The chemical and electrical microenvironment of neurons within the central nervous system is protected and segregated from the circulation by the vascular blood–brain barrier. This barrier operates on the level of endothelial cells and includes regulatory crosstalk with neighboring pericytes, astrocytes, and neurons. Within this neurovascular unit, the endothelial cells form a formidable, highly regulated barrier through the presence of inter-endothelial tight junctions, the absence of fenestrations, and the almost complete absence of fluid-phase transcytosis. The potent psychostimulant drug methamphetamine transiently opens the vascular blood–brain barrier through either or both the modulation of inter-endothelial junctions and the induction of fluid-phase transcytosis. Direct action of methamphetamine on the vascular endothelium induces acute opening of the blood-brain barrier. In addition, striatal effects of methamphetamine and resultant neuroinflammatory signaling can indirectly lead to chronic dysfunction of the blood-brain barrier. Breakdown of the blood-brain barrier may exacerbate the neuronal damage that occurs during methamphetamine abuse. However, this process also constitutes a rare example of agonist-induced breakdown of the blood-brain barrier and the adjunctive use of methamphetamine may present an opportunity to enhance delivery of chemotherapeutic agents to the underlying neural tissue. The vasculature pervading the nervous system is exceptionally specialized and because of its restrictive nature has been dubbed the blood brain barrier (BBB). The BBB controls the ionic microenvironment for the central nervous system (CNS). It excludes neurotoxic plasma proteins and yet enables cellular and molecular crosstalk between the periphery and the brain parenchyma  with neurons and non-neuronal cells including pericytes, astrocytes, perivascular macrophages, and microglia Daneman, . The actOn the abluminal side cerebral ECs interact with surrounding pericytes with which they share a basement membrane. Pericyte coverage at the BBB and blood-retinal barrier is the highest of all vascular beds, with EC to pericyte ratios typically ranging between 1:1 and 3:1  have been identified in BBB ECs. CLDN5 contributes to BBB function in that it regulates paracellular transport of small  blood solutes  transporters, solute carriers, transcytosis, and transendothelial migration of leukocytes  is a highly addictive CNS stimulant with demonstrated neurotoxicity. Long-term damage to monoaminergic nerve terminals is caused by excitotoxicity, mitochondrial dysfunction, and increased production of reactive oxygen and nitrogen species  opening of the BBB, suggests that METH and related compounds could be used to enhance the transport of other drugs to the CNS. Indeed, this is an idea proposed as early as 2007 Kast, . HoweverResearch into the effects of METH on the BBB should be considered topical and highly relevant, and not just with regard to understanding METH abuse and its neurological consequences. METH affects barrier function of BBB ECs directly but also induces a response, which is reminiscent of neuroinflammation, in that it involves glial activation, cytokine, and MMP release as well as oxidative stress. Mechanistic insights into METH action are likely to enhance our understanding of the BBB, particularly the molecular and cellular mechanisms of its pathophysiological regulation, as well as reveal vital insight into the specificity of BBB efflux pumps for monoamines. Finally, research into METH may even facilitate our reach toward disease targets currently hidden behind the BBB.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.


Transplantation of dopamine- (DA-) rich foetal ventral mesencephalic cells emerged as a promising therapy for Parkinson's disease (PD), as it allowed significant improvement of motor symptoms in several PD patients in open-label studies. However, double-blind clinical trials have been largely disappointing. The general agreement in the field is that the lack of standardization of tissue collection and preparation, together with the absence of postsurgical immunosuppression, played a key role in the failure of these studies. Moreover, a further complication that emerged in previous studies is the appearance of the so-called graft-induced dyskinesia (GID), in a subset of grafted patients, which resembles dyskinesia induced by L-DOPA but in the absence of medication. Preclinical evidence pointed to the serotonin neurons as possible players in the appearance of GID. In agreement, clinical investigations have shown that grafted tissue may contain a large number of serotonin neurons, in the order of half of the DA cells; moreover, the serotonin 5-HT1A receptor agonist buspirone has been found to produce significant dampening of GID in grafted patients. In this paper, we will review the recent preclinical and clinical studies focusing on cell transplantation for PD and on the mechanisms underlying GID.  Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by intraneuronal inclusions of aggregated proteins (named Lewy bodies and Lewy neurites) and degeneration of the dopamine (DA) neurons of the substantia nigra pars compacta. The reduction of DA levels in the striatum results in the appearance of motor symptoms, including bradykinesia, rigidity, postural instability, and resting tremor. Although PD has usually been considered a pure motor disease, this pathology is now recognized as heterogeneous, and a significant variety of nonmotor symptoms such as depression, constipation, pain, and sleep disorders have been described in PD patients . To dateCell transplantation for treating PD was first tested as a potential therapy in 1985 . In the Clinical benefits in PD grafted patients have been observed in open-label trials. Accordingly, an improvement of motor symptoms, particularly of hypokinesia and on-off fluctuations, has been described in several grafted patients , 7–9, whThe effect of foetal DA neuron transplants on LID has been variable; in some patients a significant reduction of LID has been observed, while in others dyskinesia has been unaffected or even made worse , 10, 18.GID has emerged as a potentially serious adverse effect induced by DA neuron transplants in 15 to 57% of patients (depending on the study) that have undergone cell replacement therapy , 20, 23.Development of GID has been proposed to be caused by several different mechanisms, such as extent of DA neuron degeneration , excessiThese promoting factors have been examined in the past years in GID patients. However, given the low number of grafted patients available for these studies, the development of a preclinical animal model of GID, represented by 6-OHDA-lesioned rodents subjected to striatal foetal ventral mesencephalic cell grafting, has provided a useful tool to investigate the mechanisms responsible for the appearance of GID. Studies using this animal model have shown that grafted DA neurons can survive, reinnervate the lesioned striatum, and provide significant improvement of motor function . HoweverAnalysis of patient data and the use of this animal model have allowed researchers to propose several risk factors for the development of GID, although, so far, no single most important factor has been identified. No differences in striatal [18F]DOPA uptake were observed between transplanted patients who did or did not develop GID , 23, sugConversely, in grafted rats, it has been demonstrated that the transplant size may have an impact on the induction of GID. In fact, rats with large grafts displayed more severe GID (amphetamine-induced) compared to rats with smaller grafts . MoreoveFurthermore, the severity of preoperative LID was correlated with the severity of GID. Animals with severe preoperative LID receiving intrastriatal DA grafts showed pronounced reduction of LID, but at the same time all animals developed GID; by contrast, in rats with mild preoperative LID, which showed mild reduction of LID after grafting, the expression of GID was usually mild or absent . This asHost immune response is one of the major debated issues in this field. Despite the fact that for many years the brain has been thought to have immune privilege, cell transplantation trials have demonstrated that this is not fully correct . AccordiRecent studies suggest that GID development could be related to the composition of the grafted tissue , 39, 57.The presence of serotonin neurons into the grafts and their role in the development of GID represents another important aspect that has emerged from recent preclinical and clinical observations. In fact, serotonin neurons develop in close proximity to the DA neurons of the ventral mesencephalon; since the availability of embryonic tissue has been a limiting factor in the previous trials, the landmarks of dissection have been usually broad, so to collect a high number of transplantable cells. However, this procedure caused the inclusion of a significant number of serotonin precursor cells in the graft .It is well known that the serotonin neurons possess the machinery to take up exogenous L-DOPA, to convert it into DA, and store DA into vesicles. This mechanism plays a role in the release of L-DOPA-derived DA when the vast majority of the DA neurons have degenerated; indeed, it has been suggested that abnormal release of DA from the serotonin terminals may be responsible for the excessive swings in striatal synaptic DA levels, which underlie the appearance of LID –66. AccoThus, given the role of the serotonin neurons in the development of LID, the inclusion of this neuronal population in the grafted tissue has been proposed to be another risk factor for the development of GID , 42, 47.The balance between serotonin and DA grafted neurons may be important for the severity of LID and GID. Indeed, in partial 6-OHDA-lesioned rats it has been demonstrated that serotonin-rich grafts were effective in inducing a widespread serotonergic hyperinnervation of the host striatum , 70, 71 The involvement of the serotonin system in GID has been investigated in several preclinical studies. Using 6-OHDA-lesioned rats subjected to striatal VM grafting, significant dampening of GID (induced by amphetamine) was seen following treatment with very low doses of the 5-HT1A receptor agonist buspirone  or combination of the 5-HT1A and 5-HT1B receptor agonists 8-OH-DPAT and CP94253, which all inhibit transmitter release from serotonergic neurons , 43. TheInterestingly, Shin and colleagues  have demClinical studies showed the efficacy of transplantation of DA neurons in restoring motor functions in PD patients over a long-time period. However, the variability of the results and the appearance of side effects represented by GID in a significant percentage of grafted patients have hindered the pursuit of this approach.There is now a general consensus that improved outcome and reduced risk of developing GID might derive from a refinement of the dissection method used to collect the foetal tissue for transplantation. Avoiding the inclusion of serotonin neurons, or other types of cells, might be one of the essential points. Furthermore, increasing the distribution of DA neurons throughout the striatum using multiple injection sites might optimize the coverage of the denervated striatum by DA transplanted cells and prevent “hot-spots” of DA release , 27. In Better results may also derive from a more accurate selection of the patients; indeed, patients poorly responsive to L-DOPA treatment are less likely to benefit from this approach and should not be included in such studies. Moreover, performing PET imaging before surgery may be useful for the correct placement of the graft in each subject , 53.Important knowledge is expected to be acquired with the ongoing double-blind clinical study using foetal cell transplantation in PD patients (TRANSEURO). This study will be important not only to provide a conclusive answer about the ability of foetal cell transplantation to ameliorate the motor symptoms of the disease, but also to pave the ground for future larger studies using stem cells.


