Functional gastrointestinal disorders (FGID), such as functional dyspepsia (FD) and irritable bowel syndrome (IBS) are characterized by chronic abdominal symptoms in the absence of an organic, metabolic or systemic cause that readily explains these complaints. Their pathophysiology is still not fully elucidated and animal models have been of great value to improve the understanding of the complex biological mechanisms. Over the last decades, many animal models have been developed to further unravel FGID pathophysiology and test drug efficacy. In the first part of this review, we focus on stress-related models, starting with the different perinatal stress models, including the stress of the dam, followed by a discussion on neonatal stress such as the maternal separation model. We also describe the most commonly used stress models in adult animals which brought valuable insights on the brain-gut axis in stress-related disorders. In the second part, we focus more on models studying peripheral, i.e., gastrointestinal, mechanisms, either induced by an infection or another inflammatory trigger. In this section, we also introduce more recent models developed around food-related metabolic disorders or food hypersensitivity and allergy. Finally, we introduce models mimicking FGID as a secondary effect of medical interventions and spontaneous models sharing characteristics of GI and anxiety-related disorders. The latter are powerful models for brain-gut axis dysfunction and bring new insights about FGID and their comorbidities such as anxiety and depression. Functional gastrointestinal disorders (FGID), such as functional dyspepsia (FD) and irritable bowel syndrome (IBS) are highly prevalent, occurring in 10–30% of the general population depending on the criteria used, and represent an important part of the workload of gastroenterology and primary care clinical practice. Those syndromes are characterized by chronic abdominal symptoms in the absence of an organic, metabolic or systemic cause that readily explains these complaints. Based on the Rome IV criteria, FD is defined as bothersome postprandial fullness, early satiation, epigastric pain and/or epigastric burning , 3. The Over the last 20 years, many animal models, which originally focused on one particular pathophysiological factor, have been developed for FGID. These models have contributed a lot to the understanding of the pathophysiological mechanisms behind the symptoms and were also used to test and validate therapeutic targets and potential therapies . HoweverIn this review, we will summarize the state of the art concerning the most relevant and most commonly used pre-clinical models that have been developed for FGID and highlight their contribution to the understanding of FGID pathophysiology. Most of the published studies use rodent models, due to the possibility of genetic manipulations and the quick turnover of the models through fast reproduction and a large number of pups in one litter, in comparison to larger animal models such as pig models. Therefore, this review will largely focus on rodent models.Functional gastrointestinal disorders have long been regarded as purely psychosomatic conditions. In the last decade, however, evidence for a low-grade mucosal inflammation, dominated by mast cells, eosinophils, and T-lymphocytes –8, as weAs mentioned above, FGIDs are currently understood as disorders of the brain-gut axis, i.e., the neurohumoral communication system between the brain and the gastrointestinal tract, leading to gastrointestinal hypersensitivity and dysmotility . HoweverStress is biologically defined as a physiological response to a stimulus that allows organisms to adapt to their environment . HoweverPsychological stress and anxiety, often reported by FGID patients, influence the onset of symptoms and predict the clinical outcome . RecentlIn this section, we made a distinction between models of stress applied to adult animals and those involving stress around birth , i.e., early life stress (ELS). The stress models presented in this section are summarized in In the last decade, data coming from clinical psychiatry showed that women's health status before and during pregnancy is a determining factor for the development of psychiatric disorders, including schizophrenia and depression, socio-emotional problems or altered stress response of the children later in life . HistoryThe most frequently studied and used model of ELS is the maternal separation (MS) model in which pups are separated from the dam and the rest of the nest every day during their first weeks of life. Multiple variations of the MS procedure have been described, with changes in the duration of the separation and the number of days of separation. Variations include MS until weaning whereas other protocols use a 24 h separation between post-natal day 3 (PND3) and PND4 . The mosPups who underwent the maternal separation, display an increased intestinal permeability as well as changes in the microbiota composition at the time of weaning which are associated with an increase in the basal level of corticosterone . ChangesThe separated pups develop an increased reactivity of the HPA axis in response to stress during adulthood , as showAs mentioned above, the socio-economic status of caregivers affects the onset of FGID in children. To mimic poverty and precarious conditions in humans and also to limit the intervention of an external experimenter which is subject to variability, a model of limited cage bedding, mostly applied between the PND2 and PND9, was developed first in rats and later also in mice , 99. SomMany mammals including rodents are born blind and deaf, and to stay warm and obtain the food and care needed to survive, they need to learn the odor of their caregiver, e.g., their mother. For this purpose, during the first 9 days of their life, pups display an enhanced capacity for odor preference learning through a stimulated release of norepinephrine, produced in the locus coeruleus, which binds to its receptors in the olfactory bulb . This alDeveloped by and mostly used in the Greenwood-Van Meerveld lab, the model of odor shock-conditioning consists of predictable and unpredictable odor-shock pairings which mimic attachment to an abusive caregiver. This model uses the association between an odor and a modest electrical shock to the tail to reproduce the pup-dam interaction and creates an olfactory attachment to the conditioned odor in response to predictable or paired odor/shock. In practice, pups are exposed from PND8 to PND12 to an odor associated with an electrical shock 2 minutes after the odor exposition while controls are only exposed to the odor , 107–109Water avoidance stress (WAS) is one of the most frequently used models of stress in adult rodents either alone or combined with the maternal separation model. Several studies over the last decade have used it to characterize acute and chronic stress-induced GI symptoms and to study the effect of treatment, including nutritional and probiotic interventions , 111. DuAs for the maternal separation, several models of physical constraint have been developed over the years, among them three versions of physical constraint: the partial restraint stress, full restraint stress and cold restraint stress. The extent and the duration of the stressor differ amongst the protocols. With a full body restraint stress applied in rats for 30 min, studies have shown neuronal activation over several brain structures and nuclei including the supraoptic nucleus, locus coeruleus, the ventrolateral medulla, the medial division of the central amygdaloid nucleus, nucleus of the solitary tract and even the dorsal nucleus of the vagus nerve, structures involved in food intake and the stress response , 128. InOne of the important findings of the last century in the field of psychobiology is the stress-buffering effect of social relationships, with an important role for oxytocin . Social However, the positive effect of this social buffering depends on the nature of the relationship between individuals as well as on the social organization of the species and/or gender. Many species, including humans and rodents, live for almost their entire life in a group with a strong hierarchy. As a result, any disturbance in this social order or abuse is a potential source of stress. In rodents, stress models used either the isolation of one animal from the rest of the group, i.e., social isolation stress, or, at the other end of the spectrum, an overpopulation within a small area, i.e., crowding stress. Often used as models for anxiety and depression-related disorders alterations of the GI physiology have also been studied in rodents submitted either to the social isolation stress or the crowding stress models. Crowding induces a strong competition for space, food, and water and leads to a strong increase of the corticosterone concentration in the first days associated with early transient alterations in the nitrergic system in the hippocampus, prefrontal cortex and hypothalamus, an increase of iNOS expression in all structures and increased of nNOS in the hippocampus and hypothalamus. These changes are normalized over time through a habituation process but remains higher than in normal housing conditions. This protocol is used for 2 to 9 weeks depending on the strain and type of rodent and is often combined with other types of a stressor to simulate the combination of chronic and (sub)acute stressors that naturally occurs in humans. Work from our group demonstrated that 14 days of crowding stress in Wistar rats induced increased permeability in the jejunum which correlated with plasma corticosterone levels. However, mast cell density was only increased in the colon . In WistConversely, in the social isolation stress (SIS) model, the animal is isolated from the rest of the cage. Often applied just after the weaning, SIS modifies the development of the brain and influences the nitrergic system in several brain areas such as the hippocampus, the frontal cortex by increasing the nNOS expression in the hippocampus and hypothalamus and iNOS in the prefrontal cortex . A decreAlthough the various animal stress models developed over the last years have provided critical information about the influence of stress on physiology, humans are usually not exposed to only one stressor during their life and a combination of stressors is often present in patients with FGID. Also, physiology can adapt to one homotypic stressor in humans and rodents, leading to habituation and absence of effect after repeated exposure. As a variety of animal models of stress are available, a wide range of stress combinations can be used to better understand the pathophysiological mechanisms behind stress-induced FGID symptoms. Combinations of unpredictable mild stress are also often used with a rotation between different stressors such as light/dark cycle, isolation, crowding, predator odor exposure, shock, cold environment, restraint stress….An often-used combination in rats is the