Online technologies may reduce barriers to evidence-based mental health care, yet they also create numerous ethical challenges. Recently, numerous professional organizations and expert groups have produced best-practice guidelines to assist mental health professionals in delivering online interventions in an ethically and clinically sound manner. However, there has been little critical examination of these international best-practice guidelines regarding appropriate electronic mental health  service delivery via technologies such as videoconferencing (including Skype), particularly for specific, vulnerable populations. Further, the extent to which concordance exists between these guidelines remains unclear. Synthesizing this literature to provide clear guidance to both mental health professionals and researchers is critical to ensure continued progress in the field of e-mental health.This study aims to review all currently available ethical and best-practice guidelines relating to videoconferencing-delivered mental health treatments in order to ascertain the recommendations for which international consensus could be found. Additionally, this review examines the extent to which each set of guidance addresses several key special populations, including children and young people, and populations living with illness.This systematic review examined guidelines using a two-armed search strategy, examining (1) professional organizations’ published guidance; and (2) MEDLINE, PsycINFO, and EMBASE for the past ten years. In order to determine consensus for best-practice, a recommendation was considered "firm" if 50% or more of the reviewed guidelines endorsed it and "tentative" if recommended by fewer guidelines than these. The professional guidelines were also scored by two raters using the Appraisal of Guidelines for Research and Evaluation II (AGREE-II) criteria.In the study, 19 guidelines were included, yielding 11 specific "firm" and a further 123 "tentative-level" recommendations regarding the appropriateness of e-mental health, competence, legal and regulatory issues, confidentiality, consent, professional boundaries, and crisis management. International consensus yielded firm guidance across almost all areas except professional boundaries and some aspects of determining the appropriateness of e-mental health. Few guidelines specifically addressed special populations. Overall guideline quality varied; however, 42% (8/19) of the guidelines scored at least 5 out of 7.This synthesis of guidelines provides a foundation for clinicians and researchers utilizing e-mental health worldwide. The lack of specific guidance relating to special populations is an area warranting further attention in order to strengthen mental health professionals’ and researchers’ capacity to ethically and effectively tailor e-mental health interventions to these groups. The increasing burden of mental health disorders has focused attention on the need for evidence-based psychological services . InternaEarly evidence across case studies, randomized controlled trials (RCTs), and meta-analyses point to e-mental health’s potentially equivalent therapeutic benefits to FTF formats -12. TherDespite this burgeoning literature, many recent critical examinations argue that there is a lack of high-level, gold-standard evidence to support the efficacy of e-mental health. Most studies have compared e-mental health interventions to waitlist controls, rather than the gold-standard attention, in vivo control ,20,21. AWhile a range of e-mental health modalities exist, each with unique clinical applications and ethical implications, videoconferencing has emerged as an especially promising modality with growing research and clinical use. Videoconferencing has already been employed for many years -12, in bAs e-mental health interventions become increasingly advanced, researchers have begun to consider their vast ethical implications ,9. WhileHowever, the rapid expansion of online interventions has somewhat outpaced regulatory bodies’ capacity to provide guidance around their provision ,26,27. AMental health professionals may require additional, tailored guidance regarding the provision of e-mental health services to populations regarded as high risk, including individuals with psychosis or suicidal tendencies . Other sThis review focuses on guidelines for delivering videoconferencing-based treatments , because this modality requires the active participation of a mental health professional in real-time, in the same way as a FTF session. Focusing on this modality allowed this review to generate more specific recommendations that were more easily applicable to practice. Discussions of the ethics of other forms of e-mental health are available elsewhere ,41.This systematic review aims to identify consensus among current best-practice guidelines and synthesize the recommendations regarding the ethical delivery of mental health treatments using videoconferencing technology. The review aims to answer the following two key research questions: (1) what areas of consensus in recommendations can be identified from international best-practice guidelines to guide clinicians in the ethical provision of videoconferencing-based mental health treatments? (2) What recommendations are made in best-practice guidelines regarding special populations including high risk, unwell, and young people?The review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews .In order to integrate research evidence with best-practice guidelines and capture the broadest set of current guidance, a two-armed approach was adopted involving systematic searches of (1) guidelines disseminated by international mental health-related professional bodies; and (2) the recent peer-reviewed literature for best-practice guidelines and recommendations published in scientific journals.We searched the websites of peak professional bodies in countries where videoconferencing-based mental health interventions are known to be occurring; as informed by a recent review . The webWe also undertook a targeted search of the peer-reviewed scientific literature for published recommendations generated by expert groups. Three electronic databases were searched , for human studies published in English between 2004 and 2014. The search and selection process, with detailed exclusion criteria is depicted in Best-practice guidelines were included if they (1) were published in English; (2) focused on ethical practice recommendations to deliver therapeutic mental health services using online technologies (including videoconferencing); and (3) stated that they were devised by more than one expert author and/or in consultation with a national professional organization and/or committee.The inter-rater reliability for the guideline search was acceptable 83%). The following data was extracted from guidelines: organization/lead author, year of publication and review, country of origin, guideline type, technological focus, target audience, and peer-reviewed status or involvement of consumer input . In orde3%. The fThe quality of guidelines published by professional organizations (identified through Search Two) was also assessed by two researchers (USD and HW) using the Appraisal of Guidelines for Research and Evaluation (AGREE-II) instrument . The AGRThe guideline selection and inclusion process are depicted in Scope and Purpose showed the highest overall quality, with a mean score of 76.6% across guidelines (range 33-100%), while the domain of Editorial Independence appeared the least well addressed across guidelines , which could reflect the methods of review used by professional organizations in publishing their guidelines. As previously mentioned, the AGREE-II assessment was not used as criteria for inclusion or exclusion. In addition to the AGREE-II criteria, guidelines were also examined in terms of whether their development had involved peer-review and consumer involvement. Only 32% (6/19) guidelines explicitly described processes of seeking peer-review from expert colleagues as part of their development [There was significant variation between guidelines in the breadth and depth of topics covered across guidelines . The AGRelopment ,51,53,55All of the guidelines reviewed indicated that mental health professionals overarching ethical obligations do not change when services are being delivered using online modalities. A summary of the ethical issues addressed across guidelines is shown in Although several sets of guidelines identified particular groups or individuals for whom e-mental health may be contraindicated, they differed in terms of how strongly they advised against providing e-mental health interventions to such individuals. The most strongly endorsed recommendation indicated that mental health professionals should incorporate an assessment process to determine the appropriateness of e-mental health services for an individual client  see -8. HowevNo firm recommendations emerged regarding the ethical implications of the psychological service type being offered -8. SeverRegarding general professional competence, three firm recommendations emerged. The two most strongly endorsed of these indicated that mental health professionals should provide online services within the boundaries of their competence, with an understanding of the limits and applications of different technologies , and that they should also assist clients to readily assess their competence by verifying their identity and making available their qualifications and professional endorsements  . Regardibilling, no firm recommendations emerged; the strongest tentative-level recommendation highlighted that mental health professionals should clarify up-front the nature and security of payment for sessions . Finally, in determining a client’s legal capacity to consent to e-mental health interventions, no firm recommendations emerged. However, the two strongest tentative-level recommendations highlighted that professionals should take steps to determine the age of potential clients to establish the appropriateness of e-mental health interventions, and should ensure that a parent/guardian’s consent is obtained for all minors before services proceed .Guidelines highlighted recommendations related to the psychologist’s accreditation, record keeping, billing, and establishing a client’s age to determine their  capacity to consent . Two firGuidelines examined several aspects of confidentiality, including privacy during the online session, client anonymity and establishing identity, and appropriate use and storage of electronic material . One firlimits to confidentiality, highlighting the importance of undertaking and documenting thorough consent processes consistent with relevant laws and regulations . A second recommendation further suggested that these consent processes should address numerous issues unique to e-mental health services including privacy and confidentiality in the online domain, security steps taken, technological equipment and skills requirements, limits to communication, and reliability of the connection . No firm recommendations emerged regarding clarifying contact