Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron system associated with both genetic and environmental risk factors. Infection with enteroviruses, including poliovirus and coxsackievirus, such as coxsackievirus B3 (CVB3), has been proposed as a possible causal/risk factor for ALS due to the evidence that enteroviruses can target motor neurons and establish a persistent infection in the central nervous system (CNS), and recent findings that enteroviral infection-induced molecular and pathological phenotypes closely resemble ALS. However, a causal relationship has not yet been affirmed.G85R) ALS mice were intracerebroventricularly infected with a sublethal dose of CVB3 or sham-infected. For a subset of mice, ribavirin  was given subcutaneously during the acute or chronic stage of infection. Following viral infection, general activity and survival were monitored daily for up to week 60. Starting at week 20 post-infection (PI), motor functions were measured weekly. Mouse brains and/or spinal cords were harvested at day 10, week 20 and week 60 PI for histopathological evaluation of neurotoxicity, immunohistochemical staining of viral protein, neuroinflammatory/immune and ALS pathology markers, and NanoString and RT-qPCR analysis of inflammatory gene expression.Wild-type C57BL/6J and G85R mutant superoxide dismutase 1 . Furthermore, we showed that CVB3 infection led to a significant increase in proinflammatory gene expression and immune cell infiltration and induced ALS-related pathologies  pathology and neuronal damage) in the CNS of both SOD1G85R and C57BL/6J mice. Finally, we discovered that early (day 1) but not late (day 15) administration of ribavirin could rescue ALS-like neuropathology and symptoms induced by CVB3 infection.We found that sublethal infection (mimicking chronic infection) of SOD1Our study identifies a new risk factor that contributes to early onset and accelerated progression of ALS and offers opportunities for the development of novel targeted therapies.The online version contains supplementary material available at 10.1186/s12974-022-02380-7. To d(TARDBP) –5. On th(TARDBP) , 7.Similar to carcinogenesis, the development of ALS was proposed to be a multistep process involving interactions between genetic mutations and environmental risk factors , 2. ThisPicornaviridae family that include poliovirus and non-polioviruses, such as coxsackievirus B3 (CVB3), echovirus, EV-A71, and EV-D68 [Strong evidence supporting the link between ALS and EV infection is still lacking . EVs ared EV-D68 , 16. Altd EV-D68 , 16. It d EV-D68 –19. As Ed EV-D68 –27. HoweG85R, accelerates disease progression and decreases mouse survival. This observation was accompanied by significantly increased immune cell infiltration and proinflammatory cytokine/chemokine gene expression and detection of tissue damage and transactive response DNA binding protein-43 (TDP-43) pathology in the CNS. Moreover, we discovered that application of anti-viral drug ribavirin during the acute phase of viral infection rescues virus-mediated ALS-like pathology and phenotype in mice.Given the possible limitations/challenges, such as differences in methodology for viral detection and the stage of the disease when samples were harvested , human vG85R (B6.Cg-Tg (SOD1G85R) 148Dwc/J) and its background strain C57BL/6J mice were obtained from Jackson Laboratory . The neonates (2–3 days) of SOD1G85R or C57BL/6J mice were intracerebroventricularly injected at the intersection between the superior sagittal sinus and the transverse sinus with either a non-lethal dose (5 × 102 pfu (plaque-forming units) in 2 μl volume) of CVB3 (Kandolf strain) or an equal volume of Dulbecco's modified Eagle’s medium . The viral dosage was selected based on our pilot study that tested multiple viral dosages. This amount of virus did not cause virus-related mortality in C57BL/6J mice despite the virus persisting in the CNS for months.SOD1Both female and male mice were used for the current study. For survival and motor function analysis, the total animal numbers for each group including numbers of male vs female mice are specified in figure legends . Mice that developed hydrocephalus after CVB3/DMEM injection or non-ALS/CVB3-related symptoms including developmental abnormality were excluded from this study. For gene expression and immunohistochemistry studies, different cohort of mice (3–5 mice for each timepoint and each group) was used. All animal procedures were approved by the University of British Columbia Research Ethics Board .G85R mice was performed by subcutaneously injecting either ribavirin (100 mg/kg mouse body weight) diluted in 100 μl of DMEM or an equal volume of DMEM in the neck of neonatal mice. The injections were done every 3 days starting at either day 1 or day 15 post-infection (PI) for a total of 5 injections.Ribavirin  treatment in CVB3-infected SOD1G85R (B6.Cg-Tg (SOD1G85R) 1248Dwc/J) and C57BL/6J mice underwent weekly motor function tests  until either reaching humane endpoint or 60 week post-infection [After 20 weeks of mock or CVB3 inoculation, SOD1nfection , 29. TheHindlimb reflex was performed by lifting the mice from the tip of the tail to assess and score their hind leg reflex on a scale of 4–0 . Reduction in leg extension is an early deficit observed in mutant SOD1 transgenic mice.An inverted grid test was performed by placing the mice on an inverted metal grid (10 cm above the surface) and measuring the time  for the mice to fall. The test is used to assess the arm/leg strength of the mice .Gait analysis is a test that quantifies the mouse movement based on its gait distance. In the assessment, the mouse feet were first marked by non-toxic paints and then placed on a long paper  that was covered by cardboard, allowing the mice to walk forward in one direction. After the paints were dried, the stride distance (the distance of forward movement between each stride) was measured and recorded. In addition to the gait measurements, the time in seconds (footprint time) needed for each mouse to walk over the track was recorded as an additional measurement of the animal’s motor function .Mouse brains harvested were fixed with 4% formaldehyde and paraffinized. Paraffin-embedded Sections (4 µm thickness) were deparaffinized through xylene and a series of isopropanol solutions . Hematoxylin and eosin (H&E) staining was conducted to evaluate tissue damages, while IHC staining was used for detecting the presence and localization of proteins of interest. For IHC, antigen retrieval was done by heating tissue sections in pH 6.0 citrate buffer  for 25 min at 121 °C, and then peroxidase blocked using 30 mg/ml hydrogen peroxide solution. Multiple washes with 1 × TBS pH 7.6  were performed afterward. The MACH4 Universal HRP-Polymer Detection System  was used according to the manufacture’s procedure for IHC staining experimentations. After the TBS washes, tissue sections were incubated with primary antibodies (dilution and targets are shown below) overnight at 4 °C, and counterstained at the end with hematoxylin solution Gill II .Mouse brain tissues were immunostained using the follow primary antibodies diluted in Tris-buffered saline (pH 7.6) containing 1% bovine serum albumen: VP1 , GFAP , Iba1 , TDP-43 , SQSTM1/p62 , ubiquitin , CD68 , CD19 , CD79A , CD4 , CD8 , and NK1.1 . Images were taken using the Aperio ScanScope AT .G85R was determined by unpaired student’s t test . The mRNA levels of different proinflammatory genes and viral RNA were measured via RT-qPCR using the Luna universal one-step RT-qPCR kit (New England Biolabs) on a QuantStudio 6 Pro real-time PCR system . The primer pairs used for mRNA measurement in mouse tissues are as follows: TNFA , NFKB2 , CXCL10 , CCL2 , CCL5 , IL6 , IL10 , CVB3 2A , and β-actin .Mouse brain and spinal cord tissues were homogenized using Qiagen Tissuelyzer LT, and RNAs were extracted using the Monarch total RNA miniprep kit (New England Biolabs) following the manufacturer’s instructions. Gene expression was measured using NanoString Mouse Immunology Panel  that was ran on a NanoString nCounter® Profiler  according to the manufacturer’s instructions. NanoString data were analyzed using Gene Ontology and statistical difference in gene expression between mock- and CVB3-infected SOD1Quantification of IHC images was performed using ImageJ (version 1.52p) with the combination of Colour Deconvolution Plugin as described . The optG85R) and non-transgenic C57BL/6J  mice were intracerebroventricularly inoculated with a sublethal dose of CVB3 (500 pfu) or an equal volume of DMEM (mock infection) for 10 days, 20 weeks or 60 weeks  of transgenic mice carrying human mutant SOD1  . Therefore, all results presented here were data from both sexes combined.G85R mice, we next examined the neuropathological changes in CVB3-infected mice. Similar to our previous observations in Balb/c mice [Having shown that CVB3 infection leads to exacerbated disease phenotypes and shortened survival in SOD1b/c mice , we wereG85R and C57BL/6J mice at day 10 and week 60 PI , SQSTM1/p62 (an ubiquitin-binding autophagy adaptor protein), and ubiquitin. Similar to our previous findings , we obseG85R and C57BL/6J mice after CVB3 infection. IHC staining was conducted to assess the astrocyte reactivity with anti-GFAP  antibody, microglial activation using anti-Iba1  and anti-CD68 (cluster of differentiation 68) antibodies, the presence of B cells with anti-CD19 and anti-CD79A antibodies, the presence of T cells using anti-CD4 and anti-CD8 antibodies, and the presence of natural killer (NK) cells with anti-NK1.1 (an NK cell-specific antigen) antibody.The role of neuroinflammation in the development of neurodegenerative diseases, especially in ALS, has been widely studied and emphasized –38. TherG85R mice at day 10 PI. However, upon CVB3 infection, both C57BL/6J and SOD1G85R mice demonstrated significant increases in infiltration/activation of astrocytes (GFAP), microglia/macrophages (Iba1 and CD68), B cells (CD19 and CD79A), T cells (CD4 and CD8) and NK cells (NK1.1) in the brain at day 10 PI. At week 60 PI, mock-infected SOD1G85R mice showed significantly higher levels of different immunological markers as compared to mock-infected C57BL/6J mice  analysis showed upregulated genes in the brain (105 genes) and spinal cord (141 genes) of CVB3-infected relative to mock-infected SOD1G85R mice, which can be categorized into six different functional groups related to immune responses as indicated , which are shared between the brain and spinal cord samples, in the brain of week 20 CVB3-infected compared to mock-infected SOD1G85R mice  or day 15 (late intervention) PI for a total of 5 injections  . Overall, our results suggest that EV infection serves as a risk/susceptibility factor, rather than a cause for ALS. We propose that at least three mechanisms contribute to the exacerbating effects of EV infection on ALS onset and progression . However, similar damages/immune responses in combination under a detrimental genetic mutation background, such as SOD1G85R, could contribute to the acceleration of disease progression  as indicated. The red arrows indicate SQSTM1/p62 or ubiquitin positive inclusions. Black boxes on the top right illustrate the enlarged images. Scale bar = 100 μm.Additional file 2: Fig. S2. Sublethal CVB3 infection triggers microglia/astrocyte activation and immune infiltration in mice. Representative images of CD68, CD79A, CD4 and CD8 immunohistochemical staining in the hippocampus regions of the brain from mock- and CVB3-infected C57BL/6J or SOD1G85R mice at day 10 PI (A) or week 60 PI (or humane endpoint) (B). Similar observation was made in the regions of infected cerebral cortex (data not shown). Immune infiltrations were quantified by optical density based on the IHC staining in the hippocampus and cerebral cortex regions. Black boxes on the top right illustrate the enlarged images. Scale bar = 100 μm.


