Exosomes (EXs) and extracellular microvesicles (EMVs) represent a diverse assortment of plasma membrane-derived nanovesicles, 30–1000 nm in diameter, released by all cell lineages of the central nervous system (CNS). They are examples of a very active and dynamic form of extracellular communication and the conveyance of biological information transfer essential to maintain homeostatic neurological functions and contain complex molecular cargoes representative of the cytoplasm of their cells of origin. These molecular cargoes include various mixtures of proteins, lipids, proteolipids, cytokines, chemokines, carbohydrates, microRNAs (miRNA) and messenger RNAs (mRNA) and other components, including end-stage neurotoxic and pathogenic metabolic products, such as amyloid beta (Aβ) peptides. Brain microglia, for example, respond to both acute CNS injuries and degenerative diseases with complex reactions via the induction of a pro-inflammatory phenotype, and secrete EXs and EMVs enriched in selective pathogenic microRNAs (miRNAs) such as miRNA-34a, miRNA-125b, miRNA-146a, miRNA-155, and others that are known to promote neuro-inflammation, induce complement activation, disrupt innate–immune signaling and deregulate the expression of neuron-specific phosphoproteins involved in neurotropism and synaptic signaling. This communication will review our current understanding of the trafficking of miRNA-containing EXs and EMVs from astrocytes and “activated pro-inflammatory” microglia to target neurons in neurodegenerative diseases with an emphasis on Alzheimer’s disease wherever possible.  As sucl., 2014 ; Rodrigul., 2014 ).(i) were first characterized about ~40 years ago as a heterogeneous group of cell cytoplasm-derived intracellular micro-particles (MPs) of endosomal origin ; (ii) are variable in their content and morphology, ranging in size from about ~30 to ~100 nm in diameter; (iii) are released by a variety of central nervous system (CNS) cells whose function was originally described as a cytological basis for intercellular signaling and communication : al. 1981 ; Columboal. 1981 ; Jiang eal. 1981 ; Mathewsal. 1981 ; Arbo etal. 1981 ; Martinsal. 1981 ). EXs oral. 1981 ; Arbo etal. 1981 ). Extracal. 1981 ; Webers al. 1981 ). Similaal. 1981 . Astrocyal. 1981 ; Leidal al. 1981 ). Some oal. 1981 ; Serpental. 1981 ; Vanherlal. 1981 ; Mathieual. 1981 ; Arbo etal. 1981 ; Seyedraal. 1981 ; Upadhyaal. 1981 ). As mosal. 1981 ; Urbanelal. 1981 ; Federical. 1981 ; Groot aal. 1981 ; Stahl eal. 1981 ; Upadhyaal. 1981 ; VanherlEscherichia coli and Bacteroides fragilis, species of the family Brassicaceae such as Arabidopsis thaliana, Protists, protozoa such as Amoeba proteus and slime molds such as Dictyostelium discoideum, nematodes such as Caenorhabditis, and up the evolutionary scale to a wide range of mammals including humans. The biogenesis, secretion and the release of EXs, MPs and/or EMVs into the extracellular space or external environment for the purpose of mediating intercellular communication or transmitting DNA- or RNA-encoded genetic information between different cell types and the environment is therefore a very ancient and conserved evolutionary process  tract bacteria such as l., 2014 ; Arbo etl., 2014 ). From wl., 2014 ; Cong etl., 2014 ; Avsar el., 2014 ).(i) are released under normal physiological conditions, but are also discharged from parent cells upon cellular activation, hypoxia and/or hyperoxia, senescence, apoptosis and disease via a paracrine- and endocrine-type type action to their target cells; (ii) represent one of the major biological mechanisms for genetic exchange, immune signaling and the spread of inflammation and disease between cells of the host; (iii) EX and EMV trafficking in the mammalian CNS is a particularly robust, active and dynamic process . The hul., 2007 ; Seyedral., 2007 ; Song etl., 2007 ). Indeedl., 2007 ; Vanherll., 2007 ).extracellular directional strategy” for vesicular sorting and translocation over both short and long distances . Many ol., 2015 ; Serpentl., 2015 ).(i) that these vesicular organelles consist of a unique repertoire of cytoplasmic components representing cellular, molecular and genetic information that is a direct reflection of the unique biological condition of the parent cell’s cytoplasm at the time of vesicular release; (ii) that these microparticles play important roles as enveloped proteolipids, a nucleic acid-enriched “information packet” in a complex extracellular communication network; (iii) that EXs and EMVs may reprogram recipient, adjacent cells and/or distant tissues as CNS-resident cells involved in immune-surveillance and the maintenance of normal cellular homeostasis, while also contributing to neuropathology during disease; (iv) that the molecular content and rates of production and secretion of EXs and EMVs vary greatly depending on the cell-type and physiological state of the cells of vesicular origin . Attestl., 2018 ; Barnes l., 2018 ; Leidal l., 2018 ).Atropa belladonna) and higher animals (Homo sapiens) over many billions of years of evolution  are the smallest known gene information-carrying nucleic acids yet discovered; (ii) are important posttranscriptional and epigenetic regulators of mRNA abundance, speciation and complexity in aging, development, neurological health and disease processes; (iii) play pivotal roles in the initiation, development and propagation of many human CNS disorders including progressive terminal cancers and lethal, age-related neurological syndromes with an inflammatory component; (iv) are loaded into both EXs and EMVs and are a typical component of their vesicular cargoes are soluble, amphipathic, single-stranded non-coding RNAs (sncRNAs) 18- to 25-ribonucleotides (nt) in length whose RNA sequences have been very highly selected. Some miRNAs, such as the neurologically relevant miRNA-378, are very highly conserved, and their core ribonucleotide sequence has remained virtually unchanged in plants . Abundal., 1990 ; Lukiw 2l., 1990 ; Lukiw 2l., 1990 ; Zhao etl., 1990 ; Hosaka l., 1990 ; Slota el., 1990 ; Swarbril., 1990 ; Briand Homo sapiens while only about 25–30 individual miRNA species, or fewer, are abundant and easily detected in the human brain and retina . The upl., 2016 ; Lukiw 2l., 2016 ; Zhao etl., 2016 ; Wang etl., 2016 ; summaril., 2016 ). Importl., 2016 ; Zhao anl., 2016 ).pro-inflammatory miRNAs”, including miRNA-34a, miRNA-125b, miRNA-146a, miRNA-155 and others in the parenchyma of the temporal lobe neocortex (Lukiw 2007 [(i) from the microarray analysis of mast cells involved in innate and adaptive immunity, autoimmunity, and inflammation  from studies of specific miRNAs, mRNAs and angiogenic proteins in glioblastoma and neuroblastoma tumor cells that have released EXs and EMVs , Huntington’s disease (HD), stroke and other neuro-inflammatory degenerative conditions. This indicates a definitive role for EX and EMV miRNA cargoes in neurological disease processes with an inflammatory component which may have considerable diagnostic, prognostic and/or therapeutic value  brains (Lukiw 2007 ) This inkiw 2007 ; Valadi kiw 2007 ; Hunter kiw 2007 ; Sethi akiw 2007 ; Zhao etkiw 2007 ; Hammondkiw 2007 ). The fil., 2007 ); (ii) fl., 2008 ; Briand l., 2008 ; Skog etl., 2008 ). As disl., 2018 ; Mathieul., 2018 ; Li et al., 2018 ; Serpentl., 2018 ; Upadhyal., 2018 ). Becausl., 2018 ; BarbagaAccumulating evidence continues to implicate secreted miRNAs, including EX and EMV-encapsulated miRNAs, in the pathogenic spreading of progressive, age-related and incapacitating neurodegenerative diseases with an uncontrolled or deregulated inflammatory component and synaptic deficits. These include Alzheimer’s disease (AD), age-related macular degeneration (AMD), Parkinson’s disease (PD), Huntington’s disease (HD), prion disease (PrD), multiple sclerosis (MS), Japanese and viral-induced encephalitis and many related amyloidopathies, tauopathies and synucleinopathies , hypoxil., 2016 ), by coml., 2016 ) and micl., 2016 ; Lukiw el., 2016 ; Alexandl., 2016 ). As obsl., 2016 ; Ludwig l., 2016 ).(i) the signals and pathways essential for stimulation and the origin of their formation, as well as the mechanism of their release from many different cell types in the CNS and their proficiency for modulating functions of target cells; (ii) the molecular-genetic injury and/or environmental factors which stimulate their release for this evolutionarily-conserved type of information communication system amongst astrocytes, microglia and neurons; (iii) their increased production and release during the initiation and spread of progressive age-related inflammatory neurodegeneration; (iv) the actual molecular contents, stoichiometry and packaging of the contents in the vesicles themselves; (v) the magnitude and signaling impact of their plasma membrane-packaged vesicular cargo; (vi) the regulation of their trafficking and targeting to neuronal cells via plasma membrane-mediated cell-surface reception mechanisms; (vii) the lipidomic, proteomic and transcriptomic profiles of these vesicles and what miRNA and/or mRNA encoded information these vesicles may be carrying; (viii) whether or not EXs and/or EMVs can transfer their miRNA-enriched intraluminal cargoes to other cell types and/or to other species; (ix) the role of circular RNA (circRNA) which have been shown in some cases to act as natural “anti-miRNA sponges” of specific miRNA activities , microparticles (MP), exosomes (EX) and extracellular microvesicles (EMV), the nature of their vesicular cargoes and miRNA composition. These include: kiw 2013 ; Zhao etkiw 2013 ; Fakhourkiw 2013 ; Pogue akiw 2013 ; Zhao ankiw 2013 ; Avsar ekiw 2013 ; Groot akiw 2013 ; Hou et kiw 2013 ; Li et akiw 2013 ; Ma et akiw 2013 ; Serpentkiw 2013 ). A singThe neurobiology of EX and EMV genesis, release, translocation and uptake by target cells, and their containment of select miRNA populations enriched in the CNS, indicate that they are significant components of a highly dynamic system of intercellular communication via extracellular translocation and targeting in brain cell health, aging and disease. Multiple independent studies indicate that while vesicle-mediated intercellular signaling is important in the homeostatic maintenance of brain cell functions, they have a substantial role in the proliferation of injury, cancer and inflammatory neurodegeneration signaling, as is observed in the AD brain. Many of the details of the mechanisms by which EXs and EMVs and their miRNA cargoes are generated and released by the activation of astrocytes and microglia and their trafficking to target brain cells, primarily neurons, remain to be further clarified. A greater understanding of the mechanisms underlying EX and EMV biogenesis, cargo selection and loading, vesicle release, translocation exterior to the cells of origin and targeting to adjacent or distant neural cells remains critical for unlocking the immense neurobiological and therapeutic potential for these ubiquitous organelles. Not only could an increased understanding of EX and EMV systems and their containment of miRNAs in the brain be useful in the treatment of CNS injuries and progressive age-related inflammatory neurodegeneration, but may also prove useful as delivery vehicles for therapeutic miRNAs, anti-miRNAs and both bioactive and neuroactive pharmaceuticals.


