Allium oleraceum with reported mixed-ploidy populations, revealed distribution ranges of cytotypes, assessed their niches and explored the pattern of niche change with increasing ploidy level. Altogether, six ploidy levels (3x−8x) were identified. The most common were pentaploids (53.6%) followed by hexaploids (22.7%) and tetraploids (21.6%). Higher cytotype diversity was found at lower latitudes than at higher latitudes (>52° N), where only tetraploids and pentaploids occurred. We detected 17.4% of mixed-ploidy populations, usually as a combination of two, rarely of three, cytotypes. The majority of mixed-ploidy populations were found in zones of sympatry of the participating cytotypes, suggesting they have arisen through migration (secondary contact zone). Using coarse-grained variables , we found evidence of both niche expansion and innovation in tetraploids related to triploids, whereas higher ploidy levels showed almost zero niche expansion, but a trend of increased niche unfilling of tetraploids. Niche unfilling in higher ploidy levels was caused by a contraction of niche envelopes toward lower continentality of the climate and resulted in a gradual decrease of niche breadth and a gradual shift in niche optima. Field-recorded data indicated wide habitat breadth of tetraploids and pentaploids, but also a pattern of increasing synanthropy in higher ploidy levels. Wide niche breadth of tetra- and pentaploids might be related to their multiple origins from different environmental conditions, higher “age”, and retained sexuality, which likely preserve their adaptive potential. In contrast, other cytotypes with narrower niches are mostly asexual, probably originating from a limited range of contrasting environments. Persistence of local ploidy mixtures could be enabled by the perenniality of A. oleraceum and its prevalence of vegetative reproduction, facilitating the establishment and decreasing exclusion of minority cytotype due to its reproductive costs. Vegetative reproduction might also significantly accelerate colonization of new areas, including recolonization of previously glaciated areas.The establishment and success of polyploids are thought to often be facilitated by ecological niche differentiation from diploids. Unfortunately, most studies compared diploids and polyploids, ignoring variation in ploidy level in polyploids. To fill this gap, we performed a large-scale study of 11,163 samples from 1,283 populations of the polyploid perennial geophyte  Polyploidy is a widespread phenomenon among flowering plants , which allows us to order more sample analyses, it has also become clear that mixed-ploidy populations are much more frequent than previously anticipated ones  of presumably allopolyploid origin  Is the current cytotype distribution a consequence of niche divergence? Is there a different pattern of niche shift with increasing ploidy level? (3) How frequent are mixed-ploidy populations, which cytotypes participate in their composition, and what processes stand behind their existence?Specifically, we addressed the following questions: (1) What is the diversity of Allium oleraceum is a geophyte with prolific asexual propagation by aerial bulbils formed within an inflorescence and partly also by daughter bulbs produced belowground by the mother bulb, while sexual seeds are formed less frequently and usually in small numbers . Ploidy level was assessed based on calibration using plants for which chromosome numbers were previously  was downloaded from this database. Two variables related to evapotranspiration processes and rainfall deficit for potential vegetative growth  were downloaded from Global Aridity Index and Potential Evapotranspiration Climate Database v2  were retained and used in further analyses , which were used for subsequent analyses (“habitat type”). Correspondence between aggregated habitat types and observed vegetation types is explained in the Allium population. (iv) Presence of arable land: the effect of agricultural practices on the potential dispersion of Allium propagules into a site was approximated by the distance of the population to the nearest arable field and transformed into two categories . (v) Rock proximity: populations were classified into two categories according to their proximity to rocky outcrops as follows: 0/1 = absence/presence of rocky outcrops within circle of 100 m radius around the population. (vi) Light conditions were assessed in the field according to the visually estimated proportion of full sunlight falling on the ground during late spring according to the following ordinal scale: 1 = strong shade, 2 = half-shade, 3 = light shade, 4 = full insolation. (vii) Heat load, i.e., a unitless index estimating the amount of heat absorbed by local site from solar radiation. The heat load was calculated from the slope, aspect and latitude of the site, using equation 2 in McCune and Keon  Haband Keon . The finNiche characteristics of the cytotypes were estimated with two groups of variables: (i) coarse-grained environmental (climatic and soil) data downloaded from open-source databases and (ii) field-recorded, local-scale environmental data. As the first step, georeferenced location data were spatially stratified to avoid discrepancies caused by highly unequal sampling in different parts of the species range, using R package “spThin”  which applies kernel smoothers to the cytotype presences in environmental space for the selection, combination, and weighting of environmental variables  directly from environmental niche space , i.e., proportion of the niche of either lower (Sn) or higher ploidy level (Se), shared with the other ploidy level. To decrease the effect of rare  environments on the estimation of the indices of niche change, analyses were performed in two settings, i.e., at the intersection of the 75th quantile and 95th quantile, respectively, of both compared environmental densities. All analyses were performed in the R platform  was used as the independent variable in separate univariate analyses. Mixed populations were duplicated according to the cytotype composition and duplicates were assigned in each group of participating cytotypes. Log-linear models were used for the analyses of categorical environmental variables, whereas the non-parametric Kruskal-Wallis test followed by multiple comparisons Dunn's test was used for the analyses of quantitative and ordinal variables  was used as the explanatory variable and visualized in an ordination diagram. Additionally, pairwise tests between cytotypes for environmental differences were performed using a reduced set of data matrices only consisting of populations of the respective pairs of cytotypes. Mixed populations were duplicated according to cytotype composition and duplicates were assigned to each group of participating cytotypes. Environmental differences between groups with different cytotype compositions were tested with a Monte Carlo permutation test using 999 permutations. The Bonferroni correction of α (at α = 0.05) for multiple tests was applied. Univariate analyses were performed using NCSS 9  were identified in our dataset using chromosome counting  = 1630.5, P < 0.001; DAPI dye: F = 97.2, P < 0.001; n = 3x, 4x, 5x, 6x) were reported. The ploidy evaluation of a total of 11,163 individuals from 1,283 populations , a high diversity in cytotype composition was found within populations . The maj2) was larger than that of uniform-ploidy populations . However, habitat heterogeneity was similar between uniform and mixed-ploidy populations  and most populations inhabited a single habitat.More individuals were analyzed from populations revealed as mixed-ploidy (median = 15) than uniform-ploidy (median = 8) by FCM . Also a sampling area of mixed-ploidy populations  were uniform two to three times more likely than those containing even ploidy level(s) . Among pot found .A. paniculatum complex  uniform tri- and tetraploid populations and one mixed 3x + 4x population in Russia, respectively. Moreover, two triploid populations were found in the contact zone with tetra-, penta- and hexaploids, one in southern Slovakia  and Eastern Europe  where they co-occurred in a mosaic-like pattern with tetra- and pentaploids. Specifically, two contact zones consist of (i) uniform tri- and pentaploid populations and mixed 3Both tetra- and pentaploids were widespread and their distributions largely overlapped. Tetra- and pentaploid cytotypes formed a diffuse mosaic-like contact zone with both single-cytotype and mixed-ploidy populations over most of their ranges. Tetraploids had the largest geographic range of all cytotypes, from northern Spain to the coastal areas of northern Norway and Iceland  were found in Romania. Considering mixed populations consisting of hexaploids, only 5x + 7x and 5x + 7x) were found only in the western foothills of the Western Alps and Jura in eastern and southeastern France and western Switzerland. Only two octoploid populations were discovered: one, ploidy-uniform, in central Spain, and the other, mixed with tetraploids, in central Italy  or more equivalent (4x−5x) than expected by chance. In the analysis of niche similarity, the majority of tests indicated insufficient power to make inferences regarding niche differentiation. For some pairs of cytotypes , the niche similarities were significantly higher than expected by chance, irrespective of the direction of the test.Values of niche overlap (Schoener's D) between cytotypes ranged from 0.056 for tri- and heptaploids to 0.806 for tetra- and pentaploids, and usually exceeded 0.250, suggesting a moderate to high niche overlap. Results of niche equivalency tests were inconclusive in most cases but suggested that most of the niches are equivalent . For somIndices of niche change comparing higher vs. lower ploidy levels showed complex patterns . Particux−4x, 4x−5x, 5x−6x; x) and higher  ploidy levels had lower niche breadths than tetraploids, with triploids and tri- and heptaploids having the narrowest niches along PC1 and PC2, respectively  were frequently found in open semi-natural habitats such as grassland and rocky outcrops, whereas higher ploidy levels  were frequently observed in human-affected habitats such as arable land and field margins as well as Robinia pseudoacacia forests. Populations of higher ploidy levels occupied preferentially extensively to highly human-affected sites in close contact with arable land  and all canonical axes  of constrained principal coordinate analysis revealed non-random differences in local-scale environmental conditions between groups of different cytotype compositions  on the right of the ordination diagram. Except for triploid populations, single-ploidy populations were ordered along the first canonical axis from lower ploidy levels on the left to the higher ones on the right. Most mixed-cytotype groups of sites with the participation of pentaploids were usually placed in intermediate positions between uniform-ploidy sites of participating cytotypes. Other mixed-cytotype groups were usually placed near the uniform-cytotype sites of one of the participating cytotypes. The second canonical axis represents a gradient of increasing habitat heterogeneity. Cytotype groups placed in the upper part of the ordination diagram were usually mixed-cytotype sites in addition to uniform triploid populations. Paired tests between cytotypes revealed significant differences (after Bonferroni correction) in local-scale environmental conditions between lower  and higher ploidy levels , and between penta- and hexaploids  correspond well to published chromosome counts, but significantly extends their distributional ranges  are rare within ploidy-polymorphic species  formed cytotype-uniform populations more frequently than even-ploidy cytotypes .The large-scale distribution pattern of different cytotypes throughout the species range was found to be remarkably complex. While tetra- and pentaploids were common across almost the entire species range and sympatric with other cytotypes at lower latitudes, other cytotypes occupied lower latitudes (below 52°) and were distributed parapatrically (tri- vs. hexaploids) or allopatrically  climatic conditions  ploidy levels. Consequently, the geographic ranges of most cytotypes overlapped to various extents, with wider ranges of tetra- and pentaploids due to their wider niche breadth. Also analysis of local-scale environmental variables demonstrated that higher ploidy levels  tend to occupy a narrower segment of habitats occupied by lower ploidy levels , but without any sign of niche novelty. In agreement with some recent studies in other polyploid complexes  Merino western Europe had similarly sized monoploid genome sizes (1Cx), which might suggest their (recent) autopolyploid origin , whose distributions cover both western and eastern parts of Europe  or eastern (3x) parts of the species range just show a subset of broad niches of tetra- and pentaploids  could also be explained by their more recent origin. Despite several competing hypotheses as to how species (cytotype) “age” may affect range size  certainly face competition from already established lower ploidy levels . Niche contraction in higher ploidy levels has previously been observed in several polyploid complexes  might be affected by their larger genome sizes  has a subcontinental climate and large areas covered by loess, with degraded forest-steppe landscapes, surrounded by mountain ranges  in A. oleraceum found in southwestern Europe. Such high ploidy levels could have originated via various pathways including auto- and/or allopolyploidy , with participation of polyploid species of the A. paniculatum complex , which are reported from the northwestern Mediterranean  is hardly explainable as a sampling bias (we sampled more intensively in these regions than in Eastern Europe). More likely, their absence or perhaps rareness coincides with the apparently low diversity of putative diploid progenitors in the western part of the Mediterranean  than in the eastern part  and within-population cytotype richness  Bernh.  