The pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) is widely unknown, and the role of autoantibodies is still undetermined.To identify brain-reactive autoantibodies possibly related to NPSLE, immunofluorescence (IF) and transmission electron microscopy (TEM) on rat and human brains were performed. ELISA was used to reveal the presence of known circulating autoantibodies, while western blot (WB) was applied to characterize potential unknown autoantigen(s).We enrolled 209 subjects, including patients affected by SLE (n=69), NPSLE (n=36), Multiple Sclerosis , and 82 age- and gender-matched healthy donors (HD). Autoantibody reactivity by IF was observed in almost the entire rat brain  using sera from NPSLE and SLE patients and was virtually negative in MS and HD. NPSLE showed higher prevalence , intensity, and titer of brain-reactive autoantibodies than SLE patients. Most of the patient sera with brain-reactive autoantibodies (75%) also stained human brains. Double staining experiments on rat brains mixing patients’ sera with antibodies directed against neuronal (NeuN) or glial markers showed autoantibody reactivity restricted to NeuN-containing neurons. Using TEM, the targets of brain-reactive autoantibodies were located in the nuclei and, to a lesser extent, in the cytoplasm and mitochondria. Given the high degree of colocalization between NeuN and brain-reactive autoantibodies, we assumed NeuN was a possible autoantigen. However, WB analysis with HEK293T cell lysates expressing or not expressing the gene encoding for NeuN protein (RIBFOX3) showed that patients’ sera carrying brain-reactive autoantibodies did not recognize the NeuN corresponding band size. Among the panel of NPSLE-associated autoantibodies  investigated by ELISA assay, only the anti-β2-glycoprotein-I (aβ2GPI) IgG was exclusively found in those sera containing brain-reactive autoantibodies.In conclusion, SLE and NPSLE patients possess brain-reactive autoantibodies but with higher frequency and titers found in NPSLE patients. Although many target antigens of brain-reactive autoantibodies are still undetermined, they likely include β2GPI. Systemic Lupus Erythematosus (SLE) is a complex disease characterized by immune system dysregulation. The distinctive SLE characteristic is the production of self-reactive pathogenetic antibodies targeting any tissue or forming immune complexes . MoreoveThis study aimed to investigate the presence of brain-reacting autoantibodies that could contribute to the picture of CNS manifestations in NPSLE patients. Our approach was based on immunohistochemistry (IHC) (through fluorescence and electron microscopy) to identify the brain areas, neuronal cell type/s, and subcellular areas targeted by the autoantibodies. Since humans and rats could only partially share common epitopes, we used brains from both species. In parallel, we used enzyme-linked immunosorbent assay (ELISA) to reveal the presence of known circulating autoantibodies correlating with the IHC data and western blot (WB) analysis to identify the possible molecular autoantibody targets.The human cohort enrolled in this study between January 2013 and February 2019 was composed of a total of 209 subjects, including 105 diagnosed with SLE Table 1,Demographic data, clinical features, and ongoing treatments were recorded. SLE activity was assessed with the SLE Disease Activity Index 2000 (SLEDAI-2K). Organ damage was evaluated using the Systemic Lupus International Collaborating Clinics (SLICC) Damage Index (SDI). Blood samples were collected on 10 ml serum separator tube gel and centrifuged within one hour from the sample collection at 3,500 rpm for 15 minutes at room temperature. Then several aliquots were made and stored at -80°C.A subgroup of patients underwent brain MRI 1.5 T examination within one month of sampling. Exams were performed on a 1.5 Tesla scanner  with a dedicated eight-channel neurovascular coil . Sequence parameters included isotropic DWI , multiplanar dual FSE proton attenuation, and T1WI, T2WI, FLAIR, and 3DT1W1. Brain MRI results were qualitatively classified as reported elsewhere .https://nbtr.ncl.ac.uk/), including the cerebellum  and hippocampus  used to perform the IHC were fixed, paraffin-embedded, and taken stored at -80 °C (autolysis time and pH were in a range of 16 – 81 hours and 6.47–6.96 respectively). Autoantibody reaction in the cerebellum was considered positive when immunostaining was revealed at least in 2 out of 3 human cases.Male Sprague Dawley rats (250–300g) were sacrificed by perfusion with 4% paraformaldehyde injected through the left ventricle. The rat brains were collected and stored in a 7% sucrose diluted phosphate buffer saline (PBS) at 4°C, included in optimal cutting temperature compound , then cu0C with patient sera (1:200 dilution) in a solution containing 0.1% bovine serum albumin (BSA) in PBS. Sections of the cerebellum from each human case were incubated with 24 patient’s sera, equally subdivided between SLE and NPSLE patient groups (each including6 from both positive and negative sera in rat sections) as well as 2 sera from healthy donors and 2 from MS. Detection of the sera was performed by using a Cy3-conjugated donkey anti-human antibody . An indirect immunofluorescence assay using HEP-2 cells substrate was used to determine serum antinuclear IgG antinuclear bodies (ANA). Fluorescence patterns were reported according to the International Consensus on ANA Patterns (ICAP)  in a solution containing 3% normal rat, 3% normal donkey serum, and 3% phosphate buffer saline (PBS). The sera were detected using a Cy3-conjugated donkey anti-human antibody . Sera were considered positive only when positivity was confirmed using at least three different rats at 1:60 dilution. The optical density (OD) of the fluorescent signal generated by each serum was measured with ImageJ. The OD of the healthy donors was used as the threshold to classify each SLE, NPSLE, or MS patient as positive or negative. Titers of the positive sera were performed by further dilutions up to 1:500,000. Double staining on rat sections was carried out by mixing the human sera with the commercial antibodies, such as rabbit anti-NeuN antibody 1:200; Abcam), mouse anti-VAChT , mouse anti-vGlut1 , chicken anti-GAD-67 , rabbit anti-GFAP , and rabbit anti-MBP . Autoantibodies were revealed by using a Cy3-conjugated donkey anti-human antibody , while the primary antibodies were revealed through Alexa488-conjugated donkey anti-mouse antibody , Alexa488-conjugated donkey anti-rabbit antibody , or Alexa488-conjugated donkey anti-chicken antibody . The Hoechst-33342 was used for nuclei detection at the 1:2,000 dilution. The colocalization between autoantibodies and anti -NeuN, -GFAP, -MBP, -GAD-67, -vGlut1, and -VAChT antibodies was analyzed with Fiji (ImageJ Software) using the plug-in JACoP. Human brain sections were treated with 0.1% Triton-100 for 1 hour at room temperature and incubated overnight at 4 0; Abcam,3) and fixed with a mixture of 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 hours at room temperature. They were then rinsed in cacodylate buffer with 3.5% sucrose, dehydrated, and embedded in Epon Resin . Semi-thin sections (thickness: 5 μm) were stained with toluidine blue and examined by light microscopy to check the histological appearance. Ultra-thin sections (thickness: 80-85 nm) were collected on nickel grids and processed for the IHC analysis. Before deposition of the sections, grids were hydrated with PBS solution in a moist chamber for 5 minutes. Next, the grids were treated with 1% BSA and 5% normal goat serum (NGS) in PBS solution to block non-specific binding. They were then incubated with human sera diluted 1:15, 1:20, 1:40, and 1:50 in 1% BSA and 5% NGS for 24 hours at 4°C. After rinsing in PBS, the grids were incubated for 1h at room temperature with the goat anti-human secondary antibody conjugated to 25 nm gold particles  and diluted 1:50 in 1% BSA-PBS , which do not or do express the RBFOXP3 gene (encoding the NeuN protein), respectively. Lysates were transferred to polyvinylidene difluoride (PVDF) membranes for 1 hour and 50 minutes at 200 Volt. The membranes were blocked with 10% BSA for one hour at room temperature and then incubated at 4°C for one night with human serum, including IHC-positive (2 from NPSLE and 1 from SLE) and 2 -negative sera diluted in a range between 1:8,000 to 1:50,000, and the rabbit anti-NeuN antibodies (Abcam), 1:200diluted. The membranes were washed three times with tris-buffered saline + 0.01% tween-20 (TBST). After washing, membranes were incubated at room temperature for 1 hour with the opportune secondary antibody . The membranes were washed three times with TBST and incubated at room temperature for 30 minutes with streptavidin conjugated with horseradish peroxidase (HRP), diluted at 1:10,000, and finally washed 3 times with TBST. The antigen-antibody reaction was revealed using ThermoScientificTMPierce Enhanced chemiluminescence WB Substrate. ImageQuantTM LAS 4000 was used to detect the chemiluminescence.Western blot (WB) was performed using either HEK293T and The serum quantification of C3 and C4 complement fractions, anti-ribosomal-P protein, anti-Ro/SSA, anti-La/SSB, anti-RNP, anti-Sm , anti-cardiolipin (aCL) IgG and IgM , anti-β2glicoproteinI (aβ2GPI) IgG and IgM , was performed following the guidelines of the commercial ELISA kits. Anti-β2GPI and aCL IgG and IgM positivity was considered for titers higher than GPL and MPL 40 U/ml, respectively. The quantification of anti-NR2 antibodies was performed by homemade ELISA using the DWEYSVWLSN peptide , as described by Putterman & Diamond . We usedp values were > 0.05 in all cases; hence, sample variances were measured with the F-Test for equality of variance, while individual or pooled variances were used for the two-tailed Student’s t-test. Pearson’s coefficient (rp) was used to measure the colocalization between the labeling patterns generated by sera autoantibodies and the other commercial antibodies used for this study. The result is +1 for perfect colocalization, 0 for no colocalization, -1 for perfect anti-colocalization, and >0.5 for good colocalization. The Chi-squared test, the Fisher exact test, and the Mann–Whitney U-test were used to compare data between NPSLE and SLE patients and between positive and negative patients for brain-reactive autoantibodies detection. Statistical analyses were carried out using the StatistiXL software. Odd ratio (OR) and 95% confidence intervals (CIs) are reported where relevant; p-values < 0.05 were considered to represent statistical significance.Continuous variables are presented as mean (SD) or median and IQR. Absolute and relative frequencies are reported for categorical variables. For each IHC experimental set, the normality of data distributions was preliminary checked using the Goodness-of-fit test. The resulting versus 1:60 and 1:60,000 in SLE serum patients; p < 0.5), NeuN antibody and brain-reactive autoantibodies stain the same cells (p > 0.5), confirming that brain-reactive autoantibodies labeled neuronal targets. Colocalization profiles with NeuN were found within the entire brain cortex , in the majority of pyramidal and granular cells of the hippocampus, and in almost all of the cerebellar granule cells . None of the rat-IHC positive sera did recognize the bands corresponding to the NeuN protein using RBFOX3-HEK293T cell lysates  their distribution was investigated. All these autoantibodies were present in both the rat-IHC positive and negative sera, suggesting they were not responsible for the labeling we observed in rat brain , but no significant difference in the distribution of MRI abnormalities according to brain-reactive autoantibodies status was detected between groups Table 2.The diagnostic performance of brain-reactive