition, there is the risk of a high immune rejection . While aRegardless of the stem cell source , stem cell therapy as with other treatments, has a number of side effects that patients’ experiences when they pass through the recovery stage. The effects of stem cell therapy after hematopoietic allogeneic transplantation, a common stem cell transplantation in humans, can be divided into two categories non-malignant and malignant . The nonin vitro cultivation of stem cells which enhances the tumorigenicity risk  must be considered in order to have safe clinical stem cell therapies. Stem cell therapies have a perfect beginning and hold tremendous potential for treatment of multiple challenging diseases. However our understanding is inadequate to apply stem cells as clinical treatments in humans. Stem cell therapy has a number of issues that include ethical, efficacy, side effects, and safety. Ethical considerations can be solved by changing the sources of stem cells, by using ASCs from the patient or IPSC derivation instead of embryonic cells. However, serious biosafety and side effects remain for stem cell therapy, which require further bioanalyses.


The prevalence of past-year suicidal ideation at age 16 years was 15% (95% CI: 14-17%). Compared to offspring of mothers with minimal symptoms, the greatest risk of suicidal ideation was found for offspring of mothers with chronic-severe symptoms [OR 3.04 ], with evidence for smaller increases in risk of suicidal ideation in offspring of mothers with sub-threshold, increasing and mild symptoms. These associations were not fully accounted for by maternal suicide attempt or offspring depression diagnosis. Twenty-six percent of non-depressed offspring of mothers with chronic-severe depression symptoms reported suicidal ideation. Risk for suicidal ideation should be considered in young people whose mothers have a history of sustained high levels of depression symptoms, even when the offspring themselves do not have a depression diagnosis.Depression is common, especially in women of child-bearing age; prevalence estimates for this group range from 8% to 12%, and there is robust evidence that maternal depression is associated with mental health problems in offspring. Suicidal behaviour is a growing concern amongst young people and those exposed to maternal depression are likely to be especially at high risk. The aim of this study was to utilise a large, prospective population cohort to examine the relationship between depression symptom trajectories in mothers over the first eleven years of their child’s life and subsequent adolescent suicidal ideation. An additional aim was to test if associations were explained by maternal suicide attempt and offspring depressive disorder. Data were utilised from a population-based birth cohort: the Avon Longitudinal Study of Parents and Children. Maternal depression symptoms were assessed repeatedly from pregnancy to child age 11 years. Offspring suicidal ideation was assessed at age 16 years. Using multiple imputation, data for 10,559 families were analysed. Using latent class growth analysis, five distinct classes of maternal depression symptoms were identified ( Suicidal ideation is common in adolescence and is one of the most salient risk factors for later suicide . TherefoEvidence from existing community studies suggests that maternal depressive disorder is associated with an increased risk of later suicidal ideation in offspring ,5. UsingThe majority of previous literature has examined links between a lifetime diagnosis of maternal depression and offspring suicidal ideation ,5. HeterThe reasons for an association between maternal depression and offspring suicidal ideation also remain unclear. The association could be confounded by socio-demographic risk factors that are known correlates of maternal depression  and are The present investigation examines the association between maternal depression symptom course over the first eleven years of their child’s life and subsequent offspring suicidal ideation at age 16 years in a large population based birth cohort. The primary hypothesis is that variation in maternal depression symptom course from pregnancy to child age 11 years will be associated with subsequent offspring suicidal ideation at age 16 years over and above potential socio-demographic and familial confounders, with greatest risk for offspring of mothers with severe and chronic depression symptoms. However, it is also expected that sub-threshold maternal depression symptoms that persist over time will be associated with increased risk of offspring suicidal ideation. The secondary hypothesis is that the associations observed will be attenuated, but not completely explained through maternal suicide attempt or a diagnosis of depression in the offspring. Analyses will also examine whether findings are similar when examining risk for offspring lifetime suicide attempt and whether patterns of associations differ by gender.st April 1991 and 31st December 1992. Of the 14,062 live births, 13,617 were singletons and were alive at one year of age. The sample is broadly representative of the UK population, however, mothers enrolled in ALSPAC were more likely to live in owner-occupied accommodation and have a car, more likely to be married and less likely to be non-white .In order to examine whether the increase in prevalence of suicidal ideation at age 16 years with increasing severity of maternal depression symptoms trajectories was solely due to an increase in prevalence of offspring depressive disorder, the pattern of findings was examined in non-depressed offspring. A similar pattern of results was found when the percentage of offspring with suicidal ideation, but no previous DSM-IV diagnosis of MDD was examined .Next, a logistic regression analysis was performed between classes of maternal depression symptoms . The overall prevalence was similar in fully imputed models taking account of missing