Nigella sativa, TQ has shown anticancer activities through the inhibition of cell proliferation, migration, and invasion. The anticancer efficacy of TQ is being investigated in several human cancers such as pancreatic cancer, breast cancer, colon cancer, hepatic cancer, cervical cancer, and leukemia. Even though TQ induces apoptosis by regulating the expression of pro- apoptotic and anti-apoptotic genes in many cancers, the TQ effect mechanism on such cancers is not yet fully understood. Therefore, the present review has highlighted the TQ effect mechanisms on several signaling pathways and expression of tumor suppressor genes (TSG). Data from relevant published experimental articles on TQ from 2015 to June 2020 were selected by using Google Scholar and PubMed search engines. The present study investigated the effectiveness of TQ alone or in combination with other anticancer therapeutic agents, such as tyrosine kinase inhibitors on cancers, as a future anticancer therapy nominee by using nanotechnology.To date, natural products are widely used as pharmaceutical agents for many human diseases and cancers. One of the most popular natural products that have been studied for anticancer properties is thymoquinone (TQ). As a bioactive compound of  Nigella sativa (N. sativa), black seed, or Alhabba Al-Sauda . A. A122]. TQ is potent enough to regulate miRNA expression ,103. ActOxidative stress and inflammation could lead to cancer . Hossen Furthermore, TQ has shown a dose-dependent anti-inflammatory effect on carrageenan-induced rat hind paw edema and cottonseed pellet granuloma compared with the reference drug indomethacin . TQ–pipeOverexpression of the protein tyrosine kinase (PTK) gene has increased the activity of PTK and modulated its downstream signaling pathways, promoting cell proliferation disorders and ultimately leading to tumor formation . ClinicaSeveral studies had proven TQ’s potential to activate tumor suppressor genes by a demethylating process for many methylated genes in cancer cells ; PTEN isN. sativa that has been intensively investigated in vitro and in vivo and shown to have several therapeutic properties, including anticancer activity. Its effectiveness on cancers is demonstrated in murine model studies in which TQ enhances higher survival rates, reduced tumor volume, reduced pro-cancerous molecules and elevated anti-tumorigenesis biomarkers. Meanwhile, in in vitro studies, TQ has shown the ability to inhibit cancer staging such as migration, proliferation, and invasion or apoptosis induction by repressing the activation of vital pathways, such as JAK/STAT and PI3K/AKT/mTOR. However, the TQ effect mechanism on cancers is still not fully understood. It is important to note that the discovery of selective anticancer agents such as tyrosine kinase inhibitors (TKI) represents a revolution in cancer treatment. However, the resistance to such therapeutic agents is one of the main challenges in cancer treatment, and different mechanisms to develop such resistance have been postulated. Activation of signaling pathways and epigenetic methylation of tumor suppressor genes (TSG) plays a crucial role in acquiring resistance to TKI. Re-expression of TSG by using demethylating agents represents a critical approach in cancer treatment. Based on the present review, it is recommended that more intensive studies of TQ’s effects, alone or in combination with TKI, on the expression of TSG and different signaling pathways that represent critical strategy in cancer treatment, are conducted. It is also recommended that nanoparticle technology and analogs be used to improve TQ drug effectiveness.TQ is the main bioactive constituent in


The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations. The annual TSMC symposium is designed to enable information sharing between DoD scientists and leaders in the field of microbiome science, thereby keeping DoD consortium members informed of the latest advances within the microbiome community and facilitating the development of new collaborative research opportunities.The 2020 annual symposium was held virtually on 24–25 September 2020. Presentations and discussions centered on microbiome-related topics within four broad thematic areas: (1) Enabling Technologies; (2) Microbiome for Health and Performance; (3) Environmental Microbiome; and (4) Microbiome Analysis and Discovery. This report summarizes the presentations and outcomes of the 4th annual TSMC symposium. The Tri-Service Microbiome Consortium (TSMC) was founded in 2016 to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations and to facilitate resource, material and information sharing among consortium members. Towards those goals, and to discuss applications and implications of microbiome research, the TSMC hosts an annual symposium that includes subject matter experts from federal/state agencies, DoD-affiliates, academic institutions, and industry . The 202www.ues.com/tsmc2020This report details those activities in an effort to foster potential collaboration across scientific communities. The meeting was also recorded and can be viewed here: The Fourth Annual TSMC meeting was originally scheduled to be hosted at the US Army Engineer Research and Development Center – Cold Regions Research and Engineering Laboratory, and so the virtual meeting was opened by their Director, Dr. Joseph Corriveau. Dr Corriveau extolled the virtues of the agenda and emphasized that this meeting aligns with the strategic objectives of the DoD in Biotechnology, especially in providing a forum for stimulating innovative collaborations between the DoD service laboratories. Some of these collaborative efforts were highlighted during the TSMC overview, provided by the TSMC Chair, Mr. Jason Soares (DEVCOM SC), and TSMC Co-Chair, Dr. Michael Goodson (AFRL). As well as the annual meeting, TSMC knowledge products include: meeting reports –4, revieE. coli to explore what is produced [B. thetaiotaomicron [The enabling technologies session was initiated by a Keynote presentation from Prof. Christopher Voigt (MIT). Prof. Voigt discussed how his research group mines microbial genomes to find pathways and gene clusters that produce chemicals of interest and then expresses them in heterologous hosts. He demonstrated their ‘sequence to molecule’ pipeline that they use to identify the 14,000 unexplored gene clusters in the human gut microbiome, how they reconstruct them with synthetic control, and then how they express these in produced . To dateaomicron , 9.Bacteroides strains take them up through conjugation.The three technical talks that followed focused on technological advances that are advancing our understanding and utilization of microbial consortia. Dr. James Comolli (MIT-Lincoln Laboratory) described methods to engineer endogenous microbes in situ by modifying microbes using mobile DNA elements and then applying these to a mixed microbial community. Whereas other have used broad host range mobile elements , Dr. ComContinuing the theme of utilizing microbial consortia, Mr. Robert Jones (ERDC-CRREL) discussed the conductive properties of melanized fungal biomasses. Mr. Jones described how melanin is produced in the cell membrane, and is then extruded extracellularly between the fungal cell membrane and cell wall to form a sheath around the fungus. It is thought that this has evolved to protect the fungus from radiation damage by scavenging free radicals. However, melanin is also an ion and electron charge carrier potentiating it is a living conductive structure that could connect biology to electric devices.The final talk in this session focused on technology that aids in the understanding of microbes at the individual level. Dr. Armand Dichosa  discussed how single cell genomics (SCG) allows the ‘unknowns’: the novel, recalcitrant, and underrepresented bacteria to be investigated. SCG uses flow cytometry to isolate single cells from any microbial consortium, and employs the rolling circle technique to amplify the genome. Coupling SCG technology with gel microdroplet (GMD) techniques has allowed previously unculturable bacteria to be investigated , 12. BriProf. Eran Elinav, Weizmann Institute of Science, Israel, was the keynote speaker for the microbiome for health and performance session. Prof. Elinav described the tools used for mechanistic understanding of microbiomes to avoid a ‘one-size fit all’ microbiome treatment approach. He then highlighted the ways in which his lab is developing microbiome treatments for health. Since diet is the dominant modulator of the gut microbiome, nutrition is a potential avenue to effect health benefits from the gut microbiome. However, there is high variability in the response to dietary changes across populations. To investigate this, Prof. Elinav’s research group initiated the ‘personalize nutrition project’ where subjects were monitored for diet, sleep, glucose levels, gut microbiome composition, blood metabolites, and mood. Using these data, individualized response to certain foods could be predicted, and when personalized diets were prescribed to 200 pre-diabetic individuals, their outcomes were improved compared to comparable diets suggested by current guidelines . MicrobiThe technical presentations within the microbiome for health and performance session included three talks that separately explored the dynamic relationships between the gut microbiota and the brain, bone, and gastrointestinal tract. Dr. Allison Hoke  focused on a connection between the brain and gut microbiota by describing changes in fecal microbiota composition in response to a “cage-within-cage resident-intruder” model of psychological stress shown to elicit post-traumatic stress disorder- (PTSD) like traits in mice . PTSD isDr. Xiaomeng You  explored relations between age-related gut microbiome dysbiosis and age-related bone loss. She presented results of several studies that used a combination of mouse models including aged mice, germ free and specific pathogen-free mice, and fecal microbiota transplant experiments. The gut microbiome is known to play a role in bone homeostasis via modulation of immune, growth and bacterial factors that directly and indirectly interact with pathways regulating bone remodeling . FurtherCampylobater jejuni is one of several TD-causing pathogens, especially in Southeast Asia. Dr. Blake Stamps (Air Force Research Laboratory) presented findings from a controlled human infection model of C. jejuni which explored whether the gut microbiota may explain inter-individual variability in susceptibility to and recovery from campylobacteriosis. Results from this preliminary study identified several commensal taxa that were associated with