Phase-contrast enhanced micro-computed tomography reveals huge discontinuities at the interfaces between dental fillings and the tooth substrate. Despite the complex micromorphology, gaps in bonding could be visualized and quantified in 3D. ImageJ. PCE-CT at sub-micrometre resolution provided images with an impressive increased contrast and detail when compared with laboratory micro-computed tomography. The interface between the dental adhesive and the tooth was often strongly disrupted by the presence of large debonded gaps . The thickness of the gaps spanned 2 µm to 16 µm. There was a large variability in the distribution of gaps within the bonding area in each sample, with some regions around the canal exhibiting up to 100% discontinuity. Although only several micrometres thick, the extensive wide gaps may serve as gateways to biofilm leakage, leading to failure of the restorations. They can also act as stress-raising ‘cracks’ that are likely to expand over time in response to cyclic mechanical loading as a consequence of mastication. The observations here show how PCE-CT can be used as a non-destructive quantitative tool for understanding and improving the performance of clinically used bonded dental restorations.Bonding of resin composite fillings, for example following root-canal treatment, is a challenge because remaining gaps grow and lead to failure. Here, phase-contrast-enhanced micro-computed tomography (PCE-CT) is used to explore methods of non-destructive quantification of the problem, so that countermeasures can be devised. Five human central incisors with damaged crowns were root-filled followed by restoration with a dental post. Thereafter, the crowns were rebuilt with a resin composite that was bonded conventionally to the tooth with a dental adhesive system (Futurabond U). Each sample was imaged by PCE-CT in a synchrotron facility  with a pixel size of 650 nm. The reconstructed datasets from each sample were segmented and analysed in a semi-automated manner using  To fix this, the dentist restores the tooth shape and function using a combination of bonded composite materials that need to tightly adhere to the substrate. Biomaterials are typically chosen for mechanical durability and for aesthetic considerations, while matching the mechanical behaviour to the remaining tooth structure. Such dental biomaterials are used to return the tooth into function for patient satisfaction, and specifically to make sure the reconstruction withstands the repeating loads of mastication for many years. In fact, a main objective of contemporary dental treatment is to establish strong and continuous bonds between the filling and the tooth substrate. Current treatment protocols advocate bonded sealing for a variety of reasons. These include increased mechanical stability of the restoration due to an improved distribution and resistance to stresses, as well as prevention of bacterial percolation at the interface  of the crown  to attributes of the bonding system    to increase contrast. 3D measurements of whole, root-treated teeth have previously been demonstrated using PCE-CT, revealing different density dental materials at micrometre resolution  accentuates interfaces due to the combined effects of high flux ‘partial-coherence’ X-rays. This facilitates the differentiation between materials with similar density  by the Ethical Review Committee of the Charité Universitätsmedizin Berlin, Germany, and were stored in an antiseptic solution prior to the experiment. Each tooth had its root canal treated following a standardized protocol  each tooth was restored using a fibreglass dental post. This was cemented into the root canal with a self-adhesive resin cement, following manufacturers recommendations . Such d2.4.Each tooth crown was restored immediately after post cementation. For that, the exposed tooth cervical area was acid etched for 10 s, followed by rinsing and air drying. A dental adhesive system was applied following manufacturers recommendations, including light curing for 10 s Table 1. A stand2.5.After tooth restoration, each sample was mounted in a transparent vial , padded with wet foam to maintain humidity and to avoid dehydration during imaging.ImageJ 1.52d, National Institute of Health, USA; Amira ZIB-Edition, Konrad-Zuse-Zentrum für Informationstechnik Berlin, Germany). The cervical areas, including the rim between root and the crown restoration were selected for imaging by PCE-CT in a synchrotron. Each sample was scanned on beamline ID19 of the European Synchrotron Radiation Facility  using inline propagation-based contrast microtomography   was used to first image each specimen . Following reconstruction  the architectures of the restorations were examined in both 2D and 3D ]. The resulting binary images had their different sets of connected pixels and voxels individually numbered and labelled using the ‘Connected components Labelling’ algorithm. The different components localized to the adhesive layer were then visually selected, discarding the irrelevant (outside of the interface) labels by using the ‘Select Labels’ function of MorpholibJ. The final Boolean volume comprising only the interfacial gaps between adhesive and tooth was overlaid onto the original volume , is due to the cutting lines, used to remove excess soft cement during restoration construction: a sharp straight scalpel was used to clear overflow prior to crown construction. Thus PCE-CT even reveals laying steps during restoration fabrication. With 2D slices containing the conditioned tooth area and gap, the percentage of gaps at the interface was calculated. Radial, orientation-specific variations in the non-bonded regions were quantified to assess the distributions of gaps at the interface. With the centre of the restoration/post defined as a pivot, the tooth and gap areas were divided in 18 equal segments (sectors) spanning 360° around the tooth long axis, each extending 20° on the tooth surface. For each sector, the ratio of gaps to the area of bonding was quantified and plotted against the azimuthal angular axis.To isolate debonding and interfacial gaps, the datasets were processed using the free extension package me Fig. 5 to verifme Fig. 5. The ext3.Laboratory µCT scans generated datasets that reproduced the approximate geometry and the main components of the tooth restoration within the root and the reconstructed crown. In such data, however, there was no difference in contrast between the resin composites used for crown reconstruction and the resin cement Fig. 5, nor wasPCE-CT at sub-micrometre resolution provided 3D datasets with an impressive contrast and a remarkable amount of detail . The micromorphology of the tooth and restoration were fully visible, revealing micrometre-diameter dentin tubules, highlighting the presence of fillers in the different resin composites and well reproducing the layout of single fibres within the fibreglass post. Example overviews and slices in the datasets are shown in a longitudinal slice in Fig. 6e.g. Fig. 6The enhanced contrast brought about with PCE-CT highlights gaps between the adhesive Fig. 6, tooth sThe results from the analysis of 18 sections of each sample, shown in the graphs of Fig. 74.in vitro, it is extremely difficult to produce a predictable sealed bonded interface. From a biological standpoint, establishing a continuous interface between tooth and adhesive is a major objective of treatment. It is a clinical objective aimed at preventing damages associated with water sorption, infiltration of bacteria and bacterial by-products, reportedly associated with secondary caries and/or re-infection of the root canal system  at the interfaces in adhesively restored human teeth. Of great concern is the observation that in each tooth at least 50% of the visible section of the outer imaged rim exhibited some form of gap, as seen in the central column of Fig. 7et al., 2019et al., 2004et al., 2005et al., 2019et al., 2014et al., 2015et al., 2019et al., 2014et al., 2014et al., 2019Due to the lack of contrast and resolution in the laboratory µCT images, it was not possible to observe the adhesive layer or the interfaces between materials. PCE-CT resolved this problem as it is extremely effective and may be the only possible means to non-destructively image interfacial gaps between the low-density polymer material and other structures within the tooth restorations (Soares et al., 2004et al., 2005et al., 2019et al., 2014et al., 2015et al., 2019Discontinuities at the interface between restorative materials and tooth substrate have been reported using high-contrast dyes such as silver nitrate. This radiopaque liquid has been widely used in dental research due to its high contrast in radiographs (Mollica et al., 2015et al., 2011et al., 2019et al., 2018The use of semi-automated image segmentation presented here will benefit from further improvements but already has many advantages. Since the data are obtained non-destructively, effects of artefacts and filtering can be tested and the 3D data can be quantified in a repeatable, quantitative operator-independent way (Carrera et al., 1996et al., 2007et al., 2004et al., 2005et al., 2011et al., 2014et al., 2016et al., 2016et al., 2016et al., 2019et al., 2011et al., 2018et al., 2020Synchrotron µCT imaging is an effective tool for examining different biological samples. The high photon flux density and almost parallel beams with a wide range of energies is suited for imaging both low- and high-density structures. Phase-contrast imaging (Cloetens et al., 2017in vitro performance record, comparable with other universal adhesive systems (Chen et al., 2015et al., 2015et al., 2010The adhesive used for testing here was Futurabond U, a clinically used universal adhesive system. It can be deployed either with or without prior acid etching of the tooth substrate (Sofan 5.in vitro. It is also a valuable tool for imaging interfacial gaps between thin polymer adhesives and tooth substrate in restored hydrated, treated teeth in 3D. Flaws in the thin polymer bonding layer were quantified non-destructively. Although a possible quantitative data processing pipeline was proposed, much more can be done for improved image segmentation and analysis. The use of PCE-CT will assist efforts to systematically assess the integrity of contact between different clinically used dental materials.The present work provides details about steps needed to measure gaps and the areas that they affect, without the use of any tracer dye and while strictly adhering to clinically used materials and procedures allowing reproducibility and comparability. The observations here clearly establish the use of PCE-CT for advancing our understanding about the ability of clinically used dental adhesive systems to form continuous interfaces with tooth tissues when measured


