Schistosoma mansoni 14 kDa fatty acid-binding protein (Sm14) + glucopyranosyl lipid A in squalene emulsion (GLA-SE). Shown here are the results of Phase Ib, an open, non-placebo-controlled, standardized-dose immunization trial involving 10 healthy 18–49-year-old women. Fifty micrograms of the Sm14 protein plus 10 µg GLA-SE per dose was given intramuscularly thrice at 30-day intervals. Participants were assessed clinically, biochemically, and immunologically for up to 120 days. In preambular experiments involving vaccinated pregnant female rabbits, we did not find any toxicological features in either the offspring or mothers, and the vaccine induced adaptive immunity in the animals. In women, no adverse events were observed, and vaccination induced high titers of anti-Sm14 serum IgG antibody production. Vaccination also elicited robust cytokine responses, with increased TNFα, IFNγ, and IL-2 profiles in all vaccinees on days 90 and 120. The completion of Phase I clinical trials, which were performed to the highest standards set by Good Clinical Research Practice (GCP) standards, and preclinical data in pregnant rabbits enabled the vaccine candidate to proceed to Phase II clinical trials in endemic areas.We report the successful closure of Phase I clinical trials, comprising Phases Ia and Ib, of the vaccine candidate against human schistosomiasis: the  Schistosomiasis is a parasitic disease that affects populations living in areas with very poor sanitary conditions, where they are infected through contact with contaminated water during their daily work, domestic, or leisure activities. Children are the main target of the infection, which strongly compromises their physical and cognitive development ,2. This Presently, the so-called Mass Drug Administration (MDA) program against schistosomiasis is the strategy adopted by WHO, in which populations in endemic areas are treated annually with Praziquantel without a previous diagnosis. This strategy actually led to an improvement with regard to the pathology associated with schistosomiasis . HoweverThe Sm14 vaccine candidate was developed at the Oswaldo Cruz Foundation  as a humanitarian vaccine to be used in populations living in areas at risk of infection ,8,9.S. mansoni, as described elsewhere [The Sm14 recombinant protein is currently the basis for an antiparasitic molecular vaccine associated with GLA-SE adjuvant and was developed as an anthelmintic bivalent vaccine directed against fasciolosis and schistosomiasis. Sm14 is a member of the fatty acid-binding protein family, originally isolated from lsewhere ,11,12, alsewhere ,16,17.S. mansoni and a significant reduction in the adult worm burdens of vaccinated/infected animals, evidenced by the mean values of adult worm loads and additional measures of the distribution of the frequencies of sequential worm burden rates in Swiss-Webster mice [During the experimental phase of Sm14 vaccine development, focused on the vaccination of outbred animals, it was possible to demonstrate consistent protection against challenge infection with the cercariae of ter mice ,19,20,21ter mice ,23.The major objective of the Sm14 vaccine project is to promote health access through a safe and immunogenic vaccine capable of blocking the transmission of the disease. This will prevent populations from needing to receive repeated doses of chemotherapeutic drugs during childhood and youth. The design of the vaccine foresees its large-scale use, combined with the other known control measures, such as chemotherapy with Praziquantel, sanitation, health education, etc., capable of promoting the control of the disease and mitigating the suffering historically produced by this disease in tropical countries.The Brazilian National Sanitary Surveillance Agency (Anvisa) approved the start of the Clinical Protocol for Phase Ia Study (CE# 990768/10-8) in a non-endemic area of Rio de Janeiro, Brazil, with 20 healthy male volunteers, which was completed with success and validated. The validation was performed with the use of a Data Bank containing all records of the study for calculations and detailed and descriptive statistical analyses of the results . In ordeThe step related to the process of vaccination in Phase Ib  was conducted by the Evandro Chagas National Institute of Infectology (INI/Fiocruz) using the same batches of Sm14+GLA-SE used in Phase Ia.The immunogenicity induced by the Sm14+GLA-SE vaccine in women was also approached by means of a platform made available by the Access to Advanced Health Institute (AAHI) (previously Infectious Disease Research Institute IDRI), Seattle, USA, where sera and cells collected at different points of the study from volunteers were shipped. The steps needed to test the safety and immunogenicity of the vaccine and the validation of the study were completed and are described in the present work.For the studies carried out in rabbits as well as the clinical trial, the Sm14 vaccine used in the study was produced at the Ludwig Institute for Cancer Research (LICR) and Cornell University, NY, USA, under good manufacturing practice conditions (cGMP). Hermetically closed vials with individual doses (0.55 mL), labeled with the product name, Sm14, lot number (PBR-0057-002), date of manufacture, and “only for investigational use”, were shipped to Brazil under import authorization from the National Council for Scientific and Technological Development (CNPq) and Anvisa. Vials were kept at 2–8 °C under continuous temperature control from the origin at Fiocruz (Rio de Janeiro) until use, with regular inspections to ensure that contents remained liquid and transparent without suspended particles.The Infectious Diseases Research Institute/Access to Advanced Health Institute (IDRI/AAHI) provided the glucopyranosyl lipid-A adjuvant in squalene emulsion (GLA-SE) in vials containing 0.4 mL at a concentration of 40 µg/mL, also produced under GMP conditions and labeled with the lot number (Part # 0037-09M001) and manufacturing date. Shipping and storage followed the same procedures as described above.The batches of Sm14 and adjuvant were filled/finished as single doses and mixed at the time of immunization.To formulate the vaccine antigen dose at the time of administration, 0.4 mL of a 200 µg/mL Sm14 suspension was aspirated using a 1 mL sterile syringe. Subsequently, the volume of the syringe was injected into the vial containing the GLA-SE adjuvant, obtaining a mixture with a total volume of 0.8 mL. After mixing by inverting the vial, 0.5 mL doses containing 50 µg of Sm14 protein and 10 µg of adjuvant were administered by the intramuscular route to both animals and women volunteers. The dose used in these studies was based on a clinical protocol approved by Anvisa for the Phase Ia study in male volunteers . The twoThe study in pregnant rabbits was conducted in accordance with the protocol provided by Anvisa. It was reviewed and approved by the Ethics Committee on Animal Research of Fiocruz (CEUA/Fiocruz) (n° L0063/08—LW 19/2013).Reproductive toxicity tests were performed with the same vaccine lot further used in Phase I clinical trials, which was produced under current good manufacturing practice (cGMP) conditions by LICR/Cornell University, NY, USA. Vials containing individual doses  were labeled with the product name (Sm14) and lot number (PBR-0057-002). Vials were kept at +2–8 °C under continuous temperature control until use, with regular inspections to ensure that the contents remained liquid and transparent without suspended particles.Twenty-four NZW pregnant rabbits, with the same age and weight at the beginning of the experiment, were divided into two groups just after mating: one group comprised twelve rabbits immunized with three equal doses of 50 µg of Sm14 + 10 µg GLA-SE/dose, with a 7-day interval between doses, and the other was the control group, comprising twelve rabbits intramuscularly injected with PBS on the same days. Injections of 0.5 mL/dose for each dose were at the external middle third of the right and left thighs alternately and administered on days 0 (day 9 of pregnancy), 7 (day 16 of pregnancy), and 14 (day 23 of pregnancy). The pregnancy period of these animals is approximately 30 days. Necropsy and anatomopathological evaluations were performed on day 29 of pregnancy.All animals had the inoculation area shaved and marked before each immunizing dose to further assess possible local reactions, which can include erythema and edema, both being scored according to standard protocols . The daiBiochemical and blood cell evaluations were performed following current laboratory practices and included liver, renal, and pancreatic functions, as well as red and white blood cell counts and coagulation parameters.All animals were anesthetized with thiopental sodium and euthanized by exsanguination. Detailed anatomopathological analyses of all organs were carried out following euthanasia. All animals were subjected to a macroscopic inspection of the organs and cavities. Samples of organs were collected in 10% buffered formalin for later microscopic analysis. The ovaries, uteri, and fetuses were collected and fixed for further analysis, such as number, size, weight, corpora lutea, placenta, and living fetuses. Paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope .Serum samples were collected from all rabbits before the start of pregnancy and on the date of euthanasia. The total anti-Sm14 IgG antibody titers in rabbit serum samples were determined by ELISA  in accorp-values below 0.05 considered to be significant.All statistical analyses were carried out by using GraphPad Prism software version 4.0. All analyses were conducted by applying the two-tailed test, with The methodology used to conduct the Sm14+GLA-SE candidate vaccine Phase Ib clinical trial in women was essentially the same as described by Santini-Oliveira et al.  for PhasTwenty healthy female volunteers from a non-endemic area of Brazil (City of Rio de Janeiro) were enrolled and registered with numbers from #1 to #20. Among them, 10 were selected and proceeded to the end of the study. The inclusion criteria comprised a negative pregnancy test, availability during the whole duration of the trial, no evidence of any acute infection, no previous or ongoing serious chronic disease, and normal findings for organ functions, as demonstrated by laboratory tests and the physical examination of the cardio-respiratory and brain/neurological systems, abdominal organs, skin, and lymphatic system. Exclusion criteria included pregnancy/breastfeeding, abnormal hematology or blood chemistry counts or data, any kind of ongoing infection, including infection with the human immunodeficiency virus (HIV), any chronic disease, alcohol/drug abuse, or a history of medication or vaccination in the preceding month. After clinical screening, 10 participants aged 26–48 years  were selected for the trial, and their demographic characteristics are depicted in Full physical exams and laboratory tests were performed at baseline and on days 0, 7, 30, 37, 60, 67, 90, and 120 . ParticiTM Cryo 1 °C freezing container  overnight. On the following day, vials were transferred to liquid nitrogen and later dry-shipped to the Infectious Disease Research Institute , where they were stored in liquid nitrogen until use.Samples of peripheral blood mononuclear cells (PBMCs) and of sera were collected from all subjects at baseline and on days 0, 30, 60, 90, and 120. Immediately after collection, PBMCs were separated by centrifugation at room temperature in Ficoll-Paque Plus  and mixed with Fetal Bovine Serum (FBS)  and 10% DMSO. Cells were subsequently transferred to 2 mL cryovials and frozen