Purpose: The interplay of inflammation and immunity affects all stages from tumorigenesis to progression, and even tumor response to therapy. A growing interest has been attracted from the biological function of MICALL2 to its effects on tumor progression. This study was designed to verify whether MICALL2 could be a prognostic biomarker to predict kidney renal clear cell carcinoma (KIRC) progression, inflammation, and immune infiltration within tumor microenvironment (TME).Methods: We firstly analyzed MICALL2 expressions across 33 cancer types from the UCSC Xena database and verified its expression in KIRC through GEPIA platform and GEO datasets. The clinicopathological characteristics were further analyzed based on the median expression. Kaplan-Meier method, univariate and multivariate analyses were applied to compare survival outcomes. ESTIMATE and CIBERSORT algorithms were performed to assess immune infiltration, and a co-expression analysis was conducted to evaluate the correlation between MICALL2 and immunoregulatory genes. Enrichment analysis was finally performed to explore the biological significance of MICALL2.Results: MICALL2 was highly expressed in 16 types of cancers compared with normal tissues. MICALL2 expression increased with advanced clinicopathological parameters and was an independent predictor for poor prognosis in KIRC. Moreover, MICALL2 closely correlated with inflammation-promoting signatures and immune infiltration including T cell exhaustion markers. Consistently, MICALL2 involved in the regulation of signaling pathways associated with tumor immunity, tumor progression, and impaired metabolic activities.Conclusion: MICALL2 can function as a prognostic biomarker mediating inflammation, immune infiltration, and T cell exhaustion within the microenvironment of KIRC. Tumor microenvironment (TME), a fertile soil for cancer progression, is composed of immune cells, bone marrow-derived inflammatory cells, fibroblasts, various signal molecules, extracellular matrix, and blood vessels Accumulating evidence has shown that the molecule interacting with CasL (MICAL) family participates in cytoskeleton dynamics, which is composed of two MICAL-L homologues  and three MICALs (MICAL1-3) Figure , we evaluated MICALL2 differential expressions across 33 cancer types and their corresponding normal tissues. Then we conducted the preliminary exploration on the role of MICALL2 expression in pan-cancers. Based on the significant results, we hypothesized that MICALL2 expression had close relation to patient prognosis, immune infiltration, and tumor progression in kidney renal clear cell carcinoma (KIRC) which was mainly investigated in the follow-up bioinformatics analyses. In addition, the potential biological functions or pathways of MICALL2 were screened using the gene set enrichment analysis (GSEA). Finally, two Gene Expression Omnibus (GEO) datasets were introduced for further validation analysis. All these findings revealed that MICALL2 could serve as a prognostic biomarker to predict the inflammatory and immune response within the TME, providing theoretical support for the precision treatment of KIRC.As shown in the flow process diagram http://xena.ucsc.edu). KIRC dataset with normal kidney tissues (N) = 72, and KIRC samples (T) = 539, was provided by TCGA database . The inclusion criteria can be found on the website (https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000178.v3.p3). GSE53757  and GSE40435  datasets were obtained from Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/).For pan-cancer analysis, the clinicopathological and transcriptome data containing 33 cancer types were provided by UCSC Xena database (http://gepia.cancer-pku.cn/), was used to compare MICALL2 expression levels between normal samples and tumor samples by matching TCGA normal and Genotype-Tissue Expression (GTEx) data .GEPIA platform  37℃ 30 min incubation with the secondary antibody. Next, DAB color rendering and hematoxylin redye were performed for 5-10 min and 3 min, respectively. The measurement of integrated optical density (IOD) was conducted by Image Pro Plus 6.0 image software.Based on MICALL2 median expression, the patients were divided into high-MICALL2 and low-MICALL2 groups. Kaplan-Meier (K-M) method was introduced to analyze Overall Survival (OS), Disease-Specific Survival (DSS), Progression-Free Interval (PFI) by R packages “survival” and “survminer”. Cox regression was further applied for the impact assessment of clinicopathological factors , and MICALL2 expression on survival based on univariate and multivariate analyses.The ESTIMATE algorithm The GSEA software (version 4.1.0) was applied to perform functional enrichment analysis of MICALL2 in KIRC. As the reference, the annotated “c2.cp.kegg.v7.4.symbols.gmt” was introduced to explore the potential biological pathways that MICALL2 may regulate in KIRC.The GSE53757 and GSE40435 datasets were used to validate MICALL2 expression difference between KIRC samples and normal tissues. For GSE53757 dataset, the differentially expressed genes (DEGs) were identified to screen the biological functions and signaling pathways between low-MICALL2 and high-MICALL2 expression samples by “limma” package . The enrichment tools such as Gene Ontology (GO) term, and Kyoto Encyclopedia of Genes and Genomes (KEGG) were then introduced to analyze the DEGs using R packages clusterProfiler, enrichplot, and ggplot2.The comparisons of between-group MICALL2 expressions were conducted by Wilcoxon test. The correlation concerning MICALL2 expression was analyzed by Spearman's correlation test. K-M method, univariate and multivariate analyses were introduced to compare survival outcomes. Statistical analyses were performed with GraphPad Prism (version 8.3) and R software (version 4.1.0). Statistical differences were confirmed when P values were less than 0.05.Figure ). Our results suggested that MICALL2 was highly expressed in 16 types of cancers  compared with their corresponding normal tissues. In contrast, MICALL2 levels were downregulated in PCPG tissues relative to normal tissues. We next preliminarily explored the role of MICALL2 expression in pan-cancers. Based on the significant results, we hypothesized that MICALL2 expression had close relation to patient prognosis, tumor progression, and immune infiltration in KIRC which was mainly investigated in the follow-up bioinformatics analyses. Then we performed paired comparison analysis on KIRC dataset from TCGA database, showing that MICALL2 was upregulated in KIRC samples compared with paired normal samples . Consistently, higher MICALL2 expressions in KIRC than normal tissues were validated by IHC staining , GEPIA platform , GSE53757 , and GSE40435 datasets . The heatmap by pairwise comparison also presented the significantly differential expression of MICALL2 in GSE40435 dataset .UCSC Xena database was introduced to analyze the differential expression of MICALL2 across 33 cancer types and their corresponding normal tissues (Table 1). MICALL2 expressions in groups of G3/G4, Stage III/Stage IV, T3/T4, and M1 were significantly higher than those in groups of G1/G2, Stage I/Stage II, T1/T2, and M0, respectively . But there was no statistical difference in MICALL2 expression between ≤65 and >65, between Female and Male, between N0 and N1 . Therefore, our findings revealed that MICALL2 expression was positively correlated with tumor progression.Differential MICALL2 expression was further examined according to the clinicopathological characteristics including age, gender, pathological grade, clinical stage, T classification, M classification, and N classification  compared with those with higher MICALL2 levels. Cox regression was further applied for the impact assessment of clinicopathological factors and MICALL2 expression on survival based on univariate and multivariate analyses. In univariate analysis, the significant predictors of survival included age , pathological grade , clinical stage , T classification , M classification , and MICALL2 expression  (Table S1). Furthermore, these clinicopathological factors and MICALL2 expression were included in multivariate analysis, showing that age , pathological grade , and MICALL2 expression  were important independent predictors for poor prognosis in KIRC patients .To reveal the association between MICALL2 levels and prognosis in KIRC patients, we performed K-M survival analyses, finding that KIRC patients with lower MICALL2 levels had a longer OS, DSS, and PFI (Table S2). Among the co-expressed immunoregulatory genes, the majority were positively correlated with MICALL2 . Furthermore, the heatmaps also revealed the positive correlation between MICALL2 and T cell exhaustion markers, such as programmed cell death protein 1 (PD-1/PDCD1), CD160, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG3), and T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT). Therefore, we further analyzed the expression difference of immunoregulatory genes between high-MICALL2 and low-MICALL2 KIRC. Among the immunoregulatory genes with significantly different expressions, the majority, such as T cell exhaustion markers, had a higher expression in high-MICALL2 group compared with low-MICALL2 group .To explore the role of MICALL2 in immunoregulation, we firstly performed a pan-cancer co-expression analysis on the immunoregulatory genes encoding MHC, immunosuppression, immune activation, chemokine receptors, and chemokines proteins , and ESTIMATE score (P <0.001). But it was negatively correlated with Tumor purity (P <0.001), and there was no significant difference in Stromal score (P =0.054). MICALL2 expression had a positive correlation with the expression of common checkpoint genes such as CTLA4, PDCD1, LAG3, TIGHT, inducible T cell costimulator (ICOS), and indoleamine 2,3-dioxygenase 2 (IDO2) . But there was a negative correlation between MICALL2 expression and programmed cell death ligand 1 (PD-L1/CD274) expression , and no significant difference between MICALL2 expression and IDO1 expression . Next, we applied the CIBERSORT tool to analyze the correlation of MICALL2 expression with TIICs (22 immune cell types). Among them, 3 kinds of TIICs were positively correlated with MICALL2 expression, including T cells regulatory (Tregs), T cells follicular helper, and T cells CD8, while 6 kinds of TIICs were negatively correlated with MICALL2 expression, including T cells CD4 memory resting, B cells naive, Macrophages M2, Dendritic cells resting, Mast cells resting, and Neutrophils .The results in Figure , the immune cell fractions of Tregs, T cells follicular helper, and T cells CD8 were higher in the high-MICALL2 group compared with the low-MICALL2 group. In contrast, the immune cell fractions of T cells gamma delta, Macrophages M2, Dendritic cells resting, Mast cells resting, Eosinophils, and Neutrophils were lower in the high-MICALL2 group than the low-MICALL2 group.We also assessed the ratio differentiation of 22 immune cell types. As shown in Table S3). The immune signatures of MHC class I, APC co stimulation, CCR, checkpoint, inflammation-promoting, T cell co-stimulation, and Type I IFN response, had higher ssGSEA scores in the high-MICALL2 group than the low-MICALL2 group. In contrast, the ssGSEA score of Type II IFN response in the high-MICALL2 group was lower than that in the low-MICALL2 group .We further analyzed 11 immune signatures including pathways, checkpoints, and functions . The genes in low-expression group were significantly enriched in metabolic pathways related to Biosynthesis of unsaturated fatty acids, Butanoate metabolism, Citrate cycle TCA cycle, Glycolysis/gluconeogenesis, peroxisome, Propanoate metabolism, Proximal tubule bicarbonate reclamation, Pyruvate metabolism, Steroid biosynthesis, Valine, leucine and isoleucine degradation . These findings revealed that MICALL2 could involve in the regulation of signaling pathways associated with tumor immunity, tumor progression, and cancer metabolism.To analyze the biological significance of MICALL2, KIRC samples from TCGA database were divided into two groups. Then GSEA was introduced to assess those enriched KEGG pathways between these two groups. The genes in MICALL2 high-expression group were mainly enriched in Cytosolic DNA-sensing pathway, Glycerophospholipid metabolism, Hedgehog signaling pathway, Homologous recombination, MAPK signaling pathway, JAK-STAT signaling pathway, Notch signaling pathway, Phosphatidylinositol signaling system, Spliceosome, VEGF signaling pathway , a total of 1299 DEGs were identified with 582 downregulated genes and 717 upregulated ones, of which the top 30 ones were visualized on the heatmap based on |logFC| values . The GO enriched DEGs significantly focused on T cell activation, regulation of immune effector process, regulation of cell-cell adhesion, neutrophil activation, and positive regulation of cytokine production . In KEGG pathway analysis, the DEGs mainly focused on chemokine signaling pathway, cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, NF-kappa B signaling pathway, and cell adhesion molecules .In the GSE53757 dataset, the patients were divided into high-MICALL2 group and low-MICALL2 group based on MICALL2 median expression level. As shown in the volcano map  and interleukin 2 (IL-2) to the more efficacious and less toxic immune normalization with programmed cell death 1 (PD-1) or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibodies in advanced KIRC Growing evidence suggests that chronic inflammation increases cancer risk, but their specific association remains unclear Unlike traditional Warburg effect, the subversive findings reveal that cellular programming drives immune cells and cancer cells to preferentially obtain glucose and glutamine, respectively To date, this is the first report to demonstrate that MICALL2 could be a prognostic biomarker associating with cancer progression, inflammatory, and immune signatures of KIRC. Due to the highly heterogeneous, adaptive, and dynamic TME, more basic and preclinical studies are expected to further verify the specific molecular mechanism of MICILL2 in regulating the inflammatory and immune functions, and the corresponding signaling pathways we enriched above. In addition, novel drugs targeting MICALL2 signaling should also be developed for the treatment of KIRC. It should be noted that, for KIRC, single agent therapy potentially causes drug resistance and limited therapeutic efficacy Here we reported for the first time that MICALL2 is highly expressed in multiple cancer types and can serve as an independent predictor for poor prognosis in KIRC patients. MICALL2 expression is correlated with inflammation-promoting response, immune cell infiltration, and T cell exhaustion within the microenvironment of KIRC. Furthermore, MICALL2 can regulate the signaling pathways related to tumor immunity, tumor progression, and cancer metabolism. Our findings could provide a molecular basis of MICALL2 as a prognostic biomarker for the precise and personalized treatment of KIRC.Supplementary figure and tables.Click here for additional data file.