maternal separation combined with one session of water avoidance stress in adult rats. In contrast to mice, maternal separation is not always sufficient to induce GI symptoms in rats  but incrWith the use of unpredictable stress models, which consist of applying a stressor  at unpredictable moments of the day for a few days, the involvement of nerve growth factor, endorphin, beta-adrenergic pathway, BDNF and mast cells mediators and the toll-like receptor 4 (TLR4) pathways have been demonstrated in stress-induced visceral hypersensitivity . Recent Infections and inflammation are among the best-characterized triggers for FGID symptoms. Although the pathophysiological mechanisms are not yet fully understood, a low-grade inflammation is considered as the main explanation for the symptoms in so-called post-infectious (PI) IBS and FD . EvidencLow dose injections of inflammatory factors like the bacteria-derived lipopolysaccharide (LPS), injected systemically at the dose of 1 mg/kg can also trigger FGID features such as rectal allodynia and colonic hyperpermeability. Visceral allodynia appears 3 h after injection last up to 12 h and is mediated by mast cell degranulation, IL1β and TNFa . When AuSeveral models have also been developed using a low concentration of dextran sodium sulfate (DSS) to induce low-grade inflammatory changes . This moRodent models of post-inflammatory GI disorders attempt to simulate a post-inflammatory situation mostly represented by the resolution of an acute infection. Most of the models presented below originally are models for IBD but can be instrumental to study certain aspects of the development of GI symptoms after resolution of the acute inflammation. The development of post-inflammatory GI disorders occurs in 25 to 100% of the treated animals depending on the trigger used. Furthermore, the severity of symptoms and functional alterations developed in the post-inflammatory period—except for the visceral hypersensitivity—seems to be independent of the severity of the initial inflammation .An acute treatment with a high percentage of DSS followed by a DSS-free period  creates Bifidobacterium infantis 35624) were able to rescue this phenotype (Trinitrobenzene sulfonic acid (TNBS), leads to a Th1 immune response with ulcers in rodents within the first 3 days after instillation. Two weeks after instillation, the inflammation is resolved, but a visceral hypersensitivity, motility dysfunction due to a persistent long-term smooth muscle hyperactivity to acetylcholine, an increased mast cell infiltration in the mucosa, an upregulation of the NMDA-NR1, as well as galanin and tachykinin expression in the mucosa and myenteric plexus, a barrier hyperpermeability and a decreased secretory function through a cyclooxygenase-2 (COX-2) dependent mechanism can be demonstrated . The lonhenotype . Mast cehenotype . Recentlhenotype  which pohenotype .Acid acetic, Zymosan and mustard oil are irritants administered directly into the colon of adult or neonatal animals by enema. When administered in pups, long-lasting visceral hypersensitivity has been reported for 8 to 10 weeks for Zymosan and mustard oil and up to 12 weeks for acid acetic  without Iodoacetamide is an alkylating agent administered by gavage in neonate rodents to induce a mild inflammation of the gastric mucosa that is associated with acute changes in sensory and motor function. Inhibition of the glucocorticoid receptors, adrenergic receptors, BDNF or the nerve growth factor (NGF) during the neonatal period suppressed the induced gastric hypersensitivity . The infWithin the GI tract, the host and billions of micro-organisms are co-existing, creating a unique symbiosis. Products secreted by the microflora influence the gut function by their effect on neurotransmitters, epithelial function, secretion, or muscle contraction . The comTrichinella spiralis is an intestinal parasite found in humans, rodents, and pigs and is used as a model of post-infectious IBS in mice. For this purpose, mice are infected with 200 to 300 larvae in one gavage. During the acute phase, parasites are evacuated from the organism triggering a Th2 inflammatory phase in both the mucosa and muscular layers  is an inbred colony originating from Wistar rats and have been selected for their ability to spontaneously develop type 1 diabetes . The BB-The Wistar Kyoto (WKY) rat, originally used as a control normotensive strain for the spontaneously hypertensive rats (SHR), has been studied in the last decades as a model of brain-gut dysfunction. Those rats display an exaggerated response to chronic stress compared to Sprague Dawley rats –224, assThe Flinders Sensitive Line (FSL) rat originates from selective breeding from Sprague Dawley and has been selected for their resistance to the choline esterase inhibitor, diisopropyl fluorophosphate . Used fiDuring abdominal surgery, the opening of the skin and the abdominal cavity triggers adrenergic reflexes involving a spinal loop which temporarily blocks GI motility. Considered as an iatrogenic disorder, postoperative ileus (POI) occurs in most patients undergoing abdominal surgery and is characterized by a transiently impaired GI motility. However, when recovery of bowel function is delayed for more than 3–7 days, this disorder is defined as an illness . SeveralManipulations of the central nervous system have also been described as potential models of GI disorders associated with anxiety-like symptoms. As described before, the limbic system and especially the amygdala are strongly involved in stress-induced colonic hypersensitivity associated with anxiety-like symptoms and the manipulation of this brain region is sufficient to induce colonic pain . Direct Trichinella spiralis (Genetic models of FGID include specific KO animals for several receptors, ion channels, and cellular pathways. These models have provided important knowledge of FGID pathophysiology. Mostly used to better understand the pathophysiology of colonic pain, they have demonstrated the importance of BDNF, guanylate cyclase, serotonin transporters, and ion channels. The latter have been extensively described in the literature to be involved in the intestinal mechanoreception and inflammation , 258. Thspiralis . Other kspiralis . The actspiralis , 263. Stspiralis . More gespiralis .Several studies have demonstrated a cross-sensitization between different abdominal organs. In the spinal cord and the brain, the convergence of the sensory neuronal pathways of the different organs is one of the mechanisms underlying this sensory visceral crosstalk . FurtherAlthough most of the research focuses on rodent models, some other species have been used to investigate the FGID pathophysiology. The guinea pig is a good model to study intestinal motility and the enteric nervous system. The models used are similar to what we previously described in rodents, with the use of stress models such as water avoidance and CRH injection , 270. AsIn rabbits, intracolonic infusion of Zymosan leads to colonic hypersensitivity  which isEx-vivo experiments demonstrated that CRH increased permeability via a TNF-α dependent mechanism (Pigs have a comparable GI system to humans, with an equivalent size, anatomy, development and diet preference, which are evidently very different in rodents . Also, techanism . Similarechanism . DespiteFunctional gastrointestinal disorders are complex and multifactorial disorders involving a complex interaction between biological, psychological and social variables that none of the current animal models can reproduce perfectly. The strengths and limitations of the varied models are listed in Most of the published studies still suffer from two limitations which weaken their translational relevance. First, most of the available studies focus on the lower GI tract , while iAll authors contributed to the first draft of the manuscript, critically revised subsequent drafts, and approved the final version.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.


Antimicrobial resistance (AMR) is a major concern facing global health today, with the greatest impact in developing countries where the burden of infectious diseases is much higher. The inappropriate prescribing and use of antibiotics are contributory factors to increasing antibiotic resistance. Antimicrobial stewardship programmes (AMS) are implemented to optimise use and promote behavioural change in the use of antimicrobials. AMS programmes have been widely employed and proven to improve antibiotic use in many high-income settings. However, strategies to contain antimicrobial resistance have yet to be successfully implemented in low-resource settings. A recent toolkit for AMS in low- and middle-income countries by the World Health Organisation (WHO) recognizes the importance of local context in the development of AMS programmes. This study employed a bottom-up approach to identify important local determinants of antimicrobial prescribing practices in a low-middle income setting, to inform the development of a local AMS programme. Analysis of prescribing practices and interviews with prescribers highlighted priorities for AMS, which include increasing awareness of antibiotic resistance, development and maintenance of guidelines for antibiotic use, monitoring and surveillance of antibiotic use, ensuring the quality of low-cost generic medicines, and improved laboratory services. The application of an established theoretical model for behaviour change guided the development of specific proposals for AMS. Finally, in a consultation with stakeholders, the feasibility of the plan was explored along with strategies for its implementation. This project provides an example of the design, and proposal for implementation of an AMS plan to improve antibiotic use in hospitals in low-middle income settings. Rising AMR is viewed as a significant threat to global health. Concerted efforts to stem the rise in antimicrobial resistance and to ensure effective antimicrobial therapies for future generations is seen as a priority for the global community. Injudicious prescribing and use of antibiotics are considered principal drivers of increasing resistance. Thus, the central focus of initiatives to address AMR is on antimicrobial stewardship .Whilst a global concern, inappropriate use of antibiotics and consequent problems of antimicrobial resistance are greater in low- and middle-income settings . The detDespite expansion of health insurance schemes, for many citizens, there is still no reliable funding stream to pay for care: consultation, diagnostic tests, and treatment. Dependence of many patients on out-of-pocket payments can affect prescribing decisions of prescribers and result in inappropriate and unregulated self-medication with antibiotics by patients ,14. To bMethicillin-Resistant Staphylococcus Aureus (MRSA) [Suboptimal antibiotic prescribing practices remain common in many hospitals in low and middle-income countries (LMICs), and have been linked to increased incidence of antibiotic-resistant bacteria such as s (MRSA) . Typicals (MRSA) , althougs (MRSA) . Prescris (MRSA) ,18,19, ns (MRSA)  and limis (MRSA) . Studies conducted in high-income countries have shown how antibiotic stewardship programmes can improve antibiotic prescribing in hospital settings, but these interventions have been poorly employed in LMICs  with onlEngagement and consultation with local stakeholders will facilitate the identification of challenges and opportunities, enabling the development of an AMS programme that is likely to be effective in their local context ,25. ThisThis project was conducted in collaboration with hospitals in Bayelsa state, located in the Niger Delta Region of Nigeria. Data collection for this study was conducted from July 2015 to September 2017. Nigeria is classified as a developing nation in lower/middle income group . In termWe employed a bottom-up mixed methods approach for this study. This involves distinct phases with different methods that are interlinked to explore and provide in-depth understanding of the subject. Antibiotic prescribing data was retrieved quantitatively and followed up with stakeholder interviews. The project was conducted in three stages. The first stage comprised a survey of antibiotic use across the selected study sites to establish the extent to which practices are typical of LMIC settings and a series of interviews with prescribers recruited across all hospitals, to identify important drivers of day-to-day prescribing practices as well as suggestions for interventions to improve antibiotic use. Stage 2 involved the application of an established theoretical model for behavioural change along with findings from stage 1, to develop a draft AMS strategy and plan. Stage 3 of the project comprised consultation with purposively selected stakeholders  to refine the proposals and inform an implementation plan.A simple data collection tool  was deveStage 1: Antibiotics prescribing practices and determinantsAcross the four study sites, data relating to 809 patients prescribed one or more antibiotics were analysed. This comprised 264 (33%) and 246 (30%) from the two tertiary sites and 151 (19%) and 148 (18%) from the secondary care sites. More than half of the patients (53%) receiving antibiotics were ≤16 years old, and a further 21% aged 17–29 years. In 219 (27%) cases more than one different antibiotic was prescribed. The five most commonly prescribed antibiotics, making up 75% of prescriptions were broad-spectrum antibiotics including metronidazole, amoxicillin, amoxicillin and clavulanic acid, cefuroxime and ciprofloxacin suggesting a high rate of empirical prescribing. The majority (58%) of antibiotics were prescribed by brand, rather than generic name. Relevant laboratory tests such as microbial, culture and sensitivity tests to guide antibiotic treatment decisions were employed in only 15% of cases. In some cases where antibiotics were prescribed, tests such as abdominal scans and fasting blood sugar were carried out and did the presence of a bacterial infection was not indicated. There was limited observance (33% cases) of Nigerian Standard treatment Guidelines . To examine rationale for antibiotic prescribing in the local setting, interviews were conducted with 17 physicians . InterviResponses of participants revealed an awareness of, and concern about, suboptimal prescribing of antibiotics in the study settings, as found in stage 1. In their explanations of current practices, they highlighted high level system factors, local conditions, individual perceptions and practices all as potential determinants of antimicrobial use. These included, a lack of system support in local settings, especially inadequate laboratory services, excessive workload in the clinics; costs of drugs, especially in the light of patient socioeconomic status and prevalence of out-of-pocket payments; specific demands from patients, lack of availability of products, and shortcomings in training and knowledge and reliance on long-term prescribing habits, impact of pharmaceutical companies’ marketing and incentives. Respondents, notably, expressed regret that practices were often suboptimal, but saw the challenges as complex and often beyond their control. For example, whilst interviewees displayed positive views to the use of policies and guidelines, these were reported as being unavailable, out-of-date or difficult to comply with because of other system factors such as non-availability of drugs. Poor laboratory services, especially facilities that were non-functional, unstaffed and or open only for limited or irregular hours resulted in poor availability of diagnostic services and delays in obtaining results of sensitivity tests. This was seen by many as contributing to high levels of empirical prescribing of broad-spectrum antibiotics. Patients without health insurance, necessitating out-of-pocket payments, could also be a barrier to requesting diagnostic tests.Thus, cost and affordability for patients were often cited by prescribers who reported compromising in the choice of drug to cheaper alternatives to ensure some antibiotic cover. The range of products procured and stocked in the hospitals also influenced drug choice. The negative impact of pharmaceutical companies promoting brands and incentives for prescribing was also raised. Concerns about quality and effectiveness of some non-branded products was also reported as a driver for brand-name rather than generic prescribing. Whilst, the patterns and determinants of antibiotic prescribing in these hospitals were often typical of those reported in other LMIC settings, the observations and experiences of prescribers, facilitated their engagement in proposing suggestions and recommendations for AMS interventions pertinent to their settings and challenges. Prescribers identified a diversity of macro and micro-level factors as determinants of antibiotic use and potential targets of an AMS programme. These are summarized in Stage 2: Development of an AMS plan using a framework of behaviour changeBehaviour Change Wheel (BCW) was employed [To guide the development of an AMS programme, a validated framework the employed . Theoretemployed ,31. The Using the BCW framework (policy categories and intervention functions) along with the domains and specific suggestions of prescribers and stakeholders, a draft framework for an AMS programme was constructed . In thisSome interventions could be implemented immediately, in that the available resources could support the initial start-up, and that existing committees (e.g. Drugs and Therapeutics Committee -DTC) could undertake planning and execution. These interventions may include increasing local awareness, development of guidelines and protocols, and implementation of local audit and monitoring. Other measures would require policy development and planning, at a higher (sometimes Government) level, and thus will be goals achievable as part of a longer-term strategy, e.g. allocation of additional finance, continued roll-out of health insurance to ensure affordability for patients, enhanced human resources.The final consideration in the development of the plan for AMS involved the sequencing of interventions. This will depend on contexts and priorities in different hospitals, e.g. some hospitals may be setting up a programme for the first time, others updating an existing strategy. In practice, in all sites, continual review will be required. Stage 3: Consultation with key stakeholdersThe third stage of the project was consultation with key stakeholders. The aim of this was to examine the feasibility and acceptability of recommendations from the perspectives of different stakeholders and to inform a plan for implementation relevant to, and workable in, their local settings. A purposive sample including key members of the hospital, clinical and administrative staff and other relevant stakeholders in regulatory/policy settings were recruited. Eight participants were drawn from different areas of practice and included a hospital medical director, prescribers, pharmacists, microbiologist, long-standing member of Drugs and Therapeutic Committee (DTC), chairperson of the medical association and representative from the State Ministry of Health. In this consultation, data were collected in one to one interviews. The stakeholders expressed their views on priorities for AMS, opportunities, and challenges for implementation in their settings.The draft framework for implementing AMS in the local settings was presented to the stakeholders and this was largely viewed as apposite and feasible. Their top priorities for implementation include increased awareness and education about antibiotic resistance, development, and provision of policies and guidelines on antibiotic use, monitoring and surveillance of antibiotic use, improved laboratory and diagnostic services and ensuring availability and quality of products.Some of these interventions had already been initiated, particularly in the teaching hospital in accordance with requirements for accreditation. In relation to these priorities, stakeholders’ perspectives regarding the opportunities and challenges in their settings and potential action plans were explored. The findings are reported in The proposed AMS plan encompasses the policy categories, intervention functions and sources of behaviour change as outlined in the three layers of the BCW in that it identifies the interacting roles of the government , the institutions  and individuals  the team will be responsible for developing a mission statement. Subgroups should be set up to take forward specific tasks and objectives set by the wider team. A mechanism will be required for maintaining and distributing local guidelines throughout the hospital to reach and engage relevant staff. Other activities of the AMS team will include regular review of antibiotics use, documentation of local bacterial sensitivity patterns and revisions of antibiotic formulary lists. The team will also plan for surveillance and auditing of practice , document findings and generate appropriate reports. These reports will be reviewed and presented to staff as part of communication and education, highlighting good practices as well as areas requiring improvement.The findings from this project have informed a plan for antimicrobial stewardship in a low-resource setting. Whilst this work commenced before the publication of the WHO toolkit, it provides a road map to developing an AMS programme in an LMIC with locally retrieved data. This project took a bottom-up approach combined with application of a theoretical framework of behaviour change, leading to the development of a multifaceted AMS plan. While the AMS plan developed here has been designed to be implemented in a specific LMIC setting, early stages of the project confirmed that many of the practices regarding antibiotic use, prescribing patterns, barriers, and opportunities for change are similar to those in other LMICs. Therefore, the findings presented here can be implemented in similar resource-limited settings.