at −80 °C in a NALGENE ®MAP, Merck, Burlington, MA, USA) [Sera were also stored in Brazil at −80 °C and transferred on dry ice to IDRI for analysis. At IDRI, sera were stored at −80 °C until experiments were performed. The immunogenicity analyses included checking for isotypes of anti-Sm14 antibodies and cell-mediated responses specifically induced after vaccination with Sm14+GLA-SE. It also included the detection and quantification of intracellular cytokines after the stimulation of PBMCs with purified rSm14. Immune cell responses were measured using the PBMC Luminex Kit for T cell cytokines  . IsotypiMA, USA) ,27.Descriptive analysis using the IBM SPSS v20 statistical package was performed for all variables. Boxplots for all follow-ups assessed whether observations were outside the limits specified for specific laboratory tests. The proportions of adverse events and 95% confidence intervals were calculated using the Clopper–Pearson method . For toxThe statistical analysis was performed based on the evaluation of local and systemic reactogenicity observed in the first 36 h after each dose of the vaccine. The results of chemical and hematological exams were compared to reference values in accordance with the Richet Laboratory (LR) criteria and ICTDR, including levels of toxicity as defined by the FDA .The body weights and structures of the analyzed organs of all pregnant rabbits, irrespective of being vaccinated or not, were within normal limits, according to established criteria . Macrosct-test to compare the mean values of groups. In all cases, differences were considered statistically significant at p-values of <0.05. No differences were found.Data are presented as the mean ± standard deviation. The parameters were evaluated by Student’s The skin and subcutaneous tissue were removed from the injection sites of the immunized animals. The results of the individual tests showed no significant histopathological alterations in the skin at the immunization site. In the anatomopathological evaluation of the organs, it was observed that all groups, including the control group, had small and scarce foci of calcification in 5% of the cortical and medullary tubules of the kidneys.Lastly, ELISA was used to analyze sera collected from the animals after the final injection and showed considerably high production of the anti-Sm14 antibody in the sera of vaccinated versus non-vaccinated PBS-treated pregnant rabbits. Anti-Sm14 antibody levels  were clearly high in the vaccinated group as compared to controls .The certification of Sm14+GLA-SE vaccine safety in the above preclinical study paved the way for a Phase Ib clinical trial of the vaccine in young adult non-pregnant women. The candidate vaccine Sm14+GLA-SE was shown to be safe, with no serious adverse events observed in the participants after any of the three doses. No local reactions at the site of the injection occurred, except for local pain, which affected 9 out of 10 women after the first dose, 5 out of 9 after the second, and 4 out of 10 after the third . Only one subject reported involuntary muscle contractions lasting 5 min near the site of injection after the first dose. With regard to systemic adverse events, two participants had a fever at D7, and two others had light local hyperemia and a headache at D37, probably related to the vaccine. Physical exams on all days  showed no clinical alterations or abnormalities. Laboratory exams did not show alterations that could be classified as toxicity according to the ICTDR criteria, except for ALT and GGT, with few participants showing elevated values within toxicity levels. However, these participants also had elevated values prior to the vaccination (at baseline), and since they had no other abnormal laboratory findings or clinical symptoms, they were considered to be most likely not related to the vaccine. All other single instances of values outside of the normal range were evaluated, and given that no abnormal laboratory findings or clinical symptoms were observed, they were all considered non-clinically significant . These data show that the vaccine produced very few adverse effects, and all observed events were local and mild .1 isotype. Responses were augmented after the second dose. Ninety percent of vaccinated subjects developed higher total specific IgG antibody titers 90 days after vaccination, and these titers remained just as high up to day 120. Compared to the baseline, total specific IgG, as well as IgG1 and IgG3, increases were statistically significant from day 60 .The results obtained in women confirmed the pattern of responses observed in Phase Ia with male adults . Our immPreclinical assays on pregnant rabbits revealed that the candidate anti-schistosomiasis rSm14-GLA-SE vaccine resulted in no changes concerning the reproductive status or other pregnancy parameters. Moreover, the Phase Ib clinical trial conducted among healthy young adult women confirmed that no serious adverse events were observed after three doses of 50 µg of Sm14 + 10 µg GLA-SE, administered three times at 30-day intervals, in 10 women from a schistosomiasis non-endemic area.Based on data generated from the clinical studies completed so far, severe toxicity (grade 3 or 4), either local or systemic, is not expected to occur. The administration of the investigational product is anticipated to be well tolerated. So, local reactions  may occur but are usually mild and self-limiting and subside without treatment.S. mansoni infection. It may possibly be useful against infections caused by other species of Schistosoma.As observed in this trial, adaptive cell-mediated and humoral immune responses , along with many previous preclinical results, strongly support Sm14 as an effective vaccine against S. mansoni and S. haemathobium. It is believed that the development of a safe, effective, and humanitarian vaccine will change the epidemiological profile of this disease, thus freeing countries such as Brazil and much of the African continent from one of the major stigmas of underdevelopment, namely, parasitic diseases, particularly schistosomiasis.Clinical studies are a prerequisite for licensing drugs and immunobiologicals by regulatory agencies in most countries. In this way, they are a constituent part of the process of research and development of products for human health. The Sm14+GLA-SE vaccine is at an advanced stage of development, after successfully passing through preclinical stages and clinical Phases Ia and Ib, and is presently in a Phase II clinical trial in the Senegal River Delta region in African sites that are endemic for both


Inflammatory bowel disease (IBD) places a heavy medical burden on countries and families due to repeated and prolonged attacks, and the incidence and prevalence of IBD are increasing worldwide. Therefore, finding an effective treatment is a matter of great urgency. Glycerol monolaurate (GML), which has a twelve-carbon chain, is a compound naturally found in human breast milk. Some studies have shown that GML has antibacterial and anti-inflammatory effects. However, the specific mechanism of action remains unclear.1) was administered for two weeks. QPCR and western blotting were performed to examine the inflammatory status. Mice described were subjected to flow cytometry analysis for immune cell activation.Acute colitis was established in mice using 3% DSS, and glycerol monolaurate  and increased the expression of anti-inflammatory factors (IL-10 and TGF-β). GML inhibited the activation of the MAPK and NF-κB signalling pathways, improved tissue damage, and increased the expression of intestinal tight junction proteins. In addition, LPMCs extracted from intestinal tissue via flow cytometry showed that GML treatment led to a decrease of Th17 cells, Neutrophils and Macrophages. 16S rDNA sequencing showed that GML increased the abundance of commensal bacterium such as Akkermansia and Lactobacillus murinus.We showed that oral administration of GML ameliorated DSS-induced colitis by inhibiting infiltration of Th17 cells, Neutrophils, and Macrophages, protecting the intestinal mucosal barrier and altered the abundance of commensal bacterium. This study provides new insights into the biological function and therapeutic potential of GML in the treatment of IBD. Inflammatory bowel disease (IBD) is an autoimmune disorder that includes Crohn's disease and ulcerative colitis. The clinical manifestations of IBD cause suffering in patients and include abdominal pain, diarrhoea and mucous-containing bloody stools . In addiDue to the restricted safety and reliability of current clinical drugs for long-term use in the treatment of patients with IBD, it is necessary to find a drug that not only alleviates the disease status but also has fewer side effects . GlyceroEscherichia coli . Our results indicated that GML could ameliorates DSS-induced acute colitis by inhibiting infiltration of Th17, neutrophils, macrophages and altering the gut microbiota.n = 10), DSS group (n = 20) and DSS+GML group (n = 20). All animal experiments were approved by the Animal Research Committee of Xiamen University.Male specific pathogen-free (SPF) C57BL/6 mice  were purchased from Xiamen University Laboratory Animal Center  and raised in the SPF Animal Room of Xiamen University Laboratory Animal Center. The animals were kept at a temperature of 20–24°C and humidity of 50 ± 5%. All animal experiments were conducted in accordance with the regulations of Xiamen University Laboratory Animal Center. All mice were adapted for a week before starting the study. Fifty mice were randomly divided into three groups: the Con group . Mice in the Con group received sterile water at the same dose. In addition, all mice were weighed daily (at 15:00). After the model was completed, the mice were killed, samples were collected, intestinal tissue was collected for length measurement, spleens were weighed, and mouse sera and faeces were collected for subsequent analysis.Studies have shown that higher GML treatment (450 mg kg-1) ameliorates HFD-induced metabolic disorders, supported by prevented visceral fat deposition, improved hyperlipidemia, modulated hepatic lipid metabolism, and reduced serum proinflammatory cytokine, TNF-α. Additionally, all doses of GML attenuated circulating lipopolysaccharide load and insulin resistance . So we cAfter the mice were sacrificed, the colon length was measured, and the distal colon was washed with phosphate-buffered saline , rolled into swiss curly hair with a toothpick, and embedded in paraffin. The samples were cut into 6-μm-thick sections and stained with haematoxylin-eosin (HE) . Finally, the colon morphology was observed by using light microscopy (Motic VM1). The colonic tissue was scored according to the pathological scoring criteria .After the mice were sacrificed, total mRNA from intestinal tissue was extracted from the same part of the colon with TRIzol. Then, cDNA was synthesized using the Reverse Transcription Kit (with dsDNase) . Quantitative PCR was performed using SYBR Green qPCR Mix . The level of mRNA was normalized to the GAPDH expression level, and the results were analysed using the 2–ΔΔCt method. The primers were designed and synthesized by Sangon Biotechnology (Shanghai) Co., Ltd .The levels of the cytokines TGF-β and IL-1β were measured in serum using enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturer's instructions, and the absorbance at a wavelength of 450 nm was measured using a microplate analyser.Colon tissues were added to RIPA  lysate containing 1% protease and 1% phosphatase inhibitors . Protease and phosphatase inhibit degradation of sample proteins. For Western Blot analysis, 