High incidences of liver necrosis (30%) of chicks were induced by –SE–VE, starting at day 16. The Se concentration in liver and glutathione peroxidase (GPX) activity were decreased (P < 0.05) by dietary Se deficiency. Meanwhile, Se deficiency elevated malondialdehyde content and decreased superoxide dismutase (SOD) activity in the liver at weeks 2 and 4. Chicks fed with the two Se-deficient diets showed lower (P < 0.05) hepatic mRNA expression of Gpx1, Gpx3, Gpx4, Selenof, Selenoh, Selenok, Selenom, Selenon, Selenoo, Selenop, Selenot, Selenou, Selenow, and Dio1 than those fed with the two Se-supplemented diets. Dietary Se deficiency had elevated (P < 0.05) the expression of SELENOP, but decreased the downregulation (P < 0.05) of GPX1, GPX4, SELENON, and SELENOW in the liver of chicks at two time points. Meanwhile, dietary Se deficiency upregulated (P < 0.05) the abundance of hepatic proteins of p38 mitogen-activated protein kinase, phospho-p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, phospho-c-Jun N-terminal kinase, extracellular signal-regulated kinase, phospho-mitogen-activated protein kinase, receptor-interacting serine-threonine kinase 1 (RIPK1), receptor-interacting serine-threonine kinase 3 (RIPK3), and mixed lineage kinase domain-like (MLKL) at two time points. In conclusion, our data confirmed the differential regulation of dietary Se deficiency on several key selenoproteins, the RIPK1/RIPK3/MLKL, and mitogen-activated protein kinase signaling pathway in chicks and identified new molecular clues for understanding the etiology of nutritional liver necrosis.Liver necroptosis of chicks is induced by selenium (Se)/vitamin E (VE) deficiencies and may be associated with oxidative cell damage. To reveal the underlying mechanisms of liver necrosis, a pool of the corn–soy basal diet , a basal diet plus all-rac Gpx1, Gpx4, Selenof, Selenoi, Selenok, Selenon, Selenoo, Selenop, Selenos, Selenot, Selenow, Msrb1, Txnrd1, and Txnrd2) in the liver and muscles of chicks fed with Se-deficient (0.01 mg/kg) and adequate (0.3 mg/kg) diets at week 6  mRNA levels of seven common selenoprotein genes  in liver and muscle. Nevertheless, these are the genetic changes far from after the onset of liver necrosis  deficiency, liver necrosis, was mostly discovered in rats . Each group of 40 chicks was kept in stainless-steel cages . The basal diet  were selected and allotted into 4 dietary treatment groups  were killed by decapitation to collect blood and liver samples. The remaining animals were sampled at week 6. After immediately dissected on an ice-cold surface, the liver was perfused with ice-cold isotonic saline before being minced with surgical scissors. The minced samples were divided into aliquots, snap-frozen in liquid nitrogen, and stored at −80°C until use.At weeks 2 and 4 of the study, chicks   and glyceraldehyde 3-phosphate dehydrogenase gene (Gapdh) were designed using Primer Express 3.0 .Literature search allowed us to target 25 selenoprotein genes in chicks  were loaded onto an SDS-PAGE (12.5%), transferred to polyvinylidene difluoride membranes, and incubated with appropriate antibodies  and centrifuged at 12,000 × tibodies  as previtibodies .P < 0.05.The results were analyzed by a one-way ANOVA using SPSS software . The data were presented as mean ± SE. The Duncan's multiple-range tests were used to evaluate the differences between treatments, and those differences were considered statistically significant when P < 0.05) by dietary Se deficiency (P < 0.05) hepatic Se concentrations at weeks 2 and 4  on 15 out of 25 selenoprotein genes assayed in the liver at week 2, whereas there was a main effect of dietary VE (P < 0.05) or an interaction between these two nutrients (P < 0.01) on 5 selenoprotein genes (P < 0.05) hepatic mRNA levels of Gpx1, Gpx3, Gpx4, Selenof, Selenoh, Selenok, Selenom, Selenon, Selenoo, Selenop, Selenot, Selenou, Selenow, and Dio1 than those fed with the two Se-supplemented diets. Conversely, liver mRNA levels of Selenoi were actually greater (P < 0.05) in the chicks fed with the Se-deficient diets than those fed with the Se-supplemented diets. Liver mRNA levels of Selenon were decreased (P < 0.05) in the Se-adequate chicks by dietary VE supplementation. Notably, chicks fed with the two VE-supplemented diets had lower (P < 0.05) liver mRNA levels of Selenoi, Selenos, Txnrd1, and Txnrd2 than those fed with the VE-deficient diets at both levels of dietary Se concentrations. Neither dietary Se nor VE affected liver mRNA levels of Gpx2, Selenopb, Msrb1, Txnrd3, Dio2, or Dio3.Dietary Se exerted the main effect (in genes . As showP < 0.05) in the liver by dietary Se concentrations (P < 0.05) SELENOP, but decreased (P < 0.05) GPX1,GPX4, SELENOW, and SELENON in the liver of chicks at weeks 2 and 4. Notably, chicks fed with the VE-supplemented diet had lower (P < 0.05) impact on the abundance of GPX4 in the liver in +Se chicks at two time points. Neither dietary Se nor VE affected GPX3 and MSRB1 in the liver at two time points. We observed liver tissues stained by H&E at 4 weeks  (P < 0.05)   relative < 0.05)  protein  < 0.05)  relativeek 4 and  protein The antioxidant effect of Se is mainly realized through selenoprotein, and an appropriate amount of Se can reduce the concentration of free radicals and thus protect organisms from lipid peroxidation. Furthermore, VE as a non-specific biological antioxidant could compensate and coordinate with Se in protecting cell membranes from oxidative damage  the mRNA levels of Gpx1, Gpx3, Gpx4, Selenof, Selenoh, Selenok, Selenom, Selenoo, Selenop, Selenou, and Selenow in liver at week 2. And six common genes  were downregulated by dietary Se deficiency in liver and muscle at weeks 2 and 6 . SELENOI (encoded by Selenoi) is an evolved selenoprotein specific to vertebrates and is essential for murine embryogenesis  as an important protein that protects cells against oxidative stress and redox-related calcium homeostasis  show the same trend at two time points, the expression of Gpx1 and Selenos was affected by VE supplementation at different time points in the liver. Overall, the selenoprotein gene mRNA levels have an inverse relationship with VE status, and further protein assays should be conducted in the future. In the liver, three additional selenoprotein genes  were downregulated by dietary Se deficiency at week 2. However, among the 25 genes assayed, only Selenoi mRNA level was decreased by dietary Se supplementation. Additionally, liver mRNA levels of Selenon were decreased in Se-adequate chicks by dietary VE supplementation. The gene code for protein is associated with neuroprotective function and cytosolic calcium regulation (Selenom)  had higher levels of phospho-p38 MAPK  in Se-deficient mice was sufficient to attenuate low-dose pro-oxidant-mediated JNK phosphorylation at Thr-183/Tyr-185 (Huang et al., Although Se deficiency affects gene expression in multiple tissues (Liu et al., via peroxide scavenging, controlling oxidative stress, and regulating subsequent stress signaling (Miyata et al., In summary, our data revealed the differential regulation of dietary Se deficiency on 25 selenoprotein genes, 7 key selenoproteins, and the RIPK1/RIPK3/MLKL and MAPK signaling pathway proteins in chicks and provided new molecular clues for understanding the pathogenesis of liver necrosis. The protective effects of Se are probably exerted The original contributions presented in the study are included in the article/The animal study was reviewed and approved by China Agricultural University.J-QH provided ideas for the experiment. TL and JZ completed the experiment and wrote the manuscript. Z-WZ and P-JW completed the parts of the figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Clinical genetic evaluation has been demonstrated as an important tool to elucidate the causes of growth disorders. Genetic defects of collagen formation (the collagenopathies) have been reported to be associated with short stature and skeletal dysplasias. Etiological diagnosis of skeletal abnormality-related short stature is challenging, and less is known about recombinant human growth hormone (rhGH) therapy.This is a single-center cohort study which aims at exploring the genetic architecture of short-stature children with skeletal abnormalities and evaluating the frequency of collagenopathies to determine their phenotype, including the rhGH treatment response.COL1A1, COL1A2, COL2A1, COL9A1, COL9A2, COL9A3, COL10A1, COL11A1, and COL11A2. The results were evaluated using American College of Medical Genetics and Genomics (ACMG) guidelines. Clinical characteristics and rhGH treatment response were summarized.One hundred and six children with short stature and skeletal abnormalities were enrolled who were evaluated by next-generation sequencing (NGS) to detect variants in the skeletal collagen genes including COL2A1 mutations were the most common, accounting for about 57.7%. Other frequent mutations associated with skeletal development include FGFR3, ACAN, NPR2, COMP, and FBN1 in 12.2%, 0.9%, 0.8%, 0.4%, and 0.4%, respectively, resulting in significantly different degrees of short stature. An overview of clinical features of collagenopathies showed growth retardation, skeletal abnormalities, and heterogeneous syndromic abnormalities involving facial, eye, hearing, and cardiac abnormalities. The average height of 9 patients who received rhGH treatment improved from a median of -3.2 ± 0.9 SDS to -2.2 ± 1.3 SDS after 2.8 ± 2.1 years. The most significant height improvement of 2.3 SDS and 1.7 SDS was also seen in two patients who had been treated for more than 6 years.Twenty-four pathogenic or likely pathogenic variants of collagen genes were found in 26 of 106 (24.5%) short-stature patients with skeletal abnormalities, of which A proband-based NGS revealed that distinct genetic architecture underlies short stature in varying degrees and clinical features. Skeletal abnormality-related short stature involving multiple systems should be tested for skeletal collagen gene mutation. Limited rhGH treatment data indicate an improved growth rate and height, and close monitoring of adverse reactions such as scoliosis is required. Childhood linear growth is the result of chondrogenesis at the skeletal growth plate, the structure responsible for bone elongation and therefore overall body size . RecentlGHR, Leri–Weill dyschondrosteosis (MIM #127300) related to SHOX, Noonan syndrome (MIM #163950), and Silver–Russell syndrome (MIM #180860), which are associated with short stature and various multiorgan malformations  and COMP (cartilage oligomeric matrix protein) in ECM components but also for NPR2 (natriuretic peptide receptor 2) in paracrine signaling, we reported the phenotypic and genotypic spectra and efficacy of GH therapy for height gain. For collagen, the most abundant protein in the human body, however, the prevalence of collagen gene mutation in short-stature patients with skeletal abnormalities is yet unknown, the current clinical manifestations of the disease are heterogeneous, and