Identifying biomarkers of developing mental disorder is crucial to improving early identification and treatment—a key strategy for reducing the burden of mental disorders. Cross-frequency coupling between two different frequencies of neural oscillations is one such promising measure, believed to reflect synchronization between local and global networks in the brain. Specifically, in adults phase–amplitude coupling (PAC) has been shown to be involved in a range of cognitive processes, including working and long-term memory, attention, language, and fluid intelligence. Evidence suggests that increased PAC mediates both temporary and lasting improvements in working memory elicited by transcranial direct-current stimulation and reductions in depressive symptoms after transcranial magnetic stimulation. Moreover, research has shown that abnormal patterns of PAC are associated with depression and schizophrenia in adults. PAC is believed to be closely related to cortico-cortico white matter (WM) microstructure, which is well established in the literature as a structural mechanism underlying mental health. Some cognitive findings have been replicated in adolescents and abnormal patterns of PAC have also been linked to ADHD in young people. However, currently most research has focused on cross-sectional adult samples. Whereas initial hypotheses suggested that PAC was a state-based measure due to an early focus on cognitive, task-based research, current evidence suggests that PAC has both state-based and stable components. Future longitudinal research focusing on PAC throughout adolescent development could further our understanding of the relationship between mental health and cognition and facilitate the development of new methods for the identification and treatment of youth mental health. In Australia, mental disorders were estimated to have a direct economic cost of up to $51 billion and a further $130 billion cost as a result of diminished well-being during the 2018–2019 period . Mental Traditional psychiatric approaches that focus on the identification and treatment of established mental disorders have limited application for developing psychopathology . Thus, uThe EEG has been used to analyze the rhythmic, oscillatory activity of the human brain for nearly 100 years since Cross-frequency coupling between two different frequencies of neural oscillations is hypothesized to facilitate communication between meso and microscales within regions, and synchronization and interaction between local and global networks in the brain inter-regionally . ThroughOne of the most researched areas in the context of PAC is memory. Numerous adult EEG studies have implicated theta–gamma PAC as a neural substrate of visual and auditory working memory . Theta–gOne popular model of working memory suggests that individual gamma waves represent memory items, which are “nested” in theta, enabling the retention of multiple items. See Other complex areas of cognition that have been associated with PAC in adults include language, attention, and fluid intelligence. PAC has been associated with linguistic processes such as verb generation , linguisPhase–amplitude coupling is also implicated in selective attention in adults e.g., . GonzaleOne of the most interesting developments in the PAC literature is that both temporary and lasting changes can be elicited by contemporary treatment modalities such as tACS (mentioned above), transcranial direct current stimulation (tDCS), and transcranial magnetic stimulation (TMS). mean age = 23.2 and SD = 4.9) with first-episode psychosis and adults with neuroleptic-naïve schizophrenia, respectively. Both studies identified increased theta–gamma PAC at rest within regions known to be associated with the default mode network (DMN) in patient groups compared to controls. These results suggest people with psychosis have dysfunctional hyperactivation of resting-state theta–gamma PAC within DMN-related brain regions, which may be a result of compensatory reallocation of cognitive resources due to dysfunction in the prefrontal cortex. Currently, research investigating the links between mental health and PAC is emerging. A number of studies have investigated the relationship between PAC and psychotic disorders. Another study compared theta–gamma PAC between young people with ADHD and healthy controls. The ADHD group demonstrated significantly reduced PAC within diffuse regions across the cortex during an arithmetic task compared to the controls. Deficits in PAC were associated with reduced performance on the arithmetic task. The authors hypothesized that reductions in PAC reflected deactivation of the DMN during the task, but failure to shift to attentional networks . Althougin vivo analysis of WM using contemporary MRI techniques such as diffusion tensor imaging (DTI) abnormalities in WM microstructure have been established across a range of mental disorders , there does not appear to have been any large-scale studies that have investigated PAC longitudinally, from a developmental perspective. As adolescence is known to be a critical period for the development of mental disorders, recent evidence that PAC is associated with mental health demonstrates a clear requirement for PAC research throughout this period. Understanding the emergence of pathology is critical for understanding the biological basis of mental illness. Because initial PAC research focused on task-based cognitive studies, some initial perspectives suggested that PAC is a transient, state-based measure, whereas other forms of CFC may be more steady and suited to longitudinal research. For example, amplitude–amplitude coupling (AAC) between lower frequency oscillations was suggested to be particularly suited to longitudinal research as it demonstrated trait like properties associated with motivational and emotional processes, as well as anxiety . KnyazevHowever, little is known about AAC as it has received relatively little attention in the literature compared to PAC. Recent evidence demonstrating that PAC is associated with mental health and cognition, including at rest and pre-post intervention, illustrates that PAC clearly has persistent, stable components equally suited to longitudinal research. This emerging research suggesting that PAC may be an important biomarker of mental health highlights the importance of longitudinal research investigating PAC throughout the rapid differential structural brain changes that occur during adolescence. Longitudinal research to determine whether PAC demonstrates similar non-linear growth trajectories to AAC and how these are associated with adolescent cognitive development and mental health may be crucial to better understanding how PAC research can contribute to better understanding mental health.It is important to also acknowledge potential confounds in PAC research. Specifically, the susceptibility of PAC measures to spurious identification of PAC has recently been highlighted . NumerouResearch increasingly suggests that PAC is a fundamental component of human information processing, involved across a range of cognitive processes. Recently, PAC has been implicated in mental health as well, with a number of studies identifying abnormalities in PAC as features of psychotic disorders and ADHD. Decreased symptoms of depression after TMS were also associated with an increase in PAC. More in-depth research is required to fully understand PAC’s role in these disorders, as well as similar research investigating whether abnormalities in PAC are implicated across a range of other mental disorders. However, PAC is believed to be closely associated with neuroplasticity and thus likely critical across mental disorder and treatment. Current findings suggest that better understanding PAC could potentially lead to a better understanding of mental health. While our knowledge regarding PAC is growing, the majority of research to date has focused on cross-sectional adult samples. As much of the initial PAC literature focused on task-based cognitive research, some initial perspectives suggested that PAC is useful only as a transient, state-based measure. However, it is now evident that PAC has both state-based and stable components, considering current evidence demonstrating PAC’s association with mental health at rest and changes over time pre- and post-TMS treatment.Further research is required to understand PAC during adolescence—a critical period for mental health in which early identification and treatment has been identified as a key strategy for reducing the burden of mental disorder. Longitudinal research focusing on PAC throughout adolescence could help pinpoint the development of psychopathology and the links between cognition and mental health during this period. Combining EEG PAC analysis with DTI for concurrent assessment of structural and functional connectivity may provide the first insights into the relationship between WM microstructure and PAC. For example, coupling between the phase of theta oscillations in prefrontal cortex to the amplitude of gamma oscillations in posterior visual cortex may correspond to increased structural connectivity between these regions. Such research may lead to a greater understanding of the relationship between structural and functional mechanisms underlying information processing and mental health throughout adolescent development. Considering the recently discovered association between PAC and contemporary treatment methods such as TMS, such research has the potential to facilitate a better understanding of the mechanisms underlying such treatment and lead to improved application of these treatments for mental health disorders in the future.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.