20 μg of protein of colon tissues were separated by SDS-polyacrylamide gel electrophoresis . The concentration of primary antibody was determined to be 1:2000. Then, the samples were ground into homogenate using a tissue grinder. The homogenate was centrifuged at 14,000 rpm and 4°C for 15 min to collect the supernatant. Protein concentration was measured using the BCA Protein Assay Kit  according to the manufacturer's instructions. The samples (20 μg/lane) were placed in a 4–15% gradient gel and electrophoresed at a constant voltage to isolate proteins, which were transferred to nitrocellulose membranes. Then, the membranes were blocked with 5% skim milk and incubated overnight with the primary antibody at 4°C, followed by incubation with an HRP-conjugated secondary antibody. Finally, the protein bands were detected using an ECL test kit . Primary antibodies against ERK1/2 (# 9102S), P-ERK1/2 (#4370), NF-κB (# 8242S p65), P-NF-κB , and GAPDH (# 5174S), were purchased from Cell Signaling Technology.RRID: AB 2757321) and Occludin (RRID: AB 2764486)  were used.Immunochemistry (IHC) was performed using the OCT-embedded sample. OCT sections were frozen at −20°C in OCT and cryostat sectioned (20 μm). Non-specific staining blockers were avoided with the IHC background blocker. The concentration of primary antibody was determined to be 1:2000. The samples were cut into 6-μm-thick slices and then mounted on glass slides coated with poly(L-lysine) and dried. First, endogenous peroxidase was blocked, and nonspecific staining blockers were added. Then, primary antibody was added, and the sample was incubated overnight at 4°C. A biotin-labelled secondary antibody was added the next day, and then, a Streptomyces anti-biotin protein  was added after incubation at room temperature for approximately 10 min. Finally, DAB chromogenic solution  was used for colour development, which was visualized using a bioptic biological microscope (Motic VM1). Primary antibodies against ZO-1  and mixed in the appropriate proportion, and then, 50 μL each of serum and PE Detection Reagent were added, and the mixture was incubated at room temperature away from light for 3 h. Due to the low concentration of inflammatory factors in the serum, the serum is not diluted. The mixture was washed and centrifuged to discard the supernatant. Finally, the microspheres were resuspended in 300 μL of washing solution and then detected by flow cytometry . Microsphere calibration of the instrument was required before using the machine. Finally, the results were analysed with FCAP Array software.Spleen and colorectal tissue were collected from the mice. The spleen was ground with the flat bottom of a syringe piston to homogenize the splenocytes. RBCs were lysed by using the 10X RBC lysing solution . The supernatant was discarded, and the red blood cell lysate was resuspended to dissolve red blood cells. The spleen tissue was carefully ground and filtered through a cellular filter with a diameter of 100 μm, and a single-cell suspension was prepared. Red blood cells were removed with erythrocyte lysate and then stained with Fixable Stain 510  and blocked with Fc-block for approximately 30 min, followed by staining of CD45 , CD11B , LY-6G . Finally, the samples were detected by FCM. The intestinal tissue of the mice was washed with PBS, cut into 1 cm fragments and placed in predigestion solution, which was configured using D-Hanks solution, including ethylenediaminetetraacetic acid (EDTA 1 mg/mL) and dithiothreitol (DTT 0.15 mg/mL), for approximately 20 min. After predigestion, the intestinal tissue was cut up and added to digestive juices, containing collagenase and DNase, at 37°C for 1 h and 30 min. Then, the digested tissue was filtered and centrifuged to obtain a single-cell suspension, which was passed through Percoll for collecting LPMCs. Then, the cells were stained with F4/80, Fixable Stain 510  and blocked with Fc-block for approximately 30 min. Next, CD45 , CD3 , CD4  were stained. Finally, the cells were stimulated with PMA, ionomycin, and brefeldin and then stained for IL-17  and detected by FCM. The concentration of antibody and Fc-block  was determined to be 1:100. 500,000 cells were collected using flow cytometry . Percoll , collagenase (1:200) and DNAse (1:200) were purchased from . 500000 cells were stimulated for intracellular cytokine staining. The stimulants (1:100) and brefeldin (1:100) were purchased from BD Bioscience, N.J. Cells were stimulated for 6 h. After washing and centrifugation, a membrane-breaking fixative solution (BD Biosciences) was used according to the manufacturer's instructions. Cells were fixed 15 min for intracellular cytokine staining.https://github.com/najoshi/sickle). The reference library used was the Curated MicroSEQ(R) 16S Reference Library v2013.1; sequencing data was analyzed using the QIIME 1.8.0 software package . Further analysis of 16S sequencing was done on R statistical computing platform with the use of the Vegan package (https://cran.r-project.org/web/packages/vegan/vegan.pdf).DNA was extracted from the faeces of mice using HiPure Soil DNA Kit B . The DNA concentration was measured using the Equalbit dsDNA HS Assay Kit. Then, PCR amplification and library construction were carried out using 20–330 ng of DNA as a template. In addition, PCR primers were used to amplify two highly variable regions, namely, the V3 and V4 regions , on the t-test or one-way analysis of variance (ANOVA), followed by the Tukey test for multiple comparisons, was performed to determine significance. A P-value < 0.05 was considered significant.The data are expressed as the mean ± standard error of the mean. GraphPad Prism 8.0 software  was used for visualization and analysis. Student's Experimental colitis induced by DSS is a classic model for the study of the pathogenesis of IBD and the development of treatment techniques and drugs for IBD. The experimental design is shown in the To further study the protective effect of GML on DSS-induced colitis, colon tissues of mice were collected and sliced for HE staining, as shown in figure . The conCytokines play an important role in the occurrence and development of intestinal inflammation, so to further research the effect of GML on colitis, we extracted total RNA from intestinal tissue and performed RT-qPCR. The fold change indicates the change of mRNA according to real-time qPCR. As shown in Both the NF-κB and MAPK pathways are important inflammatory signalling pathways. We further studied the effect of GML on p65, ERK and their phosphorylation levels by western blotting in the colon tissue. The p-p65 and p-MAPK levels of the DSS group were increased compared with those of the control group, but the p-p65 and p-MAPK levels of the GML group were significantly lower than those of the DSS group , indicatLymphocytes of the laminar propria of the colon were isolated and stained with Fixable Stain 510 and Fc-block. Then, the cells were stained for the cell markers CD45, CD3, CD4+, Ly-6G, CD11b, and IL-17A to study the effects of GML treatment on Th17, CD4+, neutrophils, and macrophages. Gating strategy for flow cytometric are shown in The 16S rDNA phylogenetic method was used to evaluate the colonic microbiomes of differently treated mice (97% similarity). The α diversity of the microbiome is indicated by Simpson's index . PCA basIn addition, the results of the Metastats analysis showed that Intestinimonas, Lachnospiraceae_NK4A136_group, Mucispirillum, Ruminiclostridium_9, and Oscillibacter  were significantly enriched after DSS treatment, while they were significantly depleted by the application of GML. Compared with their abundances in the DSS group, Akkermansia, Lactobacillus, Eubacterium, Coprostanoligenes_group, and Parabacteroides, which can regulate glucose and lipid metabolism, were significantly enriched after GML treatment . IntestiIBD is recognized as one of the challenging modern medical diseases. The recurrence of and prolonged failure to recover from can be physically and mentally distressing for patients, seriously affecting their quality of life and placing a heavy burden on the economy . MoreoveTo date, a colitis model induced by DSS has been widely used to study colitis and IBD. The changes in BW, colon length and spleen weight in mice are important indices for evaluating the DSS-induced colitis model. In this study, mice treated with DSS showed significant loss of BW, shortened colon length, and increased spleen weight, which indicated the successful establishment of a colitis model , 45. HowCytokines play an important role in the regulation of colitis –48. It hImpaired integrity of the intestinal barrier, which plays a key role in regulating intestinal permeability and the pathogenesis of colitis, is another cause of intestinal inflammation –51. It hIn addition, Th1, Th17 and Treg cells play an indispensable role in the occurrence and development of colitis. Proinflammatory factors secreted by Th1 and Th17 cells can induce the immune system to attack harmful pathogens and aggravate IBD to some extent –54. The Both the NF-κB and MAPK pathways, which play important regulatory roles in activating the production of inflammatory cytokines, are important inflammatory signalling pathways –57. The In recent years, with the development of high-throughput sequencing of microflora, the intestinal microecology has attracted increasing attention. Some researchers believe that the development of IBD is closely related to the intestinal microecology , 58. FurIn conclusion, these results indicate that oral administration of GML could slow down the occurrence of DSS-induced colitis by regulating the immune balance, protecting the intestinal mucosal barrier, and improving the intestinal microecology. This study provides new insights into the biological function and therapeutic potential of GML in the treatment of IBD.The data presented in the study are deposited in the NCBI SRA BioProject repository, accession number PRJNA865147.The animal study was reviewed and approved by Animal Research Committee of Xiamen University.K-JH performed the study and drafted the manuscript. Y-NH, J-HD, and X-MO conducted RNA extraction and qRT-PCR assays. YLin, GG, and X-SC carried out Western blot assays. K-JH, YLi, and JL developed animal model and performed experiments, and K-JH analyzed the data. BG and J-LR revised the manuscript and supervised the whole experiment. All authors have read and approved the final submitted manuscript.This study was supported by the National Natural Science Foundation of China (Nos. 81970460 and 81770548) in China, and the Key Laboratory of Intestinal Microbiome and Human Health in Xiamen, China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


