The polygenic nature of neurological and psychiatric syndromes and the significant impact of environmental factors on the underlying developmental, homeostatic, and neuroplastic mechanisms suggest that an efficient therapy for these disorders should be a complex one. Pharmacological interventions with drugs selectively influencing the epigenetic landscape (epidrugs) allow one to hit multiple targets, therefore, assumably addressing a wide spectrum of genetic and environmental mechanisms of central nervous system (CNS) disorders. The aim of this review is to understand what fundamental pathological mechanisms would be optimal to target with epidrugs in the treatment of neurological or psychiatric complications. To date, the use of histone deacetylases and DNA methyltransferase inhibitors (HDACis and DNMTis) in the clinic is focused on the treatment of neoplasms  and is based on the cytostatic and cytotoxic actions of these compounds. Preclinical data show that besides this activity, inhibitors of histone deacetylases, DNA methyltransferases, bromodomains, and ten-eleven translocation (TET) proteins impact the expression of neuroimmune inflammation mediators (cytokines and pro-apoptotic factors), neurotrophins (brain-derived neurotropic factor (BDNF) and nerve growth factor (NGF)), ion channels, ionotropic receptors, as well as pathoproteins . Based on this profile of activities, epidrugs may be favorable as a treatment for neurodegenerative diseases. For the treatment of neurodevelopmental disorders, drug addiction, as well as anxiety disorders, depression, schizophrenia, and epilepsy, contemporary epidrugs still require further development concerning a tuning of pharmacological effects, reduction in toxicity, and development of efficient treatment protocols. A promising strategy to further clarify the potential targets of epidrugs as therapeutic means to cure neurological and psychiatric syndromes is the profiling of the epigenetic mechanisms, which have evolved upon actions of complex physiological lifestyle factors, such as diet and physical exercise, and which are effective in the management of neurodegenerative diseases and dementia. The deoxyribonucleic acid (DNA) molecule encoding for proteins is essential for life and is predominantly responsible for immutable traits. The DNA in a cell exists in a strictly structured form that is achieved with the help of histones and architectural proteins. This structural organization defines the differential functional activity of the DNA molecule, which can be transcriptionally inaccessible in dense regions (heterochromatin) or transcriptionally accessible (euchromatin) in loose regions of chromatin .Epigenetics, on the contrary, gives a living creature the opportunity to adapt to dynamically changing environmental factors, hence, playing an important role in the balance of molecular processes, which, if violated, can lead to various diseases. An epigenetic phenomenon is a change in gene expression that is not mediated by changes in the nucleotide sequence of a DNA molecule. The alterations in DNA accessibility and transcriptional activity are mostly achieved by a few mechanisms: (1) chemical modifications (reversible methylation) of DNA, (2) chemical modifications  of histone proteins, (3) chromatin remodeling and the spatial three-dimensional (3D) organization of the genome, as well as via action of (4) non-coding ribonucleic acids (ncRNAs) on gene expression. At the cellular and systemic levels, epigenetic mechanisms controlling gene activity influence molecular, biochemical, and biological processes. Epigenetic control over the genome is essential in determining the unique properties of a cell, which develops from a single precursor via specialized gene expression programs.The upper-mentioned epigenetic functional and structural alterations normally appear in a given organism but may be transmitted to the next generation(s) due to the stabilization of the specific expression programs (with no changes in the genome sequence per se), for instance, via the preservation of the residual methylation after fertilization and via gamete-delivered RNAs ,5.Epigenetic mechanisms of all known types can be reliably monitored nowadays. For instance, histone modifications are well recognized as an integrative marker of genome regulation. Chromatin immunoprecipitation (ChIP)-sequencing is commonly used to assess the profile of histone lysine (K) acetylation and methylation. The most validated specific markers of genome suppression are histone H3 methylation at lysine in two positions: H3K9me3 and H3K27me3. In turn, H3K27ac, H3K4me3, and H3K4me1 are validated as genome activation factors. However, to date, a variety of other post-translational histone2-4 modifications, such as arginine acetylation, phosphorylation, SUMOylation, and ubiquitination, are shown to result in changes in genome activity .The sensitivity and efficiency of modern analytical methods allow one to assess the epigenetic landscape and reliably identify the promoter, enhancer, as well as associated insulator sequences at the single-cell level ,8,9. Progress in entire genome chromatin profiling and epigenome-wide association studies (EWASs)  fostered a surge of data about epigenetic states in various tissues and cells at their normal and pathological functionings. The contemporary challenges of epigenetic data management are the (1) development of open-access databases; (2) standardisation of experimental methods; (3) setting of standards for the analysis and integration of EWAS and GWAS data; (4) annotation of epigenetic markers at different levels of biological complexity; and (5) selection of a significant set of epigenetic markers in the context of a disease or treatment ,12,13,14Aside from the general constraints related to big data management, there are a few additional obstacles to understanding the role of epigenetic mechanisms in the pathogenesis of neurological and psychiatric diseases. Although a big pool of information on changes in the epigenome is amassed to date, we are still limited in detailed aspects of the dynamics of epigenetic landscapes in different neuronal cells in various brain regions. Similarly, a systematic search for reliable peripheral epigenetic markers of central nervous system (CNS) disorders has still not been accomplished. The rationale for using peripheral epigenetic markers, in addition to their easy accessibility in humans, is the involvement of peripheral systems  in the normal maintenance of brain functions and the generation of pathological endophenotypes. One of the possible platforms for finding significant correlations can be the concept of a so-called epigenetic “clock”, which assumes the age-related accumulation of methyl groups on a DNA molecule, allowing one to determine the biological age of cells, tissues, organs, or organisms. The best-validated model is Steve Horvath’s epigenetic “clock”, comprising 353 epigenetic markers of the human genome ,16.At present, the field of epidrug pharmacology is developing to serve mainly the needs of cancer therapy. However, epigenetically active substances, being, by default, multitarget and multifunctional, would represent the cluster of drugs of which specific and unspecific effects are potentially even of higher interest with respect to other nosologies or syndromes . The knoLimitations to use the epidrugs are related to (1) their chemical instability; (2) ubiquitous activity of DNMTis accompanied by the development of side effects; (3) unspecific acetylation, deacetylation, and methylation of non-histone proteins resulting in undesirable side effects ; and (4) cytotoxic effects and significant inhibition of hematopoiesis .The increase in life expectancy, changes in lifestyle, and informational load resulted in the emergence of new pathogenic factors (such as aging and stress) and the decrease in significance of old pathogenic factors . The profiling of CNS diseases in the world population shows a growing dominance of neurodegenerative disorders and psychophysiological stress-associated disorders ,41,42. SWhile we already know a number of compounds with primary epigenetic activity, their rapid transfer from preclinical studies into the therapy of the CNS is hindered by some flaws and difficulties.Permeability of the blood–brain barrier (BBB);Regionality of action ;Differential specificity of action at the level of neuronal or glial populations ;High selectivity of compounds to their molecular targets (at the level of protein isoforms);If the drug is multitargeted, then its molecular action has to be highly balanced with no or few side effects;Absence of cytotoxic effects on cells with a non-cancer phenotype;Lack of systemic immunosuppressive and hematopoiesis-inhibiting effects;Possibility of a differential therapy according to the phase of the disease .A list of the most crucial demands on epidrugs includes: Based on the requirements and recent promising findings on the mechanisms of CNS disorders, the following cues for epidrug research and development can be delineated: (1) the search for compounds with good BBB permeability among selective inhibitors of TET proteins and DNA methyltransferases; (2) the search for compounds with high potency to selectively regulate neuroimmune inflammation and neurotrophic mechanisms; (3) the epigenetic activity profiling and assessing of the possible “off-label” use of compounds with canonical effects, such as anticonvulsant and antidepressant agents or metabolites; and (4) the employment of CRISPRon and CRISPRoff gene-editing technologies, as well as more advanced CRISPR/Cas system versions (such as Prime editing). The last is of particular interest because it enables cell- and locus-specific reactivation or repression of the transcription of a single gene or a combination of genes (multiplexed gene targeting) under a variety of promoters . HoweverWhen determining the strategies of therapeutic intervention in the treatment of CNS disorders, one should take into account the ability of complex physiological factors (lifestyle factors), such as physical activity, dietary factors, and fasting, as well as cognitive activity, to modulate the epigenetic landscape. Being ministers of beneficiary action , these factors do not lead to the development of unwanted side effects. Therefore, their epigenetic sequels may be considered as a prototype to explore the potential targets for specific epigenetic therapy.The very nature of epidrugs points to their complexity and diversity of effects. On the cellular level, epidrugs