the response data of growth hormone therapy are limited. Collagen types II, IX, X, and XI are present in a growth plate important to long bone development and joint health, and type I collagen is the primary collagen in bone for bone formation, growth, and remodeling, and subsequently mineralization to form bone tissue. Mutations in genes that encode skeletal collagen are not uncommon in the genetic causes for growth defects with skeletal abnormalities  molecules , as well as the normal function of multiple intracellular processes required for chondrocyte proliferation, hypertrophy, and extracellular matrix production , 3. Somermations –6. Genets MIM #12300 relatme MIM #10860, whi MIM #16350, and SSubsequently, we analyzed 106 children with short stature by using a gene panel for short stature and whole-exome sequencing (WES) from our single-center cohort and searched for variants in the skeletal collagen genes. Variant interpretation, genotype–phenotype analyses, and the response to rhGH treatment of skeletal abnormality-related short stature were investigated.One hundred and six children with short stature and skeletal abnormalities in our endocrinology department were included, 64 of whom received WES and 42 of whom received a short stature-targeted gene panel sequencing. Skeletal abnormalities were characterized by an intrinsic abnormality in growth and (re-)modeling of cartilage and bone, including the whole-body skeleton of axial bones, limbs, and craniofacial bones, which were assessed by a professional physician through physical examination and measurements. All probands fulfilled the following diagnostic criteria: height standard deviation (SD) ≤ -2 with skeletal abnormalities, absence of abnormal findings on clinical examination or in laboratory tests that could account for short statures, such as hypothyroidism and GHD, and known Noonan syndrome and Turner syndrome. Clinical materials of the first and follow-up visits of the probands, including history-taking, physical examination, and auxiliary examination, were collected. Information about rhGH therapy was also reviewed and recorded. Peripheral blood samples of patients and their available relatives were collected, and genomic DNA was obtained from peripheral blood leukocytes by using standard techniques. The patients or guardians signed informed consent forms regarding the research, and this study was performed with the approval of the Ethics Committee of Peking Union Medical College Hospital.via multiple databases, including NCBI dbSNP, HapMap, 1000 human genome dataset, and database of 100 Chinese healthy adults.The 3-μg genomic DNA concentrations were sheared with a Covaris LE220 ultrasonic instrument  to a target of 100–500-bp average size. Then, the DNA fragments with a main fragment size of 150–200 bp were screened by magnetic beads to create a DNA library for each subject. The library was qualitatively controlled by Agilent 2100 Bioanalyzer . All amplified libraries were subsequently sent to BGI for circularization and sequencing on the BGISEQ-500 platform, and the primary sequencing data were read out. To detect the potential variants in the family, bioinformatics processing and data analysis were performed after receiving the primary sequencing data. Sequencing data were aligned to the human genome reference (hg19) using the BWA (Burrows–Wheeler Aligner) Multi-Vision software package to analyze single-nucleotide variants (SNVs) and INDEL calling  of short-stature genes was previously designed and assessed by our group. The capture panel covered all exons together with the flanking exon and intron boundaries (± 15 bp) of 466 genes. Sequencing was performed on an Illumina HiSeq 2500 or HiSeq 2000 platform in paired-end mode. In-house bioinformatic analysis was performed. The sequences were aligned to the reference human genome (HG19/HG20). The probe size was about 2.427 MB, and the theoretical capture efficiency of the probe was 98.83%.in silico prediction programs to assess . Pathogenic variants were under the protocol issued by American College of Medical Genetics and Genomics (ACMG) guidelines  mutation. Other frequent mutations in paracrine signaling of the growth plate development include fibroblast growth factor receptor 3 (FGFR3) covering 12.3% (13/106), NPR2 covering 7.5% (8/106), and fibroblast growth factor receptor 3 (PTH1R) (n = 2). The remaining causal genes were associated with a fundamental cellular process, including TRPV4 (n = 5), SHOX (n = 3), KIF22 (n = 2), TRAPPC2 (n = 1), ARSL (n = 1), RUNX2 (n = 1), CENPJ (n = 1), FAM111A (n = 1), and pathogenic copy number variant (CNV) (n = 1)  missense mutations, 3 (12.5%) splicing mutations, 2 (8.2%) nonsense mutations, and 1 (4.2%) in-frame insertion mutation. Except for 4 biallelic heterozygous mutations, all the others were monoallelic heterozygous mutations. The results are summarized in COL11A2 has been identified in 1/7,442 of East Asian chromosomes by the Exome Aggregation Consortium , and the c.580G>A in COL2A1 is present at an extremely low allelic frequency (4/140282) in gnomAD.Among this cohort, a total of 24 pathogenic or likely pathogenic rare variants in collagen genes were identified in 26 (24.5%) of the 106 short-stature individuals with skeletal abnormalities as per the ACMG guidelines, of which 5 variants were classified as pathogenic and 19 as likely pathogenic . Fifty-eight percent of cases had facial abnormalities commonly observed in patients, mainly a low nasal bridge, high-arched palate, small jaw, or big or prominent ears. Cleft palate was observed in two patients with type II and one with type XI collagen gene mutations, and blue sclera was observed in two patients with type I and one with type IX collagen gene mutations. In addition, there were a small number of patients with other system defects, such as three with heart defects, three with congenital cataracts, strabismus, or amblyopia, and two with mixed deafness or ear deformity and congenital aural atresia.COL2A1 mutations showed that the phenotypic spectrum of COL2A1 mutations included spinal deformity, abnormal cartilage development, midface hypoplasia, and ocular abnormalities. Almost all showed spine deformity including scoliosis, lordosis, dysplasia, and osteoporosis. The clinical manifestations of three boys  in our study only carrying COL9A1/COL9A2 mutation had very mild symptoms, with only mild chondrodysplasia.Our data obtained from 15 probands with 12 kinds of COL10A1 mutations, one missense variant 1766T>G (p.Phe589Cys) was de novo paternity and maternity confirmed in two identical twin brothers , and one truncating mutation c.1858_1865del CCTGTAAT  was confirmed to segregate with the autosomal-dominant short-stature phenotype in five affected family members (P.21). Both types of mutations were located in the NC1 domain. All three probands and affected relatives exhibited typical metaphyseal chondrodysplasia type Schmid (SMCD) (MIN #156500) phenotypes with short bowed limbs, valgus knees, pronounced lumbar lordosis, posterior flexion of hips, enlarged large joints, and a faltering gait, which were consistent with radiographical findings. In addition, the proband with truncating mutations was born with flexion of the legs and had more severe forms of SMCD with additional manifestations such as short neck, pectus carinatum, beaded ribs, and widened epiphysis of the ribs. The two cases caused by missense variants exhibited relatively late-onset ages at around 2 years of age and mild or moderate manifestations.In the data obtained from three probands in two independent families with COL11A1 [c.739G>T(p.Ala247Ser)] and one splice site alteration of COL11A2 (c.1557+5C>T). The girl (P.22) with the COL11A1 mutation had a phenotype consistent with mild Marshall syndrome (MIM #154780), with midfacial hypoplasia, cleft palate, a less severe ocular presentation, but striking ocular hypertelorism, and short stature with spondyloepiphyseal dysplasia  with congenital cataract, sensorineural deafness, relatively short extremities with elbows valgus and joints pain, and typical midface hypoplasia.Two novel variants in the type XI collagen gene were identified with one missense variant of ysplasia . The boyCOL1A1 and COL1A2) were identified, and their skeletal abnormalities were characterized primarily by osteoporosis, with less common abnormalities in the limb bones and skull. In addition to skeletal abnormalities, they also have abnormalities in many organs, such as cardiovascular, joints, ligaments, midface development, and ocular anomalies. One patient (P.26) with biallelic heterozygous mutations [c.2121_2122insGCTGGTCCT (p.Pro707_Arg708insAlaGlyPro) and c.3583T>C (p. Cys1195Arg)] and one (P.24) with heterozygous truncating mutation [c.1386delT (p. Ala463Leufs*78)] had more severe osteogenesis imperfecta, such as early-onset motor retardation, heart defects, reduced thoracolumbar bone density, and obvious joint relaxation, than did the heterozygous missense mutations [c.3997A>G (p. Thr1333Ala)] (P.25).Three patients with mutations in genes encoding type I collagen , or osteochondritis dissecans (MIM #165800), which was consistent with the clinical findings of the 10 patients in our cohort, presenting as mild midface hypoplasia, short neck, thoracic deformity, spine malformation, short fingers/toes, short metacarpal bones, internal rotation of the elbow , and café-au-lait spots, and none of them complained of bone or joint pain. Four patients with COMP mutations exhibited typical pseudoachondroplasia (PSACH) (MIM #177170) with severe short-limb dwarfism, joint pain and stiffness, and early-onset osteoarthritis, and 4 patients with mutations in FBN1 represented as acromelic dysplasia (MIM # 102370) shared severe short stature, short hands and feet, and joint limitations. Biallelic variations of NPR2 mutation can cause acromesomelic dysplasia, Maroteaux type (AMDM) (MIM #602875), while monoallelic variants result in short stature with non-specific skeletal deformities and Miura-type osteochondral dysplasia. Autosomal dominant mutations in FGFR3 causing achondroplasia (ACH) (MIM #100800) were found in 13 of our cohort. They appeared as short stature resulting from the shortening of the limbs with proximal segments affected disproportionally. In addition, the typical broad or protruding forehead, lumbar lordosis, and sacral kyphosis were seen in all these patients. Among the 8 cases of NPR2 mutation in our study, except for one case with compound heterozygous mutation characterized by disproportionate short stature, mesomelic limb shortening, and shortened and broadened fingers and toes, conforming to AMDM, the other 7 cases were heterozygous mutation with or without disproportionate short stature, facial anomalies, and non-specific skeletal deformities, including mesomelic limb shortening, cubitus valgus, brachydactyly, shortened metacarpals or metatarsals, clinodactyly, and cone-shaped epiphysis.The clinical phenotypes of other skeletal abnormality-related short stature were also briefly summarized