times, though the two strongest tentative-level recommendations indicated that professionals should clarify contact information, as well as the nature of and expectations around therapeutic contact at the commencement of e-mental health interventions , and that professionals should additionally clarify expected timeframes for their client receiving a response from them, as well as processes around emergency contacts . No firm recommendations emerged regarding capacity to provide consent.Guidelines regarding consent addressed the limits to confidentiality across technologies, the need to clarify expected psychologist-client contact, and a client’s capacity to legally consent . Two firRecommendations regarding professional boundaries emerged in two key areas: preventing boundary crossings, and social media, though no firm recommendations emerged . The twoRecommendations surrounding the assessment and management of acute distress and mental health crises were addressed across most guidelines  . The onlFew guidelines discussed applications to particular special populations. Only six discussed providing e-mental health services to clients with high risk features such as psychosis. Populations noted as being higher risk across guidelines included clients with cognitive impairments  and psychotic disorders . Although several guidelines indicated that it may be preferable to exclude these individuals from e-mental health services -8, one sA minority of guidelines  discussed the appropriateness of e-mental health services for young people, with only two explicitly noting its potential acceptability and capacity to increase access to appropriate services ,52. HoweThis review examined and synthesized available guidelines to guide mental health professionals in ethically delivering e-mental health services, with a particular focus on videoconferencing. A total of 19 guidelines were reviewed from seven countries/regions. Across guidelines, consensus supported 11 firm ethical practice recommendations. To our knowledge, this is the first time ethical and best-practice guidelines regarding e-mental health have been synthesized on an international scale. The resulting firm recommendations indicate that despite numerous, distinct best-practice documents, there is emerging agreement across jurisdictions.Despite this, no firm guidance could be drawn in several key areas, including how professionals should assess the suitability of e-mental health services to their client -8, recorDiscordance between recommendations was rare; although in some instances guidelines differed in how strongly they discouraged certain practices. The delivery of e-mental health services to high risk populations was one such area. Although there was some (tentative-level) guidance regarding how identifiable electronic data relating to client material should be managed, there was less consensus around the appropriateness of mental health professionals recording material from their sessions  . More riRegulatory issues in providing e-mental health to clients in different jurisdictions pose ongoing challenges for the field. The firm recommendations that mental health professionals be appropriately registered across their own and their client’s jurisdiction may be difficult to enforce in practice . One setOf the few guidelines that directly addressed special populations some discordance was evident as to whether this constituted a contraindication. One group noted that currently there is no convincing evidence identifying any populations for whom e-mental health services would be especially harmful, and cited emerging evidence that in some circumstances e-mental health could be successfully implemented with psychotic patients . EvidencThe issue of crisis and risk management was comprehensively addressed across 15 guidelines , and offOnly one guideline highlighted the applicability of e-mental health strategies to illness populations, but no guidelines addressed how  the proposed recommendations should be tailored to these groups’ needs. Despite this, the convenience of e-mental health may be especially helpful in removing barriers to care for clients experiencing significant physical symptoms, such as fatigue or pain, which may hinder access to a FTF session ,34. E-meThough firm recommendations regarding illness populations could not be drawn from this review, emerging literature indicates the acceptability, feasibility, and potential efficacy of videoconferencing-based psychological support among individuals with illness ,65. DespOnline psychological therapy programs for children and young people involve the same core components as for adults , and areIn cases where the referral or presenting problem itself makes parental or guardian consent difficult or inappropriate , an initial risk assessment should involve consideration of the young person’s vulnerability, and access to any other forms of appropriate evidence-based supports in the absence of online services . ChoosinAn issue not addressed by the reviewed guidelines is that young people may have limited access to e-mental health platforms due to their limited autonomy, particularly around finances. For example, some young people may not have independent access to the Internet or to computers in private locations to enable confidential conversations.Although guidelines tentatively suggested that routine practice should involve clients being identifiable , severalIn adapting e-mental health guidelines to children and young people, maintaining appropriate professional boundaries appears crucial. Due to the developing nature of their cognitive, emotional and social competencies, and their lack of exposure to mental-health services, young people may not necessarily understand the concept of professional boundaries. The reviewed guidelines tentatively indicated ways in which professionals can maintain boundaries, including monitoring what information is available about them online, maintaining professional language across all media , and avoiding interactions on social media.Despite predictions that online methods will soon form an integral part of mainstream psychological practice , gaps reAnother critical issue in translating videoconferencing-based methods into practice is ensuring increased exposure to the medium. Even when mental health professionals gain familiarity with videoconferencing and when it is shown to result in equivalent outcomes, higher client satisfaction and lower costs, professionals still report preferring FTF methods . Lack ofThere are currently few training opportunities in e-mental health methods , yet staThis review represents the first time best-practice recommendations for the delivery of e-mental health interventions using videoconferencing have been evaluated for consensus across internationally-available literature. The review’s two-armed methodology enabled a synthesis of available guidance generated both by professional bodies as well as expert groups’ published recommendations. This was critical to ensure that consensus was drawn across the broadest set of currently-available guidance; this also increases the applicability of this review to future clinical practice and research endeavors in e-mental health.Nevertheless, some limitations must be acknowledged. Guidelines currently in development may have been missed, and guidelines not published in English were excluded. The need to draw consensus across guidelines precluded the inclusion of great detail here, which may be useful to the clinician wishing to implement ethical guidance in their practice . This has implications for the future training of professionals in this field. National professional organizations’ capacity to create practice-based guidance for clinicians, as well as their capacity to develop and integrate practical materials into postgraduate training courses and accreditation and/or registration examinations as relevant, may be critical. As the literature base in this area develops, so too will clarity around what constitutes ethical best-practice for mental health professionals.This exciting area of development offers numerous fruitful areas for future research. There is scope for international bodies to jointly participate in a Delphi consensus process  regardinOnline therapies have been heralded as a way to overcome perceived stigma around mental health ,37. Yet This review aimed to determine the consensus among international ethical guidelines regarding the provision of videoconferencing-based mental health services. Though different regulatory bodies across jurisdictions have unique local parameters within which professionals need to practice, consensus emerged across current international guidelines to inform how mental health professionals can effectively and ethically deliver their services using e-mental health platforms. Innovations in delivering mental health services online involve numerous trade-offs and the balance of different ethical risks, but if managed carefully may have significant benefits to a variety of vulnerable client groups.


Recent studies have reported that decreased T cell infiltrate alone, or co-ordinate with increased macrophage infiltrate, correlate with decreased survival in a range of cancers, including patients with pancreatic cancer. Importantly, in mouse models of pancreatic cancer, therapies that decrease tumor-associated macrophage infiltrates improve the response to chemotherapy, matching data in mammary cancer models. We have previously demonstrated that immunotherapy with agonistic antibodies to OX40 was able to remodel tumors, resulting in increased CD8 infiltrate and as a consequence, decreased macrophage infiltrate. Similarly, others have shown that blocking antibodies to CTLA-4 result in increased T cell infiltrate to tumors, both in mouse models and in patients. We hypothesize that immunotherapies that remodel the tumor immune environment will increase the response to chemotherapy.To test our hypothesis, we used the Panc02 mouse model of pancreatic adenocarcinoma that forms a highly chemo- and radio-resistant tumor in immunocompetent mice, with extensive stromal involvement and diminished drug penetrance compared to more immunogenic tumors. We demonstrate that systemic immunotherapy with anti-OX40 and anti-CTLA4 transiently changed the polarization of macrophages in tumors as determined by decreased arginase expression, and delivery of gemcitabine chemotherapy during the window of changed macrophage polarization resulted in significantly improved tumor control and survival. However, we also demonstrate that combination immunotherapy resulted in Th2 differentiation in tumor-bearing mice, associated with IL-4 production by activated CD4 T cells, potentially driving the strong rebound of arginase expression in tumor macrophages at later time points. Inhibiting IL-4 in vivo improved the efficacy of immunochemotherapy by significantly extending survival. These data demonstrate that preparative immunotherapy is a novel treatment option to improve the efficacy of chemotherapy where the immune environment is poor, and may increase response rates in cancers with negative immunology.