autoantibodies, excluding anti-P and anti-NR2, in distinguishing between NPSLE and SLE patients was unsatisfactorily with 75.0% (95%CI 57.8% to 87.9%) sensitivity and 44.9% (95%CI 32.9% to 57.4%) specificity, resulting in 41.5% (95%CI 29.4% to 54.4%) positive predictive value (PPV) and 77.5% (95%CI 61.5% to 89.2%) negative predictive value (NPV). More stringent definition of NPSLE and SLE, by respectively excluding the 5 patients with headache and the 6 ANA negative patients at the time of sampling, gave similar results in term of sensitivity , specificity , PPV  and NPV . A positivity threshold ≤1:480 for brain-reactive antibodies led to higher specificity  but lower sensitivity .in vivo through a blood-brain barrier (BBB) or blood-cerebrospinal fluid (B-CSF) barrier leakage to bind neuronal cells, causing apoptotic cell death, microglial activation, synaptic pruning, and reduced synaptic density  and were directed against defined antigens . Although ANA can target any epitope within the nucleus, regardless of the cellular type used, we did not reveal any immunostaining reaction using rat brain sections in 44.9% and 25% of ANA HEp-2 positive sera collected from SLE and NPSLE, respectively. Furthermore, double staining experiments using the rat-IHC positive sera and commercial antibodies that mark neurons, astrocytes, and oligodendrocytes showed that brain-reactive autoantibodies labeled only neurons and not glial cells. If we could have detected by IHC the same ANA revealed through HEp-2, such autoantibodies would have labeled both neuron and glial cells. That could be explained by the differences in detecting ANA according to the substrates used because cell-based assays could reveal different epitopes than tissue-based assays. Among the known autoantibodies tested, only the anti-β2GPI were detected in the rat-positive IHC sera and were virtually absent in the negative ones suggesting they may be part of the heterogeneous autoantibody population responsible for the labeling we observed in rat brains. Indeed, the aPL and especially the anti-β2GPI could target neurons of the human brain, and patients with these autoantibodies were found to be more prone to exhibit seizures, cognitive impairment, psychosis, and depression compared to seronegative ones , 29, 30.RBFOX3) gene  gene . It playX3) gene . NeuN isX3) gene , 34. Morepilepsy  and cognepilepsy , while Risorders . Thus, oisorders . Howeverin vitro and in vivo through advanced imaging techniques. Furthermore, testing the effect of incorporating brain-reactive antibodies in the performance of algorithms for attributing neuropsychiatric events in SLE would be a further important point in the research agenda  SLE and NPSLE patients could possess autoantibodies potentially reacting to neuronal cells, with higher frequency and titers in NPSLE according to the broad spectrum of the neurological symptoms; (b) the whole brain but in particular the cortex, the hippocampus, and the cerebellum could be the target of brain-reactive autoantibodies mainly directed against unknown antigens; (c) anti-β2GPI could be part of the autoimmune reaction occurring at the brain level. However, further studies are needed to disentangle the specificity of brain-reactive autoantibodies in SLE patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All national and institutional guidelines for the care and use of animals were followed. Experimental protocols were performed in agreement with the Italian legislation, while the care and use of animals were approved by the American Physiological Society and EEC council directive of 24 November 1986 (86/609). Furthermore, the Independent Ethical Committee AOU of Cagliari (protocol number PG/2019/4522) approved the animal and human experimental design and procedures and all the subjects provided written informed consent.Study conception: CC, GF, AM, MP. Study design: CC, GF, AM, MP. Acquisition, analysis, or interpretation of the data: EM, CC, GC, MA, BN, MC, FL, MI, EC, AF, LL, LS, AC. Drafting the manuscript: CC, EM, MA, MP. Substantial manuscript revision: EM, GC, MA, MC, FL, MI, EC, AF, LL, LS, GF, CC, AM, AC. All authors read and approved the final manuscript.


Rosa roxburghii Tratt (RTT), on liver lipid metabolism disorders in db/db mice. The findings demonstrated that both RTT ellagitannin (C1) and RTT ellagic acid (C4) considerably decelerated body mass gain in db/db mice, significantly decreased fasting blood glucose (FBG) levels, and mitigated the aggregation of hepatic lipid droplets. At LDL-C levels, C1 performed substantially better than the C4 group, exhibiting no significant difference compared to the P (positive control) group. An RNA-seq analysis further disclosed that 1245 differentially expressed genes were identified in the livers of experimental mice following the C1 intervention. The GO and KEGG enrichment analysis revealed that, under ellagitannin intervention, numerous differentially expressed genes were significantly enriched in fatty acid metabolic processes, the PPAR signaling pathway, fatty acid degradation, fatty acid synthesis, and other lipid metabolism-related pathways. The qRT-PCR and Western blot analysis results indicated that RTT ellagitannin notably upregulated the gene and protein expression levels of peroxisome proliferator-activated receptor alpha (PPARα) and peroxisome proliferator-activated receptor gamma (PPARγ). In contrast, it downregulated the gene and protein expression levels of sterol regulatory element-binding protein (SREBP), recombinant fatty acid synthase (FASN), and acetyl-CoA carboxylase (ACC). Therefore, RTT ellagitannin can activate the PPAR signaling pathway, inhibit fatty acid uptake and de novo synthesis, and ameliorate hepatic lipid metabolism disorder in db/db mice, thus potentially aiding in maintaining lipid homeostasis in type 2 diabetes.A complex metabolic disorder, type 2 diabetes, was investigated to explore the impact of ellagitannin, derived from  Numerous metabolic disorders, such as inadequate insulin secretion, elevated blood glucose levels, and irregular lipid profiles, are linked to type 2 diabetes . As dietRosa roxburghii Tratt (RTT) belongs to the Rosaceae family as a perennial rose genus that is predominantly found in Southwest China [st China ,5. RTT, st China . Xiang est China  discoverst China ,9,10. Elst China . Researcst China  but alsost China . Ahad etst China  identifist China  found thst China . Howeverde novo lipid synthesis (DNL), cholesterol metabolism, and lipid uptake and transport [Lipid metabolism primarily encompasses ransport . The strransport ,19. DNL ransport ,21,22. Aransport ,23. The ransport ,25.Thus, in this research, we employed db/db mice to investigate the regulatory mechanism of RTT ellagitannin on lipid metabolism, using high-throughput sequencing (RNA-seq), aiming to provide a scientific foundation for its potential to ameliorate metabolic disorders in type 2 diabetes.RTT ellagitannin  and RTT ellagic acid  were acquired from our group, utilizing the extraction method in accordance with the technique described by Chen et al. . Fresh RFour-week-old C57/BKS-db/db and C57/BKS-db/m mice were housed in the Guizhou University animal facility, with a well-functioning ventilation system, maintaining 50–65% humidity and a room temperature of 22 ± 2 °C. The Animal Experimentation Ethics Committee of Guizhou University approved all experiments (No. EAE-GUZ-2020-P010). After mice acclimated for one week, db/m mice were used as the blank group (N), and db/db mice were divided into four groups (6 mice each): a model (M group), positive control , RTT ellagitannin , total triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were assessed using kits obtained from Nanjing Jiancheng Institute of Biological Engineering .Liver sections were removed from the paraformaldehyde fixative, rinsed with water, dried, and stained with Oil Red O for 10 min. Subsequently, the liver sections were immersed in 60% isopropyl alcohol for differentiation twice (3 and 5 s each), rinsed in pure water twice for 10 s each, and stained with hematoxylin for 5 min. The sections were then rinsed in pure water thrice , differentiated in 60% alcohol for 5 s, washed in pure water twice for 10 s each, immersed in a blue solution for 1 s, and rinsed in distilled water twice (not exceeding 15 s each). Lastly, the liver sections were mounted with glycerol gelatin.The samples were sequenced by Chengdu Nomi Metabolic Biotechnology Co., Ltd. . In the total RNA, Oligo(dT) magnetic beads were employed to enrich polyA-structured mRNA, and ion interruption fragmented RNA to approximately 300 bp. Conventional primers and reverse transcriptase were utilized to synthesize the first strand of cDNA, using RNA as a template, which was then used as a template for synthesizing the second strand. A Bioanalyzer Agilent 2100  ensured quality control of library fragment enrichment through PCR amplification. RNA was extracted and purified, and libraries were constructed, followed by double-end library sequencing based on Illumina novaseq6000 sequencing platform for bipartite library sequencing, with a sequencing data volume of 6 g and a read length of 250 bp.−ΔΔCt results and expressed as relative gene expression levels [Total RNAs of liver tissue were extracted using the TRIzol reagent kit . RNA was reverse transcribed using a cDNA kit  according to the manufacturer’s instructions. qRT-PCR was conducted using a 20 µL reaction sample with SYBR GREEN II  and the iCycleriQ multicolor real-time PCR detection system . Gene expression was normalized using β-actin as an internal control. The primer sequences for peroxisome proliferator-activated receptor alpha (PPARα), peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element binding protein (SREBP), recombinant fatty acid synthase (FASN), and acetyl-CoA carboxylase (ACC) for qRT-PCR are shown in n levels ,27.g for 10 min to obtain the total protein. The protein concentration was measured using the bicinchoninic acid (BCA) method, and then N,N,N,N-tetramethylethylenediamine (TEMED) was added to mix the gel. After 30 min of liquid sealing with ddH2O, 10% ammonium persulfate (AP) and TEMED were both added, and the mixture was stirred for 30 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was stopped upon the appearance of bromophenol blue, and the membrane was transferred to a PVDF membrane for detection.Liver tissue blocks were washed with precooled phosphate buffer (PBS) to remove blood stains until the solution was clear, and then they were cut into small pieces and homogenized. The radioimmunoprecipitation assay (RIPA) lysis solution (ready-to-use) was used for homogenization in an ice bath, and the supernatant was collected by centrifugation at 12,000× p < 0.05 indicated that the differences were significant and statistically significant.All experiments were repeated more than three times, and the results were expressed as “mean ± standard deviation (SD)”. Statistical analysis was performed using SPSS 25.0 software to process and organize the data. A one-way ANOVA was used, followed by Dunnett’s post hoc test for normally distributed data. Subsequently, multiple data means were rank ordered for comparison and indicated using different lowercase letters. A probability of The characteristic feature of type 2 diabetes is an initial weight gain that is followed by weight loss and thinning in mice. Throughout the experiment, the mice exhibited healthy growth with no mortality. p > 0.05) the P, C1, and C4 groups. Compared to the N group, the TG content in group M increased significantly by 144.02%; the liver TG contents of the experimental mice  all decreased significantly (p < 0.05) compared with group M, by 33.82%, 39.29, and 8.78%, respectively; however, there was no significant (p > 0.05) difference