Background: CrossFit® training is a high-intensity functional training program that aims to increase physical functional performance through biochemical responses, i.e., hormonal, metabolic, and inflammatory responses. Most hormonal, metabolic, and inflammatory changes induced by CrossFit® training have been reported in isolated clinical studies. The purpose of this review was to systematically explore the existing literature on characterization of hormonal, metabolic, and inflammatory responses resulting from CrossFit® training.Methods: A systematic search of the literature was conducted in PubMed, Web of Science and Scopus from August 2019 to October 2019. Studies were selected through critical review of the content. Using specific keywords, 623 articles were found, of which 597 were excluded for ineligibility, and 25 were eligible. The papers were separated according to subject area: hormonal (n = 8), metabolic (n = 19), and inflammatory (n = 6) changes. All were published between 2015 and 2019.Results: This review reveals potential effects of CrossFit® training on hormonal, metabolic, and inflammatory responses. However, studies had low levels of evidence and reliability due to methodological limitations.Conclusion: In summary, the results showed a greater volume and intensity of workouts accentuate the responses, that are of paramount importance for improving understanding of the effects of CrossFit® training and serve as a basis for prescribing future exercise protocols. The training consists of a combination of different exercise elements: cardiovascular (CV), gymnastic and weightlifting exercises , is structured into joint mobility, warm-up, a technical portion, and the main portion. WODs are performed with short or no breaks between exercises, repetitions, and rounds  is a training program that emphasizes functional movements. HIFT uses a combination of movements, and self-selected time periods of work and rest , exercise modalities , methods (for time or AMRAP), and intensities (absolute or relative load). In reviewing the literature, it was observed that most of the hormonal, metabolic, and inflammatory changes related to CrossFit® training have been reported in isolated clinical studies.Studies on the physiological changes resulting from CrossFit® training.To date, no systematic review of such changes has been performed. The purpose of this review was to systematically review the existing literature on characterization of hormonal , insulin-like growth factor 1 (IGF-1), adrenaline, noradrenaline, metabolic , and inflammatory  responses associated with CrossFitA systematic literature search was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines , metabolic  and inflammatory (IL-6 and IL-10) parameters. CK, that is biomarkers of muscle damage, was included in inflammatory responses.® training interventions.Study design: Randomized and non-randomized trials, using either cross-over or parallel groups, comparing different types of CrossFit® training; (3) a sample of men and women; (4) studies that investigated at least one hormonal, metabolic, or inflammatory/muscle damage variable relevant to the analysis in the present study. Studies were excluded in the following cases: (1) duplicate articles; (2) articles that were not in the English language; (3) articles that presented training protocols not based on CrossFit® training; (4) articles with special populations; (5) articles that were systematic reviews, conference abstracts, dissertations, theses, and book chapters.The specific inclusion criteria were as follows: (1) articles that were original research; (2) intervention based on CrossFitThe systematic literature search was carried out until October 2019 using the following databases: PubMed, Web of Science and Scopus. The articles were searched using a combination of keywords corresponding to the theme of the review: CrossFit OR “high-intensity functional training” OR HIFT. Medical Subject Headings (MeSH) was consulted to check possible entry terms related to the keywords. After combining the research results and discarding duplicate studies in the databases, two researchers (NJ and MRD) independently selected titles and abstracts to identify relevant studies. The included articles were retrieved, read in full (full text) and independently assessed for eligibility by the same two researchers (NJ and MRD) according to the criteria described above. A meeting was held, and in the case of disagreement regarding the selection of articles, a third author (JN) was consulted to resolve the disagreement.® training protocols, and main conclusions was extracted (see Standardized data extraction forms were completed by two researchers (NJ and MRD) and verified by another researcher (JN). Information on the type of study design, characteristics of the participants, sample size, time of experience in the profile, data collection, CrossFitcted see –3. The eThe Cochrane Collaboration's risk of bias assessment tool was used to evaluate the internal validity of the studies  were selected for a complete full-text review. Finally, 25 articles were selected . All were published between 2015 and 2019, and most were of a cross-sectional design.n = 21; pre-training and post-training or comparison between groups), longitudinal  or descriptive of a cross-sectional cohort .The study design was described as acute  assessment, in which the HR remained between 85.9 and 97.4% of the maximum HR . There was a consensus that lactate levels are high immediately after a CrossFiti et al. , when evGlycemia was often investigated (8 studies). Similar to lactate, several studies showed an increase after independent WOD (Tibana et al., ® training (Cadegiani et al., Cholesterol, creatinine, GOT and GPT were each investigated in only one study. Cholesterol showed no differences after training (Shaw et al., Six studies (Tibana et al., CK seemed to increase after training (Durkalec-Michalski et al., IL-6 increased after WOD-independent training, while IL-10 increased as a function of WOD characteristics (Tibana et al., ® training. The results of this systematic review showed that there are still few studies for each observed variable. Of the 25 studies analyzed, all had different training protocols regarding the training stimulus administered. This methodological difference was found because CrossFit® training is constantly varied and consists of a combination of different exercise elements: CV, gymnastic and weightlifting exercises (Glassman, ® training seem to be related to the training variables, and the protocols with more volume and intensity provided greater biochemical responses. In addition, psychological factors, such as pre-competitive anxiety, can alter the physiological status of athletes (Mangine et al., The purpose of this review was to systematically examine the existing literature on characterization of hormonal, metabolic, and inflammatory responses associated with CrossFit® training practitioners and create an environment conducive to an increase in cortisol (Mangine et al., The studies demonstrated that increases in testosterone and cortisol levels occurred after WODs, with longer recovery intervals (Mangine et al., ® training.Some limitations were observed in the study by Mangine et al. , who eva® training, while cortisol levels decreased (Poderoso et al., Chronically, testosterone levels in men rose after 6 months of CrossFitThe training dose required to cause hormonal changes is difficult to determine. Notably, training with a greater volume promoted increases in GH concentrations (Kliszczewicz et al., ® training practitioners have higher levels of testosterone and GH and lower levels of noradrenaline than practitioners with overtraining syndrome. The higher levels of noradrenaline in practitioners with overtraining syndrome may be a compensatory attempt to maintain performance during exercise due to reduced conversion of catecholamines to metanephrines (Cadegiani et al., ® training has a high metabolic component may explain the increases in GH levels (Kliszczewicz et al., On the other hand, increases in testosterone levels may be closely related to the health of the participants. In a descriptive cross-sectional study, Cadegiani et al.  showed tCatecholamines showed acute elevations after WODs (Kliszczewicz et al., ® practitioners with other sport athletes, the responses may differ. From this perspective, Arruda et al. (® training (Poderoso et al., When comparing the chronic hormonal responses of CrossFita et al.  observed® training studies was blood lactate. An acute increased lactate concentration response was observed after training sessions (Fernandez-Fernandez et al., ® training, WODs generally do not have a standard break time, i.e., as the training is “for time” or AMRAP, the intervals are self-selected according to the suitability of the participants. Therefore, this characteristic can keep lactate elevated for a longer duration after the session (Goto et al., The main metabolic marker evaluated in CrossFit® training practitioners are inconclusive.As for chronic metabolic responses, the lack of change in lactate response may be the result of the intensity utilized for each WOD. It must also be considered that pre-training lactate was not registered (Murawska-Cialowicz et al., ® training session due to increased catecholamines. The increase in the glycemic rate in response to a training session is due to the need for greater utilization of glucose to meet the energy required for the sport, which has the particular characteristic of always being performed at high intensity (Glassman, Blood glucose was another variable observed. According to Timón et al.  and Klis® training session (Shaw et al., Cholesterol response does not appear to be affected by a CrossFit® training performance and aerobic capacity. Although the primary objective of the study was to examine the effectiveness of a sodium bicarbonate supplementation protocol, the CK response was similar, regardless of the supplement, increasing after the WOD. Another study found an increase in CK level after different WODs, which continued for up to 24 h (Timón et al., Acute muscle damage responses through CK were investigated in three studies (Durkalec-Michalski et al., ® training practitioners to a higher volume and intensity of training for successive days may expose the practitioner to the risk of damage associated with muscle cell necrosis.In addition to being an indicator of muscle damage, CK levels have been shown to be high in people with rhabdomyolysis (Honda et al., ® competition. In contrast, Heavens et al. (IL-6 and IL-10 were evaluated in two studies (Tibana et al., s et al.  showed ts et al.  used mens et al.  studied s et al. .