is difficult to explain as a result of interploidy crosses or of primary formation of a higher cytotype in lower-ploidy level populations  have a relatively higher fitness than triploids due to an increased production of viable euploid gametes ranging from 1x−8x) ranks A. oleraceum among the most cytotype-diverse mixed-ploidy plant species  were detected in A. oleraceum, together with relatively frequent coexistence of multiple ploidy levels within populations, resulting in various cytotype combinations. The widespread distribution of tetraploids and pentaploids contrasted with narrower ranges of both lower (triploid) and higher ploidy levels (hexa- to octoploids), which also differed from each other in geographic distributions.Our study has provided detailed insight into the diversity and ploidy distribution patterns in populations of a geophytic species at a continental scale. Large ploidy diversity (six ploidy levels; 3A. oleraceum, and intensive vegetative reproduction of all cytotypes, which facilitates establishment and allows plants to avoid reproductive costs due to their minority status. Vegetative reproduction might also significantly accelerate colonization of new areas, including recolonization of previously glaciated habitats. Further study should primarily focus on molecular analyses to reveal the relationships between cytotypes and to identify their origin. Besides that, comparison of the niches of polyploids with those of identified diploid progenitors could also be tested.We further focused on possible evolutionary drivers of the observed spatial patterns of cytotypes. In contrast to the classical adaptive evolutionary scenario, assuming niche innovation or niche expansion in higher ploidy levels, we found niche expansion in tetraploids compared to triploids followed by gradual niche contraction in higher ploidy levels, with higher ploidy levels occupying mainly synanthropic habitats. The wide niche breadth of tetraploids and pentaploids might be explained by their multiple origins from different environmental conditions, higher “age,” and retained sexuality, which probably preserves their adaptive potential. By contrast, both lower and higher ploidy levels with narrower niches are mostly asexual and probably originated in a limited range of contrasting environments. Newly evolved higher ploidy levels have also faced competition from existing cytotypes, which might have affected a shift of their niche. Persistence of local ploidy mixtures could be enabled by the perenniality of The original contributions presented in the study are included in the article/MD designed the research and conducted field collection with the participation of MJ, LŠ, and KV. MJ, LK, and LŠ conducted laboratory analyses. JB extracted geoinformatics data and draw maps. MD performed statistical analyses and wrote the manuscript with help of JB and LK. All authors participated in the discussion and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The pipeline of biomarker translation from bench to bedside is challenging and limited biomarkers have been adopted to routine clinical care. Ideally, biomarker research and development should be driven by unmet clinical needs in health care. To guide researchers, clinical chemists and clinicians in their biomarker research, the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) has developed a structured questionnaire in which the clinical gaps in current clinical pathways are identified and desirable performance specifications are predefined. In kidney injury, the high prevalence of the syndrome acute kidney injury (AKI) in the hospital setting has a significant impact on morbidity, patient survival and health care costs, but the use of biomarkers indicating early kidney injury in daily patient care remains limited. Routinely, medical labs measure serum creatinine, which is a functional biomarker, insensitive for detecting early kidney damage and cannot distinguish between renal and prerenal AKI. The perceived unmet clinical needs in kidney injury were identified through the EFLM questionnaire. Nephrologists within our tertiary care hospital emphasized that biomarkers are needed for (1) early diagnosis of in-hospital AKI after a medical insult and in critically ill patients, (2) risk stratification for kidney injury prior to a scheduled (elective) intervention, (3) kidney injury monitoring in patients scheduled to receive nephrotoxic medication and after kidney transplantation and (4) differentiation between prerenal AKI and structural kidney damage. The biomarker search and selection strategy resulted in a rational selection of an eleven-protein urinary panel for kidney injury that target these clinical needs. To assess the clinical utility of the proposed biomarker panel in kidney injury, a multiplexed LC–MS test is now in development for the intended translational research. There is large potential for urinary biomarkers to improve patient care through early, noninvasive and precise diagnostics of early kidney injury. Precision diagnostics aims to improve patient management and outcome by stratifying patients for their risk of developing Acute Kidney Injury (AKI) and phenotyping kidney damage in the individual to enable tailored treatment , 2. To bIn general, the development of promising biomarkers to useful medical tests is a laborious and tedious process. Moreover, it is uncertain as the clinical, operational and the economic impact of a new test (panel) cannot directly be assessed during the translational phase from research to local clinical practice . A frame2 of GFR in a patient with normal baseline function will be undetectable by serum creatinine [In the case of kidney injury, the term AKI is used to indicate an abrupt (within hours) decrease in kidney function, which encompasses both structural damage  and loss of function without structural damage  , 8. The eatinine . While keatinine . Given tIn this study we pilot the EFLM unmet clinical needs questionnaire for kidney injury biomarkers and evaluated kidney care pathways with nephrologists to identify existing clinical gaps in contemporary test-treatment pathways at the Leiden University Medical Center Fig. . After iTo aid effective translation of biomarkers to medical tests, the EFLM Working Group on Test Evaluation developed a structured questionnaire to identify and verify unmet clinical needs, to validate the intended use, to assess the feasibility of the new test (panel) and its impact on clinical practice and health outcome , 12. ThiCurrently, clinicians mainly rely on markers for the glomerular filtration rate (GFR), markers for the combined effect of GFR and tubular function  and markers signifying glomerular injury (proteinuria and glomerular hematuria), to diagnose kidney injury. When AKI is suspected, after ruling out a postrenal cause, fluid resuscitation to optimize volume status is the primary action to assess reversibility of kidney function. Non-responsiveness to fluid assessment may indicate renal AKI. In case a glomerular or tubular disease is considered likely, specific laboratory tests on blood (e.g. serology for auto-inflammatory diseases), imaging and a kidney biopsy are important tools to aid in making a diagnosis and to guide treatment . In addiTo optimize patient care in the LUMC, we defined four major unmet clinical needs based on the nephrologists’ responses in the questionnaire. These needs are 1) early diagnosis of in-hospital AKI after a medical insult and in critically ill patients, (2) risk stratification for kidney injury prior to a scheduled (elective) intervention, (3) kidney injury monitoring in patients scheduled to receive nephrotoxic compounds and after kidney transplantation, (4) differentiation between prerenal AKI and structural kidney damage  admission is a clinical need. The sudden decline in kidney function is poorly predictable and occurs frequently in  ill patients. In addition, patients with AKI may need temporary or continuous RRT and have an increased risk to develop CKD. Through early recognition of kidney injury, the incidence of progression to AKI, as defined by the KDIGO criteria, and need for RRT may be reduced .Second, injury risk prediction prior to an intervention, including elective surgery or nephrotoxic medication in the general hospital population would provide patient benefit. Risk stratification for AKI is based on clinical risk factors, such as kidney function, medication and type of surgical or medical intervention. In practice, this stratification has been considered inadequate . BiomarkKidney damage monitoring during and after exposure to nephrotoxic medication is a third unmet clinical need. Early and non-invasive detection of kidney damage could enable precision medicine by preemptive dose adjustments and therapy switches in response to the course of kidney damage markers. Non-invasive kidney damage monitoring would be beneficial for instance in patients receiving cytostatic agents, nephrotoxic antibiotics or calcineurin inhibitors. In clinical practice, it might be unclear whether a serum creatinine-based kidney function decline is a result of a medical treatment, comorbidities or underlying kidney disease. For example, when the kidney function decreases in kidney allograft recipients with calcineurin inhibitor therapy for immunosuppression, this decline may be due to an acute rejection episode or acute calcineurin inhibitor toxicity .A fourth identified clinical need is the differentiation between prerenal AKI and structural damage with localization of affected tissue. Causes of AKI can be classified in either prerenal, renal or postrenal . PrerenaIn the second step of the EFLM checklist it is determined whether the development of a new test (panel) is justified. Therefore, alternative improvements of the clinical care pathway are discussed on their potential to reach similar objectives . One potA second alternative improvement could be the use of electronic health  monitoring to longitudinally and actively assess currently available laboratory parameters. For example, eHealth or AKI alert systems are available to stratify individuals with increased risk for developing kidney injury , 28. HowImprovement of conventional urinalysis is a third alternative solution . ExtensiIn the third step of the EFLM questionnaire the intended use of a novel biomarker test panel is validated by re-mapping the clinical pathway and discussing the envisioned impact on patient management decisions and health outcome . A new kBiomarker kinetics should reflect the intended use of a biomarker, such as early detection of kidney injury prior to or directly after a scheduled medical intervention Fig. . For effFor patient stratification prior to an intervention, a biomarker should have an altered concentration at baseline to be meaningful in clinical decision making. In kidney injury monitoring biomarkers in sequentially collected urine specimens should reflect stagnation or progression of damage. For kidney injury differentiation, a biomarker (panel) ideally indicates the injured nephron compartment and reflects pathological lesions seen on biopsy, such as ATN , 10, 34.For the development of a clinical test, the purpose and role should be specified, because the Clinical Performance Specifications (CPS) depend on its intended use . The tesFor early recognition