data– 8% . chronic-severe depression symptoms. However, there were also smaller increases in risk for offspring of mothers with sub-threshold, increasing and mild symptoms in comparison to offspring of mothers with minimal symptoms. This is an important finding because more than half of this population cohort of teenagers had experienced maternal depression at these levels. Associations between maternal depression and offspring suicidal ideation were not completely explained through maternal suicide attempt or a diagnosis of depression in the offspring. Results were similar when examining associations with offspring lifetime suicide attempt.In this population sample, five distinct classes of maternal depression symptoms were identified. Four classes showed stable levels of depression symptoms but differed in the level of severity and one class showed a change in severity, with increasing levels of depression symptoms over time. Variation in maternal depression symptom course was associated with subsequent offspring suicidal ideation at age 16 years, with greatest risk of suicidal ideation for offspring of mothers with This is one of the first studies to examine whether there is an association between variation in maternal depression symptom course over time and subsequent offspring suicidal ideation. The results extend the findings of previous longitudinal studies that have found an association between maternal depressive disorder and offspring suicidal ideation ,5 by exaincreasing symptoms were nearly two times more likely to have suicidal ideation at age 16 years compared to offspring of mothers with minimal symptoms. It should be noted however, that not only did mothers with increasing symptoms have the most uncertainty in group membership in the derivation of classes, but also, wider confidence intervals meant there was more uncertainty in the association with offspring suicidal ideation, especially when using only those with complete data. In addition, there was only weak evidence for increased risk of suicide attempt in offspring of mothers with increasing symptoms after adjusting for potential confounders.In this study, although maternal depression trajectories were mostly stable, one group of mothers did exhibit meaningful change in symptom levels over time. Fewer studies have identified a group of mothers with increasing symptoms over time ,28,29,37chronic-severe class. However, there was still evidence for an association between the classes of maternal depression symptoms and offspring suicidal ideation after accounting for these potential mediators. These findings extend results from studies that have found a retrospective diagnosis of maternal depression and maternal suicide attempt have independent links with offspring suicidal ideation and behaviour [chronic-severe depression symptoms were at highest risk of suicidal ideation with these offspring being over two times more likely to have suicidal ideation at age 16 years compared to offspring of mothers with minimal symptoms. In addition, there was evidence that offspring of mothers with chronic-severe symptoms were also at increased risk of suicidal ideation when compared to offspring of mothers with sub-threshold, increasing and mild symptoms. This is unsurprising as not only are chronic and severe symptoms of depression likely to indicate higher genetic risk, but also offspring exposed to chronic symptoms are more likely to be exposed to a variety of environmental risk factors such as a negative family environment [Previous studies have provided strong evidence that both a diagnosis of depression ,39 and mehaviour . Furtherehaviour ,11. In oironment . Geneticironment . In thisironment , althougironment . Howeverincreasing and mild classes after adjusting for all covariates. This pattern of findings across imputed and complete case samples has been shown previously in studies using the same sample that have reported that the association between maternal and offspring depression may be underestimated in complete case analyses [Our findings need to be considered in the light of some additional limitations. Firstly, as with most cohort studies, there was selective attrition over time; however, potential bias arising from missing data was dealt with using multiple imputation, utilising a large amount of additional information to make the assumption of missing-at-random plausible . Previouanalyses . It is achronic-severe symptoms. However, suicide risk should be considered in offspring, even when maternal depression symptoms are below clinical levels. Offspring of mothers with subclinical levels of depression symptoms are an important group to consider as these offspring may be less likely to be known to services as mothers may have never been diagnosed with clinical depression. In this sample, only half of mothers with sub-threshold symptoms over time were known to services. However, as expected, it is offspring of mothers with both chronic and severe depression symptoms that are most at risk and a priority for preventive interventions. Additionally, as 26% of non-depressed adolescents in this group reported suicidal ideation at age 16 years, this highlights the importance of enquiring about suicidal ideation in offspring of depressed mothers, even when offspring do not have a diagnosis of depression. Given that the majority of mothers from the chronic-severe class were already known to services, this would be an easily identified high risk group to target [In summary, variation in maternal depression symptoms over time was associated with subsequent offspring suicidal ideation and suicide attempt, with greatest risk for offspring of mothers with o target . These rS1 Table(DOCX)Click here for additional data file.S2 TableBold represents average posterior class probability for trajectory membership.(DOCX)Click here for additional data file.S3 TableImputed N = 10,559.(DOCX)Click here for additional data file.