® training on hormonal, metabolic and inflammatory factors. However, studies evaluating such aspects have a low level of evidence and reliability due to methodological limitations and biases that hinder the convergence of results. Apparently, hormonal, metabolic, and inflammatory stress marker levels increase after CrossFit® training, regardless of the protocol used. However, a greater volume and intensity of workouts accentuate the responses. Some parameters are inconclusive, such as blood glucose and IL-6 and IL-10 levels, due to different results and the small number of studies. Thus, this review sheds light on specific knowledge gaps that should be further investigated. Nevertheless, the results are of paramount importance for improving understanding of the effects of CrossFit® training and serve as a basis for prescribing future exercise protocols.The present review demonstrates the potentially significant effect of CrossFitAll relevant data is contained within the article. Further inquiries can be directed to the corresponding author.NJ structured and designed the research. NJ and MD carried out the review of studies. JGV and MD realized risk of bias. NJ, JN, JGV, MD, and JV contributed to the conception and writing of the article, reviewing, and editing the manuscript. DB corrected the final version and English grammar. All authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Various bacteria, archaea, and microbial eukaryotes also evolve H2 as a diffusible end product during fermentative metabolism through the activity of [FeFe]- or [NiFe]-hydrogenases (Horner et al., 2 is a facultative trait that is regulated through the expression and maturation of hydrogenases (Schwartz et al., 2 represents a substrate that organisms utilize to supplement their energy metabolism, thereby allowing for an expansion of their niche space in ecosystems where other sources of reductant are low or variable in supply (e.g., Amenabar et al., Approximately a third of sequenced microorganisms, spanning at least 70 microbial phyla, encode hydrogenases and are thus predicted to be capable of interconverting H2 in ecosystem level processes is increasingly being realized in both environmental and biomedical settings. A wide range of ecosystems have now been described where H2 cycling supports the bulk of primary production and where it forms the basis by which species interact, leading to ecologically structured communities. Much of the research on H2 metabolism to date has focused on ecosystems where H2 is present at elevated concentrations due to biological activity (e.g., anoxic sediments, gastrointestinal tracts; Sørensen et al., 2 can serve as source of reductant for aerobic soil microorganisms and that this can influence the composition of the atmosphere (Conrad, 2 metabolism is critical for the virulence of numerous pathogens, including Helicobacter, Clostridia, and Enterobacteriaceae (Kaji et al., The implications of H Conrad, . In para2 metabolism from the molecular to the ecosystem scale. In the area of anaerobic metabolism, there are articles exploring the metabolism of H2-metabolizing bacteria capable of sulfate reduction, acetogenesis, halorespiration, and fermentation. Two articles investigate H2 oxidation in sulfate-reducing bacteria using the model system Desulfovibrio vulgaris (Fauque et al., Smith et al. present a mathematical model of the growth and metabolism of this bacterium, whereas Löffler et al. investigate the kinetic isotope fractionation associated with its H2 oxidation activity. A comprehensive review led by Schuchmann et al. covers recent advances in understanding clostridial H2 metabolism; it details the discovery and characterization of multimeric electron-bifurcating [FeFe]-hydrogenases, including those associated with formate dehydrogenases (Schut and Adams, Dragomirova et al. focuses on heterologous expression of a [NiFe]-hydrogenase from dehalogenating Chloroflexi (Kublik et al., Pinske explores a third type of formate dehydrogenase-linked hydrogenase, namely the classical formate hydrogenlyase complex of Enterobacteriaceae (McDowall et al., This special issue, featuring 10 articles from 46 different authors, explores microbial H2 metabolism. Islam et al. report two other novel iron-sulfur proteins in mycobacteria, demonstrating that they are essential for the activity of the two high-affinity hydrogenases described in this lineage (Greening et al., Carere et al. meanwhile, build on the recent discovery that verrucomicrobial methanotrophs are facultative mixotrophs (Carere et al., Methylacidiphilum varies depending on H2 availability. Three articles also explore H2 metabolism at the ecosystem level. Adam and Perner explore the diversity of aerobic and anaerobic H2 metabolism in deep-sea hydrothermal vent systems, whereas Meyer-Dombard et al. investigate the influence of H2 on biogeochemical cycling in serpentinizing springs in the Philippines. Teng et al. review the previously underexplored area of H2 metabolism in bioremediation, including in the reduction of organohalides, nitroaromatic compounds, and heavy metals (Chardin et al., Several articles also investigate aerobic H2 in microbial metabolism and uncovers novel enzymes and pathways that mediate this process. This body of work highlights the intricate linkages between H2 cycling and the cycling of various other compounds, including methane, formate, carbon dioxide, sulfate, and organohalides, among others. In turn, these findings pave way for future studies on the biochemistry, physiology, ecology, and industrial applications of microbial H2 metabolism.In summary, this special Research Topic sheds light on the diverse role of HCG and EB drafted this editorial together and approve its submission.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.


Salmonella enteric serovar Typhimurium (ST), by oral consumption of a genetically modified (GM) probiotic strain in mice , and a significantly improved preventive effect was displayed. However, successful probiotic intervention of ST, one of the most prevalent foodborne pathogens, is challenging, and more work is necessary. We know that many probiotic strains have an inhibitive effect on ST by different mechanisms .ST is a Gram-negative, non-spore-forming, facultative anaerobic bacterium and can infect any warm-blooded animal  ST is rapidly expanding (Obaidat and Stringer, L. casei promotes overall bacterial species diversity and increases the abundance of Lactobacillus and Bifidobacterium in the cecum in mice (Peng et al., Lactobacillus spp. is mainly in the small intestine, and Bifidobacterium spp. is mainly in the large intestine (Donaldson et al., In chickens, ST first attaches to the cecal epithelial cells and then spreads to the liver, spleen, and oviduct. In pigs, early ST infection disrupts microbiome composition and functionality principally at the ileum (Argüello et al., Probiotics play important roles in human health. As normal commensals, they exert their prophylactic and therapeutic properties against ST in four main ways (Gut et al., L. casei modulates host immunity by regulating the expression of intestinal inflammation-related cytokines [e.g., suppressing pro-inflammatory cytokines and provoking anti-inflammatory cytokines after ST infection (Peng et al., Lactobacillus spp. modulate ST by regulating gene expression related to colonization and virulence (Muyyarikkandy and Amalaradjou, L. pentosus AT6 and its cell-free culture supernatants inhibit ST growth and its adhesion as well as invasion (Liu et al., In fact, probiotics can be modulators, producers, and residents after being administered. As modulators, probiotics effectively modulate either the host or the pathogen. L. casei or the GM counterpart has displayed a significant protective effect on ST infection (Peng et al., Salmonella enteritidis infection is efficacious in broilers, but data on prophylactic treatment timing regarding ST is not available (Higgins et al., Pre-administration of probiotics is an effective method demonstrated in animal studies. For example, 1-week pre-administration of either wild-type Bacillus spp., Bifidobacterium spp., Clostridium spp., Escherichia coli Nissle 1917, and Lactobacillus spp., yeast, and so on (Kanmani et al., Bifidobacterium spp. and Lactobacillus spp., harbor most of the well-characterized probiotic strains and are widely commercialized. These probiotics reduce more than 90% of caecal ST load, prevent invasion of organs, and even completely eradicate ST (Gut et al., Akkermansia muciniphila, Eubacterium hallii, and Faecalibacterium prausnitzii (Almeida et al., Probiotics are any non-pathogenic microorganisms that confer health-promoting properties when administered in adequate amounts (Hill et al., E. coli inhibits ST growth with improvement in fitness (Palmer et al., L. casei improves the protective effect on ST more than the wild-type strain by increasing CLA (Peng et al., Besides the traditional application of wild-type probiotics, GM probiotics are also studied. Although there are safety issues, GM probiotics attracted much interest due to their extra advantages and strengthened effects (Barra et al., Pseudomonas aeruginosa and reducing vancomycin-resistant Enterococcus by GM probiotic E. coli Nissle 1917, similar approaches might be also promising for ST prevention (Hwang et al., Along with the rapid development of synthetic biology, interest has increased on the design and construction of GM probiotics as live biotherapeutics for a range of medical applications (Chua et al., 6 CFU/g or CFU/mL viable cells may be an acceptable way. Considering the GRAS status, consumption of them can be without control. However, to cure ST infection, NG or GM probiotics are more promising. These probiotics can be formulated as concentrated pills or capsules containing more than 109 CFU/g or even more cells. They may be more suitable as over-the-counter drugs. Nevertheless, for any purposes, release of both conventional and GM probiotics as live biotherapeutical products in the market needs full assessment of safety (O'Toole et al., As pointed out, formulation of viable probiotics while enabling cost-effective biomass yield is a critical step toward product development of translational application (Almeida et al., ZS conceived the opinion. YS and JL wrote the draft manuscript. YS collected reference and drawn the figure. ZS finalized the manuscript and acquired funding. All authors discussed the content.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.