On the other hand different intravital, in vivo, and in situ microscopy techniques led to deeper insights into the role of NET formation during health and disease. This paper presents an overview of the main used microscopy techniques to visualize NETs and describes their advantages as well as disadvantages.Neutrophil extracellular traps (NETs) have been identified as a fundamental innate immune defense mechanism against different pathogens. NETs are characterized as released nuclear DNA associated with histones and granule proteins, which form an extracellular web-like structure that is able to entrap and occasionally kill certain microbes. Furthermore, NETs have been shown to contribute to several noninfectious disease conditions when released by activated neutrophils during inflammation. The identification of NETs has mainly been succeeded by various microscopy techniques, for example, immunofluorescence microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Since the last years the development and improvement of new immunofluorescence-based techniques enabled optimized visualization and quantification of NETs. On the one hand In this review an overview of the main methods and developments used to visualize NETs is summarized with their advantages and pitfalls . This oathogens , 13–15. pitfalls .The hallmark of the formation of NETs is the extracellular release of DNA associated with antimicrobial compounds. These antimicrobial components are myeloperoxidase (MPO), neutrophil elastase (NE), cathelicidins like LL-37, histones, proteinase 3, cathepsin, lactoferrin, or gelatinase , 17. WitHowever, besides these two main protective effects of NETs, entrapment of pathogens and degradation of cytokines or chemokines, increasing evidence is accumulating which describes detrimental consequences for the host when NETs are inefficiently eliminated, for example, in various autoimmune diseases, such as systemic lupus erythematosus (SLE) and systemic vasculitis (SVV), autoimmune lupus nephritis, or rheumatoid arthritis –22. Ther Streptococcus suis the nucleases SsnA and EndAsuis  and can be stored at 4°C for further analysis, for example, fluorescence microscopy (see below). Finally, it has to be mentioned that for all experiments a contamination of washing reagents or tissue culture media with nucleases, which, for example, may be present in bovine serum albumin or fetal calf serum, needs to be avoided, since this may cause artificial results as previously reported .A summary of the main NET visualization techniques used for quantification of NET degradation or induction is given in In addition to the visual assessment of NET formation by fluorescence microscopy, a spectrofluorometric method is often used to quantify NETs in a more high-throughput application. This method is also based on the fact that DNA is the major backbone of NETs. Some authors simply use SYTOX dyes to stain all dead cells and released DNA of a neutrophil population as an indicator for NETosis . In an o−/− [−/− mice [−/− and PAD4−/− mice using both methods of analysis. Furthermore, also human blood-derived neutrophils were used to detect ex vivo induced NETs. In summary, this method allows rapid and robust analysis of several thousand cells per sample and is independent of possible biased observer. Since this method is based on the specific staining with antibodies against H3cit, it has the only disadvantage to limit the detection on H3cit-positive events. Since H3cit-deficient mice are not completely deficient in NET formation in response to bacterial pathogens [Recently, two flow cytometry-based techniques to determine NETs have been characterized: Gavillet et al. described a flow cytometry-based model using antibodies against two key components of NETs, specifically citrullinated histones and myeloperoxidase in combination with DNA dyes . The met−/−  and PAD4−/− mice ) previouathogens , additioZhao et al. described a second flow cytometry technique combined with fluorescence microscopy to quantify NET formation, the so called high-speed multispectral imaging flow cytometry . The autHowever, both described flow cytometry-based methods allow automated, quantitative, and rapid analysis of neutrophil morphology and introduce the potential for using NETosis as biomarker in preclinical and clinical studies , 49.NETs have also been successfully visualized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Both techniques were used by Brinkmann and colleagues for the initial discovery of NETs and TEM by Fuchs et al. to describe the differences between necrosis, apoptosis, and NETosis , 34. Whi in vitro, a lot of scientists in the community still questioned the relevance of NETs during the last years because of the limited available in vivo data. Several research groups have successfully stained NETs in fixed tissue sections or blood samples. For this similar immunofluorescence microscopy techniques have been used to show the presence of NETs in vivo as shown for in vitro visualization of NETs based on DNA-intercalating dyes in combination with antibodies against NET components.Despite the successful visualization of NETs in vivo, in vitro, ex vivo, in situ, and intravital experiments. Furthermore an overview of selected publications focusing on in vivo and in situ NET detection is given in However, one important step for the interpretation of existing data is the definition of the experimental design. In  in situ staining was conducted in spontaneous human appendicitis where a costaining of neutrophil elastase, histone, and DNA demonstrated NET components inside extracellular fibrous material [The firstmaterial . In a stmaterial . sda1 deletion mutant of Streptococcus pyogenes M1 [ Streptococcus pneumoniae, NET detection during murine pneumococcal pneumoniae was successful [ Staphylococcus aureus-infected lungs revealed that the released NETs are decorated with antimicrobial peptides [As an example for infectious diseases, staining of NETs in mouse skin biopsies was conducted after an infection with angenes M1 . In an iccessful . Besidespeptides . in vivo, in situ, and ex vivo experiments detected less NET fibers compared to results from similar in vitro experiments, since the in vivo results may be influenced by a complex time-dependent regulatory process of NET induction and NET elimination within the host. However, all available publications contributed to a better understanding of NET function and the role of NETs in the pathogenesis of different diseases. Here some selected publications are summarized.MostThe method of choice to understand the impact of NETs and ongoing processes during NETosis is the analysis of NET release with live-cell imaging. Already in 2006, Buchanan and colleagues demonstrated a correlation between produced bacterial DNases, NET degradation, and pathogenicity by comparison of histopathological microscopy and live-cell imaging using DNA-intercalating dyes, . Later,  in situ NET detection is the live-cell imaging performed immediately after euthanasia of the animals without a fixation of selected organs. Using two-photon microscopy in Aspergillus fumigatus infected lungs, NETs were nicely detected in real time in situ [ ex vivo analysis of NETs. Using this approach, NETs were detected in synovia from patients with gouty [A special case for an in situ . Therefoth gouty . in vivo by intravital microscopy was a giant step in the investigation of NET formation. In 2007, Clark et al. demonstrated that neutrophils release NETs in response to platelet activation using intravital microscopy of the liver sinusoid [ in vivo experiment with spinning disk confocal intravital microscopy [ Staphylococcus aureus and monitored for NET formation over 2 hours. The NET detection was conducted with SYTOX dye. From this study it was concluded that NET-forming neutrophils are not immediately dead and the authors claimed the existence of the abovementioned two different mechanisms of NET formation, the suicidal versus vital NETosis [Finally, the real-time documentation of a viable NET-forming cellsinusoid . Furthersinusoid , 63, in sinusoid , and intsinusoid . As mentcroscopy : exterio NETosis . Escherichia coli were detected inside NETs using spinning disk confocal intravital microscopy (SD-IVM). In addition Tanaka and colleagues characterized NETs in blood vessels of different organs using intravital microscopy with a laser-scanning microscope [ in vivo or in situ experiments has been widely used as an excellent method to monitor the NET release in case of an infection or autoimmune disease.Lately, an intravascular detection of NETs in the bloodstream was successfully confirmed in septic mice  using a croscope . Thus, t in vivo evaluation of the role of NETs during several diseases. However, to fully understand the mechanisms mediating NET formation and the role of NETs during health and disease, improved NET visualization and quantification techniques are needed.Actual knowledge indicates that NET formation is a complex phenomenon mediated by a highly specialized population of neutrophils in response to infection and inflammation. During the last years, especially intravital microscopy techniques facilitated theSince the simple visualization and quantification of NETs using DNA-intercalating dyes has the risk of detection of necrotic cells or the generation of artificial results based on dye-blocking peptides associated with NETs, antibody-based techniques are definitely needed to visualize NETs. In general, microscopy is very specific but always has the limitation to be observer dependent and very time-consuming. Thus, there is a need for methodologies that enable robust and rapid assessment of NET-releasing cells, for example, by flow cytometry combined with imaging. Flow cytometry-based techniques include the advantage to enable further sorting of selected cell populations, for example, to differentiate between cells that undergo vital versus suicidal NETosis. In our opinion, future experiments should especially focus on single cell analysis to characterize the detailed cellular events that mediate formation of NETs in individual cells of a neutrophil population . This ma


Trait-based approaches provide a mechanistic framework to understand and predict the structure and functioning of microbial communities. Resource utilization traits and trade-offs are among key microbial traits that describe population dynamics and competition among microbes. Several important trade-offs have been identified for prokaryotic and eukaryotic microbial taxa that define contrasting ecological strategies and contribute to species coexistence and diversity. The shape, dimensionality, and hierarchy of trade-offs may determine coexistence patterns and need to be better characterized. Laboratory measured resource utilization traits can be used to explain temporal and spatial structure and dynamics of natural microbial communities and predict biogeochemical impacts. Global environmental change can alter microbial community composition through altering resource utilization by different microbes and, consequently, may modify biogeochemical impacts of microbes. Understanding the structure, dynamics and functioning of diverse microbial communities, and their effects on biogeochemical cycles has become one of the most fast-moving and exciting areas in biology. New high throughput methods of characterizing microbial community composition have resulted in an unprecedented wealth of data that can be analyzed to make inferences about the mechanisms operating in the microbial universe. One of the promising approaches gaining momentum in microbial ecology is the trait-based framework to understand and predict community composition and dynamics. As in community ecology of macroscopic organisms such as terrestrial plants, we can use trait-based approaches to seek general patterns of community organization and identify the mechanisms structuring microbial communities. In addition, key microbial traits can give us insights into the microbial impacts on biogeochemistry and how these impacts may change in the future.Microbes have extremely diverse metabolisms, occupy virtually all habitats on Earth and play key roles in major biogeochemical cycles. Despite all this diversity, there are some common underlying principles that govern the organization and functioning of microbial species and communities. Among those are the kinetics of resource utilization and competition for resources . There iresponse traits . Therefore, these traits can be classified as e traits . At the t traits . Not allt traits . It woulEco-physiological traits, including the resource acquisition and utilization traits, are often correlated with each other and these correlations may constitute trade-offs, if their increase or decrease has opposing effects on fitness. Organismal trade-offs prevent a single super-species from dominating, making them fundamental in determining community structure and diversity  and haveHere we discuss how resource utilization traits and the potential trade-offs among them can be used to explain patterns of microbial community structure, diversity, temporal dynamics, spatial distribution, and biogeochemical