between groups P, C1, and C4, but the TG levels of C1 and P were close to the level of group N. p < 0.05) in mitigating LDL-C, but it had a decreasing trend. However, no significant differences were observed in the HDL-C contents among the groups. Thus, RTT ellagitannin was more effective in reducing the levels of TC, TG, and LDL-C, thereby ameliorating lipid metabolism disorders in type 2 diabetes mice. In the LDL-C level, C1 improved significantly better than C4.The samples were up-sequenced to generate raw data, and the bases were recognized in the raw data of each sample; as shown in 2FoldChange > 1, p < 0.05) were observed, with 695 DEGs significantly upregulated and 550 DEGs significantly downregulated. In M vs. C4, 707 DEGs were identified, including 366 significantly upregulated DEGs and 341 significantly downregulated DEGs , cellular component (CC), and molecular function (MF). Based on the highest GO score, an enrichment analysis was conducted, calculating the DEGs associated with each GO term, using the gene list for each term. The top 30 GO term entries with the smallest p-value, with the BP classification in M vs. C1 and M vs. C4 accounting for 53% and 63%, respectively. Further functional analysis of DEGs in M vs. C1 and M vs. C4 for BP classification was conducted using the ClueGO plugin in Cytoscape . Furthermore, a KEGG pathway enrichment analysis was performed, with the DEGs being significantly enriched in 69 pathways for the M vs. C1 group  and 59 pThe gene and protein expression levels of PPARα, ACC, PPARγ, FASN, and SREBP were assessed using qRT-PCR and Western blot methods, respectively. Worldwide, millions of people suffer from type 2 diabetes, and after decades of research, numerous natural active ingredients from plants have been discovered to help prevent this condition . A previUtilizing transcriptome sequencing technology, our analysis discovered that, in comparison to the M group, there were 1245 and 707 DEGs in the C1 and C4 groups, respectively. Approximately 537 common DEGs were expressed in both groups, which were significantly involved in the BP classification of fatty acid metabolism, oxoacid metabolism, cellular respiration, and steroid metabolic process functional annotation of the GO term and the visual functional enrichment analysis using ClueGO. Among the KEGG enrichment pathways, 33 identical pathways were found in ellagic acid and ellagitannin groups, including steroid hormone biosynthesis, PPAR signaling pathway, fatty acid degradation, and fatty acid synthesis, which are all related to lipid metabolism.Cholesterol is crucial for steroid hormone synthesis . ProteinFatty acid metabolism involves the uptake, synthesis, and oxidation of fatty acids. When the β-oxidation metabolic capacity is insufficient to handle circulating fatty acids, esterification occurs, resulting in the storage of fatty acids as triglycerides (TGs) in the liver and adipocytes. This process, along with long-term past interactions, leads to fat deposition . The hepde novo synthesis in lipid metabolism. It promotes lipid deposition by enhancing the expression of key genes (FASN and ACC) that regulate downstream lipid synthesis, contributing to triglyceride and lipid droplet formation [SREBP gene was upregulated, and downstream genes of lipid synthesis FASN and ACC were overexpressed. Elevated concentrations of TG activated downstream forms that competed with glucose to enter the cell, resulting in lipotoxic insulin resistance. Excess free fatty acids contributed to ectopic lipid deposition, increased glycolysis, and inhibition of glycogen synthesis, ultimately leading to decreased insulin sensitivity [SREBP expression in the liver, inhibited FASN and ACC gene and protein expression, and slowed down fatty acid and triglyceride synthesis. This led to the prevention of hepatic fat accumulation and liver protection. The protein expression of SREBP in the ellagic acid and ellagitannin groups was inconsistent with the trend of gene expression. The peak of protein expression may be reached when the gene is degraded, and the difference in trend could be due to transcriptional and translational space–time sites [SREBP is primarily involved in fatty acid uptake and ormation ,44,45,46ormation . Mehmoodormation  found thsitivity ,50. The sitivity , demonstme sites ,53. CompIn conclusion, RTT ellagitannin exhibited a greater capacity than ellagic acid to improve FBG and hepatic lipid levels in type 2 db/db mice. Moreover, it was significantly enriched in the KEGG enrichment pathways of steroid hormone biosynthesis, PPAR signaling, fatty acid degradation, and fatty acid synthesis. RTT ellagitannin significantly increased the gene and protein expression levels of PPARα and PPARγ, while downregulating the gene and protein expression levels of SREBP, FASN, and ACC. These findings suggest that RTT ellagitannin could serve as an active ingredient for improving lipid metabolism disorders in type 2 diabetes.


These results suggest that the vertebrate head is patterned from anterior to posterior in a progressive fashion and may involve timed actions of the BMP signaling.How head patterning is regulated in vertebrates is yet to be understood. In this study, we show that frog embryos injected with Noggin at different blastula and gastrula stages had their head development sequentially arrested at different positions. When timed BMP inhibition was applied to BMP-overexpressing embryos, the expression of five genes:  During early development, the vertebrate embryo is patterned from anterior to posterior in a temporally progressive manner ,2,3,4,5:In frogs, a BMP/anti-BMP dependent time-space translation mechanism has been proposed for trunk-tail patterning by Hox genes ,7. In thchordin transgenic lines (Tg (hsp70:chd)), Hashiguchi et al. have shown in zebrafish that the expression of six3 (a forebrain marker) [otx2 (a forebrain and mid-brain marker) [gbx1  [hoxb1b  [In the rescue experiments mentioned above ,10, Nogg marker) , otx2 (a marker) ,18, gbx1 marker) , and hox marker) , are seq marker) . This iss (Tg hsp0:chd), H marker) . These fIn the deuterostome embryo, the front-most portion of the A–P axis is not the head, but the extreme anterior domain (EAD), a region wherein ectoderm and endoderm directly juxtapose . In frogTo test the role of BMP signaling in head patterning, we performed timed anti-BMP treatments in both wild-type (WT) and ventralized frog embryos. This resulted in sequential arrest (in WT embryos) or rescue  of head patterning at different values, suggesting that a timing mechanism, which is BMP dependent and can be converted into spatial patterns by anti-BMP signals, may be involved in patterning the vertebrate head.pSP64T-BMP4 (for BMP4 RNA) [pCS2-hSmad6GR (for smad6GR RNA) [Frog embryos were harvested from naturally mated females and staged according to Nieuwkoop and Faber . For timMP4 RNA) , and pCS6GR RNA) . To indupVZ1-xcg1 (for xcg-1 probe) [pBSSK-Six3 (for six3 probe) [pBluescript-KS-xotx2 (for otx2 probe) [pXgbx-2 (for gbx-2 probe) [pBluescript SK-xHoxLab1 (for hoxd1 probe) [When the desired stages were reached, embryos were fixed overnight in MEMFA at 4 °C. After dehydration in 100% methanol, they were stored in methanol at −20 °C until use. Whole mount in situ hybridization (WISH) was performed as previously described . The pro1 probe) , pBSSK-S3 probe) , pBluesc2 probe) , pXgbx-22 probe) , and pBl1 probe) .To examine the role of BMP signaling in head formation, we injected Noggin protein, an antagonist of BMP ,37, to tsix3, otx2, gbx1, and hoxb1b [The above observations are supported by gene expression analysis. In zebrafish, timed Chordin treatments from mid-blastula to mid-gastrula stages sequentially expanded the expression domains of d hoxb1b , suggestbmp4 resulted in complete ventralization, showing a blob of tissue that had no axis . Although the time of otx2 expression was not affected, its expression domain was slightly expanded at stage 10.During trunk patterning, collinearity causes Hox genes to be expressed in a 3′ to 5′ order, and that more 3′ genes are expressed earlier and more anteriorly than/to more 5′ ones ,47,48. Tal plate , but it njection . For exaDuring animal development, the patterning of the trunk-tail part of the axis has been known to be regulated by Hox genes ,50,51. TWhen the inhibitor of the BMP pathway, Noggin protein, was applied to the frog embryo from the mid-blastula to mid-gastrula stage, the patterning of the head was arrested at different positional values, i.e., cement gland, forebrain, mid-brain, hindbrain, and neck . Sequentsix3, otx2, gbx2, and hoxb1b) by timed Chordin treatments [bmp4-overexpressing embryos  by anti-BMP (Smad6 induction) in BMP-injected embryos (xcg-1 (EAD), and six3 (forebrain), and slightly otx2 (midbrain); induction at stage 9 stabilized six3 (forebrain) and otx2 (midbrain), but not xcg-1; and induction at stage 10 stabilized otx2 (midbrain) and gbx2 (hindbrain), but not xcg-1 and six3 (otx2, gbx2 and hoxd1. Further studies are needed to test our hypothesis further and to uncover how the head patterning process is regulated by BMP signaling. This study, as the basis and first step of our hypothesis, argues that the vertebrate head is progressively patterned, and the patterning process is likely to be regulated by BMP signaling.Although the mechanism by which head patterning is regulated by BMP signaling still needs to be elucidated, the results in this study suggest two aspects of head patterning. First, the vertebrate head is patterned in a temporally progressive manner: the EAD is patterned first, followed by patterning of the forebrain, midbrain, hindbrain, and the neck. Second, BMP signaling is involved in patterning the head in time and space, which can be seen from the progressive arrest of head formation by timed anti-BMP treatments  and from embryos . Based o embryos . A key cand six3 . This su


D-type cyclins encode G1/S cell cycle checkpoint proteins, which play a crucial role in defining cell cycle exit and progression. Precise control of cell cycle exit is vital during embryonic development, with defects in the pathways regulating intracellular D-type cyclins resulting in abnormal initiation of stem cell differentiation in a variety of different organ systems. Furthermore, stabilisation of D-type cyclins is observed in a wide range of disorders characterized by cellular over-proliferation, including cancers and overgrowth disorders. In this review, we will summarize and compare the roles played by each D-type cyclin during development and provide examples of how their intracellular dysregulation can be an underlying cause of disease. Phosphorylated pocket proteins are then hyper-phosphorylated by cyclin E-CDK2 complexes, causing them to dissociate from, and release, their E2F transcription factor binding partners. This release initiates the transcription of several key proteins required in order to advance the cell cycle and promote cell proliferation ..1].CCND1) encodes the general D-type cyclin, expressed in all tissue types except those derived from haematopoietic stem cell lines [CCND1 is composed of 5 exons, which are separated by 4 introns. Alternative splicing at the exon 4–intron 4 boundary results in the cyclin D1b (CCND1b) variant, which does not include exon 5, and acquires 33 new amino acids. Both isoforms are identical for the first 240 amino acids but differ in their C-termini. CCND1b is deficient in promoting Rb phosphorylation and lacks the LxxLL motif (aa 251–257) required for ligand-dependent interaction with nuclear receptors, as well as both the Thr-286 residue and the PEST sequence (aa 241–290) required for degradation  that increases susceptibility to colorectal cancer and multiple myeloma [Ccnd1-deficient mice display developmental abnormalities