based on our recent report –15. Hetep <.01). Their average height Z score at the last follow-up was significantly increased to -2.2 ± 1.2 (p <.001)   and paracrine signaling genes (FGFR3 and NPR2), in which a subset of the 29 patients with ACAN mutation and 21 patients with NPR2 mutation were from the literature review, based on previous reports ((p = .747), indicating that there is no difference in height between males and females. Six of the patients were adults with a height Z score of -6.8 ± 1.2, which was significantly lower than the 100 juvenile individuals with a height Z score of -3.7 ± 0.2 (p = .001), suggesting that the height impairment worsened with age and would be more severely affected in adults without any treatment. In addition, when the affected individuals were divided into extracellular matrix maintenance and paracrine signaling of the growth plate and long bone development according to physiological etiologies of short stature, there was no significant difference in their height impairment (p = .683). We compared the height Z score of collagenopathy patients with other different-causing gene mutations and found that the ACAN mutation resulted in milder short stature than the collagen gene mutation (p = .021), while the COMP mutation was the most severe (p = .009)  after 2.8 ± 0.4 years of administration. For NPR2-related short stature, height Z scores were significantly improved from -3.1 ± 0.8 to -2.0 ± 1.0 after 3.8 ± 0.6 years of treatment. Moreover, the growth response for rhGH treatment in ACAN-related short stature was better than NPR2 (p = .014)  and skeletal collagen genes accounted for 24.5% (26/106). COL2A1 mutations were detected in 15 patients (14%), which was slightly higher than that of a recent NGS study on 82 short Chinese patients with clear signs of bone dysplasia (positive rate = 11%) (COL2A1), FGFR3, ACAN, NPR2, COMP, and FBN1 are common for short stature due to skeletal abnormalities in outpatient clinics in pediatric endocrinology. More importantly, we provided initial information about the phenotypical spectrum of collagenopathies by proving that short stature with skeletal abnormalities and heterogeneous syndromic abnormalities may be caused by mutations in the collagen genes.In this study, we performed NGS in short-stature patients with skeletal abnormalities and identified causal variants of skeletal collagen genes in 26 (24.5%) of the 106 individuals, among which 15 patients (14%) carried e = 11%) . A recene = 11%) . MeanwhiCOL2A1 and COL10A1 genes, respectively. Both type IX and XI collagens are heterotrimers encoded by 3 different chains of COL9A1, COL9A2, COL9A3 genes, and COL11A1, COL11A2, and COL11A3, respectively. Type I collagen is also a heterotrimeric molecule encoded by COL1A1 and COL1A2. These genes encode procollagens, which are synthesized in the endoplasmic reticulum and contain a short N-telopeptide non-triple-helical (NC2) domain, a long triple-helical domain, and a short C-telopeptide non-triple-helical (NC1) domain, and then posttranslational modification generates mature collagens, which are secreted into the extracellular matrix  and linear columns of differentiated chondrocytes that are organized into resting, proliferating, mature, and hypertrophic zones, which are continuously replaced by trabecular bone with the increase in length , 18, 19.r matrix , 21. In n trimer . In addin trimer .FGFR3, ACAN, NPR2, COMP, and, FBN1, usually cause varying degrees of short stature, with or without other mild abnormalities, such as slight growth ratio imbalance and skeletal non-specific abnormalities   , 8. In a of life . Both of of life . Type X variants . In the syndrome .COL2A1 mutations, age at diagnosis might also be associated with disease severity, which was consistent with a previous study of COL2A1 based on a large database , and Silver–Russell syndrome (SRS), while there are limited data on the effect of rhGH treatment on children with short stature and skeletal dysplasia –31. Recemutation . A studymutation . Our stu therapy . Except terature . In addi studies , the ver studies –37. Therin silico prediction programs. Furthermore, there are signs or clinical manifestations that have not been identified in some of our patients due to young age or too short follow-up. It is also worth mentioning that, unfortunately, we did not routinely assess bone mass in some children without a clear history of fractures. Moreover, some evidence for segregation of the variants with short stature within the families is lacking in some suspect pedigrees because of some unavailable relatives, which can strongly support the mutations’ pathogenicity. Lastly, more accurate genotype–phenotype correlations and evidence for the treatment of recombinant growth hormone deserve further study in a larger cohort of skeletal collagenopathies children with short stature.Potential limitations of this work warrant consideration. First of all, in terms of molecular genetic testing approaches, WES may not detect large CNVs. Therefore, multiple ligation-dependent probe amplification (MLPA) or chromosomal microarray (CMA) analysis should be performed to screen for large deletions and insertions in genes belonging to the short-stature gene panel. In addition, some of the newly discovered variants still lack further validation, so we only evaluated their pathogenicity according to the ACMG guidelines, combined with their clinical phenotypes, pedigree verification, and In conclusion, skeletal collagenopathies are relatively frequent in syndromic-related short stature, and screening for collagen mutations should be considered in short-stature children with skeletal abnormalities. Although long-term studies evaluating rhGH treatment are insufficient and large cohort studies regarding rhGH dose, the optimal age to start treatment, and adverse events are lacking, initial information provided by our study about the efficacy of rhGH treatment for skeletal collagenopathies indicates an improved growth rate and height. Before starting rhGH treatment, patients with collagenopathy-related short stature should be extensively evaluated, and close monitoring of adverse reactions such as scoliosis is required.The original contributions presented in the study are publicly available. This data can be found here: National Genomics Data Center (NGDC), PRJCA008063.The studies involving human participants were reviewed and approved by the Peking Union Medical College Hospital Ethics Committee. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.MC and HZ designed the study. MC, HM, and HL collected the data. MC and XK conducted the data collection and analysis. FG guided the experimental study. MC and HZ drafted the manuscript. HY, LW, LD, SC, and HP interpreted the data and revised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the CAMS Innovation Fund for Medical Sciences (CIFMS 2021-I2M-1-003).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.


Rheumatoid arthritis (RA) is a chronic disease which causes joint inflammation and, ultimately, erosion of the underlying bone. Diagnosis of RA is based on the presence of biomarkers, such as anti-citrullinated protein antibodies (ACPA) and rheumatoid factors, along with clinical symptoms. Much evidence points to a link between the Epstein-Barr virus and RA. In this study, we analyzed ACPA reactivity to citrullinated peptides originating from Epstein-Barr nuclear antigens  in order to elaborate the diagnostic potential of citrullinated EBNA peptides. Moreover, ACPA cross-reactivity to citrullinated peptides from myelin basic protein (MBP) was analyzed, as citrullinated MBP recently was described to be associated with multiple sclerosis, and some degree of sequence homology between MBP and citrullinated EBNA exists. A peptide from EBNA2,  reacted with approximately 70% of all RA sera, whereas only limited reactivity was detected to EBNA1 and EBNA3 peptides. Moreover, screening of ACPA reactivity to hybrid peptides of EBNA3-A (EPDSRDQQS-Cit-GQRRGDENRG) and EBNA2-A and peptides containing citrulline close to the N-terminal confirmed that ACPA sera contain different populations of ACPAs. No notable ACPA reactivity to MBP peptides was found, confirming that ACPAs are specific for RA, and that other factors than the presence of a central Cit-Gly motif are crucial for antibody binding. Collectively, these findings illustrate that citrullinated EBNA2 is an optimal candidate for ACPA detection, supporting current evidence that EBV is linked to RA onset. Rheumatoid arthritis (RA) is a chronic systemic disease, which is characterized by inflammation in the synovial tissue of joints ,2. In inRA is diagnosed according to the EULAR/ACR classification criteria revised in 2010, which, beyond clinical disease manifestations, comprise serological biomarkers, such as rheumatoid factors (RF) and anti-citrullinated protein antibodies (ACPA) ,6. WhileACPAs are specific for citrulline, a non-genetically encoded amino acid, which is the result of a post-translational modification catalyzed by peptidyl arginine deiminase (PAD) enzymes. PADs are calcium-dependent metalloenzymes, which are inactive at resting levels of intracellular calcium, and are activated with high levels of calcium . The conACPAs are commonly detected in commercially available immunoassays employing citrullinated peptides. The first generation of ACPA assays was based on a synthetic linear citrullinated peptide derived from human filaggrin . In ordeIn the commercial ACPA detection assays, different citrullinated peptides are employed, which is in accordance with the cross-reactive nature of ACPAs. ACPA often recognize several citrullinated targets, preferably containing a Cit-Gly motif ,29,30,31It has been debated whether ACPAs are associated with the pathogenesis of RA, even though the etiology of RA still is unclear . CurrentIn relation to environmental triggering factors, smoking appears to contribute to RA onset. Smoking may be involved in the activation of PAD enzymes, which results in the citrullination of proteins, leading to the presentation of citrullinated antigens, and ultimately the generation of ACPAs ,41. MoreIn addition to genetic and virological associations, serological data strengthen the hypothesis of a link between EBV and RA . StudiesIn the current study, we analyzed the reactivity of RA sera to citrullinated EBV peptides from various EBNA proteins, to elaborate the diagnostic potential of citrullinated EBNA peptides, and to investigate the potential link between EBV and RA further.para-nitrophenylphosphate (pNPP)), Tris, Tween, sodium chloride, sodium carbonate, ethanolamine, MgCl2, D-Biotin, hexafluorophosphate azabenzotriazole tetramethyl uranium (HATU), milliQ water, trifluoroacetic acid (TFA), triisopropylsilane (TIS), and N,N-diisopropylethylamine (DIPEA) were from Sigma Aldrich . Synthetic EBNA1 and EBNA2 peptides were purchased from Schäfer-N  -conjugated goat-anti-human IgG, streptavidin, AP substrate tablets (Denmark) , and werCitrullinated peptides from