In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett’s Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9 Each pathway leads to the cleavage of the central component C3 into bioactive fragments C3a and C3b, ultimately activating the terminal pathway via C5 cleavage into C5a and C5b (Recent studies have generated strong evidence of tumor-promoting actions of early complement components in the tumor microenviroment (TME) . The com and C5b . C3a andThe early complement components C3 and C4, and the receptor C5aR have been reported as biomarkers for poor prognosis in gastric and colon cancers , with onvia endocytosis or exocytosis  , 7, a caocytosis . In thisTwo tissue microarrays were analyzed in this study. Information on the EAC tissue microarray was previous published , 18. A BRNA sequencing was performed on 9 BE, 8 EAC and 7 matched healthy esophageal (NE) tissue samples collected from participants at the Princess Alexandra Hospital, Brisbane, Australia, with written informed consent. Ethics approval was granted by the Princess Alexandra Hospital and QIMR Berghofer Medical Research Institute research ethics committees . The samples were collected and stored in RNAlater prior to RNA extraction and sequencing. RNA samples were extracted using previously published methods  and werehttps://portal.gdc.cancer.gov/), downloaded as fully processed level 3 data. TMM normalization was applied to the expression data using the R package edgeR.Gene expression array data was obtained from Gene Expression Omnibus accession number GSE72874 . Publicl2O2 in pH 7.5 Tris-buffer saline for 10 min. Antigen retrieval was carried out in a Biocare Medical Decloaking Chamber using Dako Target Retrieval Solution pH 9 for 15 min at 105°C. To eliminate nonspecific background staining, sections were blocked with 3% hydrogen peroxide in tris-buffered saline with 0.01% Tween-20 (TBS-T) for 5 minutes and Biocare Medical Background Sniper in 2% BSA for 15 min.Archival formalin fixed paraffin embedded (FFPE) tissue microarrays were sectioned at 3µm on superfrost+ slides. Sections were dewaxed in xylene, rehydrated in descending concentrations of ethanol to water and endogenous peroxidase quenched with 2% HFor chromogenic staining, Sigma Rabbit anti-human C9 primary antibody (Sigma Aldrich #SAB4503059) was diluted 1:300 in Background Sniper + 2% BSA for 60 minutes at room temperature. The specificity of this C9 antibody in IHC was previously assessed  by incubChromogenic images are typically used for pathology scoring; however, they are unsuitable for colocalization of overlapping markers, therefore we developed a simultaneous mIHF protocol for C9 and C5b-9 staining in archival-FFPE tissue specimens. 3µm slides were deparaffinized, rehydrated, and washed in TBS-T. Antigen retrieval was performed in Dako Target Retrival Solution pH 9 for 15 min at 105°C. All multiplex steps were with two TBS-T washes between each step. Prior to staining, tissue sections were blocked with 3% hydrogen peroxide in TBS-T for 5 minutes and background sniper for 10 minutes . Sequentially staining for C9 and C5b-9 negatively impacted on the staining of the second stain. By applying antibodies simultaneously and labelling them sequentially, both signals were maintained when compared to the single stains, validated on human esophageal carcinoma sections. For simultaneous staining, primary antibody Sigma C9 rabbit polyclonal (Sigma #4503059) diluted 1:1000 and C5b-9 mouse monoclonal clone aE11 (Abcam #ab66768), targeted against a C9 neoepitope, diluted 1:2000 was applied for 30min at room temperature. For visualization of C9 staining, after washing, Biocare Medical MACH2 Rabbit Polymer HRP was applied for 15minutes following 10 minutes incubation with tyramide signal amplification (TSA)-based fluorophores AKOYA Opal 520 1:100 in Opal 1xPlus Amplification Diluent for 10 minutes. For visualization of C5b-9 staining, sections were washed, MACH2 mouse polymer HRP applied for 5 minutes, then incubated with TSA-based fluorphore AKOYA Opal 620 1:100 in Opal diluent for 10 minutes. Sections were counterstained with DAPI (5mg/ml 1:35000 in TBS) for 5 minutes, washed, and coverslipped using Dako Fluorescence Mounting Media.mIHF images were obtained using Vectra 3.0 automated quantitative pathology system (Perkin Elmer). Fluorescent whole slide scan at 4x magnification was produced and visualized in Phenochart (v1.04), followed by multispectral image acquisition of each tissue microarray (TMA) core or selected high power regions at 20x magnification. Multispectral images were spectrally unmixed using InForm analysis software (v2.2.1) from Perkin Elmer.Biopsies were classified as normal squamous epithelium (NSE), Barrett’s esophageal mucosa (BE), dysplasia (low and high grade) (LGD and HGD), intraepithelial carcinoma (IEC) and EAC by a specialist gastrointestinal pathologist, and staining intensities for C9 and C5b-9 were scored. When the staining was non-uniform, the maximum intensity of staining in the epithelium was used for the score, providing at least 10% of the cells of that component stained to this intensity. Each component, if present in the tissue, was scored separately using a 4-grade assessment of intensity . Each cohort used liver as a positive C9 control for normalization between cohorts/slides.Colocalization between C9 and C5b-9 was determined using InForm Advanced Image Analysis software (Perkin Elmer), which provided a percentage of the C9 positive area that was also positive for C5b-9, and the C5b-9 positive area that was also positive for C9. Nonspecific autofluorescence signal from red blood cells was removed using Inform software, and threshold of detection was set visually for the whole TMA.Non-dysplastic BE and dysplastic esophageal cell lines CP-A, CP-B and CP-D were provided by Dr P. Rabinovitch ; BART-T cells were provided by Dr Rhonda Souza . All non-dysplastic BE and dysplastic esophageal cells were cultured in Keratinocyte Serum Free Medium (KSFM) in 2.5% heat inactivated fetal bovine serum (HI FBS) (Gibco), supplemented with 0.1 μg/mL Epidermal Growth Factor (PeproTech) and 0.7 μg/mL of Bovine Pituitary Extract (Sigma). OE33 and FLO-1 were purchased from the European Collection of Authenticated Cell Cultures (Sigma). OE33 were cultured in Roswell Park Memorial Institute 1640 medium (RPMI) in 10% HI FBS, while FLO-1 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) in 2.5% HI FBS. All cell lines were cultured in a 5% CO2 incubator set to 37°C. FBS used throughout this study was heated to inactivate complement components.2 in medium containing 10% C9-depleted human serum, with/without addition of 3µg/ml immunopurified C9. This dose was chosen to be lower than a previously published lytic dose of 20µg/ml in K562 cells , LAMP-1, (rabbit: LAMP-1: Abcam ab24170), then washed, labeled with secondary antibodies rab-647 and m-488 (1/300), then washed and stained with DAPI (1/1000). Labelled cells were analyzed under a Zeiss 780 NLO Microscope , with Z-stack analysis with each slice at approximately 0.38µm on 63x or 100x objective, image size of 1024x1024. Images and merged images were obtained with Zen software . Images were processed further for display by using ImageJ . Maximum intensity projection images were thresholded consistently for all images, C5b-9 puncta with sizes over 10 pixel2 and 37°C, with images taken every 4 hours, and images processed using Incucyte software to detect confluence and red object count as an indication of the number of dead cells.Cells were plated in 96 well tissue culture plate in appropriate media with depleted serum, with or without 3µg/ml C9. Propidium Iodide  was included to assess dead cell number. Plates were assessed in the Incucyte  for up to 80 hours, 5% CO-1and TFMBA peak at 1335 cm-1 were used for quantification of C9 and C5b-9, respectively.C9+ and C5b-9+ EV analysis was performed using an immunoassay integrated on a microfluidic device with asymmetric gold electrodes and surface-enhanced Raman scattering (SERS) read-out, as previously reported . The ele-1. The samples were run in triplicate. The raw Raman spectral data contained fluorescence and background noise that was removed using a fifth-order polynomial fitting method previously described [50 µL of cell culture supernatant sample and 20 µL SERS nanotags were sequentially added to each well of the microfluidic device. Following previously optimized conditions , alternaescribed .To evaluate esophageal tissue C9 and C5b-9 levels during the sequence normal epithelium (NE)-BE-EAC, we generated and stained a tissue microarray (TMA) from archival esophageal tissue samples from patients undergoing investigation for BE or suspected EAC, and also stained a previously established TMA of EAC cases , 18. A mTMA sections co-stained for C9 and C5b-9 were evaluated for staining intensity in regions of specific tissue phenotypes  using a semi-quantitative scoring system from 0 to 3 by a specialist gastrointestinal pathologist Figure 1The quantitative result shows increased intensity and proportion of both C9 and C5b-9 staining with disease stage , as well as our own RNAseq dataset of normal esophageal epithelium, BE and EAC tissues , HGD (CP-B and CP-D), and EAC (FLO-1 and OE-33). With the commercially available HET-1A cell line reportedly lacking characteristics of normal esophageal squamous epithelial cells  from the cells. At various time points following immunopurified human C9 treatment in the presence of C9-depleted human serum, cells on coverslips were fixed and then stained for C5b-9. Control conditions include untreated cells and cells exposed to C9-depleted human serum for 30 minutes S3. WhilIn spite of the MAC formation, C9 exposure did not lead to significant cell death up to 4 days in CP-B, CP-D, FLO-1 or OE33 cells Figure 4via endocytosis for lysosomal degradation  EVs (We previously reported the capture of intact EVs from conditioned medium using microfluidic immunoassay using surface enhanced Raman spectroscopy (SERS) read-out of EV surface markers . Here, w+ EV  capture generic EVs . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.CK, JW, MH, and AS conceived and designed the analysis. CK, YC, JW, RR, SB, and AL collected the data. AL and CW developed the staining methodologies. IB classified and scored the TMA. RK, KS, AW, and MT performed the EV analysis. SB and AB performed transcript assessment. WZ, GE, NC, BM, and AB provided expert analysis. CK and MH performed the analysis and wrote the paper. All authors contributed to the article and approved the submitted version.Translational Research Institute Spore Grant (MH); National Health and Medical Research Council APP1173669 (AW); Australian Research Council Discovery Project DP210103151 .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