of AKI after an intervention, a suitable biomarker should improve the detection rate of kidney injury, ideally by timely elevations ahead of serum creatinine rises. To achieve such clinical performance a cut-off value resulting in better sensitivity than specificity should be set  > 80–95%). The early diagnosis of kidney damage should induce preventive measures to reduce progression to AKI. Desirable health outcomes are the reduction in RRT incidence and ICU stay , 42. AltPrognostic markers are needed to classify the risk for developing AKI with the need for RRT, CKD and end-stage renal disease (ESRD). Patient stratification for these risks, should be applicable to the overall hospital population before any scheduled elective intervention with AKI-inducing adverse effects. To minimize unnecessary adjustments in scheduled treatments of non-critically ill patients, the specificity should outweigh the sensitivity in this test role.In patient monitoring, add-on testing would ideally guide therapy by initiating, discontinuing or adjusting a medical treatment. For instance, in a transplantation setting with patients receiving calcineurin inhibitors for immunosuppression, potential nephrotoxicity may be monitored with kidney damage markers in addition to therapeutic drug monitoring , 45. SucFor the differentiation of prerenal AKI and structural kidney damage, a useful test (panel) should discriminate ATN from other clinical conditions and comorbidities that affect urinary output and serum creatinine . Subsequhttps://www.proteinatlas.org/). Finally, untargeted proteomics studies were discussed to identify promising alternative biomarkers for kidney injury.Multiple biomarkers are needed to address the different clinical care gaps for kidney injury assessment. Four literature search strategies were applied for the selection of candidate protein-based biomarkers in urine  [Evidence of clinical performance for the prediction of AKI, AKI severity and RRT in critically ill patients was obtained from meta-analyses. Meta-analyses were available for the urinary biomarkers kidney injury molecule-1 (KIM-1) , neutropse (NAG) , cystatise (NAG) , 54, 59,se (NAG) , metallose (NAG) , 61–64  is typically procedure-related and occurs after cardiothoracic surgery with cardiopulmonary bypass or organ transplantation. In ischemic conditions, the complement system is activated and (pro) inflammatory cytokines and chemokines are released –71. For Ischemia may also induce structural kidney injury in the proximal tubules . TubularIndividuals with pre-existing kidney damage or CKD are at increased risk for AKI. Both conditions are characterized by increased permeability of the glomerular filtration barrier and ultimately leading to proteinuria and hematuria. This is caused by podocyte detachment from the glomerular slit diaphragm . The proLater stage CKD may be characterized by fibrosis, in which the extracellular matrix is reorganized . CurrentAKI biomarkers KIM-1, NGAL, TIMP-2 and IGFBP7, are widely expressed through the human body, including the proximal and/or distal tubules in the kidneys , 91. TisWithin the glomeruli, podocin, nephrin and nephrin-like protein 1 are highly abundant and expressed on the surface of podocytes , 93. Of In the proximal tubules, transporter proteins from the solute carrier superfamily (SLC) are expressed at epithelial linings. Interestingly, variants in the genes coding for SLC22A2 and SLC22A12 were related to susceptibility for kidney disease , 97, andIn the distal tubules SLC12A1, SLC13A3, calbindin and uromodulin  are typically enriched . Of thesUntargeted proteomics, is a powerful tool to discover novel biomarkers that are associated with a state of disease . ClinicaTo address this need, we focused on the proteomics studies in which the clinical endpoint AKI was defined –111. IntIn the follow-up of the literature study of biomarker candidates, proteins were selected for inclusion in a multiplex lab-developed test. The composition of the biomarker panel was based on potential to address all four identified unmet clinical needs. To maximize this potential, both clinically relevant and biology-driven biomarkers, often with yet unknown clinical relevance, were combined. The unmet clinical need for early diagnosis of in-hospital AKI after a medical intervention and in critically ill patients  could be targeted by well-studied early injury markers described in (paragraph “In the medical laboratory, proteins are commonly quantified indirectly by automated immunoassays. However, the development of specific and sensitive immunoassays is tedious and costly, often in uniplex test formats, and these tests are prone to several types of interferences , 114. MaA multiplex mass spectrometry-based lab-developed test is currently in development to assess its analytical and clinical performance of the here proposed biomarker panel. The panel will be compared to conventional markers, such as urine sediment analysis, osmolality, albumin and tubular dysfunction markers β-2-microglobulin and cystatin C in urine , 120. EfMedical test development is ideally driven by clinical needs in clinical care pathways, rather than by technological push. We here describe a first pilot experience with a structured translational approach to identify and verify gaps in clinical care pathways that encounter kidney injury burden. Four major clinical needs were identified by nephrologists in our academic centre Fig. . To fillAdditional file 1. EFLM unmet clinical needs checklist.Additional file 2: Table S2.1. Literature search strategy biomarkers for kidney injury. Table S2.2. Collected meta-analyses. Table S2.3. List of biology-driven biomarkers. Table S2.4. Kidney tissue-enriched proteins. Table S2.5. Collected untargeted proteomics studies identifying kidney injury biomarkers in urine.


Rapidly advancing genomic technologies and cross-disciplinary partnerships are accelerating the biological and clinical interpretation of genome-wide association studies, with some therapies developed based on these findings already being tested in clinical trials. The next decade promises further progress in understanding the function of genetic variants. Deciphering how associated variants modulate disease risk and severity, and how they impact cellular phenotypes informs mechanistic therapeutic hypotheses resulting in more effective drug discovery2. As the available genomic tools become more sophisticated and the cellular disease models more relevant, the next decade will deliver significant advancement in biological, clinical and therapeutic insights from GWAS variants. Accelerating these efforts will be achieved through close collaborative efforts and the blending of broad-scope systematic approaches with deep dives that rely on tailored assays in the context of specific biological processes. Here, we focus on some of the experimental approaches that will propel the translational impact of GWAS signals towards deeper pathomechanistic insights and improved therapies  have been established as a default tool for mapping the genetic variation underlying complex traits. These studies have associated thousands of variants with hundreds of phenotypes, furthering our understanding of the genetic architecture of complex diseases3, or helped to refine disease-causal cell types and cell states, such as the role of early activation of CD4+ memory T cells in immune-mediated diseases4. In parallel, single-cell technologies, particularly for transcriptomics and chromatin accessibility (ATAC-seq), have revolutionised our understanding of cell biology providing critical insights into the heterogeneity of primary tissues in health and disease. Emerging single-cell data from all human tissues, alongside the development of statistical methods, will allow us to zoom deeper into the precise disease-causal cell types and processes.The vast majority of GWAS variants fall into non-coding regions and often overlap enhancers, promoters and open-chromatin regions, suggesting a role in regulating gene expression which can be highly cell-type and tissue-specific. Therefore, it is critical to perform functional studies in a relevant system. However, for many diseases, these pathologically relevant cell types and cell states are unknown. Computational approaches that integrate single-nucleotide polymorphisms (SNPs) with chromatin marks or with gene expression help to identify the tissues and cell states through which the risk variants contribute to the disease. Such SNP enrichment methods have successfully guided the identification of disease-relevant cell types and have highlighted the role of unexpected pathogenic cell types, such as microglia in Alzheimer’s disease5. Expanding existing QTL maps of chromatin and gene expression regulation with additional healthy tissue types and disease states, and across a range of in vitro-induced conditions, will continue to provide a critical resource for mapping genetic effects on molecular phenotypes. As costs of single-cell transcriptome profiling decrease, this technology will become more broadly amenable to expression QTL (eQTL) studies6, creating further opportunities to gain invaluable insights into genetic regulation of cellular heterogeneity and cell–cell interactions.Once the disease-relevant cellular context is defined, a combination of statistical and functional approaches is required to fine-map GWAS loci to identify causal variants, genes and mechanisms. Statistical approaches include colocalisation of disease variants with quantitative trait loci (QTL) for chromatin activity and gene expression to identify signals driven by the same causal variants7 and human Organs-on-Chips8 systems that can mimic complex organs, such as the liver, gut or brain.The development of cellular models that closely reflect the physiological and disease context is critical for future translational approaches. The tremendous progress in differentiating human-induced pluripotent stem cells (hiPSC) into a range of cell types now enables the exploration of the effects of genetic variation and gene expression regulation in tissues previously inaccessible to in vitro experiments. Although in vitro cellular models do not perfectly capture the biology of primary tissues, their accuracy is rapidly increasing. This is further facilitated by the resolution of single-cell transcriptomics which has started to uncover previously unappreciated cellular heterogeneity in cell cultures; it allows for cell fate tracing and the monitoring of downstream effects of cell culture manipulations at the level of individual cells. This granularity also enables multiplexing, thus providing an enormous advantage for scaling up experiments and increasing reproducibility: cells from multiple individuals can be pooled, differentiated or stimulated together, and—following sequencing—be assigned to individual donors or culture conditions using genetic variants or barcodes. This opens up opportunities for population-scale in vitro experiments. Finally, it is now possible to use sophisticated cell culture models that recapitulate a diversity of different cell types and cell–cell interactions through organoidTechnical challenges and high costs of such complex models currently restrict their utility to modest sample numbers. However, multiplexing capabilities and cell culture miniaturisation will help to overcome these limitations. Combined with high-throughput cell phenotyping assays and genomic technologies, sophisticated in vitro models will represent a critical tool to link genetic variants with intermediate cellular phenotypes.