impacts at present and in the future. Obviously, there are many other microbial traits that are important for understanding the structure and dynamics of communities, such as traits determining responses to diverse environmental factors , quorum sensing traits, dormancy, etc., but we focus on resource utilization traits because they provide the most direct link between microbial community structure and biogeochemistry and are well described and modeled for many microbes.We provide examples for both prokaryotic and eukaryotic microbes, phytoplankton in particular, because many relevant phytoplankton traits have been measured, compiled, and used to infer the mechanisms of community organization. Phytoplankton are globally important microbes contributing about half of the Earth’s primary productivity and playing a major role in many biogeochemical cycles, such as carbon, nitrogen, phosphorus, and silica . RecentlR:The classical models that describe resource-dependent growth and utilization in microbes are based on Monod and Michaelis–Menten formulations. According to the Monod equation, the growth rate μ is a saturating function of external resource concentration max is the maximum growth rate and sK is a half-saturation constant for growth. The net growth then is μ - m, here m is mortality rate (including metabolic losses). The ability to effectively utilize resources in microbiological literature is often calculated as affinity . Resource uptake rate V is a function of external resource concentration R:where μmax is the maximum rate of uptake and Km is the half-saturation constant for uptake. Note that this constant is different from the half-saturation constant for growth in Eq. 1 . Often, the uptake rate also slows when the internal resource concentration is increasing, so that V may also depends on Q :where ∗R is possible from the monoculture data for a given mortality but more trait information is needed .As with the Monod equation, deriving Escherichia coli and some phytoplankton species  on investing into different physiological and ecological functions inevitably lead to trade-offs. Trade-offs between the acquisition of different resources are often postulated and shown to be among the major trade-offs contributing to species coexistence , 1990. TmaxQ, Eq. 4a), often related to cell size  resources has been hypothesized and described for phytoplankton and bacteria . There mcapacity ). This tBecause resource uptake of microorganisms strongly depends on cell size, incorporating cell size into models of resource utilization and competition is important, especially because cell size often influences other community interactions  and biogeochemical cycles  and can be considered a “master trait” . Cell siE. coli and a similar mechanism (high membrane permeability) proposed  may also trade-off with other traits, such as susceptibility to viral attacks, due to viruses entering cells through nutrient uptake sites . A tradeproposed .Heterotrophic microbes are thought to experience a fundamental thermodynamic trade-off between the rate and yield of ATP production in the breakdown of organic compounds . AlternaE. coli ; and they are subject to attack by taxonomically diverse enemies. Therefore, quantifying the cost of high performance for a particular trait may only be possible when multiple other traits are measured.In a comparative analysis of phytoplankton nutrient utilization traits, we found evidence for a three-way trade-off: competitive ability for nitrate and phosphate (as quantified by uptake affinity relative to the subsistence quota) tend to be negatively correlated for a given cell size, while competitive ability for both nutrients declines as cell size increases . FurtherThese results represent tantalizing glimpses into multivariate trait correlations and high-dimensional trade-offs. Quantifying these trait relationships will permit tighter theory-data linkages that should enhance prediction of community structure and ecosystem processes from environmental conditions. Developing this approach will require many trait measurements on many species, and an increased focus on higher trophic levels and pathogens to complement our understanding of resource utilization traits.Figure 1). For example, a rate-efficiency trade-off is observed in metabolic networks, where networks are either high yield but slower rate or vice versa . Moreover, resource utilization trade-offs can lead to an evolutionary diversification of social strategies, depending on environmental conditions  select different species based on a diverse set of trade-offs and can promote coexistence . We found that (1) better nitrogen competitors respond more positively to decreased nitrate; (2) species with higher light-limited growth rate respond more positively to decreased mixed-layer irradiance; (3) species with higher maximum growth rate response more positively to combined light and nitrogen availability  should increase in relative abundance as nitrate becomes scarce. This prediction can be tested with a statistical model that quantifies the slope of response to environmental predictors, for a group of species, and compares species-specific response slopes to those species’ traits. We applied this approach to the English Channel L4 phytoplankton time series  to measured nitrate uptake affinity in different species could determine whether such relationship exists, how significant it is and whether it could be used to predict uptake affinities. At present, however, establishing such quantitative relationships may still be out of reach because we either lack annotated genome information or do not have traits measured on a sufficient number of species. The presence of the different affinity uptake systems, their complexity and numerous genes encoding them further complicate potential inferences. Interestingly, the same phenotypic traits can be achieved by very different metabolic profiles, so that there may be higher redundancy at the subcellular level than at the level of a phenotype . Integra2 and nitrogen at much lower ratios to phosphorus than communities dominated by the prymnesiophyte Phaeocystis antarctica  invest more phosphorus into ribosomes and because ribosomes are relatively P-rich, that results in high P concentration and, consequently, low C:P and N:P ratios in biomass  and, consequently, having high N:C and N:P biomass ratios. Environmental conditions that select for fast growth  would then favor microorganisms with low N:P and C:P ratios and alter biomass stoichiometry  and, consequently, more efficient resource utilization and lower residual resource concentration in the environment (lower ∗R). Similarly, for a Monod-type growth description, the resource-dependent growth affinity (μmax/Ks) is low at low temperatures and increases when the temperatures approach optimum temperatures for growth . Note, however, that those species may still be inferior competitors compared to the overall best competitors. Evolutionary adaptation to temperature, well documented in microbes  activity or for growth, and would decline after that. Indeed, the residual nutrient concentrations were shown to increase past the optimum temperature for growth in bacterial cultures , suggestmicrobes , can furThe changing availability of resources  is another consequence of anthropogenic environmental change. Increased atmospheric nitrogen deposition, eutrophication, and “brownification” (increased leaching of organic carbon from soils into aquatic ecosystems) are among the many examples of resource alterations. Altered regimes of resource supply change microbial community composition by selecting species/groups with particular microbial resource utilization traits. For example, high or fluctuating inputs of resources may select for the fast growing or the high maximum uptake rate or the high storage capacity species . The perClassical mathematical descriptions of resource utilization and competition in microbes, as well as novel models including more realism provide a firm foundation for exploring the principles of microbial community organization at present and under future environmental conditions. Parameters of these models are key functional traits of microbes that determine their community structure, dynamics, and biogeochemical impacts. Characterizing these traits for many diverse microbial species through lab measurements using novel culturing techniques and “omic” approaches should provide rich data for model parameterizations. Trade-offs at different levels of biological organization and of different dimensionality are essential for predicting microbial community assembly and evolution and should be better characterized. Together, trait and trade-off information and realistically parameterized mathematical models, will be instrumental in increasing our mechanistic understanding of how natural ecosystems function.


Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins. At chemical synapses, neurotransmitter release takes place at presynaptic active zones (AZs). Morphologically, AZs can be identified via their electron-dense cytomatrix—an intricate network of specialized proteins precisely organized to execute and modulate exocytosis -dependent neurotransmitter release . The techniques can be subdivided based on their principle of bypassing the diffraction barrier: deterministic approaches, such as STED, use a phase mask to define the coordinates of fluorescence emission predefined in space by the zero-node, whereas PALM and dSTORM use stochastic activation of individual fluorophores and precise position determination .EM provides substantially increased resolution, though its application is restricted to lifeless, fixed and embedded biological samples. EM studies have been instrumental in recognizing the large morphological diversity of AZs  by stimulated emission of fluorophores at the rim of the PSF , p, their probability of exocytosis and q, usually taken to reflect postsynaptic sensitivity (Equation 1). This conceptual framework plays an important role in explaining synaptic function and plasticity ethane-N,N,N’,N’-tetraacetic acid) and EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid)] can differentiate between very tight  and larger distance  coupling regimes of synaptic vesicles and Ca2+ channels  as a major component of the AZ cytomatrix  relative to the Brp hub  or fluorescent protein expression and folding properties  are taken into account. By engaging dSTORM, this principle was recently utilized to study the nanoscopic arrangement of endogenous Brp proteins at AZs in tissue (Ehmann et al., Since localization microscopy is an explicit single-molecule imaging technique, it can be used to obtain quantitative information on both the spatial distribution and the copy number of labeled proteins 2+ channel-vesicle topographies.These current developments open up new perspectives for clarifying how functional properties are encoded in the protein architecture of AZs. Logical next steps could include searching for molecular determinants of vesicle release sites and quantitative ultrastructural studies of CaDespite a gradually emerging comprehensive protein catalog, we still lack basic information describing how the nanoscopic organization of proteins at the AZ gives rise to neurotransmission. Arguably, this is due to the diffraction-limited resolution of conventional light microscopy, which has hindered access to the spatial nanodomain in a physiologically relevant experimental setting.Several SRM techniques now exist that have the capacity to localize proteins on the nanometer scale and to resolve components of macromolecular assemblies in their native environment. In this context, we believe that localization microscopy is of particular value, as it can be used to provide direct access to molecular coordinates and to count endogenous protein epitopes (Specht et al., Harnessing the full potential of SRM will require expanding the repertoire of robust test samples and introducing optimized analytical tools (Bar-On et al., in vivo imaging in fully intact organisms. However, the feasibility of such applications must take into account light scattering and aberration in biological tissue, less amenable photophysical properties of fluorescent proteins compared with organic fluorophores and possible physiological alterations induced by recombinant protein expression (Sauer, Dynamic, live-cell SRM remains challenging. As a rule of thumb, spatial resolution always comes at the cost of temporal resolution. Therefore, imaging complex structures, such as the cytoskeleton of a whole cell, requires several minutes acquisition time at a lateral resolution of about 20 nm. This clearly limits the obtainable dynamic information. In contrast, modified SIM can easily resolve the movement of microtubules in entire living cells, albeit at lower spatial resolution (Chen et al., n Sauer, .Despite its capacity to resolve multiprotein structures, so far relatively few studies have engaged SRM to study synaptic AZs. We anticipate that this situation will change as SRM techniques become increasingly available and affordable (Holm 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.