Disruption in cholesterol metabolism, particularly hypercholesterolemia, is a significant cause of atherosclerotic cardiovascular disease. Large interindividual variations in plasma cholesterol levels are traditionally related to genetic factors, and the remaining portion of their variance is accredited to environmental factors. In recent years, the essential role played by intestinal microbiota in human health and diseases has emerged. The gut microbiota is currently viewed as a fundamental regulator of host metabolism and of innate and adaptive immunity. Its bacterial composition but also the synthesis of multiple molecules resulting from bacterial metabolism vary according to diet, antibiotics, drugs used, and exposure to pollutants and infectious agents. Microbiota modifications induced by recent changes in the human environment thus seem to be a major factor in the current epidemic of metabolic/inflammatory diseases . Epidemiological and preclinical studies report associations between bacterial communities and cholesterolemia. However, such an association remains poorly investigated and characterized. The objectives of this review are to present the current knowledge on and potential mechanisms underlying the host-microbiota dialogue for a better understanding of the contribution of microbial communities to the regulation of cholesterol homeostasis. Atherosclerosis is the underlying cause of the majority of cardiovascular disease (CVD) events, the complications of which can be fatal . According to WHO projections, exposure to multiple genetic and environmental risk factors and the growing number of dysmetabolic conditions  will contribute to making atherosclerotic cardiovascular disease (ACVD) a leading cause of death in the world by 2030 . Among tGenetics plays an important role in regulating the levels of TC and associated lipoproteins , yet genGenome-wide association studies (GWAS) have identified multiple human genetic variants contributing to plasma LDL-C and TC concentrations . These lReduction in body weight in severely obese subjects has a modest influence on TC and LDL-C, with each kilogram lost associated with a decrease of ∼0.8 mg/dL in LDL-C. When weight reduction is even higher , the cholesterol-lowering effect is even more pronounced . LDL-C cIn the last decade, research developments have positioned the commensal gut microbiota at the interface between living organisms and the environment and demonstrated its considerable influence on optimum metabolic functioning . One of More than 65 million years of mammalian-microbe co-evolution has led to an interdependence. The diversity of bacterial genes allows a wide variety of metabolic activities, such as energy extraction (5–10% of the daily energy requirements of the host) by digesting macromolecular complexes  that are not easily digestible by humans. Bacterial genes also allow the synthesis of vitamins , neurotrHowever, the complexity of intestinal microbial communities and their dialogue with the host’s metabolic pathways make functional connections complicated to disentangle in these pathologies. The fundamental challenge now is to understand the causal dimension of these relationships.Clostridiaceae/Lachnospiraceasefamilies being specifically associated with LDL-C  is positively associated with LDL-C levels .After birth primo-colonization of the digestive tract, the gut microbiota becomes richer and more diversified all through life as a result of environmental challenges such as those from nutritional status, cultural habits, and drug treatments . Thus, t-/-/Apoe-/-   .It is also of particular clinical interest to show the influence of the intestinal microbiota on the balance between absorption and cholesterol synthesis  since itvia the portal circulation. Each stage of this enterohepatic cycle is influenced by diet, hormonal cross-regulation, and bacterial activities that maintain a functional and non-toxic supply of bile acids in circulation. Indeed, BAs have a pro-inflammatory and cytotoxic potential when they are not regulated, due to their detergent activity and destabilization of membranes, as shown in cholestatic liver diseases (Bile acid (BA) biosynthesis is the predominant metabolic pathway for cholesterol catabolism in the human body. The conversion of cholesterol to bile acids is a process performed by a set of hepatic enzymes necessary for the conversion of the steroid nucleus of cholesterol, the elimination of the side chain, amidation on the side chain with either glycine or taurine  and eventually sulfonation or glucuronidation in the steroid backbone . Anotherdiseases Figure 1In the post-prandial period, primary BAs are released into the intestinal lumen. Due to their amphiphilic properties, primary BAs adsorb at an oil–water interface to form mixed micelles with other bile lipids , fat-soluble vitamins  and lipolysis products . In the absence of bile secretion, fat absorption is impaired . In the Another complexity in BA metabolism is the modification of the BA structure by intestinal bacteria . BAs thaIt should be noted that molecular species of BAs are involved not only in lipid metabolism but also in carbohydrate metabolism, energy homeostasis, and host immune responses through their agonistic or antagonistic activities on diverse receptors, the best characterized being the farnesoid X nuclear receptor (FXR) and the TGR5 membrane receptor ; these aIn the context of cholesterol reduction following antibiotic treatment, in humans , the preLactobacillus rhamnosus and plasma cholesterol and LDL-C levels. Interestingly, decreases in plasma cholesterol levels associated with acarbose treatment were associated with a decline in allistipes spp., in accordance with recent studies  led to improvements of glycemia and cholesterolemia . These c studies .in vivo remain difficult to disentangle, due to the large number of compounds and biological properties involved, including detergent and bactericidal activities and FXR signaling potential  are potential precursors of cholesterol synthesis that can be incorporated into the endogenous cholesterol synthesis pathway ) and the site of cholesterol conversion to coprostanol (colon) does not plead for a causal relationship. Nevertheless, normolipidemic subjects treated with Ezetimibe (NPC1L1 inhibitor) show residual absorption of cholesterol, which suggests as yet unidentified additional absorption mechanisms  inhibitors), new biotherapies targeting the microbiota are possible. Indeed, the data in the literature support the notion that the microbiota has a causal contribution to the metabolism of lipoproteins and host cholesterol. The mechanisms of this reciprocal influence need to be clarified, and the advent of functional analyses of the microbiota, the development of new technologies allowing the culture of anaerobic microbes, and the advent of more and more better-performing technologies will make it possible to specify the dynamics of the relationship of the intestinal microbiota with cholesterol metabolism.RV, PK, SB, MS, DR, MG, and PL originally conceived and wrote the manuscript. All authors read and approved the final manuscript.This study was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Sorbonne Université (SU), and the Fondation de France (00029519). PK was a recipient of a research fellowship from the French Ministry of Research and Technology and from the New French Atherosclerosis Society (NSFA). MS is supported by a fellowship from the Mexican National Council of Science and Technology (CONACYT).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.


Detection of pathogenic microbes as well as antibiotic residues in food animals, especially in chicken, has become a matter of food security worldwide. The association of various pathogenic bacteria in different diseases and selective pressure induced by accumulated antibiotic residue to develop antibiotic resistance is also emerging as the threat to human health. These challenges have made the containment of pathogenic bacteria and early detection of antibiotic residue highly crucial for robust and precise detection. However, the traditional culture-based approaches are well-comprehended for identifying microbes. Nevertheless, because they are inadequate, time-consuming and laborious, these conventional methods are not predominantly used. Therefore, it has become essential to explore alternatives for the easy and robust detection of pathogenic microbes and antibiotic residue in the food source. Presently, different monitoring, as well as detection techniques like PCR-based, assay (nucleic acid)-based, enzyme-linked immunosorbent assays (ELISA)-based, aptamer-based, biosensor-based, matrix-assisted laser desorption/ionization-time of flight mass spectrometry-based and electronic nose-based methods, have been developed for detecting the presence of bacterial contaminants and antibiotic residues. The current review intends to summarize the different techniques and underline the potential of every method used for the detection of bacterial pathogens and antibiotic residue in chicken meat. Globally, both developed and developing countries predominantly consume chicken as a meat product. According to the report of Global Livestock Counts, there are around 19 billion chickens in the world . ChickenCampylobacter and Salmonella are the common pathogenic microbes accounting for >90% cases of food poisonings associated with bacteria and are considered responsible for food safety hazards worldwide ).In the last few decades, there have been substantial improvements in the techniques used for identifying bacterial pathogens and antibiotic residue in food samples and especially in meat. Even though there are limitations associated with culture and microscopy, they are still the predominantly used detection techniques. Genetic and PCR are an effective non-culturable technique used for presently determining the bacterial pathogen, and on the other hand, MS techniques have emerged as an effective method for identifying microorganisms and detecting antibiotic residues. However, these approaches are limited to assess the pure cultures and are ineffective indeciphering the complex samples. To overcome this, chromatography-based methods like TLC and HPLC have been simplified and have eased the challenge associated with MS techniques. In the future, the progressive development and combination of these techniques and instruments will advance the ability to detect the pathogenic microbes and antibiotic residues.