The main function of red blood cells (RBC), which is mediated by Hb, is the transport of oxygen and carbon dioxide in the body. In addition, RBC are strongly involved in regulation of vascular tone especially under hypoxic conditions. Hypoxic vasodilation is a fundamental physiological process which directly correlates with the oxygen saturation of Hb in RBC. Since discovery in 1980s that NO is a biological signalling molecule, numerous studies linked NO/Hb to the mechanisms underlying hypoxic vasodilation. According to calculations, mathematical modelling, and experimental data most of NO in blood is scavenged by Hb by the reaction of deoxyHb with NO which results in formation of metHb and nitrate. It has been also proposed that NO can bind by nitrosylation reaction of conserved cysteine on Hb β-chain (β93Cystein) to form SNO-Hb. Although different reactions between NO and Hb are clear and well established, the mechanisms of NO release from Hb and how, in physiological conditions of high excess of free Hb, NO can escape from scavenging by Hb are not so well established. Importantly, till now, there was never shown directly that RBC released biologically active NO can activate soluble guanylyl cyclae (sGC) in neighboring cells (platelets or vascular smooth muscle cells).Most experiments that prove NO release from RBC are done in blood vessel relaxation or in isolated washed RBC. However, both these experimental approaches are not sufficient to clearly answer the question whether RBC can produce biologically active NO because vasorelaxation is a complex reaction which is not mediated only by NO and in isolated RBC experiments it was never shown that RBC produced NO or other NO related radicals could activate sGC. To answer this question we used two different approaches which allow to directly measure NO-mediated activation of sGC in intact cells (platelets) and in purified enzyme. Endogenous NO is a well-known and potent inhibitor of platelet activation and platelet inhibitory effects of NO are mediated solely by sGC activation. Intact platelet sGC is highly sensitive to endogenous NO and even nanomolar NO concentrations are sufficient for cGMP generation. In platelets, the main mediator of cGMP is PKG which phosphorylates multiple substrates responsible for platelet inhibition. For evaluation of platelet sGC activity we used our well-established model based on NO/sGC/cGMP/PKG dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation in human platelets and purified sGC which is also very sensitive to low NO concentrations. In our experiments RBC were mixed with platelet suspension or with purified enzyme and sGC activation was assessed by VASP phosphorylation and cGMP formation. All our experiments clearly show that RBC, in any conditions, cannot be sources of NO, they act only as a strong NO scavengers and all our data, albeit indirectly, indicate that hypoxia/RBC induced vasodilation is not mediated by NO derived from RBC.


Motivation is a key neurobehavioral concept underlying adaptive responses to environmental incentives and threats. As such, dysregulation of motivational processes may be critical in the formation of abnormal behavioral patterns/tendencies. According to the long standing model of the Reinforcement Sensitivity Theory (RST), motivation behaviors are driven by three neurobehavioral systems mediating the sensitivity to punishment, reward or goal-conflict. Corresponding to current neurobehavioral theories in psychiatry, this theory links abnormal motivational drives to abnormal behavior; viewing depression and mania as two abnormal extremes of reward driven processes leading to either under or over approach tendencies, respectively. We revisit the RST framework in the context of bipolar disorder (BD) and challenge this concept by suggesting that dysregulated interactions of both punishment and reward related processes better account for the psychological and neural abnormalities observed in BD. We further present an integrative model positing that the three parallel motivation systems currently proposed by the RST model, can be viewed as subsystems in a large-scale neurobehavioral network of motivational decision making. The term motivation as used in neuroscience refers to the processes which modulate the organism’s responses to environmental reinforcing cues, according to their perceived value  Smillie, . As it iThe basic concept regarding the consequential effects of stimuli on behavior, depending on their incentive values for the organism (reward or punishment) originated with the seminal work of Pavlov Pavlov, . EssentiThe relevance of motivation systems to abnormal mood conditions is supported by recent studies focusing mainly on reward processing. This has reflected developments in the investigation of the neural underpinnings of reward processes in terms of their neurochemistry  underlies mania or depression, respectively. Accordingly, numerous self-report studies using RST based questionnaires have shown higher BAS sensitivity in bipolar patients  indicate combined system involvement in BD (see summary in Table This pattern has been convincingly demonstrated in the study by Blumberg et al.  who inveAltogether, imaging findings strongly support our proposal for the involvement of several RST systems in BD, suggesting that rather than a deficit in BAS alone, the coupled interaction of BAS with BIS/FFFS is impaired Figure . To noteWe argue here for considering abnormal motivational processes in the pathophysiological study of