The promotion of neurological recovery by enhancing neuroplasticity has recently obtained strong attention in the stroke field. Experimental studies support the hypothesis that stroke recovery can be improved by therapeutic interventions that augment neuronal sprouting. However plasticity responses of neurons are highly complex, involving the growth and differentiation of axons, dendrites, dendritic spines and synapses, which depend on the pathophysiological setting and are tightly controlled by extracellular and intracellular signals. Thorough mechanistic insights are needed into how neuronal plasticity is influenced by plasticity-promoting therapies in order not to risk the success of future clinical proof-of-concept studies. Following ischemic stroke, neuronal networks in the vicinity and at distance to the stroke are reorganized. Axons and axon collaterals are enabled to sprout  structural surrogates of successful neurological recovery depending on the localization of ischemic lesions; (b) reorganization and plasticity processes of the cellular, subcellular and network level; (c) critical time windows for various therapeutic interventions; and (d) modes for the delivery of biologicals or drugs. We will shortly present (e) selected molecular signals that are likely mediators of plasticity processes, since we believe that understanding these signals is a major hallmark to prevent the failure of treatments in future clinical studies.Ischemic stroke can affect both gray and white matter tissue, which invariably results in diverse patterns of brain injury and stroke recovery. In both cases, successful stroke recovery goes along with parenchymal tissue remodeling, involving: (a) the survival of neurons and axons in the surrounding of the ischemic lesion, which otherwise exhibit delayed degeneration; (b) the promotion of perilesional axonal, dendritic and synaptic plasticity; (c) the outgrowth of axons and axon collaterals at distance to the stroke; and (d) the modulation of astroglial and microglial responses, which may both promote or impede neuronal sprouting, depending on their activation state  delivery after middle cerebral artery occlusion in mice, both acute, sub-acute and post-acute intravenous NPC delivery up to 1 month post-stroke enhanced neurological recovery  thus activating cell death pathways  and NogoA receptor. GSK249320 has been found to enhance neurite formation The phosphodiesterase inhibitor sildenafil, which increases intracellular concentrations of cGMP, a cyclic nucleotide involved in neuronal plasticity  which type of tissue shall be targeted , and (2) which structural features of neurons are influenced  wrote the article. All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.This work was supported by the German Research Council (HE3173/3-1).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.


Alongside the time-locked event-related potentials (ERPs), nociceptive somatosensory inputs can induce modulations of ongoing oscillations, appeared as event-related synchronization or desynchronization (ERS/ERD) in different frequency bands. These ERD/ERS activities are suggested to reflect various aspects of pain perception, including the representation, encoding, assessment, and integration of the nociceptive sensory inputs, as well as behavioral responses to pain, even the precise details of their roles remain unclear. Previous studies investigating the functional relevance of ERD/ERS activities in pain perception were normally done by assessing their latencies, frequencies, magnitudes, and scalp distributions, which would be then correlated with subjective pain perception or stimulus intensity. Nevertheless, these temporal, spectral, and spatial profiles of stimulus induced ERD/ERS could only partly reveal the dynamics of brain oscillatory activities. Indeed, additional parameters, including but not limited to, phase, neural generator, and cross frequency couplings, should be paid attention to comprehensively and systemically evaluate the dynamics of oscillatory activities associated with pain perception and behavior. This would be crucial in exploring the psychophysiological mechanisms of neural oscillation, and in understanding the neural functions of cortical oscillations involved in pain perception and behavior. Notably, some chronic pain  patients are often associated with the occurrence of abnormal synchronized oscillatory brain activities, and selectively modulating cortical oscillatory activities has been showed to be a potential therapy strategy to relieve pain with the application of neurostimulation techniques, e.g., repeated transcranial magnetic stimulation (rTMS) and transcranial alternating current stimulation (tACS). Thus, the investigation of the oscillatory activities proceeding from phenomenology to function, opens new perspectives to address questions in human pain psychophysiology and pathophysiology, thereby promoting the establishment of rational therapeutic strategy. Pain, affecting the well beings of millions of individuals and imposing a severe financial burden upon our societies, is a major public healthcare problem. Pain relief, especially for the patients with pathological chronic pain, still remains a very problematic challenge to the physicians. The progress in understanding of the neural representation of pain in humans is not only important for basic neuroscience research, but also critical to develop effective strategies for the diagnosis and management of the pathological pain conditions. Specifically, this constitutes the understandings of: (1) the physiological mechanisms of the nociceptive system in healthy populations, particularly the cortical processes underlying the perception of pain and (2) the pathophysiological mechanisms of the nociceptive system in chronic pain patients, particularly the peripheral and central mechanisms leading to chronic pain. Thus, for a better understanding of the physiology and pathophysiology of pain in humans, novel approaches should be developed to identify the neural activities related to the processing of noxious inputs in humans, as well as characterize their functional roles in subjective pain perception.In both physiological  related to the activation of nociceptive fibers have been disclosed Figure , includiThese painful stimulus induced ERD/ERS responses, occurring in painful information processing, have been suggested to be associated with the perception of pain . Nevertheless, the traditionally temporal, spectral, and spatial profiles can only partly reveal the dynamics of brain oscillatory activities. Investigating novel parameters comprehensively characterizing brain oscillations could help explore the psychophysiological mechanisms of neural oscillations, as well as the neural functions of cortical oscillations involved in sensory perception and behavior. In addition, pre-stimulus ongoing EEG oscillation could influence both post-stimulus electrophysiological activities and sensory perception  of pain related oscillations, should also be further investigated, for a comprehensive and systemic understanding of the brain oscillations associated with pain.Much of the research on oscillations in human EEG has focused on the dynamics of oscillations magnitudes. Nevertheless, the phase of the oscillatory activities at a given frequency band reflects cyclic fluctuations of a network’s excitability and varies on a much faster timescale than the sluggish amplitude fluctuations at the same frequency band  and EEG  have been proposed to reflect the coordination of neural dynamics across temporal and spatial scales  the potential relevance of CFC for understanding psychophysiological and pathological brain functions , and then the resulting TFDs are typically expressed as a percentage change relative to pre-stimulus EEG power to highlight the stimulus-induced changes in power within specific frequency bands  and pre- and post-stimulus EEG activities, e.g., based on a multivariate linear regression model with the combination of partial least square (PLS) regression  on healthy subjects, it is likely to establish an oscillatory basis of human pain perception and identify how a network of cortical areas involves in human pain experience. The identification of electrophysiological parameters or signatures encoding how the cortex processes the nociceptive inputs and how the experience of pain may emerge from this complex processing, could indeed open a window to study the cortical process underlying pain function as well as the physiology mechanism of nociceptive systems in humans. In clinical practice, this understanding also would make it possible to predict/measure subjective pain intensity objectively, and definitely help (1) explore the pathological mechanisms of chronic pain and (2) achieve pain relief by modulation the oscillatory activities using neurofeedback techniques, with the investigation of cortical oscillatory activities on chronic pain patients.In the last decades, a large number of EEG/MEG studies  pain induced gamma oscillations over S1 covaried with objective stimulus intensity as well as subjective pain intensity  the close association between time-frequency oscillatory features  with subjective pain intensity , even though the details of their functional roles remain unclear. From a methodological perspective, apart from the temporal, spectral, and spatial profiles of the oscillatory activities, it is instructive to adopt novel parameters  to comprehensively evaluate the dynamics of cortical oscillations, thus allowing a full exploration of the neuronal oscillations involved in pain perception. Identifying pain related oscillatory activities and establishing an oscillatory basis of pain perception, could lead new insights into the physiological mechanisms of nociceptive systems in humans. In clinical practice, this also offers exciting prospects for the investigation of pathological mechanisms of chronic pain, thus promoting the development of rational therapeutic strategy.WWP wrote and revised the manuscript. DDT revised the manuscript.WWP is supported by the National Natural Science Foundation of China (31500921).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.


