REC8 genes have been characterized in some plant species, little is known about them in Gossypium. In this study, 89 REC8 genes were identified and analyzed in 16 plant species (including 4 Gossypium species); 12 REC8 genes were identified in Gossypium. hirsutum, 11 in Gossypium. barbadense, 7 in Gossypium. raimondii, and 5 in Gossypium. arboreum. In a phylogenetic analysis, the 89 RCE8 genes clustered into 6 subfamilies (I–VI). The chromosome location, exon-intron structure, and motifs of the REC8 genes in the Gossypium species were also analyzed. Expression patterns of GhREC8 genes in various tissues and under abiotic stress treatments were analyzed based on public RNA-seq data, which indicated that GhREC8 genes might have different functions in growth and development. Additionally, qRT-PCR analysis showed that MeJA, GA, SA, and ABA treatments could induce the expression of GhREC8 genes. In general, the genes of the REC8 gene family of cotton were systematically analyzed, and their potential function in cotton mitosis, meiosis, and in response to abiotic stress and hormones were preliminary predicted, which provided an important basis for further research on cotton development and resistance to abiotic stress.Cohesin is a ring-shaped protein complex and plays a critical role in sister chromosome cohesion, which is a key event during mitosis and meiosis. Meiotic recombination protein REC8 is one of the subunits of the cohesion complex. Although  REC8 gene is required for correct meiosis. During meiosis in Saccharomyces cerevisiae and Schizosaccharomyces pombe, REC8 was found in the cohesion complex, which has homology to SCC 1/Mcd 1/Rad 21 proteins [REC8 mutations in S. cerevisiae and S. pombe disrupt the cohesion of sister chromosome arms and centromeres, resulting in reduced recombination, affecting the formation of the synaptonemal complex and causing premature separation of sister chromatids during the first meiotic division [Caenorhabditis elegans [Arabidopsis thaliana [Zea mays [Drosophila [Xenopus laevis [Oryza sativa [Citrullus lanatus [C. elegans, RNAi of REC8 leads to univalent formation and chromosomal fragmentation at diakinesis [Arabidopsis, syn1/dif1 plants exhibit defects in chromosome cohesion and condensation during meiosis, resulting in chromosome fragmentation and the formation of microsporophyll [AFD1 affect leptotene chromosomes [Cohesion, a muti-subunit protein complex, is essential for sister chromatid cohesion and chromatin loop structure in mitosis and meiosis . The losproteins ,6,7. RECdivision ,7,8,9. Sdivision , Caenorh elegans , Arabidothaliana ,13, Zea Zea mays , Drosophosophila , Xenopuss laevis , Oryza sa sativa , and Cit lanatus . In C. eakinesis . In Araborophyll ,20. In momosomes . Differeomosomes  but alsoomosomes , organizomosomes , and homomosomes . It is aomosomes .REC8 genes have been identified in many species; however, few studies on their functions in cotton have been reported, especially in the mechanism of fertility and the development process. In the present study, 89 REC8 genes in 16 species were identified and analyzed. Among them, 35 cotton REC8 genes were characterized, and structural diversity, phylogenetic relationships, the chromosomal distribution, and expression levels in different tissues and under different abiotic stresses were investigated. Our research provides insights that will help develop a comprehensive understanding of the biological functions of the cotton REC8 gene family in fertility and responses to various stresses.Cotton is not only a fiber and oil crop but also a high-protein food crop and important strategic material. Homologous recombination that takes place during meiosis (using the principle of homologous recombination) is a critical process for breeding and developing new varieties with traits for high yield, disease resistance, and ornamental appeal, so the study of genes related to the meiosis process is of great biological and economic significance . REC8 geG. arboretum , G. raimondi , G. hirsutism , and G. barbadense  were downloaded from COTTONGENE (http://www.cottongen.org/ (accessed on 30 June 2022)) and the Cotton Functional Genomics Database (https://cottonfgd.org/ (accessed on 29 June 2022)). The Arabidopsis REC8 genome file was obtained from The Arabidopsis Information Resource ). Genomic data for Zea mays, Oryza sativa, Sorghum bicolor, Ananas comosus, Vitis vinifera, Populus trichocarpa, Theobroma cacao, Glycine max, and Amborella trichopoda were downloaded from the Ensemble database (http://plants.ensembl.org/index.html (accessed on 6 August 2022)). Genome sequence data of Volvox carteri and Chlamydomonas reinhardtii were downloaded from the National Center for Biotechnology Information (NCBI).Genome sequences file of −5). Candidate amino acid sequences were determined using the Hmmsearch program and Conserved Domain Database (CDD) in NCBI to search for the REC8 domain (PF04824 and PF04825) with default parameters. REC8s in Zea mays, Oryza sativa, Sorghum bicolor, Ananas comosus, Vitis vinifera, Populus trichocarpa, Theobroma cacao, Glycine max, and Amborella trichopoda were identified following the same process.We used AtREC8 amino acid sequences as query sequences to search the four cotton species’ protein databases for candidate sequences by the blastp program ), and the subcellular localization of these genes were predicted by Cell-PLoc (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ (accessed on 4 January 2023)).Then, the physical and chemical properties of https://itol.embl.de/upload.cgi (accessed on 26 October 2022)).The REC8 protein sequences from 16 species were aligned using MUSCLE, and the neighbor-joining (NJ) phylogenetic tree of 16 species was constructed using MEGA 7 , with a G. arboretum, G. raimondii, G. hirsutum, and G. barbadense) was obtained from the reference cotton database. The distribution of REC8 genes on the four cotton chromosomes was drawn using TBtools [REC8 genes. The collinearity map was drawn by using Basic CIRCOS in TBtools (v1.108).The chromosome location information of four cotton species  and synonymous substitution rates (Ks) were calculated using TBtools software (v1.108). A Ka/Ka ratio > 1 means positive selection, a Ka/Ka ratio = 1 indicates neutral selection, and a Ka/Ka ratio < 1 represents negative selection.CDS sequences of cotton http://memesuite.org/ (accessed on 25 June 2022)) with the p-value of each motif on each protein being lower than 1 × 10−5. The exon–intron structure of REC8 genes was analyzed using TBtools. Phylogenetic trees with motif, domain, and gene structure were visualized using TBtools.The conserved motifs of REC8 proteins were identified using Multiple Em for Motif Elicitation (MEME) (REC8 genes were extracted. cis-acting elements were then predicted by PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 28 September 2022)). A phylogenetic tree with identified elements was visualized using TBtools.The 2000 bp upstream promoter sequences of REC8 genes under different tissues and stress , the transcriptome data were downloaded from the Cotton Omics Database (http://cotton.zju.edu.cn/10.rnasearch (accessed on 2 July 2022)) [To analyze the expression pattern of y 2022)) . The heaG. hirsutum cv. Coker 312 (R15) was grown in a chamber . Plants at the third-leaf stage were treated with 100 μM MeJA, 2 mM SA, and 200 μM ABA. The leaves of treated plants were collected at 0 h, 3 h, 6 h, and 12 h and were frozen in liquid nitrogen immediately for further analysis.GhUBQ7 was used as the internal reference gene, and the data were analyzed using the 2−∆∆Ct method [Total RNA was extracted from the frozen leaves using RNAprep Pure Plant Kit . HiScript III Reverse Transcriptanse  was used to convert RNA to cDNA. The qPCR analysis was performed using Taq Pro Universal SYBR qPCR Master Mix  on the ABI 7500 Fast Real-Time PCR system . Three biological replicates were performed in all analyses. t method . The priREC8 genes in the 4 cotton species and the other 12 species, we selected candidate genes from a search and comparison using HMMsearch and blastp programs. Then, the obtained sequences were filtered via CDD. Listed in REC8 genes found in G. hirsutum, 11 in G. barbadense, 7 in G. raimondii, 5 in G. arboretum, 3 in A. comosus, 4 in Arabidopsis, 7 in maize, 4 in rice, 4 in A. trichopoda, 9 in P. trichocarpa, 6 in G. max, 5 in S. bicolor, 7 in T. cacao, 3 in V. vinifera, 1 in V. carteri, and 1 in C. reinhardtii. Twice as many REC8 genes were found in G. hirsutum and G. barbadense as in G. raimondii and G. arboretum, which indicated that the tetraploid cotton is obtained by hybridizing the diploid A genome and the diploid D genome, followed by chromosome doubling. In addition, the characteristics of REC8 proteins in cotton were analyzed, including the protein sequence size, molecular weight (MV), isoelectric point (pI), and instability coefficient    with 3 mA repair , and los meiosis ,33. GhREREC8 genes on the chromosomes. One GaREC8 gene was on each of three chromosomes , and two were on Chr13 (GrREC8 genes were mapped on each of two chromosomes (Chr02 and Chr09), and Chr13 had three . The G. barbadense genome had 11 GbREC8 genes