CaBBX members were identified using the pepper reference genome database. According to the gene structure, conserved domains, and the phylogenetic tree, 23 CaBBX genes were divided into four groups, wherein the analysis of the promoter region indicated the presence of cis-acting elements related to plant development, hormones, and stress response. Interspecies collinearity analysis showed that the CaBBXs had three duplicated gene pairs, and the highest gene density was found on chromosomes 2 and 7. Transcriptome RNA-seq data and quantitative polymerase chain reaction (qRT-PCR) analysis of pepper plants spanning the entire period showed that more than half of the CaBBX genes were widely expressed in diversity tissues of pepper. Co-expression network analysis indicated that the CaBBXs and the anthocyanin structural genes had a close co-expression relationship. Thus, it was reasonably speculated that the CaBBX genes may be involved in the regulation of anthocyanin biosynthesis. Overall, this study involved the genome-wide characterization of the CaBBX family and may serve as a solid foundation for further investigations on CaBBX genes involved in the anthocyanin synthesis mechanisms and development in pepper.The transcription factors, B-box (BBX), belong to a subfamily of the zinc finger family of proteins and exhibit multiple biological functions in plant growth, development, and abiotic stress response pathways. In this study, a total of 23  Capsicum), an important species of the Solanaceae family, has significant popularity and economic value. It is widely cultivated in almost all areas with arable land, owing to its high nutritional value and complex germplasm diversity , the DcMYB7 gens regulate the inheritance of anthocyanin pigmentation in purple and non-purple carrot roots (Liquidambar formosana Hance), the R2R3-MYB transcription factor gene, LfMYB113 is positively correlated with anthocyanin content in the leaves  and modulate the anthocyanin content in pepper  that are further responsible for vacuolar acidification, thereby imparting the red color to the anthocyanins  as a typical representative is known to positively regulate anthocyanin biosynthesis. In ot roots . In the e leaves . In tranynthesis . In peppr fruits . Simultan pepper . In addiocyanins . An anthr fruits .ELONGATED HYPOCOTYL 5 (HY5) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and further, mediates their regulatory activities (Oryza sativa), anthocyanin biosynthesis is induced and fine-tuned by OsBBX14 and OsHY5. The ectopic expression of OsBBX14 in Arabidopsis thaliana results in a significant increase in the accumulation of anthocyanins in its seedlings  is highly induced by white light irradiation, and the transient overexpression of PpBBX16 in the pear skin increases the accumulation of anthocyanins, while the virus-induced PpBBX16 gene silencing reverses this trend substantially (Populus L. spp.), BBX23 directly binds to the promoter regions of proanthocyanidins and anthocyanin-specific genes and interacts with HY5 to enhance their activation , and a Chinese inbred derivative, ‘Zunla-1’, were used in this study. The HMMER SEARCH3.0 with cutoff E value ≤0.01 and SMART database were used to confirm the conserved domains among the members of the BBX family (http://www.softberry.com).To identify the BBX family of transcription factors, the hidden Markov model (HMM) profile of the B-box-type zinc finger domain (PF00643) was downloaded from the Pfam database . The refX family . TheoretX family . The SWIX family , and theCaBBXs , the sequences of SlBBXs were obtained from the tomato reference genome “SL4.0” (https://solgenomics.net/organism/Solanum_lycopersicum/genome), and those of AtBBXs were obtained from the Arabidopsis reference genome (https://www.arabidopsis.org/). MEGA-X was used to construct an unrooted neighbor-joining phylogenetic tree of CaBBXs, SlBBXs, and AtBBXs proteins with the bootstrap test replicated 1,000 times  and these domains were aligned using DNAMAN . The Gene Structure Display Server was used to predict the gene structures of CaBBXs . The ful00 times .CaBBXs was performed with MCScanx  vazyme kit  was used to synthesize the reverse cDNA. The raw data for the RNA-seq analysis were downloaded from Pepper Hub  was used to extract total RNA, and HiScriptpper Hub , and a hpper Hub  and the pper Hub . HISAT2 pper Hub , and Feapper Hub . Standarpper Hub , and theCaBBXs and anthocyanin structural genes in the extracted matrix  was used to analyze the FPKM value from RNA-seq data. The WGCNA package in R v3.6.1 language was used for the analysis . The TOMd matrix .p-value <0.05. LightCycle * 96 Real-Time PCR System  was used for performing qRT-PCR. A 50 μl reaction system was set and three biological repeats and three technical repeats were performed for each sample based on HiScript®ⅡQ RT SuperMix for qPCR (+gDNA wiper) vazyme kit . The 2−△△Ct formula was used to calculate the relative expressions. The primers for CaBBXs and actin control (Capana04g001698) were listed in GO enrichment analyses were conducted using Plant Transcriptional Regulatory Map  and WEGOCaBBX genes were identified in the pepper Zunla-1 genome, however, 22 CaBBXs were present in the pepper CM334 genome. Based on their conserved motifs, 23 CaBBX genes were named as CaBBX1 to CaBBX23 within the gene subfamilies. The length of the coding sequences ranged between 564 (CaBBX23) to 1476 (CaBBX22) amino acid residues, while their molecular weights (Mw) varied greatly between 20.33293 KDa (CaBBX23) and 54.98339 KDa (CaBBX22). The isoelectric points (PI) of the CaBBXs ranged between 4.29 (CaBBX23) and 9.17 (CaBBX21), and most of them were weakly acidic proteins. Prediction software for subcellular localization suggested that these CaBBX proteins were mainly localized in the nucleus; five members, including CaBBX7, CaBBX19, CaBBX21, CaBBX22, and CaBBX23 were found to localize to extracellular structures, such as chloroplasts and mitochondria (According to the hidden Markov model (HMM)-based profile , the genome-wide annotated proteins were screened and verified using both SMART and Pfam databases. A total of 23 chondria .2-C-X7–8-C-X2-D-X-A-X-L-C-X2-C-D-X3-H-X2 -N-X4-H (indicated in red color), and B-box2 of C-X2-C-X8-C-X7-C-X2- C-X4-H(N)-X6–8-H (indicated in green color). Protein sequence alignment showed that the two B-BOX domains had highly similar conserved sequences  contained one B-box1 domain, while two genes  had only one B-box2 domain. The remaining 18 genes existed in both two B-box domains, and nine members contained the CCT-conserved domain.The conserved sequence of the zinc finger domain of B-box was found to exist in two forms, namely B-box1: C-Xequences . For exaCaBBX family of genes in pepper was between one and five. Among them, 12 (52.17%) CaBBXs had three exons, five (21.74%) CaBBXs had two exons, two CaBBXs had four exons, and three CaBBXs had five exons. CaBBX21 only had one exon  phylogenetic tree was constructed based on the CaBBX protein sequences in pepper, AtBBX protein sequences in Arabidopsis, and SlBBX protein sequences in tomato. According to the phylogenetic tree, all BBXs were divided into four clades, wherein, each clade contained a similar number of BBX genes from the three species. Almost all these four clades corresponded to their designated domains, while some CaBBX proteins with similar domains (such as CaBBX2 in Clade II and CaBBX6 in Clade III) were clustered into different clades , and they all contained two conserved B-box domains. CaBBX9, CaBBX16, CaBBX20, CaBBX12, CaBBX18, CaBBX15, CaBBX13, and CaBBX14 genes were in the Group II. CaBBX21, CaBBX19, and CaBBX23 were classified as Group III, while Group IV included CaBBX11 and CaBBX17 proteins . The genone exon . To examt clades .CBBXs to predict the cis-regulatory elements. A total of three cis-element types were identified in the promoter regions of the CaBBox gene family. A few genes had cis-elements related to cellular development, such as flavonoid biosynthetic genes regulatory elements. In addition, many CaBBX genes contained cis-elements related to stress response pathways, including responsiveness to light, anaerobic induction, defense and stress responsiveness, and circadian control. Six typical hormone-related cis-regulatory elements in the promoter regions were identified, including abscisic acid response elements, salicylic acid, and gibberellin, and MeJA response elements. Specifically, there were 13 cis-elements for light responsiveness, including Box 4, G-Box, TCT-motif, GT1-motif, GATA-motif, I-box, AE-box, chs-CMA1a, Sp1, ATC-motif, MRE, GA-motif, and Box II -upstream of the coding region was selected as the promoter sequence in the d Box II .According to the physical positional information for the CaBBX gene family in the annotated zunla-genome, the 23 CaBBXs were found to be widely distributed in all 12 chromosomes, “01 g” to “12 g”, of pepper. Two CaBBX genes (CaBBX11 and CaBBX21) were located on chromosome “00”. Chromosomes 7 and 12 contained the most number of CaBBX genes, each harboring four CaBBXs. Chromosomes 8, 12, and 13 each contained two genes, and the remaining chromosomes each harbored one CaBBX member .Only three segmental duplicated pairs and no tandem duplicates were observed for CaBBXs in pepper. In particular, CaBBX1-CaBBX2 and CaBBX2-CaBBX6 were pairs of mutually duplicated genes, while CaBBX1-CaBBX7 were present as duplicates. CaBBX6 and CaBBX7 were located on the same chromosome and were in close physical proximity. The selection pressure in duplicated genes was estimated using the values of non-synonymous mutation (Ka), synonymous mutation (Ks), and their ratio (Ka/Ks). The Ks values ranged between 1.76 and 3.16. The Ka/Ks values for all duplicated gene pairs were less than 1.00 and between 0.128 and 0.5. It was reversed that the three groups of CaBBX duplicated genes underwent purifying selection during the process of evolution. The minimum Ks and maximum Ka/Ks values were between the duplicate gene pair, CaBBX1-CaBBX2, and these two genes may have undergone greater purifying selection .To gain insight into the potential functions of the CaBBX gene family spanning the entire developmental stage of the pepper plant, the raw BAM data of RNA-seq for various organs and tissues