diarrhea severity, supporting the prospect that certain gut microbiota taxa or communities may confer protection against C. jejuni infection and potentially other TD-causing pathogens. The study also demonstrated the utility of mining data from controlled human infection model studies to identify novel taxa or communities that may protect against TD. Those microbes can then be validated in large cohort studies, and possibly facilitate development of novel probiotics for TD prevention.Travelers’ diarrhea is a leading cause of non-battle injury in deployed warfighters and identifying strategies to prevent TD is a top military priority , 19. CamThe environmental microbiome session included an invited talk and three topical presentations discussing microbial communities found in frozen soils, contaminated soils, and aircraft vehicles. Dr. Kristen DeAngelis (University of Massachusetts-Amherst) discussed the effects of climate change on carbon dynamics, carbon storage, and greenhouse gas emissions in the context of plant–microbe interactions. Dr. DeAngelis uses the Harvard Forest, a deciduous forest in Massachusetts, to study the short-term and long-term effects of warming on carbon cycling in temperate forest soils . Soil orMs. Stacey Doherty (ERDC-CRREL) expanded on this topic to the effects of climate change on frozen terrain through investigation of ecological processes structuring microbial communities during permafrost thaw in Abisko, Sweden . PermafrRhodococcus jostii strain RHA1 to detect PFAS in laboratory media as a proof-of concept [Emerging contaminants such as per- and polyfluoroalkyl substances (PFAS) and polyfluorooctanoic acid (PFOA) pose significant risks to human health because they are both toxic and recalcitrant in the environment. Cadet Jackson Harris (US Air Force Academy) discussed techniques to isolate microorganisms capable of degrading these emerging contaminants through a laboratory incubation study where PFAS and PFOA were added over a six-week period. After observing a shift in the microbial communities due to the addition of the contaminants, bacteria and fungi capable of degrading PFAS were isolated to determine the classes of microorganisms and enzymes to optimize PFAS degradation at contaminated military sites. Cadet Harris, a member of the United States Air Force Academy’s International Genetically Engineered Machine team, helped to develop a technology to use a genetically engineered  concept . A novelPapiliotrema laurentii is a fungus that is capable of degrading a polyurethane coating [Environmental microorganisms can be detrimental to military equipment by corroding and deteriorating the coatings on aircraft and vehicles because they harbor hydrolase enzymes capable of degrading polymer coatings . For ins coating . Dr. VanCritical to microbiome research are the advancements in instrumentation, techniques, and analysis that enable visualization of microscopic organisms. An invited talk and three topical presentations discussed cutting-edge metagenomics and proteomics techniques used in environmental and human microbiome analysis and discovery. Prof. Pieter Dorrestein  opened the session by discussing the development of mass spectrometry approaches for microbiome data analysis of complex biological systems, which includes a discovery workflow . Prof. DDr. Matthew Perisin, US Army Combat Capabilities Development Command—Army Research Laboratory, discussed challenges associated with microbial corrosion on Army equipment, which results in $20 billion dollars of damage each year. Microorganisms, regulated by nutrient access levels , colonizDr. Sophie Colston, US Naval Research Laboratory, investigated the impact of diving on the gut microbiome of US Navy divers in an effort to improve warfighter performance during complex missions. Ongoing projects are determining the effect of hypo- and hyperbaric conditions on the gut microbiome by investigating fecal microbiome diversity and metabolomics.Dr. Ritesh Kumar (DuPont Nutrition and Biosciences), discussed efforts to characterize over 5,000 strains of bacteria from lean individuals to discover new probiotics to combat obesity. Using a mouse model, Dr. Kumar determined that the addition of four bacterial species improved insulin, glucose, and leptin levels. These studies highlight how advancement in microbiome analysis in discovery improves our ability to understand the role of the microbiome in human and environmental health.In this session, the themes centered on unique microbial properties including cold and soil adaptations and plastics degradation potential. The first talk from Dr. Robyn Barbato (ERDC-CRREL) focused on a new environmental intelligence platform called DRTSPORE, which predicts soil respiration activity across landscapes with respect to moisture and temperature. DRTSPORE was modeled on heterogeneous soil types, preliminarily tested on landscapes near CRREL with promising results, and will soon be validated and applied to arctic environments. Alison Thurston (ERDC-CRREL) presented on microbial changes based on 16S rRNA sequencing in Alaskan permafrost samples before and after thawing in the laboratory. Microbial communities were differentiated by sample site before thawing but interestingly microbial communities were not as distinctly clustered by sample site after thawing. Whether or not thawing is the driver in these results is next being explored. Flora Cullen (ERDC-CRREL) described a unique and novel psychrophile (growth < 20 °C) and psychrotroph (growth < 10 °C and > 20 °C) library of organisms collected from Toolik Lake, Permafrost Tunnel, and Greenland. So far, 68 organisms have been preserved and two of which showed growth at − 5 °C. Several organisms from the library produced pigments which can potentially be leveraged for coating materials for protection against extreme UV-irradiation as presented by Elizabeth Corriveau (ERDC-CRREL). Several of their genomes were sequenced using the Oxford Nanopore Technologies MinION and genes involved in pigmentation, such as in carotenoid production, were identified. Dr. Angelina Angelova  showed with metagenomics sequencing from marine environments that similar microbial biofilm communities reside on different plastics regardless of their chemistry or composition. While genetic composition and metabolic potential were not significantly different between plastic types, many plastic-degrading enzymes were identified such as hydrolases and monooxygenases, demonstrating that these communities likely have general plastic-degrading potential.Lactobacillus reuteri), significant decreases in C-reactive protein were observed versus controls as well as improvements in stress response during the challenging Trier Social Stress Test. As radiation exposure also elevates the inflammatory response and is thought to similarly modulate the composition and activity of the gut microbiome, Dr. Nabarun Chakraborty  demonstrated that the microbiome composition of mice exposed to radiation was marked by an increase in alpha-diversity, an increase in the ratio of Firmicutes to Bacteroidates, and a beta-diversity reflecting a reduction in evenness over time. Also, PCA analysis of metabolomic panels show a time-dependent variation post-exposure to radiation. Toward a better understanding of the microbial ecology in the gut, Dr. Karley Mahalak  presented her group’s engineered in vitro reactor systems. This reactor is designed to mimic and control the conditions known to be present in the gut and provide a platform for comparative microbiome studies where input conditions, such as changes in diet and the introduction of antimicrobials, are varied. USDA is collaborating with the Army’s DEVCOM SC toward understanding the effects of nutritional supplements such polyphenols and dietary fibers on gut microbial activity toward the development of food supplement bars that can enhance gut and cognitive health for the Warfighter. In a separate study from the Army DEVCOM SC utilizing in vitro fermentation, Dr. Ida Pantoja-Feliciano (CCDC-SC) examined the effects of a sudden change from a habitual diet (HAB) to 21 days of a diet consisting of Meal, Ready-to-Eat (MRE) U.S. military combat ration on the population and functional dynamics of the human gut microbiome. With resistant starch as a fermenter substrate, there was no discernable impact on community dynamics when comparing fermentation seeded with fecal samples from MRE vs HAB diets; however, there was a slight increase in microbial species associated with short chain fatty acid production and glycoside hydrolase activity when the fermenter contained fecal samples from an MRE diet (vs HAB diet). Dr. Jordan Zambrana (EPA – Indoor Environments Division) discussed his Agency’s strategies for improving indoor air quality (IAQ) to protect human health with an appreciation for how the link between indoor air chemistry and the indoor microbiome affects IAQ. Given this link between the chemistry and microbiology, discussion focused on outstanding scientific questions of interests to the EPA related to an understanding of indoor microbial ecologies, the nutrients available to sustain the indoor microbiome and a better understanding of how environmental chemicals in turn affect human physiology including the human microbiome.Lightning Talk Session #2 focused on a variety of studies that relate to the effects of dietary and environmental inputs on the activity and outcome of the human microbiome. Dr. Lisa Brenner (Rocky Mountain MIRECC) who has interests in understanding the mechanisms underlying posttraumatic stress disorder (PTSD) and mild traumatic brain injury (mTBI), has tested the hypothesis that these conditions are in part the result of chronic inflammatory disorders caused by reduced exposure to the microbial environment. When individuals afflicted with persistent PTSD and mTBI were administered a probiotic  but it was agreed that a general framework to follow for all DoD microbiome research data collection and analysis would be beneficial. These points were emphasized as a key part of the DoD Biotechnology Modernization pillar. Finally, gaps in DoD microbiome research were discussed focusing in on human microbiomes other than the gut. While DoD funds some research into non-human gut microbiomes , it is not a DoD service laboratory focus currently. The meeting highlighted that, as in microbiomes themselves, ‘it takes a village’, and that sharing capabilities and expertise, and, perhaps most importantly, continually communicating the latest advances to avoid redundancy and maximize resources will remain critical to realizing the full impact of microbiome-based solutions to advance DoD interests.