Physiological tools provide a mechanistic basis for understanding fundamental and applied ecology of tunas, billfishes, and pelagic sharks. In this synthesis, we review several templates for the interdisciplinary interactions between physiologists and fisheries scientists and highlight three areas of successful collaborations that directly benefit pelagic fisheries management. Populations of tunas, billfishes and pelagic sharks are fished at or over capacity in many regions of the world. They are captured by directed commercial and recreational fisheries (the latter of which often promote catch and release) or as incidental catch or bycatch in commercial fisheries. Population assessments of pelagic fishes typically incorporate catch-per-unit-effort time-series data from commercial and recreational fisheries; however, there have been notable changes in target species, areas fished and depth-specific gear deployments over the years that may have affected catchability. Some regional fisheries management organizations take into account the effects of time- and area-specific changes in the behaviours of fish and fishers, as well as fishing gear, to standardize catch-per-unit-effort indices and refine population estimates. However, estimates of changes in stock size over time may be very sensitive to underlying assumptions of the effects of oceanographic conditions and prey distribution on the horizontal and vertical movement patterns and distribution of pelagic fishes. Effective management and successful conservation of pelagic fishes requires a mechanistic understanding of their physiological and behavioural responses to environmental variability, potential for interaction with commercial and recreational fishing gear, and the capture process. The interdisciplinary field of conservation physiology can provide insights into pelagic fish demography and ecology  by uniting the complementary expertise and skills of fish physiologists and fisheries scientists. The iterative testing by one discipline of hypotheses generated by the other can span the fundamental–applied science continuum, leading to the development of robust insights supporting informed management. The resulting species-specific understanding of physiological abilities and tolerances can help to improve stock assessments, develop effective bycatch-reduction strategies, predict rates of post-release mortality, and forecast the population effects of environmental change. In this synthesis, we review several examples of these interdisciplinary collaborations that currently benefit pelagic fisheries management. Thunnus spp.; Fig. Xiphias gladius; Fig. Stocks of many large, highly migratory pelagic fishes  to deeper sets primarily targeting swordfish and bigeye tuna , billfishes (Istiophoridae), and pelagic sharks  are required, because the large body sizes and high oxygen demands of these fishes make them extremely challenging to safely control, possess and successfully maintain at sea. Many of these species are thus also exceedingly difficult to transport to, and maintain in, captivity. There are only a few research facilities in the world with the proximity to pelagic habitats and the logistical resources to maintain these fishes; even then, billfishes and many species of pelagic sharks have eluded successful captive husbandry.Where there are challenges, however, lie opportunities. New technologies inspire new directions. With the continuing evolution of electronic tags, laptop and hand-held computers and tablets, and portable blood analysers, interdisciplinary physiological studies are increasingly moving between the laboratory and ship, each erecting and assessing hypotheses generated in the other. Conservation physiology of pelagic fishes thus represents a rapidly expanding interdisciplinary growth area for the fields of physiology, conservation, and fisheries science, replete with profound socioeconomic and management implications.Collectively, modern assessment and spatial approaches that incorporate environmental variation into pelagic fish stock assessments improve population and distribution estimates. We posit, as have others previously e.g. , that suInterdisciplinary collaborations between the mechanistically driven physiological sciences, the pattern-oriented behavioural sciences, and the quantitatively driven applied fisheries sciences will greatly advance the synoptic understanding of the environment–pelagic fish–ecosystem interface. An earlier synthesis addressed interactions between mechanistic physiology and field-based and quantitative ecological sciences in the service of the interdisciplinary field of marine and freshwater fisheries science  and thus cannot prefer it (A mechanistic understanding of habitat selection by pelagic fishes therefore considers the following tenets: (i) individuals experience only their immediate surrounding environment ; and (ii) individuals can only truly prefer an environmental variable they can sense and where there is a direct relationship between receptor and/or afferent nerve activity and the physical variable (A corollary concern which involves the application of HBS standardizations of CPUE data for pelagic fish stock assessments is their underlying assumption that feeding motivation (and thus catchability) is proportional to time spent at depth . Such meBehaviours are often directed by the need to maintain physiological homeostasis in the face of environmental variation, coupling the physiology of a species to its ecology. Several physicochemical variables have received attention as mechanistic drivers of the aerobic scope and behaviour of pelagic fishes. In this section, we describe interdisciplinary collaborations between fish physiologists and pelagic fisheries scientists that have combined laboratory and field-based approaches to examine how pelagic fishes interact with driving variables. Powerful examples of this union include the explanation of the vertical movement patterns of tunas and other pelagic fishes via the effects of temperature on cardiovascular performance , largely demonstrate the epipelagic pattern, limiting the majority of their vertical movements to the upper 8°C of the water column between sea surface temperature and the thermocline (reviewed by Alopias superciliosus) demonstrate the mesopelagic pattern, inhabiting much deeper and cooler waters below the thermocline for most of the day and ascending at night , and opah (Lampris guttatus), making it arguably the most widespread form of regional endothermy in fishes  and in potential differences in gill structure that allow mesopelagic guild fishes to extract more oxygen from their oxygen-poor habitat  keeping the fishing gear away from the bycatch species ; and (ii) keeping bycatch species away from the fishing gear (i.e. making the gear less attractive). The latter bycatch-reduction technologies  represent an applied interdisciplinary forum for collaboration. Species-specific sensory insights can be used to enhance the performance or attractiveness of gear for target species (i.e. improving target catchability), a concept that warrants further development. However, although this approach may change target:non-target catch ratios, it is unlikely to change the magnitude of discards or bycatch. Thus, it is an ineffective conservation strategy for commercial pelagic longline, gill net, and purse seine fisheries that interact with protected billfishes and sea turtles. Overly conservative management measures that function by keeping gear away from the bycatch species (e.g. time–area closures) can be economically undesirable. A more economically desirable alternative involves improving selectivity by keeping the non-target species away from fishing gear.An integrated approach to making gear less attractive to non-target catch requires exploitable differences in the sensory biology and/or behaviours of target and non-target species that allow gear modifications to deter the latter but not the former, lest target catches decline . UnderstOnce baseline sensory data are obtained and exported to studies of field-based performance of deterrent and attractant stimuli, the efficacy of bycatch technologies can be iteratively tested with field gear modification trials that generate new hypotheses that can then be addressed in the laboratory , visual, and electroreceptive senses  caught in the pelagic longline and purse seine fisheries  and in recreationally caught tuna , metabolites (glucose and lactate), blood haematocrit, and heat shock proteins following longline capture; + concentrations to be a predictor of mortality. Taken together, the results of Novel applications combine field-based tagging and laboratory approaches in the study of fish post-release ecophysiology, and iteratively verify assays with field-based measures and telemetric technologies. In a study of 11 shark species, Many studies investigating the effects of angling on the post-release survival of pelagic fishes overlook the fundamental relationships between drag, metabolism, and endurance, assuming that physiological perturbations are proportional to the duration of exertion. Significant positive relationships between stress and the duration of the capture event have been demonstrated in a diverse group of fishes  across this continuum. Pelagic habitats and their fishes are remote frontiers for mechanistic research, because access to them is both logistically difficult and expensive. Many of these species cannot tolerate captivity. Despite these challenges, new technologies and interdisciplinary collaborations are inspiring new research directions, disciplinary Rubicons are being crossed, and insights are being obtained from the cellular to the population level. Perhaps the greatest collaborative successes in pelagic fish conservation physiology have resulted from studies of fish–environment relationships, bycatch reduction, and assessment of the factors that affect rates of post-release survival. The iterative testing by one discipline of hypotheses generated by the other is beginning to enclose the fundamental−applied science continuum, leading to the development of robust mechanistic insights that lead to informed management outcomes.The future of pelagic fish conservation physiology lies in interdisciplinary interactions of the perspectives and toolkits possessed by the disparate disciplines of physiology and fisheries science and their integration with the needs of fisheries managers and stakeholders Fig. . OvercomWe believe, as have others , that fiA.Z.H. acknowledges support from the NOAA Living Marine Resources Cooperative Science Center and NSF Educational Partnership in Climate Change and Sustainability. S.J.C. is supported by NSERC, Ocean Tracking Network Canada, and the Canada Research Chairs Program.