influence fundamental processes, such as central metabolism, autophagy, cell survival, and the reprogramming of pluripotent or cancer cells. On the tissue level, the effects of epidrugs appear as an activation of neuroprotective and neuroplastic mechanisms as well as of the modulation of neuroinflammation, which involves the intercellular interaction and reorganization of the extracellular matrix. At the next level of complexity, epidrugs target particular endophenotypes and syndromes. A final achievement would be the correction of pathologies on the organismal level. To make an inventory of the available substances with epigenetic activity and to overview the contemporary tendencies in preclinical and clinical studies of their applicability for CNS treatments, we scrutinized respective publications based on the following logic.Substances belonging to the continuously growing cluster of epidrugs were, first, stratified based on the main known biochemical/epigenetic mechanisms.Second, epidrugs from each cluster with defined biochemical mechanisms were looked for in reports on the results of preclinical or clinical studies of therapeutic effects. Then, they were clustered in groups, which were formed with respect to the pathologies and pathology-related mechanisms that should be targeted .We lined up the results of our search with respect to the number of published studies, which reflect the current interest in the respective epidrugs as therapeutic means for treating neurological and psychiatric disorders.We also stressed the role of complex epigenetic regulators in the therapy of certain diseases, assuming their actual benefits.The strategy for treating cancer with epigenetically active compounds implies their use as adjuvant therapy . To thisNonselective HDACis are currently undergoing phase II clinical trials as a component of complex therapy for brain cancer ,68,69,70The spectrum of pharmacological activity of nonselective HDACis may be the determinant of a complex pharmacotherapy for brain cancer. For example, panobinostat, vorinostat, and romidepsin inhibit glycolysis in cancer cells by enhancing the oxidative metabolism  and peroxisome proliferator-activated receptor beta (PPARδ)). Since the cancer oxidative metabolism is mainly driven by fatty acid oxidation, a combination of fatty acid oxidation inhibitors and HDACis might be effective in cancer treatment, as it was proved in a patient-derived xenograft model of glioblastoma in mice .BET inhibitors show cytotoxic activity against glioblastoma cells in vitro . The synDNMTis are also studied as adjuvant components in the treatment of brain cancer ,79.The experimental evidence and results of clinical trials accumulated to date strongly suggest that epidrugs may be effective in the treatment of brain neoplasms.Cell death, which involves a wide spectrum of scenarios, can, besides being a part of the pathogenesis of neurodegenerative disorders, be assumed to be a leading component of degeneration in brain and spinal cord injuries, brain hemorrhage, ischemic stroke, chronic insufficiency of cerebral circulation, and toxicity-related damage (including that related to toxic metabolic syndromes).HDACis showed neuroprotective effects in models of traumatic brain injury. Among the few mechanisms involved, the decrease in the neuroinflammatory response is particularly noteworthy . Suppres+) toxicity model [The neuroprotective effect of nonspecific HDACis in models of ischemia, oxidative stress, and glutamate neurotoxicity is associated with the transcriptional suppression of pro-apoptotic factors, such as p75(NTR)-dependent caspase-3 and ubiquitin-conjugating enzyme E2N Ube2n ,95,96. Aty model .Several observations further illustrate the importance of selective modulators of histone acetylation and deacetylation in realization of a neuroprotective effect ,105. TheA nonspecific DNMTi, 5-aza-deoxycytidine, potentiated MPP+ toxicity in dopaminergic neurons . AnotherBET inhibitors, especially those selective for BRD2/4 proteins, exhibit a characteristic suppression of the expression levels of pro-apoptotic factors and cytokines regulating inflammation , C-X-C motif ligands 1 and 10 , and C-C motif ligand 2 (CCL2)), as well as matrix metalloproteinase 9 (MMP9), the extracellular cleaving enzyme involved in the maintenance of the extracellular matrix and membrane receptors ,115,116.Overall, known epidrugs facilitate the neurotrophic and neuroplastic processes, inhibit the mechanisms of programmed cell death, and suppress neuroinflammation. These features prompt researchers to look at the applicability of certain epidrugs as neuroprotectors and stimulators of neuronal regeneration but also suggest the domain of neurodegenerative disorders as a potential area of applicability.Epidrugs potentially can be used in the treatment of neurodegenerative disorders because they activate neuroprotective mechanisms, suppress neuroinflammation, and evoke the mechanisms of synaptogenesis and neuroplasticity . ConsideThe most prevalent neurodegenerative diseases, such as Huntington’s disease , ParkinsDespite the differential regulation of histones in neurodegenerative disorders, it is assumed that the use of HDACis can compensate for these pathological changes ,126 via HDAC1/2 inhibitor BG45 enhances the level of synaptic proteins PSD95, spinophilin, and synaptophysin in the model of toxic β-amyloid expression in vitro . In turnHTT expression) [(Hdh) knockout mice (HdhQ111-KO), which is accompanied by an improvement in cognitive functions [The use of HDACi LBH589 (panobinostat) in the early postnatal period in the genetic model of Huntington’s disease leads to a significant improvement in behavioral and neurochemical endophenotypes , which pression) . The HDAunctions . Besides interference with histone acetylation, DNA methylation is also considered as a target for epigenetic therapy of neurodegenerative disorders . A diffeIl-1b, Il-6, Tnfa, Ccl2, Nos2, and Ptgs2) in 3xTg mice [The BETi JQ-1 affects pathological behavioral and molecular biological endophenotypes in a model of Alzheimer’s disease in mice. JQ-1 reduces Tau protein phosphorylation, an expression of pro-inflammatory factor genes  complex, and an epigenetic enzyme recruited by it, is of increasing interest. The expression of such constructs allows a locus-specific reactivation (CRISPRon) or repression (CRISPRoff) of DNA transcription ,147,148.The design or selection of epidrugs for the treatment of psychiatric disorders is determined by the characteristic biological features of psychiatric syndromes: (1) high polygenicity with small effect sizes of individual risk variants; (2) strong influence of external and internal environmental factors on the development and dynamics of psychopathological endophenotypes; and (3) overlap of pathogenetic mechanisms between psychopathological syndromes and neurodegenerative diseases .It is also important to note that known psychoactive pharmacological substances are being proven to have epigenetic mechanisms of action. In turn, it is not surprising that many epigenetic drugs mediate psychical activity because they affect neuroplasticity, which is essential to maintaining functional homeostasis and adaptive changes in the brain.Bdnf gene promoter and BDNF expression [Antidepressants, antipsychotics, and drugs with antiepileptic activity are constantly confirmed to have epigenetic effects. These epigenetic activities may be the main determinants of the clinical effectiveness of these drugs, instead of their initially proposed modes of action. For instance, imipramine was introduced for depression treatment as early as 1957. As shown more recently, its antidepressant effect in a chronic stress mouse model is mediated by an increase in H3 acetylation in the pression . Valproipression ,152. Thepression ,54. Togepression ,155,156.Effects of HDACis and BETis in the CNS are well known to be associated with an increase in the transcription of genes regulating neurogenesis, synaptogenesis, and neuroplasticity, a decrease in cytokine production, and the suppression of microglial activity. An increasing number of evidence indicates the effects of HDACis and BETis on the expression of neurotransmitter and neurohormone receptors and transporters  ,160,161.Furthermore, DNMTis hold promise for their use in treatments of stress-associated psychiatric diseases. Global and locus-specific DNA methylation reflects the active response of neuronal tissue to stress  and/or iNotably, DNMTis are expressed at different levels in various brain regions and are usually present in neurons and not glial cells . MoreoveThe BETi JQ-1, on the other hand, shows contradictory effects in mouse models of PTSD. It reduces the extinction of traumatic memory , decreasThe role of epigenetic mechanisms in developmental genetic disorders has been widely acknowledged . The effThe BETi JQ-1 showed to be beneficial in the model of Rett syndrome . The BETChanges in the epigenome have been demonstrated during the development of addiction to alcohol, nicotine, cocaine, amphetamine, cannabinoids, and opiates ,189,190.Preclinical test results indicate the potential for using HDACis to treat drug addiction ,196,197.The potential of BETis in the treatment of drug addiction is generally poorly studied, although the possibility for their use in the treatment of cocaine addiction has been discussed .Neuroplastic changes involved in forming addiction to different drugs seem to be an attainable target for epigenetic therapy. However, since the answer to whether treatment should preferentially affect neuroplasticity in a selected neuronal population is “yes” , any sucIt can also be stressed here that epigenome profiling is crucial to determine the mechanisms leading to sensitivity, resistance, and tolerance to pharmacological drugs ,200.The effects of epidrugs are mainly rapid. At the same time, they are fundamentally reversible. That makes the therapy of epilepsy and pain syndromes another potential field of epidrug application . Indeed,Epigenetics is likely to play a major role in the interaction between the environment  and gene expression. The term “lifestyle” is defined as a complex of modifiable habits and a typical way of living for an individual. It includes factors such as diet, behavior, stress, physical and cognitive activity, working habits, smoking, and alcohol consumption, all of which are shown to alter epigenetic landscapes . MoreoveIt is worth mentioning that due to the systemic effect that specific lifestyle interventions and epidrugs themselves have on