Gout is an inflammatory arthritis, which causes intense, acute pain due to the buildup of uric acid crystals in synovial fluid. The gold standard for gout diagnosis consists of synovial fluid analysis by polarized light microscopy, which is costly, time-intensive, and technique-dependent, therefore meriting a more efficient, inexpensive, and accessible method for diagnosis. We previously developed and validated a novel colorimetric gout detection method and device based on the reduction of silver nitrate by uric acid; here, we clinically validated our method and device using arthroscopically obtained synovial fluid samples from gout patients. We successfully identified uric acid crystals in clinical samples via our colorimetric method, visualized uric acid crystals in synovial fluid via handheld microscopy, and determined that silver nitrate stain did not interfere with the microscopic visualization of uric acid crystals necessary for diagnosis. We also developed and validated a method of processing turbid clinical samples for use in our device to prevent the obscuration of uric acid crystals by suspended material. Our method and device will clinically facilitate the immediate colorimetric diagnosis of gout and the subsequent bedside visualization of uric acid crystals in both ideal and turbid synovial fluid samples, allowing for a point-of-care diagnosis of gout. Gout is an inflammatory arthritis characterized by the buildup of uric acid crystals in synovial fluid, which can cause long-term destruction of joints if severe or recurrent. Ordinarily, uric acid is excreted either through the kidneys or through the gastrointestinal tract, but in patients with high blood levels, uric acid crystallizes with sodium, causing monosodium urate crystals to deposit in joints, tendons, and surrounding tissues . Gout afOn initial presentation of an acutely swollen, painful joint, differential diagnoses include—but are not limited to—gout, pseudo gout, and septic arthritis. Accurate diagnosis is imperative to provide adequate management of the patient’s condition and to prevent recurrence as well as to rule out more serious pathology, such as septic arthritis, which can progress to systemic involvement. As urate crystals in synovial fluid are not visible to the naked eye, polarized light microscopy of arthroscopically obtained synovial fluid is used to diagnose gout . SynoviaThe method of gout diagnosis via polarized light microscopy is expensive, as proper microscopy equipment and trained technicians are required for synovial fluid analysis. Although the method is 100% specific for gout in the proper clinical setting, it is only 85% sensitive. One barrier associated with the technique’s sensitivity may be related to technician/physician error in identifying urate crystals, as crystals are only identified correctly 81% of the time when inspected by trained professionals . AdditioVarious spectroscopic uric acid detectors are on the market which identify elevated serum uric acid levels . HoweverWe also sought to ensure that our method would account for the potential turbidity of the samples, as samples of synovial fluid that are obtained in the hospital or clinic can have suspended material, or debris, present within the fluid. Synovial fluid consists of plasma proteins such as cytokines and binding proteins and lubricant macromolecules including hyaluronic acid and PRG4, but erythrocytes, leukocytes, and shed lining cells can also be present in synovial fluid depending on the etiology of the arthropathy . As gout3 to zero valence, giving a distinct change in color. Ammoniacal silver nitrate solutions of varying concentrations were prepared and tested for effectiveness of detecting urate crystals both in aqueous and stained form, as silver nitrate is reduced to elemental silver upon the addition of uric acid. Alizarin red was also tested for effectiveness of distinguishing gout and pseudo gout, as alizarin red will react with calcium pyrophosphate crystals but not with urate crystals. Silver reduction catalysts were also tested for efficacy, including ammoniacal and nondiamine silver stains, to determine which would most quickly facilitate our method of urate crystal detection. Matrix beds of both gelatin and agar were also developed and tested to allow for ease of use via an immobilized detection matrix. Gelatin gave a clear matrix bed even when stored with AgNO3 for weeks, making it the right material for the matrix. Each of these aspects of our experimentation protocols culminated in images acquired via a Promobile microscope (10–200X magnification) attached to an iPhone. Finally, a low-cost method of this device was designed with an attached magnifying lid for use in resource-limited areas.A description of the development of our novel rapid gout detection method and kit  is briefSummary of approach: An Institutional Review Board (IRB) approval was obtained for the project “Efficacy and Clinical Application of a Portable Diagnostic Test in Arthrocentesis for Crystals” (SBU-IRB: 1178449-4). This clinical trial was designed as a prospective study with the objective of validating our bedside gout detection method. Clinical samples from 5 patients earlier diagnosed with gout by Stony Brook Orthopedic Practice were obtained via arthrocentesis in either an orthopedic clinic or hospital setting and were tested via both our rapid gout detection method and the standard-of-care of polarized light microscopy. In this study, we attempted to: (a) determine the efficacy of mobile microscopic imaging in detecting uric acid crystals; (b) ascertain whether the silver nitrate stain used for crystal detection would interfere with crystal imaging; and (c) develop a method for analyzing turbid clinical samples. Study population and informed consent: Subjects presenting to the Stony Brook Orthopedic Division Clinics or Stony Brook University Hospital Emergency Department were enrolled if deemed to have high clinical suspicion of inflammatory process; differential diagnoses included gout vs. pseudo gout vs. septic arthritis. Inclusion criteria were as follows: (a) patients aged eighteen and over; (b) patients with a diagnosis of gout, pseudo gout, or septic arthritis confirmed by synovial fluid analysis; and (c) patients with site-specific confirmation of gout. Exclusion criteria included: patients aged under eighteen, patients with diagnosis of inflammatory arthritis, patients lacking documented laboratory studies consistent with gout, patients with no history of gout diagnosis, or patients unable to provide informed consent. Informed consent was obtained and documented by members of the study team, which included attending orthopedic surgeons and orthopedic residents. Upon acquisition of consent, consenting subjects’ medical and radiographic imaging was reviewed to collect basic demographic and gout-relevant information. Clinical sample collection and diagnosis: We obtained IRB approval to collect samples from designated gout, pseudogout, and septic arthritis patients. In this study, we confined our analysis to synovial samples collected from previously diagnosed gout patients to validate the point-of-care uric acid crystal detection method. Upon aspiration of an affected joint from consenting study subjects earlier diagnosed with gout, half the volume of the clinical samples were marked for standard synovial fluid analysis using polarized light microscopy for standard-of-care diagnosis, and the other half was tested with our rapid gout detection method for comparison. Diagnosis of gout was confirmed to be the visualization of needle-shaped MSU crystals with negative birefringence utilizing a polarizing filter at 400X magnification. Diagnoses were not made based upon the rapid gout detection method prior to its validation. All patients received standard-of-care treatment regardless of participation in the study. No additional joint aspirate was collected in study participants; the excess remaining fluid that would normally be discarded after synovial fluid analysis and diagnosis was instead used to evaluate our rapid diagnostic method. If the rapid gout detection method that we developed does not work, then we will not be able to proceed with further studies. Differential analysis studies for detecting gout (using silver), pseudogout , and sepsis (using fluorescent dyes) are ongoing with clinical samples and will form the basis for the next report.Microscopy images of uric acid crystals stained with silver nitrate: To verify whether silver nitrate interaction with uric acid crystals interferes with acquiring microscopy images, pure uric acid crystals stained with ammoniacal silver nitrate were imaged via laboratory-based polarized light microscopy  at 400X magnification. As previously described [escribed , 20% silRapid gout detection in clinical samples: For this study, we procured synovial fluid from designated gout patients who were undergoing treatment at Stony Brook Hospital. A total of 20 ul of synovial fluid was placed on a silver nitrate/gelatin bed matrix and images were acquired with an iPhone 9 , Plugable Digital Microscope—200X , and Accu-Scope Gout Microscope—400X. Crystal images were observed with a particular aim to determine whether silver nitrate interfered with the imaging and whether the quality of the images obtained was adequate using the handheld microscope compared to laboratory-based crystal images.Turbid sample analysis: Often, joint arthrocentesis results in obtaining turbid synovial fluid. For gout diagnosis, a clear synovial fluid sample is ideally desired. To ensure that the rapid gout detection method would be efficacious in evaluating even less-than-ideal samples, 200 microliters of a turbid clinical sample was centrifuged at 100 g in a desktop centrifuge  resulting in solid components and liquid components. The liquid component of the sample was separated from the solid using a pipette and the pellet was re-suspended in phosphate-buffered saline (PBS). Interaction of AgNO3 and NaCl (in PBS) results in the formation of a white turbid product which is counteracted with ammonia to form a coordination complex which is much more soluble and can interact with uric acid readily. The PBS–solid solution was then mixed and tested as described above on silver nitrate pre-coated wells, and images were obtained.Microscopy images of uric acid crystals stained with silver nitrate: The reaction of urate crystals with ammoniacal silver nitrate used for staining does not affect the ability to detect and image the crystals under polarized light microscopy at 400X. As demonstrated in Comparative analysis of gout diagnosis: Clear synovial fluid samples procured from designated gout patients were imaged after 10 min of placing the samples on the detection matrix with an iPhone , and imaged the PBS re-suspended pellet layered onto the silver nitrate matrix bed. Images were obtained after 10 min of placing the turbid sample on the detection matrix with an iPhone  Though clear synovial fluid analysis for detecting gout is extremely easy with our method, turbid samples entail additional processing. (b) With our method, the presence of uric acid crystals interacting with silver can be visualized even with the naked eye, but to visualize distinct crystals, devices such as smart phones/tablets with additional optical device attachments are needed. (c) Gout diagnosis can be confirmed by the method that we developed, but if concurrent pseudogout and/or sepsis is to be diagnosed, additional detection methods are needed. Future work will include the additional collection of clinical samples and the use of these clinical samples and simulated synovial fluid in silver nitrate matrix chambers for the determination of the time-based stability and sensitivity of our diagnostic method and device.Our rapid gout detection method will be compared to the gold standard of polarized light microscopy, and receiver operating characteristic curves will be generated. A high level of sensitivity is expected for our rapid gout detection method due to the colorimetric nature of the test, as only a small sample of synovial fluid is required to identify uric acid crystals. A prospective clinical trial is underway and upon further validation, we anticipate that our novel rapid point-of-care gout detection method will become the standard of care as new smartphones, electronic tablet microscope attachments, and handheld digital microscopes with higher magnification are introduced. This report focuses on the validation of our method to rapidly detect uric acid crystals in clinical samples. Gout diagnosis and eliminating whether the patient has sepsis or pseudogout is the key test characteristic of the method that was developed. Based on rapid gout diagnosis at the bedside, appropriate intervention and further testing can be planned by the healthcare provider. Our ongoing studies using clinical samples to rapidly detect pseudogout and sepsis will lead to the development of a one-step bedside detection method/kit to diagnose all three conditions.