Extracellular vesicles (EVs) are membrane-bilayered nanoparticles released by most cell types. Recently, an enormous number of studies have been published on the potential of EVs as carriers of therapeutic agents. In contrast to systems such as liposomes, EVs exhibit less immunogenicity and higher engineering potential. Here, we review the most relevant publications addressing the potential and use of EVs as a drug delivery system (DDS). The information is divided based on the key steps for designing an EV-mediated delivery strategy. We discuss possible sources and isolation methods of EVs. We address the administration routes that have been tested in vivo and the tissue distribution observed. We describe the current knowledge on EV clearance, a significant challenge towards enhancing bioavailability. Also, EV-engineering approaches are described as alternatives to improve tissue and cell-specificity. Finally, a summary of the ongoing clinical trials is performed. Although the application of EVs in the clinical practice is still at an early stage, a high number of studies in animals support their potential as DDS. Thus, better treatment options could be designed to precisely increase target specificity and therapeutic efficacy while reducing off-target effects and toxicity according to the individual requirements of each patient. The therapeutic efficacy of any pharmacological treatment depends on achieving an optimal drug concentration at the site of action. This is strongly influenced by the absorption, distribution, metabolism and excretion of therapeutic agents. Moreover, these processes do not exert a constant and predictable influence on the drug concentration at the site of action, but, on the contrary, are subject to a dynamic regulation. Exposure to other xenobiotics, changes in the diet and metabolism, and disease states may affect, for instance, the expression and activity of drug transporters and metabolizing enzymes and, ultimately, influence the concentration of a drug at its active site and thus, its therapeutic efficacy ,2. In thEV-based therapies could offer a significant advantage in the delivery of drugs to specific targets. This would contribute to the already on-going shift from the old paradigm of one treatment for all the patients with the same disease to a new paradigm where each patient receives a tailored therapy based on individualised parameters. This potential is reflected in the immense expansion of knowledge within this field in the past years, with high impact papers being continuously released and showing the diverse possibilities offered by EVs in different animal models. In this article, we review the latest  and most sound studies on this novel application of EVs as a drug delivery system (DDS). Reviewed articles comprise publications describing the use of EVs as drug carriers and studies where EVs represented the therapeutic agent, as important information on the biodistribution and tissue specificity can also be gained from the latter studies. Information is presented based on the logic work-flow followed towards designing an EV-based therapy. First, the techniques for isolation and purification of EVs will be presented. Afterwards, different strategies to load EVs with therapeutic agents will be discussed. Following, we will describe possible administration routes for EV-based therapy and how tissue specificity and bioavailability can be enhanced. These different phases of an EV-based therapy are summarized in EVs comprise a heterogeneous group of nanoparticles surrounded by bilayered phospholipidic membranes, which are released by eukaryotic and prokaryotic cells into the extracellular microenvironment . In eukaEVs carry proteins, nucleic acids, lipids and small molecules, whereby their specific cargo depends on the microenvironment and the pathophysiological state of the cell . The tra+ and CD4+ T-cells  link. After intravenous administration, the peptide-coated EVs exhibited a significant enrichment in the tumor tissue and displayed higher antitumor efficacy as compared to other nanoparticles or even free-doxorubicin . It mustThis strategy was also used as therapeutic alternative for glioblastoma multiforme. Both the blood-brain-barrier and glioma cells overexpress the low-density lipoprotein receptor on their surface. EVs were loaded with the anti-cancer agent methotrexate and conjugated with a peptide that targets the overexpressed lipoprotein receptor. Glioblastoma-bearing mice that were treated with these modified EVs showed a longer median survival period compared to the control groups including methotrexate alone and EVs loaded with methotrexate but without coating . NoteworSurface proteins play a key role in the interaction between EVs and target cells. For instance, intravenous injection of EVs from cardiac progenitor stem cells overexpressing the C-X-C chemokine receptor type 4 (CXCR4) improved cardiac function in a rat model of ischemia/reperfusion injury when compared with control EVs without CXCR4 overexpression . Additio+ breast cancer cells when administered intraperitoneally to mice [Another related strategy is the addition of antibodies to the EV surface. For instance, the display of an anti-HER2 antibody on the surface of the vesicles increased the delivery of EVs to HER2 to mice . A simil to mice . Similar to mice .DNA and RNA molecules displayed on the cell surface may also be used to increase target specificity. For instance, a DNA aptamer displayed on EVs from bone marrow stromal cells was used to enhance the delivery of EVs to the bone in mice which received EVs intravenously . SynthetThe glycosylation of surface proteins plays an important role in EV-cell interaction. It was described that the treatment of EVs with neuraminidase, which removes the terminal sialic acid residues of surface proteins, affects the distribution of EVs after administration to mice. A higher enrichment in the lungs was observed when neuraminidase-treated EVs were administered intravenously respect to the untreated EVs. Similarly, when neuraminidase-treated EVs were administered into the hock, the distribution among the lymph nodes displayed a different pattern respect to untreated EVs .A different strategy is modifying the surface of the EVs with cationized pullulan, a polysaccharide that consists of maltotriose-units. Tamura et al.,  used catTranslation of the previously described findings, mostly obtained in experimentation animals, to the clinical practice requires standardized good manufacturing practices (GMPs). Several challenges arise at all the steps of the production and have been recently reviewed by Wiest et al., . The selSo far, only a few studies described the production and characterization of clinical-grade EVs following GMPs. Previously, Kamerkar et al.,  highlighProduction of large volumes of high purity EVs is an important challenge. Here, a combination of tangential flow filtration (TFF) and size exclusion chromatography (SEC) exhibited significant advantages respect to other isolation methods . EVs carThe previous studies established production pipelines for clinical grade EVs and provide a proof-of-concept on the feasibility of different processes. Other studies described extensive quality controls (QCs) that would be required for industrial production. For instance, the study of Andriolo et al., describing GMP-compliant EV production from cardiac progenitor cells, described exhaustive QC tests. First, the stability of the cell source was controlled by establishing a post-production cell bank  only for QC purposes. Comparisons between this cell bank and the master cell bank using in the regular production constitute an important QC. Furthermore, the authors perform an extensive QC on two batches of EVs in terms of sterility, presence of endotoxin, particle concentration and size, expression of exosome markers and total protein. Also, functional checks  displaying comparable results with both lots of EVs were presented . LikewisHitherto, a wide variety of vesicles have been used to target different tissues and organs in experimentation animals. However, only a few of these vesicle types have been validated for clinical grade production. One critical aspect that needs special attention is, besides those presented above, the effect of interindividual variability of the cell donors on the batch-to-batch variability of the produced EVs. In addition, while GMP-grade culture media and supplements and TFF-mediated isolation led to the production and validation of different EV suspensions, it is yet unclear whether this constitutes a cost-effective alternative. Finally, more comparative studies addressing the impact of the different strategies for the preparation and storage of clinical grade EV formulations on their safety and potency are also required.Different high-quality studies performed in animal models support the potential of EVs as a DDS from the point of view of the safety and efficacy at the preclinical level. At the clinical level, the application of EVs is still at a very early stage. So far, 18 clinical trials involving therapeutic use of EVs have been registered at the NIH website  Table 3Table 3. In the last ten years, the understanding of the role of EVs in cell communication and their potential as a source of biomarkers have been acknowledged and further explored. More recently, there has been an increase in the body of evidence addressing EV secretion, biodistribution and specificity. These findings point to a significant potential of EVs to be used as a DDS. This novel delivery strategy shows high promise, although many questions remain unanswered regarding EVs, along with technical and experimental challenges that hinder the translation from the laboratory to clinical trials.One still remaining challenge is assuring the safety of EV-based treatments. Although several studies in animal models indicate a high tolerability and ongoing clinical trials will evaluate this in humans, further research should be performed to rule out potential deleterious effects of the carrier EVs in cell-cell communication. Hereby, the use of plant EVs may have an advantage by carrying a natural cargo less related to human EVs. While the delivery potential would still be present, the chance of triggering physiological or pathophysiological responses  may be lower.Another challenge regarding EV research, is target specificity. So far, several engineering possibilities have been evaluated. Unfortunately, while many studies nicely describe an increased targeting of certain organs and tissues, off-target delivery is not always evaluated. Furthermore, future research regarding surface molecules mediating the interaction between EVs and target cells will contribute towards increasing the delivery specificity for EVs. Hereby, the characterization of the surface proteome of the EVs and the surface glycan profile of target cells could significantly improve EV target specificity.In summary, the design of therapeutic strategies using EVs as a DDS will require the optimization of all the steps of involved in the EV pipeline. Safe and efficient sources of EVs, along with isolation and loading techniques that are cost-efficient and allow to scale production, in combination with the validation of proper administration routes and EVs that are specific for a particular target, remain the cornerstone in EV research. In this way, EVs could be proposed as a new alternative in precision medicine, aimed at increasing therapeutic efficacy and decreasing side effects, resulting in improved quality of life for the patients.