such as reduced body size, hypoplastic retinas, and pregnancy-insensitive mammary glands, as well as increased premature mortality within the first 3 weeks of life ,39. Recef CDK4/6 . No huma myeloma ,42,43,44CCND1 compromises the S-phase checkpoint, inducing forced progression of the cell cycle, disrupting DNA replication, and promoting DNA damage and genomic instability, resulting in oncogenesis [CCND1 is more frequently deregulated than CCND2 and CCND3 in both solid and haematological cancers [CCND1 is strongly tied to its cellular levels, as tumour cells with high CCND1 levels exhibit uncontrolled cell proliferation. Overexpression of CCND1 can be caused by the amplification of CCND1, chromosomal re-arrangement, or the stabilisation of the CCND1 protein via impaired degradation, often caused by mutations at or around the Thr-286 phosphorylation site, leading to accumulation in the nucleus [CCND1 mRNA transcript result in shorter, more stable transcripts [As an oncogene, dysregulation of ogenesis . CCND1 i cancers , and is  cancers , mantle  cancers , pancrea cancers , melanom cancers , non-sma cancers , gastric cancers , colorec cancers , endomet cancers , and ove cancers . The onc nucleus . Additionscripts .CCND1 plays a key role in promoting cell proliferation, cell survival, angiogenesis, and cell migration, as well as in preventing cell senescence [CCND1 can execute some of these functions independently from its association with CDK4/6. For example, CCND1 binds nuclear receptors such as eostrogen receptor alpha (ERα) and steroid receptor co-factors such as SRC1 and SRC3, enhancing oestrogen-receptor-mediated transcription in breast epithelial cells [CCND1 inhibits the activity of androgen receptors (ARs) via binding and preventing the formation of the active AR complex, as well as recruiting histone deacetylases (HDACs) to repress its transcription [CCND1 binds histone acetyltransferases such as p300/CREB-binding protein-associated factor (P/CAF), which increases the transcriptional activity of ER, and HDACs to enhance transcriptional repression, as seen with AR [CCND1 acts as a key transcription regulator of several genes through a combination of interactions with transcription factors, coactivators, and chromatin-altering enzymes.In addition to its canonical role in the Rb pathway, nescence . CCND1 cal cells ,57. Convcription . Moreove with AR ,59. IndeCCND1 has also been shown to play a role in DNA repair. Following DNA damage, CCND1 is recruited by BRCA2 to DNA damage sites, after which CCND1 interacts with Rad51, a critical recombinase involved in homologous recombination and DNA repair. This facilitates its recruitment to regions of DNA damage to promote DNA repair. Decreased expression of CCND1, but not treatment with CDK4/6 inhibitors, decreases Rad51 recruitment to DNA damage sites, confirming that CCND1 promotes DNA repair independently of CDK4/6 [f CDK4/6 .CCND1 can also affect the movement and invasiveness of mantle cell lymphoma cells by localising and accumulating in the cytoplasm. Proteomic analysis of CCND1-interacting proteins showed that many of them are involved in the regulation of cytoskeletal dynamics, migration, and invasion [CCND1 enhances cellular motility by inhibiting the signalling pathways of thrombospondin 1 (TSP-1) and Rho-activated kinase (ROCK) [CCND1 can promote angiogenesis, enabling tumour survival, growth, and metastasis [CCND1 resulted in a significant decrease in migration and invasion, indicating that cytoplasmic CCND1 is essential for chemotaxis and the invasion of mantle cell lymphoma cells [invasion . For exae (ROCK) . Additiotastasis . Blockinma cells .CCND1 alone, however, is not sufficient to drive oncogenic cell transformation without cooperating mutations [CCND1 from being phosphorylated by GSK3ß or AMBRA1, resulting in the stabilisation and accumulation of CCND1 in the nucleus throughout all stages of the cell cycle. This leads to increased formation of active cyclin D-CDK4/6 complexes, genomic instability, oncogenic cell transformation, and neoplastic growth. It has been postulated that the degradation of CCND1 is vital to maintaining genomic stability following DNA damage, and, therefore, dysregulation of its export via stabilisation is a prelude to oncogenesis rather than the underlying cause [Over-expression of utations . p.Thr28ng cause ,65.CCND2, located on chromosome 12p13, consists of 5 exons that encode the cyclin D2 (CCND2) protein [CCND1, CCND2 is a component of the PI3K-AKT-mTOR pathway, which controls the transition between G1 and S phase of the cell cycle by forming a complex with its cyclin-dependent kinases, CDK4 (CCND2–CDK4) and CDK6. The formation of this complex is a rate-limiting step for progression through the G1 phase, and heavily depends on the availability of serum in cells [ protein . Like CCin cells .CCND2–CDK4 complexes control the phosphorylation of its substrate, Rb [CCND2, increases the amount of active (hypophosphorylated) Rb. Therefore, Rb-EGF complexes can form and prevent EGF accumulation, leading to cell cycle arrest [CCND2, are expressed and regulated precisely to ensure adequate entry through the cell cycle and normal cell growth.Once assembled, rate, Rb . Phosphorate, Rb , which irate, Rb ,69. A ree arrest . It is, CCND2 is the only D-type cyclin expressed in the adult hippocampus [Ccnd2 knockout mice (Ccnd2-KO) displaying reduced hippocampal neuron production [Ccnd2-KO-associated brain abnormalities result in limited impact on behavioural phenotypes [CCND2 in adult neurogenesis is less understood, there is a well-established role of CCND2 in neurodevelopment during embryogenesis [CCND2 mutations cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH), with mutations clustering at the c-terminus. These mutations occur at and around the phosphodegron residue Thr-280 and result in resistance to proteasomal degradation in vitro, leading to significantly increased CCND2 accumulation in patient cells compared to controls [CCND2 is also observed in related neurodevelopmental overgrowth conditions caused by mutations in proteins upstream of CCND2 in the PI3K-AKT-mTOR pathway, such as AKT3, PIK3CA, and PIK3R2 [CCND2 stabilisation and/or accumulation therefore appears to be a common end point for this group of disorders, making it an excellent therapeutic target [CCND2 [CCND2 plays in early neurogenesis in terms of regulating cell cycle exit in neural progenitor cells, and how stabilised CCND2 leads to the continued entering of the cell cycle into the S phase, resulting in over-proliferation and increased brain growth.pocampus , with Ccoduction . Howeverenotypes . Althougogenesis . De novocontrols . Interesd PIK3R2 ,75,76. Ct [CCND2 . This inCCND2 variants resulting in protein truncation were found in patients with microcephaly, the inverse brain phenotype to MPPH [CCND2 was poorly understood, with only mice studies indicating that Ccnd2-KO causes a lack of cerebellar stellate interneurons [CCND2 confirm the phenotypes observed in mice and further support a crucial role of CCND2 during neurogenesis, as well as the requirement for the careful control of intracellular levels of cyclin D.Recently, loss-of-function  to MPPH . Prior trneurons , supressrneurons , and sevrneurons . These rCCND2 also regulates the cell cycle in other tissues which may also be affected in patients with stabilising mutations. For example, MPPH patients display 3- or 4-limb postaxial polydactyly, indicating a role for CCND2 in early limb bud development [CCND2 in limb development has also been identified independently in the developing limb buds of chick wings [CCND2 has been implicated in pancreatic B-cell proliferation, with MPPH or MCAP patients with CCND2 or PIK3CA variants, respectively, found to have hypoglycaemia [CCND2-stabilising mutations likely results in hyperinsulinaemia and, in turn, hypoglycaemia [In addition to its key role in neurogenesis, elopment . The cruck wings . RNA seqck wings . More relycaemia ,85. β-celycaemia . PatientCCND2 in cancers is more established. Prior to being identified in patients with MPPH, the same protein-stabilising mutations in and around Thr-280 had been identified somatically in tumours (COSMIC). Recent studies have identified CCND2 and CCND1 mutations as frequent events in myeloid leukaemia, in particular acute myeloid leukaemia [CCND2, increased phosphorylation of Rb protein, and uncontrolled cellular proliferation.While its roles in development have only recently been discovered, the role of eukaemia ,88. ThesCCND2 is the most common disease mechanism associated with CCND2-associated disorders, a number of cancers have been found to have reduced CCND2 due to CCND2 hypermethylation, particularly breast and lung cancers [CCND2-promoter hypermethylation was found at an early stage of breast cancer tumorigenesis and was associated with the silencing of CCND2 expression [CCND2 expression in breast cancer samples and resulted in reduced cancer cell growth through cell cycle arrest [CCND2, and, therefore, CCND2-associated cell proliferation, is observed in cancers, but this may be due to a compensation effect leading to the up-regulation of another cyclin, e.g., cyclin E. Another explanation may be related to the stage or sub-type of cancer. For example, in gastric cancer, some studies have CCND2 hypermethylation listed as an underlying cause of proliferation [CCND2 hypomethylation leading to increased CCND2 expression in more advanced-stage gastric carcinomas [While the accumulation of  cancers . CCND2-ppression . Adminise arrest ,91. It rferation , whereasrcinomas .cyclin D3 (CCND3), located on chromosome 6p21, consists of 5 exons, but several alternative isoforms exist with different transcriptional start sites. While each isoform contains the regulatory C-terminal region of CCND3, the alternative start sites affect the CDK4-binding region, indicating a CDK4-independent role for these isoforms. In comparison to CCND1 and CCND2, CCND3 is predominantly found in bone marrow and lymphoid tissues, with the highest protein and RNA expression found in the thymus (proteinatlas.org) [CCND3 in these tissues is comparatively low.The major isoform of las.org) . High prCCND3 is an atypical D-type cyclin that is predominantly expressed in differentiated tissues [CCND3 in mice is viable, but homozygous null mice show defects in lymphoid-derived cells, such as impaired B- and T-cell differentiation and granulocyte proliferation [ tissues ,96,97. Gferation ,99,100. feration  and, at feration ,103.CCND3−/− mice also show retarded growth, significant loss of muscle mass, and impaired muscle regeneration, suggesting an important role of CCND3 in myogenesis [CCND3 may not have a direct role during development, it does play a key role in differentiation and maturation in lymphoid and musculoskeletal tissues.In addition to an effect on lymphoid-derived cells, ogenesis ,99,104. CCND1, no human disorder has yet been associated with germline mutations in CCND3. However, a role of CCND3 in multiple cancers has been observed, with most CCND3 aberrations being gene-amplification rather than single-nucleotide variants. Due to the expression profile of CCND3, it is no surprise that lymphoid cancers are most commonly associated with dysregulation of CCND3. Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin’s lymphoma, and alterations in the cyclin D/CDK4-6 pathway are found in approximately 67% of DLBCL cases, with CCND3 overexpression accounting for 53% [CCND3 have shown lower response rates to chemotherapy and shorter survival durations compared to those with low CCND3 expression [CCND3 has also been observed in chronic lymphocytic leukaemia (CLL) cells, with RNA sequencing identifying a 38-fold increase in CCND3 in NOTCH1-mutated cells compared to NOTCH1 non-mutated cells [CCND3 was confirmed [CCND3 in B-cell lymphoblastic leukaemia (B-ALL) was also described, with CCND3 being found to be indispensable for the growth and survival of B-ALL cells irrespective of the underlying driver mutation [CCND3 expression associated with cells developing resistance to the CDK4 inhibitor Palbociclib, suggesting targeting of CCND3 rather than CDK4, may be a more effective therapeutic approach. Altogether, these findings highlight a key role of CCND3 in the regulation of lymphoid-derived cells and the consequences of its dysregulation.Like  for 53% ,106,107.pression . Increased cells . A similonfirmed . A role mutation . IncreasCCND3 have recently been identified in bone tumours. Xie et al. found that 43/357 (12.04%) bone tumours sequenced had a genomic aberration in CCND3, making it the 5th most mutated gene after TP53, NCOR1, VEGFA, and RB1 [CCND3 amplifications were mainly identified in osteosarcoma patients, with 42/43 CCND3 mutations identified being in the 227 osteosarcoma patients present in the cohort . A recurrent fusion gene between KCNMB4 and CCND3 has also been identified in a cohort of osteosarcoma, but not in a cohort of 240 other sarcomas, further suggesting a specific role of CCND3 in osteosarcoma [KCNMB4-CCND3 fusion gene showed that it promoted cell migration in SAOS-2 cells. By comparing the ages of patients, it was found that CCND3 mutations were more frequent in paediatric, adolescent, and young adult (P-AYA) osteosarcoma than in adult osteosarcoma [In addition to lymphoid cancers, somatic mutations in  and RB1 . CCND3 aosarcoma . Functioosarcoma .As D-type cyclins do not possess any enzymatic functions alone, a favoured strategy for treating cyclin D based disorders involve targeting the enzymatic activities of their partners, CDK4 and CDK6. As such, three highly specific dual-CDK4/6 inhibitors have been developed and approved by the FDA for use in the treatment of advanced or metastatic breast cancer: Palbociclib, Abemaciclib, and Ribociclib ,114. TheThere are caveats, however: potent side effects occur due to lack of selectivity, the most notable of which is neutropenia, i.e., a low neutrophil count . FurtherAnother therapeutic strategy to treat stabilised D-type cyclin disorders would be to disrupt the protein–protein interactions between cyclin D and CDK4. This would prevent the formation of active cyclin D-CDK4 complexes, arresting cell cycle progression and reducing cell proliferation in addition to also potentially inhibiting the formation of cyclin D-CDK2 complexes in some stabilised cyclin D phenotypes. However, no known inhibitors of the cyclin D-CDK4 complex have been identified. This represents an interesting opportunity to explore and investigate novel inhibitors which are able to perturb cyclin D-CDK4 complex formation.Careful regulation of D-type cyclins is crucial to ensure there is sufficient proliferation and adequate cell numbers to develop, maintain, and repair tissues throughout life. Dysregulation of D-type cyclins, either by mutation in key regulatory proteins or through hyper-activation of upstream pathways, results in a range of disorders associated with over- or under-proliferation. Therapies that target cyclin D may offer exciting opportunities to overcome the effects of any dysregulation; however, it will be crucial to ensure that they do not tip the balance from over- to under-proliferation, or vice versa. While this review focused on the three D-type cyclins and their interactions with CDK4, it is now known that cyclin D has additional roles independent of CDK4, although what these roles are remains unclear ,40,57,60


Since the discovery of SARS-CoV-2, changes in genotype and reinfection with different variants have been observed in COVID-19-recovered patients, raising questions around the clinical pattern and severity of primary infection and reinfection. In this systematic review, we summarize the results of 23 studies addressing SARS-CoV-2 reinfections. A total of 23,231 reinfected patients were included, with pooled estimated reinfection rates ranging from 0.1 to 6.8%. Reinfections were more prevalent during the Omicron variant period. The mean age of reinfected patients was 38.0 ± 6. years and females were predominant among reinfected patients (M/F = 0.8). The most common symptoms during the first and second infection were fever (41.1%), cough (35.7% and 44.6%), myalgia (34.5% and 33.3%), fatigue (23.8% and 25.6%), and headaches (24.4% and 21.4%). No significant differences of clinical pattern were observed between primary infection and reinfection. No significant differences in the severity of infection were observed between primary infection and reinfection. Being female, being a patient with comorbidities, lacking anti-nucleocapsid IgG after the first infection, being infected during the Delta and Omicron wave, and being unvaccinated were associated with a higher risk of reinfection. Conflicting age-related findings were found in two studies. Reinfection with SARS-CoV-2 suggests that natural immunity is not long-lasting in COVID-19 patients. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the virus responsible for the infection known as coronavirus disease 2019 (COVID-19) . Howeverhttp://www.prisma-sattement.org, accessed on 31 March 2023). The PubMed  and Google Scholar  databases were investigated to identify all relevant studies. We used an expanded MeSH to identify a maximum number of keyword synonyms. The current search was performed on 31 March 2023 by combining the following keywords:The Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines were applied to perform this systematic review (#1: “COVID-19” OR “SARS-CoV-2”#2: “Re-infection?” OR “Reinfection?” OR “Recurrent Infection” OR “Re-positive”#3: #1 AND #2All published studies and pre-prints (including case series and prospective cohort studies) were selected according to the following inclusion criteria:(1) Studies reporting on adult COVID-19 patients (≥18 years old) with infection confirmed by a positive RT-PCR (Reserve Transcription Polymerase Chain Reaction);(2) Due to the heterogeneity of duration between two episodes of infection, we included studies reporting on patients that met the following criteria, based on the US CDC Common Investigation Protocol for Investigating Suspected SARS-CoV-2 Reinfection :+ Two episodes of infection at ≥90 days interval in patients with negative RT-PCR results between two episodes of infection, including asymptomatic patients;+ Two episodes of infection at 60 to 89 days of interval: symptomatic patients confirmed by RT-PCR test with ≥ one intermediate negative RT-PCR test with no other reason or symptomatic patients tested with different genotypes between two episodes of infection;+ Two episodes of infection at an interval of <60 days: symptomatic patients confirmed by differences between two episodes of infection in whole genome sequencing in patients infected with different virus genotypes;Only papers in English were included in this work.We excluded single-case reports and all studies based on animal experiments. Review papers were excluded, though the reference lists were assessed to identify potentially relevant studies. We also excluded studies conducted only on immunocompromised patients with cancer and acquired immunodeficiency syndrome.Four researchers independently checked the relevant articles and data extraction. All discordant results were discussed to reach a consensus between researchers.The type of study, country, and setting where studies were performed, the number and proportion of reinfection cases, demographic information (age and sex), comorbidities and vaccination status, the time between two episodes of infection, symptoms, imaging findings, and severity outcomes  were extracted from the included articles. When available, risk factors for reinfection were reported or calculated.The quality of cohort studies was accessed using the Newcastle–Ottawa Scale (NOS) , consisthttp://www.r-project.org, accessed on 31 March 2023]. In the present study, the heterogeneity I2 > 50% was identified as representing substantial heterogeneity [All statistical analyses were performed using the open-source software R [R Core Team] R: A language and environment for statistical computing from the R foundation for statistical computing, based in Vienna, Austria, in 2020. URL: [ogeneity . The pooogeneity .https://covariants.org/, accessed on 31 March 2023) and, when available, the SARS-CoV-2 sequencing data described in each study.The most frequent variant circulating at the time of reinfection in each country was assessed using the CoVariants website  and those assessed with NOS were scored as either good quality (seven studies) or fair quality (eleven studies) .Of the 26 included studies, eight were case series ,18,19,20According to the CoVariants website, the most frequent variant SARS-CoV-2 circulating at the time of reinfection in countries where studies were conducted were Alpha variant in four studies ,24,31,35In ten studies, SARS-CoV-2 variants were identified by genetic methods for 2354 reinfected patients ,31,33,38A total of 23,231 reinfected patients were included, including 46 patients from the case series ,18,19,20The overall age of discharged patients from four cohort studies ,24,25,34The male/female ratio from six cohort studies with 3,991,528 discharged patients ,30,34,37Only one study reported on detailed comorbidities for 119,226 discharged patients with hypertension (9.3%), cardiovascular disease (8.3%) and diabetes (4.2%) being the most frequent . ComorbiOne study reported information on COVID-19 vaccination before the first infection with a prevalence of 0/207 patients  Supplem. VaccinaThe length of time between two infections was described in 19 studies, including 17 studies with a calculated mean ± SD of 178.9 ± 87.1 days, ranging from 31 to 647 days ,34,35,36Seven studies reported detailed symptoms for both episodes of infection in 168 reinfected patients ,22,23,26Only one study  reportedp = 0.59)  . Howeverp = 0.14) and 0% heterogeneity (p = 90.43)  . HoweverA total of 7/16,616 (0.4%) reinfected patients died, with information reported in 18 studies ,34,35,37Seven studies addressed this issue ,34,37,38An early systematic review, mostly based on Chinese data and describing 466 reinfection cases, concluded with an estimated 15% reinfection rate . This hiThe time between two episodes of infection was about five months, as compared to two months in a previous meta-analysis based on 577 cases of reinfection ; this diWe confirm the results of colleagues regarding the highest prevalence of reinfection in female patients . This obWe found no clear evidence of different clinical patterns between primary and second infection, although our analysis was conducted on a small sub-sample of only 168 patients .Dhillon et al. reported a higher proportion of transfer to the ICU during reinfection , which dData on vaccination status are too heterogeneous to allow strong conclusions about the effectiveness of vaccination in lowering reinfection rates, though some studies suggest a potential protective effect ,30. NeveWe acknowledge that our review has some limitations. Firstly, high heterogeneity was observed in all studies analyzed. These results might have been caused by various factors, including methodology, sample size and, especially, the periods when studies were conducted, given the prevalence different SARS-CoV-2 variants at different stages in the COVID-19 pandemic.Reinfection with SARS-CoV-2 does occur, suggesting that natural immunity is not long-lasting in COVID-19 patients. Female patients seem more likely to experience reinfection than male patients. Thus far, no clear evidence for a difference in severity between the first infection and reinfection has been observed.