+ T cells for their regulatory activity using MPRAs9. This revealed an immune disease-associated variant that disrupts NF-κB binding and, as a consequence, reduces the expression of TNFAIP3 leading to increased T-cell-driven inflammation.Rapidly advancing genome-editing technologies are becoming a powerful complementary approach for dissecting the effects of genetic variants and providing important capabilities for both forward screens and validation. CRISPR screens  and interference (CRISPRi)) coupled with high-throughput sequencing and massively parallel reporter assays (MPRAs) will catalogue the effects of regulatory elements and non-coding variants across different cell types, facilitating the identification and validation of causal variants and mechanisms. Bourges et al., for example, tested immune disease variants in 14 gene deserts that overlap super-enhancers in CD4IL2RA expression10. This study highlighted six new regulatory regions associated with IL2RA upregulation and one of these regions was found to contain a variant associated with type 1 diabetes and Crohn’s disease. CRISPR screens combined with single-cell RNA sequencing (scRNA-seq) are a powerful tool for mapping thousands of enhancers to target genes while maintaining cell-type-specific context of gene expression regulation. A recent study used CRISPRi coupled with scRNA-seq to target over 5900 human candidate enhancers and identified 664 enhancer-gene pairs with evidence of regulatory interactions11.Complementary approaches can include tiling of enhancers at GWAS risk loci. Simeonov et al. used CRISPRa complexes to target a 178 kb region associated with immune diseases that regulate Even though CRISPR technologies are improving our understanding of the role that genetic variants play in disease they currently suffer from important limitations. Some of the disease-relevant cell types may not be amenable to genome editing, the efficiency of edits may not be sufficiently high and the genome-editing perturbations may induce non-biological effects. However, with developments that make these technologies more precise, applicable to a broader range of cell types and scalable, the simultaneous testing of thousands of GWAS signals in a relevant cellular context is within reach.To facilitate the interpretation of unknown variant effects, prospective variant catalogues can be created: possible variants are experimentally engineered and their effects on molecular and cellular phenotypes, such as cell viability, the activity of signalling pathways, cell response to external stimuli, protein abundance or drug toxicity are measured.PPARG locus, which is associated with risk for type 2 diabetes. While ~0.2% of the general population carry a PPARG missense variant, only 20% of these variants are associated with diabetes. Thus, to inform the future diagnostic interpretation of novel sequence variants, all 9595 possible PPARG amino acid substitutions were tested for their effects on the expression levels of the canonical PPARγ target, CD36, in a pooled phenotypic screen12.An example of how such catalogues could inform functional interpretation is illustrated by the www.varianteffect.org) is bringing together researchers with diverse expertise to tackle these challenges in a concerted effort.Currently, such approaches are limited to a small number of genes and cell lines and are coupled with only a limited number of cellular phenotypes, e.g., use of haploid cell lines and cell death as a readout. However, technology advances will enable the precise introduction of variants at scale, in native cellular contexts and linked to complex cellular readouts, enabling a comprehensive survey of the effects of variants across large numbers of genes. The Alliance of Variant Effects 13. The variant causes a proline-to-alanine substitution in the kinase domain of the TYK2 protein resulting in a weaker response of CD4+ T helper 1 and 17 type cells to proinflammatory signals. Despite the reduced T-cell activation response, individuals carrying rs34536443 protective alleles do not have an increased risk for infections or an otherwise impaired immune system, whereas individuals with complete TYK2 loss of function develop immunodeficiency. Such an allelic series provides a natural genetic “dose-response curve”, suggesting that reducing the activity, but not complete inactivation of TYK2, may be an opportunity for intervention in immune diseases. Indeed, a selective TYK2 inhibitor is currently being tested in numerous clinical trials for the treatment of various immune diseases .Coding variants only account for a small proportion of GWAS signals, but successful functional studies increasingly provide valuable insights into disease biology highlighting their translational potential. One example is the rare coding variant rs34536443 in TYK2 coding variant are still rare, the increased power of upcoming large-scale case–control and biobank sequencing studies will enable the identification of more rare coding variants. Therefore, fast interpretation of coding variants will be critical in progressing swiftly from variant identification to function and potential therapeutic intervention.Although examples such as the The progress in advancing disease cellular models combined with the increasingly precise genome-editing toolkit is rapidly enhancing the translational value of experimental in vitro studies. Our ability to more accurately interpret genetic signals and understand their mechanism of action will help with the development of tailored therapeutic approaches, driven by academic research and pharma industry alike.www.opentargets.org), Accelerating Medicines Partnership (www.nih.gov/science/amp/index.htm) or the International Common Disease Alliance (www.icda.bio) bring together a diversity of expertise including genetics and genomics, clinical, molecular, and pharmacology, to jointly accelerate the progress of variant-to-function efforts.The next 10 years will undoubtedly deliver a further significant increase in the number of phenotype-variant associations from GWAS. Fully harnessing the translational value of these association signals towards improved and safer therapies will require interdisciplinary efforts. Private–public partnerships such as Open Targets (


The rating of perceived exertion (RPE) is a subjective load marker and may assist in individualizing training prescription, particularly by adjusting running intensity. Unfortunately, RPE has shortcomings  and cannot be monitored continuously and automatically throughout a training sessions. In this pilot study, we aimed to predict two classes of RPE (≤15 “Somewhat hard to hard” on Borg’s 6–20 scale vs. RPE  Several objective  and subjective load parameters  exist to estimate a runner’s physiological response to an exercise stimulus. Recent articles provide an overview of objective and subjective load markers ,2. SubjeThe rating of perceived exertion (RPE)  is a widIndependently of gender, age, and fitness status, RPE is closely related to metabolic  verify a runner’s rating during exercise and (ii) adjust exercise intensity. In other contexts, machine learning algorithms based on wearable-derived time series data already allow one to predict other load markers . So farHere, we aimed to (i) estimate classes of RPE : six runners  were classified as trained runners, and the remaining six as recreational runners . All participants gave their consent to participate in this study after being informed about all testing procedures as well as the potential risks and benefits involved. The local university’s ethical committee pre-approved the study design, which was in accordance with the Declaration of Helsinki.Twelve male runners participated in our study. These runners were separated into two groups based on their peak oxygen consumption , whereas heart rate was measured by a GPS-enabled heart rate watch .Each participant reported to the testing facilities twice. On the first visit, all participants performed a ramp like protocol to determine VOOn the second visit (at least 72 h after the first visit) all participants performed a long continuous submaximal run wearing the GPS-enabled heart rate watch  after a standardized 10 min warm-up. All participants ran at a self-selected but constant velocity without applying a pacing strategy until full exhaustion on a predefined, flat, outdoor course. The running velocity was monitored and controlled with GPS.All runners reported their RPE, ranging from 6–20, to the an investigator, who was following the runner on a bicycle. The trained runners ran a flat 5 km-course and reported their RPE scores after each full round. The untrained runners ran a flat 2 km-course and reported their RPE scores each kilometer. “Somewhat hard to hard” (non-exerted): RPE (exerted): RPE This pilot study incorporated several models for predicting two classes of RPE from multivariate time series during moderate-intensity continuous running. Models included are two baselines (majority vote and Borg’s classification), three classifiers using distance metrics to calculate similarities . We used symbolic Fourier approximation, Symbolic Fourier approximation: Symbolic Fourier approximation (SFA) [on (SFA)  transforon (SFA) , Figure on (SFA)  algorithNearest neighbors classifier: Nearest neighbors classifiers are voting-based classifiers. A special case of this supertype is the k-nearest neighbors classifier (kNN) [k nearest time series according to a distance function or metric. Classification labels are assigned via a majority vote within this neighborhood. Prominent metrics for directly measuring distances between multivariate time series include dynamic time warping (DTW) [k, the locality aspect of this classifier is superseded and lastly diminished to the majority vote baseline, if er (kNN) . For anyng (DTW) , the (sqSupport vector machine: Support vector machines (SVM) [es (SVM)  belong tes (SVM) . Since sAnother class of classifiers employed in the present investigation was the neural network class. Due the multivariate nature of the collected time series, we considered 1D-convolutional neural networks (CNN) and gated recurrent units (GRU) , becauseGated recurrent units: Recurrent neural networks (RNNs) extend feed forward networks by introducing feedback connections within layers. This makes RNNs especially suited to handle sequential data such as time series, as preceding time stamps are included into the calculations with decaying weight. The most influential parameters of the GRU are the number of units (neurons) in use and the output function of each unit. Furthermore, we can regulate the linear and recurrent dropout [ dropout  within eConvolutional neural networks: In a convolutional neural network, a filter (matrix) is convolved with the input matrix. Each filter functions as a feature extractor. The higher the number of filters, the more feature maps generated, which regulates the number of output weights. After each convolution, these filters are moved across the input by a fixed number of elements (strides) in order to detect their specific features at different locations. After each layer of convolution, we use an intermediate batch normalization layer [global maximum pooling instead of global average pooling.on layer . Batch non layer , we usedAdam optimizer with the default parameters  two succinct labels overlapped or ii) the assumption of a constant fatigue score within that interval was void. A window length of L = 60 s showed the best results for our setting; therefore, we only report classification accuracies using this observation length. Using this combination  yields 20,000 training samples in the 5-fold cross-validation and 22,000 within the evaluation setting.With that, we labeled windows of length For significance testing, we used McNemar’s test  with a sn times) k-fold cross validation was used. Overall there were 6! = 720 , achieved accuracies of One limitation of our study is the small amount of collected RPE values. This likely impairs selecting the best machine learning classifier for the given task, as some of these techniques need more input data. Yet, achieving an accuracy of up to 86% using CNN with small amount of input data shows the feasibility to estimate classes of RPE based on data obtained from a commercially-available smartwatch and machine learning classifiers. To improve the accuracy of machine learning algorithms, future work should evaluate the use of individual HR-related intensity thresholds .Our pilot study proves that the limitations of collecting RPE values in the daily work of coaches with athletes  may be overcome by data obtained from a commercially-available smartwatch and machine learning classifiers. Future studies should aim to improve the number of classes of RPE which can be estimated. This may enable coaches to prescribe more personalized training by individually adjusting exercise intensity to ensure optimal performance enhancement and to avoid overuse.To the best of our knowledge, this is the first study using easy-to-use, low-cost, and commercially-available sensors to estimate classes of RPE in trained and untrained runners.While some use inertial measurement units (IMUs) comprise an accelerometer and a gyroscope, others use customized shoes to assess walking patterns.Buckley et al.  placed IZhang et al.  employedBoth of the aforementioned induced fatigue using different methods (pacer test , squattiEskofier et al. ,32, in cBased on our extensive data analysis, we showed that smartwatch-derived data and machine learning algorithms proved to estimate two classes (i.e., “Somewhat hard to hard” and of RPE with high accuracy. Of all applied algorithms and within our study design, 1D-CNN revealed the highest accuracy when predicting the two classes of RPE in trained and untrained runners.