EBNA1, EBNA2, EBNA3, and MBP were tested for reactivity . PeptideCitrullinated peptides originating from EBNA3 were synthesized under standard protocols by automated microwave-assisted SPPS using the “Liberty Blue” by CEM corporation  . Pre-loaCleavage of the peptides, along with the removal of side-chain protecting groups, was conducted using 95% TFA, 2.5% MilliQ water and 2.5% TIS. The resins were treated with the cleavage mixture under magnetic stirring for 90 min, whereafter the solution containing the cleaved peptide was filtered and treated with isopropyl ether at 4 °C to precipitate the crude peptide. The filtered solution was centrifuged at 3500 rpm (2 × 5 min), whereafter the supernatant was discarded. The pellet was vacuum-dried and dissolved in 50:50 ACN/MilliQ water. Following peptide lyophilization, the peptides were characterized using reverse phase ultra-high-performance liquid chromatography and electro-spray ionization mass spectrometry .n = 28) and healthy donor (n = 28) serum samples ) were from the Biobank at Statens Serum Institut . The samples were tested anonymously, therefore not requiring ethical consent. MS serum samples (n = 7) were from the Multiple Sclerosis Clinic, Department of Neurology, Rigshospitalet Glostrup . The samples were tested anonymously, therefore did not require ethical consent.RA serum samples  with pNPP (1 mg/mL) was added to each well, and AP activity was determined by measuring the absorbance at 405 nm with background subtraction at 650 nm .Microtiter plates were coated with free peptide (1 µg/mL) in carbonate buffer , and incubated overnight at RT on a shaking table (ST). All blocking (30 min), washing (3 × 1 min), and incubation (1 h) steps were conducted using TTN  buffer. Wells were rinsed and blocked, whereafter sera (1:200 dilution) were added to each well, and incubated for at RT on a ST. Next, wells were washed, and AP-conjugated goat-anti-human IgG (1 µg/mL) was added to each well and incubated at RT on a ST. Finally, AP substrate buffer  in carbonate buffer, and incubated overnight at 4 °C. Biotinylated peptides (diluted 1 µg/mL in carbonate buffer) were added to each well, and incubated for 2 h at RT on a ST. The following steps in the experiment were carried out as described above.n = 20), in order to ensure that all results were directly comparable. All samples in the RA positive pool were selected based on a high ACPA titer. A peptide-specific cut-off was introduced based on preliminary screenings, tolerating a non-specific reactivity of 5% and an intra assay variation of maximum 15%. Readings above the cut-off were regarded as positive, whereas samples below the cut-off were regarded as being negative. Inter assay variations below 15% were acceptable. All samples were tested in duplicate.Absorbances of all the results were normalized to a positive RA control pool . No significant difference in RA reactivity was found to EBNA1-B and EBNA1-C compared to HC samples (p = 0.6310 for EBNA1-B and p = 0.2044 for EBNA1-C), as only 11 and 18% of RA sera reacted to EBNA1-C and EBNA1-B, respectively. In total, 50% of the RA sera reacted to one of the EBNA1 peptides, whereas 15% reacted to two peptides, and only one RA sample reacted to all three EBNA1 peptides. A total of 28% of RA samples tested did not react with any of the EBNA1 peptides tested.p < 0.0001 for EBNA2-A, p = 0.3844 for EBNA2-B and p = 0.0977 for EBNA2-C).In contrast, 75% of the RA sera reacted to at least one of the citrullinated EBNA2 peptides, whereas 31% reacted to two peptides (primarily EBNA2-A and EBNA2-C) b, and 25Finally, RA reactivity to citrullinated EBNA3 peptides was analyzed. Limited antibody reactivity was detected, as only 4–7% of the RA sera reacted to the EBNA3-1, 2, 4 and 5 c. A totaMoreover, none of the HC samples showed specific reactivity to the 11 citrullinated EBNA peptides tested a–c.p < 0.0001), yielding a sensitivity of 39%, and to EBNA3-E (p = 0.0067). Moreover, 29% of the RA sera reacted with EBNA1-A and EBNA2-A; however, no correlation in antibody reactivity could be determined between the two peptides (r = 0.1030) (results not shown).When comparing ACPA reactivity to the EBNA peptides, 86% of the RA samples reacted to at least one of the citrullinated peptides, whereas 14% of the samples did not. When comparing between the most reactive EBNA peptides , EBNA2-A obtained the highest sensitivity of all peptides tested (68%) d. ACPA rCollectively, these results illustrate that RA patient sera have specific antibody reactivities to citrullinated EBNA peptides. Moreover, citrullinated EBNA2-A is significantly recognized by RA sera when compared to the remaining EBNA peptides examined, making the EBNA2-A an important ACPA substrate, compared to the remaining EBNA peptides tested.n = 21), HC (n = 15) and MS (n = 7) sera reactivity to the selected peptides was determined in a direct ELISA using peptides coating directly to the polystyrene surface of microtiter plates.It has previously been proposed that amino acids C-terminal to citrulline are essential for ACPA reactivity ,28,31. TAs presented, RA sera reacted to all of the peptides tested a comparep = 0.0575 for EBNA3-EBNA2, p = 0.7504 for EBNA2-EBNA3) compared to EBNA2-A, although a trend indicated a reduced reactivity to EBNA3-EBNA2. In fact, the EBNA2-EBNA3 peptide reacted with 76% of the RA samples tested, whereas 67%, 47%, and 38% of RA sera reacted to EBNA2-A, EBNA3-EBNA2, and EBNA3-A, respectively, indicating that this overcrossing peptide is a slightly better substrate than EBNA2-A in this assay. When testing for possible correlations between the antibody reactivities, a moderate negative correlation was found between EBNA2-A and EBNA2-EBNA3 (r = −0.4722) (p = 0.0307) (No significant difference in antibody reactivity was found to the overcrossing peptides ( 0.0307) d (resultp = 0.0091), whereas no significant difference in antibody reactivity was found between EBNA3-A and EBNA3-EBNA2 (p = 0.7066).In contrast to EBNA2-A, antibody reactivity to EBNA2-EBNA3 was significantly elevated when compared to EBNA3-A (p = 0.0022), indicating that other factors than the presence of the C-terminal of EBNA2-A in combination with the crucial Cit-Gly motif are essential for antibody reactivity.As presented, antibody reactivity to NCit-EBNA2-A was significantly reduced compared to EBNA2-A . Nevertheless, RA EBNA1 IgG level was significantly elevated compared to EBNA2 IgG for RA and HCs (p < 0.0001).As presented in Next, the serologic levels of the biomarkers RF IgA and IgM were analyzed by ELISA . In thisp < 0.0001). Moreover, a moderately positive correlation was determined between IgM and IgA RFs titers (r = 0.5979) (p = 0.0016).A mean of 3237 IU/mL was found for IgM RFs for RA sera, which was significantly elevated compared to HC sera with an average of 64 IU/mL (No correlation was determined between EBNA1 IgG and IgA RF (r = 0.006223), EBNA1 IgG and IgM RF (r = −0.06233), EBNA2 IgG and IgA RF (r = −0.02009), and EBNA2 IgG and IgM RF (r = −0.1015) (results not shown).In addition to full-length proteins, a potential connection between RFs and EBNA2-A was examined, which was the peptide with the highest sensitivity among the peptides screened .As presented in Recently, it was reported that elevated citrullination levels are found in MS ,58. To aRA is a chronic inflammatory disease, where the serologic biomarkers ACPA and RF are central for the diagnosis and monitoring of RA. A total of 40 to 60% of early RA cases are positive for RF, indicating that RF appears before the clinical symptoms, although the specificity of RF is not impressive . In contA total of 11 peptides originating from different EBNA proteins were tested for reactivity with RA, HC, and MS samples. Whereas three EBNA1 and three EBNA2 peptides had been previously tested ,27, antiIn fact, screening of antibody reactivity to NCit-EBNA2, overcrossing EBNA peptides and MPB peptides, confirmed that other factors than the presence of Cit-Gly are essential for ACPA reactivity. Although NCit-EBNA2 and EBNA1-A contained a Cit-Gly motif in the same position, ACPA reactivity to NCit-EBNA2 was reduced when compared to EBNA1-A and EBNA2-A . A possiCollectively, results obtained support that other factors than the Cit-Gly motif, e.g., secondary structures are essential for ACPA reactivity. This remains to be elaborated; for instance, it would be interesting to evaluate the folded structure of the peptides by performing circular dichroism analyzes.Another explanation to the difference in reactivity is the presence of overlapping ACPA populations. ACPA has previously been described to contain a group of overlapping and non-overlapping reactivities, which most likely is ascribed to backbone and side-chain dependent antibody reactivity, respectively ,28,61. BComparing the ACPA reactivity pattern to the EBV cycle transcription program, our results regarding EBNA3 peptides are surprising, because while EBNA1 is expressed throughout the lifecycle of the virus, EBNA2 and EBNA3 proteins are all expressed during the latency III stage, where the growth transcription program occurs . For thiAs the peptides from EBNA1 and EBNA2 performed well in our assays, the RA and HC cohorts were tested for reactivity to full-length EBNA1 and EBNA2 proteins, and elevated absorbances were found for EBNA1 protein compared to EBNA2 . This reRF titers of IgM and IgA isotypes were determined and sensitivities of 96% and 92% in the RA cohort were revealed, respectively . RF titeFinally, screening of MBP peptides with MS, RA and HC sera illustrated that ACPA reactivity is specific for RA as no antibody reactivity to citrullinated MBP was found in the MS sera tested, although it recently was described that MBP was citrullinated in MS disease ,58. In general, autoimmune rheumatic diseases proposed to be associated with EBV do not express ACPA, or at least only in very low levels . WhetherPreviously, ACPA seropositivity of SLE and SjS samples to the selected EBNA1 peptides, tested in this study, were determined ,17. SpecSpeaking of diagnostic tools, several assays for ACPA and RF determination exist ,63. As aAnother thing separating RFs and ACPAs is their association with disease course. Individuals with ACPA-positive RA often experience a progressive disease course with cartilage destruction and bone erosion compared to ACPA-negative RA, which often is associated with a milder disease outcome, and may indicate that ACPAs in theory are pathogenic, although the pathogenesis of ACPA still remains to be determined ,65,66. SCollectively, these findings indicate that citrullinated EBNA2-A in combination with RFs may provide a good tool for serologic diagnostics of RA, and that EBV to some extent may contribute to the onset of RA. Moreover, current findings indicate that secondary factors and the optimal location of Cit may vary between peptides of different origin. Although current findings confirm that ACPAs are specific for RA and good diagnostic markers, the actual role of ACPA still remains to be determined in detail.