Background: Neuroimaging and neuropsychological studies have suggested that common features characterize both Gambling Disorder (GD) and Alcohol Use Disorder (AUD), but these conditions have rarely been compared. Methods: We provide evidence for the similarities and differences between GD and AUD in neural correlates of executive functions by performing an activation likelihood estimation meta-analysis of 34 functional magnetic resonance imaging studies involving executive function processes in individuals diagnosed with GD and AUD and healthy controls (HC). Results: GD showed greater bilateral clusters of activation compared with HC, mainly located in the head and body of the caudate, right middle frontal gyrus, right putamen, and hypothalamus. Differently, AUD showed enhanced activation compared with HC in the right lentiform nucleus, right middle frontal gyrus, and the precuneus; it also showed clusters of deactivation in the bilateral middle frontal gyrus, left middle cingulate cortex, and inferior portion of the left putamen. Conclusions: Going beyond the limitations of a single study approach, these findings provide evidence, for the first time, that both disorders are associated with specific neural alterations in the neural network for executive functions. In the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV)  and its Several studies have demonstrated the co-occurrence of one or more addictive behaviors , as well as cross-dependence  ,5,6. AccAlcohol Use Disorder (AUD) involves deficits in controlling alcohol consumption, extreme concern about alcohol, substance consumption even when it causes problems, the need to drink increasing amounts of alcohol to achieve the same effect, or the presence of withdrawal symptoms  when the individual quickly reduces or stops drinking . HarmfulFollowing the recent inclusion of GD among the spectrum of dependencies in the DSM-5 , a numbeThe present literature review suggests that the definition of “executive functions” includes a large umbrella of multiple processes and several, different definition of executive function exist, which refer to different cognitive and neuropsychological models. Therefore, we found that in studying executive functions in GD and in AUD, authors referred to different models and, accordingly, used different tasks to analyze executive functions. The problems of the absence of a homogenous definition of executive function and the large variety of tools used to assess them in the clinical population has already been underlined in other meta-analyses .Here, we provide a revision of the current neuropsychological and neuroimaging literature on AUD and GD, evidencing possible differences or common features of the two conditions. The above-reported literature suggests that both conditions yield alterations in executive functions and the associated brain networks. Thus, we perform a meta-analysis on the functional magnetic resonance imaging (fMRI) studies assessing executive functions  in individuals with AUD and GD with two main aims, namely (1) summarizing previous findings about the neural correlates of the two conditions in terms of the activation and/or deactivation of the brain areas devoted to executive control, and (2) providing new evidence for common and distinct neural mechanisms in these two conditions.The pathophysiology of GD, which seems to have common traits with drug addiction  and obseLikewise, patients with AUD have shown a compromised inhibitory process. The effects of alcohol on the brain are different: with low doses, it activates the pleasure areas linked to the release of endorphins; with increasing doses, however, alcohol has a depressive effect on the central nervous system . In partDifferent fMRI studies have analyzed neurofunctional alterations in decision-making processes in individuals with GD. In three studies, the Iowa Gambling Task was used ,48,49, wFurthermore, in contrast to HC, patients with GD showed greater activation in the right putamen before high risk choices than before safe choices. Gelskov and colleagues  found thWe used a systematic approach to review the literature and select appropriate articles for the present meta-analysis. The search for relevant literature was performed using PubMed and Web of Knowledge (Web of Science); the data included in this meta-analysis have been collected from early 2019 to February 2020 and the search was run simultaneously on these databases, using the following keywords and their combinations: fMRI AND “Gambling” OR “Alcohol” AND “Decision Making”, AND “Gambling disorder”, OR “Alcohol Abuse Disorder”, OR “MRI”, OR “Functional MRI”. This search produced 2461 papers. There were 2152 total records remaining after the duplicates were removed.These articles were then screened according to our a priori inclusion criteria for papers, which were as follows: (1) whole-brain activity analysis performed using functional magnetic resonance imaging (fMRI); thus, we excluded positron emission tomography (PET) studies and excluded papers reporting only results from region of interest (ROI) analyses; (2) the coordinates of the activation foci were provided either in the Montreal Neurological Institute (MNI) or Talairach  referencThe flowchart of article selection is shown in http://brainmap.org/ale). It is a powerful tool to integrate the neuroimaging literature where relevant regions of activations are identified across a series of studies. Specifically, peak coordinates are obtained from studies that share a common feature of interest, such as specific tasks or processes. The ALE algorithm has been applied in many aspects of normal brain function [The meta-analysis was performed using the activation likelihood estimation (ALE) approach ,97,98; Afunction ,100,101,function ,103. A cfunction , thus emfunction .p < 0.001 and a cluster-level threshold of p < 0.05 Familywise Error (FWE)corrected [The Talairach coordinates of the studies included in the ALE meta-analysis were automatically converted into MNI coordinates using GingerALE. According to the modified procedures of Eickhoff and colleagues , the ALEorrected . For eacThe ALE meta-analysis on GD  revealedThe ALE meta-analysis on AUD  revealedExecutive functions encompass several cognitive processes, such as decision-making, response inhibition, conflict monitoring, cognitive flexibility, and their possible relationship with reward-related decision-making processes . ExecutiOur results revealed clusters of abnormal activation in GD bilaterally in the head and the body of the CN, in the right LN (including the putamen), and the right MFG. These results are in accordance with previous studies ,107,108.The striatum has been widely reported to be involved in the subjectively discounted value of delayed rewards and in the anticipation of monetary rewards ,48,54,83In this vein, GD may arise from the imbalance between the dopaminergic system, including limbic motivational areas and neural pathways connecting the frontal lobe regions with the basal ganglia , which results in impaired cognitive control, leading to progressive loss of control over gambling behaviors .The present results revealed three clusters of activation in individuals with AUD, mainly located in the PCC extending to the precuneus, in the right MFG, and in the left putamen. The highest activation we detected in the left putamen may have been related to the computed value and/or hedonic elements of making an enhanced risky choice as monetary earnings in individuals with AUD. These areas receive both glutamatergic and dopaminergic inputs and have been involved in developing and maintaining alcohol dependence, consistent with behavioral evidence that alcohol-dependent individuals show a greater sensitivity to earning experiences in support of the future risky search for a reward. The greater activation we found in the occipital areas might be explained by the fact that those with AUD needed to recruit more resources in the visual area to inhibit processing of alcohol-related pictures . We alsoThe most accredited neurobiological hypothesis for addictive behavior is dysfunction in the “cortico-striatal reward pathway”, which includes areas such as the vs. and mPFC . fMRI stOur results also provide converging evidence for the deactivation in AUD of different brain circuits, including areas of the mPFC, the superolateral prefrontal cortex (sPFC), the ACC, and the striatum. These results are consistent with the theory of reward deficiency syndrome (“RDS”) , which hHere, we detected interesting findings about the direction of functional alterations in individuals with AUD and GD that have not been previously described. We found that GD was mainly associated with the higher activation of the fronto-striatal circuit . In contrast, AUD was associated with the higher activation of the occipital cortex and of the superior portion of the putamen, as well as with the deactivation of the fronto-striatal network . Thus, GD seems to be associated with an activation of the reward network, whereas AUD seems to be associated with both the deactivation and activation of the different nodes of this circuit. The direct comparison between the two conditions  is currently unfeasible due to the paucity of the studies meeting the inclusion criteria for an ALE meta-analysis. In addition, the state of the literature does not allow a clear distinction between the different executive processes involved, just as it is not possible to determine which is the most appropriate task to measure them. For such a reason, it has not been possible to distinguish between different sub-processes of the executive functions, because often the available studies do not describe what aspect of the executive functions has been investigated. This is due to the paucity of studies, and it represents both a limitation of the present study and the current modelling of executive function itself. Future studies should clarify and shed light on which aspects of the executive functions are really lacking in GD and AUD.


Force attained during concentric contraction (active shortening) is transiently enhanced following eccentric contraction (active stretch) in skeletal muscle. This phenomenon is called stretch-shortening cycle (SSC) effect. Since many human movements contain combinations of eccentric and concentric contractions, a better understanding of the mechanisms underlying the SSC effect would be useful for improving physical performance, optimizing human movement efficiency, and providing an understanding of fundamental mechanism of muscle force control. Currently, the most common mechanisms proposed for the SSC effect are (i) stretch-reflex activation and (ii) storage of energy in tendons. However, abundant SSC effects have been observed in single fiber preparations where stretch-reflex activation is eliminated and storage of energy in tendons is minimal at best. Therefore, it seems prudent to hypothesize that factor(s) other than stretch-reflex activation and energy storage in tendons contribute to the SSC effect. In this brief review, we focus on possible candidate mechanisms for the SSC effect, that is, pre-activation, cross-bridge kinetics, and residual force enhancement (RFE) obtained in experimental preparations that exclude/control the influence of stretch-reflex activation and energy storage in tendons. Recent evidence supports the contribution of these factors to the mechanism of SSCs, and suggests that the extent of their contribution varies depending on the contractile conditions. Evidence for and against alternative mechanisms are introduced and discussed, and unresolved problems are mentioned for inspiring future studies in this field of research. When we attempt to jump as high as possible, we naturally make a countermovement  before the main movement , force after stretch (transient force) increases, a phenomenon referred to as stretch activation . BecauseIn addition to the cross-bridge kinetics, evidence seem to suggest that there are other factors that contribute to the SSC effect. For example, in preparations in which stretch-reflex activation and tendon compliance were eliminated, some SSC effect was still present even after introducing a long pause between the stretch and shortening phases of SSCs ,b. This Residual force enhancement is a property of skeletal muscle characterized by an increase in the steady-state isometric force after an active stretch compared to the corresponding purely isometric steady-state force attained at the same muscle length and activation level . AlthougSince RFE is induced by stretching an active