Our unique case demonstrates the use of an over-the-scope Padlock clip for closure of a sharp foreign body induced gastric perforation, avoiding the need for surgical intervention. A 47-year old female presented with a two-day history of abdominal pain with nausea. Abdominal CT scan revealed a linear density in the distal body of the stomach extending outside the lumen. Endoscopic evaluation revealed a toothpick perforating through the wall of the gastric antrum. Endoscopic removal was performed, and closure of the full-thickness defect was achieved with an over-the-scope Padlock clip. The patient subsequently made an uneventful recovery, with no reported complications at two-year follow-up. Early endoscopic removal and closure of gastric luminal perforations by over-the-scope Padlock clips are viable alternative treatments for defects previously considered only amenable to surgical repair. Endoscopic treatment of gastrointestinal perforations has shown to decrease the morbidity and mortality associated with more invasive surgical procedures. Toothpick ingestion is a relatively rare occurrence and often an accidental event related to food consumption. The most common locations of toothpicks prior to retrieval were small intestine 41%), large intestine (37%), stomach (20%), and esophagus (2%) [%, large Sharp-pointed objects may not be apparent on radiographic evaluation and it is recommended that these objects warrant urgent or emergent endoscopic removal depending on size and location ,3. ManagOTSCs are a category of novel clipping devices made of an elastic alloy, Nitinol, which has demonstrated clinical and technical success in the treatment of gastrointestinal hemorrhage, perforation, fistulae, anastomotic leaks, defect closure following endoscopic resection, and stent fixation . The twoA 47-year-old female with no significant past medical history presented with two days of progressively worsening sharp, intermittent, post-prandial abdominal pain associated with nausea. On examination, she was afebrile, hypertensive, and with diffuse abdominal tenderness, particularly in the epigastrium. She had a normal CBC, comprehensive metabolic panel and lipase. Initially, an abdominal ultrasound revealed a linear echogenicity traversing through the pylorus, possibly suggestive of a foreign body. Patient subsequently underwent abdominal x-ray which was unremarkable for foreign bodies. An abdominal CT scan revealed a 4 cm linear density in the distal body of the stomach which appeared to extend outside the lumen, without pneumoperitoneum Figure . It was Patient underwent an upper endoscopy revealing a toothpick in the gastric antrum perforating through the wall of the stomach Figure . EndoscoPost-procedurally, the patient recalled eating tacos three days prior with fragments of toothpicks in her food. Accidental ingestion caused gastric perforation with the sharp tip of the toothpick. The patient had an uneventful recovery and was discharged home.A follow-up endoscopy two years later revealed complete healing of the full-thickness defect with an intact Padlock clip Figure . An abdoOur unique case highlights the importance of early endoscopic recognition and closure of a sharp foreign body induced gastric luminal perforation using an over-the-scope Padlock clip avoiding the need for major surgery. It was felt that an OTSC would be the preferred method of closure, as opposed to the use of an endoclip or endoscopic suturing, given the ability to achieve full-thickness closure, not requiring a change of endoscope, ease of use, and rapid deployment. Although emergent surgery had been the standard of care for foreign body removal, surgical interventions may be associated with higher morbidity and mortality than endoscopic therapies. In a retrospective review of 136 case reports, 49% of the ingested toothpicks were removed via surgical laparotomy and were associated with higher mortality compared to endoscopic removal. The overall mortality in these cases was 9.6%; however, those successfully treated endoscopically had a mortality of 0% (p = 0.011) [Endoscopic closure of luminal perforations using an OTSC device can be achieved with similar efficacy to a surgical intervention. In an ex vivo porcine stomach experiment, the acute strength of OTSC closures of gastric incisions were non-inferior compared with the gold standard of hand surgical suturing ,10. The Early endoscopic recognition and removal of sharp foreign objects can help prevent the need for surgical interventions. Successful endoscopic closure of luminal perforations by OTSC devices can be achieved and should be considered in defects previously considered only amenable to surgical repair. Endoscopic treatment of gastrointestinal perforations has shown to decrease the morbidity and mortality associated with more invasive surgical procedures.


Targeted amplicon deep sequencing of RhCMV glycoprotein B and L genes revealed that one of the three strains present in the viral inoculum (UCD52) dominated maternal and fetal viral populations. We identified minor haplotypes of this strain and characterized their dynamics. Many of the identified haplotypes were consistently detected at multiple timepoints within sampled maternal tissues, as well as across tissue compartments, indicating haplotype persistence over time and transmission between maternal compartments. However, haplotype numbers and diversity levels were not appreciably different between control, standard-potency, and high-potency pretreatment groups. We found that while the presence of maternal antibodies reduced viral load and congenital infection, it had no apparent impact on intrahost viral genetic diversity at the investigated loci. Interestingly, some minor haplotypes present in fetal and maternal-fetal interface tissues were also identified as minor haplotypes in corresponding maternal tissues, providing evidence for a loose RhCMV mother-to-fetus transmission bottleneck even in the presence of preexisting antibodies.Human cytomegalovirus (HCMV) infection is the leading non-genetic cause of congenital birth defects worldwide. While several studies have addressed the genetic composition of viral populations in newborns diagnosed with HCMV, little is known regarding mother-to-child viral transmission dynamics and how therapeutic interventions may impact within-host viral populations. Here, we investigate how preexisting CMV-specific antibodies shape the maternal viral population and intrauterine virus transmission. Specifically, we characterize the genetic composition of CMV populations in a monkey model of congenital CMV infection to examine the effects of passively-infused hyperimmune globulin (HIG) on viral population genetics in both maternal and fetal compartments. In this study, 11 seronegative, pregnant monkeys were challenged with rhesus CMV (RhCMV), including a group pretreated with a standard potency HIG preparation  is the most common infectious cause of birth defects worldwide. Knowledge gaps remain regarding how maternal immunity impacts the genetic composition of CMV populations and the incidence of congenital virus transmission. Addressing these gaps is important to inform vaccine development efforts. Using viral DNA isolated from a monkey model of congenital CMV infection, we investigated the impact of passively-administered maternal antibodies on the genetic composition of the maternal virus population and that transmitted to the fetus. Our analysis focused on regions of two CMV genes that encode glycoproteins known to facilitate viral cellular entry and to be targeted by CMV-specific antibodies. We investigated viral haplotypes across sampled maternal tissues and amniotic fluid, finding no impact of preexisting CMV-specific antibodies on CMV genetic diversity despite the observation that antibodies reduce viral load and can confer protection against congenital transmission. We further found that some minor haplotypes identified in fetal and maternal-fetal interface tissues were also present in paired maternal tissues. This finding indicates that a large number of viral particles likely passed from dam to fetus during congenital transmission. Human cytomegalovirus (HCMV) is a member of the β-herpesvirus family and a ubiquitous pathogen that establishes lifelong infection in its host. Seroprevalence rates for HCMV range from 45% in developed nations to 100% in developing nations –4. IndeeCongenital CMV infection during pregnancy can result from either primary infection or viral reactivation and/or superinfection (secondary infection). While congenital infection could be seeded from the maternal genital tract ,6, most De novo point mutations are also thought to play a contributing role in the generation of observed intrahost genetic diversity 27]. Cell(PDF)Click here for additional data file.S2 TableAll listed samples had two successfully sequenced replicates and underwent nested PCR.(PDF)Click here for additional data file.S1 AppendixEach row corresponds to a unique haplotype. Each haplotype has listed the strain it belongs to , an identifying haplotype number , the number of mutations between it and the canonical reference strain, and the types of these mutations (synonymous or nonsynonymous).(CSV)Click here for additional data file.S2 AppendixEach row corresponds to a unique haplotype. Each haplotype has listed the strain it belongs to, an identifying haplotype number, the number of mutations between it and the canonical reference strain, and the types of these mutations.(CSV)Click here for additional data file.S3 AppendixSamples are defined by the monkey ID, tissue of origin, and collection week post-RhCMV infection. The relative frequency of each haplotype in a sample is provided.(CSV)Click here for additional data file.S4 AppendixSamples are defined by the monkey ID, tissue of origin, and collection week post-RhCMV infection. The relative frequency of each haplotype in a sample is provided.(CSV)Click here for additional data file.S5 AppendixgB or gL). Each column corresponds to a distinct frequency threshold at which haplotypes are called: 0.22% (left), 0.436% (middle), and 0.88% (right). Top panel: The number of minor UCD52 haplotypes called per sequenced sample, by tissue and week post-infection. Bottom panel: The dynamics of minor UCD52 haplotypes identified in more than one sample. Each dot represents a recurrent haplotype. Lines connecting dots indicate recurrent haplotypes in the same tissue. Haplotypes that recur in different tissues are not connected by lines. These figures together indicate that a subset of minor haplotypes persist over time and are shared between compartments. This result is robust across a large range of haplotype-calling thresholds (0.22–0.88%).Each page shows the number and recurrence dynamics of minor UCD52 haplotypes in a single dam (or dam-fetus pair) at a single locus ((PDF)Click here for additional data file.