MECP2 gene were identified as causing Rett syndrome in 1999. Over the past 20 years there has been an abundance of preclinical research with some studies leading to human clinical trials. Despite this, few viable therapeutic options have emerged from this investment of effort. Reasons for this lack of success as they relate both to preclinical research and the clinical trial landscape are discussed. Considering what needs to be done to promote further success in the field, we take a positive and constructive approach and introduce the concept of clinical trial readiness and its necessary ingredients for Rett syndrome. These include: listening to the needs of families; support from advocacy groups; optimising use of existing clinic infrastructures and available natural history data; and, finally, the validation of existing outcome measures and/or the development and validation of new measures. We conclude by reiterating the need for a collaborative and coordinated approach amongst the many different stakeholder groups and the need to engage in new types of trial design which could be much more efficient, less costly and much less burdensome on families.Rett syndrome is associated with severe functional impairments and many comorbidities, each in urgent need of treatments. Mutations in the The online version contains supplementary material available at 10.1186/s13023-022-02240-w. MECP2 gene that subsequently propelled RTT to the world stage in 1999 . Li. Li87]. Several recent clinical trials, not just in RTT but also in other neurodevelopmental disorders,  have faiMecp2 in peripheral mechanosensory neurons is associated with tactile and social interaction deficits in mice [Mecp2 heterozygote awake adult female mice exhibit abnormal spontaneous pupil fluctuations, indicating impaired cholinergic neuromodulation and altered arousal state [There are accumulating data indicating that individuals with RTT and animal models display atypical responses on electrophysiological assessments of brain function. Sensory evoked potentials and EEG analysis have been utilized in several preclinical and clinical studies in RTT . Work b in mice , but it al state . By traial state  typicallBiomarkers can be used as an objective proxy for (a) predicting response prior to treatment (biomarker suggests patient will respond) and/or (b) anticipating future response to treatment (biomarker predicts that treatment is working before change seen in measure) and/or c) biomarker change correlates with measure change after treatment. To date only a few RTT intervention trials have employed biomarkers and tried to correlate them with clinical improvements, as in the third scenario, with mixed results. The placebo-controlled clinical trial of mecasermin (rhIGF-1) (NCT01777542) used EEG-based biomarkers and a number of parent-completed scales . In thisIn summary, several studies to date provide objective evidence for deficits in sensory processing across multiple modalities in RTT and provide the scientific premise for pursuing whether these noninvasive and objective biomarkers can be informative in future clinical trials. However, as with other neurodevelopmental disorders, further research is still needed outside the confines of a clinical trial to confirm relationships between biomarkers and clinical outcome measures including those that are parent-reported.: The clinical severity scores currently in use[Clinical severityly in use were orily in use, 16. Howly in use, a subsely in use. Validatly in use. Seventely in use. AdditioThe Clinical Global Impression scales describe severity or change of severity for specific symptoms and have recently been adapted to RTT . This prSocio-emotional domain: The Rett Syndrome Behaviour Questionnaire (RSBQ) was developed as a diagnostic tool to clinically differentiate females with RTT from those with other severe intellectual disability . IndividFunctional ability domains: Some measures for specific domains are also available. The 15-item Rett Syndrome Gross Motor Scale (RSGMS) although not frequently used, has undergone substantial validation including evaluation of factor structure and test–retest reliability . It has The Mullen Scales of Early Learning (MSEL) is a generic measure of developmental skills and was a secondary outcome measure for the phase 2 rhIGF-1 trial (NCT01777542) . Modest The Communication and Symbolic Behavior Scale-Developmental Profile (CSBS-DP) measures early communication and symbolic skills in children under two years . It was Quality of life (QOL) is a concept that articulates how well an individual is living, reflecting closely patient feeling and functioning, and hence it is a critical outcome for evaluating interventions. Generic QOL measures have been used in RTT clinical trials but may not be appropriate because their development was not based on the domains of QOL important for children with intellectual disability . There aIt is clear that a core set of outcome measures is lacking for RTT and that development and validation studies are lagging behind preclinical and clinical research. For most outcome measures in frequent use, poor validation data are available. Thus far also, few comorbidities beyond epilepsy have been evaluated  and the Many of the ingredients for clinical trial readiness are already in place: there are active programs of pre-clinical research, a powerful history of database development which has enabled the availability of some trajectory data, and there is growing clinical trials experience, particularly in the US. Considerable work is, however, still necessary to achieve a core set of validated outcome measures for RTT.It is important to note that RTT is not alone in the challenges faced in translating advances in understanding the biology of neurodevelopmental disorders into effective treatments in the clinic. For example, the need for rigorous execution and transparent reporting for data from preclinical models, dynamic and clinically meaningful outcome measures and use of biomarkers to demonstrate target engagement apply not only to RTT but also to many genetic disorders in which novel therapeutics are being developed. In an ideal world, development of outcome measures, especially MCIDs, must be established prior to therapeutic trials. However, the current situation where lack of the ideal does not prevent otherwise good trials from proceeding will continue. The risk is more likely that modestly helpful but important therapeutic interventions will be interpreted as failures. The lessons learned from RTT apply to a number of genetic conditions associated with autism and intellectual disability, such as Fragile X Syndrome and Tuberous Sclerosis Complex. Furthermore, given the rapid growth in diagnostic tools for genetic disorders, the foundational knowledge gained would be instructive to the process of N-of-1 studies which continue to attract considerable attention in the rare disease space . ConversFor the future, it is imperative that the RTT community, including industry, consider alternative ways of working together for agile evaluation of new therapeutics. For example, there are increasing demands on available funding, the SARS-CoV-2 pandemic requires different models of conducting business and the recent Newron STARS trial (NCT02790034) illustrated capacity to work across multiple continents to achieve the required sample size. Supported primarily by the US National Cancer Group (thecogfoundation.org), the Children’s Oncology Group is a collaborative international group dedicated to overseeing clinical trials and contributing to improving clinical care and outcomes for children’s cancer. The Clinical Trials Committee established by the National Fragile X Foundation could also be an important model for Rett syndrome . Could sResearchers, clinicians and the RTT community acknowledge that traditional, free-standing, parallel-group RCTs are time-consuming, burdensome and costly, and it is challenging to achieve adequate sample size for the evaluation of subgroups . APTs ofFinally, rare disease communities are hungry for translational scientific evidence to provide relief to the burden imposed by RTT. When transformative therapeutics are identified, how will manufacturing capacity be ensured to meet demand and how will the cost be managed to enable broad accessibility across groups within and between countries, beyond the principle that goods should only be available to those who can pay? Progress along the pipeline of discovery has been strong since RTT was first described in 1966. In this current era of neuroscience and clinical trials, there is an imperative to learn from errors of the past, build a well-validated set of outcome measures and biomarkers, and consider novel ways of collaborating to challenge the current boundaries for current clinical management of RTT.Additional file 1: Table S1. The use of outcome measures in clinical trials in Rett syndrome.Additional file 2: References for Table S1.


Despite significant resource mobilization to address antimicrobial resistance (AMR), a persistent ‘action gap’ remains between AMR governance and the realization of societal change. Across international and national governance networks, AMR is widely recognized as an issue that goes beyond human health, requiring considerable and urgent attention from all sectors of society. In contrast, AMR awareness campaigns remain predominantly focused on framing AMR as a human health issue, with messages targeting individualistic antimicrobial consumption. In this article, we make the case that AMR awareness campaigns should frame the issue as one that goes beyond human health, in order to engage a greater number and broader array of stakeholders from across society. We argue that this could support efforts to reduce the AMR action gap, by making the issue more relevant to a wider range of stakeholders, expanding the sphere of responsibility and moving the focus from individualistic to societal-wide change. ,,,,The COVID-19 pandemic has demonstrated not only the far-reaching impact of a single untreatable infectious pathogen but also the scope for rapid, societal-wide change in response to such a threat. Antimicrobial resistance (AMR) has received considerable international attention and resources, and potentially poses a much greater long-term clinical, economic, social and environmental threat than COVID-19,,,,,,Widespread public support has been shown to be key for both bringing policy development to the fore as well as for successful implementation,,,,,,,The international AMR community has called for more research into optimizing AMR communications for local contexts, to improve understanding of localized AMR risk in order to evaluate/prioritize interventions and tailor solutions.