among 7 chromosomes .Based on the genomes of the four cotton species, we analyzed the location of the on Chr13 a. Two Grad three b. The chrbadense c,d. The REC8 orthologs in cotton, we performed a synthetic analysis on four cotton species. A total of 35 REC8 genes of cotton have synthetic relationships, including 12 from G. hirsutum, 11 from G. barbadense, 7 from G. raimondii, and 5 from G. arboretum . The results showed that the number of orthologous gene pairs between G. hirsutum and A. thaliana and T. cacao was 2 and 5, respectively  (GhREC8-04-D/GbREC8-04-D and GhREC8-06-D/GbREC8-03-D) were larger than 1, indicating that these gene pairs experienced rapid evolution. A total of 23 gene pairs had a Ka/Ks ratio less than 0.5, and 22 gene pairs were between 0.5 and 1, leading us to hypothesize that these gene pairs have undergone strong purifying selection.We calculated the Ka/Ks ratio of 47 duplicated imondii) . The resREC8 genes in cotton, we analyzed the sequence structure, motif, and conserved domains of cotton REC8 genes based on the phylogenetic tree  in subclade II had 22 exons, 1 (GhREC8-04-D) had 11 exons, and the other genes had 12 exons. In subclade III, 4 REC8 genes in cotton  and 1 in A. thaliana (AtREC8-04) had 13 exons, and 3 in cotton  had 8 exons , MeJA (methyl jasmonate), SA , and ABA (abscisic acid)) and stress responses  and development were predicted using the PlantCARE tool. Promoters of 14 genes contained an IAA-responsive element, 17 contained a GA-responsive element, 16 contained an SA-responsive element, 20 contained an ABA-responsive element, and 19 contained a MeJA-responsive element. Among stress elements found in the REC8 promoters, low-temperature response elements were found in promoters of 24 REC8 genes, drought response elements in those of 18, and defense and stress response elements in those of 15. Seven genes  involved in low-temperature and drought responsiveness were found  were highly expressed in almost all tissues and stages. GhREC8-04-D and GhREC8-05-A were similar in being highly expressed during ovule development in leaves, roots, and stems. There were high levels of GhREC8-05-D expression in anthers, bracts, the filament, pistils, and torus. Compared with fiber development, GhREC8-02-D showed relatively higher expression in other issues and stages. These results indicated that most GhREC8 genes were expressed in vegetative tissues.To evaluate the potential role of the 12 nd torus . Six GhRREC8 genes in response to abiotic stresses in upland cotton, we used public transcriptome data to analyze the effect of stresses from high temperatures, low temperatures, PEG, and salt and treatment with hormones  peaked by 6 h after treatments, and two (GhREC8-01-A and GhREC8-05-D) were downregulated. GhREC8-04-D was upregulated after MeJA and SA treatments, with respective maximum expression at 3 h and 6 h  belonging to the Rad21 gene family in A. thaliana have been identified. SYN2/4 are involved in mitosis, while SYN1 is essential for meiosis [SYN3 is essential for megagametogenesis [SYN1 (REC8) lead to male and female sterility in A. thaliana [Osrec8 mutant had abnormal homologous chromatid pairing and telomere bouquets [ClREC8 in watermelon [REC8 genes in 4 cotton species, and tetraploid cotton had twice more REC8 genes than diploid cotton, which reflected the fact that tetraploid cotton descended from the hybridization of A- and D-genome ancestors. Phylogenetic tree analysis showed that GhREC8 genes  were in the same clade as SYN1-4  and OsREC8-02(OsREC8) may be used for cloned gamete production in cotton.Heterosis is widely used in agriculture and has made a great contribution to world food security. The artificial creation of apomiesis may be another effective way to achieve heterosis fixation. The production of cloned gametes is one of the key steps in artificial apomiesis. In previous studies, the bidopsis ,40. REC8Arabidopsis and four cotton species (REC8 genes (REC8 gene family members. The promoters of REC8 genes contained cis-acting elements associated with phytohormone, stress, and defense responses (GhREC8 genes under MeJA, SA, and ABA treatments, and most were upregulated after hormone treatments, especially after SA treatment, indicating these genes might be involved in hormone regulation. ABA could interact with the JA and SA signal pathways [GhREC8 genes were induced by MeJA, they may be related to fertility (GhREC8 expression. Most genes were not affected by the abiotic stresses, indicating that these genes did not have an important role in plant responses to these stresses. The expression of GhREC8-01-A, GhREC8-05-A, and GhREC8-05-D, however, was sharply induced by PEG (REC8 genes likely had multiple functions in the hormone and stress responses and development of plants. Our next step is to study the role of the REC8 genes in cotton fertility and responses to stresses, which may contribute to the achievement of apomiesis and a further understanding of the mechanisms of resistance to stresses.Rad21/Rec8 proteins from different species share a conserved N-terminal domain and C-terminal domain, which are involved in the interaction with Smc1-Smc3 heterodimer during cohesion establishment ,41. Simi species c. MoreovC8 genes d, suggesesponses . Then, wpathways . JA actspathways ,44. Becaertility . The abid by PEG , and theREC8 family genes in 16 species, and these genes were divided into 6 subfamilies. Tissue-specific expression pattern analysis indicated that GhREC8 genes were expressed variously in cotton, indicating that they played different roles in cotton growth and development. An analysis of promoter cis-elements and expression levels under abiotic stresses and hormone treatments showed that GhREC8 genes responded to different stimuli. This study provides valuable information about the cotton REC8 gene family, which lays a foundation for further revealing the biological roles of REC8 genes in cotton meiosis, mitosis, and stress adaptation.In this study, we identified 89


Dear Editor,BMAL1 , CLOCK (clock circadian regulator), PER1/2/3 (period circadian regulator 1/2/3) and CRY1/2 (cryptochrome circadian regulator 1/2)  . Accumultor 1/2) .in situ survey (in situ survey) and mouse gut microbiome, and then further investigated the possible effects of metabolite involved in the function on the host.Gut microbes also have their own circadian rhythm and recent advances in mice reveal that the composition, function, and biogeography of gut microbiota undergo circadian oscillations, which are controlled by the timing of food intake and diet compositions and have the potential to influence the gastrointestinal tract and other organs by bacterial antigens and metabolites , 2016. Su survey , which iHelicobacter bilis and in terms of metabolic pathways, S-adenosyl-l-methionine cycle I, methylerythritol phosphate pathway I, l-lysine biosynthesis III, and seleno-amino acid biosynthesis showed significant differences between day and night  and nocturnal mice (night), and reversely in the sleep period  and PER2 (S-adenosyl-l-methionine (SAM) . Among these, only 471 were shared by cells across groups and a systematic increase in modified sites after methionine treatment  , we utilne (SAM) , and we reatment . Also, 6reatment . Then, treatment  and 1I.AHCY (adenosylhomocysteinase) gene , protein product of which was recently reported to be essential for cyclical H3K4 trimethylation and recruitment of BMAL1 to chromatin and promoting subsequent circadian transcriptional activity  > 1). Then, many rhythm-related genes with increased m6A (29/900) and 5mC  levels were identified by querying in GO databases . More imactivity . Likewised cells . AltogetAnother important reported consequence of circadian rhythm disruption  is the accumulation of intestinal reactive oxygen species (ROS), a major detrimental substance causing cellular oxidative damaging and necrosis . AdditioWe then tested the antioxidant activity of methionine in a sleep-deprived (SD) mouse model. We employed four groups of mice, one control group with normal diet and sleep-wake cycle , and three SD groups with dietary methionine manipulation  subjected to 3 days of SD in a restriction chamber with a constantly and gently rotating bar   . Firstly2O2 level of intestine, liver and brain and observed a similar effect pattern of dietary methionine in the examined tissues except for brain as that of ROS in the small intestine  staining, in which we found that, the SD mice  showed significantly increased intestinal ROS level, in line with previous report . Furtherntestine . Thus, mPer2, Per3, and Dbp (D-box binding PAR bZIP transcription factor). Enrichment analysis demonstrated that small intestines of SD mice had multiple biological processes related to circadian rhythms up-regulated  > 2, Padj < 0.05) and deleterious microbes, such as Influenza A virus and Dorea spp increasing. Moreover, potentially harmful microbes, like Lactobacillus murinus, a species which has been reported to related to intestinal dysbiosis and biotin deficiency  ; overallof genes . Consistof genes . Then, wl levels . Here, wl levels , thus itpwac021_suppl_Supplementary_MaterialClick here for additional data file.