Resveratrol is a natural polyphenol that has anti-aging and anti-inflammatory properties against stress condition. It is reported that resveratrol has beneficial functions in various metabolic and central nervous system (CNS) diseases, such as obesity, diabetes, depression, and dementia. Recently, many researchers have emphasized the connection between the brain and gut, called the gut–brain axis, for treating both CNS neuropathologies and gastrointestinal diseases. Based on previous findings, resveratrol is involved in glucagon-like peptide 1 (GLP-1) secreted by intestine L cells, the patterns of microbiome in the intestine, the 5-hydroxytryptamine (5-HT) level, and CNS inflammation. Here, we review recent evidences concerning the relevance and regulatory function of resveratrol in the gut–brain axis from various perspectives. Here, we highlight the necessity for further study on resveratrol's specific mechanism in the gut–brain axis. We present the potential of resveratrol as a natural therapeutic substance for treating both neuropathology and gastrointestinal dysfunction. Resveratrol is a polyphenol that is secreted by grapes and berries -activated protein kinase (AMPK) pathway  and phosphodiesterases (PDEs) Chung,  Figure .SIRT1 gene, considered an anti-aging related gene, in the duodenum and also rescues insulin resistance and improves neuronal networks in the brain , an incretin hormone and a major hormone of the gut–brain axis, is linked to the control of energy homeostasis and the development of obesity , and finally lead to improved cognitive function  is expressed in both the CNS and gastrointestinal tracts, and currently 5-HT has been considered as an important target in the gut–brain axis.5-HT is a growth factor, a paracrine factor, and an enteric neurotransmitter  synthesis through 5-HT receptors (Prasad et al., Considering previous data, 5-HT derived from gut and brain contributes to nervous systems globally, and the circulation of 5-HT in the body mediates the gut–brain axis (Yano et al., 2C receptor-dependent signaling (Peng et al., A current study proved that resveratrol regulates the gut–brain axis by controlling the 5-HT-dependent pathway in an irritable bowel syndrome rat model and specifically that resveratrol influences various organs including brain hippocampus, ileum, and colon through 5-HT axis (Yu et al., Another recent study demonstrated that resveratrol could increase the expression of 5-HT, leading to the improvement of brain function (Nabavi et al., Several studies have mentioned the neurological role of resveratrol in depression and anxiety (Yu et al., One current study reported that the inhibition of 5-HT release attenuates the activation of GLP-1 receptor signaling and highlighted the relationship between GLP-1 and 5-HT serotonin system (Anderberg et al., Another study mentioned that GLP-1 receptor agonist liraglutide could reduce the expression of 5-HT2A receptor and subsequently reduces body weight and inhibits serotonin synthesis in mice model (Nonogaki and Kaji, Ripken et al. suggested that serotonin treatment could boost GLP-1 release, and the blocking of 5-HT receptor could affect the production of GLP-1 (Ripken et al., A recent study proved that 5-HT enterochrnomaffin cells in gut regulates gut microbial metabolism and homeostasis and is affected by the activation of GLP-1 (Lund et al., Further, ghrelin, known as a hormone for regulation of motivation and reward system among brain function, has been interacted with GLP-1 and the monoamine transmitter 5-HT (Currie et al., GLP-1 derived from brain mainly is produced by the nucleus tractus solitarius in brain (Alhadeff et al., Given previous evidences, resveratrol can control 5-HT and its receptor and also modulate release of 5-HT through GLP-1 regulation. Ultimately, resveratrol could control the neuropathology of neurological diseases such as depression and stress-induced anxiety. Also, resveratrol can regulate gut dysfunction in irritable bowel syndrome via 5-HT. Thus, we emphasize the necessity for further study of the specific mechanism and cellular pathways regulated by resveratrol and mediated by 5-HT to fully understand the gut–brain axis.Resveratrol is involved in the gut–brain axis through another mode in addition to the GLP-1 pathway and 5-HT system.Recently, gut microbiota is emerging as an important node in the gut–brain axis (Louwies et al., trans-stilbene have been reported to be the major microbiota-derived metabolites made from resveratrol (Juan et al., Resveratrol administration could be metabolized by the liver, intestinal tract, and gut microbiota Walle, . A recenBifidobacteria infantis and Lactobacillus acidophilus are strongly linked to piceid production from resveratrol (Basholli-Salihu et al., Enterococcus faecalis and increasing the Lactobacillus and Bifidobacterium populations (Qiao et al., Specifically, dihydroresveratrol as a metabolite of resveratrol is produced in the intestines such as the cecum, colon, and rectum through fermentation by the gut microbiota (Amri et al., Recently, resveratrol has been reported to improve gut microbiota in bowel diseases under harsh oxidative stress (Hu et al., A clinical study has reported that resveratrol treatment exerts cardiovascular and anti-obesity effects by ameliorating gut microbiota diversity (Bird et al., These previous findings demonstrate that resveratrol and gut microbiota influence each other. Furthermore, resveratrol could enhance the gut microbiota diversity and the gut barrier's homeostasis. These effects of resveratrol should be investigated further to determine the specific gut bacteria that affect the gut–brain axis.Here, we reviewed previous significant evidence of the effect of resveratrol on the gut–brain axis . We summJC, J-HJ, and JS contributed to the writing of the text. JC and J-HJ made and revised all figures. JS wrote and finalized the revised manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ten years ago, Archives of Toxicology has issued an Editorial by R.D. Combes  entitledIn the following, important research lines of computational toxicology were the development and refinement of computational models for relevant toxicological endpoints, such as liver injury, cardiotoxicity, renal toxicity and genotoxicity Ekins . Best prAs far as regulatory acceptance is concerned, a decisive breakthrough was the integration of (Q)SAR methodologies into the guideline ICH M7 “Assessment and control of DNA-reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk”, issued by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human use (Amberg et al. Advanced computational methodologies (Kusko and Hong Perspectives are emerging for computational approaches to predict the toxicity of nanomaterials Buglak  and of cComputational toxicology continues to assist in refining PBPK modelling (Savvateeva et al. A burst in manuscript submissions to “Archives of Toxicology” covering the in silico/computational field is noticed since 2019, signaling both increased scientific importance and regulatory relevance in the twenty-first century of this research area (Krewski et al. A current tendency, both in the United States (Kosnik et al. Given this exciting development, further submissions of manuscripts from these fields to Archives of Toxicology are highly encouraged!


This is one of the few polymers that the Food and Drug Administration (FDA) has approved for human administration due to its biocompatibility and biodegradability [Poly/poly(lactic acid) (PLGA/PLA) carriers were prepared through spray-drying to incorporate the microcrystals that were previously prepared by ultra-sonication. In vivo testing in rat models was demonstrated to prolong drug retention in joints. The TA remained there for over 28 days, which was more 21 days compared with the TA-free group. Furthermore, these nanocarriers were demonstrated to be stable for one year.In the first research study presented in this Special Issue, Ho et al. developed polymeric microspheres which contain micronized triamcinolone acetonide (TA) in order to increase the drug retention time in joints after intra-articular administration . Poly(laThe group of Peula-García used PLGA nanoparticles to carry bone morphogenetic protein (BMP-2) . The nanIn another study, Hwang et al. fabricated PLGA carriers combined also with a hydrogel matrix. They produced oxaliplatin-loaded PLGA microparticles using a double emulsion technique and then loaded them into hyaluronic acid and carboxymethyl cellulose sodium-based cross-linked hydrogels . This drKim et al. developed an original system to be used in the topical delivery of trolamine salicylate (TS), a topical anti-inflammatory analgesic used for the treatment of small joint pain . Here, tDuse et al. used PLGA nanoparticles to encapsulate curcumin, a well know natural compound that present anticancer benefits . It was Another interesting study proposed by Varga and colleagues, who contributed with an interesting study of nanoparticle design and optimization where the (±)-α-Tocopherol (TP) with vitamin E activity was encapsulated in PLA and PLGA nanoparticles . To stab2) cells.Morelli et al. improved paclitaxel delivery in the gastro-intestinal tract by encapsulating the drug in PLGA nanoparticles coated with PEG . The nanWith the objective to overcome the undesired lag time of the commercially available risperidone, Janich et al. encapsulated this drug in PLGA–lipid microcapsules and PLGA–lipid microgels . The carA research work using PLGA nanoparticles for ocular application was also collected. Ryu et al. produced rapidly dissolving dry tablets containing alginate and dexamethasone-loaded PLGA nanoparticles . These nAn interesting approach based on a combination of cell and drug delivery for the treatment of Huntington’s disease (HD) was proposed by André et al. . The autTwo works lead by Roing and Wacker present new theranostic PLGA-based nanoparticles. In the first one, biodegradable and photoluminescent polyester (BPLP) with PLGA polymer was used to fabricate biocompatible photoluminescent nanocapsules . AdditioPLGA toxicity was investigated by Bakhaidar et al. . Here, tAnother relevant study was performed by Operti et al., who used microfluidics technology as a tool to manufacture particles in a highly controllable way . In theiFinally, a research study regarding the importance of new techniques to characterize PLGA nanoparticles was included in this special edition. Shmool et al. investigated the dynamics of PLGA microspheres prepared by freeze-drying . The watThe papers presented in this Special Issue represent a small part of the research that is ongoing in the field of PLGA nanocarriers all over the world. The huge potential of PLGA nanoparticles make them a promising drug delivery system with outstanding properties and with much more potential for exploring in the coming years. With this Special Issue, the editors expect that the readers from the field find it stimulating and contributing more ideas or methodologies for their future work.


Parkinson’s disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. However, other non-dopaminergic neuronal systems such as the serotonergic system are also involved. Serotonergic dysfunction is associated with non-motor symptoms and complications, including anxiety, depression, dementia, and sleep disturbances. This pathology reduces patient quality of life. Interaction between the serotonergic and other neurotransmitters systems such as dopamine, noradrenaline, glutamate, and GABA controls the activity of striatal neurons and are particularly interesting for understanding the pathophysiology of PD. Moreover, serotonergic dysfunction also causes motor symptoms. Interestingly, serotonergic neurons play an important role in the effects of L-DOPA in advanced PD stages. Serotonergic terminals can convert L-DOPA to dopamine, which mediates dopamine release as a “false” transmitter. The lack of any autoregulatory feedback control in serotonergic neurons to regulate L-DOPA-derived dopamine release contributes to the appearance of L-DOPA-induced dyskinesia (LID). This mechanism may also be involved in the development of graft-induced dyskinesias (GID), possibly due to the inclusion of serotonin neurons in the grafted tissue. Consistent with this, the administration of serotonergic agonists suppressed LID. In this review article, we summarize the interactions between the serotonergic and other systems. We also discuss the role of the serotonergic system in LID and if therapeutic approaches specifically targeting this system may constitute an effective strategy in PD. Parkinson’s disease (PD) is one of the most common neurodegenerative disorders, which is characterized by the progressive loss of dopaminergic neurons in the substantia nigra compacta (SNc). Dopamine replacement therapy using the precursor L-DOPA is the main treatment for the disease. However, long-term use of L-DOPA leads to the development of dyskinesias and non-motor manifestations  contains the largest group of serotonin-producing neurons, and changes in DRN function have been implicated in neuropsychiatric diseases and movement disorders Hornung, . Classic1–7) and at least 15 receptor subtypes have been identified  may have a constitutive activity, which may be associated with pathophysiological conditions  appear particularly interesting for PD , which was reduced by dopaminergic and serotonergic lesions and suppressed by NMDA glutamate receptor antagonists, suggesting that stimulation of glutamate receptors is essential for the observed neuronal response , which is the main brain neurotransmitter mediating inhibitory signals. Deficiency in brain serotonin results in alterations in the GABAergic system  in striatal dopaminergic terminals. However, in advanced stages of the disease, the dopaminergic denervation is almost complete and other cell types showing AADC activity convert exogenous L-DOPA into dopamine, including serotonergic terminals (Arai et al., 2A antagonists (Meco et al., 1A agonist have been shown to reduce LID without compromising L-DOPA efficacy (Meadows et al., Compounds acting through the serotonin system such as anpirtoline, (Bézard et al., 1A agonists such as buspirone (Politis et al., 3 receptors also reduced LID (Kwan et al., 1 agonists with drugs that modulate the glutamatergic function (Tison et al., Clinical trials with serotonergic drugs are ongoing, revealing the promising antidyskinetic effects of 5HT1A receptor agonist buspirone produced significant dampening of GID in grafted patients. However, this effect could also be explained by the dopamine D2 receptor partial antagonistic effects of the drug (Politis et al., 1A and 5-HT1B agonists, suggesting that the effect of these drugs on GID is mediated by the activation of presynaptic host-derived receptors (Shin et al., 6 receptors (Aldrin-Kirk et al., Clinical trials using transplants of fetal dopamine neuroblasts have shown promising results, although many patients have developed GID (Freed et al., Interactions between serotonergic and other neurotransmitter systems reveal that serotonin plays a crucial role in the control of movement by the basal ganglia. These interactions are of great interest for understanding the pathophysiology of PD and to develop novel therapeutic strategies. Manipulation of the serotonergic system represents a valuable target to treat LID and GID in PD patients. However, further investigation is required to clarify mechanisms of neurotransmitter interactions and to determine optimal compounds and doses for effective therapies.All authors have contributed to this work and approved its final version for submission. AM developed the idea for this review and wrote the manuscript. AL-L, CL, and JL-G prepared the figure and were involved in the literature review and preparation and revision of the manuscript.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.