Precision medicine allows a dramatic expansion of biological data, while there is still an urgent need to understand and insight the exact meaning of those data to human health and disease. This has led to an increasing wealth of data unanalyzed. The concept of precision medicine is about the customization of healthcare, with decisions and practices tailored to an individual patient based on their intrinsic biology in addition to clinical “signs and symptoms”. Construction of a standardized model for the integration of data from various platforms is the central mission of the ‘New Disease Management Model’. The model is helpful for the development of new taxonomy of diseases and subtypes, to personalize therapy based on patient genetic profiles. A rapid progression of precision therapy has been made recently. Clinical trials have shown the therapeutic efficacy of discovered and developed therapeutic agents has improved. However, next-generation drugs would be designed for disease subtypes with more specificity, efficacy and lower toxicity. An enormous amount of biological data are generated and continues to expand dramatically, with the completion of Human Genome Project and the introduction of the next-generation sequencing. The efficiency of compiling, organizing and analyzing the data to extract a true understanding of fundamental biological processes of human health and disease has not kept pace, leading to an increasing wealth of unanalyzed data . In 2011Precision medicine aims to harness large amounts of data available from the human genome and the subsequent research wave of the molecular basis of disease. This allows the customization of healthcare and integration of electronic medical information on both an individual and larger global scale. With decisions and practices being tailored to the individual patient based their intrinsic biology in addition to traditional physical signs and symptoms . There iWith advances of information technology, such as electronic medical records, it is possible to acquire detailed clinical data about individual patients and computational tools for analyzing large sets of data provide a means to search for unexpected correlations with enormous data. Recent development of large-scale and multilevel omics-based biotechnologies, e.g. proteomics, metabolomics, genomics, phenomics, diverse cellular assays, provides precision medicine with powerful methodological bases and creates great potentials for improving healthcare . PrecisiTranslational medicine research is the main component of precision medicine’s integration of large sets of bioinformatics data with medical practice. Numerous translational research centers are emerging constantly and launch translational projects. In 2012, the International Personal Genome Project published an integrated map of genetic variation from 1092 human genomes, which revealed substantial genetic variation among populations. These variations will be helpful to understand genetic variations among different ethnic and racial backgrounds as well as how these variants affect drug metabolism , 7. In 2In addition, other large-scale projects, such as The Cancer Genome Atlas (TCGA), Encyclopedia of DNA Elements (ENCODE), or Human Proteome Project (HPP), were launched , 10. TCGA secretome as complete sets of secreted proteins is an important part of the proteome. Secretomes encode approximately 10 % of the human genome and play important roles in cellular immunity, adhesion, communication, and various drug delivery mechanisms . AnalyseThere still remains the need to build a linked library network as well as new taxonomy profiles of diseases for the better implementation of precision medicine, although enormous data have been generated and shown contribution to precision diagnosis, pharmaceutical development. The network should be integrated for the convenience of searching information including individual genome, transcriptome, proteome, metabolome, phenome, data from clinical signs and symptoms, experimental tests, environmental exposures, and socioeconomic factors. This provides a deeper understanding of disease mechanisms, pathogenesis, treatment, and drive forward the development of new taxonomy to define disease subtypes. However, data from different platforms are not standardized. Building a standardized model for the integration of data is a major challenge.Bioinformatics data provide great insights to the understanding of the biological processes of human diseases and health. Computational data mining methods are important tools for the prioritization of disease candidate gene. The gene network-based guilt-by-association is the basic principle from which genes with the same interaction partners or expression data, most likely share the same biological function . There aThe development of target-specific drugs has made great progression in improving therapeutic response of various diseases, on basis of a deeper biological understanding of diseases and an efficient validation of clinical therapeutic effects. For example, the epidermal growth factor receptor (EGFR) pathway has been targeted for the treatment of patients with somatic EGFR mutant non-small-cell lung carcinoma (NSCLC). EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib have confirmed significant improvements in the ‘progression-free survival’, in advanced EGFR mutated NSCLC , 20. OthHowever, a large number of challenges should be considered and overcome during the development and clinical application of targeted agents. Various toxicities and developments of new targeted molecular therapies were revealed , includiIn conclusion, precision medicine has shown great potentialities while catering to the requirements of bioinformatics and medical practice development. Building a standardized model for the integration of data from various platforms is the central mission which is helpful for the development of new taxonomy of disease subtypes and personalized therapy based on patient genetic profiles. Furthermore, there has been progress towards the discovery and development of targeted therapeutic agents with evidence in clinical trials of the improvement of therapeutic efficacy. Next-generation drugs would be designed for diseases subtypes with more specificity, efficacy and lower toxicity.


In response to bacterial and fungal infections in insects and mammals, distinct families of innate immune pattern recognition receptors (PRRs) initiate highly complex intracellular signaling cascades. Those cascades induce a variety of immune functions that restrain the spread of microbes in the host. Insect and mammalian innate immune receptors include molecules that recognize conserved microbial molecular patterns. Innate immune recognition leads to the recruitment of adaptor molecules forming multi-protein complexes that include kinases, transcription factors, and other regulatory molecules. Innate immune signaling cascades induce the expression of genes encoding antimicrobial peptides and other key factors that mount and regulate the immune response against microbial challenge. In this review, we summarize our current understanding of the bacterial and fungal PRRs for homologous innate signaling pathways of insects and mammals in an effort to provide a framework for future studies. Innate immune systems serve as the first line of defense to protect host organisms from a wide variety of infections through mechanisms that can be activated rapidly upon recognition of a foreign threat . With thThe Toll, Immune deficiency (Imd), and Janus Kinase and Signal Transducer and Activator of Transcription (JAK/STAT) pathways are the three major signaling pathways responsible for the distinctly robust innate immune response of insects . Each paMammals induce a rapid innate immune response that converts the recognition of a pathogen into the recruitment of leukocytes and inflammatory cells to the site of infection in order to eradicate the pathogen and simultaneously to trigger the production of antibodies . There aThis review will focus on the innate pattern recognition receptors (PRRs) responsible for detecting bacterial and fungal infections. In Toll, Imd, JAK/STAT, and the mammalian counterparts, the PRRs are imperative for inducing the stimulatory signal to the intracellular mechanisms. Between mammals and insects, the structure and function of many of the receptors are very different, even within homologous pathways. In certain pathways, such as Toll, the presence of extracellular circulating receptors for pathogens is critical to mount the necessary response, while mammalian counterparts do not possess homologous mechanisms . In the Drosophila embryo, but it was subsequently shown that Toll is also required for innate immune signaling .The Toll pathway in insects is mainly responsible for the recognition of fungi and Gram-positive bacteria and the induction of certain AMPs that are secreted into the insect hemolymph . Toll isignaling . Drosophl ligand . Upon Grl ligand . WithoutDrosophila cell lines, each carrying a mutation, led to the identification of the gene, semmelweis (seml), because the mutant line was deficient in the production of drosomycin, but could produce diptericin in response to Gram-negative bacteria  and is found in the hemolymph, and it functions to detect Gram-positive bacterial cell wall components prior to Toll activation. Fly mutants for PGRP-SA/seml are highly susceptible to Gram-positive bacteria, but their susceptibility to Gram-negative bacteria is unaffected and similar to wild-type controls, further confirming the importance of PGRP-SA as a recognition molecule that functions in the Toll pathway -type PG and induces hydrolysis  with peptide chain crosslinkers, which contain the Lys-type residues in the third position of the stem peptide  and PGRP-SD, that function in cooperation with PGRP-SA. GNBPs were first isolated from the silkworm, tebrates . In parttebrates . In addifaecalis . Howeversubtilis . The ratnfection . In addi peptide . In summAlthough the PGRP-SA/GNBP1 complex is a very effective detection system leading to the activation of Toll, certain Gram-positive bacteria activate the Toll pathway in a PGRP-SA/GNBP1-independent manner, indicating an alternative bacterial recognition mechanism. Loss-of-function mutants for another short extracellular PGRP, PGRP-SD, exhibit extreme susceptibility to Gram-positive bacteria that is exacerbated in PGRP-SA/GNBP1 mutant phenotypes . This suDrosophila, Toll receptors only bind cleaved Spaetzle, which is only produced as the result of pathogen recognition by the receptors described above. Mammalian TLRs interact with the pathogen directly in conjunction with other surface proteins and co-receptors .In the mammalian system, ten TLRs have been identified and studied for specific binding tendencies . While cDrosophila Toll receptor that associates with surface molecule, MD-1, which participates in protein-protein interactions -glucan recognition proteins , which is activated upon recognition of virulence factors produced by live fungi  that binds to Death related ced-3/Nedd2-like caspase (DREDD). Imd is cleaved by DREDD and is subsequently activated by K63-ubiquitination . The insect Imd pathway is homologous to the mammalian TNF signaling pathway based on the intracellular mechanisms, although the recognition receptors are different . Imd is