Implementation research is expanding because Evidence-Based Programs (EBPs) are not adopted in many medical domains. However, rigorous implementation research is needed in nonclinical, community-based settings, which often have low capacity that undermines implementation quality and outcomes. This presentation describes Enhancing Quality Interventions Promoting Healthy Sexuality, a 5-year, cluster Hybrid Implementation RCT (Type II) testing how well a community-based setting  conducts an EBP called Making Proud Choices (MPC) that aims to prevent teen pregnancy and sexually transmitted infections, with and without an implementation support intervention called Getting To Outcomes (GTO). GTO is a 10-step model of program operation grounded in implementation theory and an intervention of written tools, training and bi-weekly onsite technical assistance.The trial compares 16 BGCs implementing the MPC program for two years, in the fashion typical of community settings, with 16 BGCs implementing MPC augmented with GTO. Capacity to carry out key program tasks prescribed by GTO's 10 steps is measured using ratings made from a standardized, structured interview with program personnel at all 32 BGC sites after each year of implementation. Fidelity of MPC is assessed at all sites by observer ratings of adherence. Youth sexual health outcomes  are assessed via surveys before, immediately following, and 6-months after MPC.After one year, GTO sites had significantly higher capacity ratings. Between groups, MPC fidelity ratings were similar and youth improved similarly on condom attitudes and behavior and sexual knowledge. Conclusions: This study is the first that assesses an implementation intervention's impact on capacity, implementation quality, and individual outcomes simultaneously and in both study conditions. GTO improved capacity, but fidelity and sex outcomes improved similarly. Methodological challenges' impact on these early results will be presented and potential solutions offered.The study is novel in that it tests the impact of an implementation support intervention in low capacity settings and assesses capacity, implementation quality, and individual outcomes simultaneously and in both intervention and control conditions. Also, this test of GTO's implementation intervention has revealed a number of methodological challenges that are not often discussed in the implementation literature, but need to be addressed.


Superparamagnetic iron oxide nanoparticles (SPION), as magnetic resonance (MR) imaging contrast agents or magnetic targeting carriers, have potential applications in diagnostics, imaging, cell and drug/gene delivery for cardiovascular diseases. SPION are highly magnetic particles that cause magnetic field perturbations, which can be identified on T2* weighted images via oxidative stress and biphasic inflammatory responses in Wistar rats In vitro studies have also suggested that iron oxide nanoparticles mediate activation of microglia in the brain via antioxidant poly (trolox) nanoparticles binding to and internalizing in endothelial cells Superparamagnetic iron oxide nanoparticles’ biocompatibility with the target organ is the first prerequisite for clinical translation, and iron oxide nanoparticles have long been believed to have low toxicity and are well-tolerated in the human body. However, with the expanding application of SPION, toxic effects, such as oxidative stress and inflammatory reaction, have increasingly attracted attention. Iron oxide nanoparticles accumulate in lysosomes , in which the low pH breaks the iron oxide core down into iron ions. It has been reported that iron oxide nanoparticle inhalation exposure may induce lung cytotoxicity in situ for a prolonged period of time due to the lack of blood flow and mechanical contraction in the ischaemic or necrotic region via venous drainage, and subsequently increase the heart stay by approximately 3–10-fold Iron oxide nanoparticles with systemic administration were mainly cleared by the reticuloendothelial system and renal excretion, resulting in cytopathological effects on the lungs, liver and kidneys, while the heart and brain remain free from adverse effects because of limited iron deposits via the Fenton reaction via activation of the oxidative stress system Another important question is whether SPION accumulation has toxicity effects on ischaemic myocardium. Although there is little information concerning the biological effects of SPION on myocardial tissues, the myocardium toxicity of excess non-SPION iron have been extensively explored. First, both primary haemochromatosis  and secondary hemochromatosis  can result in iron overload cardiomyopathy, with the pathogenic mechanism of that myocardial iron overload induces the formation of reactive oxygen species (ROS) via the sarcolemmal L-type Ca2+ channel via the Fenton-catalysed Haber-Weiss reaction, which damages the lipid-rich cell membrane, and is known as lipid peroxidation. Cellular lipid peroxidation produces polyunsaturated fatty acids and increases toxic aldehydes. The aldehyde products can form a covalent link to proteins , rendering the loss of cell membrane integrity. Structures located on the cell membrane, such as Na+-K+ ATPase and 5′-nucleotidase, were affected thereafter. Oxidative stress-mediated iron toxicity also affects other cellular organelles and their functions. Consequently, iron-induced myocardial injury occurred.The free radical-mediated pathway is the principal mechanism of iron toxicity in cardiomyocytes via oxidative stress-mediated iron toxicity, and undermining therapeutic effects. This hypothesis could be confirmed in an animal study. First, SPION-mediated therapeutic agents  are intramyocardially injected into peri-infarcted zones in an acute myocardial infarction rat model. Second, it should be investigated whether SPION deposition in the heart causes myocardiocyte loss and deteriorates the structure and function of the ventricle. For example, T2-star magnetic resonance (MR-T2*) was used to accurately evaluate cardiac iron status and detect early global ventricular dysfunction; lipid peroxidation products  in the myocardium reflect the oxidative stress mechanism; and histology was performed to examine myocyte apoptosis, inflammation and fibrosis. Third, the efficacy of novel SPION coated with antioxidants (such as N-Acetylcysteine or Trolox) was investigated in attenuating oxidative stress-mediated cardiac injury, further validating the SPION’s adverse effects and its mechanism.Based on the available studies, it is logical to assume that local myocardial delivery of SPION-mediated therapeutic agents might produce myocardial iron overload, resulting in deterioration of myocardial injury and exacerbating cardiac function The evaluation of SPION compatibility with myocardium, particularly with the ischaemic myocardium, is an urgent problem that needs to be resolved before the clinical translation of SPION in the cardiovascular field. If our hypothesis is true, then protective measures should be taken into consideration before developing clinical applications. Given that SPION toxicity mainly stems from oxidative stress, surface modification with an antioxidant (such as N-Acetylcysteine or Trolox) may be a new method used to suppress oxidative damage and injury.In conclusion, local delivery of SPION-mediated therapeutic agents might produce massive and persistent iron overload in ischaemic myocardium, consequently deteriorating myocardial injury. Thus, antioxidant coating may be a new strategy used to suppress the harmful properties of SPION.


Recent advances in brain imaging have allowed researchers to further study the networks connecting brain regions. Specifically, research examining the functioning of these networks in groups with a genetic predisposition for alcoholism has found atypical circuitry in the brains of such individuals. Further research with larger sample sizes and multimodal method integration are necessary to confirm these intriguing findings. Advances in human neuroimaging have expanded our ability to understand the functioning of the brain, with particular recent advances fostering our analytic capacity to examine networks between the brain’s nerve cells  and neuroconnectivity . Relevant to the field of alcoholism, several researchers recently have applied these strategies to groups at genetic risk for alcoholism, in hopes of identifying neurobiological, and specifically neuroconnectivity, phenotypes underlying this risk. This article provides an overview of the methods used to study connectivity and highlights research detailing the application of these methods to studying populations at risk for alcoholism.With the aim of understanding network functioning in the brain, one analytic strategy has been to examine the correlations between activation in regionally disparate brain regions during functional magnetic resonance imaging (fMRI).Psychophysiological interactions (PPI) analysis is another functional connectivity method used to analyze the coupling of neuronal activity between distinct brain regions while an individual is engaged in a task. This is different than other functional connectivity methods  in that it allows one to assess the impact of task condition (or context) on the functional connectivity of two distinct brain regions. The functional connectivity of the brain also has been measured using resting-state functional connectivity (RSFC) during fMRI. With this technique, functional connectivity is measured by correlating blood-oxygen-level–dependent (BOLD) signal, an indirect measure of neuronal activity, across the brain in an individual who is resting. Regions of the brain are thought to be functionally connected if they share a temporally correlated neurophysiologic response. Spontaneous brain fluctuations persist across a variety of states, such as sleep or anesthesia, as well as in animal species , suggestSCA requires a priori selection of a voxel, cluster, or anatomical region, usually based on previous fMRI literature delineating relevant regions of activation or through anatomical delineation. From this a priori selection, time series data are extracted and used as regressors in a linear correlation  analysis from which whole-brain, voxel-wise functional connectivity maps are derived that co-vary with the seed region , which provides an indirect measure of white matterOne major risk factor for developing an alcohol use disorder (AUD) is having a family history of alcoholism . NeuroimTask-based functional connectivity has been used in two studies of youth with family history of alcoholism to examine connectivity during working-memory tasks. In a substance-naïve sample of 12- to 14-year-olds, Some hypotheses regarding familial risk for alcoholism propose that youth with a family history of AUD may be at greater risk for alcohol abuse as a result of a developmental delay . This hyAlcoholics exhibit abnormalities in reward-related structures and atypical reward processing , suggestFronto-cerebellar abnormalities, including atypical connectivity between the frontal lobes and cerebellum, consistently have been reported in alcoholics . BecauseAlthough more research on familial risk for alcoholism and brain connectivity has focused on functional