at different developmental stages were downloaded from the Pepperhub online database and used for analyzing the expression profiles. As shown in CaBBXs were used enrichment analysis and conducted on the assigned GO terms with the corrected p-value <0.05. Three GO categories were assigned, including cellular component, molecular function, and biological process. In cellular component, the cell part and cell were highly enriched in 23 CaBBX genes. In molecular function, CaBBXs were assigned in binding and cellular process. In biological process, CaBBXs were widely assigned in metabolic process, multicellular organismal process, developmental process, regulation of biological process, biological regulation, response to stimulus, reproduction, negative regulation of biological process, reproductive process, signaling, rhythmic process, cellular component organization or biogenesis, and positive regulation of biological process   were mostly co-expressed with anthocyanin biosynthesis. In the early anthocyanin synthesis stages, CaCHS, and CaCHI were highly co-expressed with CaBBX1, CaBBX20, CaBBX21, and CaBBX23. CaF3H were co-expressed in the network but showed low co-expression with the genes in the anthocyanin synthesis pathway. In the late anthocyanin synthesis stages, CaDFR was also highly co-expressed with CaBBX1, CaBBX20, CaBBX21. And CaANS was highly co-expressed with CaBBX9, CaBBX15, and CaBBX16. Besides, R2R3-MYB transcription factors CaAN1 and CaAN2, which involved in anthocyanin biosynthesis were also co-expressed with CaBBXs. CaBBX1, CaBBX20, and CaBBX21 were co-expressed with CaAN2, but CaBBX9, CaBBX15, and CaBBX16 were co-expressed with CaAN1. In co-expression network, CaBBX3, CaBBX4, CaBBX5, CaBBX6, CaBBX7, CaBBX11, CaBBX14, CaBBX15, CaBBX17, CaBBX18, and CaBBX29 were indirect co-expression with key seven CaBBX genes or anthocyanin structural genes to affect anthocyanin biosynthesis. In addition, there was a close co-expression relationship between the four CaBBX genes .According to the expression levels of 23 *+) was the endogenous control. Expressions of CaBBX3, CaBBX4, CaBBX5, CaBBX7, CaBBX8, and CaBBX13 were analyzed, which suggested that the qRT-PCR and RNA-seq expression level findings for CaBBXs were similar. Among them, CaBBX3, CaBBX4, and CaBBX8 were highly expressed in fruits but showed lower expression in flowers. Conversely, CaBBX7, and CaBBX13 were highly expressed in the flowers but showed low expression in fruits. CaBBX5 was highly expressed in both flowers and fruits. The CaBBX8, and CaBBX13 were also expressed in other tissues such as leaves and stems. Moreover, expressions of four anthocyanin biosynthesis structural genes  and two typical regulation genes (CaAN1 and CaAN2) were also analyzed in different tissues. The related expressions of CaDFR, CaF3H, CaAN1, and CaAN2 were highly in flowers, while the CaCHI, and CaANS were highly expressed in leaves. The related expressions of CaANS, CaDFR, CaAN2 were also expressed in other tissues such as roots, fruits, and stems  and verification in the cultivated pepper “6421”. Actin gene (GenBank Accession No. Contig0023nd stems . This inCapsicum spp.) in 2014, many families of transcription factors have been identified   . Among t to date . Potato d genome . SpeciesCaBBX16 gene with one B-box and one CCT domains . In addition, these four genes contained two conserved B-box domains, however, their gene structures were different. Both CaBBX1 and CaBBX7 contained five CDS regions, while CaBBX2 had three introns, and CaBBX6 had only two exons. Although these were duplicated genes, which may arise from the same ancestor, the functions could have diverged due to structural differentiation. Bioinformatic prediction of subcellular localization showed that the CaBBX1, CaBBX2, and CaBBX6 genes were localized in the nucleus, while CaBBX7 had extracellular localization. The localization of CaBBX7 may support its ability to transport across membranes, however, the specific functions of the CaBBXs need further experimental proof. Simultaneously, nuclear localization of the BBX family of genes has been reported in other species    . ATBBX18bidopsis , and MdB Borkh.) . Howeverynthesis . The homlowering . In addisicaceae . COP1-mebidopsis . The remormation . The CaBromoters . In thisCaBBXs and anthocyanin structural genes were significantly associated. Seven CaBBX genes were closely co-expressed with anthocyanin structural genes  gene which controls the expression of MYB10 in apple fruit, thereby affecting the synthesis of anthocyanins and regulating the color of the red skin (BBX23 in poplars (Populus trichocarpa) activates the expression of MYB transcription factors and structural genes in the flavonoid pathway, thereby promoting the accumulation of procyanidins and anthocyanins (CaBBX gene family was systematically studied by both RNA-seq and qRT-PCR. CaBBX3, CaBBX4, and CaBBX5 were identified as three candidate anthocyanins regulatory genes in fruits, while the CaBBX6, CaBBX7, and CaBBX13 were three candidate anthocyanins regulatory genes in flowers. However, the specific regulatory mechanism and function of these candidate genes need to be verified by further molecular biology experiments. This study provided a strong reference for screening the transcription factors involved in the regulation of anthocyanin synthesis in pepper and laid a solid foundation for the further functional characterization of the CaBBXs.In particular, eight family members were specifically expressed in flowers, and eight were specifically expressed in fruits, accounting for 69.56% of the total expression . Specifial genes . It was red skin . The oveocyanins . In this


Results shown that germination of triple mutants were delayed and were much more sensitive to salt stress than WT plant, which can be rescued by application of SNP. qPCR analysis revealed that expressions of abscisic acid (ABA) catabolism gene, OsABA8ox1, was suppressed in triple mutants under salt stress, resulting in an elevated ABA content. Similar to SNP, application of nitrate also rescued seed germination under salt stress, which, however, was blocked in the triple mutants. Further study revealed that a nitrate responsive transcript factor, OsNLP2, was induced by salt stress, which thus up-regulates the expression of OsNRs and NR activity, resulting in promoted salt tolerance during seed germination. In addition, nitrate-mediated salt tolerance was impaired in mutant of aba8ox1, a target gene for NLP2. Transient trans-activation assays further revealed NLP2 can significantly activate the expression of OsABA8ox1 and OsNR1, suggesting that NLP2 activates expression of ABA catabolism gene directly or indirectly via NR-associated NO. Taken together, our results demonstrate that NLP2-NR associated NO was involved in salt response by increasing ABA catabolism during seed germination and highlight the importance of NO for stress tolerance of plants.Salt stress has the most severe impact on plant growth and development, including seed germination. However, little is known about the mechanism of NR (nitrate reductase)-associated nitric oxide (NO) regulates salt tolerance during seed germination in rice. Herein, we shown that inhibition of seed germination by salt stress was significantly impaired by sodium nitroferricyanide (SNP), a NO donor. Then a triple mutant,  More than 20% of the cultivated lands are threatened by high salinity, which is aggravated by the growing population, poor agricultural practices, as well as salt intrusion in coastal zones, making salt stress an increasingly serious problem worldwide ,2. Salt t stress ,13, suggnia1/nia2, resulted in reduced content of both NO and nitrite in Arabidopsis [+ uptake [cyp707a2 mutant, a key gene for ABA catabolism [nia1/nia2 [Nitric oxide (NO) is a gaseous, free-radical and redox-signaling molecule which has multiple functions and acts as molecular messenger during plant growth and development. NO also plays important roles in response to different stresses, including drought stress, temperature stress, salt stress and heavy metal stress ,15,16,17bidopsis . Numeroubidopsis ,21. In sbidopsis , while pbidopsis ,24. It h+ uptake , promoti+ uptake ,27, inte+ uptake ,29. Duritabolism . Seed geia1/nia2 , suggestArabidopsis [CYP707A2, an ABA catabolism gene in Arabidopsis, and thus was essential for nitrate-promoted seed germination [OsNR and OsNiR genes and thus regulate the nitrogen use efficiency. OsNLP4 bind to the promoter of OsNiR gene and transactivate [OsNR genes and increase their abundance [OsNLP1 and OsNLP3 but reduced in nlp1 and nlp3 mutants.Seed germination is a key developmental stage during the life cycle and is tightly controlled in plants ,32. Antabidopsis . Recentlmination , suggestactivate  while Osbundance ,38. NR aMoreover, both nitrate-promoted seed germination and stimulating effect on seedling growth by nitrate were blocked by application of NO-scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) ,40. In aOsNR genes, which displayed a delayed germination phenotype and was much more sensitive to salt stress than seeds of WT plant. qPCR analysis revealed that expressions of ABA catabolism genes, OsABA8ox1 and OsABA8ox2, were suppressed in triple mutants, especially under salt stress, resulting in an elevated ABA content. Similar to SNP, application of nitrate also promoted seed germination under salt stress, which, however, was blocked in the triple mutants. Further study revealed that a nitrate responsive transcript factor, OsNLP2, was induced by salt stress, which thus up-regulates the expression of OsNRs and NR activity, resulting in promoted salt tolerance during seed germination. In addition, nitrate-mediated salt tolerance was impaired in mutants of aba8ox1 and aba8ox2, two target gene for NLP2, suggesting NLP2 might activate expression of ABA catabolism gene directly or indirectly via NR-associated NO. Altogether, our results demonstrate that NLP2-NR associated NO was involved in salt response by increasing ABA catabolism during seed germination and highlight the importance of NO for stress tolerance of plants.In this study, we generated a triple mutant of three Arabidopsis [Stylosanthes humilis [Salt stress was demonstrated to significantly inhibit seed germination in many