highly homologous to the mammalian RIP1, which binds to the cytoplasmic domain of the TNFR , was identified through bipartite complementation assays showing that JAK homodimerization is abolished in the absence of Dome . These glycosylated molecules were originally implicated in mutants that displayed small eyes and abnormal wing development phenotypes, but were later shown to induce tyrosine phosphorylation of Hop, the Drosophila homolog of JAK  and complement-like thioester proteins, in insect hemocytes, that are dependent on Dome/upd3 signaling for expression ; ; . Howeverure 3B); . In concThe insect innate immune system is capable of mounting a robust response upon infection with bacteria and fungi through the release of AMPs, cytokines, and many other immune-response proteins. The insect immune signaling pathways described in this review rely on the presence of properly functioning extracellular and intracellular PGRPs, GNBPs, cytokine receptors, and their associated protein complexes that initiate signaling. In particular, the insect immune response against Gram-positive bacteria is orchestrated through the individual and associative functions of PGRP-SA, GNBP1, and PGRP-SD, which are able to recognize Lys-type bacteria, bind directly to the fragmented PG monomers and polymers and induce a serine protease cascade in the hemolymph that induces Spaetzle/Toll binding to trigger intracellular signal propagation. GNBP3 and Psh also induce and facilitate the serine protease targeting Spaetzle, Toll activation, and subsequent NF-κB-mediated AMP synthesis by binding specifically to fungal β-glucan structures. On the other hand, mammalian TLRs function directly as the PAMP recognition receptors that bind and transduce the information on pathogen detection to the intracellular signaling complex. More specifically, mammalian TLR4 and TLR2 are the direct homologs of insect Toll, which bind to LPS and zymosan components, respectively. TLR4 also shows similar abilities to PGRP-SA for forming complexes, in that the protein requires CD14 and MD2 to aid in LPS sensing mechanisms, however, the structures of PGRPs and TLRs are very different and lead to significantly different physical interactions with the PAMPs.There are also similarities between the insect Imd pathway and the mammalian TNF pathway. Insects rely on PGRP-LC and PGRP-LE to target specifically DAP-type PG structures that are signature PAMPs of Gram-negative bacteria and relay the information through Imd-specific adaptor proteins to the transcription factor Relish for inducing AMP synthesis. The mammalian TNF pathway is activated by the TNFR1 protein, which is very different structurally from the PGRP receptors of insects. TNFR1 recognizes cytokines, mainly TNF-α, which is released from leukocytes in order to recruit inflammatory cells to the site of injury and infection. However, the homology between the TNF pathway and the Imd pathway is based on the intracellular branching of the signaling pathway leading to the transcriptional activation of genes that are mediated by NF-κB and JNK homologs, which demonstrates clear conservation between the insect and mammalian systems. The receptors, TNFR1, and the PGRPs are the most structurally and functionally divergent of the three pathways described in this review. One of the major differences between the insect and mammalian pathways is that the mammalian pathways are activated by cytokines that stimulate an amplified inflammatory response and can lead to an adaptive response, whereas the insect pathways detect the presence of the pathogen, which activates the production of antibacterial or antifungal molecules that simply act to clear the Gram-negative bacteria as quickly and efficiently as possible. Finally, the JAK/STAT pathways of insects and mammals show the greatest homology of the three signaling pathways discussed herein. Insect and mammalian JAK/STAT pathways are structurally and functionally homologous to bind to interferon and IL molecules and stimulate the downstream phosphorylation of JAK, dimerization of STAT, and activation of pro-inflammatory molecules that can act to inhibit microbial infections, repair septic damage, or induce the production of antibodies.Overall, signal transduction is a powerful mechanism that is employed by both the insect and mammalian innate immune systems. While the homologous intracellular mechanisms are essential for these functions and have remained unchanged between these two groups of animals, the receptors that function in recognizing and targeting the infectious molecules are of equal importance and show both conserved and quite different attributes between mammals and insects. Through the evaluation and comparison of these receptors, a greater understanding is achieved for the common immunological goals of each organism, which encompasses both the conservation of innate proteins, and the divergences. The evolutionary variations in the mammalian pathways are all connected to the activation and functions of the vertebrate adaptive immune system. For mammals, eradicating the pathogen is essential, but in certain pathways, such as TNF and JAK/STAT, slight divergences from the insect systems show new obligations that serve to amplify cellular signals and stimulate the adaptive system that have become the staple of mammalian immunity. Although insects lack an adaptive immune system that is homologous to the V(D)J recombination mechanisms, PRRs are entirely responsible for the integration of relevant information for appropriate immunological responses to classes of pathogens with the outcome of efficient reactions of insects to microbes that has proven to be an exceptional strategy for the their survival and evolutionary success.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.


AbstractPageBreakhost plants in several tropical setups. Moreover, we propose that approaches similar to ours, establishing the inventories of two ecologically inter-related and species-rich groups of organisms, such as insect herbivores and their angiosperm host-plants, can serve as the foundational stone to anchor a comprehensive assessment of diversity, also in tropical environments, by subsequent addition of trophic levels.Biodiversity assessment has been the focus of intense debate and conceptual and methodological advances in recent years. The cultural, academic and aesthetic impulses to recognise and catalogue the diversity in our surroundings, in this case of living objects, is furthermore propelled by the urgency of understanding that we may be responsible for a dramatic reduction of biodiversity, comparable in magnitude to geological mass extinctions. One of the most important advances in this attempt to characterise biodiversity has been incorporating DNA-based characters and molecular taxonomy tools to achieve faster and more efficient species delimitation and identification, even in hyperdiverse tropical biomes. In this assay we advocate for a broad understanding of Biodiversity as the inventory of species in a given environment, but also the diversity of their interactions, with both aspects being attainable using molecular markers and phylogenetic approaches. We exemplify the suitability and utility of this framework for large-scale biodiversity assessment with the results of our ongoing projects trying to characterise the communities of leaf beetles and their  This inventorying urge promoted in turn the creation of Museums, Zoos and Botanical Gardens in developed countries, places to keep and share with the public the records of the catalogue . Following this tradition, the word biodiversity evokes a display of life forms, or a list or catalogue of species names, ideally ranked following some system. In this context, biodiversity is tightly linked to the practice and development of Taxonomy, after all the science in charge of recognising, describing and naming organisms. Biodiversity inventories thus benefit from every conceptual and methodological advance that has contributed to the maturation of Taxonomy, from the consolidation of evolutionary thinking to the debates on species concepts, from ultrastructural analyses under the microscope to the study of gene differences among individuals or metagenomic analyses of complex environments. However, as we said, the concept of biodiversity is an all-encompassing idea that should reflect any possible way in which life is organised, including supra-organismal assemblages, such as antagonist or mutualistic associations and behaviours, food-webs, communities and biomes, their combination in ecosystems, and so on. This is essentially the diversity of ways in which life forms can interact, an aspect of biodiversity traditionally approached from Ecology, with a boost in recent times thanks to the progress made in the field of community ecology. The diversity of interactions is perhaps a less intuitive idea attached to biodiversity than the composition of a community per se , but both are the complementary angles that shape the all-unifying concept of Biodiversity .These simplified approaches are defensible from an academic point of view, and they are also well adjusted to the serious underfunding for most biodiversity assessment initiatives. However, they are clearly inefficient to tackle the biological, cultural and moral problem dubbed as the Biodiversity Crisis . Again, The task ahead is titanic. The goal is to unravel the Earth’s biodiversity as fast as possible against the ever-growing extinction rates due to habitat disappearance, fragmentation and alteration, the combined effect of climate change, overexploitation and the impact of biological invasions . And nonIn recent years, and as a reaction to the biodiversity crisis there has been a proliferation of initiatives aiming at large-scale biodiversity assessment. This is just to say initiatives that aim at a comprehensive (with constraints) characterisation of biodiversity, with a large regional, ecological and/or taxonomic scope. Large-scale biodiversity assessment has been a traditional practice in ecology, particularly in tropical ecology, whereby scientists sample more or less indiscriminately certain environments, providing with thousands of specimens to museums and academic laboratories around the globe. In some cases, specimens are prepared and sorted, becoming amenable for identification and cataloguing when taxonomic expertise is available. However, most typically sorting reaches a relatively high taxonomic rank, too high for meaningful community analyses, and detailed biodiversity assessment stretches indefinitely in time, depending on the interest of experts and accessibility to these collections. Today, large-scale biodiversity assessment, particularly in the context of the race against the doom to extinction of many organisms, is intimately associated to what has been referred to as rapid biodiversity assessment, in other words, quickly collecting information on the species present in a given area  have built upon this tree-based concept for many years now. Thus, in this field, researchers exploit fast maximum likelihood phylogenetic analyses of query prokaryote 16S sequences against curated taxonomic references for this marker, e.g. workbench of Greengenes, SILVA and others . InspirernH data .PageBreakof their food-plants can be staggering in any given tropical environment  and at least one putative