connections present across task-related BOLD response, recent investigations have examined the intrinsic functional connectivity of brain regions in FHP youth see , specifiRecently, another study used RSFC to examine intrinsic connectivity of the amygdala in FHP adolescents and found relationships between behavior in an emotion-cognition task and functional connectivity between the left amygdala and left superior frontal gyrus (SFG) b. WeakerA number of DTI studies have identified white-matter pathways that are altered in high-risk youth and adults a, suggesAnother DTI study found risk by alcohol exposure effects related to reduced FA in some of the same white-matter pathways previously reported to be altered in FHP youth, including superior and inferior longitudinal fasciculi a. BecausAdditional support for lower FA in a variety of white-matter tracts in frontal and parietal regions was recently reported in a large sample of 80 FHP youth, who also had lower FA, compared with their peers in anterior, superior, and posterior corona radiata , with thAs shown, several studies have used neuroconnectivity methods to identify atypical circuitry in the brains of those at familial risk for alcoholism, albeit generally with small sample sizes, which is a limitation of the available research. Overall, these studies have demonstrated abnormalities in connectivity between frontal regions with parietal, ventral striatal, cerebellar, and limbic regions of the brain in these populations, suggesting that these methods may be particularly useful in uncovering neurobiological risk phenotypes. Larger sample sizes and multi-modal method integration are critical to confirm these intriguing findings. Although studies of family history risk for alcoholism have reported atypical functional connectivity using seed-based resting-state and task-based connectivity approaches, none have used graph theory to examine network characteristics of alcoholism-related risk, an analytic strategy which may prove particularly useful for increasing our understanding of the interactions between these networks. Given recent work documenting the amenability of brain functioning to change in response to treatment , identif


Cardiovascular diseases (CVDs) are still the principal cause of death worldwide. Weakened endothelial function followed by inflammation of the vessel wall hints at atherosclerotic lesion formation that causes myocardial infarction and stroke. Heart failure can arise as consequence of large myocardial infarctions. In its more severe stages, heart failure patients have a life anticipation that is parallel to destructive cancers. Accordingly, the increase in risk factor load by metabolic diseases and age augments the incidence for vascular and cardiac diseases and provides a challenge for developing efficient treatments. There is widespread proof to show that drug treatment of conventional risk factors is effective in reducing cardiovascular events. More effective treatment of CVD with various classes of antihypertensive drugs has been associated with greater benefits, but some recent studies suggest we may be reaching the optimal level of treated blood pressure in some patient groups. Apart from the treatment of cardiovascular risk factors with pharmacological agents and the use of antithrombotic drugs, there is growing awareness of the role of dietary factors and herbal medicines in the prevention of CVD and the possibility of their use in treatment. Investigators from different places of the world like China, Taiwan, Bangladesh, Pakistan, and so forth contributed to this special issue by presenting tremendous papers. These papers deliver an analysis in this field and create innovative contributions concerning the mechanism of action of bioactives and traditional herbal medicine for the treatment of cardiovascular/cerebrovascular diseases. Terminalia arjuna, Cactus grandiflorous, Crataegus oxyacantha, and Piper nigrum through curative and preventive mode of treatment analysis and this paper reported preadministration and postadministration of herbal mixture restore the levels of biomarker of cardiotoxicity, which includes cardiac marker enzymes, lipids profile, and antioxidant enzymes. Similarly, another paper in this issue reports the cardioprotective effects of Sundarban honey on cardiac troponin I, cardiac marker enzymes, the lipid profile, lipid peroxidation products, and histoarchitecture of the myocardium against isoproterenol-induced myocardial infarction in Wistar rats. Pinggan Qianyang recipe (PQR), a Chinese medicine recipe, has long been used for calming the liver. It has also been used to treat essential hypertension with satisfactory results. Consistent with this concern, this special issue published a paper that reports PQR exerts its antihypertensive effect through deterioration of the vascular remodeling process. The mechanism might be associated with regulating differentially expressed miRNAs in aorta tissue.Some interesting papers in this special issue address the cardioprotective effects of Chinese herbal medicine and natural compounds. For instance, a paper summarized the synergetic cardioprotective potential of herbal combination of four plants, namely, Antrodia camphorata (A. camphorata), a fungus generally used in Chinese folk medicine for the treatment of viral hepatitis and cancer, has shown neuroprotective effects in embolic rats. This effect may correlate with the downregulation of the iNOS, HO-1, Bax, and activated caspase-3 and the inhibition of OH° signals. Another study shows alpha-lipoic acid attenuates middle cerebral artery occlusion-induced cerebral ischemia and reperfusion injury via insulin receptor-dependent and PI3K/Akt-dependent inhibition of NADPH oxidase. Moreover, an interesting study in this special issue established the effects of tetramethylpyrazine (TMP) on functional recovery and neuronal dendritic plasticity after experimental stroke. In this study, the authors have shown that enhanced dendritic plasticity contributes to TMP-elicited functional recovery after ischemic stroke.Despite the fact that there are major developments in treating ischemic stroke over the last decade, stroke is still a serious concern for which effective drug therapy is not yet available. In the search for neuroprotective agents from natural sources, a number of plant extracts and several natural products were isolated and reported to provide neuroprotection against ischemic stroke. A few papers in this special issue report the neuroprotective effects of Chinese herbal medicine and natural compounds. For instance, Chamaecyparis taiwanensis. It is involved in multiple biological activities, including antimicrobial and antitumorigenic activities. Although hinokitiol has been reported to inhibit inflammation, its immunological regulation in lymphocytes remains inadequate. With this context, a well-designed study reported that hinokitiol downregulated cyclin D3, E2F1, and Cdk4 expression and upregulated p21 expression in concanavalin A- (ConA-) stimulated T lymphocytes. It further demonstrated that hinokitiol upregulates p21 expression and attenuates IFN-γ secretion in T lymphocytes from the spleens of mice, thereby arresting the cell cycle in the G0/G1 phase. These authors concluded that hinokitiol provides benefits in treating patients with autoimmune diseases. We expect that this special issue grants inventive awareness to increase the therapeutic value of herbal and/or Chinese medicines for treatment or prevention of cardiovascular and ischemia-reperfusion injury-related disorders.Hinokitiol is a naturally occurring compound isolated from the wood ofJoen-Rong SheuJoen-Rong SheuPitchairaj GeraldinePitchairaj GeraldineMao-Hsiung YenMao-Hsiung Yen


Extracorporeal membrane oxygenation (ECMO) is a modified cardiopulmonary bypass (CPB) circuit capable of providing prolonged cardiorespiratory support. Recent advancement in ECMO technology has resulted in increased utilisation and clinical application. It can be used as a bridge-to-recovery, bridge-to-bridge, bridge-to-transplant, or bridge-to-decision. ECMO can restitute physiology in critically ill patients, which may minimise the risk of progressive multiorgan dysfunction. Alternatively, iatrogenic complications of ECMO clearly contribute to worse outcomes. These factors affect the risk : benefit ratio of ECMO which ultimately influence commencement/timing of ECMO. The complex interplay of pre-ECMO, ECMO, and post-ECMO pathophysiological processes are responsible for the substantial increased incidence of ECMO-associated acute kidney injury (EAKI). The development of EAKI significantly contributes to morbidity and mortality; however, there is a lack of evidence defining a potential benefit or causative link between ECMO and AKI. This area warrants investigation as further research will delineate the mechanisms involved and subsequent strategies to minimise the risk of EAKI. This review summarizes the current literature of ECMO and AKI, considers the possible benefits and risks of ECMO on renal function, outlines the related pathophysiology, highlights relevant investigative tools, and ultimately suggests an approach for future research into this under investigated area of critical care.  Extracorporeal membrane oxygenation (ECMO) is being increasingly used in patients with cardiorespiratory failure whilst even more novel applications continue to evolve. ECMO may reduce mortality in patients with cardiopulmonary failure of multiple aetiologies, although conclusive evidence is still lacking –5. ECMO An increase in the scope of indications has led to the implementation of ECMO in numerous critical care settings. These include the emergency department, specialized interhospital retrieval services, cardiac catheterisation lab, and during perioperative cardiac surgical management . DespiteEqually, acute kidney injury (AKI) is also common in patients on ECMO, with incidence as high as 70%–85% . AKI in ECMO can be implemented as a venoarterial (VA) or venovenous (VV) configuration, determined by the clinical context  8, 13]., 13.8, 1AKI is characterized by an abrupt loss in renal excretory function leading to the retention of nitrogenous waste products and dysregulation of extracellular volume and electrolytes . The incRecently, consensus on the definition of AKI by the Acute Dialysis Quality Initiative has been achieved . The oriDespite subtle differences between RIFLE and AKIN , it is nNeither RIFLE nor AKIN is perfect and a number of recognized limitations exist which may affect their efficacy in ECMO. Both utilise surrogate and delayed markers of renal impairment  to diagnose and stage AKI. Serum creatinine (sCr) is an insensitive and unreliable biomarker for early detection of kidney injury and recovery . ChangesIt is clear that the pathophysiological features of EAKI are complex, multifactorial, and time dependant and likely to be synergistic. There is limited literature assessing the pathophysiological mechanisms of AKI and ECMO; however there is research that describes CPB-associated AKI. CPB produces a predominantly pulseless system, VA ECMO typically has limited pulsatility, and VV ECMO is always pulsatile. However, due to other similarities between CPB and ECMO, it is likely that there are at least