Pericytes play an indispensable role in various organs and biological processes, such as promoting angiogenesis, regulating microvascular blood flow, and participating in immune responses. Therefore, in this review, we will first introduce the discovery and development of pericytes, identification methods and functional characteristics, then focus on brain pericytes, on the one hand, to summarize the functions of brain pericytes under physiological conditions, mainly discussing from the aspects of stem cell characteristics, contractile characteristics and paracrine characteristics; on the other hand, to summarize the role of brain pericytes under pathological conditions, mainly taking ischemic stroke as an example. Finally, we will discuss and analyze the application and development of pericytes as therapeutic targets, providing the research basis and direction for future microvascular diseases, especially ischemic stroke treatment. Later, 1.1.Pericytes are mainly distributed around capillaries and microvessels, extending along capillaries, pre-capillary arterioles and post-capillary venules, but are not commonly found in lymphatic capillaries . They ar1.2.Previously it was impossible to accurately distinguish between pericytes and other vascular-associated cells because light and electron microscopies were the only technologies capable of visualizing these cells, limiting the information obtained. With the emergence and development of fluorescence microscopy confocal microscopy and genetic tools (such as fate lineage tracing), more molecular markers have been discovered for identifying pericytes, such as PDGFRβ, neuroglial antigen 2 (NG2), melanoma cell adhesion molecule (CD146), etc . Under nin vitro culture etc. Therefore, the most advanced pericyte identification relies on well-preserved tissue morphology and a combination of two or more pericyte markers ; (b) 4 wall cell subtypes , including 1,564 pericytes and 418 smooth muscle cells (SMCs); (c) 3 perivascular fibroblast subtypes with different marker genes (n = 793); (d) 4 vascular-coupled (vc) neuron and glial cell subtypes , including vc-neurons, vc-oligodendrocytes, vc-astrocytes and vc-microglia NG2(+) αSMA(+) pericytes form small clusters and multicellular spheres and differentiate to express neuronal and glial cell markers, eventually fully differentiating into neural lineage cells . Wojcinschnology . Similarchnology . Under ichnology .3.2.2+), reactive oxygen species, regulatory factors, and cellular proteins. We will also discuss the contractile characteristics of brain pericytes from these aspects , α-SMA, vimentin, desmin, myosin, nestin) and cytoskeletal proteins , regulator of G-protein signaling-5 (RGS5), CD146). An increase in the ratio of F- to α-actin not only strengthens the submembrane actin lattice but also promotes actin cross-bridging by extending the length of α-SMA filaments transmitting the generated contractile force to the extracellular matrix to contract blood vessels . Subsequ aspects .3.2.1.2+ is closely related to the rhythmic contraction of blood vessels  and SK(Ca)] channels cause vascular dilation and induce endothelial cell membrane surface hyperpolarization. This hyperpolarization is conducted along endothelial cells through homocellular gap junctions and transmitted to overlying SMCs and pericytes through heterocellular gap junctions. Then this hyperpolarization affects the contraction of smooth muscle or pericytes by controlling the activity of voltage-gated calcium ion channels ) channels are present in both vascular smooth muscles and dense capillary networks composed of capillary endothelial cells and pericytes. This ion channel can robustly control the membrane potential of vascular cells  is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infects host cells by binding to the transmembrane receptor angiotensin converting enzyme 2 (ACE2) through the viral Spike (S) protein. Studies have found that ACE2 expression in human brain vascular pericytes increases after they are exposed to the S protein, which is accompanied by enhanced expression of contractile and myofibrogenic proteins, such as α-SMA, fibronectin, type I collagen and neurogenic locus notch homolog protein-3 (NOTCH3). The enhanced expression of these proteins causes profound changes in pericyte phenotype and is associated with cell contractile morphology . As ment3.3.In the brain, pericytes actively communicate with neurovascular unit cells and make fine adjustments in response to stress stimuli . They in3.3.1.During CNS development and vascular remodeling, pericytes can regulate vascular budding, vascular stabilization, and vascular maturation . The proDuring the process of vascular budding, pericytes  and endothelial cells  secrete VEGF-A activating the VEGFR2 pathway on endothelial cells promoting endothelial cell migration, thereby promoting vascular budding . Local r3.3.2.Pericytes can control the expression of tight and adherens junction proteins, the arrangement of tight junction proteins and vesicle endocytosis at the blood–brain barrier, most of which depend on molecular pathways between endothelial cells and pericytes . As summ3.3.3.in vivo and ex vivo organ culture situations  can induce mouse brain microvascular pericytes to release NO, IL-12, IL-13, IL-9, IL-10, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, eosinophil chemotactic factor (eotaxin), chemokine ligand (CCL)-3, and CCL-4 among other cytokines , amyotrophic lateral sclerosis (ALS) and type 2 diabetes mellitus (T2DM)-associated microvascular and retinal lesions. Pericyte dysfunction is also associated with human immunodeficiency virus (HIV)-associated dementia, epilepsy, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) with subcortical infarcts and leukoencephalopathy, and brain cancer . It has 4.1.Stroke is the second leading cause of death and disability, accounting for 5.2% of global death. In 2016, there were 13.7 million new cases of stroke and approximately 87% of them were ischemic stroke patients. Among them, at least 10–20% are due to occlusion of large arteries . Accordi4.2.In the CNS, vascular pericytes interact with endothelial cells to regulate the formation of tight junctions at the BBB. Under normoxic conditions, pericytes improve the endothelial integrity by inducing occludin and ZO-1 mRNA expression through angiogenin-1. Under hypoxic conditions, pericytes reverse the reduction in occludin protein expression and secrete GDNF which upregulates claudin-5 expression, thereby promoting brain or peripheral nerve regeneration .2 concentration, they have a positive effect after prolonged exposure and have more effective barrier protection function than astrocytes at 0.1% O2 concentration (Although pericytes accelerate barrier destruction in the short term at 1% Ontration . Pericytntration . Within ntration . All of 2 concentration) and VEGF (10 ng/mL) treatment significantly increase Ang1 mRNA expression, which reaches the maximum level 2 h after starting the treatment, followed by attenuation. The expression of angiogenin-2 (Ang2) mRNA does not change, but Tie2 mRNA is significantly upregulated, which persists for 12 h  gene which serves as the main form of VEGF receptor. Overexpression of VEGF not only promotes EC proliferation and budding but also promotes recruitment of pericytes by increasing n-cadherin expression on brain microvessels, enhancing endothelial cell coverage by pericytes thereby promoting formation and maturation of new blood vessels, increasing the density of capillaries and the blood flow in the infarct zone, reducing the infarct volume, mitigating inflammation after middle cerebral artery occlusion, enhancing integrity of the blood–brain barrier after ischemia, thereby improving brain tissue survival . The TGFfor 12 h .2 and pH 6.5 acidification have no such effect. When the FGF receptor (FGFR) inhibitor SU5402 and PDGFR inhibitor Sunitinib are used, bFGF-induced pericyte proliferation and migration are significantly inhibited  pericytes to express stem cell/undifferentiated cell markers and subsequently differentiate into various neural cells, including phagocytic microglia, thereby exerting neuroprotective effects . The PDGnhibited . Since pnhibited .2+ concentration in the cytoplasm of pericytes after stroke activates Ca2+ gated anion channel TMEM16A, promoting chloride ion efflux, depolarizing the cell membrane, opening voltage-gated calcium channels, thereby resulting in a large increase in intracellular calcium ions and pericyte contraction  are recruited by SDF-1/CXCR4 axial endothelial cells and induced primarily by TGFβ to become pericytes, which are typically involved in supporting vascular functions and tumor