Preclinical models for the definition of anti-cancer drug safety and efficacy are constantly evolving. Indeed, tumor development in humans is not always fully reproducible and predictable in other animals. In turn, two-dimensional (2D) cultures, used for many years to test drug effects, are limited by the lack of tissue structure and architecture, which can influence both pharmacokinetics and pharmacodynamics, thus impairing the prediction of anticancer drug efficacy. An interesting and reliable alternative is represented by three-dimensional (3D) culture systems, including spheroids and matrix-based collagen or synthetic scaffolds, which have been validated by the EU Reference Laboratories as preclinical models to overcome at least some of the above-mentioned drawbacks.This thematic issue collects studies involving both advances in the techniques used to create and analyze some 3D culture systems and their applications in different cancer models for drug combination testing. The switch from 2D to 3D culture changes several conditions for cell growth, the first being a reduced surface for direct targeting, with the inner cell layers being protected from the external environment. At the same time, inner cells can suffer a reduced nutrient or oxygen supply, drastically changing their metabolism to a hypoxic one, causing the transcription of genes typically involved in drug resistance. This picture is even more complicated when co-cultures of different cell types are generated.A. R. Holub et al.  used an The bioprinting approach tested by Seokgyu Han et al.  allowed Ezequiel Monferrer et al.  comparedAlthough these models of scalable complexity allow us to investigate the interactions between tumor spheroids and the tissue microenvironment, their analysis is usually confined to classic microscopy approaches. Optical microscopy shows intrinsic limits that can be usually outdone only by high cost instruments. However, Eliana Steinberg et al.  have demAnother approach to increasing the information derived from 3D models has been described by Tarapong Srisongkram et al. , applyinAlthough new techniques allow researchers to extract an increasing amount of data from 3D cultured cells, the more immediate application of these models is still drug testing. This goal was pursued by Sadaf E. Pustchi et al. , testingA combined therapeutic regimen was also investigated by Layla Mohammad Hadi et al.  in 2D anIn summary, 3D cultures have contributed deeply to improving the reliability of in vitro pre-clinical models in anti-cancer drug testing. Moreover, with the establishment of organoids from various types of epithelial cancers, co-cultures of cancer organoids and immune cells have become a highly informative strategy for the development and testing of cancer immunotherapy, representing an additional useful application of 3D culture systems in cancer treatment.


Bacterial proteins exhibiting two or more unrelated functions, referred to as moonlighting proteins, are suggested to contribute to full virulence manifestation in pathogens. An expanding number of published studies have revealed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to be a multitasking protein with virulence impact in a number of pathogenic bacteria. This protein can be detected on the bacterial surface or outside the bacterial cell, where it interacts with host proteins. In this way, GAPDH is able to modulate various pathogenic processes. Moreover, it has been shown to be involved in non-enzymatic processes inside the bacterial cell. In this mini review, we summarize main findings concerning the multiple localization and protein interactions of GAPDH derived from bacterial pathogens of humans. We also briefly discuss problems associated with using GAPDH as a vaccine antigen and endeavor to inspire further research to fill gaps in the existing knowledge. Although bacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classic glycolytic enzyme catalyzing the conversion of glyceraldehyde-3-phosphate to 1, 3-bisphosphoglycerate , two distinct pathways participate in GAPDH secretion. In cells grown in Dulbecco's modified Eagle's medium, the GAPDH secretion was mediated by the type III secretion system generally involved in translocation of various effector proteins into the infected cells. Additionally, the interaction of GAPDH with CesT, a specific chaperone for type III effectors, was established. The associated chaperone may stabilize the GAPDH molecules and prevent them from interacting with other cytosolic partners, thus enabling their targeting to the type III secretion apparatus. The other secretory pathway has not been further described and is responsible for GAPDH secretion in EPEC and in probiotic E. coli strains grown in Lysogeny broth medium . Its binding specificity differs among individual pathogens, but plasminogen is the most common target . PathogeMycobacterium tuberculosis secrets iron-binding molecules known as siderophores  that compete with the host's iron-transport proteins (De Voss et al., M. tuberculosis utilizes surface-localized GAPDH to capture the human transferrin and then internalizes the transferrin–GAPDH complex. Three years later, the same research group (Malhotra et al., M. tuberculosis has even greater binding affinity to lactoferrin. M. tuberculosis GAPDH thus acts as dual receptor for both transferrin and lactoferrin. The ability of surface-localized GAPDH to bind transferrin has newly been demonstrated for Streptococcus agalactiae (Nagarajan et al., Staphylococcus ssp. Whereas, Modun and Williams (Streptococcus pneumoniae utilizes hemoglobin or heme instead of transferrin or lactoferrin as an iron source. GAPDH of this pathogen can bind both these proteins and has been proposed to participate in iron scavenging for bacterial needs (Yang et al., The ability of an invading bacterial pathogen to survive and proliferate within a host organism also depends on the availability of several trace elements, such as iron, an essential cofactor for diverse biochemical reactions. In a healthy mammalian organism, almost all the iron is bound to the transport proteins transferrin or lactoferrin or is stored in ferritin, because free iron catalyzes the production of toxic free oxygen radicals. The free ionic iron both in extracellular fluids and inside the cells is thus far too low to support bacterial growth. Bacterial pathogens have developed several strategies, however, to exploit iron from those iron-binding proteins (Cornelissen and Sparling, Williams  identifiWilliams . StreptoListeria monocytogenes. The surface-localized GAPDH interferes with the host Rab5a protein (Alvarez-Dominguez et al., Listeria monocytogenes escapes rapidly from the phagosomal compartment to the cytosol, where it replicates (Vázquez-Boland et al., L. monocytogenes GAPDH evidently has the ability to ADP-ribosylate the Rab5a protein, thus blocking its function in phagosome–endosome fusion. As a consequence of this strategy, L. monocytogenes delays the phagosome maturation and gains time for escape from the vacuole prior to its fusion with endolysosome that would result in the pathogen's destruction (Alvarez-Dominguez et al., Extracellular localization of GAPDH has also been demonstrated for bacteria with intracellular life cycles. One can therefore assume that intracellular bacteria might use GAPDH for manipulating some host cellular processes in order to customize the host cell milieu for their successful survival and proliferation. So far, the only study supporting this hypothesis was performed on intracellular Gram-positive S. agalactiae, S. pyogenes, and Staphylococcus aureus was reported to induce apoptosis in murine macrophages (Oliveira et al., GAPDH derived from extracellular pathogens seems to affect host cellular processes, too, as GAPDH secreted by Streptococcus pyogenes is one of few known bacteria able to bind and inhibit the C5a component of the complement system. As an integral part of the innate immunity, the complement system acts in early defense against pathogens prior to the activation of acquired immune response. It promotes cell killing by the formation of a membrane attack complex and production of molecules that stimulate the function of phagocytic cells and contribute to the inflammation manifestation. The C5a component is a potent anaphylatoxin and chemoattractant for neutrophils and macrophages (Kajita and Hugli, 2O2 production in neutrophils (Terao et al., S. agalactiae, the secreted GAPDH exerts a stimulatory effect on B lymphocytes and to a lesser extent also on T cells. GAPDH-induced production of the anti-inflammatory cytokine interleukin-10 suppresses neutrophil recruitment in mice, which finally contributes to successful host colonization by this pathogen (Madureira et al., Immunomodulatory activities of surface proteins represent another strategy for promoting a pathogen's survival in its host organism. S. agalactiae (Madureira et al., S. pneumoniae (Sun et al., Bacillus anthracis (Matta et al., L. monocytogenes (Calderon-Gonzalez et al., L. monocytogenes, the GAPDH peptide was tested as a component of a cell-based vaccine against listeriosis. Dendritic cells were used as adjuvants for immunostimulation. Despite those promising results, the high GAPDH sequence homology between the bacterial strains and humans should be taken into consideration in vaccine development. Homologous sequences can be responsible for inducing cross-immune responses that result in deleterious effects on human health (Perez-Casal and Potter, S. agalactiae or suffering from autoimmune diseases which excludes as potential candidates for a subunit vaccine (Razim et al., Surface localization plus its role in virulence together with the binding to ECM proteins make GAPDH a suitable vaccine candidate for preventing infectious diseases. The host immune responses triggered by bacterial GAPDH have been studied extensively, and as a vaccine component GAPDH was able to induce protection against several pathogenic bacteria (Argiro et al., In summary, it is evident that our current knowledge concerning bacterial GAPDH remains quite limited and provides much space for further research. It is indisputable that this protein has other functions unrelated to its enzymatic role, and it meets the criteria to be included into the family of moonlighting proteins. Data from previously published studies indicate that multiply localized GAPDH participates in other non-glycolytic processes : CytosolAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by age-dependent motor dysfunction and degeneration of the midbrain dopaminergic neurons. The deposition of neuronal inclusion, named Lewy body (LB), in the affected regions is a pathological feature of PD and related disorders such as dementia with LB (DLB). Lewy body formation is thought to begin with α-synuclein aggregation and fibrillation. Experimental studies based on the knowledge obtained by epidemiological and genetic studies continue challenging researchers to make PD risk predictable and surmountable. In this context, the development of experimental models of PD has contributed to the understanding of PD etiology and the development of therapeutics. The current 11 contributions that comprise this Special Issue highlight the PD-associated phenotypes and their evaluation methods and the development of therapeutic strategies using animal models of PD .+) by glial monoamine oxidase B is transported to dopaminergic neurons probably through the dopamine transporter and inhibits the mitochondrial respiratory complex I subunits [+ and its precursor, MPTP, to make animal or cellular models of PD. Kinoshita et al. [The discovery of the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) focused the spotlight on the roles of mitochondria in dopaminergic neurons [a et al. . Another neurotoxin, 6-hydroxydopamine (6-OHDA), is a dopamine analogue which produces selective damage to dopaminergic neurons by generation of reactive oxygen species (ROS). Unlike MPTP, 6-OHDA does not cross the blood–brain barrier and is used to induce the degeneration of the nigrostriatal pathway by intracerebral stereotactic injection. 6-Hydroxydopamine-induced rodent models are generally unilateral lesion models and exhibit a rotation response by apomorphine. Rosa et al.  reportedThe pesticide rotenone is known to inhibit the mitochondrial complex I, generating ROS. Chronic systemic exposure to rotenone has been reported to reproduce selective nigrostriatal dopaminergic degeneration with LB-like α-synuclein-positive inclusions in rat . HoweverHyposmia, constipation, and rapid eye movement sleep behavior disorder are considered prodromal symptoms of PD that often precede motor symptoms. These phenotypes are particularly important in developing disease-modifying therapies that prevent the onset or control progression of PD. Taguchi et al.  reviewedA synaptic vesicle-binding protein, α-synuclein, is a key protein to produce PD symptoms, forming LBs in the associated neurons. Recent cellular and animal model studies have revealed that α-synuclein has a prion-like property, ascending from peripheral to central neural circuits. Mori et al.  reviewedPINK1 and Parkin genes cause early-onset familial PD [Mutations of ilial PD ,15. The ilial PD . Torii eilial PD  reviewedDrosophila is a powerful tool for genetics and has revealed the molecular relationship between PINK1 and Parkin in mitochondria [Drosophila is now commonly used as PD models to evaluate genetic association. Elvira et al. [Drosophila. Historical perspective of overall PD models was also well summarized by Chia et al [chondria . Drosopha et al.  reportedia et al .In summary, all articles appearing in this Special Issue cover the interesting and current topics in PD model studies. Although most PD models do not faithfully reproduce all aspects of this disease, PD model studies would advance our knowledge and promote the development of drugs and therapeutic strategies, receiving new inputs from clinical studies. This Guest Editor would like to thank all of the authors for their contributions to this Special Issue and expects significant advancement to our knowledge of PD in future studies.