Human intestinal microbiota comprise of a dynamic population of bacterial species and other microorganisms with the capacity to interact with the rest of the organism and strongly influence the host during homeostasis and disease. Commensal and pathogenic bacteria coexist in homeostasis with the intestinal epithelium and the gastrointestinal tract’s immune system, or GALT (gut-associated lymphoid tissue), of the host. However, a disruption to this homeostasis or dysbiosis by different factors  can cause brain dysfunction given the communication between the gut and brain. Recently, extracellular vesicles (EVs) derived from bacteria have emerged as possible carriers in gut-brain communication through the interaction of their vesicle components with immune receptors, which lead to neuroinflammatory immune response activation. This review discusses the critical role of bacterial EVs from the gut in the neuropathology of brain dysfunctions by modulating the immune response. These vesicles, which contain harmful bacterial EV contents such as lipopolysaccharide (LPS), peptidoglycans, toxins and nucleic acids, are capable of crossing tissue barriers including the blood-brain barrier and interacting with the immune receptors of glial cells  to lead to the production of cytokines and inflammatory mediators, which can cause brain impairment and behavioral dysfunctions. The human gastrointestinal tract contains a diverse range of bacterial species called microbiota that play a key role in the body homeostasis. This complex ecosystem is involved in the immune system, energy efficiency, the synthesis of essential components or even the emotional state. Increasing evidence has associated gut microbiota with both gastrointestinal and extra-gastrointestinal diseases such as the central nervous system (CNS) in which bidirectional communication between the CNS and gut microbiota or the gut-brain axis has been of special interest in recent years. Under healthy physiological conditions, beneficial bacteria can protect against different diseases such as asthma, food allergy and type 1 diabetes  but alsoRecent studies have demonstrated participation in gut-brain communication on different pathways such as the autonomic nervous system, endocrine routes, the hypothalamic-pituitary-adrenal axis and the immune system . HoweverIn this review, we focus on the role of EVs from gastrointestinal microbiota in modulating the immune response during neurodevelopmental processes of brain dysfunctions. We also review how bacterial EVs are capable of crossing intestinal and brain barriers and their multiple molecules  or DNAs), to activate immune signaling cascades by engaging Toll- or NOD-like receptors (TLRs or NLRs) in glial cells, which leads to a neuroinflammatory immune response that can trigger brain and behavior dysfunctions.The intestinal microbiota is made up of 100 trillion cells , more thThe healthy human microbiota performs similar functions on taxa and metabolic pathways in all humans and even the existence of a common bacterial core has been hypothesized. Some microbial species are present in all human individuals . HoweverCertainly, microbiota homeostasis can be broken, whose term is dysbiosis, which is characterized by the loss of beneficial microorganisms, the appearance or increase of harmful microorganisms or the loss of overall microbial diversity . DysbiosThe inner intestine surface is made up of a single layer of cells with a permeable outer mucosa that allows the exchange of nutrients, which makes the intestine a vulnerable tissue against pathogens. The gastrointestinal tract’s immune system is composed of GALT (gut-associated lymphoid tissue), the most important structural component of mucosa-associated lymphoid tissue, with Peyer’s patches, which are lymphoid follicles located in the mucosa that play an important role in not only removing pathogens in the gut but also in maintaining a balance with the commensal and pathogenic microbiotas in the gut . CommensBifidobacterium infantis can exert beneficial therapeutic effects on mental disorders [The link between the gastrointestinal and nervous systems has been theorized since Ancient Greece but the gut-brain axis term was postulated by Ivan Pavlov in 1902, who demonstrated a cephalic phase that interacted with both gastric and pancreatic secretions . In the isorders . Conversisorders  and treaisorders .Lactobacillus rhamnosus is a probiotic bacterium that modulates the GABAergic system with beneficial effects on depression and anxiety behavior by using the vagus nerve as the main modulatory pathway between it and the CNS [One of the main pathways of potential communication between the gut microbiota and the brain is the autonomic nervous system, which includes the enteric nervous system and the vagus nerve. This nerve in intestines regulates the contraction of smooth muscles and glandular secretion and takes sensory information to the brain by playing a role in psychiatric disorders . Bacteri the CNS . Indeed, the CNS . The ent the CNS . Further the CNS . In this the CNS . They ar the CNS . The hyp the CNS . This ac the CNS . However the CNS .Finally, the last pathway with a strong interaction between the gut microbiota and the brain is the immune system. This communication is performed by cytokines , IL-17 or IL-10)  through One of the characteristics conserved in all organisms is the ability to secrete several types of membrane vesicles or extracellular vesicles (EVs). When EVs were discovered, their function was initially thought to be the removal of unnecessary compounds from cells . HoweverThese microvesicles might contain inflammatory molecules such as pro-inflammatory cytokines, TLR4, inflammation-associated RNAs and miRNAs, which contribute to the pathogenesis of different disorders such as cancer and inflammatory, autoimmune or neurodegenerative diseases, among others . For insAs in other organisms, bacteria can also produce EVs but their biogenesis and structure differ from the EVs released by eukaryotic cells see . As the All Gram-negative bacteria can produce outer membrane vesicles (OMVs), which are spherical and bilayered vesicles that derive from the outer membrane through bulging and pinching it off . These vUntil recently, it was thought that Gram-positive bacteria could not produce EVs because it would be impossible for them to traverse the thick walls found in these organisms. However, vesicles have been recently isolated and analyzed from Gram-positive bacteria. These vesicles contain lipid bilayer-enclosed structures that are morphologically similar to Gram-negative bacteria. Nevertheless, Gram-positive EVs can still be distinguished from OMVs because they lack LPS and encapsulate periplasmic components  . AlthougPorphyromonas gingivalis OMVs by mediating the coaggregation of different species in oral biofilms [The wide variety of strains and diversity of environments for which vesiculation and the release of bacteria vesicles are important processes are quite remarkable and are also necessary, advantageous and common for all Gram-negative bacterial growth and survival . Indeed,biofilms , which cbiofilms . Currentbiofilms .http://microvesicles.org/index.html, accessed on 19 April 2021) [Vibrio cholera strains, which, at a low cell density, produce OMVs with less peptidoglycan inside than at a high cell density, present a weaker and strong immunogenic effect, respectively [Recent studies have evidenced that the content in EVs differs between distinct bacterial species and even the same bacteria can produce different vesicles when external conditions change . Bacteriil 2021) . More thil 2021) . The virectively . These dectively .Pseudomonas aeruginosa activate a pro-inflammatory response  in lung epithelial cells [Moraxella catarrhalis secretes OMVs by carrying the ubiquitous surface protein UspA1/A2, which mitigates the mechanisms of serum resistance [Porphyromonas gingivalis help other pathogenic bacterial species to aggregate [Mycobacterium tuberculosis uses exosomes from its infected cells to transport lipoproteins and glycolipids to adjacent uninfected cells [Neisseria gonorrhoeae can transfer plasmids to carry antibiotic resistance genes to other Gram-negative bacteria by using OMVs [Helicobacter pylori contribute to chronic gastritis and autoimmune diseases given the presence of LPS on their surface [Campylobacter jejuni OMVs improve the interaction and invasion between the microorganism and intestinal epithelial cells due to their proteolytic activity [Shigella flexneri produces OMVs that can be fused with the outer membrane of other Gram-negative bacteria and their content is released in the recipient’s periplasm [One important factor to consider is that something positive for a parasite organism is negative for the host and vice versa. When the host reacts to an infection to eradicate it, the stressful environment can cause the production of bacterial EVs to increase. EVs contain virulence factors such as proteins  and non-protein antigens  and these EVs are able to deliver these virulence components to both prokaryotic and eukaryotic cells . OMVs fral cells , Moraxelsistance . OMVs frggregate , Mycobaced cells . Neissering OMVs . Bacteri surface , protect surface  and incr surface . Campyloactivity  while Sheriplasm .TLRs are a family of innate immune system receptors and play key roles in protecting against microbial infections . These rThe second group of TLRs , which reside in intracellular compartments , detects MAMPs in the nucleic acids deriving from bacterial and viral pathogens. TLRs 3, 7, 8 are able to recognize different types of RNAs from viruses but TLR9 recognizes the unmethylated CpG-containing DNA of a bacterial and viral origin  Figure . BacteriHelicobacter pylori increase the IL-8 production and generate a low degree of gastritis with no significant differences between Helicobacter pylori strains insofar as starting cytotoxicity, which suggests the presence of Helicobacter pylori LPS as a probable mechanism for this IL-8 up-regulation [Bacteroides fragilis produces OMVs with a polysaccharide that interacts with dendritic cells and enhances regulatory T-cells and anti-inflammatory cytokine production [Bacteroides vulgatus strain mpk induces a tolerant semimature phenotype in BMDCs using OMVs [Bifidobacterium longum KACC 91563 strain induce the apoptosis of mast cells [Helicobacter pylori OMVs are not only able to induce the release of both pro-inflammatory IL-6 and anti-inflammatory IL-10 but also induce T-cells apoptosis [Several reports have demonstrated the involvement of the activation of the immune response by OMV components. One of the most studied components is LPS, which is present on the OMV surface. OMVs from gulation . Conversoduction . Furthering OMVs  while EVst cells . Helicobpoptosis .Helicobacter pylori) and commensal  bacterial species induces a cytosolic innate immune response mediated by NLR family member NOD1 [Salmonella typhimurium OMVs activate dendritic cells in a TLR4-dependent and TLR4-independent manner through LPS and antigens are recognized by Salmonella-specific B- and T-cells [Shigella dysenteriae and Escherichia coli O104:H4 and O157 contain the Shiga toxin [In addition to the activation of the immune response of the TLRs, bacterial EVs are able to activate other immune pathways such as cytosolic NLRs . The pepber NOD1 ,87. Salm T-cells . OMVs frga toxin ,90,91, aga toxin .Shigella dysenteriae Shiga toxin production, whose presence in vesicles increases as does the number of vesicles released by the bacteria [Finally, the release of bacterial EVs can also be influenced by antibiotics. For instance, Mitomycin C is an antibiotic that is able to induce in bacteria .The gastrointestinal microbiota plays pivotal roles in neurodevelopmental processes and brain functions through the microbiota-gut-brain axis. The dysregulation of this axis by endogenous and exogenous factors such as aging and stress accelerates the occurrence of psychiatric disorders such as depression, anxiety, autism and Alzheimer’s disease ,93. For Salmonella enterica OMVs are able to pass through the Salmonella-containing vacuole and be exocytosed by infected macrophages [Campylobacter jejuni OMVs that cleave E-cadherin and occludin [Recent evidence demonstrates that bacterial EVs can directly or indirectly promote neural pathologies ,96 but urophages . In otheoccludin  and facioccludin .Aggregatibacter actinomycetemcomitans, fluorescein-conjugated EVs more strongly accumulated in the hippocampus than fluorescein-conjugated LPS and, after vagotomy, only the transport of fluorescein-conjugated EVs was blocked. These results suggest that vagotomy lowered the bacterial 16S rDNA levels in the hippocampus of mice orally administered with bacteria