Even though neurons are the main drivers of information processing in the brain and spinal cord, other cell types are important to mediate adequate flow of information. These include electrically passive glial cells such as microglia and astrocytes, which recently emerged as active partners facilitating proper signal transduction. In disease, these cells undergo pathophysiological changes that propel disease progression and change synaptic connections and signal transmission. In the healthy brain, astrocytic processes contact pre- and postsynaptic structures. These processes can be nanoscopic, and therefore only electron microscopy has been able to reveal their structure and morphology. However, electron microscopy is not suitable in revealing dynamic changes, and it is labour- and time-intensive. The dawn of super-resolution microscopy, techniques that ‘break’ the diffraction limit of conventional light microscopy, over the last decades has enabled researchers to reveal the nanoscopic synaptic environment. In this review, we highlight and discuss recent advances in our understanding of the nano-world of the so-called tripartite synapses, the relationship between pre- and postsynapse as well as astrocytic processes. Overall, novel super-resolution microscopy methods are needed to fully illuminate the intimate relationship between glia and neuronal cells that underlies signal transduction in the brain and that might be affected in diseases such as Alzheimer’s disease and epilepsy. Information processing in the central nervous system occurs mainly at synapses, the connection points between neurons. An intimate relationship between neurons and glial cells underlies proper synaptic function. Astroglia in particular play essential roles in information processing. These cells are not only needed for structural support but astroglia also fulfil several other indispensable functions. These include clean-up of brain debris, digestion of dead neurons and pruning of synapses  as well 2+ waves and through the aforementioned syncytium [In the brain, astrocytes adopt a sponge-like morphology, featuring a few stem processes and thousands of thin protrusions that emanate from these stem processes ,14. Whilyncytium . Throughyncytium ,21.The fine protrusions can either form astrocytic endfeet and encircle endothelial cells as part of the neurovascular unit of the blood–brain barrier or they can enwrap excitatory synapses throughout the brain. These processes are often called perisynaptic astrocytic processes (PAPs) and form part of the so called tripartite synapse, together with pre- and postsynaptic structures . DependiDue to the nanoscopic nature  of the PAPs, researchers have had to rely on electron microscopy (EM) to visualise and investigate tripartite synapses ,30,31. EPAP coverage of synapses depends on several factors and differs depending on the physiological state, local neuronal activity, synaptic plasticity, or certain behaviours .Confocal microscopy, which is diffraction limited, has been used to explore the dynamic nature of PAPs in real time . The dynIn this review, we will provide an overview and discussion of recent developments in tripartite synapse imaging using super-resolution methods. We will also provide a short introduction into different super-resolution methods and recent advances in revealing the nanostructure of neuronal synapses. However, for further information we point the reader to some excellent, recent reviews on super-resolution microscopy and also its use in neuroscience research .As mentioned above, confocal microscopy (as well as other types of light microscopy) is diffraction limited, meaning that discerning individual molecules can be difficult. EM, which has traditionally been used in order to image nanoscopic molecules, comes with several disadvantages, including its exclusive use for fixed tissue specimens. In addition, it is labour and time intensive, and correlational comparisons between different samples are difficult to attain and interpret. This is also true for some of the super-resolution methods described below.Super resolution imaging allows the user to image beyond the diffraction limit of light while using traditional sample immunostaining methods associated with fluorescent light microscopy. Several super resolution methods have been developed, each with its own advantages and disadvantages. However, the discussion of these is beyond the scope of this review and the authors again point to some excellent reviews of this topic . Most wiSTED microscopy relies on a second, doughnut-shaped excitation beam which depletes excitation at the periphery of the primary excitation beam, hence narrowing the emission spot and allowing resolution beyond the diffraction limit . One of SIM relies on bar code-like patterns which are shifted and rotated in the excitation path . This crSMLM encompasses several different techniques that all rely on a similar molecular mechanism - pinpointing the point source of fluorescence (which can be one fluorophore/molecule) in each imaging cycle through repeated stochastic excitation of only a small, sparsely distributed subset of fluorescent molecules . After aExM uses literal expansion of the tissue by a factor great enough to separate individual molecules of interest beyond the diffraction limit, followed by imaging with conventional microscopes such as confocal microscopy ,60. ThisSeveral advances have been made in our understanding of neuronal synapses using super-resolution microscopy .in vivo and in vitro [in vivo and in vitro [Especially STED microscopy has been employed to analyse the composition of presynaptic structures and vesicle dynamics within, both in vitro , showingin vitro . SMLM wain vitro .STED as well as SMLM have been used to image dendritic spines and postsynaptic structures in cultured cells and brain slices . RecentlSeminal work published in 2010 provided a detailed three-dimensional map of the nanoscale structure of excitatory synapses in fixed murine brain sections using SMLM . The groUsing different super-resolution microscopy techniques, it was shown that AMPARs are packed in nanoscopic clusters in cultured cells . These c2 in cultured spinal cord neurons, and that in situ about three times as many gephyrin molecules are packed more densely [Likewise, the nanoscale clustering of the scaffolding protein gephyrin as well as of glycine and gamma-aminobutyric acid (GABA) type A receptors at inhibitory synapses has been investigated . Using q densely .Recent beautiful work combined super-resolution microscopy imaging with plasticity induction and revealed a trans-synaptic organisation of scaffolding molecules both at excitatory and at inhibitory synapses in organised tissue and in cultured cells ,93,95–97As discussed above, astrocyte processes and especially PAPs display nanoscopic features. Early super-resolution studies did not focus on the relationship between synapses and astrocytic molecules but rather on the subcellular localisation and clustering behaviour of individual proteins of interest. For example, Verkman and colleagues investigated the clustering behaviour of aquaporin 4 and of the inwardly rectifying potassium channel Kir4.1 using SMLM techniques in cultured cells and in brain sections . MoreoveRecent studies have investigated translation events that occur locally in astrocytic processes in the vicinity of synapses . EM and Using STED and SIM, researchers have shown that neuronal activity increased connexin 30 expression and localisation in perisynaptic processes in hippocampal astrocytes in mouse brain slices . The samin vivo chemico-genetic approach that applies a cell-surface fragment complementation strategy, Split-TurboID and STED discovering that neuronal cell adhesion molecule (NRCAM) is expressed in cortical astrocytes where it localises to PAPs and restricts their neuropil infiltration [In a recent beautiful publication, researchers have used a plethora of techniques including an ltration . Astrocyltration .Recently, researchers visualised bestrophin-1 (Best1) expression at tripartite synapses using lattice SIM and found that, in brain tissue from wild-type mice, astrocytic Best1 localises closer to glutamatergic synapses than to GABAergic synapses . In APP/In a recent preprint, Südhof and colleagues used SMLM in mouse brain sections to decipher the role of astrocytic neurexin-1 in synapse function and maturation . The autRecent work utilising STED imaging showed that in spinal cord sections 56% of synapses (PSD95 and vGluT2 or vGluT1 pairs) are association with GLT-1 (the major astrocytic glutamate transporter), 30% with glial fibrillary acidic protein and 14% with phosphorylated ezrin . PSDs asIn a recent publication, we used fluorescence recovery after photobleaching in organotypic rat hippocampal sections to demonstrate that 70–75% of GLT-1 molecules dwell on the astrocyte surface, recycling with a lifetime of ∼22 s . Using SRecent attempts to study astrocyte processes and glutamate uptake have utilised ExM of mouse brain sections ,119. TheTo further analyse the distribution of glutamate transporters surrounding excitatory synapses, we used a 3D SMLM protocol of brain sections ,120,121.2+ transients using STED microscopy in living organotypic brain slices , a technique, in which the extracellular space is fluorescently labelled and cellular structures appear as dark shadows D–G 104,,122. Thein vivo counterparts. This is especially true for astroglia as their morphology and expression profiles are very different in culture and in intact tissue. Additionally, PAPs appear to be very sensitive to classical tissue fixation protocols, with a significant difference of PAP-positive synapses found in the adult murine neocortex when comparing chemical and cryo fixation  [in vivo imaging platforms will help evaluating the true morphology of astrocytes and hence tripartite synapses. We are only just beginning to fully explore the potential of super-resolution microscopy technology and its future capabilities, especially in the field of diagnostics.Super-resolution microscopy has helped further our understanding of neuronal and glial cell morphology and molecular dynamics. Most findings were acquired in fixed cell culture or brain section preparations. However, it is known that cultured cells differ from their yo: 34%) . Adoptinin vivo nanoscopy possible. Further adaptations and the integration of adaptive optics [As mentioned above, recent developments using STED microscopy have made e optics  will impe optics , STED [1e optics ,129 and e optics .Astrocytes found in higher primates are larger and more complex, and show unique functions and different abilities to cope with stressors and disease responses when compared with murine astrocytes . Super-rIt is well documented that glial cells undergo drastic changes under pathological conditions . Super-rHence, the use of super-resolution will be a valuable means for both clinicians and researchers in deciphering the exact pathophysiological changes occurring in and around synapses, and in evaluating novel therapeutic options.