IFNGR1, IFNGR2, IFNG, IL12RB1, IL12RB2, IL23R, IL12B, ISG15, USP18, ZNFX1, TBX21, STAT1, TYK2, IRF8, IRF1, CYBB, JAK1, RORC, NEMO, and SPPL2A) over the last 20 years. MSMD confers a selective susceptibility to infections with weakly virulent mycobacteria, including the M. bovis Bacille Calmette-Guerin (BCG) vaccines and various environmental mycobacteria in patients, primarily children, without classical immune defects. These patients may also present severe forms of tuberculosis, and about half of them might develop non-typhoidal salmonellosis. In some cases, patients also suffer from chronic mucocutaneous candidiasis (CMC), while in others, patients also present severe viral, parasitic, fungal, and/or bacterial diseases. Despite this clinical and genetic heterogeneity, almost all genetic etiologies of MSMD alter the interferon-gamma (IFN-γ)- mediated immunity by impairing or abolishing IFN-γ production or the response to this cytokine. It was proven that the human IFN-γ level is a quantitative trait that defines the outcome of mycobacterial infection. The study of these monogenic defects contributes to understanding the molecular mechanism of mycobacterial diseases in humans and to the development of new diagnostic and therapeutic approaches to improve care and prognosis. For example, MSMD patients with impaired production of IFN-γ may benefit from injections of human recombinant IFN-γ, while for patients with abolished response to this cytokine, hematopoietic stem cell transplantation (HSCT) and promising gene therapy are the only current therapeutic options. These discoveries also bridge the gap between simple Mendelian inheritance and complex human genetics.The constant progress of genomics and the establishment of new functional tests have paved the way for identifying monogenic defects conferring a selective predisposition to infections by certain microbes as a new type of inborn errors of immunity (IEIs). Mendelian susceptibility to mycobacterial diseases (MSMD) is the most characterized of these IEIs, with 36 different disorders found in 20 distinct genes (


Additionally, there was an increase in ROS production in the aged hippocampus, leading to the activation of antioxidant signaling, specifically the Nrf2 pathway. It was also observed that aged animals had deregulation of calcium homeostasis, with more sensitive mitochondria to calcium overload and deregulation of proteins related to mitochondrial dynamics and quality control processes. Finally, we observed a decrease in mitochondrial biogenesis with a decrease in mitochondrial mass and deregulation of mitophagy. These results show that during the aging process, damaged mitochondria accumulate, which could contribute to or be responsible for the aging phenotype and age-related disabilities.Aging is a physiological process that generates progressive decline in many cellular functions. There are many theories of aging, and one of great importance in recent years is the mitochondrial theory of aging, in which mitochondrial dysfunction that occurs at advanced age could be responsible for the aged phenotype. In this context, there is diverse information about mitochondrial dysfunction in aging, in different models and different organs. Specifically, in the brain, different studies have shown mitochondrial dysfunction mainly in the cortex; however, until now, no study has shown all the defects in hippocampal mitochondria in aged female C57BL/6J mice. We performed a complete analysis of mitochondrial function in 3-month-old and 20-month-old (mo) female C57BL/6J mice, specifically in the hippocampus of these animals. We observed an impairment in bioenergetic function, indicated by a decrease in mitochondrial membrane potential, O Aging is a natural process characterized by physiological dysfunction in response to accumulative damage. Physiological impairment is a consequence of the deterioration of diverse organs and tissues, including the brain . In the 2+ homeostasis, and redox balance [2+ in response to increased cytosolic Ca2+ concentration [2+ uptake opens the mitochondrial permeability transition pore (mPTP) [One of the most studied age-related cell defects found in the literature is mitochondrial dysfunction. Mitochondrial dysfunction is a hallmark of aging . Mitocho balance , all ess balance , and the balance  necessar balance . Mitochontration , mainly ntration ,11. Howee (mPTP) , leadinge (mPTP) . Cyclophe (mPTP) , and othe (mPTP) , adeninee (mPTP) ,16,17. Fe (mPTP) . In addie (mPTP) . Mitochoe (mPTP) ,21,22. AWith regard to aging, diverse studies have reported that mitochondria lose their functions and that their morphology is aberrant in both the central and peripherical nervous systems in mice ,24,25,26Aging research is possible with in vivo study using experimental models, from invertebrate to vertebrate models, which have varied lifespans . Within The main function of mitochondria is to produce energy in the form of ATP, which is fundamental to the proper functioning of several processes within the cells. In neurons, this is especially important for the synaptic process for vesicle recycling, neurotransmitter release, and ATP-dependent ion pumping . Since t2O2) and superoxide (O2−) [2O2 by the mitochondrial superoxide dismutases (SODs) [2− into H2O2. We observed an increment in the mRNA levels of gclc, hmox1, and sod1  . Superoxs (SODs) . We evals (SODs) . We evals (SODs) ; Heme Oxs (SODs) ; and Supand sod1 E–G in thand sod1 H, which Calcium is an important second messenger that is essential for synaptic communication since it is involved in the control of membrane potential and action potential in neurons ,41. SpecThe balance between fusion and fission is essential for maintaining mitochondrial morphology and function. It has been reported that during age-related neurodegenerative diseases, such as AD, there is an increase in fragmented mitochondria as a result of an increase in the fission process ,52. HoweOther important processes that contribute to the proper functioning of mitochondria as a mechanism of quality control are mitochondrial biogenesis, which generates new mitochondria, and mitophagy, which eliminates damaged mitochondria . SeveralRegarding mitophagy, it is a specialized autophagy responsible for the degradation of dysfunctional mitochondria to maintain mitochondrial functionality . There aTaken together, these data show a complete analysis of mitochondrial dysfunction during normal aging. We have described bioenergetic failure, redox imbalance, deregulation of calcium homeostasis, an imbalance in dynamic processes and impairment in quality control processes, such as mitochondrial biogenesis and mitophagy, which could finally be a contributing factor to the aging phenotype and the development of age-related neurodegenerative diseases.Understanding aging events at a cellular level is of the utmost importance to tackle healthspan during the later stages of life. Here, we studied mitochondrial-related impairments in female C57BL/6J aged mice to gain a deeper understanding of the mechanisms that may underlie aging processes in the hippocampus. We reported that hippocampal mitochondria were dysfunctional in the aged mice (20 mo) in comparison to the adult mice (3 mo), indicated by a decrease in ATP production and MMP, an increase in calcium sensitivity, an increase in ROS production and protein oxidative damage, and also an imbalance in mitochondrial fusion and fission proteins, as well as altered biogenesis and mitophagy, showing, for the first time in the same study, a complete analysis of the age-related mitochondrial changes in the hippocampus .We previously reported that hippocampal mitochondria from aged C57BL/6J mice  show impaired bioenergetic function, due, in part, to a decrease in the expression of complexes I and IV and decreased MMP, evidencing depolarization of hippocampal mitochondria in aged animals ,61,62. S2O2 production compared to female rats [The decrease in bioenergetics is correlated with redox imbalance since the production of ROS is mainly due to complex I and III ; thus, dale rats .An important antioxidant pathway is governed by the Nrf2 transcription factor. Nrf2 translocates to the nucleus under oxidative stress, promoting antioxidant gene expression, such as GCS, HO1, and SOD1 ,87. Ther+/Ca2+ exchanger (NCLX) [Another important function of mitochondria is to regulate intracellular calcium concentration via the transient opening of the mitochondrial permeability transition pore (mPTP), mitochondrial calcium uniporter (MCU), and Nar (NCLX) ,93. In ar (NCLX) , but calr (NCLX) . We obser (NCLX) ,96. Furtr (NCLX) . Therefor (NCLX) . We propr (NCLX) . Cyp-D dr (NCLX) . FurtherMitochondria form an interconnected network in the cells and are regulated by fusion and fission processes in response to diverse conditions in the cells, such as changes to nutrient availability, signaling, and different stress conditions . Many rePGC1α is a transcriptional coactivator that binds to transcription factors such as Nrf2 to promote mitochondrial biogenesis . Our resThe clearance of damaged mitochondria by mitophagy is of the utmost importance to maintain cellular homeostasis . PINK1 aAnimals. Female 3-month-old mice and 20-month-old mice C57BL/6J were obtained from Fundación Ciencia and Vida and the Institute of Public Health (ISP) in Chile. These animals were housed and maintained at 24 °C on a 12:12 h light–dark cycle, with food and water provided ad libitum. The animals were handled according to the National Institute of Health guidelines . The experimental procedures were approved by the Bioethical and Biosafety Committee of Universidad San Sebastián. The animals were anesthetized using isoflurane in an anesthesia chamber and then euthanized by decapitation. The experimental procedures were approved by the Bioethical and Biosafety Committee of Universidad San Sebastián. This study was carried out in compliance with the ARRIVE guidelines.TM Green FM , MitoTracker Red CM-H2Xros , CM-H2DCFDA , Rhod-2 AM, cell-permeant , and VECTASHIELD Antifade Mounting Medium with DAPI .Reagents and antibodies. The primary antibodies used were mouse anti-β-actin , rabbit anti-GAPDH , mouse anti-Nitrotirosine , rabbit anti-Nrf2 , mouse anti-catalase , mouse anti-SOD1 , mouse anti-Glutathione reductase , mouse anti-VDAC , mouse anti-OSCP , mouse anti-CYP-D , mouse anti-ANT , rabbit anti-OPA1 , mouse anti-MFN1 , rabbit anti-MFN2 , rabbit anti-pDRP1 , mouse anti-DRP1 , rabbit anti-MFF , mouse anti-biogenesis cocktail , mouse anti-PGC-1α , mouse anti-PINK1 , and mouse anti-PARKIN . The fluorescent dyes used were: MitoTracker2P2O7 30 µM, Na3VO4 100 mM) using a homogenizer and then sequentially passed through syringes of different calibers. The protein samples were centrifuged at 14,000 rpm for 20 min at 4 °C. The protein concentrations were determined using the BCA Protein Assay Kit . Samples were resolved by SDS-PAGE, followed by immunoblotting on PVDF membranes. The membranes were incubated with the primary antibodies and anti-mouse or anti-rabbit IgG peroxidase-conjugated antibodies (Pierce) and visualized using an ECL kit .Immunoblotting. The hippocampus of the 3- and 20-month-old (mo) mice was dissected on ice and immediately processed as previously described ,96,117. g for 10 min at 4 °C. The supernatant was centrifuged at 8000× g for 10 min; the newly enriched-mitochondrial pellet was washed twice in MSH without EDTA. Protein concentration was determined by using a standard BCA kit .Isolation of an enriched-mitochondrial fraction from the hippocampus. As previously described, a fraction enriched in mitochondria was isolated from the hippocampus ,61,87. Hn = 3) and immediately processed as previously described [Transmission Electron Microscopy (TEM). The 3- and 20-month-old mice were perfused with paraformaldehyde 4% and then their brains were removed  and incubated for 45 min at 37 °C. After incubation, slices were washed three times for 5 min in PBS and mounted with fluorescent mounting media with DAPI .Hippocampal slice staining with mitochondrial fluorescent dyes. The brains of the 3- and 20-month-old mice were dissected and immediately frozen at −150 °C. The frozen brains were mounted using an optimal cutting temperature compound (OCT compound) in a cryostat at −22 °C; then, coronal 25 µm-thick slices of unfixed hippocampal tissue were obtained. Hippocampal slices were mounted on glass slides and incubated as previously described with mitochondrial fluorescent dyes ,96,120. 2 Consumption Assay kit . In this assay, the fluorescence obtained is proportional to O2 consumption generated by the mitochondria.Oxygen Consumption Assay. Oxygen consumption was measured in 50 ug of proteins from an enriched-mitochondrial fraction incubated with oxidative substrates for 30 min a 37 °C, using an Extracellular OMeasurement of ATP content and MMP. ATP concentration was measured in tissue lysates obtained with HEPES buffer  using a luciferin/luciferase bioluminescence assay kit  . The amog for 10 min at 4 °C. After this time, mitochondrial ROS production was measured in BioTek Synergy HT  [Measurement of ROS production. ROS production was measured using the fluorescent dye CM-H2DCFDA . Briefly, a fraction enriched in mitochondria (25 μg of protein) was isolated from the hippocampus and diluted in 100 μL of KCl respiration buffer with pyruvate and malate as oxidative substrates and the addition of 25 μM DCF. Then, it was incubated at 37 °C for 30 min and centrifuged at 8000×  530 nm) .2 was added, and the response was evaluated for 15 min. Next, we added 200 μM CaCl2 and measured it for 15 min [Measurement of the calcium response. The mitochondrial response to calcium overload was measured by absorbance to 540 nm (30 °C)  for 3 mir 15 min .2 was added. The fluorescence from cytosolic calcein is quenched by the cobalt; in contrast, in a condition of closed mPTP, mitochondrial fluorescence is maintained. However, when the mPTP is open, loss of green mitochondrial calcein fluorescence occurs [Mitochondrial permeability transition pore (mPTP) assay: Mitochondrial permeability transition pore (mPTP) openings were measured in a fraction enriched in mitochondria (50 μg of protein), which were loaded with the acetoxymethyl ester of calcein dye, calcein AM, that passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. In addition, CoCle occurs .Mitochondrial DNA measurement. The mitochondrial DNA was measured in a fraction enriched in mitochondria  incubated with Quant-iTPicoGreen dsDNA reagent. This reagent emits a fluorescent signal upon binding to nucleic acids .Hmox1, Gclc, Nfe2l2, Ppid, Slc25a5, Atp5o, Vdac1, Ppargc1a, Cycs, and Sod1 genes. The cDNA was quantified by qPCR using Brilliant II SYBR Green and Kapa SYBR fast . The qPCR analysis was performed in duplicate from a reverse-transcribed product using the Rotor-Gene Q . Expression changes were calculated following the 2-ΔΔCt method using cyclophilin-A (Cyc) as a normalization control. The primers used are listed in Gene expression by quantitative real-time PCR. Total RNA was isolated from the whole hippocampus. Samples were homogenized in Trizol according to the manufacturer’s instructions. Total RNA was precipitated and treated with a turbo DNase I unit  to eliminate DNA contamination. Five micrograms of total RNA were reverse transcribed using Super-Script IV reverse transcriptase kit  with random hexamers and Oligo d(T)18 to measure the relative expression of Image analysis. All slides were photographed and scanned under the same magnification, laser intensity, brightness, and gain. Images were processed using Fiji software (NIH Image), adjusting the fluorescence threshold intensity in every picture.t-test. p-values > 0.05 and ≤0.05 were regarded, respectively, as not statistically significant and as statistically significant. In the figures, p-values between 0.01 and 0.05 are marked with one asterisk, p-values between 0.001 and 0.01 with two asterisks, and p-values less than 0.001 are shown with three asterisks. All statistical analyses were performed using Prism software .Statistical analysis. The results are presented as bar graphs indicating the mean ± standard deviation. Statistical significance was determined using a This study is of great interest since it shows a great variety of alterations in the hippocampal mitochondria of female C57BL/6L mice at a certain age (20 mo) and allows us to conclude that, during aging, mitochondria lose their capacity to produce energy, regulate ROS production and oxidative stress, and to balance calcium levels. This, together with deficiencies in mitochondrial dynamics and quality control processes, triggers the accumulation of damaged mitochondria in the hippocampus, which could contribute significantly to synaptic and cognitive impairment observed at advanced age.