Patient monitoring is indispensable in any operating room to follow the patient’s current health state based on measured physiological parameters. Reducing workload helps to free cognitive resources and thus influences human performance, which ultimately improves the quality of care. Among the many methods available to assess perceived workload, the National Aeronautics and Space Administration Task Load Index (NASA-TLX) provides the most widely accepted tool. However, only few studies have investigated the validity of the NASA-TLX in the health care sector.This study aimed to validate a modified version of the raw NASA-TLX in patient monitoring tasks by investigating its correspondence with expected lower and higher workload situations and its robustness against nonworkload-related covariates. This defines criterion validity.In this pooled analysis, we evaluated raw NASA-TLX scores collected after performing patient monitoring tasks in four different investigator-initiated, computer-based, prospective, multicenter studies. All of them were conducted in three hospitals with a high standard of care in central Europe. In these already published studies, we compared conventional patient monitoring with two newly developed situation awareness–oriented monitoring technologies called Visual Patient and Visual Clot. The participants were resident and staff anesthesia and intensive care physicians, and nurse anesthetists with completed specialization qualification. We analyzed the raw NASA-TLX scores by fitting mixed linear regression models and univariate models with different covariates.P<.001). Staff physicians rated significantly lower workload scores than residents (P=.001), whereas nurse anesthetists did not show any difference in the same comparison (P=.83). Standardized distraction resulted in higher rated total raw NASA-TLX scores (P<.001) and subscores. There was no gender difference regarding perceived workload (P=.26). The new visualization technologies Visual Patient and Visual Clot resulted in significantly lower total raw NASA-TLX scores and all subscores, including high self-rated performance, when compared with conventional monitoring .We assessed a total of 1160 raw NASA-TLX questionnaires after performing specific patient monitoring tasks. Good test performance and higher self-rated diagnostic confidence correlated significantly with lower raw NASA-TLX scores and the subscores (all This study validated a modified raw NASA-TLX questionnaire for patient monitoring tasks. The scores obtained correctly represented the assumed influences of the examined covariates on the perceived workload. We reported high criterion validity. The NASA-TLX questionnaire appears to be a reliable tool for measuring subjective workload. Further research should focus on its applicability in a clinical setting. The World Health Organization considers attentive anesthesia providers to be essential to prevent perioperative disability and death . To mainHence, accurate assessment of workload is of great importance to manage stressors. Various methods for quantifying perceived workload have been described, which can be divided into the following two large groups: subjective assessment through questionnaires and objective physiological assessment of variables such as heart rate, galvanic skin resistance, breathing rate, pupil diameter, and blinking frequency ,9.The National Aeronautics and Space Administration Task Load Index (NASA-TLX) provides the most widely accepted and validated tool to measure overall workload after completing a task -12. It wPatient monitoring can be generalized as continuous observation of a condition or certain parameters, regardless of the method used ,20. In pOnly few studies have investigated the validity of the raw NASA-TLX score in the health care sector -27. TherThe leading ethics committee  reviewed the study protocols for all four included studies and issued a declaration of no objection for each one of them . Reference number 2017-00795 covers two of the studies, as they were performed simultaneously and included the same participants. Additionally, before participation in this study, we obtained written informed consent from all participants to collect data for scientific purposes and publications.To validate the NASA-TLX, we analyzed data from four different studies all performed by anesthesia and intensive care providers at the University Hospital Zurich and Cantonal Hospital Winterthur in Switzerland, and University Hospital Frankfurt in Germany ,23,28,29In all included studies, we used the original formulation of the NASA-TLX questionnaire provided by the official NASA website. However, we modified the raw NASA-TLX surveys from six to only five dimensions by removing the physical demand question as our tasks did not require any physical effort. Visual Patient technology is a situation awareness–oriented visualization tool that displays up to 11 of the most frequently used patient vital signs in the form of a patient avatar in addition to the standard monitoring screen ,22. VisuA relevant limitation for both visualization technologies is the absence of quantification values. Both avatars only display the following three states: too low, within range, and too high. This limitation defines the intended use of the technologies, which is the situation awareness–oriented supplementation of conventional monitoring methods. They were not designed to replace numerical values. Both visualization technologies were invented, prototyped, and patented by our research group at the Institute for Anesthesiology of the University Hospital Zurich. We are developing Visual Patient into a product under a Joint Development and Licensing Agreement with Royal Philips NV and Philips Medizin-Systeme Böblingen GmBH. Regarding Visual Clot, we have signed a letter of intent with Instrumentation Laboratory. Both technologies are in the prototype stage of development and neither Visual Patient nor Visual Clot is currently CE certified as a medical device.We assessed the perceived workload using the raw NASA-TLX questionnaire after performing patient monitoring tasks using either one of the newly developed visualization tools  or respective conventional monitoring alone. The monitoring scenarios appeared in randomized order for predefined relatively short periods. Furthermore, the participants had to rate their diagnostic confidence level on a four-point Likert scale ranging from “very unconfident” to “very confident” to further assess uncertainty as a psychological stress factor. In the four mentioned studies, we generated workload through specific tasks as follows:In the primary Visual Patient study , we dispThe Visual Patient central monitoring study  includedThe same participants  also toFinally, in the Visual Clot study , we showWe investigated the robustness of the NASA-TLX questionnaire based on criterion validity, which can be further divided into predictive and concurrent validity. Predictive validity indicates the extent to which an assumption under investigation can be predicted . ConcurrIn all included studies, we had consistently recorded 11 different covariates. In this pooled analysis, we determined the expected impact on workload from these measured covariates, using both literature research and logical deductions. It was described that more professional experience should result in lower cognitive workload ,32. SincWe validated the raw NASA-TLX score and the different subscores by fitting mixed linear regression models with a random intercept per person (to cover repeated measurements) and a random intercept per study. We fitted univariate models with binary test performance (task correct or incorrect), binary confidence (unconfident or confident), distraction, center, gender, profession, binary daytime (above or below the median), binary playback sequence , and central monitor performance as covariates.We analyzed interactions of some covariates with the technology variable to explore more in depth why the NASA-TLX and its subscores improved in the case of the new visual technologies. To explore which subscore benefited the most from the new visualization technologies, we calculated univariate models for each subscore that included only the technology variable, and we compared the size of the estimated coefficients. To characterize the individuals who benefited the most from the introduction of the new technologies, we fitted a joint model for the total NASA-TLX with the technology variable and several other covariates. In one additional model per variable, we included an interaction term between technology and the respective covariate to see if the impact of certain variables was particularly strong in the case of the new technologies.P value <.05 to indicate statistical significance.All analyses were performed using R version 3.6.2 . We considered a We provide the complete data used for this study in In all four evaluated studies ,23,28,29In P<.001). Compared with the University Hospital Zurich, participants from the University Hospital Frankfurt had significantly lower total workload scores . Further, the second half of the playback sequence had a significant positive effect on lowering the perceived workload . Regarding the job position, resident physicians served as the comparison. Staff physicians  rated significantly lower workload than residents, whereas nurses did not show any significant difference compared with residents (P=.83). Distraction and the Cantonal Hospital Winterthur compared with the University Hospital Zurich correlated significantly with higher rated NASA-TLX scores . The other listed covariates did not show any relevant difference.The total workload score analysis showed that participants’ test performance and higher confidence correlated significantly with lower NASA-TLX scores (both P<.001). Staff physicians also differed significantly from resident physicians with lower workload scores in every subcategory, except the frustration level . Distraction was the only covariate to show a relevant effect on increasing the workload in every subscore of the NASA-TLX. Comparing the participants at the different centers with those at the University Hospital Zurich, the participants at the University Hospital Frankfurt had less mental (P=.02) and temporal (P=.002) demands, and less perceived effort (P=.003). The participants at the Cantonal Hospital Winterthur had more mental (P=.007) and temporal demands (P=.02), and more required effort (P=.03). We observed no gender difference in all subscores. Showing several patients simultaneously on a central monitor correlated significantly with increased mental demand (P=.05) and increased effort (P=.01) to fulfill the task. We observed no effect for temporal demand , frustration level , and self-rated performance  using a central monitor. Playback sequence showed its only significant effect on the temporal demand subscore with lower perceived workload in the second half of the task .P<.001). Performing the same tasks, we compared the conventional monitoring devices with the respective visualization technologies. The total NASA-TLX score and all listed subscores correlated significantly with lower workload scores when using Visual Patient and Visual Clot . Furthermore, staff physicians had significantly lower total workload scores compared with residents. During clinical residency training, inexperienced physicians go through a period of professional and personal growth. They acquire knowledge and skills [The analysis of 1160 raw NASA-TLX questionnaires showed that self-rated high confidence correlated significantly with lower overall raw NASA-TLX scores and its subscores . This greatly reduces the influence of confounding variables if the main factor responsible for the difference remains the interface modality shown, and it further increases the quality of the study.For patient monitoring, this study validated a modified version of the raw NASA-TLX questionnaire, in which the physical dimension had been removed from the scale owing to the nature of the given tasks. The obtained scores correctly depicted the assumed influences of the covariables that affect perceived workload. This provided a high extent of criterion validity. The modified raw NASA-TLX questionnaire appears to be a reliable tool for measuring the subjective workload of anesthesia providers who monitor patients inside the operating room. Further research is needed to investigate the applicability of the NASA-TLX questionnaire in the clinical setting and its transferability to personnel working in intensive care units. Moreover, a true validation study for the subjective workload assessment should correlate the NASA-TLX scores with objectively measurable stress characteristics such as heart rate and pupil diameter.