Mounting evidence shows genetic overlap between multiple psychiatric disorders. However, the biological underpinnings of shared risk for psychiatric disorders are not yet fully uncovered. The identification of underlying biological mechanisms is crucial for the progress in the treatment of these disorders.We applied gene-set analysis including 7372 gene sets, and 53 tissue-type specific gene-expression profiles to identify sets of genes that are involved in the etiology of multiple psychiatric disorders. We included genome-wide meta-association data of the five psychiatric disorders schizophrenia, bipolar disorder, major depressive disorder, autism spectrum disorder, and attention-deficit/hyperactivity disorder. The total dataset contained 159 219 cases and 262 481 controls.We identified 19 gene sets that were significantly associated with the five psychiatric disorders combined, of which we excluded five sets because their associations were likely driven by schizophrenia only. Conditional analyses showed independent effects of several gene sets that in particular relate to the synapse. In addition, we found independent effects of gene expression levels in the cerebellum and frontal cortex.We obtained novel evidence for shared biological mechanisms that act across psychiatric disorders and we showed that several gene sets that have been related to individual disorders play a role in a broader range of psychiatric disorders. We used precomputed LD scores that were provided by LD score regression, which were calculated using the European panel of the 1000 Genomes Project. No constraining of the intercept was applied.SNP heritability  gene sets (n = 5917) and canonical pathways (n = 1329). The GO gene sets contain genes annotated by the same GO term and cover biological processes, cellular components, and molecular functions. The canonical pathways are representations of biological processes that are compiled by domain experts. Second, we selected 126 expert-curated gene sets that have been tested to date for one of the five disorders in studies based on whole genome approaches . These gene sets cover different types of expert-curated sets: sets of functionally related genes, sets of co-expressed genes, and sets representing protein complexes or networks (online Supplementary Table S1). Third, we obtained gene-expression values of 53 tissues from the GTEx project v7 . The expression values of all genes were Winsorized at 50 reads per kilobase of transcript per million reads mapped (RPKM) and subsequently log2 transformed with pseudocount 1  derived from publicly available databases, and (2) expert-curated, i.e., genes annotated by experts in a specific research field that commonly involves an extensive experimental or methodological design and interpretation. In addition, we investigated tissue-specific gene-expression values as gene properties . First, we selected from the molecular signature database . Considering the significantly associated gene sets, the three calcium channel sets and the gene set involved in membrane depolarization showed equal association strengths, while the other gene sets present small to moderate reduced associations when taking the direction of SNP effects into account. This implies that the effects of SNPs related to calcium channels, or more broadly related to action potentials and depolarization, increase the risk on the different disorders in the same direction, while the effects of genetic variants related to the other identified functions partly act in opposite directions across disorders. In addition to this post hoc analysis, we performed a gene-set and gene-property analysis weighting the disorders according to sample size. These results were highly consistent with the initial unweighted analysis .We compared the results of these analyses – which are based on meta-analyzing gene-based associations of the individual disorders – with a gene-set and gene-property analysis including as input the meta-analyzed SNP associations of the five disorders. Hence, the direction of the SNP effects is taken into account. The results of these analyses and the initial analyses showed strong correlations [Pearson's correlation of logTo investigate the contribution of the association signal of the individual disorders to the cross-disorder association, we performed the gene-set and gene-property analyses for the individual disorders as well , indicating that the associations of the identified gene sets are not merely because they comprise genes expressed in the brain.FMRP targets (Darnell et al., GO high voltage gated calcium channel activity, GO postsynapse, MIR137 targets (Lewis et al., GO modulation of synaptic transmission, GO membrane depolarization during cardiac muscle cell action potential, GO gamma aminobutyric acid signaling pathway, and GO transcription factor activity RNA polymerase II core promoter sequence specific showed independent effects. However, the associations with the gene sets GO synapse part, GO synapse, GO neuron spine, GO voltage gated calcium channel complex, and GO excitatory synapse could fully be accounted for by the other identified gene sets. The large overlap of genes in these sets with the other identified gene sets (Third, we applied the forward selection procedure to the gene sets to shed light on the relations between their associations. We excluded the highly-brain-expressed gene set from this step as gene expression levels are already captured by conditioning on the average, cerebellum and frontal cortex tissue expression levels included in the previous step. The eight gene sets ene sets  clearly et al., et al., et al., The current gene-set analyses revealed various new sets of genes – in particular related to the synapse and neuronal functions – and gene-expression profiles of multiple brain tissues that play a role in shared genetic risk across five psychiatric disorders. The most strongly associated gene set was the highly-brain-expressed genes, which has previously been related to ASD (Pinto et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., de novo mutations (Iossifov et al., et al., et al., et al., et al., et al., et al., In addition, we identified multiple gene sets related to the synapse, which aligns with synaptic functions of several identified genes for multiple individual psychiatric disorders (Schizophrenia Working Group of the Psychiatric Genomics Consortium, Of note, the biological annotations of gene sets comprise a complex and challenging process, e.g., due to the multiple functions of many genes and incomplete knowledge. The construction of gene sets is in general based on different approaches such as shared cellular mechanism, co-expression patterns, protein-protein interaction, or co-localization. Hence, sets of genes may be based upon different inclusion criteria, creating an overlap between gene sets, as also illustrated by the current study. Clearly, it is important to recognize the impact of particular annotations on gene-set analysis results and their biological interpretation.et al., et al., To address this issue of confounding and redundancy in gene sets, we applied conditional analyses. This provided insight in how different gene-set associations relate to each other, and whether identified functions may not be biologically relevant to the disorders but rather induced by confounding factors (de Leeuw et al., Our cross-disorder gene-set and gene-property analyses are built on a meta-analysis of the gene-based associations with the individual disorders, therefore possible opposite effects of genetic variants are not taken into account. To explore if genetic variants are related to multiple disorders but with opposite effects, we performed an SNP-based meta-analysis of the five disorders and conducted a gene-set and gene-property analysis based on those results. In this analysis, genetic variants with opposite effects across disorders are cancelled out. Although these results showed strong correlations with the original analysis, we detected differences in association strength that point to partial differences in direction of SNP effects between the disorders for most identified gene sets. Interestingly, the effects on calcium channel activity are unidirectional across disorders. The outcome of different effects across disorders is supported by the recent finding that the highly correlated disorders SCZ and BD are differentiated by several genetic loci with opposite directions of effects (Bipolar Disorder and Schizophrenia Working Group of the Psychiatric Genomics Consortium, et al., et al., We note that the associations of our identified gene sets were to a large extent driven by SCZ. This is in line with previous studies that reported multiple gene sets associated with SCZ (Ripke In conclusion, the current study provides novel evidence for shared biological mechanisms that act across psychiatric disorders based on gene-set and gene-property analyses. We showed that several gene sets that previously only had been associated with individual disorders also play a role in a broader range of psychiatric disorders, supporting the view of a common pathogenesis across disorders. This indicates that the genetic overlap between disorders is not randomly distributed, but can be explained by specific biological mechanisms. The strongest evidence in our results was for the involvement of synaptic functions, and gene expression profiles of the cerebellum and frontal cortex. The genetic data collection of additional psychiatric disorders is rapidly increasing and will make it possible to extend our analyses to other disorders in the near future. Understanding the shared biological mechanisms between psychiatric disorders may provide a hint towards a general vulnerability for multiple psychiatric disorders, and could result in potential treatment for a broad spectrum of psychiatric disorders.