species, such as bidopsis , soybeanbidopsis , cotton bidopsis , and Sty humilis . In riceOsNR1, OsNR2 and OsNR3, was generated by CRISPR/Cas9  seed. Whereas under salt stress, exogenous nitrate has significantly promoted seed germination   a–c, espeberellin . In addi [Salt stress is well known as one of the most devastating abiotic stress factors in agriculture all over the world. Numerous studies in the past have demonstrated that NO was involved in salt tolerance, acting as ROS scavenger, signaling molecule, and interacting with other molecules ,47. Applenous NO . Germinaenous NO . Salt stenous NO ,53, whicenous NO . It is wnsis L.) , which iAtNOS1 simultaneously has led to a reduced NO production, inhibition of seed germination and reduced plant size in Arabidopsis. Application of SNP was able to significantly, but not fully, promote seed germination of this triple mutant [In plants, there are several NO origins which can be divided into enzymatic pathway and non-enzymatic pathway . Many ste mutant . Here inArabidopsis displayed an increased dehydration tolerance by efficiently regulating stomata close when compared with WT plant [In contrast with NO, the function of NR in salt tolerance has not yet been well demonstrated. Nevertheless, it was reported that NR-dependent NO production was important in response to salt stress in cucumber root when subjected to 50 mM sodium chloride  and in sWT plant , suggestOsNCED3 gene by salt  belongs to the transcript factor family which act as master regulator in nitrate response by binding to nitrate responsive cis-element (NRE) in many nitrogen responding genes in bidopsis ,59. In ranalysis . OsNLP1,se genes ,38,46. Tmination b, accomp with different concentrations of NaCl, Sodium Nitroprusside (SNP) and 1 mM Sodium Nitrate accordingly . Seeds were placed in a growth chamber in continuous darkness at 28 °C to facilitate germination. Germination (based on radicles > 3 mm) was recorded every 12 h or daily, depending on the experiment. For each germination test, approximately 50 seeds per genotype were used, and three experimental replications were performed. The average ± SE (standard error) of triplicate experiments were calculated.ACTIN7 as an internal control. Three biological replicates were performed for each experiment. Primer sequences for qRT-PCR are shown in Germinating seeds at different time points were sampled and stored in liquid nitrogen immediately for gene expression and enzyme activity analysis. Total RNA was extracted using Plant RNA Purification Reagent for plant tissue according to the manufacturer’s instructions (Invitrogen) and genomic DNA was removed using DNaseI (TaKara). Then RNA quality was determined by 2100 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Quantitative real-time PCR was performed using the 2× SYBR Green qPCR Master Mix (Yeasen Biotechnology) using a StepOnePlus™ Real-Time PCR System (Applied Biosystems). Gene expression was quantified at the logarithmic phase using the expression of the housekeeping gene NR was extracted and its activity was assayed according to Scholl et al. , which hv/v/v). The combined extracts were evaporated to dryness under nitrogen gas stream, reconstituted in 80% (v/v) methanol, and filtrated . The sample extracts were analyzed using an LC-ESI-MS/MS system .ABA content was detected by Wuhan MetWare Biotechnology Co., Ltd. . Seed samples were frozen in liquid nitrogen, ground into powder, and extracted with methanol/water/formic acid  to form the reporter construct. Then, the CDS region of the OsNLP2 was amplified and cloned into the pGREENII-62-SK vector by the one-step cloning kit (Vazyme) to form the effector construct. Finally, the two constructed vectors were mixed well for the transient expression assay in the rice protoplast. This transient expression assay was performed as described previously [The 2000-bp sequence of the native eviously .t-test, p < 0.05 or p < 0.01 was considered to be statistically significant.The SPSS 23 software  was used to analyze significance, data was presented as mean ± SE. Post-hoc comparisons were tested using the Student’s OsNR1, LOC_Os08g36480; OsNR2, LOC_Os02g53130; OsNR3, LOC_Os08g36500; OsABA8ox1, LOC_Os02g47470; OsABA8ox2, LOC_Os08g36860; OsABA8ox3, LOC_Os09g28390; OsNLP1, LOC_Os03g03900; OsNLP2, LOC_Os04g41850; OsNLP3, LOC_Os01g13540; OsNLP4, LOC_Os09g37710; OsNLP5, LOC_Os11g16290; OsNLP6, LOC_Os02g04340; Actin7, LOC_Os01g64630.Genes and their accession number used in this study are shown as followed: OsNLP2, plays key role in regulating ABA content by NLP2-NR module-dependent NO production and by activating the expression of OsABA8ox1 gene during seed germination under salt stress (OsABA8ox1 and OsNR1/2, resulting in an enhanced ABA catabolism and accumulation of NO production. The elevated NR-dependent NO will then mitigate the inhibition effect of salinity by further promoting ABA catabolism.In conclusion, our results demonstrate that nitrate responsive gene, t stress . Seed ge


A critical role in coronavirus disease 2019 (COVID-19) pathogenesis is played by immune dysregulation that leads to a generalized uncontrolled multisystem inflammatory response, caused by overproduction of proinflammatory cytokines, known as “a cytokine storm” (CS), strongly associated with a severe course of disease. The aim of this study is to identify prognostic biomarkers for CS development in COVID-19 patients and integrate them into a prognostic score for CS-associated risk applicable to routine clinical practice.The authors performed a review of 458 medical records from COVID-19 patients (241 men and 217 women aged 60.0 ± 10.0) who received treatment in the St. Petersburg State Budgetary Institution of Healthcare City Hospital 40 , from Apr. 18, 2020 to Nov. 21, 2020. The patients were split in two groups: one group included 100 patients with moderate disease symptoms; the other group included 358 patients with progressive moderately severe, severe, and extremely severe disease. The National Early Warning Score (NEWS) score was used alongside with clinical assessment, chest computed tomographic (CT) scans, electrocardiography (ECG), and lab tests, like ferritin, C-reactive protein (CRP), interleukin (IL)-6, lactate dehydrogenase (LDH), and D-dimer.9/L; positive test result for replicative coronavirus (SARS-CoV-2) RNA; age 40 years and over) is a useful tool to identify patients at a high risk for cytokine storm, requiring an early onset of anti-inflammatory therapy.The basic risk factors for cytokine storms in COVID-19 patients are male gender, age over 40 years, positive test result for replicative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, absolute lymphocyte count, dynamics in the NEWS score, as well as LDH, D-dimer, ferritin, and IL-6 levels. These clinical and instrumental findings can be also used as laboratory biomarkers for diagnosis and dynamic monitoring of cytokine storms. The suggested prognostic scale (including the NEWS score dynamics; serum IL-6 greater than 23 pg/ml; serum CRP 50 mg/L or greater; absolute lymphocyte count less than 0.72 × 10 Coronavirus disease 2019 (COVID-19) infection caused by SARS-CoV-2 coronavirus remains a global challenge for the world’s healthcare system. Most people infected with SARS-CoV-2 will experience mild illness. Some patients, however, develop a dysregulated immune response, inducing severe lung injury. This is manifest as acute respiratory distress syndrome (ARDS) which precedes acute respiratory failure, extrapulmonary organ dysfunction, and high risk of death. COVID-19 disease is usually associated with elevated inflammatory biomarkers, cytokines, and chemokines, especially in severe cases. Moreover, lymphocytopenia and neutrophilia often emerged, with particularly declined counts of CD8+ T cells, CD4+ Т cells, and natural killer (NK) cells . In all The study design is an observational clinical trial. We utilized 458 medical records from COVID-19 patients who received treatment in the St. Petersburg State Budgetary Institution of Healthcare City Hospital 40  from Apr. 18, 2020 to Nov. 21, 2020; the patients tested positive for SARS-CoV-2 RNA by polymerase chain reaction (PCR) amplification of nucleic acids from clinical material and presented clinical manifestations and symptoms , features of viral pneumonia seen on unenhanced lung CT scan  .2 <95%. The second group included 358 (78.2%) patients with progressive, moderately severe, severe, and extremely severe course of disease. Clinical and radiological criteria for severe course were respiratory rate more than 30/min, SpO2 93%, PaO2/FiO2 300 mmHg, progression of changes in the lungs typical for COVID-19 pneumonia according to CT data, including an increase in the prevalence of revealed changes by more than 25%, as well as the appearance of signs of other pathological conditions, changes in the level of consciousness, unstable hemodynamics , and qSOFA >2 points. The criteria for an extremely severe course were signs of ARF with the need for respiratory support (invasive ventilation), septic shock, and multiple organ failure. Thus, patients with mild and moderate and severe and extremely severe course are shown in In accordance with the International and Russian Recommendations for the Prevention, Diagnosis and Treatment of New Coronavirus Infection (COVID-19), all patients were divided in two groups of comparable age , 8. The Symptoms at admission were fever in 365 (80%) patients, cough in 329 (72%) patients, dyspnea in 265 (57.86%) patients, myalgia in 43 (9.39%) patients, general fatigue in 344 (75.11%) patients, headache in 36 (7.86%) patients, a sore throat in 29 (6.33%) patients, a runny nose or rhinorrhea in 46 (10.04%) patients, chest pain in 51 (11.14%) patients, diarrhea in 34 (7.42%) patients, nausea and vomiting in 13 (2.84%) patients, and impaired taste and smell in 40 (8.73%) patients. Four hundred fifty (98.25%) reported one or more disease-specific symptoms. CT imaging manifestation of pneumonia was detected in 458 (100%) patients.2, respiratory distress); calculation of the National Early Warning Score (NEWS), a recommended scoring system for use in COVID-19 patients  inhibitors , IL-1 inhibitors , JAK inhibitors , Bcr–Abl tyrosine kinase inhibitor (radotinib), and glucocorticoids for some