or their EVs [On the BBB, it has been revealed that bacterial EVs can cross this barrier, which increases their permeability by inflammatory or infectious processes. For instance, there are reports of the presence of bacterial nucleic acids in postmortem brains of patients with Alzheimer’s disease. This genetic material could have arrived in vesicles by taking advantage of the weakened BBB in Alzheimer’s patients , which wheir EVs . Aggregatibacter actinomycetemcomitans, a periodontal pathogen, could cross the mouse BBB and affect the brain immune response. Indeed it has been proposed that Paenalcaligenes hominis EVs can cause an impairment of the cognitive function by translocating to not only the brain through the vagus nerve but also to blood [How bacterial EVs can indirectly promote neuronal pathologies through their cargo is well-established. The presence of EVs in the CNS would probably induce an inflammatory response apart from the other possible side effects. However, as far as we know, the problem as to whether gut microbiota EVs can leak into the systemic circulation, cross the BBB and directly modify the brain still stands . A recento blood .In addition, bacterial EVs may also use transport to the brain as intracellular agents during activated leukocytes trafficking to the brain. The brain is constantly surveyed by the trafficking of activated lymphocytes and macrophages, which provides a Trojan horse mechanism for microbial entry into the nervous system across the BBB. In fact this mechanism is a well-recognized route by which viruses infect the brain . BacteriThe importance of EVs in infection is well-demonstrated with the delivery of active virulence factors and immunomodulatory molecules as well as their defensive roles, which all serve to enhance pathogenesis . Thus, iAggregatibacter actinomycetemcomitans OMVs to mice is able to cross the BBB and the secreted extracellular RNA cargos derived from OMVs. These vesicles can be taken up by the host macrophage cells and sRNAs inside OMVs by activating TNF-α expression via the TLR8 and NF-κB signaling pathway. Increased TNF-α expression in the mouse brain induced by these OMVs may cause inflammatory diseases such as Alzheimer’s disease [Aggregatibacter actinomycetemcomitans can successfully deliver extracellular RNAs to brain monocyte/microglial cells to cause neuroinflammation associated with the up-regulation of IL-6 through NF-κB activation [Aggregatibacter actinomycetemcomitans promotes the activation of the NF-κB response via TLR8 in macrophage U937 cells [It is common knowledge that bacteria express many molecules that activate immune signaling cascades by engaging receptors  and lead to an immense capacity for influencing the brain function . However disease . Similartivation . These e37 cells . These fPaenalcaligenes hominis caused cognitive impairment in aged mice whereas an oral gavage of both led to severer cognitive impairment These effects are associated with hippocampal inflammation by inducing TLR4- and IL-1R-mediated NF-κB activation in innate immune cells such as microglial cells and successively suppress NF-κB-mediated BDNF expression in the hippocampus. Paenalcaligenes hominis proliferation in the intestine of elderly individuals and aged mice can accelerate the absorption of its LPS into the brain via blood and the translocation of its EVs into the brain. This scenario suggests that cognitive function impairment by Paenalcaligenes hominis may attribute to the combined effects of its EVs and LPS [The excessive generation of gastrointestinal bacterial products due to gut dysbiosis such as LPS and kynurenine causes gastrointestinal and systemic inflammation, which lead to inflammatory bowel disease and neuroinflammation ,112. How and LPS .Proteobacteria phylum [Escherichia coli K1 can form part of the adult gut microbiota but can also provoke neonatal meningitis at a high mortality rate by invading brain microvascular endothelial cells to penetrate into the CNS [Escherichia coli OMVs [Escherichia coli K1 invasion of brain microvascular endothelial cells. These results suggest that EVs might also be involved in neonatal meningitis as Escherichia coli is able to cross the BBB without altering the integrity of the endothelial monolayer in vitro and BBB permeability in vivo [Although the brain has been classically considered a sterile organ with no microorganism under normal conditions, a few populations dominated by a phylum  and othe the CNS . The int the CNS  and alsooli OMVs . OmpA, b in vivo .Considering the importance of understanding the interaction between the gut microbiota and brain disease, a recent metagenomics study of bodily microbiota in an Alzheimer’s disease mouse model identified microbiota species from blood EVs whose composition altered in these animals. These results suggest that blood EVs are useful markers for characterizing the microbiota composition in Alzheimer’s disease or other neurodegenerative diseases .Recent evidence has demonstrated the role of bacterial EVs from the gut during neurodevelopmental processes and brain dysfunctions by modulating the immune response. These vesicles are able to cross the BBB and interact with the immune receptors of the TLRs of glial cells, which leads to the production of cytokines and inflammatory mediators and alters the brain function . However


Pregnant women are susceptible to various physiological and pathological skin and body habitus changes during pregnancy due to the alterations that occur in a multi-organ-system fashion. Pregnancy can be the onset of different dermatological diseases and can exacerbate pre-existing cutaneous conditions. Moreover, management of dermatologic diseases during pregnancy might be challenging as it requires special attention to both mother and fetus. We aim to assess the most common cutaneous changes and conditions that occur during pregnancy by reviewing the previous studies conducted on this subject.The medical literature was explored through PubMed and Google scholar databases starting from 2015 to 2021. The included searching terms were a combination of "Cutaneous changes and pregnancy," Dermal conditions and pregnancy," Pregnancy-associated dermal conditions," and "Dermatological changes and pregnant women." The inclusion criteria included original articles conducted on pregnant women and full text- articles.A total of 134 articles were obtained, 11 articles were eligible for the inclusion criteria. The 11 studies included a total number of 14,813 pregnant women and covered four countries. The most common cutaneous conditions experienced by pregnant women were primarily physiological skin changes, pregnancy-specific dermatopathologies, and exacerbations of other common skin diseases.All in all, this systematic review concluded that pregnant women are more vulnerable to various dermatological conditions during pregnancy. These changes were more commonly physiological such as hyperpigmentations. However, pathological pregnancy-specific skin conditions and exacerbations of pre-existing dermatoses like atopic eruptions were also reported. Pregnancy is associated with multiple physiological changes involving different organ systems like the endocrine, vascular, metabolic, and immune systems of gravid women resulting in several cutaneous changes which can be physiological or pathological . These cPhysiological skin changes during pregnancy primarily involve alterations in the degree of skin pigmentation and skin laxity mainly due to the effect of the elevated hormones . PregnanPregnant women are mostly aware of most of the common physiological skin changes that occur during pregnancy, and they commonly tend to seek medical advice for actual new-onset pathological skin conditions or exacerbations of pre-existing inflammatory skin disorders . HoweverMethodThe PRISMA checklist guidance for systematic review and meta-analysis  was follSearch strategySeveral keywords were used for searching purposes, including a combination of “Cutaneous changes and pregnancy,” “Dermal conditions and pregnancy,” “pregnancy-associated dermal conditions,” and “Dermatological changes and pregnant women.” All the titles and abstracts produced from this primary exploration were revised thoroughly to prevent missing potential studies. The findings were then examined to choose only original research articles evaluating the dermatological changes during pregnancy. All full-text articles written in English were defined as articles of relevance, which were then included in the second stage.Eligibility criteriaThe second step was deciding on the inclusion criteria to select the eligible studies. Abstracts were assessed manually to select the relevant studies for revision. The inclusion criteria were studies conducted on pregnant women and investigated the physiological changes and cutaneous conditions during pregnancy. Reviews and studies that had incomplete or overlapped data were excluded. Also, unavailable full-text articles or inappropriate study designs were excluded. The full description of the search strategy is shown in Figure Data review and analysisA specially designed excel sheet was used for data extraction. The chosen data from eligible research articles were then revised via the excel sheet. Any research articles published by one research group examining similar variables were reviewed for any potential duplication.ResultsThis systematic review included 11 articles that met the eligibility criteria and were published between 2015 and 2021 (Table Physiological skin changes are the predominantly recognized dermatologic manifestations of pregnancy as eight studies reported -18, threDermatosis of pregnancy or pregnancy-specific dermatosis is a major cause of morbidity during pregnancy and its prevalence has ranged from 2% ,17 to 40DiscussionDuring pregnancy, several changes occur to optimize fetal and maternal well-being which should eventually result in the delivery of a healthy baby, these changes include changes in the function of the endocrine system to regulate the various hormone secretions, immune system, and the various metabolic pathways that ensure delivery of nutrients and metabolic fuel to the fetus. Some of these changes contribute to a spectrum of physiological and pathological physical changes seen during pregnancy, such as the development of various skin conditions . In the Physiological dermatological changes in pregnancy involve pigmentary changes such as hyperpigmentation, which occurs among 90% of pregnant women. It usually occurs in a localized area and may be due to the regional differences in the density of melanocytes within the epidermal layer of the skin. However, generalized hyperpigmentation can occasionally occur ,20. A daBesides pigmentary changes, striae gravidarum or stretch marks are common structural skin changes commonly seen in pregnancy due to the rapid change in weight and the effects of elevated hormones on the integrity of collagen and other skin constituents ,18. StriDermatoses specific to pregnancy are skin pathologies that commonly occur in pregnancy or exacerbations of pre-existed dermatologic diseases seen in gravid women. The most common pregnancy-specific dermatosis was the atopic eruption of pregnancy which was seen in 4% to 72.72% of women with pregnancy-associated dermatosis ,13 and iAtopic eruption of pregnancy is a benign pruritic condition that is characterized by popular lesions or eczema in patients with a history of atopic dermatitis or predisposition to atopic dermatitis or even with new-onset atopic dermatitis during pregnancy. Its prevalence was stated to be in the range of 5%-20% . PruritiThis systematic review has few limitations, such as the majority of the studies included were from India; however, these studies were included as they met the inclusion criteria.Pregnant women are more prone to several cutaneous conditions during pregnancy, such as physiological skin changes, pregnancy-specific dermatoses, and flares of pre-existing chronic skin conditions. The most common physiological skin changes are hyperpigmentary changes, whereas atopic eruption is the most common dermatoses form. Eczematous eruptions have been the most common exacerbation of a chronic skin condition during pregnancy. These pathological skin conditions can be a source of considerable distress to pregnant women and may warrant immediate interventions, though their diagnosis and management can be challenging and require a thorough knowledge of their different presentations and special treatments to ensure maternal and fetal safety. Therefore, a physician's knowledge about the profile of these various dermatoses during pregnancy is necessary in order to plan preventive measures and provide comprehensive care for the mother and her baby.