The resulting genomic damage gives rise to a senescent cell burden that is comparable to that of a naturally aged mouse. In fact, the expression of senescence and senescence-associated secretory phenotype (SASP) markers in 4-5-month-old -/ΔErcc1 mice, along with other measurements of senescence, were equivalent and never exceeded the extent of that found in naturally aged mice. Furthermore, many features of both natural murine aging and human aging are present in -/ΔErcc1 mice. An emerging use of these mice is the ability to study age-related signaling pathways, including identifying different types of senescent cells and their key senescent cell anti-apoptotic pathways (SCAPs). Most importantly, this model represents a rapid, cost-effective mouse model for the evaluation in vivo of senolytic drugs and other gerotherapeutics.Progeroid murine models represent an emerging tool to investigate mechanisms of aging in an expedient and efficient manner. One prominent mechanism of aging is the accumulation of DNA damage and subsequent increase in cellular senescence, leading to age related pathologies.  These mice mimic the human progeroid syndrome XFE and age roughly six times faster than WT mice due to a deficiency in the ERCC1-XPF DNA repair endonuclease complex [-/ΔErcc1 [-/ΔErcc1 mice is that enhanced endogenous DNA damage promotes cellular senescence, which in turn drives aging [Aging is the progressive decline in physiological functioning with increasing age, and multiple mechanisms attempt to explain such a phenomenon. The field of geroscience, defined as the intersection of basic aging biology, chronic disease, and health, is always seeking to develop new models of aging or to refine existing ones. The  complex . DNA damErcc1-/Δ . These mes aging –6.in vitro and in vivo. Consistent with prior findings, naturally aged mice display elevated senescence and SASP marker expression in multiple tissues including in peripheral T lymphocytes that are used for measuring biological age in humans. Remarkably, increased expression of senescence and SASP genes was found in those same tissues of 4-5-month-old -/ΔErcc1 progeroid mice, often to a comparable extent to, but never exceeding the level found in old WT mice. These findings were supported by the increase in percentage of cells positive for senescence-associated ß-galactosidase staining in multiple tissues of -/ΔErcc1 and old WT mice. Elevated levels of multiple SASP factors were present in the blood of both naturally aged mice and -/ΔErcc1 mice relative to their young littermate controls. The progressive increase in senescent cell burden was measured by cross-sectional and longitudinal predictor using both gene expression analysis and in vivo bioluminescence imaging of a p16INK4a-luciferase transgenic reporter [-/ΔErcc1 mouse model of accelerated aging experiences a similar breadth and depth of senescent cell burden as do naturally aged mice, but which accumulates at an accelerated rate.The senescent cell burden is known to impair tissue homeostasis, enhance age-associated pathologies, and shorten both health span and lifespan –12. Sene-/ΔErcc1 mice, which have a comparable profile of cellular senescence in comparison to naturally aged mice, can also be used to further study SCAPs and identify senolytics which are effective against specific tissue or cell populations. In fact, many senolytics have already been tested in -/ΔErcc1 mice [-/ΔErcc1 mice with fisetin reduced expression of senescence markers in multiple tissues leading to reduced age-related pathology, and an overall health span improvement [-/ΔErcc1 mice are a proper model of accelerated aging, allowing for efficient, cost effective experimentation and evaluation of senotherapeutics for aging studies. Utilization of these mice should accelerate the identification of new therapeutic targets and the development of age-related interventions.This model of accelerated senescence and aging can be used to further study the close relationship between senescent cells and aging with the intention of developing techniques for predicting health span and treating age-related disease. Due to the fact that there are tissue-specific senescence profiles in multiple tissues, which utilize different senescent cell anti-apoptotic pathways (SCAPs), no one senolytic will be pancellular. -/Δ mice –16. The rovement . Altoget


Ferroptosis is a new type of oxidative regulated cell death (RCD) driven by iron-dependent lipid peroxidation. As major sites of iron utilization and master regulators of oxidative metabolism, mitochondria are the main source of reactive oxygen species (ROS) and, thus, play a role in this type of RCD. Ferroptosis is, indeed, associated with severe damage in mitochondrial morphology, bioenergetics, and metabolism. Furthermore, dysregulation of mitochondrial metabolism is considered a biochemical feature of neurodegenerative diseases linked to ferroptosis. Whether mitochondrial dysfunction can, per se, initiate ferroptosis and whether mitochondrial function in ferroptosis is context-dependent are still under debate. Cancer cells accumulate high levels of iron and ROS to promote their metabolic activity and growth. Of note, cancer cell metabolic rewiring is often associated with acquired sensitivity to ferroptosis. This strongly suggests that ferroptosis may act as an adaptive response to metabolic imbalance and, thus, may constitute a new promising way to eradicate malignant cells. Here, we review the current literature on the role of mitochondria in ferroptosis, and we discuss opportunities to potentially use mitochondria-mediated ferroptosis as a new strategy for cancer therapy. Ferroptosis is a non-apoptotic, iron-dependent form of regulated cell death (RCD) occurring when the intracellular levels of lipid reactive oxygen species (L-ROS) exceed the antioxidant activity of glutathione-dependent peroxidase GPX4) thus leading to the collapse of cellular redox homeostasis . Ferropt thus leaUnlike other RCDs, ferroptosis appears more like a cellular “sabotage” than a pro-active “suicide” mechanism . While tThe essential role of cellular metabolism in ferroptosis is currently widely investigated. Mounting experimental evidence has demonstrated that numerous metabolic pathways, including cellular respiration  cycle and electron transport chain (ETC)), lipid metabolism, and amino acid metabolism contribute to ferroptosis through the generation of L-ROS ,13. Of nAs master regulators of oxidative phosphorylation (OXPHOS), mitochondria are the main intracellular producers of ROS . Mitochom), and ATP depletion . E. E168]. Following import, mitochondrial iron primarily acts as a cofactor in Fe–S cluster-containing proteins  and heme-containing proteins  all of which are components of the IMM complexes of the ETC . The bioMitochondria contain a labile iron pool which is extremely redox active . In physCISD1 knockdown mice exhibit many features of Parkinson’s disease . Th. Th27]. LONP1. Conversely, LONP1 inhibition leads to the activation of NRF2/KEAP1 signalling pathway and to the up-regulation of GPX4, thus inhibiting ferroptosis [Lon peptidase 1 (LONP1) mediates the selective degradation of misfolded or oxidatively damaged polypeptides in the mitochondrial matrix and maintain the integrity of the mitochondrial genome  186]. I. I186]. roptosis .An overview of the crucial mitochondrial actors involved in ferroptosis induction are reported in Ferroptosis occurs when lipid hydroperoxide detoxification mediated by GPX4 activity is reduced to such an extent that it becomes insufficient to restrain iron-dependent membrane PUFA oxidation and toxic ROS accumulation . As a main source of cellular ROS, mitochondrial metabolism is likely to play a pivotal role in the execution of ferroptosis . A surveSuggestive evidence of the ferroptosis/mitochondria crosstalk is represented by the strong iron dependency of this RCD ,38. IntrMetabolic plasticity is a critical property that gives cancer cells the edge for expanding, persisting after therapeutic hits and evading immune surveillance . RecentlOverall, these findings provide a clear support for the potential use of mitochondria-mediated ferroptosis in cancer treatment. Future studies exploring the effects of mitochondrial metabolic rewiring in in vivo models of ferroptosis would be necessary to confirm the role of this cell death as new exciting frontier in cancer biology.


Chamelea gallina from the Holocene sedimentary succession to present-day thanatocoenosis of the Po Plain-Adriatic Sea system . Comparing the Holocene sub-fossil record to modern thanatocoenoses allowed obtaining an insight of shell variations dynamics on a millennial temporal scale. Five shoreface-related assemblages rich in C. gallina were considered: two from the Middle Holocene, when regional sea surface temperatures were higher than today, representing a possible analogue for the near-future global warming, one from the Late Holocene and two from the present-day. We investigated shell biometry and skeletal properties in relation to the valve length of C. gallina. Juveniles were found to be more porous than adults in all horizons. This suggested that C. gallina promoted an accelerated shell accretion with a higher porosity and lower density at the expense of mechanically fragile shells. A positive correlation between sea surface temperature and both micro-density and bulk density were found, with modern specimens being less dense, likely due to lower aragonite saturation state at lower temperature, which could ultimately increase the energetic costs of shell formation. Since no variation was observed in shell CaCO3 polymorphism (100% aragonite) or in compositional parameters among the analyzed horizons, the observed dynamics in skeletal parameters are likely not driven by a diagenetic recrystallization of the shell mineral phase. This study contributes to understand the response of C. gallina to climate-driven environmental shifts and offers insights for assessing anthropogenic impacts on this economic relevant species.Understanding how marine taxa will respond to near-future climate changes is one of the main challenges for management of coastal ecosystem services. Ecological studies that investigate relationships between the environment and shell properties of commercially important marine species are commonly restricted to latitudinal gradients or small-scale laboratory experiments. This paper aimed to explore the variations in shell features and growth of the edible bivalve  Evaluating how marine ecosystems could respond to near-future global warming is critical to design proper conservation and management strategies, especially in coastal areas with increasing urbanization and resource overexploitation.In the marine realm, calcifying macroinvertebrates such as corals, brachiopods and mollusks produce hard structures for support and protection that constitute high-resolution archives recording the environmental conditions that have prevailed during their life , 2. ThroQuantifying the effect of near future climate change on marine calcifying organisms requires long-term multi-generational studies for assessing their adaptability to changing environmental conditions . Neverthi.e., Holocene climate optimum HCO), with significantly higher temperatures than today at high latitudes  (p to 4°C ). Holocep to 4°C –21). HenChamelea gallina seems to be particularly sensitive to environmental changes, showing shell morphology variations in response to environmental change . Ma. MaC. ga Click here for additional data file.S1 Appendix(XLSX)Click here for additional data file.