cases. Respiratory therapy, modified antibacterial therapy, extracorporeal membrane oxygenation, sepsis, and septic shock treatment  were performed in a stepwise fashion according to indications .C-reactive protein (CRP), IL-6, etc.) and normality of sampling distribution. We used Pearson’s Chi-squared and Fischer’s exact nonparametric tests to assess the frequencies of qualitative data . Classification Trees were utilized to identify cutoff thresholds for age, NEWS score, and lab test values. The odds ratio (OR) for developing the CS—the ratio of probabilities of a certain event for different groups—was calculated for a 2 × 2 contingency table using a standard formula and the associated confidence interval. We applied the Haldane’s correction, if the 2 × 2 table contained a zero cell.Data were analyzed using STATISTICA for Windows  software. Since all data (except age) did not follow a normal distribution, we applied the Mann-Whitney, Kolmogorov-Smirnov, median Chi-square, and ANOVA tests to evaluate between-group differences in quantitative parameters . Although moderately severe lung involvement (CT score 2) prevailed in group 2, at admission, these patients reported manifestations of progressive respiratory failure and fever Table 3.In addition, prevalence of comorbidities in our cohort was greater than reported by other authors for adult patients with COVID-19 (31%) . The higComparative analysis of clinical, imaging, and lab characteristics in recruited cohorts of patients has revealed most critical CS-related variables Table 4.р < 0.001). There are reliable between-group differences in lab variables , which correlate with the NEWS score dynamics at admission until the start of CS treatment.The NEWS score dynamics across different patient groups exhibits qualitative differences: the NEWS score decreased from baseline in group 1  and increased in group 2 with progressive course of disease    Figure 2We outlined the following risk categories of patients to enhance clinical relevance of our prognostic model: category 1 (0 to 1 risk factors) = CS-associated risk is near zero; category 2 (two to three risk factors) = CS-associated risk escalates dramatically to 55%, augmenting 35.5-fold vs. category 1; category 3 (four and more risk factors) = CS-associated risk exacerbates to 96%, augmenting 718-fold vs. category 1. The obtained results are consistent with findings by other authors on the evaluation of risk factors for COVID-19-associated CS , 15. OurConsidering there are no reliable prognostic criteria for COVID-19-induced CS available today, we selected 458 patients with a different course of disease to evaluate prognostic power of available clinical, instrumental, and lab parameters; the most useful parameters for assessing outcome were collated into coherent domains, or clusters, and their prognostic power was evaluated. The clinical data collected from patient histories and at hospital admission included signs and symptoms, demographic, epidemiological, and clinical parameters, assessment of disease severity using the NEWS score, severity of COVID-19, comorbidities, dynamic evaluation of lung involvement (ground-glass opacities ± areas of consolidation) based on a standard imaging protocol for unenhanced chest CT  recordedThe basic risk factors for cytokine storms in COVID-19 patients include male gender, lactate dehydrogenase level, age over 40 years, positive test result for replicative SARS-CoV-2 RNA, absolute lymphocyte count, D-dimer and ferritin levels, dynamics in the NEWS score, and plasma IL-6 concentration.Absolute lymphocyte count, LDH, CRP, ferritin, D-dimer, and IL-6 levels are the most critical lab parameters for diagnosis and dynamic monitoring of cytokine storms.9/L; positive test result for replicative coronavirus (SARS-CoV-2) RNA; age 40 years and over) is a useful tool to identify patients at a high risk for cytokine storm, requiring an early onset of anti-inflammatory therapy.The suggested prognostic scale (including the NEWS score dynamics; serum IL-6 greater than 23 pg/ml; serum CRP = 50 mg/L or greater; absolute lymphocyte count less than 0.72 × 10The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by City Hospital No. 40, Saint Petersburg. The patients/participants provided their written informed consent to participate in this study.SS: concept development and research; guidance, choice of treatment strategy, and patient assessment; and analysis of the studied groups. AA, EG, and DK: treatment and examination of patients. SM and SU: literary search. AA and SM: formation of the base of the studied groups, statistical analysis, and writing an article. OK: statistical analysis. EM, ZA, and OG: performing X-ray, pathomorphological diagnostics and testing biological samples of patients for the presence of SARS-CoV-2 coronavirus. OG and AC: editorial of the article. SA: biobanking of materials. All authors contributed to the article and approved the submitted version.The research was financed from the budget of the St. Petersburg State Budgetary Institution of Healthcare City Hospital 40 and a grant from the funds of St. Petersburg State University ID PURE: 75253103.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.


FMR1 is located on chromosome X. Accordingly, the condition has more severe manifestations in males. FXS results from the loss of function of FMR1 due to the expansion of an unstable CGG repeat located in the 5′′ untranslated region of the gene. About 50% of the FXS males and 20% of the FXS females meet the Diagnostic Statistical Manual 5 (DSM-5) criteria for ASD. Among the individuals with ASD, about 3% test positive for FXS. FMRP, the protein product of FMR1, is a major gene regulator in the central nervous system. Multiple pathways regulated by FMRP are found to be dysfunctional in ASD patients who do not have FXS. Thus, FXS presents the opportunity to study cellular phenomena that may have wider applications in the management of ASD and to develop new strategies for ASD therapy.Autism Spectrum Disorder (ASD) represents a distinct phenotype of behavioral dysfunction that includes deficiencies in communication and stereotypic behaviors. ASD affects about 2% of the US population. It is a highly heritable spectrum of conditions with substantial genetic heterogeneity. To date, mutations in over 100 genes have been reported in association with ASD phenotypes. Fragile X syndrome (FXS) is the most common single-gene disorder associated with ASD. The gene associated with FXS,  Neurodevelopmental disorders are a broad group of neurological and psychiatric conditions wherein the development of the central nervous system is disrupted. These disorders are typically recognized early in life and often persist into adulthood.Of the many neurodevelopmental disorders, ASD has come to the forefront of social awareness. Recent estimates place the prevalence of Autism Spectrum Disorders (ASDs) at approximately 1 in 54 children, . These sTwin studies have shown significant heritability of ASD ,7, thus https://gene.sfari.org/).Nevertheless, a major obstacle in improving our understanding of the underlying pathology of ASDs has been the absence of identifiable biomarkers. With the development of advanced genomic technologies, many chromosomal copy number variants (CNV), as well as monogenetic mutations, were associated with ASD ,9,10,11.In addition to genetic links, exposures while in utero or during early development, such as toxins , medications , or stress, are strongly associated with neurodevelopmental disorders ,15,16. RA reliable biological definition of ASD would provide strong, distinct relationships between genomic variants, pathophysiological processes, and clinical phenotypes. However, many of the identified risk genes or environmental exposures are only found in a subset of patients and often share correlations with multiple disorders ,19,20. AA number of syndromic monogenetic neurodevelopmental disorders such as Fragile X Syndrome (FXS), Rett syndrome (RTT), and tuberous sclerosis (TSC), have strong associations with ASD ,23. On tEven though monogenetic neurodevelopmental disorders are distinct clinical entities, these disorders have provided important insights into potential common-shared pathophysiology and may provide a guide for unraveling the molecular underpinnings of non-syndromic autism .fragile X mental retardation 1 (FMR1) gene mutation, the mutation responsible for FXS, is recommended as a first-tier genetic test in the current expert guidelines for ASD management [One of the most important examples of syndromic ASD is Fragile X Syndrome. Approximately 30 to 50% of individuals diagnosed with FXS meet the criteria for a diagnosis of ASD, with FXS-ASD patients composing approximately 3% of all cases of ASD ,28,29,30nagement .FMR1 gene. The overall prevalence of FXS is approximately 1 in 7000 in males and 1 in 11,000 in females [FMR1 gene [FMR1 gene which, as in the other 95% of cases, result in absent or markedly decreased production of the fragile X mental retardation protein (FMRP). FMRP regulates the translation of approximately 4% of fetal brain mRNA and directly regulates several classes of ion channels [Fragile X syndrome (FXS) is a neurodevelopmental disorder due to an X-linked mutation in the  females ,33. In mMR1 gene . The remchannels ,35,36,37channels .FMR1 gene was identified in 1991 by positional cloning [FMR1 gene typically contains less than fifty-five CGG trinucleotide repeats and is located at Xq27.3 . This protein is highly conserved among vertebrates (~92% similarity between human and chicken homologs) and thus speaks to the importance of its biological activity [The product of the activity . FMRP isactivity ,44,45. Tactivity . The levFMRP is diffusely expressed throughout the brain and varies widely among subpopulations of neurons . The levWithin a given neuron, FMRP localizes to the soma, axon, and dendritic compartments . FMRP asCanonically, FMRP’s main function is that of an mRNA binding protein, responsible for the localization, stabilization, and translation of approximately 4% of fetal brain mRNA ,39,58,59+ (BKCa) channels, and Slack [V1.2, CaV1.3 and CaV2.2, [V1.2, and CaV2.2, FMRP directly complexes with these channels to regulate their function.Non-canonical functions of FMRP, such