Epistatic interactions and negative heterosis have been shown to be associated with interchromosomal interactions in wheat. Physical gene-gene interactions between co-regulated genes clustered in “transcription factories” have been documented, and a genome-wide atlas of functionally paired, interacting regulatory elements and genes of wheat recently produced. Integration of these new studies on gene and regulatory element interactions, co-regulation of gene expression in “transcription factories,” and epigenetics generates new perspectives for wheat breeding and trait enhancement. Rates of wheat yield improvements have slowed in recent years to 0.5–0.9% per year, and there is much concern about achieving the additional yields necessary to feed the world using less land and with fewer inputs . There iEuropean wheat breeders studying the genetic interactions underlying heterosis for yield found that the heterosis was mostly due to epistatic interactions . DigenicAlthough few digenic epistatic QTL interactions between non-homologous chromosomes were found in crosses between the high-yielding elite lines , some inHeterosis as defined by the geneticist has interactions at its heart. During the past decade, we have become aware of specific interactions on a massive scale between loci closely linked, between genes mapping further apart, and even on different chromosomes due to the development of new technologies that enable nearby sequences within nuclei to be identified e.g., . Such inIn hexaploid wheat interphase nuclei, folded, condensed chromatin segments enriched in cytosine methylation and histone H3K27me3 are common. Some 32,000 such structures, comprising 51% of the genome, were found by Because loops and loop complexes are associated with RNA Polymerase ll, it is concluded that they form “transcription factories” (TFs) . These fin trans interactions occur within TFs involving enhancers, promoters, RNA polymerase, regulatory proteins, coding and non-coding RNA transcripts, R loop formation, and RNA processing systems (The coordinated expression of genes in associated loops (in the same transcription factory) implies that  systems  to mutua systems . Perturb systems  or one w systems . Such pe systems . Several systems . The bioThe efficiency with which a gene loop is formed in a transcription factory is determined by its epigenetic state, among other factors, which in turn is determined by its DNA sequence and the transacting factors which specify the histone methylation/acetylation and DNA methylation status of the local chromatin . Geneticcis-linked regulatory elements. These results have significant implications for haplotype breeding approaches  are established in the egg and early zygote divisions when the parental genomes are first brought together and are inherited through subsequent somatic cell divisions until another meiosis or different regulatory situation is imposed. Thus, the epigenetic chromatin state of an allele reflects the outcome of its activity relative to other loci when they are first active together in the F1 individual plant. Resulting siRNAs may change the chromatin structures of alleles differentially and, consequently, an allele’s ability to be incorporated into and expression in a TF until other developmental regulatory factors emerge to alter its epigenetic state.The conclusion that selections in European wheat in recent decades have purged epistatic sources of negative dominance effects  prompts Genetic variation on which plant breeding is based will be predominantly found in genes and their regulatory elements, including the regulatory elements in transposable elements . The avaKnowledge of the physical interactions of genes and regulatory elements, including those more distant in transposable elements  in TFs, 1 If genes within a factory are co-selected and coordinated to contribute to the same trait, then studying the variation between genotypes for a given TF would provide a rapid way of defining targets for trait improvement, either by genetic intervention using CRISPR/Cas technologies or by selection using defined DNA markers. These are testable hypotheses. Comparisons between diverse individuals of genes and their regulatory elements in functionally equivalent TFs and the haplotypic segments associated with them should enable alleles that poison or enhance factories to be identified and also become targets for elimination, exchange, modification, or selection. In a polyploid like wheat, any such identified genes could perhaps be deleted or inactivated without major detriment using CRISPR/Cas-based technologies, thereby deleting sources of negative heterosis in otherwise excellent parents of F1 hybrids. This is a testable approach. Where detrimental sequences are too numerous to be deleted, then it may be possible to edit the favorable sequences into other lines to create a genetic gain. The hypothesis that genes not in factories are much less likely to be involved in determining heterosis can also be tested. Genes belonging to more than one factory may be particularly interesting and important to characterize.Because genes in a given TF are co-regulated, uncovering genes within a transcription factory may help establish hypotheses that link genes, transcription factors, factories, and traits and extend the data on gene networks in KnetMiner.Interacting gene-containing loops in TFs are most often from the same chromosome, which implies that the loops are genetically linked. Recombination between them will therefore be infrequent in breeding programs. This limits the opportunities for exploiting homologous recombination to modify the content of TFs in pure line breeding. Hybrids would be better for creating new combinations of alleles/genes in TFs, especially where intrachromosomal interactions are the most frequent. Because TFs are likely to include unlinked genes, including genes on homologous and non-homologous chromosomes, variant TFs created in breeding programs are most likely to result from combining variant genes and their regulatory elements from different chromosomes. Given the above logic, it is not surprising that much/most of the variations underpinning heterosis in wheat are epistatic  and arisvia CRISPR/Cas editing to delete or correct the remaining genes/regulatory elements that epistatically inhibit yield, thereby increasing the segments’ value even more. Deletion breeding based on precision-targeted editing needs to be explored. It also may be the means by which new variation can be continuously brought into elite breeding pools without negative yield losses, thereby boosting continuous yield gains over long periods. In addition, the deployment of new enhancers to genes that are incorporated into TFs using CRISPR/Cas or transgenic technologies may also provide new opportunities to enhance the expression of the multiple genes within the TF, thereby enhancing the traits in a more efficient way than by changing one gene at a time.While breeders are often reluctant to lower the performance of elite breeding pools by making wide crosses, there are many examples of yield gains being achieved by the incorporation into domesticated wheat varieties of a trait-enhancing chromosomal segment or segments from wild relatives . Yet, itThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Clonal bacterial populations exhibit various forms of heterogeneity, including co-occurrence of cells with different morphological traits, biochemical properties, and gene expression profiles. This heterogeneity is prevalent in a variety of environments. For example, the productivity of large-scale industrial fermentations and virulence of infectious diseases are shaped by cell population heterogeneity and have a direct impact on human life. Due to the need and importance to better understand this heterogeneity, multiple methods of examining single-cell heterogeneity have been developed. Traditionally, fluorescent reporters or probes are used to examine a specific gene of interest, providing a useful but inherently biased approach. In contrast, single-cell RNA sequencing (scRNA-seq) is an agnostic approach to examine heterogeneity and has been successfully applied to eukaryotic cells. Unfortunately, current extensively utilized methods of eukaryotic scRNA-seq present difficulties when applied to bacteria. Specifically, bacteria have a cell wall which makes eukaryotic lysis methods incompatible, bacterial mRNA has a shorter half-life and lower copy numbers, and isolating an individual bacterial species from a mixed community is difficult. Recent work has demonstrated that these technical hurdles can be overcome, providing valuable insight into factors influencing microbial heterogeneity. This perspective describes the emerging microbial scRNA-seq toolkit. We outline the benefit of these new tools in elucidating numerous scientific questions in microbiological studies and offer insight about the possible rules that govern the segregation of traits in individual microbial cells. Although bacteria grown from a single cell or colony are often treated as uniform, heterogeneity in clonal bacterial populations has been observed since the dawn of bacteriology. Koch and Cohn independently discovered sporulating subpopulations of cells via microscopy in 1876, and Roland Thaxter reported that morphologically distinct cells form intricate structures in isolated cultures of myxobacteria in 1892 . DespiteDespite advancements aided by fluorescence microscopy, studies that rely on fluorescent measurements in single cells are not truly high-throughput and typically require a hypothesis-driven approach that relies on tedious construction of a small number of reporters to answer a specific set of questions. Even in the cases in which genome-wide fluorescent reporter strains exist (for example The emergence of single-cell RNA sequencing (scRNA-seq) techniques make it now possible to observe transcriptome-wide heterogeneity agnostically, without any preconceived bias introduced in studies when choosing a limited number of genes studied simultaneously. Mammalian scRNA-seq techniques, such as InDrop , DropSeqEukaryotic single-cell techniques cannot be directly applied to microbial cells without overcoming several obstacles. Firstly, bacterial mRNA does not contain the 3' poly-adenosine (poly-A) tail that is present in eukaryotic mRNA . The mosAnother difference between bacterial and mammalian transcription is the stochasticity of bacterial gene expression , which iin situ hybridization techniques  reads (97%). In a more high-throughput approach, two other laboratories independently developed similar split-pool indexing techniques to profile the transcriptomes of tens of thousands of bacterial cells  but limited the transcript coverage to 105 genes  only in the later OD time points, a deeper analysis of that subpopulation was not discussed (B. subtilis grown in minimal media (In bacterial systems, there are clues that such temporal ordering can be studied, though no analysis has focused on this aspect thus far. scRNA-seq has demonstrated changes over time in bacterial gene expression and suggests that some cellular processes may be more prone to heterogeneity than others . One stuiscussed . In anotal media . This suUsing time course and pseudotime studies, it is possible to uncover the order in which different regulatory features are expressed therefore providing insight about the mechanisms of development. When pseudotime algorithms, such as Monocle, Slingshot, P-Creode , or otheExperimental evolution studies have uncovered many rules of natural selection in many organisms . ExperimIn several systems, it has been demonstrated that a fraction of the cellular population may dynamically switch their phenotype when subject to changing environments. Such switching involves cells entering and exiting physiological states in which specific gene sets are expressed and have been shown to have benefits for the community in some instances . The exiAnabaena, this sequestration is achieved by differentiation of a cell fraction into a specialized nitrogen-fixing cell type (heterocyst; see In some cases, it has been demonstrated that cellular processes must segregate into either distinct cells, locations, or specific times in order to avoid the deleterious effects of having metabolically incompatible processes co-occur in a single cell. For example, oxygen radicals are known to destabilize nitrogenase, so aerobic respiration is sequestered from nitrogen fixation in microorganisms . In the In addition to growth in suspension, microenvironments may drive cellular differentiation through the accumulation and depletion of nutrients and byproducts. While the specific microenvironments that effect different microbes vary greatly, as well as the evolutionary strategies developed by microbes to address environmental obstacles that arise inherently with growth, it is interesting to speculate that there are some strategies that recur throughout biology.Microbes are exposed to high environmental variability, raising the question whether distinct cell types are truly conserved throughout the bacterial world? Our hypothesis is that the space for bacterial heterogeneity across the entirety of microbiology is vast; however, we posit that even within this space there are rules that govern which processes segregate and that these rules can be identified via high-throughput characterization of single cells that compose communities.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.MB and AR came up with the idea for this perspective and wrote the manuscript. All authors contributed to the article and approved the submitted 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.