Somatosensory-evoked potentials (SEP) represent a non-invasive tool to assess neural responses elicited by somatosensory stimuli acquired via electrophysiological recordings. To date, there is no comprehensive evaluation of SEPs for the diagnostic investigation of exercise-induced functional neuroplasticity. This systematic review aims at highlighting the potential of SEP measurements as a diagnostic tool to investigate exercise-induced functional neuroplasticity of the sensorimotor system by reviewing studies comparing SEP parameters between athletes and healthy controls who are not involved in organized sports as well as between athlete cohorts of different sport disciplines.A systematic literature search was conducted across three electronic databases  by two independent researchers. Three hundred and ninety-seven records were identified, of which 10 cross-sectional studies were considered eligible.Differences in SEP amplitudes and latencies between athletes and healthy controls or between athletes of different cohorts as well as associations between SEP parameters and demographic/behavioral variables  were observed in seven out of 10 included studies. In particular, several studies highlight differences in short- and long-latency SEP parameters, as well as high-frequency oscillations (HFO) when comparing athletes and healthy controls. Neuroplastic differences in athletes appear to be modality-specific as well as dependent on training regimens and sport-specific requirements. This is exemplified by differences in SEP parameters of various athlete populations after stimulation of their primarily trained limb.Taken together, the existing literature suggests that athletes show specific functional neuroplasticity in the somatosensory system. Therefore, this systematic review highlights the potential of SEP measurements as an easy-to-use and inexpensive diagnostic tool to investigate functional neuroplasticity in the sensorimotor system of athletes. However, there are limitations regarding the small sample sizes and inconsistent methodology of SEP measurements in the studies reviewed. Therefore, future intervention studies are needed to verify and extend the conclusions drawn here. Exercise induces widespread adaptations in the human body. Such adaptations lead to strong health benefits, especially reduced all-cause mortality, reduced (co-) morbidity, and improvement in overall well-being . SEPs represent neural responses elicited by external somatosensory stimuli recorded via electrophysiological methods , free of injury or neuronal diseaseIntervention: measurement and comparison of SEP parameters (short- and long-latency SEP) between athletes and controls or between different athlete categoriesControl: age-matched healthy controls, not participating in organized sport or regular exerciseOutcomes: latency and amplitude of SEP waves, HFOStudy design: intervention or cross-sectional studiesArticles not meeting the inclusion criteria were excluded from this systematic review.A systematic literature search was performed by two independent researchers  in the electronic databases PubMed, Web of Science, and SPORTDiscus into all available sources with publication year until March 2021. The reference lists of the included studies were also scanned to generate a broader scope of the search. Only studies published in the English language were reviewed and included in the systematic review.Searches were performed in PubMed , Web of Science , and SPORTDiscus  using the keywords “somatosensory evoked potentials” AND “athletes” OR “sports” OR “exercise.”Records were screened and selected by two review authors  independently based on previously defined PICOS eligibility criteria  independently collected the following data items from the included studies:Methods: study design Participants: number, mean age, age range, gender, inclusion criteria and exclusion criteria, sports discipline, history of sports participation.SEP application: SEP stimulation sites (extremity and nerves), pulse characteristics, motor task during stimulation, and stimulation conditions.Outcomes: primary and secondary SEP outcomes specified and collected, and time points reported.Notes: funding for studies and notable conflicts of interest of authors.Disagreements were resolved by reaching a consensus or by involving a third person (PR).Assessment of methodological quality of eligible studies was performed by two review authors  independently using the JBI Critical Appraisal Checklist for Analytical Cross-Sectional Studies , no athlete population (n = 10), wrong comparison (n = 3), wrong species (n = 1). Furthermore, 1 study was identified through citation searching of screened studies. Finally, a total of 10 studies were deemed eligible for inclusion in this systematic review . Furthermore, it is noteworthy that all studies examined provided incomplete information on the inclusion and exclusion criteria of the recruited participants (Question 1 of the JBI Checklist). Methodological quality assessment results were summarized and visualized in The majority of the studies examined can be described as being of high methodological quality. However, heterogeneous results can be observed, with two studies showing considerable methodological problems due to incomplete specification of participant characteristics  and inappropriate statistical methods  investigated exclusively male participants Stimulus rate: 3–5 HzNumber of averaged pulses: 500 Peripheral recording electrodes: Erb's point, C5 (skin over the spinous process)Cortical recording electrodes: Fz, C3'/C4' (10/20 system)SEP components: N9, N13, P14, N20, P20, N30Tibial nerve stimulation:Stimulation site: tibial nerve at the ankle Pulse length: 0.2–0.3 msPulse intensity: motor threshold (sufficient intensity to cause a plantar flexion of 1–2 cm)Stimulus rate: 3–5 HzNumber of averaged pulses: 500 Peripheral recording electrodes: popliteal fossa, L1 (skin over the spinous process)Cortical recording electrodes: Fz, Cz' (10/20 system)SEP components: N8, N22, P30, P39The result of this evaluation is visually summarized in Stimulation sites of SEP measurements for upper and lower limb responses were evoked by either median nerve stimulation at the wrist  of peripheral nerve stimulations were fixed in some of the identified studies, i.e., at 333 ms  during application of the stimulation and signal recording  did not observe differences in short-latency SEP parameters between athletes and healthy controls or between athletes of different sports  also revealed differences between athletes and healthy control participants or between different athlete cohorts  appear to be enhanced in athletes following stimulation of the limb that was primarily trained  have been identified and may be responsible for heterogeneous results across studies. It is known that, for example, stimulus rates above 3 Hz can lead to peak attenuation i.e., a decrease in the amplitudes of peaks with more than 30 ms latency . Another limitation in deriving neurophysiological features from SEP signal amplitudes involves the fact that in group comparisons, differences in underlying tissue properties between groups potentially affect SEP amplitude. In longitudinal studies, this issue is addressed by normalizing SEP amplitudes to baseline values and then comparing changes in relative SEP amplitude between different groups (McGregor et al., All studies examined were cross-sectional, which prevents causal inferences about the interplay between training-induced neuroplasticity in the somatosensory system and concomitant SEP modulations. The question of the relationship between nature and nurture thus remains unanswered, so the results reviewed here should be interpreted as preliminary and with caution. Crucially, future intervention studies in form of randomized controlled trials investigating different sports are needed to unravel the causal relationship between athletic training and specific SEP modulation as well as differential SEP modulations according to different training regimes.Furthermore, the collective findings might not be sufficiently representative of both genders due to the overall small sample size and the even smaller number of female athletes included in the identified studies. In general, differences in brain structure and function between males and females have been revealed which might be reflected in differences at the behavioral level (Grabowska, Finally, the small to moderate sample sizes in the studies reviewed here, ranging from 5 to 16 athletes per group, may be responsible for some apparent null findings. The problem of studies with insufficient sample size is a common challenge in sport and exercise science (Lohse et al., Based on the available evidence from cross-sectional studies, we propose that SEP measurements can be a valid and cost-effective neurodiagnostic tool for assessing functional neuroplasticity in the context of athletic performance. The potential of this method is supported by its ease of use and high inter-session reliability. Crucially, however, long-term interventions are required to validate the conclusions derived in this review that long-term athletic training leads to specific SEP modulations that are directly related to superior motor performance.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.TM and SH independently performed the literature search. TM wrote the manuscript. SH, RK, and PR provided critical revision. All authors interpreted the data, contributed to the manuscript, reviewed, approved the content of the final version, and agree to be accountable for all aspects of the work.We acknowledge support from Leipzig University for Open Access Publishing.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.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.


Neurodegenerative diseases are characterized by the progressive loss of structure and/or function of both neurons and glial cells, leading to different degrees of pathology and loss of cognition. The hypothesis of circuit reconstruction in the damaged brain via direct cell replacement has been pursued extensively so far. In this context, stem cells represent a useful option since they provide tissue restoration through the substitution of damaged neuronal cells with exogenous stem cells and create a neuro-protective environment through the release of bioactive molecules for healthy neurons, as well. These peculiar properties of stem cells are opening to potential therapeutic strategies for the treatment of severe neurodegenerative disorders, for which the absence of effective treatment options leads to an increasingly socio-economic burden. Currently, the introduction of new technologies in the field of stem cells and the implementation of alternative cell tissues sources are pointing to exciting frontiers in this area of research. Here, we provide an update of the current knowledge about source and administration routes of stem cells, and review light and shadows of cells replacement therapy for the treatment of the three main neurodegenerative disorders . Stem cells, present in every living organism, are cells characterized by the unique ability to proliferate indefinitely by using a particular asymmetrical division, by self-renewal properties, and by the competence to drive their differentiation towards specific cell phenotypes in, virtually, all tissues . In thisStem cells guarantee the physiological process of tissues regeneration and can be classified according to either their developmental competence  or tissue origin  . In partEmbryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of mammalian blastocysts. These cells show pluripotent properties and can easily differentiate into tissues from the three primordial germ layers , finally constituting the complete soma of the adult organism . The priIn 2007, Takahashi and Yamanaka introduced a new technology able to reprogram mouse somatic cells into pluripotent embryonic-like cells by transferring through viral vectors four transcription factors  responsible for pluripotency ,16 and cFetal stem cells can be isolated from a direct biopsy of the fetus or from fetal annexes  ,20. AmonAdult stem cells, also known as somatic stem cells, are present in specialized niches of all tissues, where they act as key regulators of homeostasis, driving the cell fate between self-renewal and differentiation. Among this broad subgroup, both MSC and hematopoietic stem cells (HSC) have been widely investigated and applied for over 60 years in clinical practice paving the way for bone marrow (BM) transplantation . InteresAlong with stem cell sources, administration routes can influence migration, distribution, and the amount of transplanted cells in the target area . In addiIntracerebroventricular (ICV) of stem cells appeared for years to be the most precise delivery route for neural stem cells (NSC) implantation . HoweverIntravascular infusion, both intravenous and intra-arterial, represents a valid, safe, and less invasive