metabolism and the immune system, their overall positive impact on brain function and homeostasis of nervous tissue can be, at least in part, a secondary phenomenon.Both lifestyle factors and systemic neuroinflammation have been linked to several pathological conditions, including psychiatric and neurodegenerative disorders ,210,211.Chronic low-grade inflammation induces a multitude of epigenetic changes. Meta-analysis of EWASs of serum C-reactive protein (CRP), a sensitive marker for low-grade inflammation that is also a proxy for IL-6 levels, found differential methylation at 218 CpG sites to be associated with CRPs . AuthorsImportantly, increased systemic inflammation caused by an energy-dense Western diet is reversible by a change in diet, as switching back to normal food rescues the inflammatory profile in mice susceptible to atherosclerosis . A reducFurthermore, obesity also increases oxidative stress through different mechanisms, including hyperleptinemia, chronic inflammation, low antioxidant defense, and postprandial reactive oxygen species (ROS) generation . ROS andDietary change is presumably the most obvious lifestyle intervention to tackle obesity or metabolic dysfunction, and thus modulates systemic inflammation, oxidative stress, and potentially neuroinflammation. At the moment, the dietary factors that have epigenetic effects can be grouped into three classes: single nutrients, particularly vitamins and polyphenols ; microRNThe involvement of central metabolites in the regulation of the activity of epigenetic enzymes provides a mechanistic link between dietary and calorie intake and changes in the epigenetic landscape . For exaIn turn, calorie restriction is an appreciated longevity factor, and mechanisms behind its action are mostly found to be linked to reduced nutrient signaling . FastingNeurodevelopmental factors are particularly relevant in the etiology of psychiatric disorders . SomewhaOn the other hand, a maternal high-calorie diet is also associated with the development of both metabolic and psychiatric diseases in offspring through epigenetic mechanisms ,250,251.Several dietary factors exert epigenetic effects and modulate psychopathological conditions independent of the fatty acid or caloric content of food. The dietary factor acetyl-L-carnitine , which iExercise increases the activity of histone acetyltransferases and histone deacetylases, reduces the level of global DNA methylation, and thus sets back the “time” of the epigenetic clock ,270,271.TET proteins perform hydroxylation of DNA at 5-methylcytosine (5mC) , therefoThe most important regulators of the epigenome are non-coding RNAs. They are mostly represented by microRNAs (miRNAs), or enhancer and long non-coding RNAs (eRNAs and lncRNAs) which show a complex tertiary structure. MicroRNAs control transcription and intranuclear and cytoplasmic processing of genetic information; enhancer RNAs mainly mediate changes in the structural organization of DNA . There iThere is ample evidence of significant associations between specific miRNA expression profiles and psychopathological conditions ,288,289.To assess the current state of clinical trials for substances with epigenetic activity in the treatment of pathological conditions of the CNS, an analysis of the US National Institutes of Health database   was carrUpon analysis, the breadth of the goals of clinical trials with valproic acid or its salts used as monotherapy or as a component of complex therapy was noteworthy. In addition to the most common pathologies targeted with valproic acid , the therapeutic efficacy of this drug is being studied in the treatment of pain syndromes, depression and PTSD, Alzheimer’s disease (one study), spinal muscular atrophy (six studies), and oncological diseases of the brain . Hydralazine is being studied as a primary therapy for Alzheimer’s disease in one database-registered clinical trial . Its effIt is well accepted that epigenetic mechanisms are the core links between pathological environmental factors and disease. In analogy, it is true that interfering with the epigenetic mechanism might be the way to prevent the development or slow down the progress of neurological and psychiatric diseases. Therefore, epigenetic therapy of diseases of the CNS appears to be a promising field in pharmacology.Need for inhibitors and activators specific to different types and isoforms of epigenetic enzymes that also have good BBB permeability;Discovery of compounds with bimodal or complex activity;Understanding the changes in genome–epigenome interactions;Expansion of the bioinformatical annotation epigenetic markers at different biological levels;Evaluation of the applicability of the CRISPRon and CRISPRoff tools in humans;Further development and optimization of the cell-specific delivery of compounds or genetic constructs using viral vectors, anti- or nanobodies, or PEGylated immunoliposomes.The development of epigenetic therapy for CNS diseases nonetheless faces several challenges:The expanding amount of data from studies interrogating the mechanisms of action and effects of epigenetically active drugs and compounds in the CNS strongly suggests a potential for the therapeutic use of epidrugs in brain cancer. The present review particularly outlines the prospects of using HDACis, DNMTis, and BETis for the therapy of brain neoplasms . Analysis of the CNS-specific targets and effects of epidrugs with respect to functional regulation of brain activity reveals that they also often target glial elements, affecting microglia differentiation and secretory activity. Glia provide support, protection, and, at the same time, foster neuroinflammation processes, thus regulating them negatively and positively. Although the epigenetic biomarkers of neuroinflammation in neurological (and psychiatric) disorders are not comprehensively described ,296,297


Intestinal transplantation (ITx) remains a lifesaving option for patients suffering from irreversible intestinal failure and complications from total parenteral nutrition. Since its inception, it became obvious that intestinal grafts are highly immunogenic, due to their high lymphoid load, the abundance in epithelial cells and constant exposure to external antigens and microbiota. This combination of factors and several redundant effector pathways makes ITx immunobiology unique. To this complex immunologic situation, which leads to the highest rate of rejection among solid organs (>40%), there is added the lack of reliable non-invasive biomarkers, which would allow for frequent, convenient and reliable rejection surveillance. Numerous assays, of which several were previously used in inflammatory bowel disease, have been tested after ITx, but none have shown sufficient sensibility and/or specificity to be used alone for diagnosing acute rejection. Herein, we review and integrate the mechanistic aspects of graft rejection with the current knowledge of ITx immunobiology and summarize the quest for a noninvasive biomarker of rejection. Intestinal transplantation (ITx) remains a lifesaving option for patients suffering from irreversible intestinal failure and complications from total parenteral nutrition. Since the technique was established and became a clinical option in the late 1990s, it has been recognized that one of the outstanding features of this procedure was the strong allogenic response triggered by the graft, which has to be contained using more aggressive immunosuppressive regimes compared to other types of solid organ transplantations . ConsideTraditionally, it has been considered that there are several possibilities to elicit an allogenic immune response that will contribute to graft rejection . The so-Some years later, the semi-direct pathway was described ,17,18, wIrrespective of the pathway that is eliciting the allogenic response, one feature that has been clearly established in experimental models of organ transplantation is the importance of the secondary lymphoid organs as sites of allorecognition and T cell activation. In the absence of secondary lymphoid organs in mouse models, the kinetics of alloresponse are much slower or absent, depending on the type of solid organ considered . ITx hasAdditionally, in human ITx, it has also been shown a fast turnover (more than 50% of the population at days 3 to 5 post-transplant) of the recipient-derived lymphocytes emerging through the mesenteric lymph  and alsoRecent evidence indicates the existence of a new activation pathway, the so-called inverted direct pathway, that is responsible for early de novo DSA formation. This pathway operates by the interaction of donor-derived CD4+ T lymphocytes with recipient B cells that may present in the context of recipient MHCII molecules and consequently act as direct alloactivators of donor T lymphocytes, operating in a way similar to the direct pathway with the particular feature that, in this case, donor vs. recipient cell roles are inverted . From anIn the past few years, with the possibility of performing sequencing techniques that allow the identification of individual T cell clones and a clever combination of allostimulation experiments , pioneerResident memory T cells (Trm) are a subset of memory cells phenotypically and transcriptionally different from circulating effector and central memory T cells, which are mainly characterized by their non-circulating behavior, remaining long term, particularly in barrier organs such as skin, gut, lungs and reproductive tract, with the subset numerically the largest T cell memory subset. In recent years, it has been clearly established that in addition to its role in infectious immunity, this subset can be particularly relevant in the process of graft rejection . InteresAuthors have also transcriptionally and functionally characterized the profile of the alloreactive CD4+ Trm, which are mainly Th17-like cells in this model. In the field of clinical ITx, Kroemer et al. (2021) recently described Th17 cells as central players in keeping the progress of thymoglobulin-resistant graft rejection . The chaFurthermore, considering the above-mentioned two-way alloreactivity pathway, it is important to consider that the intestinal graft harbors an important population of resident memory T cells ,42 that Although the main drivers of alloresponse have been characterized, as described above, there are several mechanisms that have been described on homeostatic/physiopathogenic circuits in the intestinal mucosa that have not been explored in the context of intestinal transplantation.MHC class II expression by intestinal