DeepG4 is very accurate to predict active G4 regions in different cell types. Moreover, DeepG4 identifies key DNA motifs that are predictive of G4 region activity. We found that such motifs do not follow a very flexible sequence pattern as current algorithms seek for. Instead, active G4 regions are determined by numerous specific motifs. Moreover, among those motifs, we identified known transcription factors (TFs) which could play important roles in G4 activity by contributing either directly to G4 structures themselves or indirectly by participating in G4 formation in the vicinity. In addition, we used DeepG4 to predict active G4 regions in a large number of tissues and cancers, thereby providing a comprehensive resource for researchers.DNA is a complex molecule carrying the instructions an organism needs to develop, live and reproduce. In 1953, Watson and Crick discovered that DNA is composed of two chains forming a double-helix. Later on, other structures of DNA were discovered and shown to play important roles in the cell, in particular G-quadruplex (G4). Following genome sequencing, several bioinformatic algorithms were developed to map G4s in vitro based on a canonical sequence motif, G-richness and G-skewness or alternatively sequence features including k-mers, and more recently machine/deep learning. Recently, new sequencing techniques were developed to map G4s in vitro (G4-seq) and G4s in vivo (G4 ChIP-seq) at few hundred base resolution. Here, we propose a novel convolutional neural network (DeepG4) to map cell-type specific active G4 regions . In 2000, the Human Genome Project revealed the widespread presence of G4s in the genome using algorithms. To date, all G4 mapping algorithms were developed to map G4s on naked DNA, without knowing if they could be formed in a given cell type. Here, we designed a novel artificial intelligence algorithm that could map G4 regions active in the cell from the DNA sequence and chromatin accessibility. Moreover, we identified key transcriptional factor motifs that could explain G4 activity depending on cell type. Lastly, we used our new algorithm to map active G4 regions in multiple tissues and cancers as a comprehensive resource for the G4 community. Deoxyribonucleic acid (DNA) is a complex molecule carrying genetic instructions for the development, functioning, growth and reproduction of all known living beings and numerous viruses. In 1953, Watson and Crick discovered that DNA is composed of two chains forming a double-helix . G4 sequG4s were found enriched in gene promoters, DNA replication origins and telomeric sequences , 7. AccoG3+N1−7G3+N1−7G3+N1−7G3+, or the corresponding C-rich motif  [i.e. regions comprising the G4(s) along with flanking sequences. For instance, Quadron—a machine learning approach—was proposed to predict G4s based on sequence features (such as k-mer occurrences) from a region of more than 100 bases, and trained using in vitro G4 regions with G4-seq [e.g. G4 activity).Following the Human Genome project , computagorithm) , 20. Usigorithm) , or by sgorithm) . Recentlgorithm) , and G4sgorithm)  as regioh G4-seq . By combh G4-seq . Anotherh G4-seq . Thus, ae.g. ATAC-seq). For this purpose, DeepG4 exploits the genomic context (a 201-base region) of a G4, which comprises the potential G4 forming sequence, but also other DNA motifs that may play a role in G4 activity. Moreover, adding chromatin accessibility, which is publicly available for most cell lines, tissues and cancers, into the model allows to predict G4 regions that are active depending on the cell-type, since it was previously shown that in vivo G4 peaks strongly colocalize (98%) with regions identified by either FAIRE-seq or ATAC-seq, or both [Here, we propose a novel method, named DeepG4, aimed to predict cell-type specific active G4 regions (regions that were mapped both in vitro and in vivo in a given cell type) from DNA sequence and chromatin accessibility. DeepG4 implements a CNN which is trained using a combination of genome-wide in vitro (G4-seq) and in vivo (G4 ChIP-seq) peak DNA sequences, together with chromatin accessibility measures ( or both . DeepG4 https://pypi.org/project/MACS2/). We downloaded G4P ChIP-seq (similar to G4 ChIP-seq) peaks already mapped to hg19 for A549, H1975, 293T and HeLa-S3 cell lines from GEO accession number GSE133379 [We downloaded G4 ChIP-seq data for HaCaT, K562 and HEKnp cell lines from Gene Expression Omnibus (GEO) accession numbers GSE76688, GSE99205 and GSE107690 , 28, 29.SE133379 . We usedSE133379 . We usedWe defined positive DNA sequences (active G4 region sequences) as forming both in vitro and in vivo G4s as follows. We only kept G4 ChIP-seq peaks overlapping with G4-seq peaks. We then used the 201-bp DNA sequences centered on the G4 ChIP-seq peak summits.https://cran.r-project.org/web/packages/gkmSVM).As negative (control) sequences, we used sequences randomly drawn from the human genome with sizes, GC content (% GC), and repeat content (tandem repeat number from Tandem Repeat Finder mask from hg19 genome) similar to those of positive DNA sequences using genNullSeqs function from gkmSVM R package .We used position weight matrices (PWMs) for transcription factor binding sites from the JASPAR 2018 database , kernel size (20 bp), kernel activation (relu), pool size (12 bp), drop-out (0%), epoch number (20), number of neurons in the dense layer (100) and the optimizer choice (rmsprop) were selected by Bayesian optimization . In S1 FThe first layer of DeepG4 contains kernels capturing specific sequence patterns similar to DNA motifs. In order to obtain DNA motifs from the first layer  of DeepG4, we proceeded as follows see . For a gEach kernel PFM was then trimmed by removing low information content positions at each side of the PFM (threshold >0.9). PFMs whose size were lower than 5 bases after trimming were removed. PWMs were next computed from PFMs assuming background probability of 0.25 for each DNA letter as done in JASPAR.http://rsat.sb-roscoff.fr/) with parameters: median, cor = 0.6, ncor = 0.6. We used PWM cluster centers as DNA motifs for further analyses.Because many PWMs from DeepG4 were redundant, we used the motif clustering program matrix-clustering from RSAT suite (https://keras.rstudio.com/). DeepG4 is available at https://github.com/morphos30/DeepG4. All fasta files used for training and predictions were also deposited.DeepG4 was implemented using Keras R library . We first browsed the genome where G4 regions were mapped by ChIP-seq as active in K562. For instance, we looked around the oncogene KRAS known to be regulated by a G4 in its promoter . Removing chromatin accessibility significantly lowered cell-type specific prediction accuracy. For instance, the AUROC of HaCaT (independent) was 0.939 for DeepG4* as compared to 0.986 for DeepG4, which represented an important difference . We alsoThese results thus demonstrated the ability of DeepG4 to accurately predict cell-type specific active G4 regions from DNA sequences and chromatin accessibility. Moreover, results also revealed the importance of incorporating chromatin accessibility into DeepG4 for cell-type specific predictions.The first layer of DeepG4 convolutional neural network encapsulated kernels that encoded DNA motifs predictive of active G4s. Hence, we extracted from the first layer the kernels and converted them to DNA motif PWMs to better understand which motifs were the best predictors of G4 activity. DeepG4 identified 900 motifs, many of them were redundant. To remove redundancy, we clustered the motifs using RSAT matrix-clustering program and kept the cluster motifs  for subsequent analyses. Cluster motifs could be divided into two groups: a group of de novo motifs and a group of motifs that resembled known TFBS motifs. To distinguish between these two groups, we used TomTom program (MEME suite) which mapped the cluster motifs to JASPAR database. DeepG4 motifs matching JASPAR were considered as known TFBS motifs, while motifs that did not match were classified as de novo motifs.We first assessed the ability of DeepG4 motifs to predict active G4 regions. Hence, we computed DeepG4 cluster motif variable importances using random forests and found strong predictors . In orde−10). Other strong predictors, such as cluster 4, matched KLF5 motif MA0599.1 . It was very interesting to observe that such motif corresponding to one half of a canonical G4 motif also matched a known TFBS motif, which supported the complex interplay between G4s and TFBS protein binding [Many strong predictors were de novo motifs which ressembled the G4 canonical motif or parts of the canonical motif. For instance, cluster 1 comprised 4 stretches of GG+, thus almost forming a canonical G4 motif . Cluster binding .We then assessed the enrichment of DeepG4 cluster motifs around active G4 regions and around canonical G4 motifs . Motifs These observations revealed the important role of TFBS motifs that could act directly in G4 activity as part of G4 structure, as previously shown for SP1 in vitro , or coulUsing DeepG4, we could map active G4 regions genome-wide in many different tissues and cancers for which no G4 ChIP-seq experiments were available, but for which we could find publicly available chromatin accessibility data (ATAC-seq or DNase-seq). Hence, we made the mapping available on the DeepG4 Github repository as a resource for the G4 community.p = 0.011, p = 0.063, We first browsed the genome at known oncogenes and looked at predicted active G4 regions . In MYC,Since G4s are known mutagenic regions when unresolved, we then looked at the link between G4 activity and mutation rates in BRCA breast cancer . We founi.e. regions that are detected both in vitro and in vivo, unlike previous algorithms that were developed to predict G4s forming in vitro (naked DNA). For this purpose, our method exploits the genomic context of G4s, which comprises the G4(s) as well as other motifs in the vicinity that may play a role in G4 activity (i.e. transcription factor motifs). Moreover, adding chromatin accessibility into the model allows to predict active G4 regions depending on the cell type. Our novel method which maps active G4 regions in a cell-type specific manner at 201-bp resolution is complementary to existing algorithms based on regular expression (e.g. quadparser) and scores (e.g. G4Hunter), which map the exact location of potential G4 forming sequences and propensities. Moreover, DeepG4 provides a useful tool for mapping active G4 regions for cell lines, tissues and cancers for which no experimental data are available to date. Therefore, DeepG4 comprehensive predictions in tissues and cancers will represent a useful resource for the G4 community.In this article, we propose a novel deep learning method, named DeepG4, to predict active G4 regions from DNA sequence and chromatin accessibility. The proposed method is designed to predict active G4 regions G3+N1−7G3+N1−7G3+N1−7G3+) or even parts of it. Most notably, many motifs corresponded to half or 3/4 of the canonical motif. The combination of these G4 parts, which is captured by DeepG4 as a deep neural network, brings flexibility in G4 modeling. Strikingly, some motifs completely or partly matched known TFBS motifs including KLF5 motif MA0599.1 and FOS (AP-1) motif MA0476.1, suggesting that they could contribute directly to G4 structures themselves or participate indirectly in G4 activity in the vicinity through the binding of transcription factors. In line with this result, it was previously found that G4s are enriched in the vicinity of the architectural protein CTCF at 3D domain  borders [DeepG4 uncovered numerous specific DNA motifs predictive of active G4s. Many motifs resembled the canonical G4 motif  is that it requires a region of several hundred bases, thereby restricting the resolution of G4 mapping. Once an active G4 region is mapped, methods such as G4Hunter or pqsfinder have to be used to identify the exact position of the G4(s) within the region. Our model could be improved by adding novel neural layers in order to find as well the exact location of potential G4 sequences. Second, DeepG4 does not process the DNA sequence in a strand-specific manner, thus a given motif could be redundantly encoded in both strands within the convolutional layer. However, post-processing of DeepG4 motifs using methods such as matrix-clustering alleviates such problem by mapping complementary motifs (same motifs on different strands) to each other to merge them into cluster motifs. Third, the prediction performance of DeepG4 strongly depends on existing datasets that are limited, potentially inaccurate and biased, especially regarding in vivo mapping. Once more techniques for in vivo G4 mapping will be developped, DeepG4 will need to be retrained in order to improve prediction accuracy. Moreover, since DeepG4 was trained based on human data, predictions on non-mammalian genomes are expected to be less accurate. Fourth, DeepG4 is limited to predict active G4s but a similar approach could be used to predict any active non-B DNA structure using permanganate/S1 nuclease footprinting data .S1 FigFor each hyper-parameter, the optimum is marked as a red triangle.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.