Lilium spp.) is an important cut flower around the world. Flower senescence in lilies is characterized by the wilting and abscission of tepals, which results in a decrease in flower quality and huge economic loss. However, the mechanism underlying flower senescence in lilies is largely unknown. In this study, single-molecule, real-time (SMRT) and Illumina sequencing were carried out in L. oriental ‘Siberia’. Sequencing yielded 73,218 non-redundant transcripts, with an N50 of 3792 bp. These data were further integrated with three published transcriptomes through cogent analysis, which yielded 62,960 transcripts, with an increase in N50 of 3935 bp. Analysis of differentially expressed genes showed that 319 transcription factors were highly upregulated during flower senescence. The expression of twelve NAC genes and eleven senescence-associated genes (SAGs) showed that LoNAC29 and LoSAG39 were highly expressed in senescent flowers. Transient overexpression of LoNAC29 and LoSAG39 in tepals of lily notably accelerated flower senescence, and the promoter activity of LoSAG39 was strongly induced by LoNAC29. This work supported new evidence for the molecular mechanism of flower senescence and provided better sequence data for further study in lilies.Lily ( Senescence is a natural process in which organs lose the capacity for cell division and gradually die. During vegetative growth in plants, under nutrient deficiency or moderate stress, old leaves initiate the process of senescence, allowing plants to use limited nutrients to sustain young leaves or to resist environmental stress . During Tulipa gesneriana), flower senescence is accompanied by large amounts of ethylene synthesis. ACS is a key gene in ethylene synthesis, which encodes 1-aminocyclopropane-1-carboxylate (ACC) synthase. Silencing of ACS significantly delays tepal senescence in tulips [L. oriental ‘Stargazer’ [Plants can be divided into ethylene-sensitive and -insensitive types . For ethn tulips . Most ofargazer’ .Gladiolus palustris) [In addition to ethylene, hormones such as abscisic acid (ABA) also play a role in promoting flower senescence. ABA promotes flower senescence in both ethylene-sensitive and -insensitive plants. The increase in ABA content in lily flowers is closely related to the senescence of tepals . This phlustris) .NAC genes were involved in leaf senescence; inhibition of a NAC gene AtNAP significantly delays leaf senescence [Ipomoea nil), elimination of one NAC gene, EPHEMERAL1, by CRISPR/Cas9 significantly delays flower senescence [Rosa hybrida) [R. hybrida), RhERF4 regulates pectin metabolism via direct regulation of the pectin degradation-related gene β-GALACTOSIDASE 1 (RhBGLA1), which leads to petal abscission [Downstream of the hormone signal, many transcription factors, and functional genes are involved in the regulation of flower senescence. NAC , Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC)) is a large transcription factor family in plants. It was reported that many nescence , which ihybrida) ,11,12. Dhybrida) . In rosescission . HoweverPetunia hybrida), and their function was verified by virus-induced gene silencing (VIGS) [In order to study the mechanism of transcriptional regulation, transcriptome sequencing is widely used to screen key genes. Through Illumina sequencing, several transcription factors, such as Ethylene Responsive Factor (ERF), Auxin Responsive Factor (ARF), basic Helix-Loop-Helix (bHLH), HD-Zip, and MADS-box, were found to be related to flower senescence in petunia (g (VIGS) . In lilig (VIGS) ,17, verng (VIGS) , and flog (VIGS) . Illuming (VIGS) .L. spp.) is a perennial bulb flower of the Lilium genus, and one of the top four cut flowers in the world [L. oriental ‘Siberia’, in this study, SMRT and Illumina sequencing were carried out and, through Cogent analysis, the sequencing data were integrated with three published sets of transcriptome data in lily. Hence, a better version of sequence data was generated. Additionally, many differentially expressed genes related to flower senescence, color, and scent were obtained. LoNAC29 was found to be a key transcription factor involved in flower senescence, partly by regulating the expression of a senescence-associated gene LoSAG39. This work provided a better understanding of flower senescence and established a good basis for further study in lilies (L. spp.).Lily  was grown in a greenhouse located in the campus of Huazhong Agricultural University in the spring. The room temperature ranged from 15 to 25 °C. The air humidity was kept at 60–70%. The development stages of the lily flower were defined according to Shi et al. [Lily  were bought from Nanhu flower market and transported to the laboratory within one hour. The flower stems were cut to a length of 30 cm and the leaves at the stem base were removed; the flowers were then placed in vases with distilled water.As for transient overexpression in lily tepals, cut flowers of lily (L. asiatic ‘Orange Matrix’) was bought from Nanhu flower market before the flowers were opened and further cultivated in the greenhouse. Growth conditions were the same as with lily . The flower opening process was also divided into four stages as above  [RNA was extracted from lily samples with the hot borate method . The conay 2019) , and theay 2019) , followeay 2019) . Next, tThe clean data in each sample were mapped to the reference sequence (non-redundant transcripts) using the Bowtie2  and RNA-Seq tools of Expectation-Maximization (RSEM) software , and the read counts were generated. Based on the read counts, the DEGs were analyzed using DESeq  with theCogent analysis was carried out with default parameters, according to the method described in . The k-mTransDecoder analysis (default parameters) was carried out according to the method described in . FirstlyL. oriental ‘Siberia’), as listed in ACTIN in L. oriental ‘Siberia’ (LoACTIN) was used as the internal control gene, according to [−ΔΔCT method [t-tests  were used for statistical analysis.For qRT-PCR, the cDNA template was synthesized with 1 μg RNA by using cDNA Synthesis SuperMix  according to the manufacturer’s instructions. The primers used for qRT-PCR were designed based on the SMRT sequencing data in lily -PCR [The sequences of PNI)-PCR . The seqLoNAC29 and LoSAG39, the ORF carrying the proper restriction site was amplified by PCR, digested, and inserted into the pSuper1300 vector, which was derived from pCAMBIA1300 . For promoter activity analysis, the promoter of LoSAG39 harboring the appropriate restriction site was digested and inserted into the PBI121 vector.For overexpression (OE) of LoNAC29 and LoSAG39 in lily tepals was performed as previously described [LoNAC29, pSuper-LoSAG39 or pSuper (empty vector) were transformed into Agrobacterium tumefaciens GV3101 separately. After culture overnight, the cells were harvested and resuspended in infiltration buffer . OD600nm was adjusted to 0.8 and incubated in the dark without shaking for two hours. Lily tepal discs with a diameter of 12 mm were obtained using a hole punch and infiltrated with A. tumefaciens GV3101 cells under a vacuum (0.6 atm). The discs were then washed in deionized water and cultured on a Petri dish with wet filter paper in the dark at 8 °C for three days. After that, the Petri dish containing the discs were taken out, and the time point was defined as 0 day. Thereafter, the discs were cultured in Petri dishes at 23 °C and 60% relative humidity under 5000 lux light.Overexpression of escribed  with minpSuper::LoNAC29, and reporter PBI121 empty vector, or PBI121 harboring the promoter of LoSAG39, were transferred into A. tumefaciens GV3101. After overnight culture, cells were collected and resuspended in the infiltration solution as above, and the density was adjusted to OD600 nm = 1.6. Agrobacterium carrying the effector or reporter were mixed with the ratio 1:1 (v/v) and incubated in dark at 23 °C without shaking for two hours. Agrobacterium were then injected into the back of N. benthamiana leaves using a 1 mL syringe (needle was removed before injection). After cultivation in the dark at 23 °C and a relative humidity of 40–60% for three days, the leaves of treated N. benthamiana were collected, immediately frozen in liquid nitrogen, and stored at −80 °C.The effector pSuper1300 empty vector, or N. benthamiana were ground in liquid nitrogen, and the soluble protein was extracted with extraction buffer. To measure the GUS activity, 4-Methylumbelliferyl β-d-glucuronide hydrate (4-MUG) was used as a substrate. The fluorescence of the 4-MU product was measured with a fluorescence spectrophotometer  at 365 nm excitation and 455 nm emission. The GUS activity was defined as the amount of 4-MU produced per minute (unit: μM 4-MU mg−1 (protein) min−1).Determination of GUS activity was performed according to . BrieflyElaeis guineensis homolog genes, followed by Phoenix dactylifera , Ananas comosus (3415 unigenes), Musa acuminata (3277 unigenes), Asparagus officinalis (2417 unigenes), and Anthurium amnicola (2350 unigenes)  sequences were obtained, with a mean length of 2397 bp and N50 of 3240 bp. Based on the 5′ primer, 3′ primer, and poly A, 310,912 FLNC sequences were found in CCS, with a proportion of 78.42%. After removing the redundancy, 73,218 non-redundant transcripts were obtained, with the total number of 203,392,716 nucleotides and N50 of 3792 bp . The resnigenes) .Several Illumina sequencing data in lily were published recently ,17,18, wThe integrated sequencing data were further analyzed with TransDecoder. The result showed that in the original SMRT sequencing data, 43,998 sequences (60%) had the full length, 12,938 sequences (18%) were 5prime_partial, 699 sequences (1%) were 3prime_partial, and 15,583 sequences (21%) were internal and others which were missing both the 5′ and 3′ ends or the score of alignment in UniProt and PFAM databases were lower than −20. After Cogent analysis, 42,027 sequences (67%) were full length, 7774 (12%) and 548 (1%) were missing their 5′ and 3′ end, respectively, and 12,611 (20%) were internal and others .By using DESeq software, DEGs  were determined between different opening stages in lily. Stage 1 versus stage 4 pairwise had the most DEG genes, 5071 genes were upregulated and 10,172 genes were downregulated. In contrast, stage 2 versus stage 3 had the least DEG genes . The DEGNAC unigenes, as well as 1 HB-HD-ZIP unigene and 1 ERF unigene, were homologous to several published senescence-associated genes, including ANAC029(AtNAP) [ANAC046 [ANAC032 [ANAC087 [ANAC022(NAC1) [ANAC002(ATAF1) [Ipomoea nil EPHEMERAL 1 (NAC092) [RhHB1 [RhERF113 [Through SMRT and Illumina sequencing, 2081 TF and transcription regulators were obtained. Of those, 319 genes were highly expressed at stage 4 (senescence), while 511 genes were notably downregulated with senescence of the flowers . Most of[ANAC046 , ANAC032[ANAC032 , ANAC087[ANAC087 , ANAC02222(NAC1) , ANAC0022(ATAF1) , Ipomoea) [RhHB1 , and RhEL. oriental ‘Siberia’, the tepals were green, and with the opening of flowers, they gradually turned white  [Flower opening is often accompanied with a release of flower scent. Terpenoids are major components in the flower scent of stage 3) . KEGG paSAG genes sharply increased in stage 4  flowers. The result showed that the expression of NAC2 rapidly increased more than 1500 times in stage 3 