epithelial cells (IECs) was described in the 1980s ,44, and As of today, there are no noninvasive biomarkers showing adequate sensitivity and specificity to stand alone as diagnostic assays for intestinal acute cellular rejection (ACR). However, a number of biomolecules have been suggested as potential rejection markers, with some of them, such as plasma citrulline or fecal calprotectin, which are in clinical use. These biomolecules show altered expression during ACR, either due to tissue injury, the accompanying immune response activation and/or bystander inflammation or the ensuing metabolic changes in the rejecting intestinal graft . UnfortuBelow, we summarize the most significant findings and hypotheses explored so far according to three main pathophysiological mechanisms: direct graft injury secondary to the acute rejection process, local and systemic inflammation triggered by the alloimmune response and altered local metabolism following damage to the intestinal graft, explored via high throughput technologies. Many molecular events and subsequent cellular and molecular alterations are intertwined and dependent on the intensity and gravity of the immune response.Intestinal mucosa is the main target of the rejection process, and ultimately enterocyte loss ensues. Circulating levels of citrulline, a nonprotein amino acid produced mostly by enterocytes as the end product of glutamine metabolism, have been suggested as a measure of functional enterocyte mass. Citrulline is stable and relatively easy to analyze in plasma and dried blood spot samples . In ITx Several other biomolecules involved in enterocyte metabolism have been explored in the setting of intestinal ACR. Rodent studies have indicated that the intestinal fatty-acid binding protein (I-FABP), present primarily in mature, villus enterocytes which are normally undetectable in the serum, increased during ACR (196 ± 24 ng/mL), and rejection treatment with cyclosporine consistently reversed rejection and decreased I-FABP in rats . HoweverThe activity of histamine-degrading enzymes diamine oxidase and histamine N-methyltransferase (HNMT) was found to decrease in the mucosa of rejecting rat allografts and revealed a strong negative correlation with the histological rejection score, whereas isografts showed rather constant tissue enzyme activity up to eight days after ITx . These tEnterocyte apoptosis is a central mechanism of tissue injury during intestinal ACR as one of the key effector mechanisms of cytotoxic T lymphocytes (CTL). Its increased occurrence is ubiquitous in different species, and its magnitude correlates with the severity of rejection ,63,64. IIncreased serum levels of suppressor of tumorigenicity 2 (ST2), a regulatory decoy receptor of IL-33, have been observed during inflammatory bowel diseases . In a smA recent murine study using proteomics revealed significant alterations in the tissue expression of ninety-four proteins in the intestinal grafts that developed moderate rejection, compared to non-rejecting isografts . InteresThe initial inflammation in the early stages of allorecognition initiates multiple phenotypic changes in the graft. Earlier experimental studies have indicated numerous alterations, including progressive mucosal pro-inflammatory activation ,72, heatCytokines and their receptors play key roles in the activation and propagation of the alloimmune response through their chemotactic effects, inducing the expression of adhesion molecules, promoting the proliferation and differentiation of specific alloreactive T and B cell clones or having a direct cytotoxic effect on the allograft cells ,76. SeveIn spite of the early enthusiasm around the potential of various cytokines to diagnose intestinal ACR, there seems to be a gap between the experimental and clinical data, and only a few clinical reports have been published and are based on a small number of analytes and patients ,82,83,84A small pilot study found an increase in plasma regenerating islet-derived 3-alpha , a C-type lectin antimicrobial peptide synthesized by enterocytes and Paneth cells, during intestinal acute rejection . As the A relatively recent and still evolving paradigm in the follow-up of ITx patients is the monitoring of the development of humoral immunity against the graft. De novo production of antibodies against the graft’s human leukocyte antigens (HLA), the so-called donor-specific HLA antibodies (DSA), has been linked with a higher risk of ACR in several types of organ transplants, including ITx ,92,93,94A different and more pragmatic approach has been represented in the analysis of the ostomy effluent. Whereas this approach is less used in rodents, it allows easy, non-invasive access to abundant biological material originating in or having direct contact with the intestinal mucosa, the same site where rejection occurs. Fecal calprotectin levels (FCL) have been routinely used in practice over the last few decades to initially differentiate between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), as well as to assess mucosal healing or the recurrence of inflammation in the follow-up of IBD patients. Calprotectin is a calcium- and zinc-binding protein of the S-100 protein family, mainly found within neutrophils, and its presence in feces is due to neutrophil infiltration into the gastrointestinal tissue secondary to an infection or an inflammatory process. Fecal calprotectin is homogenously distributed in stools and is very resistant to in vivo degradation  and in vitro degradation . The first article on the analysis of calprotectin in the stomal fluid of intestinal transplant recipients reported increased FCL both during ACR rejection and non-specific enteritis and found an optimal cut-off level for calprotectin in predicting the presence or absence of intestinal allograft rejection at 92 mg/L with 77% specificity and 83% sensitivity . This inA combined approach using a multiplexed analysis of 17 cytokines and high-throughput proteomics of the ostomal effluent of 16 intestinal transplant recipients found increased levels of five innate immune cytokines early post-transplantation -8, tumor necrosis factor (TNF)-alpha, IL-1beta and IFNγ), but only IFNγ levels were significantly higher in samples with rejection . ProteomThe presence of a very large, complex and dynamic microbial microenvironment makes the intestinal graft unique. The recent advances in sequencing technology have allowed the high-resolution analysis of the intestinal microbiome, unraveled the profound effects of intestinal microbiota on their hosts and involved the occurrence of multiple, highly diverse diseases, such as asthma , diabeteIn a clinical study on 19 ITx recipients, comparisons between samples from non-rejecting ITx and ITx with ACR revealed major alterations in the proportions of multiple bacterial taxa associated with active rejection . At the Although a direct cause–effect relationship between the microbiome alterations (including the enterococcus predominance) and the ACR and its role in graft monitoring remains to be established, these initial findings have important potential clinical implications in terms of patient management, as future targeted strategies to control the degree of intestinal Enterococcus colonization could have a beneficial impact on graft and patient outcome.The last four decades of experimental and clinical research have identified hundreds of molecules that may be altered during the intestinal ACR, with some having the potential to assist with its diagnosis, prognosis and follow-up. Unfortunately, very few, if any, have shown true value in a clinical setting. This may be due both to the differences between experimental and clinical settings, the small size and inherent heterogeneity of clinical studies, as well as the technical and biological limitations of the approaches used. Nowadays, it is believed that either an entirely new, intestine-specific assay or a panel of less specific tests would be necessary to increase the accuracy of non-invasive rejection monitoring. The application of new analytical and bioinformatics techniques would allow a comprehensive analysis of complex biological samples and the simultaneous detection of a large number of chemically diverse analytes. This has already been applied in kidney transplantation, where a composite metabolite–mRNA signature in urine was diagnostic of ACR with high accuracy ,112. In In 36 ITx patients receiving different types of visceral allografts, metabolomics of the stomal effluent or feces demonstrated different metabolomic profiles between rejection and nonrejection . A totalA second high-throughput analysis using quantitative proteomics with iTRAQ-labeling and mass spectrometry performed in rejecting murine intestines and mentioned earlier  identifiThe intestinal graft appears to have a unique immunology due to its contact with external antigens and the exposure to the intestinal microbiota, the gut-associated lymphoid tissue following with the graft, as well as the abundance of epithelial cells. Graft rejection occurs more frequently than in any other type of organ transplant, in spite of potent immunosuppression, and may rapidly result in the loss of the mucosal barrier, life-threatening sepsis and patient death. At the same time, the transplanted intestine lacks reliable, specific, noninvasive biomarkers, allowing non-invasive rejection surveillance. As most of the non-invasive assays tested to date in ITx are the same as those used in the management of inflammatory bowel disease, they all lack specificity by default.Future research should continue the quest to improve our understanding of two-way allogenicity, with an emphasis on the generation of regulatory populations that may reduce the burden of immunosuppression. Additionally, further research efforts should continue to explore the development of assays capable of determining the involvement of memory T cells in the graft rejection process and their potential translational use. Likewise, advances in the monitoring of other types of allografts, such as circulating cell-free donor  DNA or the alterations in the microbiota that may herald or favor ACR, should be tested in adequately sized, multicenter studies on either prospectively collected material or on existing biobank samples. The continuous quest for the identification of well-defined alterations occurring during rejection in the main targets of the immune response, that is, the intestinal mucosa and its microenvironment, could ultimately reveal the long-sought and badly needed rejection biomarker.