diet marker (either trnL or psbA-trnH); and (4) phylogenetic inference for beetle species delimitation and host-plant identification.Over the past few years (since 2007) we have thus developed on the notion that we can significantly contribute to an enhancement of biodiversity studies by targeting the fast characterisation of complex leaf beetle (or other herbivore insects) communities in the tropics as well as their ecological associations by using a combination of DNA-barcodes, tree-based species delimitation and forensic characterisation of food plants, with a robust and automatable analytical set-up. As a general proposition, we advocate that, when attempting large-scale biodiversity studies, where both delimitation and identification of species represent a challenge, the most efficient approach involves the use of DNA sequence data (only one or few ‘barcodes’) and phylogenetic approaches. Thus, our general workflow for large-scale biodiversity assessment of tropical leaf beetle communities includes four distinctive stages: (1) indiscriminate sampling of chrysomelid beetles in a particular environment or region; (2) non-destructive DNA extractions and specimen preparation for future reference; (3) DNA sequencing of at least one beetle mtDNA marker  in the case of bPTP, both desirable characteristics for rapid biodiversity assessment.We mentioned above that DNA-enhanced species delimitation has achieved fundamental progress over the past few years in great part thanks to the development of powerful phylogenetic methodologies to deal with gene tree incongruence as well as conceptual advancement on how to integrate taxonomically relevant data. However, these procedures are time and resource consuming, benefiting from the analysis of multiple genes and generally from a good taxonomic knowledge of the group of interest. These tree-based procedures find a good use in systematic research but are impractical for large-scale, rapid biodiversity assessment. Instead, our methods of choice, with a good trade-off between economy and speed of analysis (including data acquisition) and robustness and accuracy of results are the Generalized Mixed Yule-Coalescent model , and an additional mtDNA locus in the case of New Caledonian Eumolpinae . In every case, the individuals characterised from a genetic viewpoint were preserved and mounted dry, with their genitalia dissected. Vouchering specimens from such large-scale biodiversity studies is essential for fulfilling the inventorying angle of biodiversity assessment, particularly when the lack of readily available taxonomic expertise or the weak taxonomic knowledge of the focal group, hampers the immediate naming of species. The amount of new species for Science in understudied tropical faunas can be high, and subsequent in-depth taxonomic work to name species usually reveals undescribed diversity. As will be seen below, the non-destructive treatment of samples is crucial to allow for species descriptions and instantly provides with standard type material (besides the DNA sequences used to speed up their discovery). Preparation of our processed specimens has yet another short-term practical advantage, which is allowing for a fast complementary assessment of species diversity based on the concept of morphospecies, i.e. groups of individuals that look alike. A comparison between the two pragmatic strategies for rapid species PageBreakassessment, DNA-based GMYC-groups versus morphospecies, can assist in the evaluation of performance of the first, objective method  or the cassidines Coptocyclaleprosa (Boheman), Omoceruscaeruleopunctatus (Boheman) and Parorectisrugosa (Boheman) in Nicaragua  or the whole Chrysomelidae community in a National Park in southern Vietnam, both analysed using a similar accumulation-curve approaches as in New Caledonia, reveal that our samples may represent between 53–69% of the total leaf beetle diversity in the studied biomes. Thus, a continued sampling effort should recognise in the order of 500-600 Chrysomelidae species in the abovementioned subfamilies in the dry Pacific side of Nicaragua, or the same number of chrysomelids in a 10 sq. km. forest patch across a slight elevation gradient in southern Vietnam.Once there is a sound estimate of species numbers resulting from a sampling effort of known intensity, it is possible to investigate how representative the measure of biodiversity is of the total expected diversity. For example, we used a strategy based on rarefaction curves representing accumulation of objectively delimited species across sampling events for New Caledonian f choice . PrelimiThe experience gained from this type of studies shows that the main limiting factor for robust diversity assessment is obtaining sampling densities representative of the studied environment always, i.e. fieldwork. Once samples are available, laboratory methods can be optimised in weeks or few months, depending on the number of samples used and smoothness of PCR protocols, and a similar or slightly longer time for standardised analytical procedures.PageBreak In our approach, conversely to the mainstream DNA-barcoding stance of these contemporaneous and other subsequent studies, one of our main motivations was to extract taxonomically relevant information from processed food in the face of an incomplete reference database, by exploiting molecular phylogenies as the most rigorous and powerful tool for taxonomic assessment.We stressed already that there is one quantitative advantage of molecular characters to aid biodiversity assessment: speeding up the rate of species delimitation and also diagnosis. Additionally, these characters have at the same time the potential to contribute an extremely important qualitative advantage: the possibility to investigate complex systems and processed samples, which is the door to community ecology and the study of food-webs. In 2009, simultaneously with the studies of ecology inside a vial. In recent years, we have been particularly interested in the analysis of the host trophic ecology, but the same samples are amenable to studies of different trophic levels  template DNA from the host. In a DNA metabarcoding framework, this can be achieved by using primers specifically designed to target a specific taxonomic group of potential diets e.g., . One sucand matK . These aH spacer . SequencChrysomelinae , although resolution dropped at lower taxonomic levels . Robust phylogenetic analyses provided a sound identification shortcut relying on information available in public sequence databases, and despite lower accuracy at infrafamilial taxonomic levels, we could refine our inferences, sometimes down to the species level, thanks to detailed floristic catalogues for the areas where the beetles had been collected. Yet public database incompleteness is a severe problem and inference power greatly benefits from availability of a local reference database for meaningful comparisons . Automatically drawing taxonomic conclusions from trees was a challenge that we met exploiting the taxonomic hierarchy attached to Genbank data (inspired by PageBreakPageBreak(http://www.ibe.upf-csic.es/SOFT/Softwareanddata.html) makes it possible to boost large-scale biodiversity assessment both in its inventorying angle, but also in the study of interactions if applied to the identification of ecology-in-a-vial associations or metabarcoding studies.These approaches are becoming standard in many studies of tropical biodiversity, including studies on leaf beetles Table  and othen is low . Thus, wChrysomelinae, the Mexican Calligraphathermalis Gómez-Zurita associated to the composite Perymeniummendezii (Alticinae in the genus Arsipoda with one of them associated to Myrsinaceae (Taophila (Eumolpinae) together with an assessment of their dietary breadth , or the Mo’orea Biocode Project, on the homonym island in the Tahiti archipelago (http://mooreabiocode.org). While these initiatives exceptionally mobilise millions of dollars and hundreds of scientists for comprehensive biological prospection, and are built with the right spirit, they typically yield a very modest global output. The reason is the currently existing bottleneck of available taxonomic expertise for extracting meaningful biodiversity information from these surveys, which remains the most serious challenge for large-scale biodiversity research  and commitment of politicians and funding agencies alone can provide already a quantitative advantage for biodiversity research. As noted before, the emphasis for effective biodiversity research needs to be put again on funding expeditions and environmental sampling, pretty much with the same spirit as in the original voyages of discovery, but with the benefits of technology and trained specialists in different groups. Initiatives of this kind exist, most notably targeting insular systems, e.g. SANTO 2006, targeting the island of Espiritu Santo, the largest in the archipelago of Vanuatu (research . This tohttp://www.gobi.org), Center for Tropical Forest Science (http://www.ctfs.edu), or the Global Genome Biodiversity Network (http://data.ggbn.org/index.php), among others. These initiatives address some of their objectives by bringing genomics, taxonomy and ecology together through the combination of strategic sampling and massive sequencing technologies, when possible.Besides these fundamental limitations, there are still others of technical and conceptual nature which need to be dealt with, such as devising creative and efficient ways to incorporate new technologies for the improvement of large-scale biodiversity assessment. These should include for instance the use of next-generation sequencing technologies and environmental metagenomics, or more specifically in the case of insects the recently developed ‘metagenome skimming’ approach , which p