alternative route of administration compared to ICV . The pecTo date, the intranasal delivery route of administration represents a promising strategy to circumvent the BBB and to deliver drugs straight to the brain. This non-invasive way reduces the likelihood of adverse events compared to the intravascular one. Several studies have shown that stem/progenitor cells, administered intranasally, migrate to the brain through the nasal cavity and lead to positive findings in PD , malignaSeveral studies reported evidence about the ability of stem cells to mediate tissue repair not only by a direct replacing of the damaged tissue but also acting as a key modulator of the local environment by influencing immune/inflammatory milieu and providing anti-apoptotic/cytoprotective effects on the resident cells ,47. ThisStem cell-derived paracrine therapy could represent a new exciting pharmacological approach in avoiding immune compatibility and largely reducing the cost and time associated with expansion and maintenance of cell lines. Several studies have highlighted the contribution of stem cell transplantation in immunomodulation. For example, Wolbank et al.  reportedAD represents, arguably, the most significant social, economic, and medical crisis of our time . This prSeveral drugs, focused on facilitating amyloid clearance or preventing amyloid production, have been developed and investigated in clinical trials with results far from satisfactory, having failed to improve cognitive and functional ability of AD patients . On 7 JuRecently, researchers are attempting a multifaceted approach that includes stem cells and neuro-regeneration to shed light on a better understanding of cellular mechanisms of neurodegeneration involved and to study the possibility of development of efficient cell-based therapies. Stem cell-derived neurons may provide a drug-screening platform to identify small molecules able to improve well-established cellular phenotypes linked to Aβ and tau pathologies . In partDespite promising results of preclinical data, numerous unresolved safety issues need to be overcome prior to transferring this technology from the bench to the bedside and human clinical trials are still in their infancy regarding stem cell therapy in AD  80]. Th. Th80]. In conclusion, although several challenges should be taken into account, stem cells carry enormous promise to enhance our understanding of AD molecular basis and provide a platform for discovery of novel therapeutic options. Since the clinical applications of stem cell technology are still in their early stages, this strategy is likely to become crucial and contribute significantly to the treatment of AD in the near future.SOD1), TAR DNA-binding protein (TARDBP), fused in sarcoma (FUS), amyotrophic lateral sclerosis 2 (ALS2), senataxin (SETX), janus-faced spatacsin (SPG11), vesicle-associated membrane protein-associated protein B (VAPB), and angiogenin gene (ANG) [C9orf72), a gene also involved in cognitive dysfunction as frontotemporal dementia [ALS, also known as Lou Gehrig’s disease, is a progressive neurodegenerative disease, with a multifactorial etiology, characterized by motor neurons’ loss in the spinal cord’s ventral horn and in the motor cortex . The worne (ANG) . In addidementia . The SALToday, no effective therapies were shown to provide a substantial clinical benefit for ALS patients. FDA approved only two treatments: riluzole, which prolongs median survival by about only 2 to 3 months , and edaPrevious preclinical stem cell transplantation studies have been performed in mice and rats ALS model expressing mutant SOD1, reporting motor neurons degeneration, muscle atrophy and motor dysfunction. For example, Chen R. and Ende N. , by admiRegarding clinical trials, the first one approved by FDA employed NPCs in 18 affected ALS-patients with the scope to assess the safety of this kind of cells into the spinal cord . NumerouPD is one of the most frequent chronic neurodegenerative disorder affecting aged people ,100. Thihttp://www.transeuro.org.uk/, accessed on 21 June 2021) [Despite the advances in the treatment of PD, there is still no specific cure for this condition. Conventional medications  are highly effective in the early stage of PD in relieving motor symptoms, but systemic administration is often associated with adverse long-term side effects, such as drug-induced dyskinesia . More rene 2021) . Completne 2021) .A useful candidate stem-cell equivalent source might be represented by MSCs that have been shown to reduce dopamine depletion and rebuild the damaged striatal dopaminergic nerve terminal network in a PD animal model . A previStem cell technological improvements are opening new therapeutic perspectives for the treatment of several neurodegenerative diseases that, currently, lack of effective pharmacological therapy. The use of stem cells and their application in the replacement therapy is based both on their highly proliferative capability with peculiar differentiative properties, as well as on their cytoprotective effects, mediated by release of bio-molecules on the endogenous tissue environment. Following this standpoint, over the past few years, there has been progressive development of experimental procedures to generate human-derived neurons and glial cells, useful for cell replacement therapy. Although stem cell therapy could represent a ray of hope for patients suffering from neurodegenerative disorders, several concerns still need to be addressed to improve their application in the clinical setting . To thisIn addition to these preeminent scientific issues, another substantial matter that needs to be evaluated is represented by the high running costs of stem cell therapy. Indeed, the production phases must comply with specific GMP standards , which i


Sepsis survivors experience high morbidity and mortality, and healthcare systems lack effective strategies to address patient needs after hospital discharge. The Sepsis Transition and Recovery (STAR) program is a navigator-led, telehealth-based multicomponent strategy to provide proactive care coordination and monitoring of high-risk patients using evidence-driven, post-sepsis care tasks. The purpose of this study is to evaluate the effectiveness of STAR to improve outcomes for sepsis patients and to examine contextual factors that influence STAR implementation.This study uses a hybrid type I effectiveness-implementation design to concurrently test clinical effectiveness and gather implementation data. The effectiveness evaluation is a two-arm, pragmatic, stepped-wedge cluster randomized controlled trial at eight hospitals in North Carolina comparing clinical outcomes between sepsis survivors who receive Usual Care versus care delivered through STAR. Each hospital begins in a Usual Care control phase and transitions to STAR in a randomly assigned sequence (one every 4 months). During months that a hospital is allocated to Usual Care, all eligible patients will receive usual care. Once a hospital transitions to STAR, all eligible patients will receive STAR during their hospitalization and extending through 90 days from discharge. STAR includes centrally located nurse navigators using telephonic counseling and electronic health record-based support to facilitate best-practice post-sepsis care strategies including post-discharge review of medications, evaluation for new impairments or symptoms, monitoring existing comorbidities, and palliative care referral when appropriate. Adults admitted with suspected sepsis, defined by clinical criteria for infection and organ failure, are included. Planned enrollment is 4032 patients during a 36-month period. The primary effectiveness outcome is the composite of all-cause hospital readmission or mortality within 90 days of discharge. A mixed-methods implementation evaluation will be conducted before, during, and after STAR implementation.This pragmatic evaluation will test the effectiveness of STAR to reduce combined hospital readmissions and mortality, while identifying key implementation factors. Results will provide practical information to advance understanding of how to integrate post-sepsis management across care settings and facilitate implementation, dissemination, and sustained utilization of best-practice post-sepsis management strategies in other heterogeneous healthcare delivery systems.NCT04495946. Submitted July 7, 2020; Posted August 3, 2020.The online version contains supplementary material available at 10.1186/s12913-021-06521-1. Contributions to the literatureSepsis survivors experience high morbidity and mortality, and healthcare systems lack effective strategies to address patient needs after hospital discharge.The STAR program extends nurse navigator support during post-sepsis care to facilitate targeted, evidence-driven recommendations using an integrated, patient-centric telehealth approach.The proposed study combines a pragmatic randomized trial framework with longitudinal implementation science and prospective economic evaluations to compare usual care versus care delivered with added STAR program support for improving post-sepsis care and outcomes.Research will add new practical information to advance understanding of how to best integrate effective post-sepsis management across care settings and heterogeneous healthcare systems.Sepsis is a common, life-threatening condition defined by organ dysfunction due to a dysregulated response to infection . Recent We developed the Sepsis Transition and Recovery (STAR) program to address persistent morbidity and mortality for sepsis survivors. The STAR program uses telehealth nurse navigation to deliver a bundle of best-practice care elements for longitudinal post-sepsis care up to 90 days. These care elements are directed towards the specific challenges and sequelae following a sepsis hospitalization and include: 1) identification and treatment of new physical, mental, and cognitive deficits; 2) review and adjustment of medications; 3) surveillance of treatable conditions that commonly lead to poor outcomes including chronic conditions that may de-stabilize during sepsis and recovery; and 4) focus on palliative care when appropriate . Prior oThe objectives of the Engagement and Collaborative Management to Proactively Advance Sepsis Survivorship (ENCOMPASS) trial are to concurrently evaluate: i) the effectiveness of the STAR intervention for reducing mortality and hospital readmission assessed 90 days after hospitalization among high-risk sepsis survivors, and ii) contextual factors related to the real-world implementation of the program. Furthermore, because implementing support programs such as STAR requires health system investments, we will also evaluate the effect of these investments on costs and cost-effectiveness.ENCOMPASS is an effectiveness-implementation hybrid type I trial. The evaluation component is designed as a two-arm, pragmatic, stepped-wedge cluster randomized controlled trial conducted at eight hospitals in which each participating hospital begins in a usual care control phase and transitions to the STAR program intervention in a randomly assigned sequence, with one of eight hospitals assigned to transition at each four-month interval , and the trial protocol adheres to the Standardized Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines [The hybrid type I trial design combining comparative effectiveness evaluation with collection of implementation outcomes is optimal for ENCOMPASS because STAR satisfies previously described design considerations , includiidelines   , 36. We We will test the STAR program intervention within the course of providing usual care among a large and diverse population of post-sepsis patients admitted to eight hospitals within Atrium Health (AH), one of the largest, vertically integrated health systems in the US. The eight acute care hospitals participating in this study use the same EHR, which connects across all points of care, including outpatient practices, urgent care locations, emergency departments and hospitals. Additionally, study hospitals vary in size and provide care to diverse regions of North Carolina, representing seven different counties Table . We deliPrior to the initiation of recruitment for ENCOMPASS, the study team worked with key stakeholders to define sepsis patients with high post-sepsis care needs likely to benefit from the STAR program, based on clinical sepsis criteria and coexisting risk profiles . ConsistWe will exclude patients: 1) with a change in code status  within 24 h after index presentation due to presumed limitation of aggressive treatment and exposure to STAR program components; 2) reside > 2.5 h drive time from the treating hospital due to the maximum reach of the community services leveraged within the STAR program and the general assumption that these patients may have less comprehensive utilization tracking within AH record systems for study outcomes; 3) are actively participating in a different AH care management program  documented