The paper evaluates the possibility of using dissimilar materials joined by welding technology in the construction of agricultural machinery. The desire to design larger and more efficient structures requires designers to combine materials with different mechanical and structural properties. In such a case, it is very important to properly select welding parameters so that, on the one hand, the quality of the joint meets the standard requirements, and on the other, the welding process is not too energy-intensive. In this paper, overlay joints connecting S355 steel with Strenx 700 steel were analyzed in terms of strength for three different values of welding parameters and different thicknesses. The starting point was the reference parameters recommended by the company’s welding technologists, which were reduced by 10 and 20% according to the linear welding energy. The study compared the strength, ductility and macrostructure of the joints, as well as the energy intensity of the process. The proposed dissimilar joints achieved approximately a 10% increase in the strength limit of the components in comparison to the previously recommended welding parameters. Additionally, finite element analysis calculations of the improved designs showed significant weight reduction (up to 40%) for the relevant agricultural machinery components. The development of agriculture is dictated primarily by the need to produce higher quantities of food while enhancing its quality. These changes are significantly influenced by new technologies and process innovations used in the production of machinery and equipment for agriculture. They contribute to an increase in the efficiency and productivity of agrotechnical operations. In the era of the widely understood Agriculture 4.0 linked to the balanced agriculture system, there is a trend toward the use of high-performance machinery with large working widths. Due to the steadily decreasing natural resources and growing global population, it is necessary to guide agricultural production toward more balanced and efficient use of natural resources. In order to ensure food security, the share of large and very large farms in the agro market is increasing. For their operation, these enterprises need high-performance, cost-effective, large-width machinery that allows precision farming. To meet the expectations of the agro market, manufacturers of agricultural machinery must use unconventional combinations of construction materials at the stage of designing high-performance machinery dedicated to specific agrotechnical operations, to ensure a high level of reliability and operational durability of machinery implementing the concept of Agriculture 4.0.In an era of rising agricultural costs and falling prices for agricultural products, the world is facing a new challenge. In particular, this challenge is the search for cost savings in the means of production of agricultural machinery, so as to provide the farmer with economical and relatively inexpensive solutions. In the case of cattle, horse, sheep and biomass producers for energy needs, to achieve increased efficiency and economy in the green fodder harvesting chain, it is necessary to provide machinery solutions with increased operating productivity while reducing the times of individual agrotechnical procedures. Cheaper, more economical and more efficient machines provide a lower depreciation threshold which allows for a faster increase in profitability and earned income. The time saved and increased income enable farmers to reallocate capital to other sectors of the farm.Rising production costs associated with, among other things, high prices of raw materials including steel, electricity and labor, force machine manufacturers to optimize manufacturing processes in order to provide the end user with a highly efficient, reliable product at an attractive price. Structures of high-performance machines made of typical construction materials, such as S355 steel, are subject to damage during extreme operation by cracking of their components. Examples of such damage can be found in the structures of green forage harvesting machines .Typically, these cracks are fatigue cracks, caused by cyclic operating loads resulting from the variety of field layouts and the nature of agricultural tractor driving. Examples of the levels of operational loads occurring during the operation of the mower set are shown in Welded joints in the context of high-strength steel materials have received significant attention from both scientific and industrial sectors, with applications extending to various fields, including agricultural machinery. The utilization of dissimilar welds, where different materials are carefully selected and joined, offers distinct advantages over constructing machinery from a single material. Incorporating dissimilar joints enables the use of materials with higher strength properties in localized stress concentrators without uniformly increasing section thickness, promoting a more even stress distribution and thus enhancing operational durability. This approach can yield machinery designs that achieve a delicate equilibrium between longevity, weight optimization, and cost efficiency. By carefully choosing materials for specific components or sections, engineers can utilize the superior properties of each material, such as strength, durability, or corrosion resistance, to meet the specific requirements of different parts of the machinery. This not only enhances the overall performance and longevity of the equipment but also allows for more efficient utilization of resources, making it a compelling choice for modern manufacturing processes. These advantages have contributed to the acceptance of dissimilar joints in industries such as shipbuilding, chemical, energy, automotive, and machinery, underscoring its practical and economic benefits.Optimizing parameters is vital for any modern manufacturing method. However, to do so assumes even greater significance in welding processes, as these processes directly influence the mechanical behavior of materials due to variations in welding parameters. This is even more true for dissimilar joints, where the correct parameter adjustments are imperative to ensure the structural integrity and quality of the welds. These direct improvements to welding parameters enhance equipment performance and longevity, reduce maintenance costs and downtime, and conserve resources. Moreover, optimized parameters ensure high-quality, defect-free welds, ensuring structural integrity and product safety. In summary, parameter optimization is vital for efficient, cost-effective, and eco-friendly manufacturing processes.Due to the previously elaborated considerations, the subject of dissimilar welds of high-strength materials has gained increasing importance in recent years. By extension, there has been a growing interest in the Strenx, a highly durable and lightweight steel, and its welded joints. In order to provide an overview of the current state of the research in this area, a short literature review was conducted, including studies on the welding of high-strength steels with a particular focus on Strenx 700 and their dissimilar welds.Autogenous laser welding of Strenx 1100MC and laser-welded joints of Strenx 700MC steel  highlighEvaluations that were extended to dissimilar welding by performing quality assessments of dissimilar welded joints of Hardox 450 and Strenx 700 involving non-destructive testing and microstructural analysis unveiled the significance of welding consumables in achieving the required quality of welded joints . MoreoveThe impact of varying heat input on the microstructure and mechanical properties of dissimilar high-strength steels (S700MC/S960QC) joined using undermatched filler material and gas metal arc welding was investigated in . The resIn summary, this literature review provides a brief examination of the current state-of-the-art in welding high-strength steel materials, covering various welding processes, parameters, and their impacts. However, it is evident that there exists a noticeable gap in the literature regarding the investigation of dissimilar welds involving Strenx 700. Recognizing this gap, the present research paper aims to contribute significantly to this area by addressing the limited knowledge available on the welding of Strenx 700 with dissimilar materials, specifically focusing on optimizing the welding parameters necessary for joints with desirable mechanical properties.In order to ensure an adequate level of reliability in the design of agricultural machinery for harvesting green fodder, research is needed to reduce the weight of machines while increasing their strength and stiffness. Therefore, there is a reasonable need to look for solutions that increase the load-to-weight ratio and reduce production costs.To achieve this goal, the study initially focuses on attaining mechanical reliability in dissimilar joints. Once joints with adequate mechanical properties are achieved using the recommended welding parameters, the study investigates the mechanical properties of joints that were welded with lower welding energy. This approach effectively reduces production costs by minimizing energy consumption while still enhancing the mechanical properties of the welded joints.The improved design of currently available agricultural machinery is realized through the implementation of these dissimilar joints and welding parameters, resulting in a significant reduction in weight without sacrificing structural reliability.In order to reduce the energy consumption of the welding process of machine components manufactured by the company Samasz, Zabludow, Poland, the first step was to analyze the types of welded joints used during the construction of typical machines: a disc mower a, a rotaThe solutions for the structural nodes of the analyzed machines, which are particularly prone to deformation, are designed as framed-box structures. These structures are crafted using welding techniques with formed steel sheets. The most common choice for the structural material in these solutions is S355 steel, which is frequently susceptible to damage resulting from local overloads . IncreasBased on an analysis of machine design, In order to carry out the planned experiments, the test joints from dissimilar materials  were prepared. The base material was structural steel S355 in the thickness range of 6–14 mm, sheets of which were joined by double-sided overlays made of a 5 mm thick sheet of Strenx 700 material. Welds were made using MAG technology. A fusible electrode in the form of Lincoln Supramig HD ISO 14341-A-G46 4 M21 wire was used for welding . The welI—average value of welding current [A], U—average value of arc voltage [V], V—welding speed [mm/s], k—thermal efficiency factor .Strenx steel is a high-alloyed material with a fine-grained structure, unlike S355 steel. Using welding parameters dedicated to one of these two materials can adversely affect the properties of the joint. Therefore, through the experimentation of the linear welding energy parameter, the optimal values of current, voltage and welding speed were determined for the adopted quality criterion which is the strength of the joint. The welding linear energy  was deteFor all the welding parameters analyzed, the joints that were prepared have passed visual examinations. The welded joints underwent a visual inspection (non-destructive testing) following the application of specific welding parameters prior to the final sample cutting. This inspection encompassed an assessment of the weld’s appearance, geometry (dimensions), quality, shape, surface condition, and the presence of potential welding irregularities such as open cracks, assembly discrepancies, delaminations and adhesions, as well as any discernible bubbles and inclusions. In all analyzed instances, plates welded with the parameters designated in this study consistently met the established criteria, making them suitable for the extraction of the requisite test samples. Waterjet cutting technology was utilized to ensure correct shapes for the sample joints, and specimens that were used in the strength tests were prepared in accordance with l = 0.001 mm/s. An MTS 828 servohydraulic testing machine (2) was used for the tests. Measurement of specimen strain was carried out using an Aramis 4M vision system (3). During the test, the force and displacement of the specimen’s measurement base were recorded continuously. In addition, using the ARAMIS 4M vision system, the deformation of the specimen during load build-up was observed using the digital image correlation (DIC) method. Using the Aramis 3D 4M made it possible to register the deformation process and to locate critical failure deformations.The experimental setup is shown in The tests conducted were aimed at determining the strength characteristics of the welded joints obtained, as well as determining the strain distributions in the test specimens analyzed. Each test was repeated three times, and the result was the arithmetic average of these repetitions. Similar to the geometry of the samples, the testing procedures followed the guidelines specified by the respective standards of PN-EN ISO 4136:2022-12 for butt joints and PN-EN ISO 9018:2016-01 for overlay joints.Rm), yield strength (Re) and specimen elongation to break (A%). In addition, the fracture of the samples itself was analyzed to detect weld inconsistencies such as cracks, undermelting, fusion deficiencies, slag, and blister nests. Based on the observation of the fracture area, the appropriateness of the selection of the materials to be joined and the choice of welding parameters were determined. Experimental investigations consisted of determining the effects of the process and welding parameters on the mechanical properties of the material, including tensile strength . The results of the study are shown in Based on the ground microhardness profiles of the weld joint, intermetal areas with increased hardness near the fusion zone on the S355 steel side were observed. This transition from ductile to brittle fracture behavior is a critical concern, especially in the context of high load variations in agricultural machinery, posing a significant safety risk. In the boundary zone between the native Strenx