growth  is a highly lethal vascular brain tumor with rapid progression and frequent recurrence . Despiter growth . Studiesr growth . Therefor growth . Zhang er growth . Mechanir growth . Fibrotis system .6.Pericytes play a crucial role in neurological diseases and they may be therapeutic targets for a variety of diseases, including stroke, glioblastoma, traumatic brain injury, migraine, epilepsy, spinal cord injury, diabetes, Huntington’s disease, AD, multiple sclerosis, glioma radiation necrosis, and ALS . We beliJF: Visualization, Writing – original draft, Investigation, Methodology, Software. HL: Visualization, Writing – original draft, Writing – review & editing. PY: Data curation, Investigation, Visualization, Writing – review & editing. ZW: Investigation, Methodology, Software, Visualization, Writing – original draft. PZ: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The present study was supported by a First-Class Construction Project of Medical Key Discipline of Yangpu District (YP19ZA08). The funder has no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Conflicting studies suggest these drugs promote the differentiation of neural progenitors to oligodendrocyte lineage, while others report antipsychotics inhibit the proliferation and differentiation of oligodendrocyte precursors. Here, we utilised in-vitro (human astrocytes), ex-vivo (organotypic slice cultures) and in-vivo (twitcher mouse model) experimental study designs of psychosine-induced demyelination, a toxin that accumulates in Krabbe disease (KD), to investigate direct effects of antipsychotics on glial cell dysfunction and demyelination. Typical and atypical antipsychotics, and selective D2 and 5HT2A receptor antagonists, attenuated psychosine-induced cell viability, toxicity, and morphological aberrations in human astrocyte cultures. Haloperidol and clozapine reduced psychosine-induced demyelination in mouse organotypic cerebellar slices. These drugs also attenuated the effects of psychosine on astrocytes and microglia and restored non-phosphorylated neurofilament levels, indicating neuroprotective effects. In the demyelinating twitcher mouse model of KD, haloperidol improved mobility and significantly increased the survival of these animals. Overall, this study suggests that antipsychotics directly regulate glial cell dysfunction and exert a protective effect on myelin loss. This work also points toward the potential use of these pharmacological agents in KD.The role of altered myelin in the onset and development of schizophrenia and changes in myelin due to antipsychotics remains unclear. Antipsychotics are D Schizophrenia is a chronic condition involving the complex interplay between genetic and environmental factors and is thought to be associated with aberrant neurodevelopment ,2. Schiz2 receptor subtype antagonism and atypical antipsychotics have additional serotonin  receptor antagonism [2 receptors, of which expression levels increase following combined oxygen and glucose deprivation [2 receptors can affect myelin formation by regulating the development and function of oligodendrocytes [2 receptor antagonists, studies show that D2 agonists increase the number of oligodendrocyte progenitor cells and protect oligodendrocytes against oxidative injury [2 receptor agonists. Moreover, studies suggest the protective effect of D2 agonists can be diminished by D2 antagonists, suggesting that D2 receptor activation may play an important role in oligodendrocyte protection against injury, whereas typical antipsychotics may restrict oligodendrocyte recovery from injury. Contradictory, haloperidol and clozapine prevented apoptotic cell death in oligodendrocytes that were cultured under glucose-deprived conditions, highlighting their potential protective effects [Antipsychotic treatment of patients with schizophrenia has also been suggested to directly regulate the expression of genes associated with oligodendrocyte maturation and myelination, thus influencing white matter integrity ,6. The etagonism . It has rivation . Dopamindrocytes ,15. In adrocytes . Paradoxe injury , which m effects .galc gene, which codes for the enzyme galactosylceramidase (GALC). Psychosine  is a toxic lipid that accumulates abnormally in those with KD. It has been shown to be toxic to oligodendrocytes and cause demyelination by various proposed mechanisms [Krabbe disease (KD), also known as globoid cell leukodystrophy, is a rare autosomal recessive disease caused by mutations in the chanisms . Given tCompounds were reconstituted in dimethyl sulfoxide  at stock concentrations of 5 mM and 20 mM depending on solubility, and were as follows: Clozapine , Olanzapine , Amisulpride , Quetiapine , Risperidone , Aripiprazole , Haloperidol , Chlorpromazine , Sulpiride  Eticlopride  and Volinanserin . Psychosine  was prepared as 10 mM stock concertation in DMSO. Working concentrations from stock compounds were made using serum-free media prior to treatments. Primary antibodies (1:1000 dilution) were mouse anti-vimentin , chicken anti-glial fibrillary acidic protein , mouse anti-myelin oligodendrocyte glycoprotein , rabbit anti-myelin basic protein , chicken anti-neurofilament H , rabbit anti-ionized calcium binding adaptor molecule 1  and mouse anti-neurofilament H non-phosphorylated . Secondary antibodies (1:1000 dilution) were goat Alexa-549 anti-mouse , goat Alexa-633 anti-chicken  and goat Alexa-488 anti-rabbit . Hoescht (4 µM) was used to stain cell nuclei .6 per well and cultured for 24 h until greater than 80% confluent. For MTT assays, after treatment, media was removed and replaced with 100 µL of fresh serum-free media supplemented with 10 µL of 12 mM MTT Formazan  and plates were incubated for 2.5 h at 37 °C. Subsequently, 75 µL of media was removed, and 50 µL of DMSO was added per well. Cells were incubated for ten minutes, and the absorbance was read at 540 nm. For LDH assays, cytotoxicity was measured on aliquots of the cellular supernatant using the CyQUANTTM LDH Cytotoxicity Assay Kit following manufacturer instructions. LDH activity was measured using 490-nm absorbance. The immunocytochemistry and fluorescent microscopy of human astrocytes was carried out as we have outlined previously [Human astrocytes  were cultured exactly as we have described previously ,26,27,28eviously ,26,27,28® , and the edges were closed with varnish. Slides were stored in the dark until imaged. Images of slices were acquired using scanning confocal microscopy  at 10× and 20× magnification. The mean fluorescence for each treatment group was analysed by examining regions of interest (ROI). Image analysis was carried out using ImageJ (https://imagej.nih.gov/ij/) and Imaris® software.Organotypic cerebellar slice cultures were prepared exactly as we have described previously ,29,30,31www.transnetyx.com) using real-time PCR. Haloperidol was given at a dose of 1 mg/kg/day in drinking water. Mice were kept in grouped cages, had constant access to food and water and were under a 12-h light/dark timetable. Humane endpoints, weight measurements, behavioural observations, twitching, immobility and locomotor scores were all as per established protocols previously published by our group [All animal work was carried out in compliance with EU legislation approved by Trinity College Dublin ethics committee and in accordance with guidelines from the Health Products Regulatory Authority (HPRA) under project authorisation number AE19136/P123. A colony of heterozygous twitcher mice obtained from Jackson Laboratory, Cambridge, UK  were maintained for breeding under pathogen-free conditions. Ear punch samples were genotyped by TransnetYX  were fixed at p < 0.05 *, p < 0.01 **, p < 0.001 *** and p < 0.0001 ****.Statistical analysis was performed using GraphPad Prism 9 , and the values shown are means +/− standard error of the mean. The sample size was calculated for one-way ANOVA by referencing previously published astrocyte psychosine toxicity experiments by our group ,23,24,312 and 5HT2A receptors, except aripiprazole which is a partial agonist (2 (Eticlopride) and 5HT2A (Volinanserin) were used to determine their effects on psychosine-induced toxicity. MTT  C57BL/6J mice, cultured for 12 DIV and treated with or without psychosine in the presence or absence of haloperidol or clozapine, with analysis conducted at 14 DIV a. Psychop values denoted as *). At PND 25, haloperidol showed no significant effect in twitcher mice on any measurement tested. At PND 28 and PND 30, haloperidol treatment induced subtle but significant  improvements in distance (m) .The typical antipsychotic haloperidol was given at a dose of 1 mg/kg/day to twitcher mice starting from PND5 onwards a. Halopeance (m) f; mean sance (m) g; time mance (m) i; centreance (m) j and corance (m) k, but noance (m) h in twitance (m) l nditions , which enditions , regulatnditions , activatnditions  and altenditions , enhancenditions  and redunditions . Contrasnditions . (iii) Mnditions ,43,44. Anditions , while onditions . Lastly,nditions  and cyclnditions  have beeIn this study, we used psychosine-induced in vitro and in vivo models to investigate the effects of antipsychotics on glial cell dysfunction and demyelination. Psychosine is a toxin that aggregates in the brains of those afflicted with the neurodegenerative disorder globoid cell leukodystrophy, Krabbe disease (KD) ,50. KD iin-vitro human astrocyte studies relied on MTT assays of cell viability, and there are confounding factors that can influence such assays [ex-vivo slice culture experiments used the cerebellum, as it is rich in white matter and myelin-producing oligodendrocytes, and this area has been shown to be prone to psychosine-induced toxicity in previous studies [We acknowledge that our h assays . To addr studies ,23,24,292 antagonist (Eticlopride) and 5HT2A antagonist (Volinanserin) reduce psychosine-induced toxicity and morphological changes in human astrocytes, with inhibition at D2 receptors showing stronger efficacy. We also show that haloperidol and clozapine attenuate psychosine-induced demyelination in cultured organotypic cerebellar slices and reduce psychosine-induced loss of GFAP and vimentin expression. Regarding microglia, in agreement with previous studies [Here we show that psychosine  induces cell toxicity and reduces cell viability in human astrocytes, in agreement with previous studies ,23,24,29 studies ,23,24,29 studies ,23,24,29The main points presented here are the following: (i) antipsychotics with dopamine and serotonin antagonism directly regulate glial cell dysfunction and exert a protective effect on myelin loss in cell cultures and organotypic cerebellar slices, and (ii) haloperidol, an antipsychotic with mainly dopamine antagonism improved survival and had a positive effect on behaviour in twitcher mice, an established demyelinating murine model of KD. Taken altogether, our findings indicate that antipsychotics are myelin protective and suggest the potential therapeutic benefit of agents with similar antipsychotic pharmacology in Krabbe disease.


Despite the increasing global population of individuals with intellectual and developmental disabilities (IDD), this population remains especially vulnerable to health disparities through several factors such as a lack of access to sufficient medical care and poor determinants of health. To add, numerous studies have shown that healthcare professionals are still insufficiently prepared to support this population of patients. This review synthesizes the literature on current pre-graduate IDD training programs across healthcare professions with the goal of informing the creation of evidence-based curricula.Four major databases were searched for current pre-graduate IDD training interventions for healthcare professionals. The Preferred Reporting Items for Systematic Reviews and Meta-Analysis flow diagram and the Best Evidence Medical Educations systematic review guide were used to frame our collection and analysis.Of the 8601 studies screened, 32 studies were identified, with most studies involving medical students (50%). Of note, 35% of studies were interprofessional. Most interventions utilized multiple pedagogical methods with a majority including clinical experiences (63%) followed by theoretical teaching (59%). Kirkpatrick levels showed 9% were level 0, 6% were level 1, 31% were level 2A, 31% were level 2B, 19% were level 3, 3% were level 4A, and none were level 4B.There is a paucity of formally evaluated studies in pre-graduate health professional IDD education. As well, there are a lack of longitudinal learning opportunities and integration into formal curriculum. Strengths identified were the use of multimodal approaches to teaching, including interprofessional approaches to optimize team competencies.The online version contains supplementary material available at 10.1186/s12909-023-04259-4. Persons with intellectual and developmental disability (IDD) are vulnerable to health disparities. Lack of access to sufficient medical care, poor determinants of health, and exclusion from public health and preventive care are all related to poor health outcomes in this population. Various reports highlight gaps in healthcare for persons with IDD globally –3. DespiUnfortunately, studies on formal pedagogical structures directed at health providers in IDD care are limited. In a systematic review of post-graduate medical training in intellectual and developmental disabilities a paucity of objectively evaluated research in this area and a potential for specialized, interprofessional, competency-based education programmes were highlighted . While tThis study aimed to conduct a systematic review to describe the characteristics and educational outcomes of recent pre-graduate IDD training across various health care professions. The purpose of our review was to synthesize the literature on current pre-graduate IDD training interventions across healthcare professions, with the goal of informing the creation of evidence-based curricula.Our aim was to synthesize the literature on current pre-graduate IDD training interventions for healthcare professionals. To do so, we used the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) flow diagram and the Best Evidence Medical Education (BEME) systematic review guide to frame our collection and analysis.The literature was first searched on June 21st 2021, followed by a second search on March 8th, 2023 to provide the latest findings. Ovid and Webofscience were used to search the literature. In particular, Ovid was used to search the Medline, Embase and Psychinfo databases. The search was conducted using subject keywords “or” combinations of student*, trainee*, interprofessional*, and healthprofession* with “or” combinations of developmental disab*, intellectual disab*, ASD, autis*, learning disab*, mental retard*, asperger* with “or” combinations of education*, curricu*, and training. The search was limited to English language, peer-reviewed articles published from 2011 to current, to account for recent and relevant interventions only. Following the initial search, articles of interests’ references were scanned for additional publications.Studies were included if they were an educational intervention aimed at improving IDD knowledge, skills, self-efficacy, and/or attitudes for any group(s) of pre-graduate health professional trainees. Pre-graduate health professional trainees were defined as trainees within their pre-licensing years of a professional program. Interprofessional interventions that included graduate health professionals or other areas of study were included if they included pre-graduate health professionals as well. No sample size cut-off was employed as the relative paucity of work in this research area was expected. Those studies that included an intervention but had no formal evaluation outcomes, and that evaluated learner’s attitudes, knowledge, skills, and/or self-efficacy without a corresponding intervention were excluded.The initial database search identified 8601 studies in which, after removing duplicates, reviewing titles and abstracts for relevance yielded 249 articles. After applying inclusion and exclusion criteria, and searching reference lists of significant articles, 32 papers were included in the final review. Study flow is outlined in Fig. With guidance from all other authors, one author (L.V.) analyzed the core papers, and extracted data from the 32 studies into a table classifying data according to (i) year of publication, (ii) country of origin, (iii) pre-graduate training speciality, (iv) learner level of participants, (v) instructor type, (vi) setting of instruction, (vii) timeline, (viii) pedagogical approach, (ix) focus of content, (x) evaluation method (xi) evaluation outcomes, (xii) Kirkpatrick level, and (xiii) BEME quality of evidence score. Additional file Instructor data was classified into the following categories: (1) faculty members, (2) non-faculty professionals, (3) patients, parents, or caregivers, (4) senior students, and (5) unclear. The setting of intervention was classified as: (a) specialized clinical setting, (b) non-specialized clinical setting, (c) non-clinical setting, (d) clinical setting , and (e) unclear. Next, the timeline of the intervention was classified as: (i) single session, (ii) short-term, less than 1 month, (iii) 1–3 months, and (iv) longitudinal of longer than 3 months. The pedagogical approach