A paradigm shift in neuroscience was the discovery that new neurons are constantly produced in the adult mammalian brain of several species, including Homo sapiens. These new-born cells are formed in some main neurogenic niches, including the subventricular zone (SVZ) at the margin of the lateral ventricle and subgranular zone (SGZ) in the hippocampal dentate gyrus (DG). In the DG, neuroblasts derive from SGZ progenitors and migrate to the hippocampal granular layer becoming adult granule cells, which are integrated into functional adult circuits. It has been confirmed that adult hippocampal neurogenesis (AHN) is a long-lasting phenomenon in the human brain. The functions of hippocampal new-born cells are not fully established. Experimental studies suggest that they have unique electrophysiological properties, including hyperexcitability, which enable them to regulate adult granule cells. Their specific function depends on the anatomical hippocampal location along the hippocampal dorsal-ventral axis. Dorsal hippocampus plays a more defined role on spatial learning and contextual information, while the ventral hippocampus is more related to emotional behavior, stress resilience and social interaction. Several reports suggest a role for AHN in pattern separation, cognitive flexibility, forgetting and reversal learning. It has been proposed that deficits in AHN might impair normal DG function, including pattern separation and cognitive flexibility, which could play a role on the etiology of affective disorders, such as depression, anxiety and post-traumatic stress disorder (PTSD). In this paper, we review recent scientific evidence suggesting that impairment of AHN may underlie the pathophysiology of affective disorders even in humans and that neurogenesis-inspired therapies may be a promising approach to reduce symptoms of affective disorders in humans. One of the most remarkable discoveries in neuroscience was that new neurons are constantly produced in the adult brain of several mammalian species , includiSeveral scientific evidences point to an important role of adult hippocampal neurogenesis (AHN) on the normal DG function. These functions include pattern separation, cognitive flexibility, forgetting and reversal learning . Based oIn this paper, I review recent scientific evidence suggesting that impairment of AHN may underlie the pathophysiology of affective disorders, even in humans. I emphasize that modulation of AHN by pharmacological or non-pharmacological therapies (including aerobic exercise) may become a pivotal approach to diminish mental suffering in people afflicted by affective disorders. I also discuss the importance of physical activity for mental health as consequence of our evolutive history and a new hypothesis to explain the human correlate of enriched environment.While the existence of adult neurogenesis in the human SVZ is yet debatable , AHN hasA paradigm-shift insight is that impairment of AHN in the ventral hippocampus (VH) may affect the normal physiology of DG, contributing to affective disorders . It has Cognitive flexibility is the ability to adapt to a new situation when a previous paradigm is changed, or to a changing environment in general . It is nIt has been recently suggested that a reduction in the AHN caused by chronic stress, mainly in the VH, might impair pattern separation and cognitive flexibility, contributing to affective disorders, including depression, anxiety and PTSD, even in humans . ReducedAffective disorders are likely hyperactivation or hypoactivation of physiological neural circuits with downstream neurochemical changes. Projections from VH to medial prefrontal cortex mediate anxiety, context aversion and behavior inhibition in mice , while bImpaired adult neurogenesis following chronic stress might dysregulate such neural pathways, contributing to anxiety and depression . SeveralRodent studies suggest that diminished DG inhibition by adult-born neurons might result in hyperactivation of hypothalamus–pituitary–adrenal (HPA) axis resulting in increased levels of glucocorticoid release in a vicious cycle detrimental to AHN . A contrImpaired pattern separation and cognitive flexibility have been reported in depressed humans . CognitiAlthough this paper discusses the etiology of affective disorders, such as de-depression and anxiety, as related to impairment of memory-associated DG functions like pattern separation and cognitive flexibility, anxiety behavior and other affective disorders might be related to other factors, including malfunction of reward processing and innate components . This haHomo erectus, evolved to walk in a bipedal posture and were hunter-gatherers for million years  and posterior semicircular canal radii (r = 0.66). These data suggest a correlation between traits related to endurance activity and brain evolution.The semicircular canal system, which is important for specific ocular, head and body movements allowing stability, was also shaped for allowing better performance during endurance running and other energy-consuming aerobic activity . For exaAn important question to address is how millions of years of hunter-gatherer life and daily endurance activity influenced brain developing and cognition? It has been proposed that intense and continuous aerobic activity influenced brain cognition by shaping systems of growth factors . There iVoluntary and forced running up regulates levels of brain-derived neurotrophic factors (BDNF) in rodent and human brains, which contributes to neuronal survival and increased adult neurogenesis . Long phin vivo experiments suggesting that levels of brain growth factors and brain size in different mammalian species correlates with aerobic physical activity performance , an approach developed by Tracey Shors and colleagues at the department of psychology of Rutgers University . This approach relies on meditation techniques such “mindfulness,” and continuous aerobic exercise, for the treatment of affective disorders, such as depression, anxiety and PTSD  for 20 min in a sit position, followed by 10 min of walking meditation. Finally, patients were asked to perform aerobic exercise for 30 min in a funny way involving dancing activities, including Zumba and Jazzercise. Sessions were performed twice a week for 8 weeks . AccordiThe neurogenesis-inspired therapies, such as MAP-training, are very promising, but they do not prove that improvement of symptoms of patients with depression and anxiety are due to enhanced AHN. Innovative technology to monitor adult neurogenesis in the human brain are needed and they are not available at the moment.Future studies should confirm that normal DG physiology is impaired due to impaired adult neurogenesis in humans and their contribution to the pathophysiology of affective disorders. Therapies aiming at enhancing AHN may be feasible in the future to treat depression, anxiety and other affective disorders . PhysicaThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Postoperative Ileus (POI) remains an important complication for patients after abdominal surgery with an incidence of 10–27% representing an everyday issue for abdominal surgeons. It accounts for patients’ discomfort, increased morbidity, prolonged hospital stays, and a high economic burden. This review outlines the current understanding of POI pathophysiology and focuses on preventive treatments that have proven to be effective or at least show promising effects.Pathophysiology and recommendations for POI treatment are summarized on the basis of a selective literature review.While a lot of therapies have been researched over the past decades, many of them failed to prove successful in meta-analyses. To date, there is no evidence-based treatment once POI has manifested. In the era of enhanced recovery after surgery or fast track regimes, a few approaches show a beneficial effect in preventing POI: multimodal, opioid-sparing analgesia with placement of epidural catheters or transverse abdominis plane block; μ-opioid-receptor antagonists; and goal-directed fluid therapy and in general the use of minimally invasive surgery.The results of different studies are often contradictory, as a concise definition of POI and reliable surrogate endpoints are still absent. These will be needed to advance POI research and provide clinicians with consistent data to improve the treatment strategies. Postoperative ileus (POI) is a common problem encountered by surgeons after abdominal and even non-abdominal surgery . AlthougThe underlying mechanisms of POI are a complex interaction of inflammation, neural reflexes, neurohumoral pathways, and pharmacologic effects. While interstitial cells of cajal (ICC) provide the rhythmicity of gut motility by their pacemaker activity, the enteric nervous system is the key player in influencing gut motility with mediation by parasympathetic and sympathetic pathways , 12. It The identification of predisposing factors associated with POI could allow for a targeted surveillance or even prophylaxis for patients at risk. Most meta-analyses searching for independent risk factors for POI have been published in a certain surgical field, commonly in patients undergoing colorectal surgery. The varying definition of POI hinders comparability of single studies and conclusions thereof . For gasThe main clinical features of POI are abdominal distension, nausea and vomiting, and the absence of flatus or stool passage. While there might be mild tenderness on palpation, usually, no muscular defense is present in clinical examination. Laboratory tests typically show no specific alterations , 27. As The risk factors collected over the years of POI research and the growing understanding of its pathophysiology have led to various prevention strategies. Those that have a promising effect on POI development are summarized in Table The infusion of fluids is supposed to compensate for the intraoperative fluid loss. Whether to opt for crystalloids or colloids is discussed widely, recently, no superiority was identified for either method , 44. TheOpioid use in postoperative analgesia leads to activation of central and peripheral opioid-receptors that facilitate their analgetic effect but their side effects as well. The μ-opioid receptor subtype located in the central nervous system is mainly responsible for analgesia, whilst peripheral μ-receptors mediate gastrointestinal dysfunction by inhibition of enteric nerve activity and propulsive motor activity. The development of μ-receptor specific antagonists  that do not pass the blood-brain-barrier allowed for a specific inhibition of these side effects without affecting the analgesic potency . Whilst The opioid-mediated detrimental influence on POI led to opioid-sparing analgetic regimens. As a so-called multimodal analgesia, these protocols use a combination of different therapies  block, NSAR), thus reducing opioid administration and have been implemented in various schemes into fast-track practices , 59.The placement of an epidural catheter with continuous administration of a local anesthetic led to an accelerated recovery of gastrointestinal function as shown by a Cochrane review and its consecutive update , 60. AsiOne of the most common alternatives to epidural catheters nowadays is the TAP block where a long-lasting local anesthetic  is injected or a catheter is placed (for continuous application) between the internal oblique and transverse abdominis muscle layers guided by ultrasound providing a regional anesthesia . As thisThe role of COX-2 produced prostaglandins is known to play a central role in the development