material and its heat-affected zone, there is a local 10% reduction in microhardness compared to the original material. This reduction enhances the material’s ductility in the zone of large structural changes, which is a desirable phenomenon. Similarly, reducing the welding parameters by 20% (V3) in terms of linear welding energy compared to the recommended welding parameters of the V1 joint results in a decrease in microhardness in the heat-affected zone by 2%.By analyzing the structure of the obtained welded joint and relating the size of the HAZ to the size of the areas of the joint obtained with the reference parameters, it can be concluded that reducing the welding parameters by 10% results in a reduction in the size of the HAZ in the analyzed joint on the S355 steel side by 5%, and on the Strenx 700 side by 10%. Reducing the welding parameters by 20% results in a reduction of the heat-affected zone by 25% on the S355 side and by 20% on the Strenx 700 side. Subsequently, based on optimized welding parameters, structural nodes were selected in machines in which, in order to increase their ad hoc strength and reduce weight, joints of dissimilar materials were used with selected welding parameters representing 80% of the value of the recommended welding linear energy. Redesigned according to these recommendations, the machines are currently in the field testing phase and, for the time being, show increased operational reliability.Meanwhile, to confirm the assumptions made about the use of dissimilar materials in the construction of agricultural machinery, numerical calculations using finite element analysis (FEA) were carried out for the selected mower made by Samasz. A spatial discrete FEA model of mower constructions was made, taking into account all components affecting their stiffness and strength. Due to the specifications of the structural elements, the discretization was carried out using plate-shell elements  characterized by linear displacement functions and linear beam elements (CBEAM2). Separable connections were modeled using CBEAM2 elements and multi-point constrain (MPC) elements of RBE2 and RBE3 types. Calculations were performed using MSC software AFEA (Marc v2020). The preliminary results of the numerical calculations have been presented to demonstrate the uniformity of stress levels within the structure when different material grades are selected and applied. It has been observed that, subsequent to the modifications, stress values throughout the structure generally exhibit consistency, albeit with localized concentrations attributed to the structural design. The process of selecting different material grades also necessitated adjustments to the thickness of individual structural components, inadvertently resulting in a reduction in the overall stiffness of the calculated model. Consequently, modifications were implemented within the spatial structure of the model, including adjustments to the dimensions of the closed profiles created for the subject structure.It is imperative to emphasize that the FEA calculations presented in this study are of a preliminary nature, primarily intended to illustrate the stress behavior of components manufactured from dissimilar materials. For a more comprehensive FEA, a more extensive approach to finite element modeling would be required, considering the regions with elements composed of varying material grades within the structures under examination and the changes in the stiffness of the calculated construction. However, this extends beyond the scope of the current paper and remains a subject for further research consideration by the authors.Boundary conditions for individual models of discrete structures were adopted in accordance with their working conditions . In all As a result of the numerical calculations, the distributions of half stresses of the analyzed structure were determined. The results show the degree of effort of the structure after a given load and illustrate the possibility of using different materials with different strength properties. Utilizing dissimilar welded joints at critical points in green harvesting machine design enhanced structural strength while reducing system weight.The non-separable joining of S355 and Strenx 700 steels with the MAG welding method, featuring a 20% reduction in welding parameters based on linear welding energy, significantly increased static joint strength (up to 10%) and flexibility. This improvement was due to reduced thermal energy input, leading to slower grain growth in the weld zone.Optimized welding parameters reduced the energy intensity of welding these materials by up to 20%, directly cutting production costs. Using the MAG technique with the adopted welding parameters and the filler metal, both butt and overlay welding of S355 and Strenx 700 steels allowed for the creation of high quality joints concerning macrostructure and strength properties.Strength tests revealed that cracking occurred in the heat-affected zone on the S355 steel side.The joint exhibited slightly higher strength and elongation values in comparison to the weaker of the joined materials, with a local increase in the joint’s strength limit of up to 10%.The use of base material with lower strength characteristics, reinforced by overlays of higher-strength steels, improved the strength and stiffness of the resulting structural element.Simultaneously, this approach reduced weight and extended the operational lifespan of the welded joints.Implementing such measures in the manufacturing of agricultural machinery reduced production costs and facilitated the design of larger, more efficient, and reliable solutions.The recommendations presented in the paper, focusing on the utilization of dissimilar joining materials through optimized welding parameters, resulted in design modifications in Samasz-manufactured agricultural machinery. These modifications specifically targeted the mower, tedder, and rake systems used for multi-row forage harvesting.Operational data gathered from these adapted machines revealed a decreased incidence of failures and structural damage, particularly concerning issues related to cracks, in contrast to older solutions.This collection of evidence served as concrete validation of the proposals presented in the paper.Developing Agriculture 4.0 machinery is vital for boosting agricultural productivity and efficiency, and is especially in alignment with the European Green Deal and related international strategies. High-performance, circular-economy-based machinery should incorporate energy-efficient, carbon footprint-optimized technologies, achieved through thoughtful material selection and low-energy welding. Research into unique welding parameters enhances economic benefits, reduces carbon footprint, and improves operational reliability. Based on the conducted research, the primary conclusions of the study are as follows:


Recent morphological data on human brain development are quite fragmentary. However, they are highly requested for a number of medical practices, educational programs, and fundamental research in the fields of embryology, cytology and histology, neurology, physiology, path anatomy, neonatology, and others. This paper provides the initial information on the new online Human Prenatal Brain Development Atlas (HBDA). The Atlas will start with forebrain annotated hemisphere maps, based on human fetal brain serial sections at the different stages of prenatal ontogenesis. Spatiotemporal changes in the regional-specific immunophenotype profiles will also be demonstrated on virtual serial sections. The HBDA can serve as a reference database for the neurological research, which provides opportunity to compare the data obtained by noninvasive techniques, such as neurosonography, X-ray computed tomography and magnetic resonance imaging, functional magnetic resonance imaging, 3D high-resolution phase-contrast computed tomography visualization techniques, as well as spatial transcriptomics data. It could also become a database for the qualitative and quantitative analysis of individual variability in the human brain. Systemized data on the mechanisms and pathways of prenatal human glio- and neurogenesis could also contribute to the search for new therapy methods for a large spectrum of neurological pathologies, including neurodegenerative and cancer diseases. The preliminary data are now accessible on the special HBDA website. Human brain atlases have been growing rapidly, driven by the increasing availability of modern methods for image visualization and digitization of new and already existing datasets ,2,3. As Brain atlases make it possible to define the organization of brain structures, their shape, relative localization, etc. ,7. They The history of human brain atlas development is reviewed in many papers ,6,7,8. IThe first digitized brain atlas was developed in the 1970s. It was a natural step forward in brain atlasing, making it possible to overcome the limited functionality of printed books. Recently developed neurobiology methods make it possible to construct spatial brain maps and atlases extending from gross morphology to tissue and cell resolution, including specific transcriptional maps ,8.Atlases of the developing brain should be considered separately. Brain growth and maturation are highly complicated processes. Neurodevelopment determined by morphogenetic, genetic, and environmental forces shapes brain morphology, neural architecture, and synaptic circuitry . Cortex Current developmental biology contains a variety of data on the differentiation of stem and embryonic cells, intercellular interactions with the definition of molecular mechanisms of auto-and paracrine regulation, transcriptome features of differentiated cell lineages, and other information at the intersection of embryology, molecular biology, and genetics. The majority of such studies are performed on model animals, cell cultures, and living sections. However, data from model experiments cannot be directly extrapolated on humans and need validation . The devEvaluation of normal human neurogenesis is necessary to understand the mechanisms of regenerative potential in postnatal ontogenesis. The underlying causes of many brain disorders are still not well understood at cellular and tissue levels. Moreover, a lot of psychiatric and neurological pathologies have prenatal origins and cannot be adequately studied in model experiments ,21. In aThe majority of modern publications on normal and pathological human brain development are the data obtained using noninvasive techniques, mainly magnetic resonance imaging (MRI) and its modifications—functional MRI and diffusion tensor imaging ,35,36,37Unfortunately, the resolution of noninvasive techniques is still insufficient for the study of human brain development at the level below macromorphological, and the correlation between such data and real anatomical and histological characteristics of the developing brain still remains a separate field of research ,46,47,48Therefore, the development of fetal brain atlases is limited compared to that of neonatal, paediatric, and adult brain atlases ,44. The Several studies have provided the human brain transcriptome profile with an expression of developmental dynamics . Even nTherefore, a histological and immunohistochemical examination is currently the only technique that actually maps the brain directly .Studies on human brain development at the level below macromorphological are mainly related to the names of researchers developing PaskoRakic’s ideas, and/or specifically dedicated to the problem of postnatal neurogenesis ,54,55,56Brain maturation is a complex morphogenetical process. A detailed study of the early development of the human brain is necessary to understand the normal function of the mature human brain and the causes, manifestation, and progression of pathological processes. Currently, there is a great lack in the fundamental knowledge of human developmental neurology. There is no consistent periodization of the development of fetal brain structures or integrative gyrification hypothesis. This deficiency is related to the fact that the majority of studies in the field of neurobiology are designed as model experiments.Studies on human autopsy material are limited due to the complex reasons. Furthermore, intrauterine fetal death often leads to poor condition of the material even for a routine pathology examination. There are also other limitations, both legally established and in the field of individual and professional ethics. Thus, there are relatively few research groups over the world dealing with fetal human material. Only the printed Bayer and Altman Atlas ,83,84,85Most of the current publications on the development of the human brain are based on non-invasive research materials , which provide information mainly at the macromorphological level. Histological research materials cover in more detail the embryonic and pre-fetal periods of development, while data on the fetal period are presented mainly by macro-preparations and schemes ,84,85,86Another consequence of the small number of scientific groups dealing with fetal autopsy material is the lack of research performed at the modern methodological level. Current advanced research is concentrated mainly on the features of transcriptome functioning in the developing brain ,51,56. BWorldwide, there is a growing interest in the accumulation of large-scale scientific and reference data into databases available for general use. Morphology, including immunomorphological tissue research, generates large amounts of of digital images, which provide valuable information. However, data are mostly provided only as quantity tables or conclusions and do not fully contribute to publishing data in original research papers. Complete and detailed data are not available to a wide range of experts, including neuroscientists and neurologists.The integration of newly obtained and existing information remains the main problem: “the gap between the molecular basis of development and already established ideas on the organogenesis, between ultrasonography results and anatomy, data from the dated textbooks and guides on histology and embryology, obstetrics and neonatology, and the current level of understanding of human intrauterine development