some common underlying factors with VV and VA ECMO. Progression of preexisting multisystemic disease, pre-ECMO management, activation of proinflammatory mediators, alterations in renal macro/microvasculature, ischaemia-reperfusion, haemolysis and oxidative stress, disruption of the glycocalyx, impaired renal autoregulation, iatrogenic nephrotoxicity , and iatrogenic coagulation abnormalities are possible factors involved in the pathogenesis of EAKI . These pα) are believed to be the main contributors to this process [Activation of proinflammatory mediators caused by the underlying disease process, combined with the interaction of blood on the nonendothelialised ECMO interface, may initiate and amplify a systemic inflammatory response. Proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-8, and tumour necrosis factor-alpha  fluid and diuretic therapy, high PEEP strategies (pre-ECMO), regular adjustments in inotrope/vasopressor administration, and variations in ECMO circuit pressures (post-ECMO initiation) commonly occur. In the pre-ECMO setting, these interventions are often prescribed at the expense of renal function as the clinician attempts to delay or prevent the initiation of ECMO. The subsequent delay in the commencement of ECMO with potentially harmful therapy may compound the rapid fluctuations in microvascular blood flow leading to reperfusion EAKI , 17, 69.Reperfusion injury is likely to be a key pathogenic event in the microcirculatory dysfunction leading to EAKI. It occurs due to inflammatory and oxidative stress-mediated injury, with leukocyte activation and production of reactive oxygen/nitrogen species (RNOS), following restoration of circulation to previously hypoxic cells and subcellular structures –72. WithAs mentioned, excess IV fluid therapy before and during ECMO is likely to aggravate this pathophysiological process. Fluid boluses cause recruitment of these hypoxic-shunted renal microcirculatory units and resuscitation of the mitochondria. This “washes/flushes” the hypoxic and acidotic capillary detritus into the circulation, further disrupting oxidative phosphorylation . This alHaemolysis and subhaemolytic damage (leading to erythrocyte deformability), induced by the shear stress of the ECMO circuit, is also likely to contribute to EAKI through oxidative/nitrosative stress –88. AmonUltimately, the homeostatic imbalance caused by ECMO-induced haemolysis causes oxidative/nitrosative mediated kidney injury by altering the microvasculature and destruction of the glycocalyx , 87, 106The nonendothelialised ECMO interface and destruction of the glycocalyx may, in isolation or in combination, lead to a hypercoagulable state, which results in the formation of microemboli and microthrombi within the renal vasculature, particularly with VA ECMO , 122. Im cor pulmonale, shock, and organ dysfunction. The primary disease process in conjunction with ongoing lung injury and altered haemodynamics induced by ventilation may further exacerbate organ injury and increase the risk of EAKI [Patients supported on VV ECMO with respiratory failure and receiving lung protective ventilation have often had prolonged hypercapnia. Despite correction by ECMO, this induces significantly altered haemodynamics and RBF . This ca of EAKI . To this of EAKI , 127. HoAs mentioned, the centrifugal ECMO pumps are preload dependant and deficient in the Frank-Starling mechanism; thus maintenance and bolus IV fluid administration are necessary to maintain adequate ECMO flow and minimise “suckdown,” particularly in the immediate days following ECMO initiation . HoweverDespite the pulsatility differences between VV and VA ECMO, provided that ECMO circuit flow rates are in the range of premorbid cardiac output, there is a lack of evidence to suggest that a nonpulseless system increases the risk of EAKI. Initiation of peripheral VA ECMO can lead to profound reperfusion and hyperaemia, which may exacerbate ischaemia-reperfusion injury. On the contrary, among other mechanisms, overall renal perfusion may improve as a result of the reduced venous pressure induced by the negative drainage pressure in the IVC.An increased duration of support with ECC is an independent risk factor causing an increased incidence of AKI and subsequently higher morbidity and mortality . This hiA number of tools can detect and define the consequences that the various pathophysiological processes associated with EAKI have on renal function. Methods for determining AKI can be categorised into broad groups: physiological, biochemical, radiological, histological/immunohistochemical, and biomarker evaluation .2-isoprostanes and isofurans, which correlate with oxidative stress-mediated kidney damage [In current clinical practice, the use of broad physiological end-points  and biochemical  are used. These indices are synthesised using RIFLE, AKIN, and calculations to determine the fractional excretion of sodium (FeNa) and urea (FeUrea) , 29, 131y damage .Advanced physiological parameters can be obtained through invasive procedures. Renal and pulmonary artery transit-time flow probes with oximetry capability may facilitate a deeper understanding of EAKI. Moreover, they may allow accurate measurement of macro/microvascular flow, renal cortical and medullary tissue perfusion, and renal function throughout the entire course of ECMO in animal models , 133. ThImaging studies may be utilised to define the presence and severity of AKI in patients supported by ECMO. Renal ultrasound ± Doppler is the most commonly utilised imaging modality for the assessment of patients with AKI . AlthougContrast enhanced ultrasonography (CEU) uses contrast agents composed of microbubbles with an injectable gas in a supportive shell of phospholipids or proteins . CEU canRenal biopsies in order to assess the presence, nature, and extent of AKI are not performed in patients on ECMO due to an unfavourable risk : benefit ratio; however, its use may be helpful in experimental animal models. Important pathophysiological factors may be identifiable through histological and immunohistochemical assessment of nephrons. Conventional staining methods combined with various immunohistochemical staining, apoptosis quantification, and gene expression for glycocalyx disruption and major controllers of vessel tone may delineate the cause and extent of EAKI. Among other methods, transmission electron microscopy (TEM) with ruthenium red, ferritin, lanthanum, and cupromeronic blue staining may help to specifically identify renal glycocalyx disruption . AdditioWhilst the role of novel biomarkers in AKI has shown promise they have been underutilised for patients on ECMO. In 2005, the Council of the American Society of Nephrology recommended that the highest research priority be the standardization and discovery of new biomarkers of AKI . FollowiNeutrophil gelatinase-associated lipocalin (NGAL) and cystatin C (CystC) have the greatest depth of research for the detection of AKI. Other promising biomarkers include kidney injury molecule-1 (KIM-1), renal-liver type fatty acid binding protein (L-FABP), insulin-like growth factor-binding protein 7 (IGFBP7), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Combining these biomarkers may collectively improve the sensitivity and specificity of detecting AKI in patients supported by ECMO . Novel bEarlier detection of kidney injury using new biomarkers has led to the recognition of a subgroup of patients with “subclinical” or “incipient” AKI , 180–182 in vivo animal models provide a valuable means to undertake detailed, systematic research into complex clinical scenarios and pathophysiological changes associated with ECMO [ in vivo models with large animal subjects may help to delineate and define the relative risk of EAKI. An in vivo ovine model has many advantages [The multitude of complex, interrelated variables present in patients on ECMO are difficult to control in a study setting. As a result, clinical research in patients on ECMO is challenging. Simulated ECC andith ECMO . Furthervantages , 184. Fivantages , 185–187vantages –190. in vivo, ovine model of VV ECMO may successfully mimic the severity of patient illness and reproduce key haemodynamic and immunological aberrations. It can also serve as its own control, mimic histological findings in relevant organs, and provide a means to gain insight into factors contributing to interpatient variability [ in vivo model will allow a thorough investigation into the renal response to various clinical conditions. Translational clinical investigations into EAKI will lead to a more accurate assessment of the relative contribution of ECMO circuit factors on renal function. Moreover, implementing novel renal monitoring modalities based on relevant pathophysiology will provide novel strategies for preventing/treating EAKI.Aniability . Ultimat in vivo animal model with the goal to improve the understanding of the changes observed during ECMO and, subsequently, optimise the process. A better understanding of this will help to define optimal renal support for patients during therapy, ultimately reducing the risks associated with ECMO and improve patient survival.There are limited data within the literature exploring EAKI. Furthermore, there are no studies using RIFLE/AKIN in conjunction with advanced methods for investigating AKI in patients supported by ECMO. The high incidence rate of AKI in patients supported on ECMO and the subsequent mortality that emerges as a result of AKI is unacceptable. It is likely that EAKI exists as a dependent entity; however it is equally possible that initiation of ECMO serves to protect renal function. Understanding the patient-device interface and its role in the progression of normal renal function to AKI is imperative. This, in conjunction with earlier detection with the use of novel investigative methods, is fundamental step towards reducing EAKI. These issues can be investigated using an