as nuclear functions, microRNA interactions, and protein–protein interactions have been identified ,35,36,37nd Slack ,63,64, c CaV2.2, ,66,67, a CaV2.2, ,69 are mThere are many paradigms that can be used for developing research questions about the etiology of ASD. Currently, there is a vast array of relationships between ASD and disruptions in developmental processes due to genetic or environmental insults. Many of these perspectives provide useful frameworks for guiding research into potential causative mechanisms of ASD. One of the major areas of disruption in both FXS and ASD is found at the synapse ,72,73,74FMR1 mutation comes from the “mGluR theory” of FXS [Fmr1-KO mouse, an mGluR5 regulated form of synaptic plasticity, long-term depression (LTD) is exaggerated [Fmr1-KO mice is reflected in both FXS and non-syndromic ASD patients, as alterations in mGluR5 expression are seen in postmortem ASD brains [Homer1a, Shank3, Ngln3 produced phenotypes that approximate those seen in FXS and ASD [The loss of FMRP results in excess and dysregulated mRNA translation, delocalization of FMRP regulated proteins, and thus profound changes in the structure and physiology of the synapse ,57. A pi” of FXS . Type 1 ” of FXS . Stimula” of FXS . In the ggerated . The patD brains ,80. AddiD brains . Since d and ASD ,83,84. IFMR1 mutations are in mechanisms of plasticity, such as α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor expression, N-methyl-D-aspartate (NMDA) receptor localization, and endocannabinoid (eCB) signaling [Fmr1-KO mouse model and importantly, genetic variants for these proteins are found in non-syndromic ASD patients and are predictive of an ASD diagnosis [Many of the synaptic deficits which occur due to ignaling ,86,87,88ignaling ,85,87,89iagnosis ,91,92,93FMR1 and ASD. More specifically this review highlights the relationship between FMR1 mutations and ASD with regard to the dysfunctional regulation of presynaptic activity by the endocannabinoid system (ECS), BKCa channels, and CaV2.1 and CaV2.2 channels.Synaptic dysfunction can be subdivided into various subcategorizations  ,94. EachFMR1 mutations and ASD, those which are imperative for appropriate presynaptic activity have a substantial body of clinical and preclinical evidence implicating them in the pathology of both disorders. At the synaptic level, presynaptic dysregulation results in aberrant neurotransmitter release and altered synaptic plasticity, which underlies the hyperexcitability seen at the circuit level [FMR1 mutations and ASD: the endocannabinoid system (ECS), and BKCa channels, and, P/Q and N-type Ca2+ channels [Of the many pathophysiological processes associated with it level ,64,65,87it level ,96,97. Hchannels ,99,100.In essence, the presynaptic hypothesis posits that presynaptic dysregulation causes computational errors at the synaptic level, resulting in circuit level and systems-level brain network dysfunction that manifests as neurodevelopmental pathology.V2.1 and CaV2.2 channels are regulated by FMRP activity [V2.1 and CaV2.2 channels, FMRP interacts with these channels directly to inhibit calcium entry into the cell [V2.1 and CaV2.2 channels [FMR1 mutations, or when there is loss of function in the aforementioned FMRP associated synaptic components, dysregulation of calcium dynamics occurs resulting inappropriate neurotransmitter release  is composed of two primary cannabinoid receptors, cannabinoid type 1 receptor (CB1) and cannabinoid type 2 receptor (CB2), and two primary ligands, arachidonoyglycerol (2-AG) and N-arachidonoylethanolamine (AEA) ,115,116.mGluR), activation of group I mGluR which activates phospholipase C β (PLC-β) to cleave phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the 2-AG precursor, 1,2-diacylglycerol (DAG). This is hydrolyzed by the serine lipase, diacylglycerol lipase α (DGL-α) in central neurons and diacylglycerol lipase β (DGL-β) in immune cells , to form 2-AG [2+ via NMDA receptors (eCBNMDA) [2+ dependent manner and produces the precursor, DAG, needed for the production of 2-AG by DGL-α [2+ dependent manner, in response to an influx on intracellular Ca2+ causes cleavage of phosphatidylethanolamine (PE) by N-acetyl-transferase into the AEA precursor, N-arachidonoyl-PE (NAPE), which is then cleaved by the NAPE-hydrolyzing phospholipase D (NAPE-PLD) into AEA. Metabolism of AEA is carried out by fatty acid amide hydrolase (FAAH) which hydrolyzes AEA into AA and ethanolamine (EA). Once synthesized, 2-AG and AEA diffuse retrosynaptically and interact with CB1 receptors located on the presynaptic neuron [Of the two endocannabinoid ligands, 2-AG is the most abundant found in the mammalian CNS and is a full agonist at CB1 ,121,122.orm 2-AG ,124. TheeCBNMDA) ,126. PLCby DGL-α . The synby DGL-α . Approxiby DGL-α ,129. Theby DGL-α ,131. AEAc neuron .i/o protein subunits of CB1. CB1 activation inhibits adenylyl cyclase and reduces cAMP production [i/o protein inhibition of presynaptic CaV2.1 and CaV2.2 channels [CB1 signaling by 2-AG or AEA can result in the activation of multiple signaling pathways mediated by the Goduction . Activatoduction . Crucialchannels .The retrograde nature of the ECS provides a unique form of synaptic plasticity called depolarization-induced suppression of inhibition (DSI) at inhibitory GABAergic synapse and depolarization-induced suppression of excitation (DSE) at excitatory synapses ,135. BriEndocannabinoids also exhibit activity at transient receptor potential cation channel subfamily V member 1 (TRPV1), G protein-coupled receptor 18, 55, and 119  ,137. WhiCNR1, the gene for CB1R [CNR1 and DAGLA, the gene for DGL-α, were significantly associated with autism [CNR1 gene modulate striatal responses and gaze duration to happy faces [GRM5, NGLN3, HOMER1A, SHANK3, and several genes coding for NMDAR subunits [GRM5, the gene for mGlur5, are risk variants for ASD [A growing body of clinical evidence associates the ECS with ASD phenotypes. Post-mortem studies of brain tissue from ASD patients indicated reduced expression of for CB1R . Additiofor CB1R . Analysih autism . A seriepy faces ,146. Twopy faces ,148. Rissubunits ,153,154. for ASD . Further for ASD ,155,156.Fmr1-KO mice consistently show evidence of ECS dysfunction [Studies with the function ,101,102.function . Loss offunction .mGluR production requires a scaffolding protein called Homer1a, which complexes mGluR5 and DGL-α [Fmr1-KO mice, interactions between mGluR5 and Homer1a are reduced and this is causal for hyperexcitability of cortical neurons and seizures [mGluR and eCBNMDA forms of 2-AG synthesis [Fmr1-KO mice and upregulation of Homer1a expression rescued cognitive deficits. Importantly, increasing the bioavailability of 2-AG normalized plasticity deficits and rescued the hyperactive, anxiety, and cognitive impairments phenotypes of the Fmr1-KO mouse [Fmr1-KO mice [Molecular and physiological studies indicated that appropriate eCBnd DGL-α ,158. In seizures . Homer1aynthesis ,160. TheKO mouse ,161. Fur-KO mice ,163,164.Pharmacological and genetic manipulations of specific ECS components strengthen the link between the ECS and ASD pathology. Mice with a targeted DGL-α deletion from direct pathway medium spiny neurons (dMSNs) of the striatum had impaired social interest and increased repetitive behaviors . Mice wiDevelopmental studies show that a temporally orchestrated pattern of ECS expression and activity is imperative for appropriate brain connectivity ,170,171.Clinically, phytocannabinoids (pCBs), plant-derived molecules with similar chemical structures as eCBs, have demonstrated success in the treatment of neurodevelopmental disorders. The pCB, cannabidiol (CBD) has FDA approval for the treatment of two forms of epilepsy: Dravet Syndrome and Lennox-Gastaut Syndrome ,182. In Mecp2 mutation [Cannabidivarin (CBDV), a propyl analog of CBD, has also shown promise as a treatment for ASDs . A smallmutation . Additiomutation . CurrentImportantly, these molecules largely avoid the undesired psychotropic side effects that result from CB1 activation, strengthening their appeal as potential treatments for ASDs . StudiesLarge conductance voltage and calcium-sensitive potassium (BKCa) channels are expressed ubiquitously throughout the body, however, regulatory subunits of these channels are tissue-specific . In the 2+ [+ ions (thus hyperpolarizing the membrane) and complexing with P/Q and N-type Ca2+ channels to inhibit Ca2+ entry and control neurotransmitter release [2+ entry is the rate-limiting step for BKCa activation [2+ sensitivity of BKCa channels through direct interactions with the α and β4 subunits [Functionally, the α subunit of the BKCa channel opens in response to membrane depolarization and intracellular increases in Ca2+ . It has  release ,206. Of tivation . FMRP resubunits ,208. ThiKCNMB4 gene, (β4 BKCa subunit), and GRM5 gene, (mGluR5) were two of the three identified genes. This is particularly important in regard to the overlap between FXS and ASD since BKCa channel activity is directly regulated by FMRP at the β4 unit and mGluR5 dysfunction in FXS has been well established [Genetic studies have uncovered a relationship between genetic variants for BKCa genes and ASD. Skafidas et al., 2014  examinedablished ,76,99.KCNMA1 and a diagnosis of autism [KCNMA1 gene were identified in two patients with ASD and intellectual disability [Two studies that investigated chromosomal abnormalities in ASD patients discovered a mild to moderate association between mutations in f autism ,210. Addsability . Genome BKCa dysfunction is also associated with other neurodevelopmental disorders. A patient with moderate to mild intellectual disability and febrile seizures was identified as having a mutation only in the β4 BKCa regulatory domain of FMRP . AnalysiFmr1-KO mouse demonstrated that loss of FMRP regulation of BKCa channels increased action potential duration [2+ influx, and neurotransmitter release. Zhang et al., 2014 [Fmr1-KO mice. Importantly, genetic upregulation of BKCa channels expression normalized the synaptic and circuit deficits in the Fmr1-KO mouse [Fmr1-KO mouse, a frequently co-morbid feature of ASD. A genetic mouse model null for the BKCa α subunit gene (Slo1) was developed to explore the role of BKCa channels in neurodevelopmental disorders [−/− mouse has