becomes wider” . This prThus, a digital database on the immunophenotypical cell profiles in the developing human brain is highly requested. The accumulation of data on human brain development, including data on the immunophenotype of various brain regions, remains an important task that has both fundamental and practical importance. The digital high-resolution histological reference annotated atlas of normal prenatal human brain development is a relevant task for human neurobiology. A separate issue is the creation of a consistent database of cortex brain development, covering all stages of intrauterine development, including the fetal period, which will present materials not only at the macromorphological but also at the histological level, taking into account the regional specific features of the immunohistochemical profile of various cortical territories of the maturing big brain. Therefore, the Human Prenatal Brain Development Atlas (HBDA) has been launched, which aims to create spatiotemporal reference maps of the whole fetal brain. The first draft of the HBDA will profile 16 fetal brains at various stages of prenatal development. It will combine histological and immunohistochemical methods. The HBDA could help answer questions regarding the histological brain structure, developmental biology, and cell fate and lineage, including neuro- and gliogenesis. This paper describes in detail a research strategy for the HBDA.The HBDA will be based on postmortem human brain autopsies from the unique Collection of the Prenatal Human Development of the Laboratory of the Nervous System Development at the Research Institute of Human Morphology. It consists of the material from the second post-conceptional week (2 pcw) until birth. The materials have been collected in the last five decades and are currently being renewed. The collection consists of more than 200 formalin-fixed human embryos and fetuses at different gestational stages. The collection contains not only fixed material but also histological, immunohistochemical, and electron-microscopic preparations. All the material was taken and handled according to national legislation and the Declaration of Helsinki. Protocols were approved by the local Ethics Committee of the Research Institute of Human Morphology. The classification of the fetal period, which is divided into four stages , early fetal (13–20 weeks), middle fetal (21–28 weeks), and late fetal (29–40 weeks) periods)), will be used . At leasMost samples are buffered formalin-fixed. For the prenatal reference Atlas, serial coronal and sagittal 5–10 μm thick paraffin sections from the whole embryo or fetal head, fetal brain, or an entire hemisphere, depending on the developmental stage, are stained with hematoxylin-eosin, Mallory trichrome, and Nissl techniques.A combination of the classic cytoarchitecture with recent immunohistochemical methods is a great advantage of the immunomorphological approach that resulted in the regional-specific immunophenotyping of brain areas at various prenatal stages. In accordance with the HBDA objectives, immunohistochemical experiments are planned using direct immunoperoxidase, double, and multiplex labeling with immunofluorescence and chromogen methods directly on a series of histological sections, followed by an analysis using morphometry. After the reference histological application, immunohistochemical experiments will be applied . Multiplex immunolabeling will be used for the spatial analysis of antigen patterns on the same slice. Thus, immunophenotypes of the whole brain structure will be obtained as a result of the procedure, and the spatial-temporal rates of the structure maturation will be accessible for examination using morphometry and statistical analysis. The mechanisms and factors of the differentiation between multipotent and oligopotent progenitor cells of the nervous system are central problems in recent developmental neurobiology. A series of immunohistochemical experiments with a wide panel of specific markers will be taken to mark the main central nervous system cell populations on the whole fetal hemisphere slice. The choice of a marker panel for the project is based not only on the authors’ own experience but also on the published data and online materials on related sources, such as The Human Protein Atlas, Allen Brain Atlas, and others. Preference in each phase of experimentation will be given to markers that are most well-established in world research and medical practice and consistently work on human fetal autopsy material. Thus, the HBDA will be useful for the comparative analysis with other research groups’ data  ,91. ThisA series of immunohistochemical experiments will be carried out in order to characterize neuroblasts of the neuronal and glial differentiation lineages. The application of antibodies against transcriptional factors associated with one of the cell differentiation lineages—neuronal, astrocyte, oligodendrocyte—is widely used in recent developmental neurobiology ,55. AntiThe functional markers may be used as indirect indicators of the functional activity in the process of cell maturation in nervous tissue. Antibodies to the functional proteins of presynaptic terminals, including synaptophysin, are classically used for the monitoring of synaptogenesis and functional maturation of neural tissue ,101,102.Annotated atlases based on the series of virtual slices of sixteen human fetal brains in different developmental stages will be a main component of the HBDA.The basic annotated atlases will contain an image library of virtual sections with a zoom tool corresponding to an objective resolution of up to ×20. Virtual slices from the serial histological preparations of human fetal brain autopsies will be made using the MECOS-C2 system, which is specially modified for large (non-standard) preparation sizes (up to 12 × 9 cm).The system collects hundreds of images in lengthwise strips, which are later stitched together to create one large image. The exposure time, white balance, and flat-field correction are set independently for each slide. The acquired SVS files will subsequently be converted to JPEG. As the initial step of the project, histological preparations from the collection will be digitized, annotated, and analyzed to create a reference base for the Atlas. New preparations will be prepared especially for the HBDA .https://brainmicroscopy.com/collection/homo/brain-development/, accessed on 28 March 2023), which is granted by the Historical Committee of the Federation of European Neuroscience Societies (FENS). The HBDA results will be organized as annotated atlases of the developing human brain, with a series of virtual sections, with a resolution of up to 20,000 × 20,000 px; moreover, new results from the fundamental neurodevelopmental study, with regional-specific immunophenotype profiles of the fetal human telencephalon, will be provided, and developmental heterochrony in human cortical development will be highlighted.The HBDA team already has experience in digitizing preparations and creating a virtual collection, including fetal brain samples. Archival preparations (2600 × 3000 px) from the Laboratory Collection are already available on the special laboratory website. Each vector area will then be associated with a certain brain structure and annotated; in this way, the collating vector allows the flexibility to create various presentation modes  .There is no one uniform nomenclature for all structures of human developing telencephalon. Structural delineations will be based on classic adult human brain morphology and special publications on the certain structure developmental course, with special references for evolutionary nomenclature principles. All published sources used for each structural complex will be provided as special comments. Differences between existing neurologic traditions will be compared and commented on, with special references to classic German, American, and Russian (Soviet Union) ontological principles and the recent “standard” of anatomical ontology provided in Nieuwenhuys et al. , TerminoThe primary goal of the HBDA is to create the immunophenotype neurogenetic map of the developing human telencephalon, which may underlie an analysis of regional heterogeneity and heterochrony under the developmental course of human normal brain maturation. The formation and maturation of the forebrain are among the main topics in recent neurobiology since the human cortex is a functional base for human cognition and intelligence. Studying the prenatal human telencephalon contributes to solving fundamental questions of developmental neuroscience and also greatly develops special human neurology. Within a great number of studies devoted to molecular and genetic mechanisms of neuroblasts migrations, basic themes such as periodization of normal development, ranges and variabilities, comparative functional morphology, and cytoarchitectonic analysis of various regions of the developing human brain remain poorly covered. There are almost no complex studies on the recent methodological level devoted to region-specific normal morphogenesis of the human brain. Published studies within the last decade are very limited and focused on certain regions of the developing brain, and are often performed on limited material , applied with regional specificity in mind, indicates the heterogeneity of, at least, the neuronal differentiation between the large regions of the forebrain, and the existence of regional specificity in the expression (transcription) of genes related to neural diseases between different brain regions ,110,111.The development of CNS malformations involves various processes, including abnormal neurulation, proliferation, migration, etc. . As mentCaenorhabditis elegans, the tadpole larva of Ciona intestinalis, and two annelid species, the polychaete Platynereis and the leech Hirudo verbena [Undoubtedly, atlases of human brain development still have a long way to go in terms of detailed resolution. For several invertebrate species, including  verbena , connect verbena ,116. ThiWith the aim of HBDA, two main steps are needed: immunophenotyping and cell lineage mapping of certain telencephalon subdivisions at various prenatal stages  followed by the presentation of project results as a reference annotated online atlas. New data on the developing brain proteome, including regional specifics, will be collected, and the digital source could become a base for further investigation on human brain development.The human cell proteome contains thousands of proteins. In this project, neuronal and glial cell lineage differentiation will be examined with main specific markers of certain cell populations, including the transcriptional factor and functional neuro-specific markers. The immunomorphological approach makes it possible to compare immunohistochemical cell profiles of different cortical areas with annotated virtual brain slices and region-specifically evaluate the maturation rates of the main cell lineages—astroglial, oligodendroglial, and neuronal—to describe the heterochrony of cortex development and analyze developmental changes and tendencies at the different stages of prenatal human ontogenesis.The main data of the atlas will be organized as macro-and microphotographs of the embryos and fetuses at different developmental stages, including annotated graphs with morphology and immunohistochemical experiment results, a series of virtual slices implemented with technology for creating digital images for remote viewing without loss of quality and resolution, as well as diagrams superimposed and overlaid with annotated “maps” of fetal brain structures. Systematization and post-processing of the obtained fundamental data involve the creation of diagrams and three-dimensional reconstructions that clearly demonstrate the processes of growth and development of the human brain. In the pilot version of the site, navigation both by the period of development of embryos and fetuses and by individual brain structures or certain key methods and markers is planned.The developers of HBDA will focus on creating a user-friendly interface with easy switching and cross-references between sections, the hierarchical organization of tabs and links, and convenient switching between them. Navigation through the source is planned separately by the stages of development, histological methods used, immunohistochemical markers, and systems, regions, and structures of the developing human brain.A reference database for the developmental research of different human brain structures. These materials are unique, which makes it possible for them to be in demand for many years.Comparative material for general neurology—non-human mammal, cultural, and other experimental studies.A reference database for the comparative analysis of non-invasive  study results, newly spatial genomic sequence technology, and high-resolution 3D-visualization of the native tissue samples with phase-contrast CT.The Atlas would eliminate the following gaps in the necessary data:(a)in obstetrics practice—for the comparison of ultrasonography and MRI/CT results;(b)in neonatology—for brain maturity criteria;(c)in prenatal and perinatal pathoanatomy—for the intrauterine and neonatal death cause analysis and searching for pathological changes in the fetal brain.For medicine practice, the results of this project will be useful:To date, many scientists all over the world use such online and offline sources in everyday practice. Such an online source would make it possible to save funding, as well as the time and efforts of specialists and researchers. Therefore, one can expect that the HBDA will be useful as an annotated guidance for fundamental research. An online atlas of the developing brain will also be useful in educational programs on embryology, histology and cytology, neurology, physiology, endocrinology, pathoanatomy, genetics, etc. .Thus, the HBDA is a research program that connects a fundamental scientific study of the prenatal human brain with a digital presentation of the project and results in an online available renewable resource. The authors believe that the digital atlas of normal human brain development will fully respond to current trends in scientific practice, its methods, and means of communication.