Ischemic heart disease is a leading cause of mortality and morbidity worldwide. We previously demonstrated that acacetin protects against myocardial ischemia reperfusion injury in rats, although the underlying mechanism remains to be elucidated. In the present study, we investigated the effects of acacetin on autophagy during hypoxia/reoxygenation (H/R) injury by exposing H9c2 myocardial cells to H/R with or without acacetin pretreatment during hypoxia. Our results show that acacetin significantly increased cell viability in a dose-dependent manner, enhanced antioxidant capacity, and suppressed protein apoptosis of rat cardiomyocytes H9c2 cells following H/R injury. In addition, lentiviral infection of H9c2 cardiomyocytes revealed that acacetin pretreatment significantly enhanced the fluorescence intensity of autophagy proteins Beclin 1, LC3-II, and p62. These results indicate that acacetin protected H9c2 cardiomyocytes from H/R damage by enhancing autophagy. Moreover, we found that application of acacetin increased activation of the PI3K/Akt signaling pathway, whereas cotreatment with the PI3K inhibitor LY294002 reversed the inhibition of apoptosis and autophagy induced by acacetin. In conclusion, acacetin mitigated H/R injury by promoting autophagy through activating the PI3K/Akt/mTOR signaling pathway. Ischemic heart disease is a leading cause of mortality and morbidity worldwide . FollowiZiziphora bungeana (Juz.), was previously demonstrated to protect against myocardial ischemia reperfusion injury , 3332, 3In summary, the results of this study suggest that acacetin inhibited apoptosis through the PI3K/Akt/mTOR signaling pathway to alleviate H/R injury in H9c2 cardiomyocytes by promoting autophagy. Thus, the clinical efficacy and safety of acacetin for reducing myocardial H/R injury warrant further study.


According to several animal and human studies, vitamin D appears to play a significant role in the development of diabetic nephropathy. However, the possible renoprotective effect of vitamin D and its influence on the reversal of already existing renal damage remains doubtful. At this moment, there are a few hypotheses concerning the underlying molecular and genetic mechanisms including the link between vitamin D and inflammation, oxidative stress, and extracellular matrix accumulation. The present review aims to investigate the potential role of vitamin D in the development of diabetic kidney disease from a translational approach. Diabetes mellitus (DM), one of the most common chronic diseases , is the 2 and 25(OH)D3. Most researchers and available guidelines  consider vitamin D deficiency in the general population as a 25(OH)D3 level of less than 20 ng/mL (50 nmol/L)  [2D3 reflects the reserve status of vitamin D [2D3 in the kidneys is regulated by the plasma parathyroid hormone and serum calcium and phosphor concentrations [Vitamin D is metabolized to 25(OH)D(OH)2D3] . 1,25(OHitamin D . The protrations . Vitamin3 in the bloodstream. The complex formation of VDBP/25(OH)D3, its filtration, and the reabsorption of this complex in the proximal renal tubular cells are critical for the retrieval and activation of vitamin D. People with renal damage, like diabetic patients with DKD, have increased urinary VDBP concentrations [VDBP is a low-molecular-weight protein of 58 kDa, which predicts the bioavailability of 25(OH)Dtrations . The effects of vitamin D are mediated through binding to its receptor (VDR), which is present in a variety of tissues in the human body, including the kidneys ,2, and mThis indicates that the kidneys play a crucial role in vitamin D metabolism by controlling the reabsorption of calcium and phosphate and by regulating the synthesis of the active form of vitamin D . The binVDR gene is located on the long arm of chromosome 12 (12q13.11), containing 14 exons [VDR gene polymorphisms with DM and its microvascular complications has become a hot topic for intensive research [These cofactors will bind to specific DNA locations, leading to a modification in the expression of its target genes, transcriptional response, and protein formation ,6,7. The14 exons ,9. Curreresearch . The aim of the present review was to investigate the role of vitamin D in the development of DKD with a focus on the possible underlying molecular and genetic mechanisms.3 concentrations are significantly lower in DN compared to healthy controls, suggesting that vitamin D plays a significant role in the development of DN [VDR gene has been observed [Several animal studies have shown that 25(OH)Dnt of DN ,16. In mobserved . More spobserved . p < 0.05) in serum creatinine and urinary albumin excretion rate (UAER) after 8 weeks compared to the DN group. Similar results were observed in another study [Studies in rats and mice have investigated whether supplementation with vitamin D might have a renoprotective role in the process of developing DN ,20,21,22er study . p < 0.05). Vitamin D supplements might reverse the histopathological changes caused by DN [2D3) showed remarkable histopathologic changes compared to DN rats without treatment (p < 0.05) [In STZ-treated DBA/2J mice, treatment with low-dose vitamin D, in the group with human transgenic VDR, almost entirely blocked the onset of DN . In anoted by DN ,21,22,23 < 0.05) ,20,24. p < 0.05). Light microscopy showed that silencing of the VDR gene eliminated the renoprotective effect of vitamin D therapy [In addition, elevated albuminuria was lowered by calcitriol treatment in DN rats  compared to healthy controls [3 concentrations compared to healthy controls [3 concentrations and HbA1c  [3 levels in those with T2DM compared to healthy controls [Multiple studies have demonstrated that the prevalence of 25(OH)Dcontrols ,31,32,33controls , a pheno 0.0001) . Howevercontrols .n = 2224) showed a significantly higher risk for development of T2DM in patients with a 25(OH)D3-deficiency (≤20 ng/mL)  [Several authors have published an association between lower levels of vitamin D and a higher risk of DM ,38,39,40< 0.001) . 3 concentration less than 20 ng/mL had a 65% higher risk for the development of microalbuminuria  compared to patients with a 25(OH)D3 concentration at a minimum of 30 ng/mL [The association between vitamin D deficiency and microvascular complications in diabetic patients is significantly higher than that in non-diabetics ,43,44,4530 ng/mL . 3 concentration (<15 ng/mL) had a faster decline in eGFR in comparison to patients with a 25(OH)D3 level of >15 ng/mL [2D3 concentration in patients with an eGFR decrease from 60 mL/min/1.73 m2 to <15 mL/min/1.73 m2 (p < 0.05), but no significant changes were observed when the eGFR decreased from >90 mL/min/1.73 m2 to 60 mL/min/1.73 m2 [3 concentration were observed with worsening renal function. In a randomized controlled trial (RCT), type 2 diabetic patients treated with renin-angiotensin-system (RAS) blockers and with a 25(OH)D15 ng/mL . Another/1.73 m2 . No sign3 concentrations seem to be significantly lower in DN patients, indicating that vitamin D deficiency is more prevalent in DN patients [n = 300), patients with T2DM and CKD were 1.7-times more likely to have a vitamin D deficiency when compared to the diabetics without CKD [3 concentrations and urinary albumin:creatinine ratio (UACR)  [Several studies have indicated that 25(OH)Dpatients ,52,53,54hout CKD . A cross 0.0001) . 3 concentrations and eGFR . In T2DM patients at different stages of DN (n = 502), significantly lower serum concentrations of 25(OH)D3 were reported in microalbuminuric (UAER 30–300 μg/mg) (n = 171) and macroalbuminuric subjects (UAER > 300 μg/mg) (n = 130) vs. normoalbuminuric patients (UAER < 30 μg/mg) (n = 201) (p < 0.01) [3 was found in the macroalbuminuric vs. the microalbuminuric group (p < 0.01). There was a strong positive significant correlation between 25(OH)D < 0.01) . A signi3 concentrations [n = 63) showed no significant correlation between the baseline serum concentration of vitamin D metabolites and UACR [n = 18) compared to the patients without nephropathy (n = 40) [Complementary to these results, different studies proved that the prevalence of DN was high in T1DM and T2DM patients with low 25(OH)Dtrations ,46,54,55and UACR . No sign(n = 40) . 3 concentration, ROC analysis showed that the optimal cut-off concentration was 10.5 ng/mL with a sensitivity of 82.6% and a specificity of 72.7%  [p < 0.001) higher in diabetic patients with DN compared to patients without DN [p < 0.001) with the 24-h urinary protein excretion was also demonstrated. UVDBP was significantly different among patients with normoalbuminuria, microalbuminuria, macroalbuminuria, and the control group (p < 0.001) [In a study investigating the potential to diagnose T2DN based on 25(OH)D4–0.849) . Some st4–0.849) ,59,60,61thout DN . A positIn addition to the higher risk of DN in patients with vitamin D deficiency, multiple interventional studies have shown a recovery of renal function after vitamin D therapy ,63,64,65p = 0.03 and p < 0.001, respectively). A prospective observational study (n = 63) examined whether an oral cholecalciferol treatment over a 4-month period , and 40,000 IU monthly in patients with vitamin D insufficiency defined as 16 ng/mL (40 nmol/L) to 32 ng/mL (80 nmol/L)) decreased the albuminuria in T2DN patients [This study showed a significant reduction in the UAER and eGFR during paricalcitol therapy compared to placebo . A prospective randomized trial investigated the effect of calcium supplementation with or without calcitriol supplements in type 2 diabetics with CKD stage 2–4 and hypovitaminosis D (n = 50) [p = 0.03), while serum creatinine remained stable in the group treated with both calcium and calcitriol supplementation . A significant reduction in UACR was seen at both time points (months 2 and 4) compared to the baseline UACR ((n = 50) . The dian = 51) showed that treatment with oral vitamin D supplements  for three months resulted in no statistically significant difference in UACR in patients with proven T2DN and vitamin D deficiency [Although several well-designed observational and interventional studies have demonstrated a causal relationship between vitamin D and the risk of the development of DN, the results are still controversial and there are still a few interventional trials that have shown no statistically significant effect of vitamin D supplementation ,66,67,68n = 50) [In a cross-sectional study with 119 T2DM patients, treatment with calcitriol (0.5 µg daily for 2 months) in those with a vitamin D deficiency/insufficiency resulted in a reduction in albuminuria, though the difference was not significant . A doubln = 50) . p < 0.0001) in the vitamin D-treated group. A group of 25 patients was treated with 1,25-dihydroxycholecalciferol  for 8 weeks, while another group of 25 subjects received a placebo. After completing the vitamin D treatment, no significant reduction in an oxidative or significant increase in anti-oxidative parameters could be detected. In addition, no significant changes were observed in eGFR and serum creatinine between intervention and placebo groups. However, there was a significant reduction in proteinuria  induction . LPS is p < 0.05) in DM2 patients with or without DN. The activated p-STAT binds to nuclear DNA of the VDR to regulate the transcription of VDR. High dose of IL-15 itself can cause a massive pro-inflammatory reaction of IL-1, IL-6, and tumor necrosis factor-alpha (TNF-α) by LPS-activated macrophages . In diabp < 0.05), and induced a massive pro-inflammatory response. In the LPS plus IL-15 treated monocytes, there was a significantly higher expression of nuclear p-STAT5 and co-expression of VDR-p-STAT5 complexes in DM2 and DN uremic patients, compared to healthy controls (p = 0.016). This upregulation was more significant in DKD patients compared to diabetic controls [In addition, the VDR expression was considerably lower in the DN group compared to non-DN diabetic patients , and p-STAT5 expression levels did not change significantly compared to healthy controls [The potential anti-inflammatory and immunomodulatory effects of 1,25(OH)h uremia . After pcontrols . 