COPI assay results revealed the presence of targeted binding relation between PCSK6 and STAT1.This study was aimed at investigating the expression and role of proprotein convertase subtilisin/kexin type (PCSK6) in inflammatory bowel disease (IBD). DSS induced mouse colitis and mucosal barrier injury, down-regulation of TJ proteins, improvement of permeability, and increases of the proportions of Th1 and M1 macrophages. After PCSK6 knockdown, the colitis in KO mice was improved relative to WT mice, the TJ protein levels increased, and the proportions of Th1 and M1 macrophages decreased. STAT1 inhibitor treatment also inhibited chronic colitis in mice. As revealed by PCSK6 binds to STAT1 to promote STAT1 phosphorylation and regulate Th1 cell differentiation, thus promoting the M1 polarization of macrophages and aggravating colitis progression. PCSK6 is promising as the new target for the treatment of colitis. PCSK6 is a kind of calcium-dependent subtilase-like endonuclease with secretory property, which can be persistently secreted outside the cells . As a kiDetection of Th1 and Th2 cell proportions : The sepF4/80+CD11b+M1 cell proportion measurement: Briefly, cells were subjected to 20-min incubation using antibodies (10 μl), which includedFITC-CD11b and PE-F4/80 monoclonal antibodies . Results were displayed in %.° C, samples obtained were later subjected to 20-min centrifugation for detection in line with ELISA kit  protocols, and calculated by standard curve approach, with results being displayed in ng/ml.For detecting cytokines in tissues, PBS was added to wash gut tissues twice, followed by liquid nitrogen grinding till no granule could be observed. Later, samples were lysed with NP-40 lysate  for a 30-min period on ice, followed by collection of supernatant protein liquid. Cell experiments were conducted to measure cytokine contents within the medium. To be specific, the isolated medium was stored under -80-/- (KO) mice  were adapted to environment for one week and later utilized in this experiment. These animals were later classified as WT, KO, WT-DSS or KO-DSS group (n=10 each). For those of DSS group, they were fed with 2.5% DSS for constructing the chronic colitis mouse model. Specifically, 2.5% DSS was administered on days 1-5, 8-12, 15-19 and 22-26, while purified water was provided on rest days in each mouse.This work built the chronic colitis mouse model through using disuccinimide octanediate (DSS) . Female To explore the underlying mechanism, mice in WT group were further classified as Control, DSS, and DSS+Cirsilineol groups. Cirsilineol, the STAT1 inhibitor, was intraperitoneally injected at 0.5 mg/kg/Day to block the STAT1 signal. Remaining treatments were the same as aforementioned descriptions.Fluorescein isothiocyanate-dextran (FITC-D) was administered into each mouse via gavage, then its level was detected, and alterations of intestinal mucosal permeability was judged. Specifically, following mouse intervention, mice were fasted for water and food for a 4-h period before they were killed. Later, FITC-D  was given into each mouse through gavage, then FITC-D content in serum was detected, and a fluorescence spectrophotometer was utilized to analyze the sample fluorescence density.° C. Afterwards, HRP-labeled IgG secondary antibody  was further added for membrane incubation. Then, chemiluminescence was conducted to detect protein bands, while Image Pro-Plus 6.0 software was employed to analyze absorbance (OD), and GAPDH was an internal control. Results represented target protein-to-control protein OD ratio.Protein content was measured, then samples were subjected to electrophoresis for protein separation, followed by transfer on PVDF membrane. Thereafter, 5% defatted milk was added to block membranes for a 2-h period, prior to overnight incubation using TBST-diluted primary monoclonal antibodies  under 45 cells/well, and transient transfection was conducted according to the HiPerFect transfection reagent  instructions to obtain the PCSK6-Si Th0 cells.Th0 cells were transfected with PCSK6 overexpression plasmid  for constructing PCSK6-OV Th0 cells. Thereafter, siRNA was used to silence PCSK6 expression, and the transiently transfected siRNA concentration was 160 nmol/L. Th0 cells were seeded in 12-well plates at the density of 2×10All data were represented by (±s). Data in two groups were compared by two independent sample t-test, while those across multiple groups by one-way ANOVA. P<0.05 (two-sided) was indicative of significant difference.The data supporting the findings of this study are available from the corresponding author upon reasonable request.-/- knockdown KO mice were utilized for experiments. DSS induced the occurrence of mouse chronic colitis. According to dynamic detection of body weight, the body weights of WT and KO mice gradually increased, and there existed no significant difference between groups. By contrast, the body weights of KO-DSS and WT-DSS mice gradually decreased, but the decrease level in KO-DSS group significantly reduced in relative to WT-DSS group, and the difference was of statistical significance  and silenced (PCSK6-Si) in Th0 cells. After induction, the proportion of Th1 cells between Control group and NC group was not significantly different, that in PCSK6-OV group significantly increased, higher than that in Control group, whereas that in PCSK6-Si group was lower than that in Control group, and the difference was significant  lymphocytes are the important component of the human immune system, making an essential role in the immunity-mediated inflammatory diseases , 11. Prein vitro. As a result, PCSK6 overexpression promoted Th1 formation, and increased Th1 cell proportion, while PCSK6 silencing decreased Th1 cell proportion. Additionally, PCSK6 promoted STAT1 phosphorylation. As observed from previous studies, STAT1 is the key signal promoting Th1 formation [In-vitro experiments also suggested that, Cirsilineol also reduced Th1 formation, similar to the effect of siRNA-PCSK6.PCSK6 protein is a common homologous protein of PCSK9, both of which belong to the PCSKs protein family. PCSK6 is extensively expressed in numerous tissues, including neuroendocrine, liver, intestinal and the brain tissues . At presormation , 25, whiPCSK6 binds to STAT1 to promote the phosphorylation level of STAT1. In IBD, PCSK6 promotes Th1 cell formation via p-STAT1, thereby indirectly promoting the M1 polarization of macrophages. PCSK6 has an important effect on IBD, and PCSK6-STAT1-Th1 is promising as a new signal of IBD treatment, while the extensive role of PCSK6 in inflammatory disease deserves further investigation.