The rapid evolution of all sequencing technologies, described by the term Next Generation Sequencing (NGS), have revolutionized metagenomic analysis. They constitute a combination of high-throughput analytical protocols, coupled to delicate measuring techniques, in order to potentially discover, properly assemble and map allelic sequences to the correct genomes, achieving particularly high yields for only a fraction of the cost of traditional processes . From a bioinformatic perspective, this boils down to many GB of data being generated from each single sequencing experiment, rendering the management or even the storage, critical bottlenecks with respect to the overall analytical endeavor. The enormous complexity is even more aggravated by the versatility of the processing steps available, represented by the numerous bioinformatic tools that are essential, for each analytical task, in order to fully unveil the genetic content of a metagenomic dataset. These disparate tasks range from simple, nonetheless non-trivial, quality control of raw data to exceptionally complex protein annotation procedures, requesting a high level of expertise for their proper application or the neat implementation of the whole workflow. Furthermore, a bioinformatic analysis of such scale, requires grand computational resources, imposing as the sole realistic solution, the utilization of cloud computing infrastructures. In this review article we discuss different, integrative, bioinformatic solutions available, which address the aforementioned issues, by performing a critical assessment of the available automated pipelines for data management, quality control, and annotation of metagenomic data, embracing various, major sequencing technologies and applications. Talking Glossary of Genetic Terms). The definition embraces the cases where either the sampling is conducted, in an environmental habitat, or the material is collected from the tissue of a particular host organism, aiming to unravel the complexity of the microbial species, which are adapted to cooperate through symbiotic modes. The scrupulous study of a metagenome  or with Next Generation Sequencing (NGS) Metzker,  methodolDespite the fact that the experimental implementation of a NGS experiment comprises a painstaking and arduous procedure, its output, namely the volumes of short sequence reads, in digitized format, represents just the initial step, for the whole analytical process, setting a point where the plethora of available data are totally illegible and non-comprehensible. In order to dig out the information hidden in these datasets, one needs to define elaborate, multi-step, bioinformatic analytical workflows that can be performed either serially or in parallel with each other. As such processing tasks are so profoundly versatile and complicated in their logical structure and programmatic development, that even an experienced team of programmers can only develop a handful of them. In this respect, the intricate nature of the various processing steps that need to be assembled together, in order to form computational workflows appropriate for different analytical tasks, strongly supports the formation of federated computational infrastructures, representing repositories of software services, that can be transparently, , integrated in the available workflows, or can compile new ones. The vision for the creation of a suitable collaborative, environment, for a long list of genomic sequence analysis tasks, representing an analog of a virtual laboratory, relies on the extent of automation, easiness in integration, transparency, and functional versatility it provides. Beneath, follows a rough account of the main processing modules, incorporated in the workflows developed for metagenomic analysis.The genomic (DNA) material, isolated from a metagenomic sample, is transformed through the complicated experimental DNA sequencing protocols into short sequence reads of variable length, according to the protocols and instrumentation applied Mardis, . This bade novo assembly. Assembly via mapping to a known genome as reference can provide very reliable results for sequencing projects dealing with single-cell samples as it can bypass performance issues originating from sequence repeats, short length of reads, low coverage of sequencing, etc.  and, if possible, constructs that contain multiple contigs (scaffolds) with reliable connections between them. Each of these constructs originates from a different DNA sequence, that can be part of or a genome by itself and can be later investigated for the detection of open reading frames (ORFs), that is genomic areas, containing gene encoding sequences. The assembly task is so far, from the aspect of computational load, the bottleneck for any sequencing project whether the data corresponds to single cell genomes or metagenomic samples. The assembly of reads to contigs (and scaffolds) is a very laborious task, requesting avidly memory processing power resources, setting an important challenge, for which numerous algorithms . The algorithms  or even terabytes (TB) in size along with a yearlong experience in order to produce reliable results. This is why, there is an earnest need for metagenomic analysis platforms introducing automated, workflows for various processing goals, integrating tools in the form of services, operative inside processing pipelines. This has resulted into the development of various pipelines : (i) CloVR-metagenomics  are utilized in order to normalize taxonomic or functional counts for clustering and for visualization purposes. The main advantage of CloVR's setup is that it provides the user with the option of using local resources or to access a cloud provider for additional computational capacity. A potential downside of the platform is the lack of quality control, assembly and gene detection tools (which are available only in the single-genome and 16S-rRNA versions of the software) making it highly dependent on the read length of the sequencing datasets.CloVR-metagenomics  is a desktop application for automated sequence analysis, which requires two different inputs; a set of fasta-formatted files (raw sequencing data), and a tab-delimited metadata file which provides sample-associated information for comparative analysis. Local installation requires a Virtual Machine (VM) player in order to boot the appropriate VM image available by their website. For a cloud-based instance, users can use the Amazon Cloud where they find an available Amazon Machine Image (AMI) from the Request Instances Wizard. The pipeline initiates by clustering redundant sequence reads with UCLUST Edgar,  and usesGalaxy) is available but a user can download and install an instance on his/her server for exploitation of local resources, tools and databases in order to create custom workflows. Local installation requires only the downloading of the latest release and the initiation of the local instance can be done by running the appropriate BASH (BASH—The GNU Bourne-Again SHell) script (run.sh) included in the downloaded directory. A Galaxy workflow for metagenomic datasets was published , (ii) sequence assembly to contigs with eight different assembly methods exploiting four different assembly tools  included in the release, which automatically handles the whole process. The modules of this pipeline make up a complete analytical workflow that includes: (i) quality control using two different tools (SQLite) format for optimal data management based on relational database technology. The results from the previous steps can be utilized for comparative metagenomic analysis of the original dataset against other metagenomes or complete genomes derived from the SEED environment. What makes this platform attractive to the user is that similar to IMG/M, it provides a user friendly GUI behind a web server that makes the handling of the data and its analysis as intuitive as possible. It also provides numerous tools both for functional analysis and for comparative genomics and it can handle both assembled and not assembled sequence data. The only thing missing from the pipeline are the appropriate modules for raw read quality control and assembly tasks but either than that it constitutes an easy to use and well established functional and taxonomic annotation system that fully exploits the potential of public sequence databases.This pipeline supports both raw sequence reads datasets or already assembled contigs, as input. Local installation is not available as it is offered as an online service for which the user must register in order to upload metagenome datasets and to create jobs. The modules of the automated pipeline comprise four main tasks: (i) normalization of the data, (ii) parallel screening of the sequences against public databases  is a metagenomic platform, which workflows enable a complete metagenomic analysis, emphasizing in the programmatic optimization so that the computational cost of the various processing tasks, is minimized. Installation requires downloading the latest version of the package which includes all the essential programs, scripts, and databases. Each of the required programs of the pipeline must then be compiled and installed separately in order to be able to be called upon by the automated pipeline. This pipeline, works with raw read datasets from one or more metagenomic samples, whose sequences are clustered together using CD-HIT  sequencing quality control (ii) metagenomic assembly, (iii) ORF/gene detection, (iv) functional annotation, (v) taxonomic analysis, (vi) comparative analysis, and (vii) data management capabilities. From the pipelines we examined, only MetAmos and SmashCommunity included analytical tools for raw sequencing data whereas the rest mainly focused on detecting and annotating putative gene coding regions, as well as providing taxonomic characterization for the generated metagenome. Assessing the complexity of an installation is a fairly subjective matter, yet as “easy” we consider the installation, where the user doesn't have to perform arduous compilation and dependencies' installation tasks, since those usually require a higher level of informatics expertise. For example we consider complex for the inexperienced user, that of RAMMCAP, as it requires a manual installation of each of the integrated tools of the pipeline contrary of the installation of MetAmos, which is handled automatically through the execution of a Python script. The number of features that constitute each of the above-mentioned pipelines are summarized in Table The field of Metagenomics holds the promise for the elucidation of the genomic and taxonomic diversity of environmental niches. The rapid advances in sequencing technologies and in the development of algorithms for massive functional annotation of the analyzed genomic content intensify the capabilities of metagenomic analysis, rendering it feasible for an ever-growing number of projects. Powerful, fully automated bioinformatic pipelines lower the entry barrier to the field, through the compilation of numerous workflows, incorporating state-of-the-art algorithms optimized for specific analytical tasks, adjusted also for integration of various datasets, by resolving compatibility issues between them. There are pipelines focusing more on functional and taxonomic analysis, omitting the data-crunchy assembly part while others offer complete solutions where the user simply inputs the data from the sequencer machine and gets a fully annotated genomic report. As expected from other areas of computer science, a trade-off between user-friendliness and efficiency or flexibility of performance is observed here too. The highest the quality and the performance superiority of the workflows, the more profound knowledge they request for their impeccable installation and operation, thus minimizing their accessibility by different scientific communities, short of these skills. On the contrary, pipelines dedicated in resolving smaller, more specific processing tasks, have matured so as to provide very intuitive GUI-based solutions, often via a web server, accessible through the Internet. The broad range of integrative analysis platforms encompasses various pipelines, addressing the pressing need for disparate, versatile, complex, processing tasks. The adopted strategy for the development of efficient workflows, adjustable to varying, yet very specific every time, processing needs, posits on the modularity and transparency of the integrated code, that is the autonomous character of these modules, together with their easiness in integration and user-friendliness in their utilization. Moreover, in order to optimize the computational cost of such processing tasks, parallel processing designs are put forward, aiming to maximally exploit, multi-processor configurations. Among the examined suites of tools Table , we beliThe 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.