in the EHR at time of hospital admission; or 4) have been previously randomized as part of this study.Our readmission risk model includes clinical and administrative data selected based on their association with post-sepsis outcomes and conceptual models of post-sepsis pathophysiology such as: physiologic measurements, lab values, basic sociodemographic characteristics, and personal medical history –48. All For patients in both arms, data from the automated, daily patient list are sent electronically to the study database. During the intervention phase at each facility , the navigator receives the list of admitted, eligible high-risk patients via secure email. The navigator contacts eligible patients at intervention hospitals by telephone and introduces the STAR process before discharge. Our team has successfully deployed these same processes during prior transitions interventions . In thisArm 1: Usual Care. When assigned to Usual Care, hospitals and their providers will not have access to the STAR program. Patients in the Usual Care group continue to receive routine clinical care throughout their hospitalization and following hospital discharge. Usual Care elements are not prescribed but, in everyday practice, typically consist of: patient education and follow-up instructions at discharge; routine recommendations for timely outpatient follow-up visits; arrangements for home health services, use of existing transitional care services for some patients  , or careArm 2: Nurse Navigator-Driven STAR. Patients admitted to hospitals in the intervention arm at time of eligibility determination receive care via the STAR program Fig. . The STAAll clinical and outcomes data are collected directly from the AH EHR system and Enterprise Data Warehouse (EDW). Data are collected during the index hospitalization and include: basic sociodemographic characteristics , past medical history , infection and disease severity , hospital procedures , and organizational variables . Additional data are collected on care delivered during follow up and include: physical, speech, and occupational therapy, mental health assessment and referrals to behavioral health, outpatient follow-up visits, documented medication reconciliation in the EHR, Care Alignment Tool completion, and palliative care consultation. Navigators also document completion of STAR workflow processes in the patient’s EHR through the care management electronic documentation form, then data is exported into a research database (REDCap) .The primary outcome is a dichotomous, composite measure of mortality and hospital readmission assessed 90 days post index hospital discharge. This combined outcome is ideally suited to our pragmatic study design because mortality and hospital readmission are widely regarded as patient-important outcomes . In addiIn ENCOMPASS, mortality is defined as any date of death documented in the AH EDW within 90 days of index hospital discharge, including events from national death record data uploaded monthly into the EDW via our institutional subscription . RehospiInternational Classification of Diseases, 10th revision diagnosis codes) related to: a) sepsis or common infection  number of days alive and outside the hospital  –60; 2) a, sepsis , pneumon, sepsis ; 5) numbWe will use an intent-to-treat approach to primary and secondary analyses, such that all patients meeting identical criteria and randomized will be analyzed, regardless of adherence to intervention assignment. This real-world approach will assess intervention effectiveness while limiting selection biases associated with adherence. Because hospitals are randomized to STAR in a staggered sequence and the outcome varies at the patient level, we will use a generalized linear mixed-effects model to compare the composite 90-day mortality and hospital readmission primary outcome measure between the intervention conditions . The intMethods to Handle Missing Data. We do not anticipate substantial missing data because all outcomes are routinely collected variables and utilization is broadly captured within our large integrated system. Values for patients who do not have health care utilization or mortality records during the study follow-up interval are assumed to be null. While utilization may occur outside AH, this is not expected to be a major limitation because of AH market share and accessibility. Specifically, AH operates three large hospitals in Cabarrus and Mecklenburg Counties, the only acute care hospitals in Anson, Burke, Cleveland, Lincoln, Stanly, and Union counties, where most of this study will be conducted, and more than 40 hospitals in the region overall. Additionally, any utilization occurring outside the system is anticipated to be non-differentially distributed between groups and thus impact treatment groups equally. Further, internal historical data indicates nearly 75% of high-risk patients are Medicare-insured . For these patients, we will have complete healthcare claims within and outside AH facilities during the study interval, as captured through participation in the local AH-managed Accountable Care Organization. We will conduct subgroup analyses within this Medicare-insured population and will use this data to explore missing data patterns that can be adjusted using pattern-mixture methods in sensitivity analyses , 70.Based on our internal data from sepsis admissions to the study hospitals between 2015 and 2017, we estimate a total of 4480 patients will be eligible for participation during the 36-month ENCOMPASS trial enrollment period . From this historical data, we observed a 90-day mortality rate of 18% and 90-day hospital readmission rate of 42%. From published literature, a small percentage of deaths (~ 1–2%) and between 22 and 42% of hospital readmissions after sepsis are preventable , 71, 72.Outcome measures for evaluation of the STAR program implementation are summarized in Table We will collect data before, during, and after implementation. For pre- and post-implementation data collection, we will conduct qualitative interviews with eight administrative leaders and eight providers regarding organizational support, culture, and recommendations for STAR implementation and then post-implementation to plan future dissemination. During the implementation phase, we will gather data at three time points  from: a) qualitative interviews and surveys with navigators and providers to assess and understand barriers to and enablers of STAR implementation and to help improve access to sepsis care, b) qualitative interviews and surveys with patients and caregivers (recruited from intervention arms) to learn about their experiences with STAR during the peri-discharge transition, opinions and perspectives on services received and not received, and preferences for care, and c) focused ethnography of the navigators to obtain a holistic and nuanced understanding of the navigator’s role in the STAR intervention , 76. IntTo increase policy impact, we will also conduct a prospective economic evaluation to provide an understanding of the resource implications of the STAR program intervention on post-sepsis health benefits and costs. We will combine primary data and mathematical modeling to examine the costs and cost-effectiveness of STAR over the 90-day study follow up as a measure of benefit from the health system perspective . We willThe ENCOMPASS trial is overseen by a Data and Safety Monitoring Board (DSMB) comprising experts in critical care, infectious disease, internal medicine, biostatistics, and epidemiology. The DSMB and study investigators agreed not to establish formal guidelines for stopping but are monitoring 90-day all-cause mortality rates and major depressive disorder using the Patient Health Questionnaire-9  as safetHealthcare systems urgently need programs to better support sepsis survivors following hospital discharge —a key priority that has been intensified as a result of the coronavirus disease 2019 pandemic and anticipated increasing number of individuals impacted by sepsis . The ENCSecond, effectiveness studies for complex healthcare problems like this one too often neglect the vital importance of evaluating and adapting implementation practices based on robust implementation science frameworks and constructs. Similarly, there has been no dissemination and implementation research specifically on post-sepsis care in the context of a determinants framework to establish effective processes and their outcomes. Our proposal is innovative in its emphasis on optimization of dissemination and implementation of post-sepsis care in heterogenous healthcare settings, while enhancing traditional implementation frameworks with focused ethnography to obtain a more nuanced understanding of the nurse navigator’s role in implementation success. By evaluating the implementation and effectiveness of the STAR intervention across eight diverse hospitals, we will generate knowledge that will immediately be relevant for a broad range of acute care settings and healthcare systems. Additionally, incorporating cost effectiveness analyses will further provide key information to stakeholders to guide future use and scale-up of the STAR program, if shown to be effective and cost-favorable. Thus, the proposed project has the potential to shift the paradigm of how post-sepsis care can be more effectively implemented and disseminate the information widely to improve care and costs for millions of sepsis survivors.Additional file 1.


Epilepsy is a complex multi-factor group of disorders characterized by periods of hypersynchronous activity and hyperexcitability within brain networks. In this scenario, inhibitory neurotransmission dysfunction in modulating excitatory transmission related to the loss of subsets of parvalbumin-expressing inhibitory interneuron may have a prominent role in disrupted excitability. Some studies also reported that parvalbumin-positive interneurons altered function might contribute to psychiatric comorbidities associated with epilepsy, such as depression, anxiety, and psychosis. Understanding the epileptogenic process and comorbidities associated with epilepsy have significantly advanced through preclinical and clinical investigation. In this review, evidence from parvalbumin altered function in epilepsy and associated psychiatric comorbidities were explored with a translational perspective. Some advances in potential therapeutic interventions are highlighted, from current antiepileptic and neuroprotective drugs to cutting edge modulation of parvalbumin subpopulations using optogenetics, designer receptors exclusively activated by designer drugs (DREADD) techniques, transcranial magnetic stimulation, genome engineering, and cell grafting. Creating new perspectives on mechanisms and therapeutic strategies is valuable for understanding the pathophysiology of epilepsy and its psychiatric comorbidities and improving efficiency in clinical intervention.Parvalbumin is a calcium-binding protein present in inhibitory interneurons that play an essential role in regulating many physiological processes, such as intracellular signaling and synaptic transmission. Changes in parvalbumin expression are deeply related to epilepsy, which is considered one of the most disabling neuropathologies.  