COBLL1 gene is associated with leptin, a hormone important for appetite and weight maintenance. Dietary fat is a significant factor in obesity. This study aimed to determine the association between COBLL1 gene, dietary fat, and incidence of obesity. Data from the Korean Genome and Epidemiology Study were used, and 3055 Korean adults aged ≥ 40 years were included. Obesity was defined as a body mass index ≥ 25 kg/m2. Patients with obesity at baseline were excluded. The effects of the COBLL1 rs6717858 genotypes and dietary fat on incidence of obesity were evaluated using multivariable Cox proportional hazard models. During an average follow-up period of 9.2 years, 627 obesity cases were documented. In men, the hazard ratio (HR) for obesity was higher in CT, CC carriers  in the highest tertile of dietary fat intake than for men with TT carriers in the lowest tertile of dietary fat intake . In women, the HR for obesity was higher in TT carriers in the highest tertile of dietary fat intake than for women with TT carriers in the lowest tertile of dietary fat intake . COBLL1 genetic variants and dietary fat intake had different sex-dependent effects in obesity. These results imply that a low-fat diet may protect against the effects of COBLL1 genetic variants on future obesity risk.The  The prevalence of obesity is steadily increasing worldwide . ObesityDietary fat contributes to obesity risk . In the Not all types of dietary fat adversely affect leptin secretion and sensitivity . A high-Inconsistent findings regarding the contribution of dietary fat in obesity exist ,22. A 12COBLL1 (cordon-bleu WH2 repeat protein-like 1) gene is associated with neural tube formation [COBLL1 rs6717858 is associated with plasma leptin levels secreted from adipose tissue and obesity-related factors, such as body mass index (BMI), central obesity, waist circumference (WC), fat mass, and lipid levels (prominent effect in women) [COBLL1 expression is associated with adipose tissue dysfunction [COBLL1/GRB14 (rs6738627) is strongly associated with body fat ratio, and the COBLL1 gene is upregulated by a HF diet [COBLL1 expression affects obesity and dyslipidemia risk by increasing lipid storage in adipose tissue [ormation , centralormation , fastingormation , type 2 ormation , blood lormation , CVD risormation , gastricormation , prostatormation , and leuormation ,32. COBLn women) ,33,34,35function ,37. Genefunction ,39. The  HF diet . Decrease tissue .COBLL1 gene and dietary fat intake in obesity [COBLL1 gene in obesity. Therefore, this study aimed to examine the interaction between COBLL1 genetic variants and dietary fat in obesity using urban–rural prospective cohort data.Several studies have examined the individual effects of the  obesity ,33,34,35n = 312; non-obese men, n = 1228; obese women, n = 315; non-obese women, n = 1200). Regardless of sex, the obese group had higher BMI and WC than those of the non-obese group . Among men, the obese group had lower mean age, metabolic equivalent of task (MET), resident in Ausung, high-density lipoprotein (HDL) cholesterol level, and carbohydrate intake and higher household income, alcohol consumption, TG, total energy, protein, and fat intake than those of the non-obese group . Among women, the obese group had higher average diastolic blood pressure than that of the non-obese group (p < 0.05).We documented 627 cases of obesity over an average period of 9.2 years. COBLL1 rs6717858 genotypes (TT vs. CT or CC) . HDL-cholesterol was higher in men with the CT or CC genotype than in men with the TT genotype . TG was lower in men with the CT or CC genotype than in men with the TT genotype . The mean age was higher in women with the CT or CC genotype than in women with the TT genotype . In women with the CT or CC genotype, average systolic blood pressure and diastolic blood pressure were higher than those in women with the TT genotype . In women with the TT genotype, fat intake was higher than that in women with the CT or CC genotype .p = 0.89 in Model 1; beta ± SE: −0.015 ± 0.009, p = 0.12 in Model 2). In women, there was no significant difference between dietary fat and BMI .In men, the highest fat intake was associated with a 41% higher incidence of obesity than that with the lowest fat intake in Model 1 . No significant difference in obesity was observed in Model 2 . In women, no significant association with obesity was observed in tertile 3 (compared to that observed in tertile 1) .We further investigated the association between dietary fat intake and obesity . The parCOBLL1 rs6717858 genotypes and incidence of obesity. In men, there was no significant difference in obesity based on the CT or CC genotype . In women, there was no significant association between the incidence of obesity and CT or CC genotype . After adjusting for age, sex, and area, there was no significant difference between COBLL1 rs6717858 genotypes and BMI in the additive model . Women in tertile 3 of dietary fat with the TT genotype had a 49% higher incidence of obesity than that of women in tertile 1 with the TT genotype . In Model 2, men in tertile 3 of dietary fat with the CT or CC genotype had a 63% higher incidence of obesity than that of men in tertile 1 with the TT genotype . Women in tertile 3 of dietary fat with the TT genotype had a 53% higher incidence of obesity than that of women in tertile 1 with the TT genotype . COBLL1 rs6717858 genotypes and incidence of obesity, stratified by dietary fat intake . Women with the CT or CC genotype had a 64% higher incidence of obesity than that of women with TT genotype in the <15% group . In Model 2, women with the TT genotype had a 34% higher incidence of obesity than that of women in the <15% group . Women with the CT or CC genotype had a 64% higher incidence of obesity than that of women with the TT genotype in the <15% group .We further investigated the association between COBLL1 genetic variants and dietary fat intake in obese patients. After adjusting for covariates, men in the highest tertile of dietary fat intake with the CT or CC genotype had 63% increased incidence of obesity compared to those men in the lowest tertile of dietary fat intake with the TT genotype. In women, those in the highest tertile of dietary fat intake with the TT genotype had 53% increased incidence of obesity compared to those in the lowest tertile of dietary fat intake with the TT genotype. Using large-scale prospective cohort data from Ansan and Ansung, we identified an interaction between COBLL1 rs6717858 genotypes and the incidence of obesity based on sex was not significant. The COBLL1 gene is associated with abdominal obesity, BMI, fat mass, WC, waist-to-hip ratio, and blood lipids [GRB14/COBLL1 rs6717858  and BMI, weight, and BMI-adjusted WC [The association between the d lipids ,27,34,35d lipids . The curusted WC . In addiusted WC , in contMen in the highest tertile of dietary fat had a 41% higher incidence of obesity compared to men in the lowest tertile of dietary fat. In women, there was no significant difference in obesity regardless of the adjusted covariates. The association between dietary fat and obesity was stronger in men because the rate of lipoprotein metabolism for dietary fat is lowered owing to the androgen-inhibitory effect ,46,47,48Fat intake also increases the risk of obesity by altering several hormones, such as serotonin and leptin ,45. DietCOBLL1 rs6717858 genotypes and sex. Fat intake in men with the CT or CC genotype and women with the TT genotype was positively associated with obesity, independent of the adjusted covariates. Fat intake in tertile 2 was positively associated with obesity in women with the CT or CC genotype, independent of the adjusted covariates. Among the women with the CT or CC genotype, there were 60 cases in total . The small number of women may have a significant effect on the lack of statistical tests.Our findings showed that the influence of fat intake on the incidence of obesity differs according to the COBLL1 rs6717858 genotypes and sex. There were no significant differences in obesity among the men. In women with the TT genotype, a fat intake of ≥ 15% increased the incidence of obesity. In women with the CT or CC genotype, a fat intake of < 15% increased the incidence of obesity. Fat intake has a different effect on obesity, depending on sex and genetic variants. Some studies have indicated that fat intake can prevent obesity [To improve public health and prevent chronic diseases, the Korean Nutrition Society recommends an appropriate fat intake of 15–30% in Korean adults . In term obesity ,51. In a obesity . In anotCOBLL1 gene expression contributes to the influence of a HF diet in obesity [COBLL1 gene expression [COBLL1 gene expression in adipocytes in 4-month-old mice [Controlling leptin levels by  obesity ,52,53,54pression . In a geold mice .COBLL1 rs6717858 genotypes differed according to sex, suggesting an effect on sex hormones. The major T allele of COBLL1 rs6717858 is associated with abdominal obesity, body fat, WC, and blood lipid accumulation, especially in women [In this study, the incidence of obesity for the in women ,27,34,35in women . Menopauin women . Sex horin women . As thisin women ,57.COBLL1 rs6717858 genotypes and dietary fat in obesity based on sex in a follow-up study. Second, the causal association between obesity and gene–nutrient factor was clear because of the prospective cohort study design. Third, several covariates that may interfere with obtaining accurate results were adjusted for. Fourth, the effects of basic covariates  on incidence of obesity, and the effects of these factors after adjusting for dietary variables, were taken into account.This study had certain strengths. First, to our knowledge, it was the first to investigate the interaction between This study also had some limitations. First, the number of women with the CT or CC genotype  was small, which may have affected the statistical power. Second, because this study was conducted among Koreans, the findings cannot be generalized to other races/ethnicities. Third, obesity is common regardless of age; however, this study focused on middle-aged adults. Fourth, because only dietary data at baseline were used during analysis, dietary changes over time should be considered.Based on these limitations, future research directions must consider three perspectives. First, because different types of dietary fat have different effects in obesity, the influence of dietary fat type on the incidence of obesity requires further investigation. Second, because obesity occurs regardless of age, its incidence should be studied across all age groups, not only among middle-aged individuals. Third, the ratio of various nutrients to total energy intake will determine the influence of dietary fat in obesity.COBLL1 in obesity among middle-aged Korean adults. Dietary fat is positively associated with obesity in men. An important finding was that dietary fat was positively associated with obesity in men with the CT or CC genotype independent of the adjusted covariates. Dietary fat intake in women with the TT genotype was positively associated with obesity independent of the adjusted covariates. These results suggest that fat intake in obesity differs according to sex and genetic variants. Our results highlight the importance of environmental factors considering individual genotypes and could help to reduce obesity using basic scientific data for personalized nutrition.In conclusion, we present new information on the interaction between dietary fat and This study used data from the Korean Genome and Epidemiology Study (KoGES) and included a total of 3055 Korean adults (1540 men and 1515 women). To identify potential risks for common diseases, KoGES, a large population-based prospective cohort study, has recruited participants since 2001 and conducted follow-up surveys on occurrence of new diseases and lifestyle changes. Participants were followed up every 2 years to determine the association between genetic and environmental factors. This study included KoGES data from baseline (2001–2002) to follow-up (2003–2014). The study protocol was examined and approved by the Institutional Review Board (IRB) of Inha University on 31 January 2020 (IRB No. 200129–1A).n = 1193), no information of area, sex, age, BMI, alcohol consumption, smoking status, metabolic equivalent of task (MET) (n = 2358), total energy, carbohydrate, protein, fat, or fiber intake (n = 128), obesity or observation period (n = 3296). Finally, this study included 3055 participants (1540 men and 1515 women)  data (5 