Semaphorins have recently been recognized as crucial modulators of immune responses. In the pathogenesis of COVID-19, the activation of immune responses is the key factor in the development of severe disease. This study aimed to determine the association of serum semaphorin concentrations with COVID-19 severity and outcomes. Serum semaphorin concentrations  were measured in 80 hospitalized adult patients with COVID-19 (moderate (n = 24), severe (n = 32), critical, (n = 24)) and 40 healthy controls. While SEMA3C, SEMA3F and SEMA7A serum concentrations were significantly higher in patients with COVID-19, SEMA3A was significantly lower. Furthermore, SEMA3A and SEMA3C decreased with COVID-19 severity, while SEMA3F and SEMA7A increased. SEMA4D showed no correlation with disease severity. Serum semaphorin levels show better predictive values than CRP, IL-6 and LDH for differentiating critical from moderate/severe COVID-19. SEMA3F and SEMA7A serum concentrations were associated with the time to recovery, requirement of invasive mechanical ventilation, development of pulmonary thrombosis and nosocomial infections, as well as with in-hospital mortality. In conclusion, we provide the first evidence that SEMA3A, SEMA3C, SEMA3F and SEMA7A can be considered as new biomarkers of COVID-19 severity. COVID-19 presents with a wide range of clinical manifestations, from asymptomatic infection to pneumonia with acute respiratory distress syndrome (ARDS) and multiple-organ failure ,3. UnderSemaphorins (SEMA) are a large family of secreted and membrane-bound signaling proteins expressed in most tissues, and divided into eight subclasses based on their Sema domain located on the N-terminal region . They exSemaphorins were initially discovered as axonal guidance molecules in the development of the nervous system . SubsequHowever, the role of semaphorins in the immunopathogenesis of infections remains to be elucidated. Studies on mouse models of sepsis showed increased concentrations of several semaphorins in serum and tissues, and a blockade of semaphorins or their receptors led to a reduction in tissue damage and better survival rates ,19. In cHere, we hypothesize that semaphorin concentrations, due to their key function in controlling immune responses, correlate with COVID-19 severity and outcomes.This study was part of a prospective, non-interventional cohort study that included consecutively hospitalized adult patients with COVID-19 at the University Hospital for Infectious Diseases Zagreb (UHID) in Croatia between April and December 2021 . At that time, the Delta (B.1.617.2 and AY lineages) SARS-CoV-2 variant predominated in Croatia (data were taken from the ECDC database on SARS-CoV-2 variants) ,23. EighAll included patients had bilateral pulmonary infiltrates on chest images. Patients who had a concomitant bacterial infection at the time of admission were excluded, as were those who began corticosteroid or antiviral medication prior to enrollment. Active malignant disease, pregnancy and immunosuppression  were other exclusion factors.Forty healthy, SARS-CoV-2-RNA-negative, age- and sex-matched healthcare workers were included as controls.All participants provided written informed consent. The study followed the Declaration of Helsinki’s ethical principles and was approved by the UHID Ethics Committee (code 01-673-4-2021).According to the National Institute of Health, COVID-19 severity was classified based on clinical symptoms, the oxygen level at admission and level of care (pandemic department or intensive care unit) . BrieflyAt admission, demographic and comorbidity data were collected, including the presence of cardiovascular disease (CVD), arterial hypertension, chronic pulmonary disease , chronic renal failure (CRF), diabetes mellitus, dyslipidemia, gastritis or gastroesophageal reflux disease (GERD) and chronic medications. All patients had their body mass index (BMI) measured.The following routine laboratory results were obtained at admission: C-reactive protein (CRP), procalcitonin (PCT), ferritin, white blood cell count (WBC), absolute neutrophil (ANC) and lymphocyte count (ALC), platelet count (Plt), bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), serum creatinine, gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH), fibrinogen and D-dimer.The disease severity scores on the admission of each patient were calculated for MEWS , SOFA 2, PSI 2727 and 4CThe patients were treated according to the standard of care at the time, which included anticoagulants, remdesivir, tocilizumab, baricitinib and dexamethasone, at the discretion of the supervising physician.Clinical monitoring, including oxygen requirements, invasive and non-invasive ventilation and complications, were assessed daily and collected in a standardized form.Semaphorins were quantified using standardized enzyme-linked immunosorbent assay (ELISA) , as suggested by the manufacturer.p-value of 0.05. Spearman’s rank correlation coefficient was used to examine correlations, which were then summarized in a correlation matrix. A receiver operating characteristic (ROC) analysis was used to compare the discriminatory performance of the laboratory variables under consideration. Time to hospital discharge or readiness for discharge stratified by biomarker levels was evaluated using the Kaplan–Meier method and hazard ratios (HR) with 95% confidence intervals (95% CI) and p-values were calculated by the log-rank test. Risk factors associated with critical COVID-19 were investigated using a univariate and subsequently multivariable logistic regression analysis. The strength of association was expressed as an odds ratio (OR) and its corresponding 95% CI. GraphPad Prism Software version 10  was used for statistical analyses.Clinical, laboratory and demographic data were analyzed and reported descriptively as frequencies and medians with interquartile ranges. To compare two groups, Fisher’s exact test and the Mann–Whitney U test were used. To compare three or more groups, the Kruskal–Wallis test with Dunn’s multiple comparisons test was used. All tests were two-tailed, with a statistically significant Eighty patients with COVID-19 ; median age 62, IQR 47–68 years) and 40 controls ; median age 59, IQR 41–67 years) were included.p = 0.0001) and a lower PaO2/FiO2 ratio  on admission.COVID-19 severity was categorized as moderate in 24 (30%), severe in 32 (40%), and critical in 24 (30%) patients. As shown in As illustrated in In the group of severe COVID-19, oxygen supplementation had a median of 9.5 L (IQR 4–15 L) and the duration of required oxygen supplementation was 6.5 days (IQR 4.7–9 days). In the critical group, all patients required HFNC , 12 (50%) of them received NIV during hospitalization (median duration of 2 days (IQR 1–6 days). Ten patients (41.67%) required invasive mechanical ventilation , 6 patients (25%) needed continuous renal replacement therapy (CRRT), and one patient was treated with veno-venous extracorporeal membrane oxygenation (vv-ECMO).Overall, 47 (58.75%) patients were treated with remdesivir, 76 (95%) with dexamethasone, 12 (15%) with tocilizumab and 4 (5%) with baricitinib.In 10 patients (12.5%), pulmonary thrombosis was diagnosed . Nosocomial infections were diagnosed in 16 patients (20%); 10 patients with critical, 5 with severe and 1 with mild COVID-19. Ten patients (12.5%) in our cohort died during hospitalization .p ≤ 0.0001), SEMA3F  and SEMA7A  concentrations compared to healthy controls (p ≤ 0.0001). There were no statistical differences in SEMA4D serum concentrations between groups .Concentrations of the serum semaphorins SEMA3A, SEMA3C, SEMA3F, SEMA4D and SEMA7A were detectable in all patients with COVID-19 . Patientcontrols . In contNext, ROC analysis was performed to determine cut-off values of serum semaphorin concentrations for differentiating patients with COVID-19 from healthy controls . All tesAs shown in As shown in Next, ROC analysis was performed to determine the cut-off value of serum semaphorin concentrations for differentiating patients with critical COVID-19 from patients with moderate/severe COVID-19 b. A cutoFinally, we performed a multivariable logistic regression analysis to identify factors associated with the development of critical COVID-19. After adjustment for potential cofounders, SEMA3F > 4.7 ng/mL , SEMA7A > 2.0 ng/mL , admission paO2/FiO2 < 150  and 4C mortality score > 9  were associated with the critical disease, while age, sex, comorbidities, CCI, obesity and laboratory parameters such as CRP and LDH were not associated with critical disease in our model .p = 0.0116), SEMA3F > 4.7 ng/mL  and SEMA7A > 2.0 ng/mL  appeared to be an efficient prognostic biomarker associated with time to recovery.We examined the impact of serum semaphorin concentrations on time to recovery, as defined by time to hospital discharge or readiness for discharge by day 28. In a survival analysis using Kaplan–Meier estimates, SEMA3A < 13 ng/mL  and SEMA7A  were significantly higher than in patients who did not require advanced respiratory support.Twelve patients required mechanical ventilation (including NIV and IMV) during hospitalization. In this group, SEMA3F , SEMA3F  and SEMA7A .Ten patients were diagnosed with pulmonary thrombosis, and these patients had significantly higher serum concentrations of SEMA3C  and SEMA7A .Next, we analyzed baseline serum semaphorin concentrations with the development of nosocomial infections as a complication of COVID-19. Patients who developed nosocomial infections had significantly higher levels of SEMA3F  and higher SEMA7A  serum concentrations.Overall, 10 patients in our cohort died during hospitalization. Non-survivors had significantly lower SEMA3A , AST , LDH  and CK . Serum SEMA3C positively correlated with SEMA4D , ANC , and negatively with CK . Serum SEMA3F positively correlated with SEMA7A , 4C mortality score , Hb , urea , LDH  and d-dimer , while negatively with ALP . Serum SEMA4D positively correlated with SEMA3C . Serum SEMA7A positively correlated with SEMA3F , 4C mortality score , CRP , procalcitonin , ANC , Plt , urea , LDH , Troponin T , fibrinogen , d-dimer , and negatively correlated with albumin level.We analyzed potential correlations among paired laboratory parameters, including semaphorin concentrations and clinical variables in patients with COVID-19, as presented in In this study, we provide the first evidence that COVID-19 patients have different semaphorin serum concentrations as compared to healthy controls. While SEMA3A was decreased, SEMA3C, SEMA3F and SEMA7A were increased in COVID-19. Furthermore, we showed an association of semaphorin levels with COVID-19 severity; SEMA3F and SEMA7A were higher in critical COVID-19, while SEMA3A and SEMA3C negatively correlated with COVID-19 severity and were lower in the critical group. Semaphorins showed equal or better accuracy in predicting disease severity than the widely used CRP, IL-6 or LDH.Firstly, we found decreased serum concentrations of SEMA3A that further decreased with COVID-19 severity. Class 3 semaphorins, specifically SEMA3A, have immunosuppressive and regulatory effects that include neutrophil migration, induce the shift of activated macrophages (M1) to the resolution phase phenotype (M2) and negatively control the T cell-mediated response predominantly via the activation of regulatory T cells (Tregs) . In patiSince the fine-tuned immune response is vital in determining the outcome of the SARS-CoV-2 infection , we can Next, we found that SEMA3C is increased in moderate, but not in critical, COVID-19. Furthermore, SEMA3C was increased in a subgroup of patients diagnosed with pulmonary thrombosis. Less is known about the immunoregulatory role of SEMA3C, but it was shown that SEMA3C regulates fibrosis, vascular development, pathological angiogenesis and the migration of tumor cells. The expression of SEMA3C is related to tumor progression and poor prognosis in lung cancer, prostate, breast cancer, gastric cancer and ovarian cancer, which makes it a potential therapeutic target for malignant diseases ,34. SEMASimilarly, SEMA3F and SEMA7A concentrations were increased and positively correlated with COVID-19 disease severity and were identified as predictors of COVID-19 outcomes, including the need for mechanical ventilation, development of pulmonary thrombosis and nosocomial infections. Both SEMA3F and SEMA7A are secreted by activated immune cells and have emerged as regulators of neutrophil migration, vascular permeability and cytoskeletal remodeling, and the initiation of an inflammatory signaling cascade in models of acute lung injury. An increased number of neutrophils in bronchoalveolar fluid with a high expression of SEMA3F and NRP2 was observed in a murine model after the LPS challenge, and neutrophil-specific loss of SEMA3F resulted in more rapid neutrophil recruitment and clearance from the lungs . By inteIn our study, there were no differences in SEMA4D serum concentrations between healthy controls and COVID-19 patients. SEMA4D regulates immune activation and inflammatory responses by modulating cytoskeleton reorganization through its principal receptor, CD72, located on T cells, B cells, macrophages and dendritic cells . In animRecent research has shown that micro RNAs (miRNA) control SEMA signaling in immune, cardiovascular and nervous systems, and malignancies, directly through their receptors, or indirectly by modulating the molecules that regulate the expression of semaphorins . SimilarThis study should be viewed within its limitations; since this was an observational study, causality could not be determined; a relatively small number of participants in COVID-19 severity subgroups limits statistical analysis and should be confirmed in a larger population; the impact of comorbidities on semaphorins and other inflammatory markers was not evaluated; the concentrations of semaphorins were determined at a single time point, and dynamic variations related to clinical outcomes were not examined. Nevertheless, we studied a well-defined cohort of patients and report the first data examining the semaphorins’ profile in patients with COVID-19. Additional studies are needed for a better understanding of the complex underlying immunopathological mechanisms including their effect on development and activation of B and T cells, and how semaphorins contribute to COVID-19 progression.In conclusion, we have shown that patients with COVID-19 have different expressions of SEMA3A, SEMA3C, SEMA3F and SEMA7A than healthy controls, which correlate with disease severity and outcomes. Due to their role in regulating inflammation, cell migration, fibrosis and angiogenesis, which have already been explored in neoplastic and autoimmune diseases, semaphorins could be new diagnostic and prognostic biomarkers and potential therapeutic targets in COVID-19, which warrant further investigation.