Epilepsy is one of the most disabling chronic neurologic disorders, significantly impacting patients’ quality of life . WorldwiDrug resistance in epilepsy also is associated with loss of productivity, employment, and significant direct and indirect health costs . It coulEpilepsy is a complex multifactorial group of disorders characterized by periods of hypersynchronous activity and hyperexcitability within brain networks. It is complex because it is not a single disease but the result of a wide range of underlying etiologies and pathologies , all shaThe transient occurrence of signs and/or symptoms due to an abnormal excessive or synchronous neuronal activity in the brain defines an epileptic seizure . DysfuncInterneurons represent a crucial evolutionary step that enables different forms of computational processing and rapid dynamics. Among them, parvalbumin-positive interneurons (PV+) are one of the master regulators of excitation-inhibition balance and timing of principal cells, supporting different oscillation patterns . Not onlTherefore, PV+ interneurons may exert a convergent role, configuring an important link in epilepsy and psychiatric comorbidities. Promising discoveries could lead to a better understanding of the pathology mechanisms and determinant advances in the treatment. Therefore, advances in potential therapeutic interventions will be discussed, as we will present key findings on the parvalbumin role on epilepsy and epilepsy comorbidities.epilepsy(ies) or seizure(s) with parvalbumin, parvalbuminergic, or parvalbumin with terms related to the respective sections. The detailed information can be found in the Considering the sui generis characteristics and its involvement in epilepsy and neuropsychiatric disorders, we integrated basic and clinical findings in a translational perspective, highlighting potential therapeutic strategies in this comprehensive revision. Using PubMed, we combined the descriptors N-terminus of the protein [https://www.uniprot.org/uniprot/P20472]. Generally, the EF-hands function in Ca2+ binding with rapid to intermediate kinetics and affinity , in the C-terminal helix length , and properties of Ca2+ and Mg2+ binding and free energy change for divalent ions  lineages, the two isoforms of parvalbumin expressed in mammals, exhibit markedly different Caent ions . Those cent ions .2+ affinity relative to β-parvalbumin isoform, as it selectively binds Ca2+ over Mg2+ by ≈10 kcal/mol. A recent study indicates that, although the intrinsic characteristics of the EF-hand contribute strongly to the selectivity of Ca2+ in α-parvalbumin, allosteric changes affecting secondary and tertiary structures play a significant role in differentiating strong from weak Ca2+ binding. The authors also report that Ca2+ affinity and selectivity against Mg2+ are properties that emerge both from local effects at ion binding sites and non-local contributions elsewhere  are compared, some classic works emphasize the similarities of the expressions of parvalbumin and calbindin . HoweverPV+ interneurons are classified into two subgroups according to the synaptic input, the axo-axonic (chandelier) and the axo-somatic (basket cells) . Axo-axoThe morphology and characteristics of PV+ interneurons were elucidated in studies with different species, indicating that in primates, PV+ interneurons were found mainly in the hilus of the dentate gyrus, in the strata oriens layer, and the pyramidal layer of the Ammon’s horn. The predominant synapses were axo-somatic symmetric and asymmetric. Multipolar basket cell-like neurons in the neocortex were prominent in CA1. Also, in this subpopulation, it was identified that most dendrites have smooth, spineless dendrites or aspiny, with synapses occurring on the central axis of their dendrites, in addition to being postsynaptic to other axonal terminals .Findings also exist for more symmetric axo-somatic synapses in pyramidal cells in the region of CA2 than CA1 and CA3. Additionally, it is understood that the connection of PV+ interneurons dendrites occurs by gap junctions along the dentate gyrus and Ammon’s horn. Descriptions of the morphology, distribution, and location of such advanced neurons that hippocampal cells form a subset of GABAergic neurons, a local circuit, and that both feedback and feedforward inhibition participate .2+ transient , consistent with precise and reliable recruitment  . They maendrites , with a rites [+ . Interesruitment .+ channels density  is the most common type of epilepsy in adult patients and one of the most challenging types for seizure control, with more than 30% of pharmacoresistance . In ordeper se are not necessarily related to either hippocampal sclerosis)  . Does sulerosis) .Pivotal studies report findings showed increased reactive synaptogenesis of fibers labeled for GABAergic interneurons in resected epileptogenic tissue. Also, it was observed preferential losses of hippocampal/CA4 hilar neurons from extrahippocampal mass lesions or idiopathic patients. Those mapped GABAergic neurons were especially basket cells and hilar interneurons, which are involved in several feedforward and feedback axon circuits onto principal cells and other interneurons . Some ofAlthough parvalbumin immunoreactive cells are only a subset of GABA and calcium-binding neurons, a loss of parvalbumin could mean an essential and specific loss of a subpopulation of inhibitory neurons. However, several studies pointed that the observed reduction in parvalbumin- immunoreactivity could be associated with the parvalbumin protein expression levels and not the loss of the parvalbumin-expressing GABAergic neurons . The redAnalysis of postsynaptic target elements of PV+ axon terminals showed that they form symmetric synapses with soma, dendrites, axon initial segments, and spines as in control, but the ratio of axon initial segment synapses was increased in the epileptic tissue. Furthermore, the inputs in initial axon segments increased about three times in the epileptic samples . At the So, additionally to the change of the specific above-mentioned local-circuit neurons, parvalbumin could also be responsible for synaptic reorganization in epilepsy by forming abnormal synapses. A recent finding demonstrates that in the resected sclerotic hippocampus of TLE patients, the sprouting of parvalbumin axons in dendrites and spines of granule cells in the dentate molecular layer was observed with the presence of ectopic PV+ neurons . The abnParvalbumin innervation appears to be restrictedly evaluated in other regions than the hippocampus. Neuronal cell densities in the basolateral complex of the amygdala were significantly reduced in the lateral nucleus (LA) in TLE patients as compared to the autopsy, followed by a parvalbumin reduction. Ultrastructure analysis revealed a reduction in perisomatic boutons and a remarkable reduction of axo-somatic GABAergic input onto excitatory cells, which correlated with a higher perisomatic fibrillary gliosis . Also, tTherefore, this brief overview of the histological and ultrastructure investigation of parvalbumin in human hippocampal epilepsy is not a simple change in the number of this interneuronal subpopulation. Instead, there is probably morphological plasticity in which increases in the initial axon segments and/or reductions or abnormal dendritic and somatic innervations contribute to excitability and synchronization . However, a model based entirely on the specific electrical manipulation of PV+ interneurons is not yet possible, making the association of genetic and optogenetic techniques necessary. Nevertheless, it is possible to stimulate areas with an intense concentration of PV+ interneurons, such as the prefrontal cortex , since Gor 5HT3A .While in the etiology of most epilepsies, a combination of both acquired and genetic factors is involved, epilepsies with a determinant genetic background (known as idiopathic) constitute only a minority of all seizure disorders  being esDysfunctions of mutated voltage- or ligand-gated ion channels have been considered to be a significant cause of idiopathic epilepsies , which wA receptors and metabotropic GABAB receptors are also significant candidates for involvement in epilepsy, considering the central role of GABA in mediating inhibition in the brain. Mutations in two of these genes have been associated with epilepsy in humans  and are associated with epilepsies (+-gated sodium (Nav) is attributed mainly to parvalbumin dysfunction . Nav channels have been implicated in numerous neurologic diseases, and Nav 1.1/SCN1A is a significant target of epileptogenic mutations. Thus, the reduced firing of inhibitory neurons through sodium channel loss of function may affect GABA release . It is suggested that Nav 1.1 is involved in the maintenance but not in the initiation of sustained fast spiking in the interneurons and probably also in regulating GABA release from the interneurons  using in vivo two-photon calcium imaging in the neocortex  2.1 (P/Q-type), could also be of great importance in sustaining parvalbumin dynamics and could be implicated in the epileptogenic process. Researchers developed an exciting new model using a conditional genetic approach to selectively ablate CACNA1A in specific subsets of cortical GABAergic interneurons. The regionally knockout animals exhibit a severe form of generalized epilepsy. It was also demonstrated that this mutation selectively impaired GABA release from parvalbumin, also leading to unreliable transmission with high failure rates and perturbed kinetics  regulates several cellular processes and death cascades by regulating mRNA translation . The mTOMutations in several transcription factors have been described to play a role in epilepsy  consistently caused SRSs (not SE) but did not persisted  injection does not increase neurodegenerative and morphogenic effects in parvalbumin–/– mice, indicating that KA effects are not altered in the absence of parvalbumin alone , parvalbumin was evaluated in the central auditory neurons. Cochlea and other auditory nuclei showed decreased parvalbumin volume and cell size at the same time it exhibited greater density. The authors interpreted this change in number and morphology as a protective mechanism to prevent cell death in the face of reduced afferent input .Wistar Audiogenic Rats (WAR) is a genetically selected seizure-prone rat strain susceptible to audiogenic seizures when exposed to high-intensity acoustic stimulation . These a2+ channel, lower levels of Ca2+-binding protein in these corresponding structures (In the Wistar Albino Glaxo from Rijswijk (WAG/Rij) rat, a genetic model of absence epilepsy, the somatosensory cortex contains a focus that initiates a cascade of events that ultimately leads to the occurrence of the bilateral and generalized SWDs . Quantifructures .2+-permeable AMPA receptor subunit expression ratio, indicating that hyperexcitability induced by NMDA receptor modulation was mediated through interneurons  in the amygdala and the hippocampus . These eImpairments of cortical PV+ interneurons are sufficient to induce generalized seizures , associaThe prevalence of anxiety in epilepsy in population-based studies is 13–23% . SeveralThe putative neurobiological substrates of these psychiatric comorbidities remain obscure, but specifically to anxiety associated with epilepsy, amygdalar impairments of excitatory/inhibitory balance seem to be central . The incIn animal models, the antiepileptic effects of benzodiazepines also treat anxiety-like behaviors . In a gePsychosis is the mainly psychiatric comorbidity related to PV+ interneurons   and loss–/– mice are less responsive to prepulse inhibition (PPI) than parvalbumin+/+ mice [–/– and parvalbumin+/+ mice  as lamotrigine, carbamazepine in a scenario of an already compromised channel function in GABAergic interneurons, could increase network excitability  . TMS is Transcranial magnetic stimulation paired-pulses selectively can activate GABA inhibitory functioning, observed by short-interval of cortical inhibition and cortical silent period measures . Not surIn vitro, repeated low-frequency TMS can induce long-term depression (LTD)  , while irneurons . These frneurons  particulrneurons . Also, trneurons .Unlike deep stimulation, the risks of TMS are reduced because they do not involve an invasive surgical procedure and can be reversible/interrupted. Thus, this therapeutic focusing on PV+ interneurons activation could be considered for patients with a high level of refractoriness.Given the challenges and restrictions in pharmacological and surgical intervention, gene therapy has been considered the most promising treatment strategy to achieve unmet needs at the bedside and dissect the circuit’s function in physiology and behavior at the benchside . In epilA receptor, as the allosteric sites (affinity for benzodiazepines drugs as zolpidem) have been genetically engineered to be rendered sensitive to pharmacological modulation in a restricted manner. Reversing this GABAA receptor knock-in by generating zolpidem-insensitive mice and then genetically imposing zolpidem sensitivity on a selected cell type enables the manipulation of specific GABAergic circuits  . Those wpression . Also, tpression . TherefoN-Oxide (CNO), an inert metabolite of the atypical antipsychotic drug clozapine together with olanzapine  . In the in vitro and in vivo studies in rodent models that chemogenetic enhancement of distinct populations of GABAergic interneurons parvalbumin activation reduced epileptiform discharges frequency in organotypic hippocampal slices model and increase in postsynaptic inhibitory input   .The authors discuss the prominent role of PV+ interneurons in postsynaptic inhibition due to their perisomatic targeting and extensive axonal trees. Therefore, individual PV+ interneurons are essential in mediating the effective inhibition of pyramidal neurons.in vivo model. The 4-AP injection to one of the hippocampi resembles the clinical situation where focal seizures are frequently initiated in an area of limited abnormal brain tissue from where they spread and propagate through regular brain networks. Recruitment of hippocampal PV+ interneurons caused a reduction in the frequency in the more severe seizure behavior scores and reduced the occurrence of all convulsive behavior, thus probably blocking the generalization  lization .in vivo (Reports show CNO pass the rodent blood-brain barrier (BBB), and a small amount of converted clozapine from systemic CNO delivery would presumably occupy CNS-expressed DREADDs in vivo . There iin vivo .For instance, some researchers have evaluated both CNO and clozapine effects in light of those new findings. In the study on parvalbumin activation in kainic acid and hippocampal kindled