of POI in a murine model . TherefoAs the administration of COX-2 inhibitors also led to reduced opioid-doses and a faster recovery of patients , 67, theIt is well known from research conducted in murine models on POI pathophysiology that the manipulation of the gut is directly associated with intestinal dysmotility. This effect was also verified in humans by the detection of tryptase and interleukin-(IL) 8 and 10 in the peritoneal fluid after open surgery which—aside from low levels of IL-8—was not seen after MIS . It mighThe administration of chewing gum as a method of sham feeding has been established especially for patients who are intended to a delayed enteral feeding. Its effect is mediated by a cephalovagal reflex resulting in prokinetic parasympathomimetic activity . The praA stimulating effect on bowel activity is contributed to coffee consumption in popular belief. Its mechanism of action is not well understood, but several pathways such as gastrin release, exorphins binding to opiate receptors, and the inhibition of adenosine receptors are hypothesized . Recent It has been hypothesized that neuromodulation can be used to promote gastrointestinal recovery according to its therapeutic uses in various diseases such as migraine and seizures. For instance, there is promising data that transcutaneous vagal stimulation reduces the inflammatory response within the intestinal wall and prevents POI in rodents via the activation of vagal efferents . Also, eIt has to be stated that once prolonged POI has manifested, there is no evidence-based therapeutic approach. Prokinetic substances like metoclopramide, erythromycin, and acetylcholinesterase-inhibitors such as neostigmine are widely used in clinical routine without showing a benefit in clinical symptoms or shortening POI in a Cochrane analysis . Also, tIn summary, the incidence of POI remains high in patients even in the era of MIS and fast-track programs and is a burden for both patients and society. A Delphi approach within the Association of Coloproctology of Great Britain and Northern Ireland even stated POI to be in the high priority list of non-cancer related clinical problems . A lot oEfforts in POI research should focus on the development of reliable and, at its best, objective criteria allowing for qualitatively better studies and their comparability . In our


As fathers are increasingly involved in childcare, understanding the neurological underpinnings of fathering has become a key research issue in developmental psychobiology research. This systematic review specifically focused on (1) highlighting methodological issues of paternal brain research using functional magnetic resonance imaging (fMRI) and (2) summarizing findings related to paternal brain responses to auditory and visual infant stimuli. Sixteen papers were included from 157 retrieved records. Sample characteristics , neuroimaging information , and main findings were synthesized by two independent authors. Most of the reviewed works used different stimuli and tasks to test fathers’ responses to child visual and/or auditory stimuli. Pre-processing and first-level analyses were performed with standard pipelines. Greater heterogeneity emerged in second-level analyses. Three main cortical networks  and a subcortical network emerged linked with fathers’ responses to infants’ stimuli, but additional areas  were also responsive to infants’ visual or auditory stimuli. This review suggests that a distributed and complex brain network may be involved in facilitating fathers’ sensitivity and responses to infant-related stimuli. Nonetheless, specific methodological caveats, the exploratory nature of large parts of the literature to date, and the presence of heterogeneous tasks and measures also demonstrate that systematic improvements in study designs are needed to further advance the field. During the past two decades, a global change in fatherhood has occurred, at least in Western societies . The chaA rapid literature search on Scopus for “fathering” or “paternal caregiving” revealed that research tagged with these keywords has started to become a stable topic in the international scientific community only after 2000, with a minimum of 25 (year 2003) to a maximum of 118 (year 2018) annually published papers. This rapidly accumulating amount of research highlights the importance of fathering and direct fathers’ engagement in daily caregiving for infants’ development, especially during the first months and years of life ,17. Gettperomyscus californicus) reported increased neural activity of the medial preoptic area (MPOA) in males following pup exposure . Th. Th25]. Second, the embodied simulation network appears to be specifically activated in the paternal brain while watching or hearing their own infants’ stimuli. This network is thought to facilitate an immediate grasping of others’ intentions and to favor an emotional resonance between two interacting individuals . Among tA third brain network—that is the emotional regulation network—was also reported. These include the IFG  and the Other brain regions not previously included in the paternal brain model emerged from this review. These include bilateral motor areas that can add to the embodied simulation network ,84. For One of the most important points raised by this systematic review is that the paternal brain is highly responsive to infant stimuli. Nonetheless, the available literature surprisingly lacks enough details on the actual time spent by fathers in direct caregiving activities during daily life. This is a major limitation of studies so far, as it dramatically reduces the possibility of making further hypotheses on the effective role played by direct experience in fathering in linking transition to fatherhood and functional changes in brain reactivity to infant stimuli. From a methodological point of view, there is substantial variability among the included studies in terms of sample size , with half of the studies involving fewer than 25 participants. This may imply potential limitations in the statistical power of the original analyses. Moreover, it should be highlighted that a recent meta-analysis failed to witness the test-retest reliability of task-functional MRI . These mThe present systematic review suggests that child-related visual and auditory stimuli activate a paternal brain network that includes, but is not limited to, three interconnected subnetworks that regulate embodied simulation, mentalization, and emotion regulation and recruit subcortical areas that have relevant roles in the socio-emotional elaboration of child-related stimuli. However, this conclusion should be considered with caution, as several shortcomings of the studies to date  have been highlighted, and different brain regions may serve multiple functions. As previous research on mothers showed that caregiving sensitivity is underpinned by regions involved in the same networks activated in the paternal brain , it is pFuture research is warranted to test how the complex paternal network is associated with fathers’ caregiving sensitivity and, in a broader sense, the quality of fathers’ caregiving behavior. Overall, the review provides a partial understanding of the activation of fathers’ brain networks, as it does not take into account the amount of father involvement in direct caregiving, the developing nature of father–child relationship, and other sensory stimuli characteristic of the father–child relationship. The focus on functional connectivity analysis could unveil the development and characteristics of complex neural networks related to fatherhood. The use of advantageous study designs, such as longitudinal studies starting from transition to fatherhood and including emotional and physical dimensions of paternal closeness, would also generate a deeper understanding of human fatherhood, useful for research advancement and clinical purposes.


Many studies have claimed bilingualism strengthens the neural mechanisms that underpin executive functions and enhances cognition in the elderly (Bialystok, In the present article, after recapping the main sources of variability in research findings , which dA main source for the considerable variability in reported results comes from the fact that researchers have investigated different types of executive functions. Early studies stated that inhibition was the specific executive function enhanced by bilingualism. Scientists argued that this advantage resulted from bilinguals' life-long practice in inhibiting their non-target language during conversation (Bialystok et al., There are several variables that could lead to ceiling effects in executive control tasks and obscure real differences between monolinguals and bilinguals. For instance, tasks with low levels of difficulty could mask a bilingual advantage in executive functions due to ceiling effects in performance (Bialystok et al., Variables related to spoken language/s such as age of acquisition, the number of languages spoken, the degree of similarity between these languages, and the relative degrees of proficiency a speaker has in each of their languages may also modulate any potential bilingual enhancement in executive functioning (Bialystok, Button et al.  estimatePublication bias has affected bilingualism research (de Bruin et al., Selective reporting is another factor that contributes to the low replicability of reported results; researchers may run analyses until they find something that matches their predictions, as pointed out by Chambers . SelectiPrevious research has already suggested that pre-registered reports and multi-center studies are appropriate measures to avoid publication bias and selective reporting of results (e.g., Paap et al., Cespón and Carreiras  reviewedA number of studies have investigated ERP differences in the neural processes involved in executive functioning in monolinguals and bilinguals (Kousaie and Phillips, There is widespread misinterpretation of N200 amplitude modulations. The fact that N200 amplitudes are higher in bilinguals than monolinguals performing executive tasks has erroneously been taken as evidence for enhanced neural processing in bilinguals (Fernández et al., There are some ERP studies that have offered partial or inconclusive evidence for enhanced executive neural processing in bilinguals relative to monolinguals. Kousaie and Phillips  observedThe existence of contradictory interpretations in studies investigating relationships between bilingualism and executive functions was also indicated outside ERP literature (Paap et al., The development of theoretically-grounded reviews in basic science (e.g., Cespón and Carreiras, Also, these “Reviews on data interpretation” could provide solid hypotheses for pre-registered studies. If these pre-registered studies become a common practice within the field, over the long-term, meta-analyses could be conducted on pre-registered studies. This would represent an interesting approach given the higher methodological quality of pre-registered compared to conventional studies and the possibility of correcting for publication bias simply by taking withdrawn pre-registrations into account.In conclusion, by clarifying how specific patterns of data should be interpreted, taking the key sources of variability into consideration, and avoiding the methodological errors reviewed here, progress can be made. These are essential steps to clarify whether and how specific experimental conditions lead to enhanced neural processing underlying executive functioning in bilinguals.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.