Protein phosphorylation on tyrosine (Tyr) residues has evolved as an important mechanism to coordinate cell communication in multicellular organisms. The importance of this process has been revealed by the discovery of the prominent oncogenic properties of tyrosine kinases (TK) upon deregulation of their physiological activities, often due to protein overexpression and/or somatic mutation. Recent reports suggest that TK oncogenic signaling is also under the control of small adaptor proteins. These cytosolic proteins lack intrinsic catalytic activity and signal by linking two functional members of a catalytic pathway. While most adaptors display positive regulatory functions, a small group of this family exerts negative regulatory functions by targeting several components of the TK signaling cascade. Here, we review how these less studied adaptor proteins negatively control TK activities and how their loss of function induces abnormal TK signaling, promoting tumor formation. We also discuss the therapeutic consequences of this novel regulatory mechanism in human oncology. Tyr phosphorylation (pTyr)-dependent signal transduction results from the combination of three molecular actions: initiation of the pTyr signal induced by the TK (the writer), propagation of the signal through recognition of the phosphorylated protein by a SRC Homology 2 (SH2) domain-containing protein (the reader) and control of the signal by phosphatases that dephosphorylate the substrate (the eraser) ..60].Finally, SLAP may also participate in cell transformation in specific situations, as reported for SOCS proteins. For instance, SLAP could be involved in the blockade of cell differentiation required for the induction of erythroleukemia by the FLI-1 oncoprotein . MechaniGRB10 expression is down-regulated in many human tumors [GRB14 gene is frequently mutated in human CRC with microsatellite instability [GRB10 and GRB14 roles in human cancer are poorly documented and not clear. GRB10 may participate in progression of some human tumors, as suggested for primary cervical squamous carcinoma  and FLT3n tumors . Moreovetability .Lnk−/− mice that resembles the myeloproliferative abnormalities found in human MPN [LNK may have tumor suppressor function as suggested by the phenotype of uman MPN . LNK ectuman MPN , 179–181uman MPN , 180. Houman MPN . Somaticuman MPN . ConsistSurprisingly, LNK is up-regulated in some solid tumors, including the mesenchymal subtype of serous ovarian cancer, and this is associated with poorer outcome . FunctioERRFI1, mice frequently develop spontaneous or chemical-induced tumors [ERRFI1, as reported in EGFR-amplified glioblastoma [MIG6 tumor suppressive role is supported by the finding that upon deletion of d tumors . Additiod tumors , 184 andd tumors , 184. MIblastoma , 185, orblastoma . MIG6 inAll this data indicates that loss of adaptor-mediated regulation of oncogenic TKs contributes to tumor formation. These novel findings may have significant consequences for cancer treatment/management, such as the discovery/development of novel biomarkers and the improvement of therapy targeting TK-dependent signaling.−/−Errfi1 mice are highly sensitive to the EGFR TKI gefitinib [The expression status of some of these adaptors in transformed cells may predict the tumor responses to drug that target oncogenic TK signaling. For instance, low expression in tumor cells of adaptors that control TK catalytic activity, such as MIG6, might predict heightened oncogenic signaling dependent of deregulated TKs, such as ERBB receptors, and therefore may be a good predicator of tumor cell response to TK inhibitors (TKIs) Figure . Indeed,efitinib . MIG6 siefitinib . A low Mefitinib . Analysiefitinib . OverallSLA was silenced [Inactivation in tumor cells of adaptors that control substrate stability, such as SLAP, would also predict heightened oncogenic signaling dependent of the deregulated TK, such as SRC, and therefore may be a good predictor of tumor cell response to TKIs. Conversely, high SLAP expression level might indicate that although SRC activity is aberrant in these tumors, its transforming activity could be hampered by SLAP-mediated destruction of important SRC substrates. This would render tumor cells less dependent on SRC oncogenic signaling and, consequently, rather resistant to a SRC-based therapy Figure . This mosilenced . TherefoDue to their prominent tumor suppressor function, forced expression of these adaptors in tumor cells could represent an alternative therapeutic strategy to target oncogenic TK signaling in human cancer. Restoration of adaptor expression in tumor cells can reduce tumor growth and/or metastasis formation. Nevertheless, gene therapy is still a technical challenge and alternative methods to modulate adaptor activity or expression might be considered. These adaptor proteins are frequently inactivated in tumors via a methylation-dependent mechanism. Accordingly, demethylation drugs, such as 5-aza-2′-deoxycytidine that exhibits clear tumor suppressive roles, could restore adaptor expression in tumor cells and may represent a potential anti-tumor therapeutic strategy. Forced expression of many of these adaptors, such as LNK  and SOCSFinally, because of their prominent role in immune response, their level of expression may dictate the tumor cell response to cytokine-based therapy. For instance, SOCS1 and SOCS3 up-regulation seem to be responsible for the unresponsiveness to IFN therapy in patients with leukemia. Indeed, SOCS1 constitutive expression has been observed in patients with CML , in agrein vitro and animal models. Similarly, while underlying mechanisms of adaptor-mediated negative feedback regulation was thought to be rather well established, recent molecular analyses challenged this idea and suggest the existence of broader mechanisms involved in their activities. For example, CBL has been reported as the main SLAP effector in the control of TK signaling through ubiquitin-mediated degradation via the proteasome or the lysosome [Since the discovery of CIS twenty years ago, this small class of adaptor proteins has emerged as an important player in the mechanisms to finely tune TK-mediated signal transduction. While their functions have been mostly investigated in hematopoietic cells due to their high expression levels and to the essential role of TK signaling in hematopoiesis, more recent data revealed additional important roles in non-haematopoietic cells, as exemplified by the SLAP tumor suppressive role in the colon. Therefore, one important perspective will be to uncover such physio-pathological activities in non-hematopoietic tissues using novel relevant lysosome , 53–55 blysosome . Additiolysosome . We thusDue to their feedback role in the control of TK signaling, the negative function of these adaptors is expected to be exacerbated upon aberrant expression of TK activities, uncovering novel tumor suppressor functions for these adaptors. This idea has been experimentally validated in myeloproliferative malignancies (e.g. LNK) but tumor suppressor and anti-metastatic activity for these adaptors have been now reported also in solid tumors (e.g. SLAP and SOCS1). Therefore, a broader tumor suppressor role for these adaptors is expected in human cancer, which will deserve more investigation in the future. Besides, we suspect that oncogenic TKs may find a way to overcome this control mechanism and in some situations divert this regulatory process to sustain tumor progression. How oncogenic TKs cope with this control mechanism is an additional important issue to be addressed in the future. Intriguingly, the function of some adaptors in cancer is more complex because their activity seems to be context-dependent. The reason for such variable activity is quite obscure, but we suggest that these molecules target additional important signals that are involved in the control of tumor development/progression, such as the immune response. It will be thus important to elucidate such tumor promoting function in order to better understand their general role in this human disease and improve TK-based therapeutic strategy according to their activities. In conclusion, this rather neglected mechanism in the control of TK signaling is emerging as an additional important mechanism for the control of human malignancy and a better understanding on the role of this adaptor family in human cancer should ultimately improve TK-based tumor therapies.


Refractory cardiac shock in the cardiac surgical intensive care unit confers significant morbidity and mortality. Extracorporeal membrane oxygenation (ECMO) has become a common intervention for refractory cardiogenic shock when other therapies have failed. However, it is difficult to predict who will benefit from this costly, resource-intensive, but potentially life-saving technology. Here, we discuss the utility of a novel biomarker, serum butylcholinesterase, in determining survival in patients supported with ECMO following cardiac surgery. The specter of post-cardiotomy shock haunts cardiac surgeons and cardiac intensivists. Whether manifest as failure to septe from cardiopulmonary bypass, persistent low cardiac output with malperfusion, or sudden hemodynamic collapse in the cardiac surgical intensive care unit (CSICU), refractory cardiac shock confers significant morbidity and mortality. When inotropes, vasopressors, and intra-aortic balloon pumps fail, extracorporeal membrane oxygenation (ECMO) has become a common intervention for refractory cardiogenic shock. The work by Distelmaier and colleagues  continueApproximately 1% of all adult patients undergoing cardiac surgery will experience post-cardiotomy shock . ECMO haPrevious studies have attempted to identify predictors of successful weaning from ECMO, predictors of mortality on ECMO, and predictors of long-term outcomes after ECMO support ,4-8. TheOther groups have evaluated cardiac biomarkers as predictors of cardiac recovery during ECMO support with disappointing results . DistelmThe mechanism by which butyrylcholinesterase mediates the association with decreased mortality is not known. This tempers enthusiasm for this biomarker, as we neither understand the function of this enzyme, nor determinants of its level, nor the consequences of its deficiency. Indeed, in a study of stroke patients, cholinesterase activity was higher in patients than in matched controls . BecauseIf butyrylcholinesterase levels truly predict ECMO-specific outcomes, this biomarker could have significant implications. Compared to other uses of ECMO ), the decision to undertake ECMO support in post-cardiotomy shock occurs in a compressed time frame. Whereas patients with severe ARDS typically decline to the point of requiring ECMO over a span of hours, post-cardiotomy cardiac failure can occur suddenly: an unanticipated failure to septe from cardiopulmonary bypass or an unheralded arrest in the CSICU. In the latter, there is little time to calculate a prognostic score such as the PRESERVE score proposed for ECMO and severe ARDS .The ability to predict ECMO outcomes prior to surgery would arm surgeons and intensivists with more data to guide emergent decisions. Indeed, if this study’s findings are confirmed, one can envision development of a pre-operative 'game plan', with support options in the case of refractory shock explicitly laid out. While the pre-operative determination of appropriate support options for a given patient has some appeal, would preclusion of ECMO support based on a single pre-operative biomarker be acceptable to physicians and patients? Therapeutic decisions based on well-characterized biomarkers are common in other fields  where mechanistic rationale and extensive clinical data exist. How strong must a biomarker, or indeed any prognostic data, be in order to justify withholding of ECMO support in an immediately life-threatening situation? These are important but unanswered questions. But as the cardiac surgical population becomes more complicated, ECMO technology improves, and mechanical circulatory support for refractory post-cardiotomy shock becomes more common, it will become critical to develop a rational framework for the use of this expensive and resource-intensive treatment. Distelmaier and colleagues have made an important step towards answering this question: not whether we could, but whether we should utilize ECMO to support a patient with post-cardiotomy shock.ARDS: Acute respiratory distress syndrome; CABG: Coronary artery bypass graft; CSICU: Cardiac surgical intensive care unit; ECMO: Extracorporeal membrane oxygenation.The authors declare that they have no competing interests.