2D3 stimulates the formation of vitamin D/VDR complexes and the exhibition of VDR DNA-binding sites. These binding sites can be attached by p-STAT5 resulting in p-STAT5/VDR complexes, which induce the formation of anti-inflammatory cytokines and inhibit the secretion of pro-inflammatory cytokines [p < 0.01) [Treatment with 1,25(OH)ytokines . These fytokines ,33. Mono2D3. Similar results were obtained in a prospective study with DM1 patients with vitamin D deficiency of insufficiency [These concentrations were not influenced by treatment with 1,25(OH)ficiency . DM1 patficiency . Vitamin D may play a renoprotective role in DN by negative regulation of the renin-angiotensin-aldosterone system (RAAS) by suppressing renin expression . An incrTreatment with losartan resulted in a reversal of these effects, suggesting that VDR activation attenuates renal fibrosis at least in part by suppressing the RAAS . In subtThese findings indicate both systemic and intrarenal RAAS activation . CombineThis therapy was more effective compared with treatment with losartan or paricalcitol alone, and reversed the decline of slit diaphragm proteins, leading to the restored glomerular filtration barrier structure and markedly reduced glomerulosclerosis. Paricalcitol may be added to treatment in patients with DN who are also receiving RAAS inhibitor therapy to further reduce albuminuria . The selective VDR activation with paricalcitol for the reduction of albuminuria in patients with type 2 diabetes (VITAL study) demonstrated the additional antiproteinuric effect of paricalcitol in diabetic patients on stable treatment of RAAS inhibition, suggesting the comparable synergistic effect in patients with DN . In pre-2D3 could occur via the toll-like receptor 4 (TLR4) and nuclear factor kappa B p65 (NF-κB p65) pathway [p = 0.006). IL-15 and TLR4 mRNA concentrations were significantly higher in the DN group compared to DM2 patients without DN (both p < 0.05).Another hypothesis suggests that the potential anti-inflammatory effect of 1,25(OH) pathway . Treatme2D3 did not affect the mRNA concentrations of IL-15, TLR4, or TLR9. After treatment with LPS and IL-15, there was a significant increase in protein levels of NF-κB p65 and a significant decrease in inhibitor of NF-κB (IκB) levels in THP-1 monocytes from DM2 patients with or without DN compared to healthy controls (p < 0.05). Pretreatment with 1,25(OH)2D3 blocked the changes of NF-κB p65 and IκB levels induced by LPS and IL-15. No significant differences were found for TLR9 mRNA levels. Pretreatment with 1,25(OH)p < 0.01). Pretreatment with 1,25(OH)2D3 resulted in a significantly decreased IL-6 secretion by monocytes, blocking the pro-inflammatory effects of LPS and IL-15. Based on these results, the mechanism of action is likely to be via the TLR4/NF-κB p65-signaling pathway [Treatment with LPS and IL-15 also resulted in a significant increase in IL-6 and MCP-1 levels in DM2 patients with or without DN compared to healthy controls ( pathway .Extracellular matrix (ECM) is a prominent hallmark of DN, which ultimately results in renal fibrosis ,24,81. IThe increased proteinuria in DN rats is also associated with an increased expression of transient receptor potential cation channel, subfamily C, member 6 (TRPC6) . In addiHuman transgenic VDR was able to attenuate these changes significantly. Upregulation of VDR restores the slit components, nephrin and podocin, by inhibiting the fibronectin synthesis caused by hyperglycemia . Comparep < 0.01) and after knocking down VDR, the effect of vitamin D therapy was partially repressed (p < 0.01) with increasing intracellular TGF-β levels [Hyperglycemic conditions stimulate the expression of TGF-β in rat glomerular mesangial cells, leading to accumulation in the ECM . TGF-β1 β levels . 2D3 inhibited high glucose-induced fibronectin production in cultured mesangial cells and increased nephrin expression in cultured podocytes [p < 0.01), which decreased significantly after treatment with 1,25(OH)2D3 (p < 0.05) [Treatment with 1,25(OH)odocytes . Vitaminodocytes . In the odocytes . High gl < 0.05) .Nephrin is a cell surface receptor that plays an important role in cell-cell adhesion and signaling in glomerular podocytes, which contributes to renoprotection. In ex vivo isolated rat glomeruli, nephrin has been associated with the p85α regulatory subunit of nephrin-phosphatidylinositol 3-kinase (PI3K). PI3K or lipid kinase produces PIP3, which is a regulator of the Akt’s translocation to the plasma membrane as a second messenger. Akt gets activated by phosphorylation modification of threonine 308 and serine 473 . Although PI3K is definitely involved in the development of DN, the exact function of PI3K in the diabetic kidney has not yet been fully understood. Different PI3K isoforms might explain the contrasting intracellular actions . In one Inhibition of the PI3K/Akt pathway ameliorates the effects of high glucose on renal tubular cells through downregulation of HDAC5 . On the p < 0.05). Similarly, nephrin and PODXL were significantly downregulated in high glucose-treated human glomerular epithelial cells compared to controls (p < 0.05), which has been linked to loss of the permselective renal barrier and proteinuria [p < 0.005). After knocking down VDR, the effect of calcitriol was inhibited for 100% and paricalcitol for 60%  is essential for normal podocyte structure and function. In DN rats, a reduction in the expression of PODXL in the glomeruli has been seen. After activation of VDR, there was a markable recovery of PODXL levels -Wnt-β-catenin-snail signaling pathway . A paricp < 0.05) [An effect of vitamin D on the immunohistological changes by macrophage switching via the VDR-peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway has been shown. In hyperglycemic conditions, RAW264.7 macrophages exhibited a significant switch from M2 to M1 phenotypes ( < 0.05) . When ma2D3 resulted in a significant upregulation of M2 markers and downregulation of M1 markers (p < 0.05). VDR and PPARγ mRNA and protein levels increased significantly in a vitamin D dose-dependent manner starting compared to control and hyperglycemic conditions (p < 0.05). After the antagonization of PPARγ, the effect of vitamin D on the switch of macrophage phenotype from M2 to M1 was significantly attenuated (p < 0.05). Treatment with 1,25(OH)2D3 effect on the macrophage phenotype switch (p < 0.05) [p < 0.005) [2D3. When knocking down VDR, there was a significant elimination of the 1,25-(OH) < 0.05) . An in v< 0.005) . In hype2-vitamin D-derived synthetic ligands inhibited renal fibrosis by reducing TGF-β/SMAD signaling [Vitamin D analogues may also exert antifibrotic effects by influencing TGF-β/Smad pathway. Activation of TGF-β/Smad signaling contributes significantly to both glomerular and tubulointerstitial fibrosis . In an aignaling . Vitaminignaling .By activating the p38MAPK signaling pathway, podocytes in DN may be structurally impaired . TubuloiTubular injury in DN may be treated by targeting the p38MAPK signaling pathway. Experimental evidence suggests that inhibition of p38MAPK may contribute to a preserved renal function and reduced albuminuria along with a reduction in tubulointerstitial lesions, including tubular atrophy, tubular cell apoptosis and increased interstitial volume in db/db mice . In DN, p < 0.01). Oxidative stress, characterized by the production of reactive oxygen species (ROS), is another crucial element in the pathophysiology of DN. ROS can stimulate the protein kinase B (Akt)/uncoupling protein 2 (UCP2) signaling pathway  and the −7 mmol/L 1,25(OH)2D3) in the high glucose group (30 mmol/L) resulted in a significant increase in SOD and a decrease in MDA (both p < 0.01) compared to the high glucose group without vitamin D supplementation. These results were significantly reversed (p < 0.01) after knocking down the VDR gene. Secondly, the effect of vitamin D on the mitochondrial membrane potential and apoptotic events was investigated. Treatment with vitamin D (1 × 10p < 0.01), which was reversed after vitamin D treatment (p < 0.01) or after knocking down the VDR gene (p < 0.01). The renoprotective effect of vitamin D therapy has also been studied via oxidative stress response pathways, such as the protein kinase B (Akt)/uncoupling protein 2 (UCP2) signaling pathway [A decrease in mitochondrial membrane potential indicates early apoptosis of the cells. In hyperglycemic conditions, there was a significant decrease in mitochondrial membrane potential ( pathway . An impop < 0.01). In contrast, UCP2 protein levels were significantly higher in hyperglycemic people than in healthy controls (p < 0.01). After treatment with vitamin D, there was a significant increase in VDR and p-Akt, and a decrease in UCP2 (p < 0.01). This effect was significantly reversed after knocking down the VDR gene. When Akt is dysfunctional, renal tubular apoptosis increases. UCP2, a mitochondrial carrier, regulates oxidative stress, mitochondrial membrane potential, and energetic cell processes. The protein levels of VDR and p-Akt were significantly lower in the hyperglycemic group compared to healthy controls and the high glucose group treated with vitamin D . The effect was partially reversed by treatment with vitamin D (p < 0.01). When knocking down the VDR gene, the vitamin D repression effect was partially inhibited (p < 0.01). The protection of the kidney against ROS injury by 1,25(OH)tic rats . A high In addition, in hyperglycemic conditions, there was a significant increase in p-JAK2, which reflects hyperglycemia-induced ROS. This effect was also decreased by vitamin D treatment. The same results were obtained for p-STAT1 and p-STAT3 levels. All these findings suggest that vitamin D treatment could exert its renoprotective role via a reduction in oxidative stress. Another possible mechanism is the inhibition of mesangial cell proliferation via the mammalian target of rapamycin (mTOR) pathway induced by DNA-damage-inducible transcript 4 (DDIT4) and tuberous sclerosis complex 2 (TSC2) protein . Diabete3, there was an increase in DDIT4 expression levels. In DN rats, there was also downregulation of TSC2 protein levels, a negative regulator of the mTOR pathway, which improved after treatment with 1,25(OH)2D3 (p < 0.05). On the other hand, the opposite pattern could be seen for p-Akt, which is a positive regulator of the mTOR pathway (p < 0.05) [The resultant increase in mTOR activity leads to enhanced cellular metabolism and growth and contributes to enhanced ECM expansion . mRNA an < 0.05) . p < 0.01). After treatment with 1,25(OH)2D3, there was a marked attenuation of RhoA mRNA and protein levels (both p < 0.05), as well as of ROCK activity compared to hyperglycemic conditions (p < 0.01).Vitamin D might also exert renoprotective effects by affecting the RhoA/Rho associated protein kinase (ROCK) pathway, which was investigated in an in vitro study with human renal proximal tubular cells . HyperglmTOR gene in the onset of DN [mTOR gene in DN