women) .COBLL1 rs6717858 genotypes and the incidence of obesity was classified into two groups: <15% and ≥15%. Few study participants consumed >30% fat (9 men and 14 women); therefore, groups with ≥15% fat intake were combined.A semi-quantitative food-frequency questionnaire (SQ-FFQ) was used to obtain dietary information at baseline. The SQ-FFQ was used to evaluate the amount and frequency of food consumption by Koreans aged 40–69 years in the past year. The SQ-FFQ excluded those with a total energy intake of <100 kcal or >10,000 kcal of energy per day. Intake of fatty foods was calculated by multiplying the nutrient content of each food unit. Dietary fat intake  was assessed as fat (g/day)/total energy  × 1000. The ratio of dietary fat intake  was calculated as fat (g/day) × 9/total energy  × 100 and classified into tertiles according to dietary fat . According to the Korean Nutrition Society in 2020, the recommended range of dietary fat intake (% energy) was 15–30% for Koreans . The ass2. Height was measured with the participants standing on a horizontal surface while wearing light clothing, with the heel, buttocks, back, and head bordered by a vertical plate and the head facing forward. The measured height was read to one decimal place in centimeters. Body weight was measured with the participants wearing minimal clothing and standing on a flat floor. The measured weight was read to the nearest 10 g. Obesity was assessed as BMI ≥ 25 kg/m2 using World Health Organization standards for Asians [The BMI for diagnosing obesity was calculated as kg/mr Asians . In thisCOBLL1 gene (rs6717858) because it has previously been linked to blood lipid accumulation, plasma leptin levels, central obesity, body fat, BMI, and WC [p ≥ 1.0 × 10−6 based on standard quality control procedures [Korean genome data were obtained using the genome-wide human SNP Array 5.0 from Affymetrix, which is commonly used in many studies on the genomes of various diseases ,60,61. W, and WC ,33,34,35ocedures .COBLL1 rs6717858  was conducted using PLINK (version 1.90 beta). Dietary fat intake was divided according to sex into tertiles, medians, and continuous types. The association between dietary fat  and BMI was also analyzed. The association between COBLL1 rs6717858 genotypes and BMI was evaluated using linear regression analysis and presented as beta and SE. The general characteristics of obesity (obese vs. non-obese) and COBLL1 rs6717858 genotypes (TT vs. CT or CC) were compared using t-tests for continuous variables and chi-squared tests for categorical variables. The HRs and 95% CIs for the interactions between COBLL1 rs6717858 genotypes and dietary fat intake in obesity were calculated using multivariable Cox proportional hazard models. Model 1 included the following variables: age, area, education level, income level, alcohol consumption, smoking status, MET, and BMI. Model 2 further included the following variables: total energy and dietary fiber. All statistical analyses were performed using SAS software . The significance level was set at p < 0.05.Genetic analysis of


Solea senegalensis, Kaup 1858), a marine flatfish species, is of commercial interest for both fisheries and aquaculture. In aquaculture, currently there are several production bottlenecks, mainly in larvae culture and rearing. The hox genes participate in cell differentiation and structuring of the anterior–posterior axis during embryonic development. In this work, using cytogenetic and genomic techniques, hox genes were isolated and sequenced to study the sequence and cluster organization, together with the cytogenetic localization. Results were analyzed from an evolutionary perspective by comparing with other species. Our findings should represent a useful starting point for further research in order to gain more knowledge of the development of Senegalese sole for planning functional analyses focused on the main concerns that characterize the development phase.The Senegalese sole , a marine flatfish, belongs to the Pleuronectiformes order. It is a commercially important species for fisheries and aquaculture. However, in aquaculture, several production bottlenecks have still to be resolved, including skeletal deformities and high mortality during the larval and juvenile phase. The study aims to characterize the hox gene clusters in S. senegalensis to understand better the developmental and metamorphosis process in this species. Using a BAC library, the clones that contain hox genes were isolated, sequenced by NGS and used as BAC-FISH probes. Subsequently the hox clusters were studied by sequence analysis, comparative genomics, and cytogenetic and phylogenetic analysis. Cytogenetic analysis demonstrated the localization of four BAC clones on chromosome pairs 4, 12, 13, and 16 of the Senegalese sole cytogenomic map. Comparative and phylogenetic analysis showed a highly conserved organization in each cluster and different phylogenetic clustering in each hox cluster. Analysis of structural and repetitive sequences revealed accumulations of polymorphisms mediated by repetitive elements in the hoxba cluster, mainly retroelements. Therefore, a possible loss of the hoxb7a gene can be established in the Pleuronectiformes lineage. This work allows the organization and regulation of hox clusters to be understood, and is a good base for further studies of expression patterns.The Senegalese sole ( Solea senegalensis, Kaup 1858) is a marine flatfish belonging to the Pleuronectiformes order and Soleidae family. It is a species of major interest in fisheries and aquaculture, mainly in the Iberian Peninsula, and is well accepted by markets throughout Europe [The Senegalese sole , but within teleosts, these become 8 clusters , which have likely arisen from a third round of genome duplication specific for the teleost lineage, called “TSGD” or 3R [hox genes, they can also be classified into four groups of paralogous genes: the anterior group , group 3 , the central group  and the posterior group  [Based on their sequence similarity, the D” or 3R . Based oes 9–13) .hox gene in fishes are scarce and are based on the genetic and genomic characterization of the hox gene clusters [hox gene clusters in S. senegalensis based on the structural arrangement and content of repetitive elements. The objective is to reveal molecular and evolutionary clues that would enable us to track possible differentiating structural markers in Pleuronectiformes. These, in turn, would allow further specific expression analyses of the larval development, which could be of interest for future breeding programs, to be carried out. In addition, phylogenetic and evolutionary research should enable interesting hox genes for such expression analysis to be found.Studies of the clusters ,39, exprclusters ,41,42, aclusters . Therefohoxab, hoxbb, hoxca, hoxda gene clusters were isolated from a BAC library of Solea senegalensis by PCR-4D methodology [hoxbb cluster, the primers were designed using available sequences in the public database SoleaDB .The hodology  using sp2, 1.5 U of NZYTaq II DNA polymerase enzyme, 0.4 mM of dNTP mix, plus 5 µL of 10× Reaction Buffer, and sterile H2O to complete. For each reaction, a negative and a positive control are included. All reagents are from Nzytech . Genomic DNA was extracted from F1 specimens of the Central Service of Marine Culture Research (SC-ICM) of the University of Cádiz.The total volume (25 µL) of PCR mix contained 50–100 ng of DNA template (BAC-DNA or genomic DNA), 4 moles µM of each primer, 4.0 mM of MgClThe PCR reaction was performed on the SimpliAmp™ thermal cycler from Applied Biosystems . The amplification cycling consisted of an initial denaturation at 95 °C for 3 min and 35 cycles of denaturation at 95 °C for 45 s, annealing at the corresponding temperatures of each primer pair  for 45 sOnce the positive clones were detected, BAC-DNA was extracted and purified to be sequenced and used as a cytogenetic probe, using the Large Construct and Plasmid Mini kits’ protocol from Qiagen , respectively. Once extracted, clones were quantified by spectrophotometry using the NanoDrop™ 2000/2000c . In addition, the BAC clone was verified by PCR using the protocol previously described.Escherichia coli str. K-12 substr. MG1655 and pCC1, using the program NGLess v1.0.0-Linux64 [BAC-DNA was sequenced by the NovaSeq 6000 method of the Illumina sequencing platform  in combination with 251 nts Paired End. Subsequently, a quality filtering was performed with the BBMap v38.36 program , proceed-Linux64 . To perf-Linux64  was used-Linux64  was usedhox clusters from three Pleuronectiformes species, S. senegalensis, Scophthalmus maximus, and Cynoglossus semilaevis as well as two other distant non-Pleuronectiformes fishes, Sparus aurata (Sparidae) and Oreochromis niloticus (Cichlidae), was carried out. The genome sequences of the five species were downloaded from the National Center for Biotechnology Information (NCBI) database: S. senegalensis (assembly GCA_019176455.1 IFAPA_SoseM_1); S. maximus (assembly ASM2237912v1); C. semilaevis (assembly Cse_v1.0); S. aurata (assembly fSpaAur1.1); and O. niloticus (assembly O_niloticus_UMD_NMBU). The hox clusters were then located and extracted from their reference genomes.An analysis of repetitive element abundance in hox cluster were the number of loci per Mb (NL/Mb) and their coverage, measured as a percentage of length occupied by repeated elements per cluster analyzed (%) [hoxba cluster, a sliding window of 3 kb was applied using TE annotations from RepeatMasker results. Different types of repetitive elements were recorded, such as simple repeats, DNA transposons, LINEs, SINEs, LTRs, and low-complexity sequences. Alignments of the S. senegalensis hoxba cluster with those of the other species were carried out with the default parameters of LASTZ software [Homolog-based searches against the database CONS-Dfam_3.4 using RepeatMasker (v4.1.2) with RepBase repeat library  and the yzed (%) ,15. To ssoftware .hox clusters were carried out using ENSEMBL and GenBank-NCBI databases in order to describe the organization within each hox cluster. One gene sequence from each hox cluster of S. senegalensis was extracted from the NCBI genome database and used for the BLAST searching of the hox cluster in another twelve representative species. These species were Cynoglossus semilaevis (tongue sole), Scophthalmus maximus (turbot), Paralichthys olivaceus (Japanese flounder), Hippoglossus hippoglossus , Seriola dumerili (greater amberjack), Seriola lalandi (yellowtail amberjack), Echeneis naucrates (live sharksucker), Oreochromis niloticus (Nile tilapia), Gasterosteus aculeatus (stickleback), Sparus aurata (gilthead seabream), Danio rerio (zebrafish), and Lepisosteus oculatus (spotted gar). The order and direction of each gene were annotated to make a schematic representation of the cluster microsynteny.Cross-species genome comparisons of the S. senegalensis larvae of 1–3 DPH, which were treated with 0.02% colchicine for 3 h, placed in a hypotonic solution of 0.4% KCL and fixed in modified Carnoy’s solution (absolute ethanol: acetic acid (3:1)). The cellular homogenizate was splashed onto slides previously heated to 50 °C and left until the Carnoy’s fixing was complete. Finally, the preparations were dehydrated in an increasing ethanol series of 70, 90, and 100%.Chromosome preparations were obtained from BAC probes were labeled with digoxigenin and/or biotin using DIG-Nick translation Mix or BIO-Nick translation Mix  following the manufacturer’s protocols. Hybridization and post-hybridization treatments were performed according to . Finallyhttps://bit.ly/2tPVbhY, accessed