muscle, it seems reasonable to speculate that REF is induced during SSCs. Previous studies revealed that the force at the end of a stretch at a given speed and final muscle length increased with increasing stretch magnitude . FurtherIn order to determine the possible contribution of RFE to the SSC effect, it is essential to distinguish between the contribution of the RFE and all the other possible sources, such as, the effects of stretched cross-bridges, stretch-reflex activation, and storage/release of energy in tendons and other series elastic elements of muscles. To achieve conditions where only RFE could contribute to the SSC effect, we conducted experiments in skinned fiber preparations where the storage/release of elastic energy is minimal and stretch-reflex activation is fully abolished. Furthermore, by inserting an appropriate pause between the stretch and shortening phase of SSCs, any effect of elongated cross-bridges could also be eliminated. Providing a pause of 2 s still resulted in greater force at the onset of shortening and greater work during shortening for SSC conditions compared to conditions where shortening was not preceded by muscle stretching  should affect the magnitude of SSC effect because the effect of pre-activation should become smaller when the shortening velocity is slow enough to develop the force after initiating the shortening without pre-activation , 2016.In addition, stretching velocity would also affect the magnitude of the SSC effect. The reason for the velocity dependence may be associated with the recent observation that high velocities of stretch that resulted in muscle slippage, were associated with a decrease in RFE compared to the RFE obtained for slow and moderate stretch velocities . Thus, iMoreover, fiber type might affect the SSC effect because of differences in cross-bridge kinetics between fast and slow twitch fibers  and possIf cross-bridge and RFE (titin) contribute to the SSC effect, the relative contribution of these factors can be identified by changing the cross-bridge kinetics while maintaining titin function or vice versa. This may be achieved, for example with so-called reduced force conditions in which cross-bridge action is submaximal due to submaximal activation, fatigue, chemical inhibition, or genetic modifications. For these conditions, one would expect the contribution of myosin cross-bridges to decrease while the contribution of RFE associated with passive structures, such as titin or myosin binding protein C, might be preserved. We recently confirmed that the magnitude of RFE was preserved in reduced force conditions including those achieved by chemical inhibition of myosin cross-bridges. Thus, it is reasonable to assume that contributions to the SSC effect not produced by cross-bridges, for example titin, become more prominent in reduced force states. This hypothesis has been tested using a myosin inhibitor, blebbistatin, and it was found that the SSC effect was still observed even though the active force was substantially decreased . SimilarIn this review, we addressed the effects of pre-activation, cross-bridge kinetics, and RFE as possible candidates for the SSC effect. However, we would like to emphasize that the laboratory experiments on isolated muscles and fibers may not necessarily reflect what happens in natural SSCs during human movement. For example, in a countermovement jump, the triceps surae muscles are passively elongated in the initial phase and then actively stretched in the latter phase of the countermovement. Although the triceps surae muscles are stretched in this countermovement phase, the corresponding muscle fibers remain isometric or may even shorten when force increases in this phase of the movement . It is uAn additional consideration that may affect the SSC effect is the muscle length at which the SSCs occur. In our experiments on single skinned fiber preparations, we typically worked on the descending limb of the force-length relationship where RFE is known to be greater than at shorter (ascending and plateau regions) fiber lengths . Muscle Taken together, these findings discussed above suggest that results obtained in reduced preparations  under laboratory conditions may not necessarily reflect the conditions experienced in SSCs that occur during natural human movements. Therefore, to gain better insight into the mechanics and force response of muscles during SSCs that occur in human movement, experiments adopting more physiological contractile and activation conditions should be performed, in parallel with basic experiments in reduced preparations and well-controlled contractile and activation conditions to elucidate the molecular mechanisms underlying the cross-bridge kinetics and/or the role of RFE in SSCs.in vivo human voluntary muscle testing. The mechanisms involving cross-bridge kinetics and RFE introduced in this brief review will need direct and independent evaluation before they can be fully accepted/rejected. However, it is our hope that some of the initial support for cross-bridge and RFE based mechanisms may inspire research in this exciting area of muscle mechanics, and may lead to a better understanding of SSCs in skeletal muscle.There is accumulating evidence that stretch-reflex activation and storage/release of energy in tendons cannot explain the entirety of the SSC effect. It appears that contractile and structural protein-based mechanisms, which have received little attention to date, may play a significant, possibly a dominant role in producing the SSC. Since protein-based mechanisms likely involve the contractile proteins actin and myosin and the structural protein titin, it may be difficult to elucidate the mechanism underlying the SSC effect using research based on All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cry for Help”. From this way, plants have been co-evolved with their microbiota adapting to local environmental conditions to ensuring the survival of the entire holobiome to improve plant fitness. Thus, the strong selective pressure of native extreme microbiomes could represent a remarkable microbial niche of plant stress-amelioration to counteract the negative effect of climate change in food crops. Currently, the microbiome engineering has recently emerged as an alternative to modify and promote positive interactions between microorganisms and plants to improve plant fitness. In the present review, we discuss the possible use of extreme microbiome to alleviate different stresses in crop plants under the current scenario of climate change.In the current scenario of climate change, the future of agriculture is uncertain. Climate change and climate-related disasters have a direct impact on biotic and abiotic factors that govern agroecosystems compromising the global food security. In the last decade, the advances in high throughput sequencing techniques have significantly improved our understanding about the composition, function and dynamics of plant microbiome. However, despite the microbiome have been proposed as a new platform for the next green revolution, our knowledge about the mechanisms that govern microbe-microbe and microbe-plant interactions are incipient. Currently, the adaptation of plants to environmental changes not only suggests that the plants can adapt or migrate, but also can interact with their surrounding microbial communities to alleviate different stresses by natural microbiome selection of specialized strains, phenomenon recently called “ According to the Organization for Economic Cooperation and Development (OECD), agriculture is considered one of the most essential economic, social and environmental activities for human beings. Until now, agricultural and livestock products has been able to respond to the growing world demand. However, it is estimated that by 2050 the world population would reach 9.7 billion people and that crop productivity should increase approximately 60–100% to meet this demand of food . This isTo date, many studies have investigated biotechnological solutions to counteract the negative effect on crop yield. Since the last 20 years, isolation through culture-dependent techniques and inoculation of cultivable Plant Growth-Promoting Microorganisms (PGPM) have substantially improved plant fitness under laboratory conditions . HoweverCry for Help” , reduce electron transport rate (ETR) and limit the productivity of some crops  combined with salycilic acid to improve Helianthus annuus resistance. Recently, Bacillus sp. and Enterobacter sp.) with a high potential to lag the effects of drought on wheat (Triticum aestivum) and corn (Zea mays) seedlings. In the case of arbuscular mycorrhizal fungi (AMF), for example, Phoenix dactylifera against water deficit, finding a significant increase in peroxidase and polyphenolxidases in the roots of mycorrhized palms enhancing the survival of plant in these stress conditions. In addition, Leymus chinensis and in C4 grass Hemarthria altissima under water deficit condition. Our current knowledge about the role of AMFs to help plants withstand drought stress is mainly involved in direct absorption of water by hyphae and their transfer to the host plant, increasing water content and eliminating the generation of ROS and the production of biomolecules and enzymes with antioxidant capacity  under low-temperature stress accelerated the biosynthesis of strigolactone to recruit AMF (Glomus intraradices). On the other hand, in a study published by Triticum aestivum subsp. aestivum, using host-mediated microbiome engineering as a strategy to improve the resistance of this crop to drought stress. therefore, this phenomenon could eventually be associated with different abiotic stress such as high radiation, freezing, among others.Currently, several studies have been highlighted the important role of root endosphere, which are able to recruit desirable microorganism from soil to improve plant fitness and yields of crops . To datensferred . Thus, pnsferred , where por Help” . AlthougPseudomonas syringae, increase the secretion of L-malic acid toward the rhizosphere, selectively recruiting the rhizobacterium Bacillus subtilis promoting the formation of biofilms in the roots and consequently increasing plant defenses  that increases the expression of L-malic acid toward the rhizosphere which caused a systemic resistance response induced in plants against pathogen by recruiting of B. subtilis. In addition, a study by A. thaliana recruit three bacterial phylum  in the rhizosphere after activation of foliar defense by the downy mildew pathogen, Hyaloperonospora arabidopsidis.The modification of the microbial communities is given by the alteration of the radical exudation profiles of primary and secondary metabolites with biocidal and/or semiochemical activities, which influences the microbiome recruiting specific microorganisms and their microbial activities . This redefenses . Some yeE. cloacae, Stenotrophomonas maltophilia, Ochrobactrum pituitosum, Herbaspirillum frisingense, Pseudomonas putida, Curtobacterium pusillum, and Chryseobacterium indologenes) identified by culture-dependent techniques. The authors showed that elimination of E. cloacae led to the complete loss of the community, suggesting an important role of the key species in the functioning of the total community. Studies conducted by Thus, currently it is well known that the survival of plants depends on the interaction with the holobiont. However, the protective function is not carried out by all microorganisms, but rather by some microbial groups that, due to additive or synergistic effects, are fundamental to the functioning of this protection network. These key microorganisms within the microbiome, called “core microbiome”, forms strong facultative and mutualistic interactions with the other microorganisms inside the microbiome to generate the protective effect. The core microbiome concept was introduced in human microbiome studies to define a relatively stable community that contributes to important biological functions . The corThe plant holobiome has been developed over the centuries to adapt to the different terrestrial biomes. Cold environments, such as Antarctic, and dry environments, such as Deserts, have aroused great curiosity regarding the assembly of microbial communities and microbe-plant interactions. Although the study of the microbiome in extreme environments is still an incipient area, some authors have begun to identify the complex interactions between the microbiome and vegetation associated with these hostile environments. For example, desert  represent about a