not been performed. Our recent pharmacological study using the BKCa channel inhibitor paxilline found that paxilline treatment induced unique social anxiety-type deficits during adulthood [Studies with the duration ,212. Spel., 2014  showed tisorders . This stdulthood .BKCa channels expressed outside the central nervous system respond to eCB signaling in vascular endothelial cells . AdditioA BKCa channel agonist, BMS-204532 (BMS), was developed in 2002 for the treatment of ischemic stroke, however, it failed to demonstrate clinically significant therapeutic effects in phase III trials . Since BFmr1-KO mouse have demonstrated promise for BMS as a therapeutic for FXS. In an initial study, BMS treatment rescued social, cognitive, and anxiety phenotypes and normalized dendritic morphology in the Fmr1-KO mouse [Fmr1-KO mouse [1/2 = 1.9) [Studies with the KO mouse . Two subKO mouse ,217. One2 = 1.9) . This woV2.2 channels are key components of neurotransmission in the central nervous system and in the autonomic and sensory nervous system, and play a key role in early development [2+ currents by reducing the expression of CaV2.2 channels via proteasomal degradation. A follow-up study found that loss of FMRP resulted in increased Cav2.2 channel currents due to increased surface expression [Caelopment ,219. Theelopment . It is lelopment . FMRP inelopment ,104. Thipression . This coV2.2. A study of 20 ASD patients identified a duplication of the chromosomal region 9q43.3, which contains the gene CACNA1B, which produces Ca2+ currents in CaV2.2 channels [2+ activity itself has been identified in ASDs. A small study of six ASD patients with six age-matched controls found significantly higher levels of Ca2+ in ASD patients [V2.2 channels and highlight their physiological importance in neuronal functions.There are numerous links with sequence variants in voltage-gated calcium channel genes and ASD. Iossifov et al., 2014 [channels . This dupatients . These fV2.2 channels in the body, and the effects that calcium channel-directed medications have on the cardiovascular system, the development of therapeutics which seek to target these channels in central neurons face significant challenges. However, the connection to FMRP, ECS, and BKCa channels have with alterations in CaV2.2 function and presynaptic activity represents an important pathway that may have therapeutic implications for ASD.Due to the widespread expression of CaFMR1 mutations and ASD. Specifically, a set of neural mechanisms disrupted by the FMR1 mutation that result in presynaptic dysregulation, and also share associations with ASD independently of FMR1. The link between these systems and neurodevelopmental disorders, specifically FXS and ASD, is a relatively recent area of research [In this article, we have reviewed a specific subset of deficits connected by research ,225,226.2+ channels. It has not been explored if eCBs have activity at BKCa channels in central neurons, however, eCBs can modulate BKCa channels in cell culture [v2.2 channel activity during critical periods in development are sorely needed. Additionally, it is unknown if specific genetic variants in these FMR1 related regulatory mechanisms are able to produce a model of the spectrum of ASD phenotypes. In the case of FMR1, unique mutations that interfere with FMRPs regulatory function on BKCa are associated with unique neurodevelopmental phenotypes associated with ASD [Future studies are needed to explore more detailed mechanistic questions surrounding the presynaptic hypothesis of ASD with regard to the ECS and BKCa channels. Questions such as, Do the ECS and BKCa channels interact? It is well established that they each regulate the same presynaptic Ca culture . Developwith ASD ,211.FMR1 mutations, or direct insults to regulatory systems.Due to the high level of heterogeneity seen in autism, it is imperative that systems that are mechanistically linked, and shown to modulate a spectrum of behavior, be thoroughly studied. This is particularly crucial in regard to ASD. Novel methods for modeling this disorder are needed, as are therapeutics. Therefore, the development of new models, and the identification of associated genetic variants in the population, is critical for improving our understanding of this complex and diverse disorder. The dysregulated presynaptic regulatory mechanisms discussed in this review present numerous targets which could be approached systematically to answer questions about presynaptic calcium dynamics and spectrum-like phenotypes that arise either due to


Phaeophyceae) have been consumed by humans for hundreds of years. Current studies have shown that brown algae are rich sources of bioactive compounds with excellent nutritional value, and are considered functional foods with health benefits. Polysaccharides are the main constituents of brown algae; their diverse structures allow many unique physical and chemical properties that help to moderate a wide range of biological activities, including immunomodulation, antibacterial, antioxidant, prebiotic, antihypertensive, antidiabetic, antitumor, and anticoagulant activities. In this review, we focus on the major polysaccharide components in brown algae: the alginate, laminarin, and fucoidan. We explore how their structure leads to their health benefits, and their application prospects in functional foods and pharmaceuticals. Finally, we summarize the latest developments in applied research on brown algae polysaccharides.Brown algae ( Algae is an important food source consumed by humans since ancient times. Marine macroalgae, in particular, are important food sources in the coastal regions of East Asia such as China, Korea, Japan, and Indonesia . The gloPhaeophyceae alone accounts for over 1800 species and 66% of the total algae consumption [Laminaria (kombu), Undaria (wakame), and Macrocystis [Macroalgae are classified into green, red, and brown algae . Brown asumption . The mosrocystis .Mannitolrocystis . It is arocystis  and apperocystis . Mixed-lrocystis .The polyrocystis . Such hed-mannuronic acid (M) and α-l-guluronic acid (G) connected through 1,4-glycosidic linkages [Ascophyllum nodosum, for instance, is about 2:1 [2+ [Alginate is the predominant polysaccharide component found in the cell walls and intercellular matrix of brown macroalgae. It is a linear polysaccharide composed of two conformational isomer residues: β-linkages . Therefolinkages . The M/Gbout 2:1 . Alginatbout 2:1  which al 2:1 [2+ . Pseudomonas sp. HZJ216 and efficiently produced AOSs with DP of 2–7 [Saccharophagus degradans 2-40T, which produces AOSs DP3–5 [Vibrio sp. W13 mainly released oligosaccharides DP of 2–5 [Cellulophaga sp. NJ-1 [Alginate oligosaccharides (AOS) are oligomers with a degree of polymerization of 2 to 25, commonly obtained by chemical degradation , physical degradation (such as microwave degradation), enzymatic degradation , or chemical synthesis. Compared with physicochemical methods, enzymatic degradation of alginate is eco-friendly, energy-saving, selective, and the products are biologically more active . AlginatSs DP3–5 . Endo-tyP of 2–5 . Zhu et sp. NJ-1 . AlginatAOSs have been reported to modulate a variety of biological activities and are beneficial to health. Studies have shown that AOSs with different degrees of polymerization have differential biological activity. Therefore, they can be used as antimicrobial, antioxidant, prebiotic, antihypertensive, antidiabetic, antitumor, and anticoagulant agents; their many applications are further discussed below. ,33,34,35Microbulbifer (DP: 2–5) was capable to scavenge free radicals , ABTS+ , and hydroxyl) and was non-toxic even at high concentrations [▪, ▪OH, and O▪−2, polymeric alginate scavenged up to 23, 46, and <1%, while monomeric alginate (represented by glucuronic acid) scavenged up to <1, 25, and 99%, respectively [AOSs can scavenge free radicals. The AOS produced by the alginate lyase from trations . Mia et ectively . Due to ectively . The posectively .2O2 stress [Compared to chitosan and fucoidan-derived oligosaccharides, AOSs showed higher free radical scavenging capacity . Studies2 stress . In mice2 stress . Further2 stress . The fre2 stress ,45. Escherichia coli, Salmonella paratyphi, Staphylococcus aureus, and Bacillus subtilis [Hu et al. found that oligo-G and oligo-M (DP: 1–5) obtained by enzymatic hydrolysis had in vitro antibacterial activity against 19 bacterial strains. The antibacterial spectrum of the M oligomer fractions was wider than that of the G oligomers. Within the former, mannuronic acid oligomers with a molecular weight of 4.2 kDa had the highest antibacterial activity and a strong inhibitory effect on subtilis .Candida albicans mycelial infiltration in an in vitro epithelial cell model. OligoG CF-5/20 reduced the expression of C. albicans virulence proteins (phospholipase B (PLB2), SAP4 and SAP6) [The drug candidate OligoG CF-5/20 is developed by the Norway-based biotech company AlgiPharma. It is a G-enriched alginate oligosaccharide composed of G (85%) and M (15%) blocks. The OligoG CF-5/20 is effective in disrupting and destroying biofilms in a dose-dependent manner. The number of colony-forming units (CFU) in the lungs of infected mice was reduced by 2.5 log; furthermore, 5% OligoG CF-5/20 significantly reduced the minimum biofilm eradication concentration (MBEC) of colistin from 512 to 4 µg/mL after 8 h . OligoG nd SAP6) , but thend SAP6) . The resnd SAP6) . Similarnd SAP6) .Pseudomonas aeruginosa. Azithromycin combined with 2 mg/mL enzymatically produced AOS inhibited the growth of Pseudomonas aeruginosa, virulence factors, and biofilm formation controlled by quorum sensing [Pseudomonas aeruginosa biofilm and colistin to maintain its antibacterial activity. OligoG CF-5/20 did not change the orientation of the alginate carboxyl groups, while mass spectrometry analysis showed the oligomers to reduce pseudomonal quorum-sensing signaling [2+, in homopolyguluronic acid, is known to induce changes in coordination of the carboxylate groups [2+, avoiding the formation of a strong biofilm, so that the colistin can better play an antibacterial effect [p < 0.001) [OligoG CF-5/20 has been proposed as inhalation therapy for the treatment of chronic bacterial respiratory diseases . The oli sensing . Pritchaignaling . The gele groups , which re groups . Howeverl effect . Tønderv< 0.001) .AOSs can enhance immune activity and regulate the function of the immune system in a variety of ways, including regulating the secretion of cytokines and immune-complement molecules. The AOSs produced by depolymerization with alginate lyase increased TNF-α-inducing activity compared to untreated alginate, including the expressions of cytokine-induced TNF-α, granulocyte colony-stimulating factor (CSF), single nuclear cell chemotactic protein-1 , granulocyte macrophages (GM)-CSF, and eosinophil chemokine . AOSs caRecent studies have also shown AOSs to have antitumor effects. They exert, for instance, an inhibitory effect on the proliferation of human leukemia U-937 cells and produced cytotoxins in human monocytes . AOSs thLaminaria and Saccharina macroalgae, although it is also found in Ascophyllum, Fucus and Undaria [d-glucopyranose residues; the majority of glucose is 6-O-branched, while a part of it has β-1,6-intrachain links [d-mannitol at the reducing end of the chain is called an M chain, while laminarin without mannitol at the reducing end is a G chain [Eisenia bicyclis has a ratio of 2:1 of (1–3) and (1–6) linkage [L. saccharina and L. digitata [A. esculenta, U. pinnatifida, A. nodosum and F. serratus  [C. thermocellum which can hydrolyze laminarin into a series of oligosaccharides (DP2 to DP7) [Vibrio breoganii 1C10, of which VbGH16C can hydrolyze laminarin to oligosaccharides of DP8 and DP9, and VbGH17A can hydrolyze laminarin into a series of laminarin oligosaccharides (DP4 to DP9) [Saccharophagus degradans 2-40T and identified as a novel β-1,3-endoglucanase (EC 3.2.1.39) and bacterial β-1,6-glucanase (EC 3.2.1.75). This bifunctional laminarinase can degrade both the backbone and branch chain of laminarin, and is also active on hydrolyzing pustulan which is a linear β-1,6-glucan. This enzyme also showed transglycosylase activity toward β-1,3-oligosaccharides when laminarioligosaccharides were used as the substrates [Laminarin is another major storage carbohydrate of brown macroalgae. It is commonly found in the fronds of  Undaria . Laminarin links . Laminar G chain   ,70,71. Tctively) ,73. Lamictively) . The endctively) . For debctively) . Endo la to DP7) . Badur e to DP9) . Wang etbstrates . The aboLaminarins and laminarin oligosaccharides are recognized for their various biological activities; they have shown to stimulate innate immunity , stimulaL. hyperborea and A. nodosum to remove DPPH free radicals effectively, with clearance rates of 87.6 and 93.2%, respectively. Compared to extracts obtained with water solvents, acid-extracted laminarin was showed to have higher antioxidant activity [Studies have shown of the crude extracts of laminarin from activity .Staphylococcus aureus and Listeria monocytogenes) and Gram-negative  bacterial strains. Notably, the inhibitory rate of A. nodosum extract against Salmonella typhimurium can reach 100%. Laminarin-rich extracts obtained using ultrasound and acid solvents had minimum inhibitory concentrations (MIC) of 13.1 mg/mL for E. coli and S. typhimurium and 6.6 and 3.3 mg/mL for S. aureus and L. monocytogenes, respectively [Laminarin-rich seaweed extracts are found to have inhibitory effects against both Gram-positive  by stimulating monocytes to produce cytokines . SpecifiLaminaria sp. extract was first reported in 1941 [Laminaria. If each glucose residue has an average of two sulfate groups, the anticoagulant activity of the preparation reaches 25–30% of that of standard heparin [The anticoagulant activity of  in 1941 . Althoug in 1941 . Many st heparin , while t heparin . A deriv heparin . This suStudies have shown that β-glucans cause reduced recruitment of inflammatory cells and decreased secretion of inflammatory mediators in liver tissues through direct effects on immune cells or indirect effects as dietary fibers . LaminarBifidobacterium animalis and Lactobacillus casei, also increasing the production of short-chain fatty acids, such as lactic acid and acetic acid [The prebiotic properties of algae polysaccharides enable them to play an important role in regulating human intestinal health . For lamtic acid .Fucus vesiculosus) has a simple composition and contains only fucose and sulfate groups  [Fucus vesiculosus contains 84% fucose, 6% xylose, 7.3% galactose, and 2% mannose [Fucus distichus is composed of 51.6% fucose, 2.7% xylose, 1.5% galactose, 0.7% mannose, and 0.2% glucose [Fucoidan is a sulfated polysaccharide that consists mainly of fucose repeating units besides several other monosaccharide residues. It is commonly found in brown seaweed ,112, andsulfate) . In addi mannose . The fuc glucose . ComprehWenyingzhuangia fucanilytica, and found that Alf1_Wf was capable of hydrolyzing α-1,4-fucosidic linkage and synthetic substrate. Besides, Alf1_Wf could act on partially degraded fucoidan [The structural variety of fucoidans is to a large extent related to the different types of brown algae they are found in. Generally, α (1→3) and/or (1→4) glycosidic bonds constitute the main chain of the macromolecules, dominating in most backbone structures. The presence of sulfate groups at the C-2, C-4 and or C-3 position is another important feature ,126,127.fucoidan . Comparefucoidan ,133.Fucoidan has significant antitumor activity against liver cancer, stomach cancer, cervical cancer, lung cancer, and breast cancer ,137,138.Dictyota mertensii, Lobophora variegata, Spatoglossum schroederi, and Fucus vesiculosus inhibit the HIV reverse transcriptase (RT) enzyme, while other studies have shown that they also reduce the amount of the HIV-1 p24 antigen [Fucoidans isolated from different seaweed species have potential antiviral activity. For instance, they can inhibit the replication of enveloped viruses, including the human immunodeficiency virus (HIV) and the herpes simplex virus (HSV) . Accordi antigen . CompareLeathesia difformis and verified the selective antiviral activity of different components against HSV-1 and HSV-2 [Undaria pinnatifida has shown antiviral activity against 32 HSV clinical strains, including 12 ACV-resistant (4 HSV-1 and 8 HSV-2) and 20 ACV-susceptible ones. Judging by the survival rate and lesion score, oral fucoidan can protect mice from HSV-1 infection by stimulating cytotoxic T lymphocytes, natural killer activity, and neutralizing antibodies [Many studies have reported the blocking effect of fucoidan on HSV infection. Fledman et al. isolated different fucoidan components from nd HSV-2 . Fucoidatibodies .The above findings clearly indicate the potential antiviral activity of seaweed fucoidans, which can also strengthen the immune response of the host and achieve multi-channel and multi-level regulation of the immune system ,152. TheFucoidan can enhance the various beneficial effects of lactic acid bacteria on immune function by improving Th1/Th2 immune balance , and canIt has been reported that the destruction of connective tissue during inflammatory diseases such as chronic wounds, chronic inflammation, or rheumatoid arthritis is a result of a continuous supply of inflammatory cells and increased production of inflammatory cytokines and matrix proteases . SelectiStudies have shown that fucoidan can also exhibit antidiabetic effects by reducing postprandial hyperglycemia and pancreatic β-cell damage, increasing insulin secretion, and regulating glucose metabolism to reduce blood sugar ,168. FucIn addition, fucoidan may have a positive effect on antidiabetics by reducing β cell damage in the pancreas and increasing insulin secretion. According to a complex mechanism, the polysaccharide enhances the activity of sirtuin 1, thereby inducing deacetylation and upregulation of FOXA2 and p-FOXO-1 to promote the expression of PDX-1 and its regulation of insulin synthesis, thereby reducing β cell apoptosis and dysfunction in mice . FurtherEcklonia kurome and Hijikia fusiforme were found to have the strongest effect in terms of thromboplastin time (TT) and activated partial thromboplastin time (aPTT) [Heparin is a highly sulfated polysaccharide found in mammalian tissues and has been used as an anticoagulant for more than 50 years . Howevere (aPTT) . The mece (aPTT) . Fucoidae (aPTT) .The posie (aPTT) .Fucus vesiculosus-derived fucoidan was found to hinder fat accumulation in 3T3-L1 adipocytes by stimulating lipolysis through increased hormone-sensitive lipase expression and reduced glucose uptake [Fucoidan also has a positive effect in treating and preventing obesity. It has been shown to suppress the formation of 3T3-L1 adipocytes, thus inhibiting fat accumulation, by downregulating fatty acid binding proteins, acetyl-CoA carboxylase, and peroxisome proliferation-activated receptor γ. . Furthere uptake .At present, there is limited information available regarding the antiallergic effect of fucoidan. Recent studies have shown that the orally administered polysaccharide suppresses allergic symptoms by promoting the expression of galectin-9 mRNA and serum galectin-9 levels, thereby preventing immunoglobulin E (IgE) binding to mast cells .This review summarized the physicochemical and structural properties of polysaccharides and oligosaccharides derived from brown algae. Their structure and composition determine their biological activity and thereby their nutritional and therapeutic potentials. Although more is now known regarding their biological activities in vitro and significant advance has been made in their extraction from natural sources and modifications, further structural-activity investigation is necessary. Sustainable technologies must be established for the purification of the polysaccharides and the production of oligosaccharides, minimizing energy and chemical consumption while allowing upscaling of consistent quality and freedom from side-effect causing impurities. Lastly, research on catalytic enzymes, including alginate lyase, laminarinase, fucoidanase, and fucosidase, with high stability and desired substrate specificity is needed to enable the production of high-purity oligosaccharides with uniform structure and degrees of polymerization. Progress in enzyme and metabolic engineering will further promote the utilization of brown algae polysaccharides in the food and pharmaceutical industries.