on 1 July 2022) and the Guidelines of the European Union Council (86/609/EU). The experiment was authorized by the Ethics Committee of the University of Cadiz .The experimental procedures were in accordance with the recommendation of the University of Cadiz  for the use of laboratory animals , thirteen Actinopterygii teleost species, one Actinopterygii non-teleost species, one Sarcopterygii species, and three Chondrichthyes species. Sequence concatenation in each cluster was done using DAMBE6 software and aligned with the MAFFT program following an iterative “genafpair” method of 1000 iterations. The PhyML 3.0 program [A phylogenetic tree was constructed for each  program  followin program  and usin program  was used program . Finally program .hoxab, hoxbb, hoxca, and hoxda clusters   , where t species .S. senegalensis hoxba cluster against sequences of the other species  to be localized in the acrocentric pair number 16, close to the centromere. Marker BAC 38B21 helps to localize BAC 39L15 (hoxbb cluster) in an internal position of the acrocentric pair 12. Marker BAC 4M14 showed that the localization of BAC 51K20 (hoxca cluster) is in the acrocentric chromosome pair 13, close to the centromere. Finally, marker BAC 36J2 allowed BAC 29F20 (hoxda cluster) to be localized in a subcentromeric position of the q arm of submetacentric pair 4.The four BAC clones isolated were observed in different chromosomes pairs . The BACribed by  , pseudogenes (in S. aurata), and absences of specific genes  appears before the Holostei species (Lepisosteus oculatus)  . Howeverculatus) .S. senegalensis [S. senegalensis were isolated from 11 BAC clones, showing a non-chromosome-specific localization [hox genes were not included in such study. The participation of the hox genes in regulation of the metamorphosis has been demonstrated in other organisms, such as lampreys [hox clusters present in S. senegalensis have been characterized. These hox clusters were also found in different chromosomes, thus also showing a non-chromosome-specific localization, as can be observed by FISH and genomic data in which all seven clusters are localized in different chromosome pairs. This is a general rule observed in other teleost species, as can be observed in the comparative genomic analysis. The localization of each hox cluster in different chromosomes is a clear evidence of the teleost-specific genome duplication (TSGD) event, which occurred approximately 350 Ma and has been cited to explain the long speciation process of teleosts [The recent advances achieved in the structural genomics of galensis ,17,59 allization , but hoxlampreys . In thisteleosts .hox clusters with the morphological complexity and development plan along the body axis [S. senegalensis has 49 hox genes, a number similar to that of other teleost species, including C. semilaevis (48), E. naucartes (49), O. niloticus (48), S. aurata (49), and D. rerio (48), among others, all organized in seven clusters. These numbers are similar to that of L. oculatus (43), a non-teleost species that did not undergo the TSGD event, so there is not a correlation with the number of paralogues and morphological complexity. Hence, the hox duplication from the TSDG event is not associated with an increase in the morphological complexity of the individuals but with a greater probability for the teleost species to diversify morphologically [Controversies have arisen concerning the association of the number of ody axis ,63,64. Sogically .hox gene losses since its divergence from teleosts [hox genes observed in teleost species are also found in the spotted gar, but there are some other hox genes only present in this species . Two of these (hoxd8 and hoxd1) have been found in other super-orders of the Teleostei infraclass, so it is probable that these two genes have been lost after the radiation of the three main teleost subdivisions: Elopomorpha, Osteoglossomorpha, and Clupeocephala (this last included in this study). However, hoxa6 and hoxd2 could have been lost before the TSGD event or before the radiation of the extant teleost linages [hox clusters in teleost with those corresponding homologue clusters in spotted gar, it can observed that one of the teleost duplicates conserves almost all hox genes that are present in the spotted gar cluster, and the other teleost duplicate has many hox gene losses. This observation suggests that one of the duplicated clusters has preferentially undergone gene losses that the other one has not. In contrast to other teleosts, D. rerio has lost the hoxdb cluster but has conserved the hoxcb cluster, which is a common characteristic among Cypriniformes [The spotted gar lineage has experienced no teleosts . Such stteleosts . All hox linages . Compariniformes .hoxba cluster shows that an Elopomorpha teleost species such as A. anguilla is not clustered within the remaining Clupeocephala species: instead it appears before the holostean L. oculatus, thus indicating that few variations in the gene sequences of this cluster have occurred since the divergence of the evolutionary lineages of these two species. Similarly, the phylogenetic results of the hoxbb, hoxca, hoxda, and hoxdb clusters showed a different grouping of the teleost clade, also indicating the high similarity among teleost sequences in each cluster. In contrast, the hoxaa and hoxab genes accumulate enough changes to differentiate clearly each clade and subclade. This result is in accordance with that obtained by [hoxa2a and hoxa2b phylogeny. Moreover, in a phylogenetic analysis carried out with different teleost species, hoxba–hoxbb, hoxca–hoxcb and hoxda–hoxdb clusters failed to separate a and b cluster duplicates but not with hoxaa and hoxab clusters [hoxa paralogues except from those of the hoxb, hoxc, and hoxd paralogue clusters, which makes the classification based on the coding sequences of these last clusters inaccurate.The phylogenetic analysis of the ained by  based onclusters , thus inS. senegalensis, it presents all the hox genes common to all teleost species included in the study, so no hox losses have occurred within the Senegalese sole lineage since the divergence of the Clupeocephala super-order. However, some polymorphisms have been detected in some hox clusters in other species of Pleuronectiformes. These polymorphisms were observed, above all, in P. olivaceus, in which there was an absence of hoxa2b, hoxb7a, and hoxc3a. However, the polymorphisms detected in P. olivaceus should be treated with caution due to the poor quality in some sequence stretches. Polymorphisms have also been detected in other Pleuronectiformes species, mainly in the hoxba cluster, the most variable cluster. Surprisingly, all of these polymorphisms were located in the 5′ half of the cluster, and they were an inversion of eve1 and hoxb13a in C. semilaevis and hoxb7a losses in C. semilaevis, H. hippoglossus, and P. olivaceus. Moreover, polymorphisms in this 5′ half region have also been observed in other non-Pleuronectiformes species, such the eve1 pseudogenization in S. aurata and hoxb8a loss in G. aculeatus. The hoxb7a loss seems to be specific to some Pleuronectiformes lineages, since it has not been observed in any other species included in the study. Hence, it could be a hox gene that is not strictly necessary to establish the body plan of Pleuronectiformes. However, the loss of hoxb7a has also been described in Tetraodon nigroviridis, Oryzias latipes, Oryzias melastigma, Takifugu rubripes, Kryptolebias marmoratus, or Xiphophorus maculatus [hoxb7a losses have occurred recurrently in particular lineages during the teleost evolution [Concerning aculatus ,68,69,70volution . This imhoxba cluster. In contrast to protostomes, repetitive elements are often completely excluded from hox clusters in chordates [hox gene clusters are strongly refractory to invasion by repetitive elements [Xenopus, chicken, zebrafish, lizard, and human hox gene clusters revealed an abundance of repetitive elements, with a larger abundance in lizard hox clusters than in other species [hox clusters is lower than whole genome coverage values [hoxba cluster, where we have reported, as in the lizard species, an increased accumulation of interspersed repeats comprised mainly retrotransposons [The analysis of repetitive sequences produced striking results in the hordates ,73, and elements ,75,76. H species . In some species . Among v species . Our rese values ,15,17 exnsposons .hoxba cluster observed in these fish species is also interesting because, while vertebrate hoxb clusters are relatively homogeneous in size  [hoxba clusters analyzed in this work were found to be substantially larger, by a factor of 2.5. This larger size has been described before in lizards of genus Anolis due to the accumulation of repetitive elements, primarily retrotransposons, which are genetic elements typically excluded from these genomic loci [Gasterosteus aculeatus and Astatotilapia burtoni show a large intergenic region (63 kb) containing repetitive elements between hoxb13a and hoxb9a [hoxb7a gene has been lost independently several times during teleost evolution. The repetitive element profile analysis in Pleuronectiformes demonstrates that an accumulation of repetitive elements exists around the hoxb7a region, so these elements could be responsible for the losses observed for this gene in Pleuronectiformes. Overexpression of hoxb7a in zebrafishes has been associated with deformation of the body axis [hoxb7a mRNA [hoxb7a gene is the only member of paralogous group 7, and so the absence of hoxb7a cannot be compensated for by other members of the same paralogue group [The size of the  100 kb) , all hoxmic loci ,78. In ad hoxb9a . The anaody axis  and the ody axis ; this exb7a mRNA . Howeverue group .hox gene clusters may have provided an ideal substrate for the evolution of phenotypic novelties. Proliferation of TEs may have contributed to genomic incompatibilities that enabled formation of different species during evolution [hox genes have been correlated with speciation rates [hoxba clusters in the different fish species reported in this study remains unknown, and their contribution to hox gene cluster rearrangements in fish needs further analysis.TEs are known to make different functional contributions in genomes that include their roles in generating mutations, in determining genome size and rearrangements, in the regulation of gene expression, and in altering chromosome structure. In recent years, TEs have been recognized as a natural source of regulatory sequences for host genes ,83. Becavolution . In addion rates ,78. The hox clusters are revealed by this study. Among them is the preferential accumulation of polymorphisms in the 5′ region of the hoxba cluster, which is associated with a larger accumulation of repetitive elements; this could play a regulatory role in the cluster. In Pleuronectiformes, hoxb7a was lost in some species but not in S. senegalensis. All these findings have allowed us to achieve the main objective of finding differentiating structural markers in Pleuronectiformes. In turn, further directed analyses of gene expression will be focused on these structural markers, with a view to determining the role of the hoxb7a gene in the development of the body plan of Pleuronectiformes.Various genetic and evolutionary characteristics of the Given that studies of this kind are scarce in flatfish, the results obtained in this work represent a useful starting point for further analysis aimed at clarifying the expression pattern of these clusters in a spatial–temporal framework as well as the role played by repetitive elements in expression regulation.