third of the planet’s biomes, which are characterized by significant absence of rainfall, extreme fluctuations in temperature, generally low nutrient status, high levels of incident UV radiation and strong winds . The micMeloidogyne incognita) to varying degrees. Moreover, Setaria italica L.), a drought-tolerant crop grown in semi-arid regions in northeast China. They observed that four isolated strains had the ability to generate ACC deaminase, as well as tolerance to drought. In the case of microbiome, some plant-associated microbiomes have been identified in deserts habitats, for example, Agave species  Gentry, and Agave deserti Engelm) distributed in central Mexico and in southern California, finding microorganisms capable of conferring resistance to high temperatures and low water availability. Moreover, Cactaceae) native to arid and semi-arid ecosystems which also are represent microorganisms capable of conferring drought resistance.Some authors have investigated the use of PGPM from these environments, for example, Deschampsia antarctica and Colobanthus quitensis  have been identified in leaves of both vascular plants that could directly or indirectly promote the fitness of host plants (C. quitensis modulate the content of salicylic acid, jasmonate, indole-3-acetate and ABA in shoot tissue of plants exposed to UV-B radiation, which would indicate that these endophytic fungi could modulate the hormonal content of C. quitensis to improve its ecophysiological performance under high UV-B radiation. Thus, the plant-associated microorganisms can play a crucial role to ensure the plant survival (To date, some studies have identified the role that extreme microorganisms play in the nutrition and survival of these vascular plants. For example, Antarctic tarctica . In the he plant . Moreovet plants . Recentlsurvival .D. antarctica that inhibit Botrytis cinerea, and Everniastrum cirrhatum lichen have important antimicrobial properties against Fusarium moniliforme, Fusarium oxysporum and Fusarium udum. On the other hand, C. quitensis and D. antarctica to improve the net photosynthetic rate and water absorption under drought conditions in cultivars of Lactuca sativa L. var. Longifolia. In addition, a recent study conducted by Arthrobacter and Planoccocus and two endophytic root fungi Penicillium chrysogenum and Penicillium brevicompactum that worked effectively to reduce saline stress in pepper, lettuce, onion and tomato plants. The results of these experiments demonstrate a high capacity to enhance crop yield under biotic and abiotic stresses by some microorganisms. However, as discussed earlier, the use of few microorganisms generally represents low success in the field due to the low adaptability and competition of introduced microorganisms with the native microorganisms.Nowadays, the use of Antarctic microorganisms to enhance the yield of agricultural crops under this new scenario of climate change is more frequent. In the case of Emerging Infectious Disease, D. antarctica and C. quitensis can modify their rhizosphere microbiome under different stress conditions. Similarly, A recent study by Azospirillum sp. improves the efficiency of nitrogen absorption in soil, influencing the metabolic activity of other microbial communities (Azospirillum sp. and Pseudomonas sp. can increase in the diversity of microbial communities in wheat crops, which produces large changes in profiles of carbon-source utilization modifying the soil carbon pools, generating labile organic matter and increasing soil fertility (In the soil, the microbial communities are responsible for nutrient cycling, absorption and release of nutrients, mineralization and processing of organic and inorganic compounds . These pmunities , 2012. Bertility .In the case of complete microbiomes, the information is limited and even more in terms of agroecosystems. Some studies have begun to analyze the effect of microbiome transferring in different scenarios. For example, The new metaomics techniques have significantly increased our knowledge about the dynamics and abundance of microbial communities in soil-plant systems. However, the complex interactions between microorganisms-plants-soil is largely unknown. In the current scenario of climate change, a new green revolution is required to achieve future food security, with new concepts and approaches to achieve a more sustainable development of agriculture. In this context, the study and use of complete microbiomes or core microbiomes from extreme environments could represent a promising alternative to increase crop yields under different stress conditions. These microorganisms could transfer their innate resistance to agricultural crops. Furthermore, news formulations of bioinoculants composed of microbiomes or core microbiomes could eliminate the ecological barriers imposed by native microbial communities in soil, increasing the persistence of added microorganisms. In addition, the formulation of bioinoculants from the natural selection imposed by plants, through the phenomenon of cry for help, where the plant in stress situations selects its microbiome represents an eco-friendly alternative for a new generation of bioinoculants taking advantage of the closed relationship between the microbiome and their hosts, which could represent an excellent alternative to improve the plant fitness to counteract the climate change effects.RR wrote the main manuscript text. PD critically revised the manuscript and approved the final version.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.


Bioelectrochemical systems are revolutionary new bioengineering technologies which integrate microorganisms or enzymes with the electrochemical method to improve the reducing or oxidizing metabolism. Generally, the bioelectrochemical systems show the processes referring to electrical power generation or achieving the reducing reaction with a certain potential poised by means of electron transfer between the electron acceptor and electron donor. Researchers have focused on the selection and optimization of the electrode materials, design of electrochemical device, and screening of electrochemically active or inactive model microorganisms. Notably, all these means and studies are related to electron transfer: efflux and consumption. Thus, here we introduce the basic concepts of bioelectrochemical systems, and elaborate on the extracellular and intracellular electron transfer, and the hypothetical electron transfer mechanism. Also, intracellular energy generation and coenzyme metabolism along with electron transfer are analyzed. Finally, the applications of bioelectrochemical systems and the prospect of microbial electrochemical technologies are discussed. Bioelectrochemical systems, revolutionary new bioengineering technologies, integrate microorganisms or other bio-based catalysts with an electrochemical method to improve the reducing or oxidizing metabolism. Generally, bioelectrochemical systems show the process of electrical power generation or achieve the reduction reaction with a certain potential poised by means of electron transfer between the electron acceptor and electron donor .Previously, bioelectrochemical systems have been widely applied in the form of microbial fuel cells (MFCs), since Michael To expand the application of bioelectrochemical systems, researchers have focused on the selection and optimization of the electrode materials, design of the electrochemical devices, and screening of electrochemically active or inactive model microorganisms. Recently, bioelectrochemical systems have been widely applied in nitrate removal, solid waste processing, desalination and materials science . NotablyHere, we first introduce the basic concepts of bioelectrochemical systems, and elaborate on the mechanisms of extracellular and intracellular electron transfer. We also analyze the hypothetical electron transfer mechanism, intracellular energy generation and coenzyme metabolism. Finally, the applications of bioelectrochemical systems and prospective microbial electrochemical technologies are discussed.An electrochemical system is a series of electrochemical models, or devices, in which electronic behaviors occur with different types of catalysts. In terms of bioelectrochemical systems, it mainly describes a series of technologies that are used for biotechnology applications including electricity generation  and the via an external circuit to be used for the reduction of oxygen, or another electron accepter, with the generation of current . MFCs, are electrochemical systems with microorganisms acting as biocatalysts in the anode chamber, have been widely used for electricity generation with various substrates . In MFCs current . Recentl current .Shewanella putrefaciens  to an electrode was achieved  mediate the direct electron transfer between cells and electrodes  through cell growth and is directed involved in the extracellular electron transfer between cell and electrodes  or the assistance of cytochromes . MoreoveGeobacter metallireducens and Geobacter sulfurreducens can achieve higher rates of ethanol consumption and better cell growth as more energy is generated  consists of a series of electron or proton carriers, including cytochromes, coenzyme Q and lots of oxidoreductases. Within the ETC, electrons are transferred from high potential electron donor to a low potential electron acceptor along with ATP synthesis .Shewanella and Geobacter have an excellent ability for intracellular electron transfer, and thus are widely used in MFCs  reduction, and nanowire used for interactions with electrodes  in E. coli can reprogram the intracellular metabolism and accelerate the intracellular electron transfer rate by 183%  and ATP play a vital role in intracellular redox metabolism, metabolite synthesis and transportation, stress responses and transcriptional regulation  and drive ATP synthesis .Compared with aerobic respiration, the energy generated by oxidative phosphorylation is not enough for cell growth at high rates under anaerobic conditions. The main reason is that due to the low supply level of intracellular ATP, as the intermediate metabolites acted as electron acceptors they were not matched by oxygen levels . GeneralThe concentration of co-enzymes, NADH or NADPH, represents the level of intracellular reducing power and are involved in many oxidation-reduction reactions. As redox reactions occur inevitably along with electron generation and consumption, the release or capture of electrons in bioelectrochemical systems will disturb the intracellular steady state environment and cause a shift of metabolic flux .Geobacter can catalyze fumarate reduction with the cathode electrode as the sole electron donor, and this reduction was catalyzed by fumarate dehydrogenase (NADH-dependent)  . For elecarriers . Meanwhicarriers .Lactococcus lactis, homolactic fermentation switched to mixed acid fermentation to keep the balance of intracellular reducing power along with the electricity power output  also uses electrochemistry to affect microbial metabolism. The electron transfer in either anodic EF or cathodic EF can regulate the ORP and the NADtabolism . RecentlL-lysine . CathodiL-lysine .Photosynthetic MFCs that combined photosynthesis and generation of electric energy, have also gained much attention recently due to their more sustainable energy production than that of non-photosynthetic MFCs . The micvia extracellular and intracellular electron transfer chains would extend the future application of bioelectrochemical systems.All types of microbial electrochemical technologies are based on the energy interchange: chemical energy into electrical energy (MFCs), electric energy into chemical energy (MECs) and solar energy into electrical energy (Photosynthetic MFCs). For all types of bioelectrochemical systems, energy conversion efficiency is the key factor that determines bioelectrochemical system performance, especially the energy conversion step in which electrical energy is involved. Electron transfer plays an important role in this step, which it is implied is a future direction for METs research. The electron’s behavior, intracellular reducing power and energy metabolism in bioelectrochemical systems is of increasing concern in the context of precise regulation of fermentation and degradation. Understanding the mechanism of electron transfer TZ, JL, and YJ wrote the manuscript. WZ, YF, FX, WD, PW, and MJ provided literature and data. JM contributed to the writing of the manuscript and the overall manuscript design.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.