was classified as: (1) experiential, sub-stratified into (a) patient/family experiences, (b) clinical, (c) workshops, (2) theoretical, and (3) interprofessional. The focus of content was classified as: (i) perspective/awareness, (ii) medical/clinical knowledge, and (iii) unclear.Evaluation methods were organized as such: (1) intervention evaluation , (2) participant evaluation , (3) learning assessment (assessment of knowledge/skills/perspective gained following intervention), and (4) other. The evaluation outcomes were synthesized from each study, and Kirkpatrick and BEME gradings were applied to all studies. The Kirkpatrick classification was chosen as it has been commonly applied to the evaluation of health professional education programs . The KirA summary of study characteristics is available in Tables Table The majority of the studies were conducted in the United States of America 44%, 14/32) %, 14/32 , 53, 54,With regards to trainee demographics, most of the studies were specifically targeted towards medical students 50%, 16/32) [ 32, 37,, 51–53. rd years  [nd years  [th years  [st years  [As for trainees’ year in their respective programs, the results were varied with the most studies including 3rd years %, 10/32 , 10/32) , 48, 50,%, 9/32) –50, 53, %, 7/32) , 47, 49,nd years %, 9/32 [%, 5/32) , 51, 53.%, 5/32) , 52, 54 %, 5/32) , 41–44.Many of the interventions included faculty members 72%, 23/32) [/32 23–2, 48–54 aThe majority of interventions were single sessions 38%, 12/32) –44, 50. %, 12/32 As for the setting of intervention, the majority included non-clinical settings 75%, 24/32) –52, 54, /32 –52, Most of the studies used experiential approaches to teaching 88%, 28/32). Experiential activities typically included a clinical experience  , 52, 53,/32. ExpeA variety of evaluation methods were used to assess intervention outcomes. Participant evaluations of their own learning were overwhelmingly used 84%, 27/32) , 43–54. /32 , 43–As for study outcomes, the Kirkpatrick model was applied to evaluate the outcomes of the educational interventions. Our review produced a mean and median of 2.16 and 2.5, respectively (if 2A = 2.0 and 2B = 2.5). In order of scoring, 9% 3/32) , 33, 45  , 33, 45Our BEME evidence-based scoring system review produced a mean and median of 3 and 3, respectively. We graded 16% 5/32) , 38, 45  , 38, 45Through this systematic review, we aimed to summarize the literature surrounding pre-graduate healthcare professional training in IDD. Our analysis has brought forward several points of importance in IDD curriculum design. In particular, we saw that many of the highest BEME scores , 52, 53 We found that the majority of interventions were a single session intervention  –44, 50. Interestingly, an interprofessional approach to education was found amongst several studies. The mean BEME scores and Kirkpatrick levels for these interventions were 2.9 and 2, respectively. While these scores reflect some gains in knowledge and/or perspective, this was less than expected given the promising literature on interprofessional education and improvements in educational outcomes –60. A poFurthermore, it is essential to analyze our findings through a critical disability lens to ensure a comprehensive and equitable interpretation. Critical disability studies view disability as both a lived reality in which the experiences of people with disabilities are central to interpreting their place in the world, and as a social and political definition based on societal power relations . InclusiThe findings of our study were limited by the inclusion of only English publications, despite an international scope. As well, we excluded studies that did not describe a clear intervention. For instance, we excluded a review of Australian medical schools’ IDD education over 20 years as it focused on summarizing the current curriculum to inform revision, rather than a discussion of intervention characteristics or educational outcomes . MoreoveIn conclusion, this review of IDD curricula in international pre-graduate health professional.education has provided an overview of published interventions and highlighted several trends. First, the literature in this field supports the use of multimodal approaches to achieve greater educational outcomes. Program developers can consider the use of multiple pedagogical methods in IDD curricula. Second, many interventions were single-session, pilot studies. There is a need for longitudinal learning opportunities and consistency through integration into formal curricula, which should also be formally evaluated. Third, interprofessional components to education are increasingly being used. Future studies can integrate team competencies and its evaluation along with IDD self-efficacy outcomes. Finally, while interventions frequently involved patients and caregivers in their design and implementation, these experiences were seldom situated within the larger systemic disparities faced by patients with IDD. To strengthen this approach, future studies could adopt a critical disability lens to gain deeper insights into patients' lived realities and to advocate for systemic change. In summary, there is an increased need for formal, effective IDD education for healthcare professionals. It is especially important that this education be directed at the level of pre-graduate training to equip health care professionals with the skills and attitudes to care for those with IDD before entering the workforce.Additional file 1: Table S1. Summary of reviewed literature on IDD teaching in pre-graduate health professional training.


B1-type ATPases related to the intracellular transport and detoxification of metals. However, due to a lack of information regarding the HMA gene family in the Cucurbitaceae family, a comprehensive genome-wide analysis of the HMA family was performed in ten Cucurbitaceae species: Citrullus amarus, Citrullus colocynthis, Citrullus lanatus, Citrullus mucosospermus, Cucumis melo, Cucumis sativus, Cucurbita maxima, Cucurbita moschata, Cucurbita pepo, and Legenaria siceraria. We identified 103 Cucurbit HMA proteins with various members, ranging from 8 (Legenaria siceraria) to 14 (Cucurbita pepo) across species. The phylogenetic and structural analysis confirmed that the Cucurbitaceae HMA protein family could be further classified into two major clades: Zn/Co/Cd/Pb and Cu/Ag. The GO-annotation-based subcellular localization analysis predicted that all HMA gene family members were localized on membranes. Moreover, the analysis of conserved motifs and gene structure (intron/exon) revealed the functional divergence between clades. The interspecies microsynteny analysis demonstrated that maximum orthologous genes were found between species of the Citrullus genera. Finally, nine candidate HMA genes were selected, and their expression analysis was carried out via qRT-PCR in root, leaf, flower, and fruit tissues of C. pepo under arsenic stress. The expression pattern of the CpeHMA genes showed a distinct pattern of expression in root and shoot tissues, with a remarkable expression of CpeHMA6 and CpeHMA3 genes from the Cu/Ag clade. Overall, this study provides insights into the functional analysis of the HMA gene family in Cucurbitaceae species and lays down the basic knowledge to explore the role and mechanism of the HMA gene family to cope with arsenic stress conditions.The heavy-metal-associated (HMA) proteins are a class of P Cucurbita (squash and pumpkins), Cucumis (cucumbers and melons), Citrullus (watermelons), and Lagenaria (bottle gourd), are among the ten most economically significant vegetable crops in the world, while numerous others have regional significance [Cucurbits are one of the major crop families with high economic value and are widely cultivated worldwide. Four genera, ificance . Neverthificance ,3.Arsenic (As) is a non-essential metalloid ubiquitous in soil at low levels; however, agricultural soils are threatened by toxic contamination from anthropogenic activities, leading to excessive accumulation of arsenic . Its pre1B-type ATPase, also known as the heavy metal ATPase (HMA), which belongs to the large P-type ATPase family, plays an important role in heavy metal transport [Plants have developed various adaptation strategies to protect themselves from harmful environmental conditions, including accumulating and transporting heavy metals and metalloids . Membranransport ,11,12.2+ and Zn2+, and distribute non-essential heavy metal ions, including Cd2+, Co2+, and Pb2+. Typical HMA proteins contain the E1–E2 ATPase domain and a haloacid dehalogenase-like hydrolase domain. Additionally, both sides of the N-terminal and C-terminal metal-binding sites may possess one or