By contrast, being married (p < 0.001), divorced or widowed (p < 0.001), and/or retired (p < 0.001) were each associated with a higher ARABRISK score. A large representative study is needed to calculate the risk of T2DM among Saudi nationals.Type 2 diabetes mellitus (T2DM) is a chronic disease with ever-increasing prevalence worldwide. In our study, we evaluated the prevalence of the risk of developing T2DM in Saudi Arabia and investigated associations between that risk and various sociodemographic characteristics. To those ends, a web-based cross-sectional survey of Saudi nationals without diabetes, all enrolled using snowball sampling, was conducted from January 2021 to January 2022. The risk of developing T2DM was evaluated using a validated risk assessment questionnaire (ARABRISK), and associations of high ARABRISK scores and sociodemographic variables were explored in multivariable logistic regression modeling. Of the 4559 participants, 88.1% were 18 to 39 years old, and 67.2% held a college or university degree. High ARABRISK scores were observed in 7.5% of the sample. Residing in a midsize city versus a large city was associated with a lower ARABRISK risk score ( According to the International Diabetes Federation, in 2021, approximately 537 million people worldwide lived with diabetes, a number that is expected to reach 700 million by 2045 [The likelihood of developing type 2 diabetes mellitus (T2DM) depends on a combination of modifiable and non-modifiable risk factors, as follows: first-degree family history of T2DM, high-risk race or ethnicity, overweight or obesity, sedentary lifestyle, being at least 35 years old, history of impaired glucose tolerance, impaired fasting glucose, elevated hemoglobin A1C, hypertension, dyslipidemia, history of cardiovascular disease, polycystic ovary syndrome, and other conditions associated with insulin resistance .Many risk scoring models for T2DM require the findings of blood tests which, due to necessitating a clinic visit or diagnostic testing, complicates the wide use of those models from a public health perspective . A methoThe systematic increase in the prevalence of diabetes, together with the high rate of undiagnosed diabetes and prediabetes , indicatAgainst that background, the objective of our study was twofold: to measure the prevalence of the risk of developing T2DM among adults in Saudi Arabia using a validated risk scoring questionnaire  and to evaluate associations between the risk and various sociodemographic variables. Notably, unlike in previous studies, we recruited participants using online snowballing via social media and included all 13 regions of Saudi Arabia.In our cross-sectional survey-based study, conducted in Saudi Arabia between January 2021 and January 2022, we recruited participants using snowballing sampling. The target population was invited to participate by accessing a web link to the survey via ads on social media platforms. To be included in the study, participants had to be between 18 and 74 years old at the time of the survey, be a Saudi national, and be able to read Arabic fluently. All participants diagnosed with diabetes at the time of the survey were excluded from the sample.In our survey, we used the Arabic version of CANRISK , a validThe primary objective of our study was to measure the prevalence of the risk of developing T2DM among adults in Saudi Arabia. By extension, the secondary objectives were to analyze the most common risk factors of T2DM in Saudi Arabia, to compare the prevalence of individuals at risk for T2DM between differently sized cities , and to explore the associations between various sociodemographic variables and an increased risk of developing T2DM.We defined a large city as a city with a population of more than 1,000,000, which in Saudi Arabia includes Riyadh, Jeddah, Dammam, Makkah, and Madinah. By contrast, midsize cities had from 300,000 to 1,000,000 people , whereas small ones had fewer than 300,000 people  . All othAmong other definitions, the risk categories were defined according to the original CANRISK questionnaire , such thAssuming a 20% frequency of individuals at risk of diabetes and the population of Saudi Arabia  based on the General Authority for Statistics , we calc2 for continuous data and Kruskal–Wallis for categorical data. Predictors of high ARABRISK scores were assessed with univariable and multivariable logistic regression models built with sociodemographic variables that were not part of the ARABRISK calculated score—that is, city size, type of insurance, marital status, and employment status. The findings of logistic regression were presented as odds ratios (ORs) with 95% confidence intervals (CIs). Any p-value of less than 0.05 was considered to indicate a statistically significant difference between groups of variables included in the logistic regression models.Data were analyzed using R software . Descriptive statistics were used for frequencies and percentages of the answers, as well as for the medians and interquartile ranges (IQRs) of total scores. Percentages of the survey categories were compared between the groups of differently sized cities, and the significance of the differences between the groups in relation to the survey categories and ARABRISK total scores were tested with one-way analysis of variance (ANOVA) or χFor the geospatial visualization of our results, we used the R-based package rgeolocate that interfaces with MaxMind GeoLite2 IP databases . Using tn = 2269) of the participants lived in a large city, whereas only 139 (3.0%) lived in a small one. The median calculated ARABRISK score for the whole sample was 16 (IQR = 10 to 25). While 92.5% of the sample had a low to moderate risk of developing T2DM , 7.5% had a high risk . The baseline characteristics of the sample and ARABRISK total scores according to groups of differently sized cities are presented in Of the 5663 participants who completed the online questionnaire, 4559 participants were included in the analysis, while the rest were excluded for having been diagnosed with diabetes and/or not being Saudi nationals . Approxin = 3414), only 4.4% of whom had given birth to a baby weighing 4 kg or more. Approximately half of the participants  had a body mass index (BMI) of less than 25 kg/m2, while 7.8% had a BMI of more than or equal to 35 kg/m2. Exercising was reported by 42.1%, while 28.9% reported eating fruits and vegetables on a daily basis. Added to those characteristics, 11.6% of the participants had hypertension, 7.2% had a history of high blood glucose readings, and more than 80% had a family history of diabetes. Last, by level of education, participants were categorized as having completed junior high school or less (2.3%) or having earned a high school degree (30.5%), a college degree (10.3%), or a university degree (56.9%).By age, participants ranged from 18 to 74 years old, and 88.1% were between 18 and 39. By sex, three-quarters of the participants were female , as was living in a small city  or rural area . However, in the multivariable model, only midsize cities maintained the statistical significance . Predictors of the occurrence of high ARABRISK scores were assessed with logistic regression modeling . The unip < 0.001), divorced or widowed , or retired , all of which maintained their statistical significance in the multivariable model. By contrast, the same model revealed that lower ARABRISK scores were associated with having private insurance  and being unemployed .Other variables significantly associated with high ARABRISK scores in the univariable model were being married  and the prevalence of prediabetes and T2DM ,27, it iBeyond that, our analysis revealed that retirement was associated with an increased likelihood of a high ARABRISK score; that outcome can be expected, given that retired individuals tend to be older, which is a known risk factor for T2DM . Last, an = 1187). Second, because CANRISK was translated into Arabic and validated by Alghwiri et al. and showed high reliability and validity in Saudi and Jordanian populations [Although our results benefit from our study’s large sample size, our findings have several limitations. First, the original CANRISK was validated for adults aged 40–74 years old but can be used among younger adults, according to the Canadian Task Force on Preventive Health Care’s recommendations for screening for T2DM in adults . On thatFifth, regarding the findings shown in In future research conducted to inform public health decision-makers in Saudi Arabia, using a different geospatial mapping technique with Global Positioning System (GPS) data and a mobile application could yield more granular data about the exact location  of participants . If applIn our cross-sectional web-based survey study, the risk of developing T2DM was relatively low. Most participants were female, were young, and had a high level of education. The key sociodemographic factors associated with having high ARABRISK scores included living in a large city, being retired, not having private insurance, and being married, divorced, or widowed. At present, Saudi Arabia faces a diabetes epidemic, and against the country’s steadily increased incidence of diabetes in recent years, the widespread use of self-administered risk scores may increase the early detection of prediabetes and diabetes and, in turn, improve its prevention and treatment.


P < 0.001], age at initial sexual activity ≤ 19 years , sexual partners > 1 , ASCUS and above , non-condom contraception , and HSIL and above . Compared with women aged 56–65 and 35–45 years  the HPV infection rate was significantly lower in those aged 46–55 years . Furthermore, ≤ high school age , not breastfeeding , ASCUS and above  were potential risk factors for cervical cancer and precancerous lesions. In women with HPV infection, ≤ high school education level, initial sexual activity at ≤ 19 years of age, number of sexual partners > 1, ASCUS and above, non-condom contraception, HSIL and above were risk factors for HPV infection. Compared with women aged 56–65 years, those aged 35–45 and 46–55 years had significantly lower HPV infection rates, and high school age and below, non-breastfeeding, and ASCUS and above were all potential risk factors for cervical cancer and precancerous lesions.Cervical cancer is the most common gynecological malignancy and screening for risk factors with early detection has been shown to reduce the mortality. In this study, we aimed to analyze the characteristics and risk factors of human papillomavirus (HPV) infection and precancerous lesions in women and provide clinical evidence for developing strategies to prevent cervical precancerous lesions and cancer in women. Furthermore, we evaluated the influencing factors for high-risk HPV infection. From April 2018 to December 2021, 10,628 women were recruited for cervical cancer screening at Liaoning Cancer Hospital, Shenyang Sujiatun District Women’s and Infants Hospital, Benxi Manchu Autonomous County People’s Hospital, and Shandong Affiliated Hospital of Qingdao University. The study participants were tested to determine if they were HPV-positive (HPV +) or underwent thinprep cytology test (TCT) for atypical squamous cells of undetermined significance (ASCUS) and above. Furthermore, colposcopies and biopsies were performed for the histopathological examination. Finally, 9991 cases were included in the statistical analysis, and the factors influencing HPV infection and those related to cervical cancer and precancerous lesions were analyzed. HPV + infection, high-grade squamous intraepithelial lesion-positive (CINII +) in cervical high-grade intraepithelial neoplasia, and early cervical cancer diagnosis rates were 12.45, 1.09, and 95.41%, respectively. The potential risk factors for HPV were education ≤ high school [odds ratio (OR) = 1.279 (1.129–1.449),  Cervical cancer is the most common gynecological malignancy and in 2020, with 604,000 new cases worldwide, with approximately 342,000 related deaths . CervicaIn contrast, developed countries have high coverage and availability of cancer screening and compliance by the target population . The incThe incidence rate of cervical cancer at a younger age is showing an increasing trend . FurtherThe German scholar Hausen first identified HPV infection as the main pathogenic factor of cervical cancer and precancerous lesions, which could be considered a milestone discovery in the prevention and treatment of this disease . A consiP < 0.05)  .The study of HPV prevalence and its subtype distribution may provide relevant information for routine vaccination and the types of HPV strains used in vaccination. HPV infection rates among women worldwide range from 11.70 to 7.20% . The higIn China, significant differences were also reported in HPV infection rates in different regions, ranging from 13 to 31.9% . HPV alsThis study was approved by the Ethics Committee of Liaoning Cancer Hospital and informed consent was obtain from all individual participants included in the study (No.: 20180106).From April 2018 to December 2021, we