Pork leanness and marbling are among the essential traits of consumer preference. To acquire knowledge about universal epigenetic regulations for improving breed selection, a meta-analysis of methylated DNA immunoprecipitation sequencing (MeDIP-seq) profiling data of mixed loin muscle types was performed in this study.MeDIP-seq profiling datasets of longissimus dorsi muscle and psoas major muscles from male and female pigs of Landrace and Tibetan breeds were preprocessed and aligned to the porcine genome. Analysis of differential methylated DNA regions (DMRs) between the breeds was performed by focusing on transcription start sites (TSSs) of known genes . All associated genes were further reviewed for their functions and predicted for transcription factors (TF) possibly associated with their TSSs.When the methylation levels of DMRs in TSS regions of Landrace breed were compared to those of Tibetan breed, 10 DMRs were hypomethylated (Landrace < Tibetan), and 19 DMRs were hypermethylated (Landrace > Tibetan), accordingly (p≤0.001). According to the reviews about gene functions, all associated genes were pieces of evidence for their roles in a variety of muscle and lipid metabolisms. Prediction of the binding TFs revealed the six most abundant binding TFs to such DMRs-associated TSS (p≤0.0001) as follows: ZNF384, Foxd3, IRF1, KLF9, EWSR1-FLI1, HES5, and TFAP2A.Common DMRs-associated TSS between the lean-type and the marbled-type loin muscles were identified in this study. Interestingly, the genes associated with such regions were strongly evidenced for their possible roles on the muscle trait characteristics by which further novel research topics could be focused on them in the future. Apart from the promoter region, which commonly locates near the transcription start site (TSS), the chromatin loop formation also allows the long-distant cis-acting elements such as enhancers and repressors to cooperate in regulation .-59.ESSSCDNA methylation patterns mutually distributed among the tissue types usually implied universal metabolic features shared in common among them . Based oESSSCG00000038672 (hCHCHD2), ESSSCG00000027257 (PSMB1), and ESSSCG00000031489 (DUSP22) were also noticeable  should also be considered for any further intensive study.The regulatory function of the acquired TSS-associated DMRs required further biological evaluation despite the knowledge of their methylation levels. Since DNA methylation could either inhibit or recruit particular TFs in various circumstances ,20,64. tIn summary, the determination of TSS regions that were differentially methylated between lean-type and marbled-type pig breeds in loin meat muscle tissues was demonstrated as a novel approach in this study. The procedure rendered the limited numbers of differentially methylated genes, but with strong concatenation with the different muscle characteristics presented between these two breeds. We also introduced a karyoplot for the available virtualization of TSS-associated DMRs along with their candidate TFs binding to them. As a result, the knowledge acquired from this study established several new research topics in lean and marbled meats to evaluate the epigenetic gene regulation. It is worth noting that this study aimed to demonstrate a beneficial strategy for porcine muscle MeDIP-Seq data analysis, which was also applicable to other biological systems.KC, TS and RK planned the study design, collected the datasets, and analyzed the data. EO and CN refined the study design and the objective. DP carried out technical coding correction and hardware maintenance. KC and RK drafted and reviewed the manuscript. All authors read and approved the final manuscript.


Secreted by the pineal gland to regulate the circadian rhythm, melatonin is a powerful antioxidant that has been used to combat oxidative stress in the central nervous system. Melatonin-based therapies have been shown to provide neuroprotective effects in the setting of ischemic stroke by mitigating neuroinflammation and accelerating brain tissue restoration. Melatonin treatment includes injection of exogenous melatonin, pineal gland grafting and melatonin-mediated stem cell therapy. This review will discuss the current preclinical and clinical studies investigating melatonin-based therapeutics to treat stroke. Melatonin (N-acetyl-5-methoxytryptamine) is secreted by the pineal gland in the brain to regulate the circadian rhythm in mammals including light-dark cycles. The hormone has been found in other parts of the body including bile , cerebroNeuroprotection via melatonin has been explored for the treatment of stroke, which is characterized by aberrant inflammation and excessive production of free radicals. In this review, we will discuss preclinical and novel clinical studies investigating melatonin-based therapies to rescue the CNS following ischemia.Melatonin is effective as a free radical scavenger and an indirect antioxidant and serves as an accepted mechanism for neuroprotection. Melatonin can scavenge hydroxyl radicals (via the Fenton reaction from hydrogen peroxide) and peroxynitrite anions ,8. Also,The use of animal stroke models has contributed to the use of melatonin as a potential therapeutic. Stroke can be induced in experimental rats that have had their pineal gland removed because it is known to cause a reduction in the circulating levels of melatonin . Mice thStudies both in vitro and in vivo suggest that melatonin serves as a protector for glial cells and leads to a functional recovery, reduced inflammatory response and improved behavioral outcomes in ischemic animals. However, damage is still present in the lateral aspect of the striatum and suggests that normalization of motor protection may only require protection of the cortex . AnotherFurther studies are needed to explore alterations in glial cells that accompany melatonin treatment. Evidence suggests that improved glial cell survival after melatonin treatment protects injured neurons, since these cells secrete trophic factors . AdditioRecent studies have provided evidence that cell replacement therapy  may be effective to alleviate a stroke . The firHowever, several comparable studies utilized a sample of rats that underwent a pinealectomy alongside the allograft transplantation and this particular group of rats did not receive any additional neuroprotection . The traThe two different treatment methods, intracerebral pineal gland grafting and exogenous melatonin treatments, differ in the targeted line of cells. Experimental evidence demonstrates that pineal gland grafts reduce ischemic tissue volume 2 to 3 days after the onset of stroke as opposed to on the first day and suggests that pineal gland grafts target secondary cell death. In comparison of both treatment plans, a pineal gland graft is very invasive and entails an extensive transplantation surgery, while the exogenous melatonin treatment is less invasive. Although, it is important to consider that a pineal gland graft may provide greater ongoing treatment with chronic stroke patients because of the massive cell death that occurs after a stroke takes place . ExogenoA potential side effect of pineal gland graft therapy to consider is graft rejection. Therefore, pineal gland graft therapies would need to be accompanied by immunosuppressive agents to ensure long-term graft survival . MelatonFurther research is required to determine the efficacy of the pineal gland grafts and their relation to melatonin. A potential research option is to explore melatonin antagonist molecules in conjunction with a graft transplant to investigate if melatonin is providing all of the neuroprotection or another contributor such as various growth factors . This coRecent advances in stem cell therapy research have highlighted the involvement of melatonin receptors in stem cell mechanisms . MelatonVascular dementia and cognitive decline are two common detrimental effects that follow stroke . Post-stAn overwhelming amount of recent evidence points to melatonin’s neuroprotective effects in stroke, specifically highlighting its anti-oxidative, anti-inflammatory and anti-apoptotic capabilities, as shown in Evidently, melatonin’s therapeutic benefits in post-stroke injury may also be due to its ability to alter microglial activity and ameliorate mitochondrial dysfunction. Melatonin can transform microglia into their advantageous form in the ischemic brain through melatonin receptors. In vitro, melatonin escalated NeuN, BDNF and MAP2 and attenuated GFAP, Iba1 and caspase-3 protein under hypoxic conditions . AnotherAlthough past research has elucidated that pineal gland grafts may be therapeutically effective in stroke due to the steady stream of melatonin it provides, current investigations into the efficacy of pineal gland transplantations is lacking. Nonetheless, recent studies examining the effects of pinealectomy in the brain have been conducted. For instance, pinealectomized rats demonstrated significant melatonin scarcity, lower levels of superoxide dismutase (SOD) and elevated lipid peroxidation (LPO). Endurance training ameliorated oxidative stress at certain time points but did not rehabilitate the lack of melatonin . PinealeCurrent research reveals the importance of melatonin receptors, as the targeted cellular mechanism for melatonin-induced neuroprotection, as depicted in On account of melatonin’s plethora of neuroprotective features, stroke-induced dementia stands as a significant therapeutic target for melatonin-based treatment. In APP/PSI mice, a model of AD, long-term administration of melatonin ameliorated spatial learning and memory loss, curtailing Aβ accumulation and mitochondrial dysfunction . OxidatiRegarding recent clinical trials, melatonin-based treatment has been utilized for stroke and neurodegenerative disease patients. 60 patients received an oral dosage of melatonin 3 days before and after carotid endarterectomy (CEA) surgery. Blood samples of the melatonin group demonstrated significant reduction of inflammatory factors, NF-κB, TNF-α, IL-6 and S100β and an elevation of antioxidant activity through Nrf2, SOD, CAT and GPx . FurtherNevertheless, further preclinical investigation into the clinical safety and efficacy of melatonin in post-stroke treatment is warranted. Experimental stroke in preclinical trials should examine the long-term effects to assess the safety of melatonin therapy, ensuring no adverse effects, such as behavioral side effects and neurological inconsistencies, are present. To further bolster the safety profile for clinical treatment, investigators should examine neuronal tissues and peripheral organs for potential toxic effects during various stages post-melatonin therapy. Stroke Treatment Academic Industry Roundtable (STAIR) and Stem Cell therapies as an Emerging Paradigm (STEP) propose guidelines to potentially transition previously discussed therapies to clinical trials ,80. BothStroke stands as a prominent agent of cognitive impairment and mortality worldwide and unfortunately has limited treatment options. Therefore, novel therapies like melatonin are crucial to attenuating stroke’s devastating effects. Melatonin acts as an antioxidant and free radical scavenger, ameliorating neuroinflammation and accelerating brain tissue restoration. Preclinical studies utilizing animal stroke models and clinical trials with stroke patients have been conducted to investigate melatonin’s therapeutic potency in the context of stroke. Experimental stroke models in vitro and in vivo have demonstrated melatonin’s neuroprotective capabilities, as melatonin reduces infarct size and enhances glial cell viability. Melatonin delivered exogenously displays substantial therapeutic effects but results fluctuate with changing melatonin levels. However, pineal gland grafts have the ability to provide long-term melatonin and therefore, may be useful for chronic treatment. Nonetheless, further examination of pineal gland transplantation efficacy is necessary. The mechanism behind melatonin-induced neuroprotection seems largely due to receptors MT1 and MT2, which can also be found in stem cells. Moreover, stem cell-based therapy for stroke and neurodegenerative disease may be bolstered by targeting the MT1 and MT2 receptors. Regarding stroke-related dementia, melatonin-based therapy may be a viable treatment option due to their neuroprotective properties, repressing inflammatory factors, oxidative stress and apoptotic pathways in stroke and AD models. In addition, melatonin enhances BDNF and CREB, alleviating cognitive impairment. Notably, recent clinical trials exploring the curative potential of melatonin in stroke and neurodegenerative diseases display encouraging results in alleviating brain injury. Nevertheless, additional preclinical studies examining the safety and efficacy of melatonin-based therapies in stroke and stroke-related dementia are warranted to ensure the treatments’ optimal clinical applications.