mental illnesses, driven by neural system sensitive to reward, punishment or their regulation during goal-conflict. Based on a theoretical framework and converging empirical evidence, we suggest that although the BAS dysregulation theory has indeed provided a parsimonious and intuitive model for the involvement of motivational processes in BD, a conceptual revision is needed to further adapt these insights to the complexity of human psychopathology. Our new model maintains that since combined activity of the three motivation systems is responsible for the varied encounters with the external environment, BD is probably underpinned by compound motivational abnormalities: depression involves a hyperactive BIS and a hypoactive BAS, while mania results from a hyperactive BAS coupled with a hypoactive BIS (as illustrated in Figure Thinking along this theoretical framework may provide useful insights into other psychiatric disorders as well. For example, similar abnormalities in motivation may also exist in unipolar depression MDD (Kasch et al., Such a complex large-scale network model has recently become a common conceptual framework in neuroscientific research (Bressler and Menon, To further investigate the core features of our model, such as dysfunction within and between motivational networks, future studies may explore the dynamics in functional-connectivity of the different systems’ key nodes using, for example, the network cohesion index (NCI; Raz et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These analyses have revealed how the initial steps of polarization, in which neurons establish an axon, occur. Interestingly, both excitatory and inhibitory cortical neurons establish neuronal polarity de novo by undergoing a multipolar stage reminiscent of the manner in which polarity formation occurs in hippocampal neurons in dissociated culture. In this review, we focus on polarity formation in cortical neurons and describe their typical morphology and dynamic behavior during the polarization period. We also discuss cellular and molecular mechanisms underlying polarization, with reference to polarity formation in dissociated hippocampal neurons in vitro.Cortical neurons consist of excitatory projection neurons and inhibitory GABAergic interneurons, whose connections construct highly organized neuronal circuits that control higher order information processing. Recent progress in live imaging has allowed us to examine how these neurons differentiate during development  Neurons are highly polarized cells that typically exhibit a single axon and several dendrites. Dendrites receive incoming signals at the synapse and convey them to the soma. These signals trigger action potentials at the level of soma, which propagate along the axon and are transmitted to target cells at a presynaptic site. A critical question in neurobiology is how neurons acquire axon-dendrite polarity, a property required for directional information flow in the nervous system.in vivo or in situ  suggest that several types of neurons establish neuronal polarity by inheriting apicobasal polarity from neuroepithelial progenitors or maintaining front-rear polarity in migrating cells -ergic. During development, excitatory neurons originate from the dorsal telencephalon  Figure . AsymmetMorphologically, bipolar progenitor cells are extraordinarily slender and extend a long apical process toward the pial surface and a short basal process toward the ventricle. During asymmetric cell division, the daughter cell destined to become a neuron assumes a multipolar shape from which emanates multiple short, thin processes in the SVZ/IZ , a new potential axon emerges in vivo or at least in situ, polarity formation primarily occurs in multipolar cells in a manner similar to that seen in dissociated hippocampal neurons. In the next section, we focus on early polarity events that occur during multipolar cell stages prior to axon formation.In contrast, as described above, in both excitatory and inhibitory cortical neurons Radial glial cells, the main progenitor population of excitatory cortical neurons, are neuroepithelial cells that exhibit apicobasal polarity. However, young neurons and intermediate progenitors appear to lose that polarity by retracting apical and basal processes during asymmetric cell division and assuming a multipolar shape (Tabata and Nakajima, Recently, several papers have reported that signaling and cytoskeletal proteins function in the multipolar-bipolar transition of excitatory neurons in the IZ (reviewed in Cooper, in situ, other processes occasionally extend up to 150–200 μm but fail to extend further, and eventually only one exceeds 200 μm in length and differentiates into an axon (Yamasaki et al., de novo. Further study examining dynamic movement of cellular components in multipolar cells during axon formation should validate this view.During multidirectional tangential migration in the MZ, inhibitory cortical neurons extend a leading process in the direction of their movement (Tanaka et al., in situ and hippocampal neurons in vitro. Although in both cases neurons initially appear multipolar, the mode of “random growth and retraction” of processes differs slightly. First, minor processes of hippocampal neurons in vitro show alternate increases and decreases in length, while those of cortical neurons in situ often show alternate appearance and disappearance of processes. Therefore, potential sites of axon initiation seem to be set at the very beginning of the polarization process