and 2000 times in stage 4, compared with stage 1. However, the change in NAC4 was not as strong as NAC2. Additionally, both NAC2 and NAC4 were induced by ABA treatment  analysis . The exp stage 4 . To confreatment .NAC2 was cloned, and phylogenetic analysis showed that it had high homology with AtNAC29 in Arabidopsis thaliana, so the gene was named LoNAC29  is very complex, as most of the cultivars are diploid or tetraploid, and some are triploid. L. oriental ‘Siberia’ is diploid (2n = 2x = 24) [Ploidy of lilies (2x = 24) . The gen2x = 24) . Previou2x = 24) ,19. HoweL. spp.). Among the 62,960 transcripts, 42,027 (67% of the sequences) were predicted to have the full length by TransDecoder analysis, which was also better than our original SMRT sequencing data , the senescence of flowers is presented as the wilting and abscission of tepals, accompanied by the yellowing and falling off of leaves. With regard to plant senescence, previous studies focused on leaf senescence—in particular, the synthesis and regulation of chlorophyll in leaves [SAG1 (LoSAG39), SAG5, SAG6, SAG7, and SAG8 had a very high expression level in senescent flowers and leaves, suggesting that they may be involved in both leaf and flower senescence , SAG1 (LoSAG39), SAG2, and SAG7 were homologous to the important senescence marker gene SAG12 in Arabidopsis, which encodes a cysteine protease [SAG4, SAG5, SAG6, SAG8, and SAG9 were similar to AT5G50260 (AtCEP1), which was involved in pathogen defense, and atcep1 knockout mutants showed enhanced susceptibility to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum [SAG unigenes might be involved in protein/nucleic acid degradation and pathogen defense during flower senescence in lilies. In 319 differentially expressed genes which were highly expressed at stage 4, except for the 11 selected SAG unigenes, some unigenes were annotated as gibberellin 2-β-dioxygenase , cytokinin dehydrogenase , cis-zeatin O-glucosyltransferase , and 9-cis-epoxycarotenoid dioxygenase   , suggestp0/1704) , which wGA20ox1, a key gene for gibberellin synthesis, thus accelerating flower senescence [In terms of the regulatory mechanism underlying flower senescence, it was reported that an ethylene-induced HD ZIP transcription factor RhHB1 inhibits nescence . On the nescence . In thisNAC gene AtNAP (ANAC029) that plays a key role in Arabidopsis leaf senescence [NAC genes in other species [NAC genes promoted leaf senescence partly through inducing chlorophyll degradation [Since the discovery of a nescence , many st species . It was radation , ABA synradation , or ROS radation .NAC gene regulates flower senescence in Japanese morning glory (Ipomoea nil cv. Violet). Knock-out of NAC gene EPHEMERAL 1 significantly delays senescence in Japanese morning glory (Ipomoea nil) [NAC gene in flower senescence is not clear.Some studies have also shown that the oea nil) . HoweverNAC unigenes, as well as 1 HB-HD-ZIP unigene and 1 ERF unigene were homologous to ANAC029 (AtNAP) [ANAC046 [ANAC032 [ANAC087 [ANAC022(NAC1) [ANAC002 (ATAF1) [Ipomoea nil EPHEMERAL 1 (NAC092) [RhHB1 [RhERF113 [LoNAC29 is a key regulator in the process of lily flower senescence, and OE of LoNAC29 significantly promotes the senescence of lily tepals. In the LoNAC29 OE samples, the expression of LoSAG39, SAG2, and SAG7 was significantly increased, and LoNAC29 strongly induced the promoter activity of LoSAG39  , ANAC046[ANAC046 , ANAC032[ANAC032 , ANAC087[ANAC087 , ANAC02222(NAC1) , ANAC002 (ATAF1) , Ipomoea) [RhHB1 , and RhE LoSAG39 . LoSAG39y tepals .L. oriental ‘Siberia’, the flower color is green at stage 1 and, with the opening of flowers, the tepals gradually turn white. White flowers are formed partly as a result of chlorophyll degradation [L. oriental ‘Siberia’ and, accordingly, the expression level of chlorophyll synthesis-related genes and photosynthesis-related genes also decreased gradually , a NAC transcription factor was found to mediate chlorophyll synthesis partly through direct activation of HEMA1 and CRD1 genes [L.oriental ‘Siberia’, most of the genes involved in chlorophyll synthesis and photosynthesis were clustered into subcluster 1, subcluster 2, and subcluster 11. These clusters contained 114 transcription factors  is also accompanied by the release of flower scent. The main components of floral fragrance are terpenoids, benzenoids/phenylpropanoid, and derivatives of fatty acids. Terpenes are synthesized by the MEP and MVA pathway. The main aroma components in L.oriental ‘Siberia’, cis-ocimene, linalool, and other terpenes, are released in large amounts at full bloom stage [Freesia hybrida) flowers [Citrus sinensis Osbeck) [Rosa bourboniana, the flower life is much shorter, compared with most R. hybrida varieties, which are non-fragrant and widely used as cut flowers [The flower opening of lilies (om stage . In this flowers  and ERF7 Osbeck) . Here, i flowers . Synthes flowers . In futuL. oriental ‘Siberia’), which provided a good foundation for the further study of gene cloning and related molecular mechanisms in lily (L. spp.). Additionally, we found that the key regulator LoNAC29 promotes lily flower senescence by inducing the expression of LoSAG39.Overall, through this study, we obtained a high-quality version of transcripts and made new insights into flower senescence in lily (


This study aimed to optimize astaxanthin extraction by supercritical CO2 technology from fermented liquor of shrimp waste and study bioaccessibility using simulated gastrointestinal digestion (GD) of the optimized extract. A Box–Behnken design with three variables  was used to optimize the supercritical CO2 extraction. The optimized CO2 extract was obtained at 300 bar, 60 °C, and 6 mL/min, and the estimated characteristics showed a predictive extraction yield of 11.17%, antioxidant capacity of 1.965 mmol of Trolox equivalent (TE)/g, and astaxanthin concentration of 0.6353 µg/g. The experiment with optimal conditions performed to validate the predicted values showed an extraction yield of 12.62%, an antioxidant capacity of 1.784 mmol TE/g, and an astaxanthin concentration of 0.52 µg/g. The astaxanthin concentration decreased, and the antioxidant capacity of the optimized extract increased during gastrointestinal digestion. In conclusion, our optimized supercritical CO2 process is suitable for obtaining astaxanthin from shrimp by-products after lactic acid fermentation.Lactic acid fermentation increases the bioactive properties of shrimp waste. Astaxanthin is the principal carotenoid present in shrimp waste, which can be found esterified in the liquid fraction (liquor) after its lactic acid fermentation. Supercritical CO Shellfish are a widely and increasingly consumed food, and shrimp are economically the most important . HoweverAstaxanthin also has neuroprotective, anti-inflammatory, anti-diabetic, and cardioprotective properties, attenuates photo-toxicity caused by UV radiation, and has recently shown anticancer activity ,12,13,14In general, to exert their bioactive properties, antioxidants must be bioaccessible and/or bioavailable. Bioaccessibility refers to the amount of a compound present in the gut after being released from the food matrix. Complementarily, bioavailability is associated with compounds that have been absorbed, distributed, metabolized, and excreted from the body. To date, not many studies have investigated the bioaccessibility and bioavailability of astaxanthin. The available information states that both free and esterified forms of astaxanthin have low bioaccessibility and bioavailability due to their susceptibility to thermal and chemical degradation ,18. Yangcis- and trans-astaxanthin can permeate lipid membranes to a different extent both in vitro and in vivo. However, conformational isomers of esterified astaxanthin have different bioaccessibility, since they are able to cross the epithelial barrier and stabilize the molecule; therefore, esterified astaxanthin could be more bioaccessible and have different health benefits [It has also been reported that the bioaccessibility and bioavailability of astaxanthin can be modified due to its conformational isomers because benefits ,12,16.2 extraction [2 extraction has shown high astaxanthin extraction yield and recovery; this technique has some advantages compared to other extraction methods, such as eco-friendliness, faster extraction, high purity, and thermolabile molecule recovery [Several methods are used for astaxanthin extraction from shrimp waste, such as lactic acid fermentation , solventtraction . Lactic traction ,22, whictraction . Howevertraction . Supercrrecovery ,24.2 extraction of astaxanthin from lyophilized liquor obtained by lactic acid fermentation of shrimp waste, analyze its antioxidant activity, and determine its bioaccessibility level using an in vitro model.Therefore, this study aimed to optimize the supercritical CO2 extraction variables on the three response variables , as shown in Three predictive models were obtained to fit the quadratic polynomial of Equation (2) to experimental data of the effects of different supercritical COp-value (<0.0001), in addition to the lack of fit being insignificant (p > 0.05). The model was dependent on the linear term of pressure , temperature , and flow rate , as well as the interaction between temperature and flow rate . Predictive models for extraction yield (YY) are shown in Equations (1) and (2).According to ANOVA , the extUsing coded variables:Using original variables:p < 0.05) in extraction yield values (2 (coefficient of determination) ≥ 0.8, a significant level p < 0.05, coefficient of variance (CV) ≤ 0.1, and lack-of-fit test > 0.1. These values indicate that the model is adequate and reproducible [This predictive model explained 90.14% of the total variation , and the lack of fit was insignificant (p > 0.05) , temperature , and flow rate , as well as the interaction between pressure and flow rate  and temperature and flow rate . Predictive models for antioxidant capacity (ACY) are shown in Equations (3) and (4).The antioxidant capacity (ABTS assay) model was highly significant for its small  > 0.05) . The modUsing coded variables:Using original variables:p < 0.05) in antioxidant capacity values  dependent on linear terms of pressure , temperature , and flow rate , as well as the interaction between pressure and flow rate . Predictive models for astaxanthin concentration (ASTY) are shown in Equations (5) and (6).The analysis of variance for astaxanthin concentration  demonstrUsing coded variables:Using original variables:p < 0.05) in astaxanthin concentration values   and gastric digestion (59.09 ± 3.01 mmol TE/g lyophilized liquor) by the ABTS and ORAC assays, respectively; the lowest antioxidant capacity was in the undigested extract.The antioxidant capacity of the optimized COnd ORAC) . The antAs shown in 2 extraction is a separation technology based on the solubility of bioactive compounds in a fluid whose density and dissolving power can be modified by different parameters such as pressure, temperature, and flow rate [2 extraction has some advantages compared with other separation methods, such as high bioactive compound purity, enhanced extraction efficiency, and low environmental impact [2 extraction [2 extraction. The process is affected by pressure, temperature, and flow rate, consistent with previous reports on the extraction