recruited 10,628 women for cervical cancer screening in Liaoning Province at Liaoning Cancer Hospital, Shenyang Sujiatun District Women’s and Infant Hospital, Benxi Manchu Autonomous County People’s Hospital, and Shandong Affiliated Hospital of Qingdao University. The average and median ages of the selected research participants were 49.61 ± 7.195 years, and 49 years, respectively.All women in the study underwent HPV testing and the thinprep cytology test (TCT). Community population and hospital outpatient opportunistic screenings were used to evaluate whether participants met the following inclusion criteria: (1) lived in Liaoning for > 3 years; (2) a history of sexual activity; (3) no sexual activity, vaginal medication, or drug flushing within 1 week before examination; (4) no serious organ dysfunction or mental illness; (5) voluntary participation and signed consent; and 5) willingness to complete the questionnaire survey. The exclusion criteria were: (1) women who were pregnant, lactating, or menstruating; (2) history of cervical surgery or hysterectomy; and (3) diagnosed with a tumor and being treated for other serious internal and external diseases. This study was approved by the ethics committee of Liaoning Cancer Hospital.TCT . The examination application forms of each patient was filled and their name and age and collection date was placed on the specimen bottle . The completely filled application form was checked to ensure the information was consistent with that on the preservation solution vial. The cervical brush was then placed in the preservation solution and rinsed by plunging it up and down 20 times, while spreading and rotating the bristles back and forth to make the cells fall into the solution.Finally, the cervical brush was rapidly rotated to release the collected cells into the preservation solution, the sampler was discarded ensuring the brush head was not left in the bottle, and then it was sent to the pathology department for liquid-based analysis. The operating procedures of the system for the TCT, which is a liquid-based cytology production process, includes the following three steps, which were strictly adhered to: cell mixing, collection, and transfer. Briefly, the sample was placed into 95% alcohol for wet fixation, the production was completed, and then the next staining and diagnosis step was performed. The negative slides should be stored for 1 year, whereas the positive slides should be stored for a long time. Finally, the diagnosis was made by a full-time cytological diagnosis doctor in the pathology department.The detection kit was purchased from Beijing Haolejie Healthcare Medical Equipment Company (Beijing). The E6/E7 mRNA detection kit was used to detect the following 14 high-risk HPV mRNAs known to cause cervical cancer , and displayed the detection results of 16 and 18 to provide more clinical guidance information. The kit detects E6 and E7 mRNA of high-risk HPV to avoid missing the detection of high-level lesions and cancer, which could result from only detecting the L1 region. Furthermore, no cross reaction occurred with low-risk HPV and there were fewer false positives, which reduced unnecessary colposcopies and over diagnosis.The questionnaire used in this study adopts the principle of voluntariness and was designed professionally by the members of our research group. The questionnaire includes questions on the participants’ personal information; relevant medical, reproductive, sexual activity, smoking, contraceptive, and sports history; educational level; and economic income.Cervical exfoliative cells were examined using cervical liquid-based thinprep cytology test (TCT). The diagnostic criteria are based on the Bethesda system (TBS) classification: atypical squamous cells of undetermined significance (ASCUS) without clear diagnostic significance, excluding ACS-cannot exclude high-grade squamous intraepithelial lesion (ASC-H), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous epithelial lesion (HSIL), squamous cell carcinoma (SCC), and atypical glandular epithelial cells (AGCs) .For HPV+, TBS classified ASCUS or above, or both, or clinically suspicious abnormalities, a colposcopy is further recommended. The examination results suggested the need for a multipoint tissue biopsy of cervical lesions and bite the tissue for pathological examination. The pathological results were the gold standard, and the diagnostic criteria included normal or inflammatory reaction and cervical intraepithelial neoplasia-intraepithelial neoplasia (CIN) and cervical cancer. CIN is divided into CIN I, CIN II, and CIN III  accordinThe quality control evaluation of the cervical exfoliative cell examination was conducted using 20 and 5–10% of the randomly selected positive and negative smears, respectively. All smears were reviewed by experts in the field and the acceptable quality rate of the smear results was 80%. The quality control of the colposcopy examination involved a spot check of 10 and 20% of the normal and abnormal reports, respectively. The results were rechecked by experts in the field and the standard quality rate of the reported results was expected to reach 90%. The quality control of the histopathological examination was performed by spot checking 10% of the pathological sections and the results were recheck by experts. The coincidence rate of diagnostic results was expected to reach 90%.2) test. The influencing factors were analyzed using univariate and multivariate logistic regression to evaluate the correlation between the relevant factors mentioned in the questionnaire and HPV infection, cervical cancer, and precancerous lesions.The statistical analysis of the relevant data was conducted using the statistical package for the social science (SPSS) version 19.0 software and the count data rate (%) was analyzed using the chi-square  and 65 (5.4%) were HPV 16 + and 18/45 + with detection rates of 1.84 and 0.65%, respectively. Furthermore, 980 (80.8%) patients were positive for other high-risk types of HPV, with a detection rate of 9.81%. Among the tested patients, 16 women had mixed HPV infections of the above three groups. The positive rate of HPV16 and HPV18/45 in the total screened population was 2.5%  .In this study, 9,991 participants were recruited for high-risk HPV screening in 2018–2021, which included 1,244 (12.45%) that were positive for high-risk HPV. The average age of the women was 49.99 (35–65) years and they were all divided into the following five age groups: 35–40, 41–45, 46–50, 51–55, and 56–65 years old. The prevalence of high-risk HPV infection had two peaks at 35–40, 55–60/61–65 years with infection rates of 13.09, 14.29, and 15.80%, respectively .Further colposcopy was performed in 1,571 cases. There were 1,015 cases with complete colposcopy report, and the referral rate was 64.61%. There were 812 abnormal cases suspected to be diagnosed by colposcopy, 673 cases of low-grade lesions, and 66 cases of high-grade lesions, with a detection rate of 0.66% .P < 0.05)  .P < 0.001], initial sexual activity age ≤ 19 years old , number of sexual partners > 1 , ASCUS and above , non-condom contraception , and HSIL and above  were risk factors for HPV infection. Compared with women aged 56–65 years, the HPV infection rate of those 35–45  and 46–55 [OR = 0.79 (0.683–0.915)] years old (P = 0.002) decreased significantly  = 1.279 (1.129–1.449), ficantly .Among the 9,991 patients included in the analysis, 1,004 had complete pathological results, including follow-up update, whereas 109 and 895 cases were CINII + and LSIL/inflammation, respectively. Furthermore, 136 and 1 patients were referred for a colposcopy because of TCT/HPV and clinically suspected abnormalities, respectively. The result of the colposcopy evaluation was negative and, therefore, no biopsy was performed. In addition, 137 patients were classified as LSIL and inflammation, and in the absence of pathological results, 8,210 women with double negative screening results (TCT-/HPV-) were regarded as having no cervical lesions. Therefore, 109 CINII + , 5 invasive carcinoma and 9, 242 LSIL/inflammation cases, respectively were included in the statistical analysis.P < 0.05). The detection rate of CINII + increased significantly with increasing age, early sexual activity, multiple sexual partners, total family income ≤ 50,000 yuan, bleeding during sexual intercourse and extramarital sex, but there was no significant difference , and the early diagnosis rate of cervical cancer was 95.41%(104/109). The results of the 28 logistic regression analyses using CINII + and diet as dependent variables showed a significant correlation between educational level, breastfeeding, and TCT positivity , P = 0.017], ASCUS and above  were potential risk factors for cervical cancer and precancerous lesions ,  lesions .In this multicenter, cross-sectional population study in women, we found that the overall prevalence of HR-HPV was 12.45%, which is lower than the national average. HPV infection can occur at any age and is related to age. In this study, the age-specific distribution showed a bimodal curve, and the first peak appeared in the 35–40-year-old age group, with the infection rate of middle-aged women showing a low trend. The second peak of high-risk HPV infection was observed in women aged 55–60 and 61–65 years, who were born in the 1960s–1970s and the economy of China has improved considerably since then. In addition, the education level of these age groups is significantly lower than that of the younger women, which contributes to their lack of knowledge about HPV infection. In addition, the low level of autoimmunity and hormones further impairs the resistance of the cervix to HPV infection, which may explain the significant increase in their HPV + rate.Presently, there is no specific or effective drug treatment for HPV infection, and although a vaccine has been developed and listed in China, it is expensive and does not prevent all subtype infections . TherefoIn this study, multicenter cervical screening was used to analyze the incidence of HPV infection, cervical cancer, and high-grade precancerous lesions in women. The results led us to conclude that high school and below, initial sexual life age ≤ 19 years old, number of sexual partners > 1, ASCUS and above, non-condom contraception, HSIL and above were all risk factors of HPV infection. The reason may be that women with a low educational level lack the awareness of cervical screening and prevention strategies . Women ≤Numerous studies have shown that an active sex life is closely related to the occurrence and development of cervical cancer. Literature reports from countries other than China state that factors such as sexual partners and frequency are closely related to cervical cancer . CondomsCervical lesions mostly occur in married women and it is the most serious cervical disease, and shows higher occurrence in younger women . CervicaP < 0.05) risk factors of cervical cancer and CIN II/III.HPV has been reported to be transmitted through direct or indirect skin or sexual contact . The infThe CINII + detection rate increased with increasing age, early sexual activity, multiple sexual partners, total family income ≤ 50,000 yuan, bleeding during sexual intercourse, and extramarital sex, but not significantly. This study has been compared with other similar related research data in the near future see . As a prCervical cancer is a prominent public health problem and proms focusing on its prevention and control should be strengthened. In this study, multicenter cervical screening of women to determine the HPV infection rate and incidence of cervical high-grade precancerous lesions, demonstrated a high school education and below, initial age of sexual activity ≤ 19 years old, more than one sexual partner, ASCUS and above, non-condom contraception, and HSIL and above as risk factors of HPV infection. Furthermore, a high school education and below, non-lactation, and a positive TCT result were identified as risk factors of cervical cancer and high-grade precancerous lesion infection. In conclusion, the investigation of risk factors for HPV infection and cervical high-grade precancerous lesions are of great significance for reducing the incidence rate of cervical cancer. Consequently, we recommend the establishment of strategies to enhance the awareness of the risk factors of high-risk HPV infection; to promote healthy sexual behavior, life, and health habits; and to improve immunity and strengthen physical exercise, which could effectively reduce the incidence rate of cervical cancer.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Liaoning Cancer Hospital (No. 20180106). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.HP and CW designed and supervised the project. DY, JZ, and XC collected clinical data samples. DY collected and processed data and data analysis, and drafted the manuscript. JM translated and polished the manuscript. All authors reviewed, discussed, and edited versions of the final report manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.