in vitro. Second, the location of a hippocampal neuron cell body in vitro appears fixed during the multipolar stage, while that of excitatory or inhibitory cortical neurons in situ does not. These activities may be due to microenvironmental differences, such as adhesive properties: hippocampal neurons interact with a positively-charged planar substrate, while cortical neurons do not. Some of the activities one sees in hippocampal neurons in in vitro might be artifacts.There are minor differences between behavior of cortical neurons In vitro and in vivo studies suggest an instructive role for centrosome positioning in axon specification (Lefcort and Bentley, in situ reveal that different mechanisms may govern axon formation in these cells (Sakakibara et al., in vitro, one minor process becomes an axon and then behaves as the dominant process. In both cases, the centrosome attracted to the growing axon. On the other hand, neurons in the CP at later migration stages exhibit a leading process oriented toward the brain surface, which then behave as the dominant process and attract the centrosome. When an axon forms at the rear of these cells in situ, the centrosome does not translocate toward the initiating axon but rather remains oriented toward the leading process (Sakakibara et al., in vivo, these observations suggest that centrosome positioning is passively controlled by a balance of protrusive activities among processes and does not play an instructive role in excitatory cortical neurons in vivo. In migrating inhibitory cortical neurons, the primary cilium, whose basal body is formed from a centriole, reportedly regulates Sonic hedgehog-mediated reorientation of the leading process (Baudoin et al., in vivo may further prompt our understanding of microtubule function underlying axonal morphogenesis.in vitro studies of hippocampal neurons, although in vivo studies validating these findings have also been reported. Extracellular mechanisms regulating cortical neuron polarization have been studied by in vivo analyses of knockout phenotypes or gene manipulation in embryonic mouse brain, although most of these studies have been confined to excitatory cortical neurons.Axon specification in cortical neurons is driven by intracellular and extracellular mechanisms Figure . IntraceCytoskeletal changes dependent on protein phosphorylation are required for axon specification; thus, axon formation can be assessed using markers recognizing differential phosphorylation states of cytoskeletal proteins (Sternberger and Sternberger, The aPKC/Par complex plays a central role in axon specification: for example, aPKC inhibition suppresses axon formation in hippocampal neurons (Zhang et al., in vitro (Schwamborn and Püschel, Several small GTPases function differentially in neuronal polarization (Arimura and Kaibuchi, 3 transport by GAKIN/KIF13B functions in axon formation, suggesting a role for accumulated PIP3 in positive feedback regulation of Rac and Cdc42 small GTPases (Horiguchi et al., Axon formation also requires directed transport of membrane vesicles and other cargos along polarized microtubules (reviewed in Conde and Cáceres, in vivo (Yau et al., Recent studies show that regulators of microtubule dynamics are required for neuronal polarization. Control of microtubule minus-end dynamics by CAMSAP2 is essential for polarization of cortical neurons in vitro, its growth rapidly increases, while that of other immature processes does not (Esch et al., in vivo. Some investigators propose that a single axon is specified via positive feedback signals that stabilize process extension (Arimura and Kaibuchi, in vivo. Because excitatory cortical neurons under the same conditions appear multipolar (Hayashi et al., in vivo.Interestingly, when the growth cone of an immature process of a hippocampal neuron encounters a preferred substrate de novo. These neurons initiate axons after assuming a multipolar stage, in which no fixed polarity is exhibited. Although these neurons appear similar during axon formation, it is important, especially in the case of inhibitory neurons, to examine the dynamics of cellular components to validate this view. Also, it will be interesting to examine whether these neuronal subtypes share signaling pathways governing polarity. It should be noted that inhibitory cortical neurons are in fact diverse and consist of multiple morphological subtypes with different spatial and temporal origins. Thus, future investigations are needed to determine whether these subtypes share a common mechanism of axon initiation. In addition, it is important to define dynamic processes governing dendrite formation by both excitatory and inhibitory cortical neurons. These analyses will likely require a combination of genetic labeling of specific excitatory and inhibitory neuronal subtypes with live imaging both in situ and in vivo.Here, we have reviewed recent evidence suggesting that cortical neurons, both excitatory and inhibitory, establish polarity in situ, care should be taken in generalizing these mechanisms to other neuronal types. Proper understanding of polarity formation in the cerebral cortex requires identification of the key processes that underlie external cue-mediated polarization in vivo.Finally, analysis of dissociated hippocampal neurons has set the foundation for our current understanding of polarization processes and their molecular basis. Although some of this knowledge is applicable to cortical neurons The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.