rats we described previously, the authors showed clozapine also significantly lowered seizure stage, shortened afterdischarges, and generalized tonic-clonic seizures. Interestingly, a seizure could not even be induced by the kindling stimulation in genetically modified drug-receptor mice that received clozapine pre-treatment (this was not observed in the CNO treated rats) . While tFood and Drug Administration (FDA) or European Medicines Agency (EMA)  . CNO metcy (EMA) . Howevercy (EMA) . In addicy (EMA) . To datecy (EMA) .in vivo   . In addiOptogenetics is a methodology that enables the modulation of neurons or pathways through light-sensitive ion channel proteins or pumps expressed in the cells of interest through genetic manipulation  can be targeted to any locus in the genome. Particularly, transcription activator-like effector (TALE) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) systems can be targeted at nucleotide resolution  production that can be easily prepared with one cloning process. The technique is time- labor-, and cost-effective. Mediated by a small guide RNA (sgRNA or gRNA), Cas9 can specifically introduce double-strand DNA breaks at pre-selected genomic loci, which (i) contain a target sequence (protospacer) complementary to the typically 20 nucleotides guide sequence of the gRNA, and (ii) are followed by a protospacer sequence adjacent motif. By the co-delivery of Cas9 and multiple gRNAs, the CRISPR/Cas9 system enables the simultaneous modification of several loci/genes .Therefore, the simplicity of sgRNA production has made it possible to generate gene libraries, gain and loss of valuable function for genetic screens and genetic therapy, which can be applied for either human, mouse, and primates studies . This deScn1a.dCas9 system targeted to GABAergic interneurons via intracerebroventricular injection into neonatal Scn1a+/– mouse pups. Electrophysiological recordings demonstrated that deficits in action potential generation in parvalbumin fast-spiking GABAergic interneurons known to be dysfunctional in Scn1a+/– mice were rescued by the Scn1a.dCas9 system. This report shows new horizons on the promising intervention by the CRISPR/Cas9 system in parvalbumin cells, which can promote a significant advance in epilepsy therapy.Given the abnormalities of GABAergic inhibitory interneurons in epileptic brain tissues, a strategy with considerable promise is to restore normal circuit function by transplanting GABAergic interneurons/progenitors to the seizure focus . InterneDuring early brain development, cortical GABAergic interneurons primarily arise from Medial Ganglionic Eminence (MGE) and Caudal Ganglionic Eminence (CGE) and then migrate tangentially over long distances to their final destinations, where they form local inhibitory synaptic connections with excitatory pyramidal neurons and other GABAergic interneurons .Nkx2.1-expressing progenitors, and a series of other transcription factors regulate the migration, differentiation, and electrophysiological properties  exhibited significantly reduced electrographic seizure activity and duration compared with untreated or vehicle-injected mutants. Immunohistochemistry analysis revealed MGE cells differentiate mainly in somatostatin and PV+ interneurons , and the antiepileptic properties were attributed to the enhancement of GABA-mediated inhibition onto host pyramidal neurons, based on electrophysiology in vitro and immunohistochemical techniques  were distributed throughout the adult hippocampus of adult epileptic mice in the pilocarpine TLE model. The group that received grafted interneurons showed a marked reduction in the electrographic seizures and restored behavioral deficits in spatial learning, hyperactivity, and the aggressive response to handling. In the host brain, GABAergic progenitors migrated up to 1,500 μm from the injection site, and in addition to expressing genes and proteins characteristic of interneurons, they also differentiated into functional inhibitory neurons and received excitatory synaptic input. In contrast with the hippocampus, from cell grafts into the basolateral amygdala, about only 10% differentiated into interneurons (the majority was somatostatin-positive). Although the group that received grafted cells rescued the hyperactivity deficit, the treatment did not affect seizure frequency .Stem cells from the anterior subventricular zone (SVZ) of postnatal F344 rat pups expressing the human placental alkaline phosphatase were grafted into the hippocampus of young adult rats at 5 days after excitotoxic injury . Analyses through the forced swim, water maze, and novel object recognition tests revealed SVZ stem cells grafting reversed the injury-induced cognitive deficit and depressive-like state. Graft-derived cells exhibited excellent survival and pervasive migration, and they mainly differentiated into astrocytes and GABAergic interneurons, including neurons and oligodendrocytes. Interneuronal types were mainly characterized by calbindin+ and PV+ reductions and abnormalities in neurogenesis by both maintaining a normal level of stem cell activity in the subgranular zone. Grafted cells expressed significant amounts of neurotrophic factors, which were believed to contribute to stimulating neurogenesis in both the local injured area and subventricular zone, probably contributing to the increased reelin secretion, an extracellular matrix protein provided by interneurons that control newly born dentate granule cell migration .Similar findings were observed in the pilocarpine chronic TLE. The research group that has previously provided a very descriptive protocol using this model  reportedInterestingly, graft-derived neuropeptide Y-positive and somatostatin-positive interneurons were primarily seen in smaller clusters, suggesting specific clones probably derived them. Grafted groups exhibited a smaller reduction of normal neurogenesis induced by SE, also displaying fewer aberrant neurogenesis. Animals submitted to cell therapy presented a smaller reduction in reelin interneurons, preserving more PV+, neuropeptide Y+, and somatostatin+ interneurons 5 months after SE. To test whether all the therapeutic effects were related to the activity of grafted cells, a different group received the transplanted cells that were genetically modified for inhibitory modulation by DREADD protocol. It has compared a period of inhibition of grafted cells by CNO administration with a previous control period. Researchers showed the seizure suppression was abolished, suggesting a direct antiepileptic impact of graft-derived interneurons through increased inhibitory neurotransmission .Comparing this study and previously published by Hunt and colleagues, as both used pilocarpine TLE models, it would be interesting to discuss the differences, implications, and the potential advances of using inhibitory human pluripotent stem cells.Shetty’s research group recently expanded the translational use of grafted cell therapy using a non-invasive intranasally administered Human MSC-Derived Extracellular Vesicles protocol. They developed a protocol for efficiently delivering extracellular vesicles, nanosized membranous particles released by neural stem cells, and mesenchymal stem cells from the bone marrow (hMSCs). Intranasally Administered hMSCs incorporation by neurons were comparable between Naïve and SE Rats in most forebrain regions, but interestingly, SE-injured lesions incorporated more cells . Extracellular Vesicles markers were mostly found inside neurons and microglia but not within astrocytes, and it was not reported if hMSCs differentiated into interneuronal subtypes . A previConsidering the significant role in regulating many physiological processes, such as intracellular signaling and synaptic transmission, changes in parvalbumin are deeply related to epilepsy. This review discussed some preclinical evidence from various animal models in epilepsy that demonstrate the translational value of interventions aiming at those interneurons.We have explored different forms of manipulations, from current antiepileptic and neuroprotective drugs acting on PV+ interneurons to cutting-edge manipulations such as DREADD and optogenetics, cell therapy, or other groundbreaking advances in neurosciences. Those techniques can be viewed as therapeutic approaches and innovations that enable us to move up to a higher understanding of the pathophysiology of the disease. The current state of DREADD has significantly advanced toward synthetic improved chemical ligands such as JHU37152 and JHU37160, more potent and selective that can be associated with high-affinity DREADD PET radiolabeled ligands and be mapped through non-invasive visualization, additionally to DREADD-assisted metabolic mapping . Those aSimilarly, there are advances in optogenetics that enable the dual control of excitation and inhibition. BiPOLES is a reliable dual-color neuronal spiking and silencing optogenetic tool that was already demonstrated in worms, flies, mice, and ferrets . The praAs previously discussed, many pharmacological treatments present a non-specific mechanism that makes it difficult to pinpoint the role and changes in parvalbumin related to the pathology and the antiepileptic effect. All drugs indicated in this review have been shown to generally act on GABAergic neurotransmission, which is the most studied and evaluated in different treatment protocols. Those are valuable and indispensable interventions to the clinic and understand inhibitory transmission in the pathophysiology, but non-pharmacological approaches may represent a new horizon of neuroscience research, including epileptology.Embryonic mesenchymal cells differentiate mainly in somatostatin and PV+ interneurons, and the antiepileptic properties were attributed to the enhancement of GABA-mediated inhibition onto host pyramidal neurons based on the many reports described here. In 2020, Cronutt, a 7-year-old male sea lion with intractable seizures due to algae intoxication, successfully received a precisely targeted injection of MGE-cell from pig source in the hippocampus. Since then, Cronutt has been seizure-free and this report has paved a new avenue for cell therapy in large mammals .Reduced numbers of parvalbumin cells, altered morphology, immature electrophysiological properties with lower firing rates, and power of gamma oscillations play a role in Epilepsy and its comorbidities. Strategies of enhancing GABAergic inhibitory neuron function either through secondary mechanisms or directly by modulating parvalbumin function may affect its activity and consequently reduce seizure susceptibility and behavioral disruption. Similarly, TMS pulses interfere with GABAergic functioning and possibly the parvalbumin fast-spiking interneurons changing synchronization and cortical excitation that may present abnormally in patients with epilepsies and/or psychiatric disorders.Finally, since parvalbumin is involved in many higher cognitive processes, its altered function is related to other psychopathologies and psychiatric comorbidities in Epilepsy. There are some challenges in addressing behavior and comorbidities in experimental epilepsy studies. Many of the described findings showed differences in some behavioral tests associated with many essential processes and not to a specific phenotype involved in anxiety, depressive, and psychotic-like behavior. Untangling differences and understanding the mechanism and changes in brain structures may contribute to a neurobiology perspective in epilepsy comorbidities. Parvalbumin functioning requires such an approach due to its intrinsic characteristics of regulating information processing through different brain regions.Considering this, chemogenetic and optogenetics approaches could provide essential insights into parvalbumin’s role in Epilepsy and psychiatric comorbidities. In rodents, stimulation of PV+ interneurons shows a complex engram of circuits related to psychiatric-like behaviors. Depressive-like behaviors are mainly related to disruption of prefrontal parvalbumin activity, but the hippocampus contributes at least in part to this process. In contrast, the hippocampus plays a fundamental role in anxiogenic and psychotic-like behaviors, with the amygdala and prefrontal cortex also associated with anxiety and psychotic-like deficits, respectively. Still, parvalbumin activity in several not-limbic structures could influence these effects and be investigated in the future. Nevertheless, it is possible to speculate that the propagation of seizures in Epilepsy may affect the functioning of PV+ interneurons in different circuits, sharing similar circuits that are involved in psychiatric symptoms exclusively.LG, TP, MR, and JLL conceived and wrote the manuscript. MR designed the figure. JPL contributed with a critical and historical revision, and with funding acquisition. All authors approved the manuscript 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.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.