Harmful practices in the management of childhood diarrhea are associated with negative health outcomes, and conflict with WHO treatment guidelines. These practices include restriction of fluids, breast milk and/or food intake during diarrhea episodes, and incorrect use of modern medicines. We conducted a systematic review of English-language literature published since 1990 to assess the documented prevalence of these four harmful practices, and beliefs, motivations, and contextual factors associated with harmful practices in low- and middle-income countries.We electronically searched PubMed, Embase, Ovid Global Health, and the WHO Global Health Library. Publications reporting the prevalence or substantive findings on beliefs, motivations, or context related to at least one of the four harmful practices were included, regardless of study design or representativeness of the sample population.Of the 114 articles included in the review, 79 reported the prevalence of at least one harmful practice and 35 studies reported on beliefs, motivations, or context for harmful practices. Most studies relied on sub-national population samples and many were limited to small sample sizes. Study design, study population, and definition of harmful practices varied across studies. Reported prevalence of harmful practices varied greatly across study populations, and we were unable to identify clearly defined patterns across regions, countries, or time periods. Caregivers reported that diarrhea management practices were based on the advice of others , as well as their own observations or understanding of the efficacy of certain treatments for diarrhea. Others reported following traditionally held beliefs on the causes and cures for specific diarrheal diseases.Available evidence suggests that harmful practices in diarrhea treatment are common in some countries with a high burden of diarrhea-related mortality. These practices can reduce correct management of diarrheal disease in children and result in treatment failure, sustained nutritional deficits, and increased diarrhea mortality. The lack of consistency in sampling, measurement, and reporting identified in this literature review highlights the need to document harmful practices using standard methods of measurement and reporting for the continued reduction of diarrhea mortality.The online version of this article (doi:10.1186/s12889-015-2127-1) contains supplementary material, which is available to authorized users. The WHOFor decades, health initiatives have targeted the expansion of ORS and ORT, including the UNICEF Growth Monitoring, Oral Rehydration, Breastfeeding and Immunization (GOBI) initiative, the USAID/CDC Africa Child Survival Initiative - Combatting Childhood Communicable Diseases (ACSI-CCCD), and the WHO Integrated Management of Childhood Illness (IMCI) initiative. Despite these efforts, a shift in global attention away from diarrhea management seems likely to have contributed to slowing – and even reversals – in progress toward full coverage for ORT , 4.Many fewer programs have specifically targeted non-adherence to other recommended diarrhea management practices, such as the restriction of fluids, breast milk and/or food intake during diarrhea episodes, and incorrect use of modern medicines. All four of these practices are associated with negative outcomes and conflict with WHO treatment guidelines. Curtailment of fluids and restriction of feeding during diarrhea can increase the risk of dehydration, reduce nutritional intake, and potentially inhibit child growth and development. The use of antibiotics and other medications is appropriate only in the treatment of cholera or dysenteric diarrhea in children. Antidiarrheal drugs and some antiemetics not only have no benefit in diarrhea treatment, but may also cause serious, even life-threatening side effects in children . We haveDetermine the documented prevalence of these four harmful practices across low- and middle-income populations, as reported in various studies since 1990;Describe how these practices have been examined and reported on previously;Explore beliefs, motivations, and contextual factors associated with harmful practices as reported through both quantitative and qualitative studies; andHighlight associations between these harmful practices and other characteristics of the episode, child, caregiver, and household.This review summarizes existing literature on harmful practices in diarrhea case management in children under five years of age, including fluid and breastfeeding curtailment, food restriction, and inappropriate use of medications for diarrhea management in children in low- and middle-income countries. The primary objectives of the review are to:Findings from this review will identify critical next steps to address harmful practices in diarrhea management and ultimately improve child survival.We searched PubMed, Embase, Ovid Global Health, and the WHO Global Health Library in September 2013. Papers were identified that included variations on the combination of the following terms within the publication’s title or abstract or as a keyword: 1) diarrhea; 2) low- and middle-income country; and one or more terms related to 3) a harmful practice or general management of diarrhea. Search terms were developed in PubMed . For all studies, information on the study design, study population, and sample size was extracted. For studies reporting prevalence of practices, data were extracted on the definition of the practice measure, the reported prevalence of the practice, and variation in the practice by other factors . For non-prevalence studies, data were extracted related to beliefs, motivations, or context directly related to one or more of the harmful practices and then classified by common themes.We summarize the results for each of the four harmful practices in the results section of the manuscript. For each practice, we: (1) describe how the practice was defined and measured in these studies; (2) summarize reported findings on prevalence, including variations by characteristics of the diarrhea episode, child, caregiver, and household; and (3) report on beliefs, motivations, and contextual factors investigated and relevant results.The initial search yielded 2,266 articles in Pubmed, 2,512 articles in Embase, 1,512 articles in Ovid Global Health, and 1,890 articles in the WHO Global Health Library. After removing duplicates, 4,270 unique articles remained. Title and abstract review and full article review were conducted by the first author (EC). After reviewing titles and abstracts, 294 articles were identified for full article review. Based on a review of the full article, 157 articles did not meet the inclusion criteria and a full text copy of 23 manuscripts could not be located. In total, 114 publications met the inclusion criteria and were included in the review  in Egypt in 2002 . Use of Multiple studies addressed variance in inappropriate medication use by factors including characteristics of the diarrhea episode, child, caregiver, and household Table . A hospiCaregivers reported using antibiotics and other drugs to treat diarrhea because they were accessible and believed to be efficacious Table . MultiplThis is the first review, to our knowledge, that addresses harmful practices related to fluids, feeding and medication use during episodes of childhood diarrhea. The findings indicate that there have been many studies – both quantitative and qualitative – that have documented these harmful practices. However, reported prevalence varies greatly across study populations, and we were unable to identify clearly defined patterns across regions, countries, or time periods. A limited number of studies looked at the variation of these harmful practices across potential influencing factors, including characteristics of the diarrhea episode and child, caregiver, or household-level traits. Findings of association differed across studies.The motivation for harmful practices during diarrhea treatment also appears to vary across populations, although studies consistently report general caregiver concern for their child’s health and caregiver action to treat the illness to the best of their knowledge and abilities. Caregivers reported that their actions were based on the advice of health care providers, community members, or elderly relatives, as well as their own observations or understanding of the efficacy of certain treatments for diarrhea. Others reported following traditionally held beliefs on the causes and cures for specific diarrheal diseases.Across studies, the measurement of harmful practices was inconsistent and not guided by a conceptual or theoretical framework. Most studies were focused on general practices in diarrhea treatment, and harmful practices were rarely a primary outcome of interest. This has limited the availability and quality of data on the topic. Variations in study design, sample populations, diarrhea episode reference periods, and measurement definitions make drawing comparisons and conclusions across studies challenging. This is further compounded by inconsistent quality in data collection and reporting. Most studies relied on sub-national population samples and many were limited to small sample sizes. The variation in treatment practices by perceived type of diarrhea highlights the importance of using local terminology in order to capture all episodes of diarrhea as perceived by the community . AlthougAlthough this systematic review highlighted limitations of existing research, the available evidence suggests that harmful practices in diarrhea treatment are common in certain populations. A multicountry analysis using MICS data from 28 countries between 2005–2007 reported the majority of mothers did not maintain their child’s nutritional intake during illness . These pThe strengths of this literature review include applying a systematic process for searching and summarizing the literature, and accessing articles during a time frame in which global efforts focused on improving coverage. This review was limited by the inclusion of only peer-reviewed literature and the exclusion of non-English language publications. Additionally, the quality of individual articles was not assessed, allowing for the potential inclusion of studies with misrepresentative findings.Harmful practices in the management of childhood diarrhea are prevalent to varying degrees across cultures and include fluid and breastfeeding curtailment, food restriction, and inappropriate medication use. Inappropriate management of diarrhea episodes can result in higher risk of mortality through increased levels of dehydration or lasting health consequences as a result of nutritional restrictions or prolonged diarrhea illness. These practices must therefore be addressed as a matter of urgency in maternal, newborn and child health programs. These programs need to target not only the behaviors of child caregivers, but the broader social network, because our findings show that these practices are often informed by traditional beliefs, popular knowledge, and the instruction of authority figures, including elderly community members and health workers. Broader health systems interventions are also needed to address the alarming findings of high rates of inappropriate use of medications during diarrhea episodes. In addition, the global health community must do a better job or measuring the prevalence of these practices in standard ways, to produce evidence that can be used as the basis for action.