Pompe disease is produced by an enzymatic deficiency that leads to aberrant accumulation of glycogen in in multiple tissues, mainly muscle, causing progressive heart, respiratory and motor failure. Dysregulations observed in these patients are derived from glycogen accumulation but also to different secondary abnormalities. The characterization of the metabolic profile associated with this disease is a valuable approach to gain a larger view of all the metabolic dysregulations caused by the disease, and its potential correlation with clinical progression and response to therapies. This article describes the metabolic alterations reported to be significantly altered in Pompe disease patients in recent years. From a clinical perspective, this information could contribute to guide in the diagnosis, evaluation of disease severity, treatment decision and monitoring of Pompe disease patients.Lysosomal storage disorders (LSDs) constitute a large group of rare, multisystemic, inherited disorders of metabolism, characterized by defects in lysosomal enzymes, accessory proteins, membrane transporters or trafficking proteins. Pompe disease (PD) is produced by mutations in the acid alpha-glucosidase (GAA) lysosomal enzyme. This enzymatic deficiency leads to the aberrant accumulation of glycogen in the lysosome. The onset of symptoms, including a variety of neurological and multiple-organ pathologies, can range from birth to adulthood, and disease severity can vary between individuals. Although very significant advances related to the development of new treatments, and also to the improvement of newborn screening programs and tools for a more accurate diagnosis and follow-up of patients, have occurred over recent years, there exists an unmet need for further understanding the molecular mechanisms underlying the progression of the disease. Also, the reason why currently available treatments lose effectiveness over time in some patients is not completely understood. In this scenario, characterization of the metabolic phenotype is a valuable approach to gain insights into the global impact of lysosomal dysfunction, and its potential correlation with clinical progression and response to therapies. These approaches represent a discovery tool for investigating disease-induced modifications in the complete metabolic profile, including large numbers of metabolites that are simultaneously analyzed, enabling the identification of novel potential biomarkers associated with these conditions. This review aims to highlight the most relevant findings of recently published omics-based studies with a particular focus on describing the clinical potential of the specific metabolic phenotypes associated to different subgroups of PD patients. Lysosomal storage diseases (LSDs) are a group of over 70 inherited metabolic disorders, frequently presented in childhood, caused by mutations in genes that affect the function of lysosomal hydrolases, accessory proteins, membrane transporters, or trafficking proteins, that finally result in the accumulation of biomolecules inside the lysosomes and lysosomal impairment. Although individually LSDs are defined as rare disorders, as a group they are relatively common, with an incidence as high as 21:100,000 live births in some countries . From a GAA gene [PD is an autosomal recessive GSD caused by mutations on the encoding gene of the acid alpha-glucosidase (GAA) lysosomal enzyme, which is responsible for the hydrolysis of glycogen to glucose. This enzymatic deficiency leads to glycogen accumulation in various tissues, including musculoskeletal, cardiac, respiratory and nervous systems ,14,15. DGAA gene . In 2015GAA gene ,22. SincGAA gene ,24,25,26From a clinical perspective, there are two main phenotypes described in PD: the infantile onset PD (IOPD) and the late onset PD (LOPD) phenotypes . MoreoveGiven the broad clinical variability and the unpredictability of different genotype–phenotype correlations in these patients, the application of omics technologies could remarkably contribute to improve the landscape of PD . Indeed,®, Sanofi-Genzyme, Cambridge, MA, USA) approval by the Food and Drug Administration (FDA) and the European Medical Agency (EMA) in 2006 completely changed the landscape for PD patients [Regarding PD treatment, enzyme replacement therapy (ERT), given as recombinant human GAA (rhGAA), has been available for more than 15 years . Althougpatients , ERT is patients ,70,71. Mpatients , includipatients ,59, limipatients  and devepatients ,74. Notapatients ,76,77, gpatients  and alsoAlthough not specific, urinary glucose tetrasaccharide (Glc4) seems to be the most important biomarker in measuring the progress of PD ,86,87,88The detection of metabolic alterations associated with the development of PD may contribute to the identification of new diagnostic biomarkers and improve the diagnosis of this disease. In this context, a range of studies have compared the metabolic profile of PD patients and healthy individuals using different omics approaches aiming at the identification of potential metabolic biomarkers that could be clinically useful in PD diagnosis . In thesIn the study conducted by Sidorina et al., data from proteomic and lipidomic analyses were combined to identify novel PD biomarkers and gain knowledge in the physiopathological mechanisms underlying PD . CompariOther studies have been focused on evaluating the performance of different analytical methods as metabolic screening tools for different LSDs causing an accumulation of oligosaccharides (OS) ,106,107.Another study conducted by de Moraes and coworkers , which iAlterations in the urinary levels of Hex7 and Hex6, in addition to Glc4, were also found in PD patients in a study conducted by Hagemeijer et al. . The indOther studies have focused on identifying metabolic changes related to a particular disease phenotype . In PD pOne of the hallmarks of PD is muscle atrophy ,115,116 The metabolic profiles of skeletal muscle from GAA-KO and wild-type mice were also compared in a more recent study conducted by Meena and colleagues . In partOther studies have reported different findings in relation to glucose metabolism observed at the transcriptome level. In the study conducted by Kinton et al., the transcriptomic profiles from healthy individuals’ and LOPD patients’ (ranging from 19 to 78 years age) muscle biopsies were compared to identify cellular processes that were specifically altered in LOPD patients . The resIncreased glycolysis was also observed using a proteomics approach in a study conducted by Moriggi and coworkers focused on gaining deeper insights into the molecular players involved in the impairment of muscle metabolism of LOPD patients . Using aA recent metabolomics study was focused on characterizing differences in the OS profile of IOPD and LOPD patients. The results from this study revealed that the urinary OS profile of IOPD patients was characterized by significant increased concentrations of Glc4 Hex5, Hex6, and Hex7, when compared to LOPD patients . NotablyOver the last years, several studies have focused on exploring metabolic changes associated with the response to ERT or gene therapy  in PD. MAlthough ERT is the standard of care for PD patients, many patients present poor outcomes due to the heterogeneous response and limited efficacy of the drug in clearing muscle glycogen storage . Late-onMetabolic alterations after ERT were also analyzed by Kinton and coworkers to investigate the early response of LOPD patients to treatment . In thisMeena et al. also evaluated the potential of ERT , based oGAA gene transfer by AAV in GAA-KO mice restored expression levels of genes involved in bioenergetics and metabolism processes, including glycogen degradation, glucose and glucose-1-phosphate degradation, and serine and glycine biosynthesis pathways. The fact that the enhancement in glucose metabolism observed in this study is opposite to the results reported more recently by Moriggi et al. [A similar animal model was used in a different study for the evaluation of the efficacy of AAV in restoring GAA enzymatic activity and reverting the gene expression dysregulations observed in the skeletal muscle and spinal cord tissues of GAA-KO mice . Comparii et al.  could beOverall, the results from these studies reflect that, following therapeutic interventions aimed at restoring GAA enzymatic activity, certain metabolic changes revert to the state observed in healthy individuals, while other metabolic changes are not affected after treatment. These results are in accordance with previous studies that have demonstrated that although ERT can improve clinical outcomes and survival of PD patients, it does not revert many other biological processes underlying this disease. In this line, several factors have been shown to contribute to skeletal muscle resistance to ERT, including defective autophagy , which iThis review summarizes the most relevant findings reported in omics-based studies focused on the characterization of metabolic alterations associated with PD-specific phenotypes. Overall, these studies have revealed that alterations in glycogen, glucose, lipids and aminoacids, nucleotide metabolism and TCA cycle are the most frequently observed . NotablyThe results from these studies highlight the presence of a deep metabolic remodeling in this disease and confirm the potential of omics-based approaches in lysosomal diseases to reveal clinical and biological associations to generate pathophysiological hypotheses. Also, these studies have demonstrated how some of these metabolic alterations can be reverted following different therapeutic approaches. Different alterations in specific metabolites and metabolic enzymes have been reported in these studies . AlthougDue to the high clinical heterogeneity among PD patients, individual phenotyping and patient monitoring are essential for optimal management of these patients. Although great advances have been achieved in recent years towards improving routine testing of many LSDs ,141,142,Future studies including larger cohorts of patients and time-series measurements for treatment monitoring will be needed to verify the biological relevance of the metabolic alterations already reported in PD patients and the following response to treatments. Also, the application of computational-based data augmentation techniques to create synthetic sets of samples that have proven to be an adequate strategy to overcome the limited number of samples often found in other LSDs datasets  could be