of lipophilic compounds from shrimp waste by supercritical CO2 extraction [2 fluid and co-solvent addition (ethanol) [Supercritical COlow rate . During low rate ,28. Supel impact ,15. For traction ,15,29,30traction ,23,31. Tethanol) ,15.2 extract obtained here showed a higher extraction yield than those reported by Sánchez–Camargo, Martinez–Correa, Paviani and Cabral [2 extract showed similar antioxidant capacity, by ABTS and ORAC assays, like that reported by Roy, Getachew, Cho, Park, and Chun [2 extract from fermented liquor of shrimp waste showed a lower astaxanthin concentration comparison with the supercritical CO2 extract from shrimp waste without lactic acid fermentation, as reported by Radzali, Masturah, Baharin, Rashidi, and Rahman [2 extraction can have different astaxanthin yields [The optimized supercritical COd Cabral , Razi Pad Cabral , and Royd Cabral  and a sid Cabral . In addiand Chun , which hand Chun ,10,15. Hd Rahman , Sánchezd Rahman , Razi Pad Rahman , and Radd Rahman . The lown yields ,15,35.The bioaccessibility of carotenoids such as astaxanthin is often limited by their interaction with food matrix constituents. For instance, astaxanthin in shrimp waste is usually found in a chemical complex with proteins linked by the -OH group of the xanthophyll molecule. The bioaccessibility of astaxanthin of marine origin has been evaluated in salmon and supplements by Chitchumroonchokchai and Failla . Contrarp < 0.05) the astaxanthin concentration and antioxidant capacity (ABTS and ORAC assays). Several studies have shown that the initial concentration of bioactive compounds can be degraded due to the physicochemical and biochemical factors in the gastrointestinal environment. In in vitro studies, these detrimental effects can be attributed mainly to pH and enzymatic conditions. Due to pH changes, bioactive compounds such as carotenoids can be partially hydrolyzed and deprotonated, affecting their reactivity toward the probes used in antioxidant studies [2 extracts increased at the end of gastrointestinal digestion. In addition, metabolic changes during gastrointestinal digestion can lead to the formation of new metabolites or deprotonated forms of the original bioactive molecule. This is yet to be elucidated, and astaxanthin metabolites during GD have not yet been reported [The simulated gastrointestinal digestion significantly affected  and carotenoids  from shrimp waste, which all have adequate and, in conjunction, good antioxidant capacity [2 extract could be due to the available carotenoids released with good antioxidant activity [Supercritical COcapacity . Therefoactivity ,37,38,39Litopenaeus vannamei) exoskeleton was purchased from a local market in Culiacán, Sinaloa, Mexico. The exoskeleton was washed with tap water and ground. Then, a commercial mixture of lactic acid bacteria  needed during cheese production  and molasses production  were used for lactic acid fermentation of the shrimp waste, according to Marcia, Malespín, Sánchez and Benavente [Shrimp , 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (Sigma 10102946001), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) (Sigma 440914), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma 238813), and fluorescein (Sigma 46955) were also purchased from Sigma-Aldrich .Analytical-grade ethanol, methanol, hydrochloric acid, sodium carbonate, phosphoric acid, potassium phosphate monobasic, potassium phosphate dibasic, and potassium persulfate were purchased from Sigma-Aldrich . Astaxanthin standard  as a co-solvent in a 95:5 ratio. According to the experimental design (g (rotor 221.22) and at 4 °C for 15 min. The collected supernatant was separated and dried using a rotary evaporator , nitrogen (gas) was used for complete samples drying in darkness to prevent the extract from oxidation. All samples were resuspended in 1 mL of ethanol for antioxidant capacity assays; other extracts were resuspended in 1 mL ethyl acetate for astaxanthin concentration and stored in darkness at −20 °C for further analysis.Astaxanthin extraction was performed according to Roy, Getachew, Cho, Park, and Chun , with mol design , the conl design . Every em (extract) is the dry mass obtained from supercritical CO2 extraction and m  is the weight of lyophilized liquor used for the extraction [The following equation was used to determine the extraction yield:traction . 2S2O8, and ethanol) and incubated for 2 h at room temperature in darkness. The absorbance at 734 nm was measured using an Epoch microplate spectrophotometer , and ethanol was used as a blank. The results were expressed as Trolox equivalent millimoles per gram (mmol TE/g) of lyophilized liquor.The 2,2-azino-di(3-ethylbenzthiazoline-6-sulfonate) (ABTS) radical-scavenging activity was evaluated according to Karadag, Ozcelik, and Saner  with sli2 extract resuspended in ethanol was mixed with 200 µL of fluorescein and 75 µL of 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH). The absorbance of elicitation and emission was set at 485 and 580 nm, respectively. The reaction absorbance was monitored for 70 min using a Synergy HT spectrometer , and a phosphatic solution  was used as a blank. The results were expressed as Trolox equivalent millimoles per gram (mmol TE/g) of lyophilized liquor.The antioxidant capacity was evaluated using the oxygen radical absorbance capacity (ORAC) assay according to Huang, Ou, Hampsch-Woodill, Flanagan, and Prior  with sli2 extracts of lyophilized liquor was analyzed according to Hu, Lu, Lv, Wang, Ding, and Wang [v/v/v) ratio. Astaxanthin identification was performed by comparing the retention time of the sample with the astaxanthin standard. A standard curve of astaxanthin (R2 = 0.992) was obtained for the injection of six concentrations . The astaxanthin concentration in supercritical CO2 extracts was calculated using the integrated area of HPLC peak areas and expressed as micrograms of astaxanthin per gram (µg/g) of lyophilized liquor.The astaxanthin concentration in supercritical COand Wang  with sli2 extraction. Pressure (X1), temperature (X2), and flow rate (X3) were considered independent variables, and extraction yield, antioxidant capacity (ABTS), and astaxanthin concentration were considered dependent variables. Individual experiments were performed in random order. A quadratic polynomial regression model was assumed to fit (Y) the obtained data. The following model form was developed to describe the three response surfaces (Y):Y is the predicted response variable value , β0 is the constant value, β1 and β2 are linear coefficients, β12 is the interaction coefficient, and β11 and β22 are quadratic coefficients. The significant terms (p ≤ 0.05) for the second-order polynomial model were recalculated to obtain a predictive model for each variable [2 extraction. The optimal levels were obtained by the numerical method and by contour plots of the graphs. The supercritical CO2 extraction at optimum conditions was carried out for quintupled. The Box–Behnken design with a three-variable set of 15 experiments  was usedvariable . All the2 extract resuspended in ethanol was subjected to in vitro simulated gastrointestinal digestion according to the static method described by Brodkorb, Egger, Alminger, Alvito, Assunção, Ballance, Bohn, Bourlieu-Lacanal, Boutrou, Carrière, Clemente, Corredig, Dupont, Dufour, Edwards, Golding, Karakaya, Kirkhus, Le Feunteun, Lesmes, Macierzanka, Mackie, Martins, Marze, McClements, Ménard, Minekus, Portmann, Santos, Souchon, Singh, Vegarud, Wickham, Weitschies, and Recio [g at 4 °C for 15 min, and the supernatant was collected and freeze-dried. For further analysis, the digestive extracts were resuspended in ethanol  and ethyl acetate (for astaxanthin concentration study).The optimized COnd Recio  with sliBioaccessibility was calculated using the formulaThe initial values are the initial antioxidant activity and astaxanthin concentration of the undigested samples. The final value is the antioxidant activity and astaxanthin concentration after in vitro gastrointestinal digestion. 2 extracts were reported as the mean ± standard deviation. Digestive extract data were analyzed by one-way analysis of variance (ANOVA), followed by Turkey’s HSD test using the statistical package Minitab 17 . Statistical differences at the level of p < 0.05 were considered significant. Each experiment was performed in quintuple or as otherwise specified. Antioxidant capacity (ABTS and ORAC), extraction yield, and astaxanthin concentration data of optimized CO2 extraction of astaxanthin from lactic-acid-fermented liquor from shrimp waste. The optimized extracts showed antioxidant activity against different free radicals, indicating their possible use as additives during the development of functional foods or food supplements. However, the simulated gastrointestinal digestion process decreased the astaxanthin concentration, mainly attributed to pH changes during each digestive stage. Although the astaxanthin concentration decreased, its content did not reflect the antioxidant capacity of the optimized extracts since they showed higher antioxidant potential. This may also suggest that optimized supercritical CO2 extracts are rich in other carotenoids that can act in synergy and could be available during gastrointestinal digestion. Furthermore, encapsulation strategies could be implemented to improve the bioaccessibility of astaxanthin and its derivatives. Finally, supercritical CO2, considered a green technology for the extraction of bioactive compounds, is recommended for future studies on the valorization of astaxanthin from waste biomass of marine origin. This study optimized the supercritical CO


The infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus causes pneumonia of coronavirus diseases 2019 (COVID-19) , 4. As oThe B.1.1.7 lineage, also known as 20I/501Y.V1 or VOC 202012/01, was first detected in the United Kingdom (UK) in December 2020; it demonstrates a substantial transmission advantage over other non-VOC variants with a 50–100% higher reproduction number and has been identified in at least 130 countries [The mutations in S protein could have profound impacts on diagnostic detection, antiviral immunity, and virus infectivity, all of which could actually contribute to the rapid spreading of above VOCs. It was shown that the deletion at positions 69 and 70 of the S glycoprotein (ΔH69-V70) of 501Y.V1 variant was associated with diagnostic test failure for the ThermoFisher TaqPath probe targeting the spike protein, a phenomenon termed S-gene target failure (SGTF) . MeanwhiIn addition to human–human transmission, animals had been implicated in the transmission of SARS-CoV-2 viruses. For example, an investigation on 16 mink farms in the Netherlands indicated that 68% of tested individuals had evidence for SARS-CoV-2 infection, some of them turned out to be infected with strains from minks. Moreover, cat, dog, ferret, hamster, tiger, and lion were also capable of being infected by the early SARS-CoV-2 strain . InteresMice are known hosts of a number of infectious diseases, such as pestis, epidemic hemorrhagic fever, bacillary dysentery, and salmonellosis. Considering that mice are widely existing and hard to be controlled, particularly in communities of poor sanitation, and may cause catastrophic impacts if being a N501Y VOCs transmitter Fig. , further