Galectin-3 reportedly participates in the inflammatory process that causes insulin resistance in the target tissues. However, the role of high plasma galectin-3 levels as an indicator of protein-energy wasting (PEW) in patients undergoing maintenance hemodialysis remains unclear. This study included 240 hemodialysis patients  from a tertiary medical center. A baseline assessment of demographic and clinical data, biochemical parameters, and body composition was conducted. Plasma galectin-3 and other biomarkers were measured using a multiplex bead-based immunoassay. Participants were then divided into two subgroups depending on the median value of plasma galectin-3. Malnutrition was identified using the geriatric nutritional risk index (GNRI) and the criteria of the International Society of Renal Nutrition and Metabolism. Independent risk factors for elevated plasma galectin-3 and malnutrition were identified by multivariate logistic regression. The high galectin-3 group was more likely to be older, have lower lean tissue mass and GNRI scores, be diagnosed with PEW, dialyze through a tunneled catheter, and have higher circulating IL-6, TNF-α, and MCP-1 concentrations than the low galectin-3 group. After multivariate adjustment, only low mean arterial pressure, dialyzing with tunneled cuffed catheters, and elevated systemic inflammatory markers correlated with high galectin-3 levels. Plasma galectin-3 concentrations also increased significantly in hemodialysis patients with PEW. However, compared with other commonly used nutritional indicators, galectin-3 did not show superiority in predicting PEW. Although the plasma galectin-3 levels correlated with PEW severity, this correlation disappeared after adjustment for potential confounding variables . In conclusion, plasma galectin-3 is a valuable biomarker for systemic inflammation but is less prominent for PEW in patients with maintenance hemodialysis. Further identification of novel biomarkers is required to detect patients at risk for malnutrition and implement appropriate interventions. Chronic kidney disease (CKD) is a major noncommunicable disease with an increasing prevalence over the past few years ,2. Most Galectin-3 is a β-galactoside-binding protein that is fundamental in a number of cellular functions, including cell adhesion, proliferation, apoptosis, signal transduction, and immune and inflammatory response regulation ,16. ThisThe role of galectin-3 in nutritional status assessment has not been explored until recently. For patients with biliary tract cancer, an increased plasma concentration of galectin-3 is associated with the risk of malnutrition . In addin = 6); (2) having a prosthetic limb (n = 2); (3) having an implanted pacemaker or cardioverter-defibrillator (n = 2); and (4) having a history of solid tumors (n = 3), bacterial infections (n = 5), or hepatobiliary disease (n = 4). Ultimately, 240 patients were included. On 5 July 2019, the Institutional Review Board of Taipei Veterans General Hospital approved this study (protocol code: 2019-07-026BC). Furthermore, this study conformed to the Declaration of Helsinki. Written informed consent was obtained before the participants entered the research.Between 1 January and 31 December of 2020 in a tertiary medical center, this cross-sectional study initially recruited 262 adult patients (≥20 years old) on maintenance hemodialysis treatment thrice weekly for at least 12 weeks. Residual renal function, defined as the renal urea clearance greater than 2 mL/min, was lost in all cases. The exclusion criteria were as follows: (1) total hemodialysis treatment time < 12 h per week .We collected fasting blood specimens before dialysis initiation on the midweek session. Plasma was separated within 30 min and stored at −80 °C for subsequent analyses. Then, the plasma levels of galectin-3, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were detected using an inhouse multiplex bead-based immunoassay (MBIA) according to a published protocol ,28. Brieurea) using the second generation Daugirdas’ equation [Using a Hitachi 7600 automated chemistry analyzer , we measured biochemical parameters such as serum albumin, blood urea nitrogen, creatinine, calcium, and phosphate. Dialysis adequacy was determined from the single pool Kt/V for urea  as a surrogate to estimate lean body mass.The body composition of patients undergoing hemodialysis was evaluated by a multifrequency bioimpedance analysis (MFBIA) device . On the day of blood sampling, patients underwent MFBIA approximately 30 min after a dialysis session. The bioelectrical impedance measurements were obtained using a set of frequencies  at the five segments of the body . Then, body composition parameters such as fat mass, fat-free mass, and ratio of extracellular water (ECW) to total body water (TBW) were measured using preprogrammed prediction equations provided by the manufacturers and normalized to the square of the individual’s height in metres. We used fat-free mass index  and the International Society of Renal Nutrition and Metabolism (ISRNM) criteria for PEW ,32. The utrition ,31.2; (3) muscle mass: muscle mass loss > 5% (muscle atrophy) measured by MFBIA on two separate occasions over a 3-month period; and (4) nutrient consumption: nPCR less than 0.8 g/kg/day. When at least three criteria were met, the patient was diagnosed with PEW.The ISRNM expert panel recommended four main categories, namely, biochemical indicators, body mass, muscle mass, and nutrient consumption, for PEW clinical diagnosis . In this2) test, Student’s t-test, or Mann–Whitney U test, as appropriate. The relationships between the studied variables were explored by Spearman rank correlation. The independent risk factors for elevated plasma galectin-3 and malnutrition were identified using multinomial logistic regression analysis. Age, sex, and variables with p < 0.1 in the analysis of univariate data were used in the multivariate analysis. Multicolinearity was identified by calculating the variance inflation factor (VIF) for each predictor in multivariable models, and a VIF ≥ 2.5 indicated considerable collinearity. Furthermore, the diagnostic accuracy of the GNRI, galectin-3, IL-6, TNF-α, and MCP-1 in predicting PEW was evaluated by constructing receiver operating characteristic (ROC) curves. The optimal cutoff point and the maximum summation value of sensitivity and specificity were determined using the Younden index. The areas under the curve (AUC) of each marker were compared using DeLong’s test [p < 0.05 was considered statistically significant. All statistical data were analyzed using SPSS Statistics 23  and GraphPad Prism 9 .The results are expressed as counts and percentages for the categorical data, and mean ± standard deviation or median and interquartile range for the continuous normally or non-normally distributed variables. The patients were classified into two different groups according to the median value of plasma galectin-3. The two groups were compared using the chi squared , 134 (55.8%), and 113 (47.1%) subjects, respectively. Around 80% of the participants dialyzed with arteriovenous (AV) fistulas. The median GNRI score was 101.3, and 62 (25.8%) individuals had a low (≤98) GNRI. According to the ISRNM criteria, 48 patients (20.0%) were diagnosed with PEW. Moreover, the median plasma concentrations of galectin-3, IL-6, TNF-α, and MCP-1 were 567.3, 11.9, 2.9 and 173.6 pg/mL, respectively.To evaluate the relationship between galectin-3 and clinical and laboratory findings in patients undergoing maintenance hemodialysis, we further divided the patients into two subgroups based on the median value of plasma galectin-3. The high galectin-3 group had a higher proportion of older people with a lower MAP and was more likely to have lower FFMI and GNRI scores and be diagnosed with PEW than the low galectin-3 group. Additionally, patients in the high galectin-3 group were more likely to dialyze through a tunneled catheter and less likely to have an AV fistula than those in the low galectin-3 group. The high galectin-3 group also had significantly lower serum albumin levels but significantly higher circulating concentrations of IL-6, MCP-1, and TNF-α than the other group.The relationship between plasma galectin-3, systemic inflammatory markers, and various nutritional parameters, such as BMI, FFMI, and fat mass index (FMI), was determined using Spearman’s rank-order correlation. Galectin-3 positively correlated with the plasma levels of IL-6, TNF-α, and MCP-1 and negatively correlated with serum albumin, GNRI, and FFMI shown in . The thrp < 0.1 in the univariate analysis, MAP, tunneled cuffed catheters, and circulating TNF-α and MCP-1 remained significantly correlated with plasma galectin-3 concentrations.The factors influencing plasma galectin-3 levels in these patients were identified by logistic regression analysis . In the We next examined whether elevated galectin-3 levels could discriminate between hemodialysis patients with and without malnutrition. The ISRNM Diagnostic Criteria for PEW were used for the nutritional status assessment. Indeed, patients with PEW demonstrated significantly higher plasma galectin-3 levels than those without PEW shown in A. FurtheIn addition, the predictive factors of PEW in these patients were determined by logistic regression analyses . AlthougThis study revealed that galectin-3, an important modulator of many biological processes, was significantly upregulated in the plasma of hemodialysis patients with elevated levels of inflammatory markers. In the ROC analysis, the diagnostic accuracy of circulating galectin-3 in predicting PEW was inferior to that of GNRI or IL-6. Additionally, although the plasma galectin-3 levels correlated with PEW severity, this correlation disappeared after adjustment for potential confounding variables. Therefore, this study does not support using plasma galectin-3 for routine nutritional assessment of these patient groups.Low-grade systemic inflammation is a common hallmark of CKD and has been consistently associated with higher risks for death and severe complications . InflammOur results showed that the plasma galectin-3 level significantly increased in hemodialysis patients at risk of PEW, although it was not an independent predictor for PEW. This finding contradicts the results of studies conducted in patients with gastrointestinal malignancy ,26, and Moreover, as noted above, an elevated plasma galectin-3 level is clearly associated with chronic inflammation in hemodialysis patients; however, it may not adequately reflect the severity of muscle and fat wasting at the patient level. Chronic inflammation can cause skeletal muscle atrophy partly via inducing local and systemic insulin resistance ,40. GaleHowever, this study has several limitations. First, because of small sample size of this study, future studies must have a sufficiently large sample size to confirm these findings. Second, the study has a cross-sectional study design, which cannot determine causal effects or analyze the long-term changes in nutritional status. Hence, the research results must be interpreted cautiously. Third, unmeasured confounding effects may still exist despite adjusting for potential confounders by multiple regression. Fourth, impaired renal clearance, cardiac inflammation, activated platelets, and other etiologies may underlie the elevated circulating levels of galectin-3 in hemodialysis patients ,45,46. AInflammation and PEW are common findings in patients undergoing maintenance hemodialysis. In these patients, the level of plasma galectin-3 is associated with an increased risk for chronic systemic inflammation but not PEW. In addition, galectin-3 did not show superiority in predicting PEW compared with GNRI, which is a well-validated nutritional screening tool for patients at risk for PEW. In future studies, identification of novel biomarkers is required to detect patients at risk for malnutrition and implement appropriate interventions to decrease the burden of CKD.