rats was observed compared to diabetic controls (p < 0.05). Treatment with vitamin D, insulin, and the combination showed significant downregulation of the mTOR genes in diabetic rats (p < 0.05) but remained significantly higher than in the control group (p < 0.05). Next to the molecular mechanisms behind the development of DN, several studies investigated possible associated genetic underlying mechanisms. Investigating the role of the et of DN , a signiGFAT) gene in the renoprotective effects of vitamin D has been found [GFAT gene was significantly lower in diabetic rats treated with 20,000 IU/kg of vitamin D compared to healthy controls and diabetic rats without treatment (both p < 0.05). The gene expression of aldose reductase (AR) and advanced glycation end product (AGE) cellular receptor (RAGE) were not significantly different in this study.A role for the glutamine-fructose-6-phosphate transaminase 1 (en found . The expPTPN2) gene. This gene is responsible for the expression of PTPN2, also known as T cell protein tyrosine phosphatase (TCPTP), which is an anti-inflammatory cytokine. In vitro analysis with human acute monocytic leukemia cells (THP-1) showed that a high glucose environment evoked a significant inflammatory response  , VDR expression did not change significantly. One of the genes closely associated with VDR is the protein tyrosine phosphatase non-receptor type 2 (p < 0.001) and was significantly positive with VDR mRNA  and 25(OH)D3  [In serum and peripheral blood mononuclear cells (PBMCs) isolated from T2DM patients, the expression of PTPN2 correlated significantly but negative with UACR  . Both VDp < 0.05). After knocking down the PTPN2 gene, there was a further significant increase in inflammatory cytokines (p < 0.05). Even treatment with paricalcitol failed to exert anti-inflammatory effects after knocking down the PTPN2 gene. There was a significant upregulation of both VDR and PTPN2 expression in THP-1 cells treated with paricalcitol (0.2 ng/mL for 6 h) as well as a reduction of inflammatory cytokines (SOCS1), SOCS3, and type IV collagen) was explored in rat glomerular mesangial cells. All three genes were upregulated in hyperglycemic conditions (p < 0.01), and the expression was repressed after vitamin D therapy (p < 0.01). The relationships between four single nucleotide polymorphisms (SNPs) (Bsml (rs1544410), ApaI (rs7975232), TaqI (rs731236) and FokI) in the VDR gene and DN have recently been investigated [To further explain the effect of hyperglycemia on JAK/STAT signaling , the inf ApaI rs775232, Tastigated ,109.p = 0.008; p = 0.015, respectively). Even more, the BB + Bb genotype and the B-allele frequency were significantly higher in the early onset DN compared with the late-onset DN . This might suggest that the BsmI phenotype could be a risk factor for early-onset DN  [BsmI polymorphisms appear to be associated with DM2 and DN in the Chinese population. In patients with DM2, the BB + Bb genotype and B-allele frequency were significantly higher compared with controls (2–5.333) . VDR gene polymorphism and the development of renal nephropathy [p < 0.002) [VDR gene and the risk for DN1. The underlying biological mechanism of this process remains unclear. However, in a Tunesian case-control study with 100 patients with chronic renal failure in ESRD and 149 healthy subjects, no association was found between the BsmI hropathy . The F-a< 0.002) . The stuThe f allele represents the restriction site of the FokI gene. When the f allele is absent, the gene is transcribed into a shortened length resulting in dysfunction of the VDR gene. However, due to the complex interaction between genes and environmental factors and other genes, the result remains undetermined . In the VDR genotype polymorphisms showed that the BBFFAATt combination of VDR polymorphisms was significantly more frequent in the DN group compared to healthy controls . The BbFFAaTt polymorphism was significantly more frequent in the DN group compared to DM2 patients  [The ApaI polymorphism showed no significant correlation with DM2 or DN . The eff2D3 due to impaired 1α-hydroxylase activity [Vitamin D deficiency has been related to DM and its complications . As vitaactivity ,111. In activity ,49,50,51activity . Complementary, vitamin D deficiency is independent of the degree of albuminuria and is not a consequence of renal deterioration in DKD patients . In contDespite the fact that vitamin D shows a crucial role in the development of DKD in multiple animal and human studies, the possible renoprotective role of vitamin D in the process of developing DKD and reversal of already existing renal damage remains uncertain. Several interventional studies have published controversial results regarding this renoprotective effect. A potential underlying reason for these controversial results is the analytical variability and the lack of standardization of 25(OH)D assays. 2 and 25(OH)D3 based on isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), making it possible to standardize 25(OH)D measurements. The lack of assay standardization is the major source of bias, thus, making it impossible to pool research results to develop consensus cut-off points . The Vit2 and 25(OH)D3, cross-reactivity with 24,25(OH)2D, and matrix and/or patient-dependent biological variations [The introduction of an international Vitamin D standardization certification program (VDSCP) has led to an impressive improvement in the number of standardized 25(OH)D assays . Howeverriations .There are currently no recommendations about the optimal dosage or timing of vitamin D analogues in DN. There is a call for more large-scale, long-term, randomized, and controlled human studies evaluating the influence of vitamin D and its analogues on the development of DKD, the delay in the decline of eGFR, the progression to ESRD, and mortality. It would be interesting to investigate the role of vitamin D in the prevention of DN in diabetic patients with a high risk of development of microalbuminuria, based on a urinary proteomic risk classifier (CKD273) score . FurtherIn agreement with the National Kidney Foundation-Kidney Disease Outcomes Quality Initiative (NKF-KDOQI) publications concerning bone metabolism and disease in patients with CKD, vitamin D regimens might consist of supplementation with ergocalciferol, 50,000 IU, for four weekly doses, and then monthly for 5 months to reach 25(OH)D levels of ≥ 30 ng/mL . There aRegarding the genetics behind DN, multiple polymorphisms  have recently been investigated, although the exact impact on DKD development has not yet been determined. Future studies should focus on genetic factors influencing vitamin D concentrations in DM patients with CKD. Research into the interindividual variability in the metabolism of vitamin D and different responses to supplementation, which might result from genetic polymorphisms, could be of interest as well .


To determine the precise spatiotemporal expression dynamics of Lsamp, Opcml, Ntm isoforms, and Negr1, in the developing brain, we generated isoform-specific RNA probes and carried out in situ hybridization in the developing  and adult mouse brains. We show that promoter-specific expression of IgLONs is established early during pallial development (at E10.5), where it remains throughout its differentiation through adulthood. In the diencephalon, midbrain, and hindbrain, strong expression patterns are initiated a few days later and begin fading after birth, being only faintly expressed during adulthood. Thus, the expression of specific IgLONs in the developing brain may provide the means for regionally specific functionality as well as for specific regional vulnerabilities. The current study will therefore improve the understanding of how IgLON genes are implicated in the development of neuropsychiatric disorders.The members of the IgLON superfamily of cell adhesion molecules facilitate fundamental cellular communication during brain development, maintain functional brain circuitry, and are associated with several neuropsychiatric disorders such as depression, autism, schizophrenia, and intellectual disabilities. Usage of alternative promoter-specific  The first sign of vertebrate brain development is the appearance of the neural plate, which gives rise to neuroepithelial cells (NECs), the precursors of neural progenitor cells (NPCs). Early histogenetic processes of neural tissue formation encompasses massive proliferation and migration of progenitor cells, followed by neuronal differentiation together with neuritogenesis and axon guidance to appropriate targets for creating synaptic connections ,2,3. The1a and 1b), leading to transcripts that encode proteins with alternative N-terminal sequences. Other members, NEGR1 and IgLON5 have instead a single promoter . Mice were housed under standard laboratory conditions of a 12 hr light/dark cycle with lights on at 7:00 a.m., and they had ad libitum access to food  and water. Mice were mated and the presence of a vaginal plug was considered to be embryonic (E) day 0.5. Breeding and housing of the mice was conducted at the animal facility of the Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia. All animal procedures were performed in accordance with the European Communities Directive (86/609/EEC) and permit  from the Estonian National Board of Animal Experiments.Lsamp 1a and universal (uni) probes were prepared as described in [Ntm 1a (285 bp), Ntm 1b (500bp), Opcml 1a (492 bp), Opcml 1b (514 bp) and Negr1 (650 bp) transcripts were cloned from a cDNA pool of C57BL/6 mouse brain and inserted into pBluescript KS+ vector . We used the following primers (containing restriction sites):Ntm1a For TATAGCGGCCGCGAGTATGAGTGGAGATAATTACGGANtm1a Rev TATAGTCGACCTTGGAAGAGGCACAGAGCCNtm1b For TATAGCGGCCGCGCTGGATTCAACCCAGCCACNtm1b Rev TATAGTCGACGTGGGTACAAGGAATAGCAGCCOpcml1a For TATAGCGGCCGCGGTGTGCCCATGCGAAGCACOpcml1a Rev TATAGTCGACGGATGAAGAGCAGGGCAGTGOpcml1b For TATAGCGGCCGCTCCTTTCTGTCAGAGACACTTGCOpcml1b Rev TATAGTCGACTGGGTACAAGGAATAGCAGCCTGNegr1 For TATAGCGGCCGCATGGTGCTCCTGGCGCAGGNegr1 Rev TATAGTCGACCAGCCTGGTCCCTTGTAATTCCATThe ribed in . Mouse cThe mouse brains were dissected immediately after decapitation and fixed for 4 days in cold 4% PFA /PBS (pH 7.4). In newborn and adult mice, one temporal lobe was removed to allow better access to the fixative. The mouse brains were cryoprotected for 2 days in 30% sucrose  in 4% PFA/PBS, and stored at −80 °C until sectioning. The non-radioactive in situ hybridization on 40-µm free-floating mouse  brain cryo-sections using digoxigenin-UTP -labeled RNA probes was performed as described in .For the whole-mount in situ hybridization, the embryos were dissected in cold PBS and fixed in cold 4% PFA/PBS (pH 7.4) for 4 days. Fixed embryos were washed with PBS containing 0.25%, Tween 20  (PBST), followed by dehydrated in 25%, 50%, 75% methanol  in PBST, and 100% methanol for 5 min at each step. Thereafter, the samples were rehydrated with methanol/PBST in reverse series, PBST washing and treatment with 10 µg/mL of proteinase K  in PBST for 10 min is followed by washing with 2mg/mL of glycine  in PBST. Embryos were then refixed in 4% PFA/0.2% glutaraldehyde  for 20 min. After washing with PBST embryos were incubated in a prehybridization solution containing 50% formamide /5X SSC (pH 5) overnight at 65 °C with gentle agitation.For hybridization RNA probe (500ng/mL) was added to the prehybridization solution and incubated over 3 days with gentle agitation at 65 °C. Samples were washed as follows: thrice with 1% SDS/5X SSC/50% formamide for 30 min at 65 °C; thrice with 2X SSC/50% formamide for 30 min and then overnight at 65 °C; thrice with TBST  for 10 min each and incubated two days at 4 °C in anti-DIG-AP-conjugated antibody /TBST. Unbound antibody was removed by three 10 min and five 60 min washes in TBST. Embryos were washed thrice with NTMT  for 10 min followed by incubation in the staining solution .For cutting 50-µm vibratome sections, the stained  embryos were inserted into 1 mL of 0.5 % gelatine Sigma Aldrich, Taufkirchen, Germany/30 % BSA Sigma Aldrich, Taufkirchen, Germany/20 % sucrose /PBS, wherein 140 µL of 25 % glutaraldehyde  was added immediately before insertion and incubated for 10 min. The sections were mounted into 70 % glycerol and micro photographed using an Olympus BX61 microscope equipped with an Olympus DX70 CCD camera .Lsamp 1b promoter was performed as described previously [Free-floating sections were stained overnight for X-Gal staining for detecting the distribution of eviously . AlternaBrains of adult mice were dissected and fixed in 4% PFA for 4–5 days followed by cryoprotection with 30% sucrose in PBS, frozenand sectioned at 40 µm. Free-floating coronal sections from the cerebellar paraflocculus region were permeabilized, blocked and immunostained with primary antibodies: mouse anti-Lsamp , goat anti-Opcml , mouse anti-Negr1 , mouse anti-Ntm  followed by secondary antibodies: Rhodamine (TRITC)-AffiniPure Donkey Anti-Mouse IgG (H+L)  and Rhodamine Red-X-AffiniPure Fab Fragment Rabbit Anti-Goat IgG (H+L) antibody . Nuclei were stained with 5 μg/mL Bisbenzimide H 33258 /PBS for 15 min. Subsequently, sections were washed with PBS and mounted with Fluoromount mounting medium , and covered with a 0.17-mm coverslip . Specificity of the immunohistochemistry was determined by incubations without the primary antibodies. Fluorescent images were obtained with the Olympus FV1200MPE  laser scanning confocal microscope. Abbreviations in all the figures representing anatomical data have been adopted from the mouse brain atlas .In conclusion, we have shown that IgLON members are involved in, and perhaps orchestrate, the early events of pallium development. The complexity of the spatiotemporal distribution patterns of the IgLONs is intimately linked to regional brain organization and to the emergence of functional distinctions in the developing nervous system. Distinct isoform-specific expression domains and gradients suggest the importance of IgLONs alternative promoter usage in helping coordinate the complexly integrated functions of neural cell proliferation, differentiation, and morphogenesis into the more specialized substructures.Importantly, IgLON expression patterns (1) may be responsible for creating the anatomical and functional boundaries between regions (which may then be stabilized by other molecules), (2) may help stabilize pre-existing boundaries between anatomical and functional regions, or (3) may be a consequence of the anatomical and functional regions, perhaps acting as tumor suppressing inhibitors of cell proliferation and apoptosis. Indeed, it would not be surprising if IgLONs were performing different roles in different brain regions. The IgLON superfamily may have evolved the alternative promoter system to produce necessary mRNA isoforms from single gene loci in the specific histogenetic fields at specific times.


T. aestivum L.) as the most grown crop worldwide is of utmost importance for global food security. Thousand kernel weight (TKW) in wheat is closely associated with grain architecture-related traits, e.g. kernel length (KL), kernel width (KW), kernel area (KA), kernel diameter ratio (KDR), and factor form density (FFD). Discovering the genetic architecture of natural variation in these traits, identifying QTL and candidate genes are the main aims of this study. Therefore, grain architecture-related traits in 261 worldwide winter accessions over three field-year experiments were evaluated.The future productivity of wheat . Further analysis reveals the four novel candidate genes potentially involved in more than one grain architecture-related traits with a pleiotropic effects e.g. TraesCS6A01G383800 gene on 6A encoding oxidoreductase activity was associated with TKW and KA. The allelic variation at the associated SNPs showed significant differences betweeen the accessions carying the wild and mutated alleles e.g. accessions carying C allele of BobWhite_c5872_589, TraesCS6A01G383800 had significantly higher TKW than the accessions carying T allele. Interestingly, these genes were highly expressed in the grain-tissues, demonstrating their pivotal role in controlling the grain architecture.Genome-wide association analysis using 90K SNP array in FarmCPU model revealed several interesting genomic regions including 17 significant SNPs passing false discovery rate threshold and strongly associated with the studied traits. Four of associated SNPs were physically located inside candidate genes within LD interval e.g. BobWhite_c5872_589  found to be inside These results are valuable for identifying regions associated with kernel weight and dimensions and potentially help breeders in improving kernel weight and architecture-related traits in order to increase wheat yield potential and end-use quality.The online version contains supplementary material available at 10.1186/s12870-021-03183-3. T. aestivum L.), the main source of protein and calories in human food is a major aim to ensure global food security. By 2050, the world population will reach 9800 billion and the annual rate of food demand will reach 1.6% surpassing the current annual genetic gains of this crop [Meeting the globally increasing demand for wheat  but new efforts are required exploring different environments and germplasm from different origins in order to enhance grain yield and quality.Exploration of crop genetic resources is useful to recognize sources of variation for agronomic and physiological traits and discovery of new alleles for improving yield potential, their components as well as grain quality traits , 29, 30.ectively , 34, 35,ectively , 36–38 aectively . Genome-ectively –43. Up tPpd-A1) at 2A (TRITD2Av1G019250), TRITD4Bv1G171270 at 4B encoding a Big Grain 1 protein, and two other candidate genes at 6B (TRITD6Bv1G005370 and TRITD6Bv1G005450) encoding an acid β-fructofuranosidase have been detected within QTL for KL and TKW in recombinant inbred lines (RILs) population [TRITD1Bv1G118820, TRITD2Av1G189400 and TRITD7Bv1G173200 encoding auxin response while TRITD4Bv1G175480 involved in auxin signaling and TRITD3Av1G012070, encoding for a Flavin-containing monooxygenase as an auxin biosynthesis. Moreover, candidate genes encoding for cytochrome P450 were found in most of the TKW QTL [In addition, most of what is known about genetic control of TKW had been carried out using bi-parental populations which present some limitations , 38, 44.TraesCS4A02G229100 which is an auxin-regulated gene involved in organ size and TraesCS4A02G2 corresponding to a polygalacturonase involved in carbohydrate metabolic process increasing kernel size and TKW [Other candidate genes as  and TKW  were fouTherefore, to gain deeper insights into the genetic basis of grain architecture-related-traits that could be of interest for the future improvement of grain yield and quality, a GWAS was undertaken in a diverse winter wheat panel of 261 genotypes tested during 3 years. For this purpose, 17,093 SNPs markers with recently known physical position were used to detect the most significant and effective SNP and for the identification of candidate genes underlying the studied traits. Our results showed 17 highly associated SNPs across 9 chromosomes of which four multi-traits associated SNPs were reported on chromosomes 1B, 2A, 5B, and 6A. These SNPs were located within the physical positions of candidate genes which are potentially associated with grain architecture-related traits and potentially involved in improving grain weight and size.The markers were distributed within the whole genome. The highest coverage of markers (51.5%) was on genome B with 8809 SNPs, genome A was covered by 38.6% of the whole markers with 6595 SNPs, while D genome presented the lowest coverage, 9.9% (1689 SNPs). The homoeologous group 1 chromosome had the highest number of SNPs (17.96%), while the chromosomes of group 4 presented only 7.02%. Chromosome 5B harboured the highest number of SNPs with 1784 markers, while chromosome 4D held only 46 SNPs , 146 genotypes from Eastern Europe-Western Asia (55.6%), and 42 North-American accessions (16%) Table . HeatmapG × Y (environment) interactions (p < 0.001) , 13.10–21.92 mm2 (2017), 12.81–21.75 mm2 (2018) and 13.26–21.52 mm2 (BLUEs). KDR ranged from 1.58 to 2.15 (2016); 1.60 to 2.15 (2017); 1.58 to 2.07 (2018) and 1.59 to 2.11 (BLUEs). BLUEs values for FFD ranged from 1.66 to 2.13. FFD varied between 1.58–2.12 (2016), 1.63–2.12 (2017) and 1.66–2.13 (2018)  varied from 31.06 to 60.16 g, showing ranges of 29.08 to 61.11 g (2016), 31.15 to 60.37 g (2017), and 31.04 to 63.05 g (2018) as shown in Table 8) Table . Boxplot8) Table .Fig. 1BP < 0.001) and positive correlation with KA (0.91), KW (0.82), KL (0.78), FFD (0.68) and a weaker association with KDR (0.13). The KA revealed a strong correlation with KL (0.90), KW (0.84) while weaker associations were detected for KDR (0.24) and FFD (0.33). The KL and KW presented a positive correlation (0.55), while a negative association was reported for KW and KDR (− 0.31) and KDR with FFD (− 0.14)  compared to those from Central and Northern Europe   and North America which presented the lowest values   presented higher TKW (48.18 g) and KA   was annotated as a protein of unknown function DUF1677 in rice (O. sativa). The candidate gene TraesCS2A01G136800 is located on chromosome 2A  and has a role in Heat shock protein DnaJ, cysteine-rich domain. The alleles of SNP Excalibur_c12169_1088  which is located within the interval of the previous candidate gene did not show a significant effect on the associated traits as shown in Fig. TraesCS5B01G274000 ; the carrying alleles are C and T where allele C affect significantly KA and KL (p < 0.05)  and encodes an oxidoreductase activity, acting on a sulfur group of donors, NAD(P) as acceptor. The SNP BobWhite_c5872_589 located within the gene  held C and T alleles, where allele C affected TKW significantly (p < 0.01) but had no significant effect on KA.The candidate gene TraesCS6A01G383800 shows the highest expression in most of the grain tissues  while genes TraesCS1B01G303200 and TraesCS2A01G136800 also showed high values. For their part, TraesCS5B01G274000 gene had very low expression compared with the other three but showed high expression values for embryo proper  which annotates Gliadin/LMW glutenin/Bifunctional inhibitor/plant lipid transfer protein/seed storage helical domain (Table Sp < 0.01) but had no effect on KA compared to allele T (Data not shown).Interestingly in Chromosome 1A, the SNP TA001286–0611-w showed a significant association with TKW and a weaker association with KA LOD = 3.18).18. This10 > FDR). FDR threshold was above than -log10 > 4.2 for the studied traits. Therefore, we reduced the number of spurious associations by only considering those SNPs, which exceeded the FDR. Markers were identified on chromosomes 1A (1), 1B (2), 2A (2), 2B (3), 3A (1), 4B (1), 5B (4), 6A (2), 6B (1)  threshold (−log (2), 2B , 3A (1),(1) Fig. .Fig. 3M10 > 5). The most significant marker (−log10 = 6.91) was Excalibur_c12169_1088 located in chromosome 2A and affected this trait by + 2.176 g (R2 = 5.2%). The phenotypic variation explained by markers (R2) ranged between 3.2 to 5.2% , 2A (1), 2B (2), 6A (1) and 6B (1). Four of these markers located on chromosomes 2A, 2B, 6A, and 6B showed highly significant associations , Excalibur_c12169_1088 on chromosome 2A  and wsnp_Ex_c1600_3051075 on chromosome 1B  were the most significant ones detected. Marker effects oscillated between 1.9 to 2.6% , 2A (1), 2B (1), 3A (1), 4B (1), 5B (1) and 6A (1) were reported. Six of these MTAs located on 1B, 2A, 2B, 3A, 5B and 6A showed -log2 reached values of 2.0 to 2.2%.Three MTAs related to kernel length were detected on chromosome5B  that showed high effects in these traits. R10, MAF, effect, related candidate genes, and their functions are shown in Supplementary Table No MTAs were detected for FFD, KDR and KL when the -log10 > FDR threshold was used. Conversely, several MTAs were reported between the -log10 > 3 and -log10 < FDR range: 5 (KA), 2 (FFD), 19 (KDR), 3 (KA), 1 (TKW), 23 (KL). Information about SNP detected for each trait, their physical position, −logFor all of these markers associated with the studied traits, we detected the closest candidate genes based on the physical position within local blocks of LD. Our results showed that four MTAs across four chromosomes are associated with more than one trait. Those MTAs were detected on 1B (TKW-KA), 2A (TKW-KA), 5B (KA-KL) and 6A (TKW-KA)  which can be used in further analysis.Our analysis detected 17 marker-trait associations related to grain architecture traits across 9 chromosomes, indicates that these are quantitative traits under polygenic control as was reported . These SIn this study, TKW among 261 winter wheat genotypes showed variability from 31 to 60 g and 7 MTAs related to this trait were detected on chromosomes 1A, 2A, 2B, 5B, 6A, and 6B. Previous studies identified QTL on chromosomes 1A , 2A 9, , 51, 2B In the same way, extensive genotypic variation and significant G × E interactions were reported for KA, KL, KW, FFD, and KDR. Although previous studies reported QTL associated with KA , 51, 56,The identification of underlying genes with annotations related to the grain architecture traits provides further reliability for the MTAs identified in the current study. Although high number of candidate genes have been detected, of which four HC candidate genes reported in our work have shown effects in other agronomical traits, none of them have been documented as effective on the size, shape, and weight of the grain and can be considered novel.TraesCS1B01G303200 (chromosome 1B) that annotated as Protein of unknown function DUF1677 (O. sativa) showed its influence on TKW and KA. Even though Gerard et al. [In this work, we found that the allelic variation at the locus of the gene d et al.  documentTraesCS2A01G136800 on chromosome 2A has a role in Heat shock protein (HSP) DnaJ, a cysteine-rich domain, which is a major protein in response to stresses  which is the most common domain of many nucleotide-binding protein folds. The energy from NTP hydrolysis is typically utilized to induce conformational changes in other molecules, which constitutes the basis of the biological functions of most P-loop NTPases. P-loop NTPases show substantial substrate preference for either ATP or GTP [The candidate gene P or GTP . AlthougTraesCS6A01G383800 (chromosome 6A) encodes oxidoreductase activity, acting on a sulfur group of donors, NAD(P) as acceptor. It has been reported that GSH-dependent protein-disulphide oxidoreductase (TPDO) increases the activity in a period of maximum synthesis of storage proteins in wheat grains, which is the third week after anthesis. There is a correlation between TPDO activity in maturing grains and dough extensibility as the enzyme reduces SS bonds in high-molecular-weight polymers [The last identified candidate gene polymers . This hipolymers  but no aTraesCS1A01G007200 gene located on chromosome 1A associated with TKW and with a weaker association with KA has a nutrient reservoir activity molecular function and annotates Gliadin/LMW glutenin//Bifunctional inhibitor/plant lipid transfer protein/seed storage helical domain. It is known that gliadin and glutenin proteins have a major role in grain quality determination [As was previously described, we found that the mination . Its rolmination , 74, itsTraesCS6A01G383800, respectively, showed the importance of allele C in increasing TKW, where most of the accessions carried allele C originated mainly from Central-Northern European countries.The highly significant SNPs within the candidate genes were analyzed using statistical comparison of alleles at each SNP with the associated traits. This approach have been used recently to explore the alleles for enhancing agronomic traits and abiotic tolerance , 75, 76.To our knowledge, our study involves the first GWAS analysis performed by FARM-CPU algorithm to identify several key genomic regions associated with TKW and five other grain architecture traits in a new and wide winter wheat panel consisting of 261 genotypes from 30 countries during three years. Genome-wide association analysis using 90 K SNP array revealed many MTAs including 17 highly associated SNPs across 9 chromosomes of which four multi-traits associated SNPs were found on chromosomes 1B, 2A, 5B, and 6A. We reported four novel candidate genes related to these traits showing high expression values in different grain tissues.Future studies should deepen the relationships between the function of these genes and their effect on grain architecture. These results will be valuable for identifying regions associated with kernel weight and its dimensions and could be useful for providing further insights for increasing grain quality, milling performance, and grain yield.Institute of Field and Vegetable Crops (IFVCNS), Novi Sad, Serbia and parental lines of Western European hybrid breeding programs were also included in this set  over three consecutive years  with three replicated blocks in 5 m2) and TKW (g) using the MARVIN Digital Seed Analyser . In addition, to evaluate the differences in grain density and the deviation of a shape from a cylindrical form, the factor form density (FFD) and kernel diameter ratio (KDR) were calculated according to Giura and Saulescu [After harvesting, 200 random kernels of each plot (per accession and year) were used to assess kernel size traits including, kernel length , kernel width , kernel area  for each trait over years was calculated using the following equation, all these calculations were accomplished using Genstat 19:σg2, σgy2, and σe2 are mean squares for genotype, genotype × year (environment) interaction and residual error, respectively, y is the number of years and r represent the replicates. Summary statistics across different years were corroborated by mean, minimum, maximum, standard deviation and coefficient of variation using Genstat 19 software. A correlation matrix between traits and boxplots of each trait and differences among geographical regions were performed using MVApp v2.0.Analysis of variance (ANOVA) for all measured traits was performed and broad-sense heritability (The restricted maximum likelihood (REML) algorithm was applied to calculate the Best Linear Unbiased Estimators (BLUEs) for each trait in each accession across the years by considering the accession as a fixed effect and the environment as a random using Linear and Nonlinear Mixed Effects Models (nlme) package in R .http://www.traitgenetics.com) the 90 K iSELECT chip [http://www.wheatgenome.org/, IWGSC RefSeq v1.1) and then used to determine the population structure, linkage disequilibrium (LD) and for GWAS calculation. The genome-wide pairwise estimates of LD were calculated as a squared correlation between pairs of polymorphic SNPs (r2) for the whole-genome using GenStat 18 [2) vs. the distance between pairs of polymorphic SNPs (Mbp) using R-package GGPLOT2 [2 = 0.2 is approximately 2.0 Mbp and we used this window to discover the candidate genes with a reasonable distance as suggested by 3. The genetic relationships among genotypes as the population structure were revealed by principal component analysis (PCA), using Genomic Association and Prediction Integrated Tool (GAPIT 3) package in R [The panel was genotyped by TraitGenetics GmbH  were considered as significant marker-trait associations (MTAs). FDR was calculated for each trait separately at the significance level of 0.01 and used in further analyses as recommended by Alqudah et al. [GWAS was performed using BLUEs values in each accession for each trait applying different statistical models through GAPIT in R. FARM-CPU model was applied by considering the random effect model (REM) and the fixed effect model (FEM) iteratively and associated markers as a cofactor that empowered us to avoid any false-negative and control the false positive associations by preventing model overfitting , 84–86. h et al. .http://www.wheatgenome.org/, IWGSC RefSeq v1.1). Because, each block of LD contains high number of candidate genes, we have selected the candidate genes which have SNPs within their physical positions. WheatMine platform (https://urgi.versailles.inra.fr/WheatMine/begin.do) was used to search for the gene ontologies (GO) and InterPro number and description. The underlying genes were further examined for their association with grain architecture traits using previously published literature.Those highly significant MTAs based on their physical positions were further used to identify the high-confidence (HC) putative candidate genes, based on their physical positions within the LD ± 2 Mbp window, using the reference genome sequence of Chinese Spring by blasting against IWGSC RefSeq annotation v1.1 (http://www.wheat-expression.com/).In-silico gene expression analysis in different grain tissues of the multi-traits candidate genes was analyzed using RNA-Seq expression data from Wheat Expression Browser , Kernel length (KL), Kernel width (KW), Kernel area (KA), Kernel diameter ratio (KDR) and Factor form density (FFD) in an experiment with 261 wheat genotypes evaluated during 3 years. Table S3. Significant marker-trait associations and candidate genes for grain architecture traits in 261 winter wheat genotypes.Additional file 2: Table S4. List of candidate genes within the most important four LD blocks.Additional file 3: Figure S1. Origin of the genotypes of the panel. Figure S2. Single nucleotide polymorphism (SNP) density on 21 wheat chromosomes. The x-axis shows the interval distance in Mb. Figure S3. Screen plot for the first ten principal components (PCs) explains the variation using 90K chip and Population structure based on their origin. Figure S4. Kinship heat map of the panel. Figure S5. The linkage disequilibrium decay in the wheat population. Figure S6. Phenotypic variation and Heritability (H2) for Thousand-kernel weight (TKW), Kernel length (KL), Kernel width (KW), Kernel area (KA), Kernel diameter ratio (KDR) and Factor form density (FFD) using BLUEs values. Figure S7. Variation on Thousand-kernel weight (TKW), Kernel length (KL), Kernel width (KW), Kernel area (KA), Kernel diameter ratio (KDR) and Factor form density (FFD) based on the origin of genotypes. C-N Europe ; E Europe-W Asia (Eastern Europe-Western Asia); N America (North America). Figure S8. Overview of significant markers trait associations identified for Thousand-kernel weight (TKW), Kernel length (KL), Kernel width (KW), Kernel area (KA), Kernel diameter ratio (KDR) and Factor form density (FFD) using BLUEs values. Multitraits MTAs are indicated with gray rectangles.