more soluble metal-binding domains (MBDs) that interact with or bind to specific metal ions [1B-type ATPases, a heavy-metal-associated regulatory domain [1B-ATPase group and the Zn/Co/Cd/Pb P1B-ATPase group [HMAs transport essential metal ions required for plant growth and development, such as Cutal ions ,13,14. Ty domain ,16. Basese group .Arabidopsis thaliana [Oryza sativa [Populus trichocarpa [Glycine max [Zea mays [Sorghum bicolor [Hordeum vulgare [Linum usitatissimum [Brassica napus [Pyrus bretschneideri [Morus alba [Medicago truncatula [Fagopyrum tataricum [A. thaliana, AtHMA1 is involved in exporting Zn from the chloroplast [AtHMA3 enhances tolerance and accumulation of Cd, Zn, Pb, and Co in plants [SpHMA3 in Sedum plumbizinccicola has been reported to confer Cd hyper-tolerance [OsHMA5 is involved in the shoot translocation of Cu2+ in rice [OsHMA1 and OsHMA3 are involved in the Zn and Cd transport, respectively [CsHMA3 and CsHMA4 have been associated with transporting cadmium, lead, and zinc from the root to the stem [HMA genes have been identified in the model and non-model plants with a different number of genes and diversification patterns, including thaliana , Oryza sa sativa , Populuschocarpa , Glycinecine max , Zea mayZea mays , Sorghum bicolor , Hordeum vulgare ,23, Linutissimum , Brassicca napus , Pyrus bhneideri , Morus arus alba , Medicaguncatula , and Fagataricum . The funoroplast , while tn plants . Likewisolerance . Moreove in rice , whereasectively . Furtherthe stem .Pteris vitatta [Citrullus amarus, Citrullus colocynthis, Citrullus lanatus, Citrullus mucusospermus, Cucumis melo, Cucumis sativus, Cucurbita maxima, Cucurbita moschata, Cucurbita pepo, and Legenaria siceraria. HMA genes were identified and studied in terms of their chromosomal location and synteny, phylogeny, conserved motifs, structure, and expression profiles in different tissues of C. pepo under copper treatments, a recognized heavy metal related to the HMA family, as well as their expression under arsenic stress conditions. Therefore, our results provide insights for future investigations into the roles of HMA genes in Cucurbitaceous plants and other species.Although, HMA genes play a vital role in heavy metal transmembrane trafficking in different plants . To date vitatta . NeverthC. amarus, C. colocynthis, C. lanatus, C. mucusospermus, C. melo, C. sativus, C. maxima, C. moschata, C. pepo, and L. siceraria), the Basic Local Alignment Search Tool (BLAST) of the Cucurbit genomics database v2  was employed using all the Arabidopsis HMA genes as queries [https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 15 October 2022) were used. Sequences with no HMA-related domains were excluded from a further analysis. Moreover, genomic, coding sequence (CDS), and promoter region sequences were downloaded from the CuGenDB for confirmed genes. Further, physiochemical parameters such as molecular weight, polypeptide length, and theoretical isoelectric point (pI) value were calculated using ExPASy ProtParam software . The identified HMA genes were named CamHMA1-CamHMA10; CcoHMA1-CcoHMA9; ClaHMA1-ClaHMA10; CmuHMA1-CmuHMA9; CmeHMA1-CmeHMA10; CsaHMA1-CsaHMA9; CmaHMA1-CmaHMA12; CmoHMA1-CmoHMA12; CpeHMA1-CpeHMA14; and LsiHMA1-LsiHMA8 using the prefix “Cam”, “Cco”, “Cla”, “Cmu”, “Cme”, “Csa”, “Cma”, “Cmo”, “Cpe”, and “Lsi” for C. amarus, C. colocynthis, C. lanatus, C. mucusospermus, C. melo, C. sativus, C. maxima, C. moschata, C. pepo, and L. siceraria, respectively, followed by “HMA” for the heavy-metal-associated domain and lastly, the progressive number according to their chromosome number and chromosomal positions. A. thaliana HMA sequences were downloaded from the Arabidopsis Information Resource .For the identification of members of the HMA gene family in Cucurbit species  in each chromosome where an HMA gene was found. Moreover, an exon–intron gene structure analysis was carried out by submitting merged General Feature Format (GFF3) files of Cucurbitaceous plants to the Biosequence structure tool in TBtools [Physical chromosome location data for each HMA gene in the Cucurbit species were obtained from the CuGenDB database and then displayed by using the MapGene2Chromosome V2 .The amino acid sequences of the Cucurbit species and Arabidopsis were imported into MEGA 7 , and mulCitrullus, Cucumis, and Cucurbita genera was carried out and visualized using TBTools with the E-value < 1× 10−10 [http://services.cbu.uib.no/tools/kaks, accessed on 15 May 2023) using the CDS of Cucurbits. The duplication date was estimated according to the following formula, Million Years Ago (MYA) = Ks/2λ, assuming a clock-like rate (λ) of 6.56 synonymous substitutions per 10−9 years [A syntenic relationship analysis of the HMA gene family between species of 1× 10−10 . Non-syn−9 years .https://meme-suite.org/meme/, accessed on 15 October 2022) to identify common and species-specific motifs in the Cucurbit proteins. The analysis was performed with a maximum number of motifs of ten and an optimum motif width from 6 to 50. To identify motif function, discovered MEME motifs were searched in the ExPASy-PROSITE database using the ScanProsite tool . Furthermore, promoter sequences (2000 bp upstream) from Cucurbit HMA genes were submitted to The PlantCARE database  to analyze potential cis-regulatory elements within promoter sequences of the Cucurbit HMA genes.HMA proteins were subjected to the Multiple Em for Motif Elicitation (MEME) tool found in the MEME suite . The amino acid sequences of HMA proteins were imported into the OmicsBox program to execute three steps: (1) BLASTp against the NCBI non-redundant protein database, (2) mapping and retrieval of GO terms associated with the BLAST results, and (3) annotation of GO terms associated with each query to relate the sequences to known protein function.The functional annotation, including cellular component, molecular function, and biological process of HMA proteins, was performed using OmicsBox Software (C. pepo exposed to copper oxide (CuO) nanoparticles, bulk CuO (100 mg kg−1), and CuSO4 (320 mg kg−1) were used to generate a heatmap and compare the expression of CpHMA1-14 genes identified by using the tydir and ggplot2 packages .RNA-seq gene expression data of CpeHMA genes were retrieved from the NCBI GEO DataSets (Accession: GSE173716) from a previously published work by Marmiroli et al. . The fraC. pepo var. cylindrica “Golden” seeds were germinated directly in the soil. Seedlings were transferred to 0, 50, 100, and 200 µM As (V) soil treatments and irrigated with the same water concentration every other day. Roots, leaves, flowers, and fruit tissues were collected at the anthesis stage, as previously reported by Obrero et al. [C. pepo according to the E.Z.N.A. Plant RNA Kit  following manufacturer instructions. An equivalent concentration of total RNA of the different tissues was used to synthesize first-strand cDNA with the SuperScript™ IV First-Strand Synthesis System . Equal cDNA concentration of samples was used for the qRT-PCR analysis. A StepOnePlus Real-Time PCR system  was employed for the qRT-PCR analysis with a final reaction volume of 20 µL containing 1 μL of cDNA template, 2 μL of forward and reverse primer at 10 μM, 10 μL of SYBR Green PCR Master (ROX) , and 7 μL of nuclease-free water. Three replicates per treatment were run to compute the average Ct values that were further analyzed with the 2−ΔΔCt method [o et al. ,44. Tisst method . The Actt method  and are C. amarus, 9 from C. colocynthis, 10 from C. lanatus, 9 from C. mucusospermus, 10 from C. melo, 9 from C. sativus, 12 from C. maxima, 12 from C. moschata, 14 from C. pepo, and 8 from L. siceraria  were found.To examine the phylogenetic relationships among the Cucurbit HMA proteins, an unrooted phylogenetic tree was constructed from alignments of the 103 full-length HMA sequences . The HMAbidopsis , CucurbiCitrullus, Cucumis, and Cucurbita species were subjected to synteny and a gene duplication analysis to confirm further the results from the phylogenetic tree , the synonymous rate (Ks), the Ka/Ks, and the duplication date using the Ks values of these pairs are presented in Citrullus duplication pairs ranges from 0.27 to 0.47 with divergent times that range from 21 to 36 MYA; similarly, in Cucumis and L. siceraria, the Ks value ranges from 0.27 to 0.44 and 0.33 to 0.44, and the divergent times from 20 to 33 MYA and 24 to 34 MYA, respectively. Nevertheless, Cucurbita duplication pairs’ Ks values range from 0.01 to 0.53 and divergent times from 1.27 to 40.88, which means a spectrum range of both, synonymous mutation, as well as diversification time. Interestingly, the tandem duplicates CmaHMA7-8, CmoHMA7-8, and CpeHMA10-CpeHMA9 