Annals of Intensive Care about the dose adjustments of vitamin C in critically patients undergoing renal replacement therapy [We have read with interest the manuscript from Honore et al. in  therapy . After aMedicine  journal in 2019 (online: 2019 November 27) [The authors included in their study the Wu et al.’s case report of a patient with hemolytic jaundice induced by pharmacological dose of ascorbic acid in glucose-6-phosphate dehydrogenase (G6PD) deficiency . This mamber 27) , becauseThe authors searched for studies and used studies published in 2020, like Fujii et al.’s study , so the We conclude that the exclusion of Wu et al.’s study does not probably affect the overall conclusions of Honore et al.’s study, but it affects directly the particular conclusion exposed previously because Wu et al.’s study failed to provide valid scientific evidence.We recommend to exclude it, but a good and accurate literature review is a key element in high-quality original studies.


Mutations in the encoding genes in patients and mouse models underlie severe phenotypes including kidney stones with CLCN5 and osteopetrosis or hypopigmentation with CLCN7. Dysfunction of those intracellular CLCs that are expressed in neurons lead to neuronal defects. Loss of endosomal ClC-3, which heteromerizes with ClC-4, results in neurodegeneration. Mutations in ClC-4 are associated with epileptic encephalopathy and intellectual disability. Mice lacking the late endosomal ClC-6 develop a lysosomal storage disease with reduced pain sensitivity. Human gene variants have been associated with epilepsy, and a gain-of-function mutation causes early-onset neurodegeneration. Dysfunction of the lysosomal ClC-7 leads to a lysosomal storage disease and neurodegeneration in mice and humans. Reduced luminal chloride, as well as altered calcium regulation, has been associated with lysosomal storage diseases in general. This review discusses the properties of endosomal and lysosomal Cl−/H+ exchange by CLCs and how various alterations of ion transport by CLCs impact organellar ion homeostasis and function in neurodegenerative disorders.The regulation of luminal ion concentrations is critical for the function of, and transport between intracellular organelles. The importance of the acidic pH in the compartments of the endosomal-lysosomal pathway has been well-known for decades. Besides the V-ATPase, which pumps protons into their lumen, a variety of ion transporters and channels is involved in the regulation of the organelles' complex ion homeostasis. Amongst these are the intracellular members of the CLC family, ClC-3 through ClC-7. They localize to distinct but overlapping compartments of the endosomal-lysosomal pathway, partially with tissue-specific expression. Functioning as 2Cl Data from patients, mouse models and cell biophysical measurements highlight an important role for Cl− which is accumulated in a secondary active transport by CLC Cl−/H+ exchangers  possess a further glutamate, referred to as “proton glutamate,” whose mutation abolishes or strongly diminishes the transport of both protons and chloride  display similar transport properties but differential subcellular localization  and the loss of VGLUT1, SV acidification was not impaired   in primary neurons from ClC-3-deficient mice was reported to be reduced, suggesting that glutamate toxicity due to excessive glutamate release contributes to the neurodegeneration  pointed to the activity of ClC-7 as a ClClcn7−/− mice , displayed conspicuous loss of hippocampal and cortical neurons. Starting in the CA3 region of hippocampus neuronal loss progressed into the dentate gyrus and by the age of 1.5 years no hippocampal structures in Clcn7lox/lox;EMX1cre mice could be detected  revealed slowed lysosomal degradation of endocytosed protein  in humans (Kornak et al., CLCN7 mutations (Cleiren et al., CLCN7-related ARO in about 50% of the cases, as well as OSTM1-related ARO, are also neuropathic with primary neurodegeneration manifesting in developmental delay, hypotonia, retinal atrophy and seizures (Steward, CLCN7 mutations, which may lead to subcellular mislocalization of the ClC-7/Ostm1 complex or the impingement of the dysfunctional subunit on the ion transport properties of the unaffected subunit (Schulz et al., Clcn7−/− mice with death after only a few weeks and neurodegeneration (Alam et al., PM/Ostm1 (Leisle et al., Clcn7+/G213R ADO2 mice also presented fibrosis in non-skeletal tissues such as lung and muscle; their brains exhibited perivascular fibrosis, β-amyloid accumulation and astrogliosis, and the animals showed behavioral abnormalities (Maurizi et al., Mutations in Steward, , which iSteward, . DominanPM/Ostm1 (Leisle et al., During the first electrophysiological analysis of osteopetrosis-causing ClC-7 missense mutations, it was surprisingly found that, besides loss-of-function mutations due to impaired ER exit or reduced ion transport, several pathogenic ClC-7 mutations accelerated the voltage-dependent activation of ClC-7de novo mutation of ClC-7 was identified in two children with hypopigmentation and delayed development (Nicoli et al., Y715C led to a drastic enlargement of late endosomal/lysosomal compartments (Nicoli et al., Y553C mutant (Polovitskaya et al., Y715C, the enlarged compartments were reported not to be acidified in contrast to surrounding smaller, hyperacidified lysosomes, which was attributed to the hyperactivity of the ClC-7 mutant (Nicoli et al., Recently, a heterozygous −/H+ exchangers on distinct, but overlapping organelles of the endosomal-lysosomal pathway (Stauber et al., Loss-of-function and in some cases also gain-of-function of the intracellular CLCs, ClC-3/ClC-4, ClC-6 and ClC-7/Ostm1, lead to neuropathies, often neurodegeneration. They all function as Cl− export from endosomes/lysosomes. At membrane voltages in the range of those measured for endosomal/lysosomal compartments, which can be up to inside-positive 100 mV (Koivusalo et al., − accumulation by CLCs and the necessity of their ion transport. Mutation of the “gating glutamate” does not only convert the exchanger into a pure Cl− conductor, but also abolishes its voltage dependence. Therefore, such a mutation may indeed represent a gain of function in respect to Cl− transport. Interestingly, while the uncoupling mutation of ClC-5 or ClC-7 leads to effects that are similar to the respective knock-out in mouse models, the heterozygous mutant of ClC-6 results in a much more severe disorder in patients than in the murine gene knock-out model (Poët et al., In some cases, it has been proposed that the CLC serves as a structural protein that functions to recruit other proteins such as components of the transport machinery by direct protein-protein interaction. Amongst others, the existence of different missense mutations which unlikely all prevent the same interactions argues against this. Although a role a role for protein-protein interactions of intracellular CLCs in vesicular trafficking cannot be excluded, the most straight-forward explanation for the importance of CLCs is their role in the regulation of the vesicular ion homeostasis. In this respect, the pronounced outward rectification of the vesicular CLCs remains enigmatic, because it would strongly favor Cl− by their Cl−/H+ exchange mechanism is pivotal –also for ClC-5, in addition to the acidification- (Novarino et al., − concentration rises to > 100 mM in the lysosome (Saha et al., − concentrations, but a normally low pH, have been found in nematode models of various neurodegenerative lysosomal storage diseases including Gaucher and Nieman-Pick A and B (Chakraborty et al., − concentration has been shown to influence the enzymatic activity of the lysosomal protease cathepsin C (Cigić and Pain, − affects vesicular function is elusive (Stauber and Jentsch, The luminal pH is of undisputed importance for endosomes and lysosomes and impaired acidification is linked to neurodegeneration (Mellman et al., −/H+ exchanger does not only –if at all– affect the luminal Cl− concentration and pH, but ion homeostasis in general, including the transmembrane voltage and concentrations of other ion species (Ishida et al., 2+ (Chakraborty et al., The presence of a Cl−/H+ exchangers impinges on the trafficking, function and possibly signaling of endosomal and lysosomal organelles and interconnected pathways such as autophagy, which in turn leads to cell death of vulnerable neurons. Future work is required to uncover in detail the molecular mechanism by which these CLCs contribute to the functioning of the endosomal-lysosomal pathway.In summary, dysfunction of the intracellular CLC ClSB, HH, and TS wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nursing workforces are strengthened in COVID-19 units to enable closer monitoring of patients’ vitals and personalized care. Yet, the healthcare teams confronted with COVID-19 are particularly strained and require active psychological support from psychologists and unit managers . Although benzodiazepines are often used as first-line treatments in case of acute agitation, after ruling out any somatic cause , randomized control trials don’t support their use (McDermott and Gruenewald, et al., Unfortunately, in case of a drug shortage, alternative treatments must be considered. Midazolam can be used either orally or by subcutaneous injection to treat acute behavioral symptoms (McDermott and Gruenewald, et al., et al., Good clinical practice for the prescription of psychotropic drugs (Livingston et al., If chemical restraint is not possible, physical restraint may exceptionally be prescribed preferably with abdominal and pelvic straps (Livingston The COVID-19 outbreak is overwhelming all healthcare systems and we need to provide new guidelines for better medical management of elderly BPSD patients and their families and preserve hospital staff by streamlining their procedures.


With an enormous prevalence worldwide, diseases of the oral cavity and respective tissues are a highly relevant global health issue . Beside n = 23) with or without depression based on the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMDs) Axis II, respectively. All study participants underwent an electromyographical examination to assess their bioelectrical resting activity of temporal and masseter muscles. The resting activity of selected masticatory muscles did not differ between the two groups. The authors concluded that these preliminary findings should lead to further research with larger sample size to gain insight the psychological factors related to bioelectric parameters in masticatory muscles. A Polish study by Gerreth et al. [The high variety and complexity of oral-systemic interactions is displayed by ten excellent articles in this special issue “Oral Health and Systemic Diseases”. Thereby, seven studies are reported, complemented by three systematic reviews. A case-control study by Gerreth et al.  compreheh et al.  examinedh et al.  examinedh et al.  was perfThree systematic reviews are part of this special issue. The systematic review by Pérez-Losada et al.  also appAltogether, the ten articles in the special issue “Oral Health and Systemic Diseases” show different aspects of oral and systemic disease interaction and provide several implications for dental care of these patients. One main conclusion is the necessity of further research, especially large-scaled prospective studies to get a better understanding of the needs of respective patient groups. Furthermore, patient centered dental special care appears recommendable for different patient groups, which were examined in the different studies. The special issue “Oral Health and Systemic Diseases” gives the reader an interesting and clinically relevant insight into oral and systemic disease interactions, an emerging field of dental and medical research.