were speculated to diverge in recent time, <3 MYA ago, in contrast to the Citrullus, Cucumis, and L. siceraria duplicates that resulted >20 Mya.In addition, gene duplication events of the HMA gene family were found in all species, the We identified ten common conserved motifs in HMA proteins by analyzing motif composition with MEME motif analysis, as shown in cis-elements that can influence the expression of Cucurbit HMA genes, 2000-bp DNA sequences upstream of the start codon (ATG) for the HMA proteins for each species were analyzed using the PlantCARE database. There were 20 common elements identified associated with environmental stresses and plant hormonal processes. cis-regulatory elements associated with stress responses and possibly involved in the activation against heavy metals were TC-rich repeats, engaged in defense and stress response, LTR, associated with low temperatures, TCA cis-elements, related to salicylic acid that has an attenuation against biotic and abiotic stresses, and associated with heavy metal toxicity. To identify common putative The GO analysis performed with Omics Box suggested the putative participation of HMA genes in multiple biological processes, molecular functions, and cellular components . For insC. pepo, we analyzed the expression profiles of CpeHMA genes. We based our analysis on their FPKM values derived from RNA-seq data acquired during copper (Cu) treatments, a heavy metal known to be linked with the HMA gene family. This initial investigation was a foundation for comparing subsequent expression results under arsenic exposure and validating our assumptions regarding gene functionality  in root, leaf, flower, and fruit tissues under 50 µM, 100 µM, and 200 µM As treatment through RT-qPCR. Notably, no downregulated genes were observed among all the evaluated HMA genes and tissues. However, genes belonging to the Cu/Ag clade exhibited significantly higher differential expression across all tissues  [C. melo) [Citrullus (425 Mb for C. lanatus) [Arabidopsis [MtHMA8 in Medicago and ZmHMA9 in Zea mays [Arabidopsis, AtHMA2 is expressed mainly in vascular tissues [Arabidopsis and Oryza sativa have demonstrated that different HMAs exhibit diverse subcellular localizations [CsHMA3 and CsHMA4 was found to confer tolerance to Cd and Zn with metal efflux tolerance and accumulation of Cd and Pb through sequestration, proving to be part of the Zn/Co/Cd/Pb clade and suggesting its role in plant translocation from the plasma membrane and bioaccumulation of these metals into the vacuoles [The HMA “heavy metal ATPase transporter” is a type of ATPase known as the Pnt cells ,16. Althbidopsis  and sevebidopsis , soybeanbidopsis , and Pop[Populus , a compr[Populus ,20,28. CC. pepo) ,48 in coC. melo)  and Citrlanatus) , indicatbidopsis . NeverthZea mays ,28. ThisZea mays . In Arab tissues ; howeverizations ,53. In Cizations . Expressvacuoles . However1B-ATPases is the presence of soluble metal binding domains (MBDs) that regulate transport activity [1B-type ATPases are capable of driving the efflux out of cells of both essential transition metal ions  and toxic metal ions , contributing to their homeostasis maintenance [Arabidopsis focused on heavy metal stress. Several genes, i.e., AtHMA4, AtHMA2, and AtHMA3, have been identified as Cd transporters involved in transporting Cd across the cell membrane and from the cytoplasm to the vacuole [Ls1 and Ls2 that transport As (III) and the peptide transporter OsPTR7 associated with the translocation of methylated-As species in Oryza sativa [Furthermore, an important feature of Pactivity . The conactivity ,57. P1B-ntenance ,32. Prev vacuole ,11. Howe vacuole . Accordi vacuole , HMA pro vacuole ,60,61. A vacuole ,63,64. Na sativa ,59,65,66Pteris vitatta in response to arsenic, which implies the role of P-type ATPase in the translocation of this metalloid [Pteris vitatta when treated with a mix of Ag-As due to the inhibition of the entrance, translocation, and the enhancement of As tolerance when Ag and As are supplemented simultaneously; it is known that Ag transport and homeostasis across plant tissues are mediated via HMA proteins from the Cu/Ag clade [Although HMA proteins have not been previously characterized in arsenic transport, transcriptomic analyses showed that P-type ATPase genes were upregulated in roots and shoots of the hyper-accumulator etalloid . AdditioAg clade ,68. HoweAg clade , and theAg clade .C. pepo genes (CpeHMA) in roots, leaves, and pollen from C. pepo under Cu treatments [CpeHMA2, CpeHMA4, CpeHMA5, CpeHMA9, CpeHMA10, CpeHMA11, and CpeHMA12, exhibited low to no expression levels in all tissues, whereas genes in the Cu/Ag clade such as CpeHMA3, CpeHMA6, CpeHMA7, CpeHMA8, and CpeHMA14 in the Cu treatments and CpeHMA3, CpeHMA6, CpeHMA8, and CpeHMA14 in the As treatments were highly expressed. CpeHMA6 showed a high upregulation in leaf tissue under Cu treatment and in root and flower tissues under As stress. It was observed from the phylogenetic tree that CpHMA6 is orthologous to AtHMA8 from Arabidopsis, which is related to the Cu transport through the thylakoid membrane [In order to understand the role of HMAs, we analyzed the gene expression levels from eatments , a well-membrane ,70,71. Cmembrane . Neverthmembrane ,73.CpeHMA3 gene, which is classified within the Cu/Ag clade, exhibited significant upregulation in response to both Cu and As treatments. Notably, the Cu/Ag clade lacks annotated HMA domains, a distinctive feature of this particular clade. The absence of highly conserved regions, particularly those associated with similar functionality observed in other species, can have an impact on both the affinity of the protein for various ionic metals and its inherent characteristics, such as heavy metal binding properties [CpeHMA8 is an orthologue of AtHMA5 and OsHMA5 in Arabidopsis and Oryza sativs, respectively, while CpeHMA14 is an orthologue of AtHMA7. AtHMA5 is located in the plasma membrane and is involved in the Cu translocation from roots to shoots or Cu detoxification of roots [OsHMA5 is involved in loading Cu to the xylem of the roots and other organs [Populus trichocarpa suggested that PtHMA5 may differ in function from AtHMA5 and OsHMA5, where it was found to have a significant role in Ag detoxification in addition to Cu detoxification [CpeHMA7 in root tissues under CuSO4 is orthologous to AtHMA7, also known as RAN1. The AtHMA7 gene has been recognized as an ATP-dependent copper transporter that interacts with the ethylene receptor ETR1, which is primarily found in the endoplasmic reticulum regulating plant growth and development [CpeHMA3, CpeHMA6, CpeHMA7, CpeHMA8, and CpeHMA14 across the different tissues under Cu and As stress, it is plausible to hypothesize that HMA proteins may play a role in the transportation and tolerance mechanisms of arsenic in Cucurbita pepo, similar to their involvement in copper transport. However, further study and confirmation are required to elucidate the specific molecular pathways with which these CpeHMA genes respond to arsenic stress.Moreover, the operties . Moreoveof roots . OsHMA5 r organs . Neverthfication . Highly elopment . Therefocis-elements identified were the ABRE, ARE elements, LTR, and TC-rich repeats, which are associated with various stress responses, such as abscisic acid stress, anaerobic induction, low-temperature stress, defense mechanisms, and oxidative stress, which may be involved in generating a response to arsenic-induced oxidative stress [Moreover, through the examination of cis-regulatory elements in the HMA gene family, it has been observed that all genes harbor multiple cis-elements associated with abiotic stress, including those related to heavy metals ,76. Othee stress ,28. ConsCpeHMA genes in various tissues/organs of C. pepo in response to both Cu and As stress indicate that the members of this gene family might be involved in transporting As metal ions across Cucurbit tissues, especially CpeHMA6. This information is valuable for functional investigation and understanding alternative molecular mechanisms responding to As stress in Cucurbits and other crops.Metal transporters play vital roles in distributing and transforming essential, non-essential, and even toxic metal ions in plants. This study comprehensively analyzed the HMA gene family in ten Cucurbit species. A total of 103 HMA genes from species of the Cucurbitaceous family were characterized and classified into two groups based on a phylogenetic analysis and their structural characteristics. According to their evolutionary metal association, the Cucurbit HMA genes had conserved or divergent gene structures, protein motif patterns, and cis-regulatory elements. The expression profiles of