The use of non-drug intervention for calcium deficiency has attracted attention in recent years. Although calcium carbonate is the preferred raw material for calcium supplementation, there are few reports on the mechanism of the combined action of chondroitin sulfate and calcium to alleviate osteoporosis from the perspective of gut microbiota and metabolomics. In this study, a rat model of osteoporosis was established by feeding a low-calcium diet. The intestinal microbiota abundance, fecal and plasma metabolite expression levels of rats fed a basal diet, a low-calcium diet, a low-calcium diet plus calcium carbonate, and a low-calcium diet plus chondroitin sulfate were compared. The results showed that compared with the low calcium group, the calcium content and bone mineral density of femur were significantly increased in the calcium carbonate and chondroitin sulfate groups. 16 S rRNA sequencing and metabolomics analysis showed that chondroitin sulfate intervention could reduce short-chain fatty acid synthesis of intestinal flora, slow down inflammatory response, inhibit osteoclast differentiation, promote calcium absorption and antioxidant mechanism, and alleviate osteoporosis in low-calcium feeding rats. Correlation analysis showed that the selected intestinal flora was significantly correlated with metabolites enriched in feces and plasma. This study provides scientific evidence of the potential impact of chondroitin sulfate as a dietary supplement for patients with osteoporosis.The online version contains supplementary material available at 10.1186/s12986-023-00726-3. In 1994, the World Health Organization proposed the concept of osteoporosis as a “progressive systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture” . DecreasCalcium supplements are generally divided into two groups based on the nature of their chelating counterbalance ions with organic calcium supplements, including negatively charged organic molecules such as malate, citrate, fumarate and gluconate, and inorganic calcium supplements relying on inorganic chelating molecules such as carbonates, phosphates and chlorides . CalciumThe combination of calcium supplements and vitamin D is considered the traditional strategy for osteoporosis treatment. But this method usually requires larger doses of calcium supplementation, which is less effective .Chondroitin sulfates (CS) are a class of sulfated glycosaminoglycans, which are linear polysaccharides composed of repeated disaccharide units comprising uronic acid and N-acetylhexosamine . In addiRecently, the role of intestinal flora in metabolic diseases has been widely investigated. Several human diseases are linked to the dysregulation or imbalance in the microbial communities . EvidencMetabolomics is a science that studies the changes in all metabolites in biological systems in response to external stimuli. Substrates and small molecule metabolites (< 1500 Da) of various metabolic pathways of products are assessed in specific physiological periods through qualitative and quantitative analysis and integration of information . Plasma In this study, using fecal and plasma metabolomics to assess gut microbes, we compared the outcomes of CS and calcium carbonate intervention in an osteoporotic rat model. Our aim was to better understand the role of CS in improving osteoporosis and its underlying mechanisms, which may provide a new approach for the remission of osteoporosis caused by calcium deficiency.Chondroitin sulfate was purchased from ORIHIRO LTD., Japan. Samples are white tablets. The recommended dose for humans is 4.2 g/60 kg. Each 2.5 g contains 17 mg of calcium and 10 mg of magnesium.Weaned female Wistar rats weighing 60–75 g, and of SPF grade at approximately four weeks of birth were selected. The rats were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd under experimental animal production license no. SCXK (Lu) 20190003 and experimental animal use license no. SYXK (Lu) 20150015. The rats were house in an environment with an ambient temperature of 22 ± 2 °C and relative humidity at 50% ± 10%. After adaptive feeding, rats were fasted for 12 h, weighed, and randomly divided into normal control group, low calcium control group, calcium carbonate control group, and Chondroitin sulfate group according to body weight, with 10 rats in each group. The normal group was fed a basal diet and deionized water, and the other groups were fed a low-calcium basal diet and deionized water. Oral intragastric was administered at 1 mL/100 g bw. The recommended intake of Chondroitin sulfate is 4200 mg per person per day, based on 60 kg weight per person, equivalent to 70 mg/kg.bw per day. The oral dose of Chondroitin sulfate was determined to be 30 times the recommended dose of human body, that is, the daily dosage of rats in each group was 2100 mg/kg.bw. In the calcium carbonate group, calcium carbonate was 525 mg/kg body weight , and the concentration of calcium carbonate solution was 52.5 mg/mL. The normal control group and the low calcium control group were gavaged with the same volume of deionized water instead of the test solution. Bone mineral density (BMD) function was tested after three months of continuous administration. This experiment was approved by Shandong University Preventive Medicine Animal Experiment Ethics Committee, and the study’s protocols adhere to the ethics and welfare requirements of the National Institutes of Health (NIH).The low calcium base feed formula is listed in Table The rats were euthanized after three months. The right femur was removed and dried in an oven at 105 °C until constant weight was obtained. The backbone was also weighed.The right femur was dried to constant weight, and BMD was measured at the midpoint of femur and distal end of femur using the xR-600 dual-energy X-ray BMD instrument (NORLAND).The right femur was dried to constant weight, placed in a crucible, charred in an electric furnace, and ash combusted in a muffle furnace at 550 °C. The calcium content of femur was determined by EDTA titration.The genomic DNA of the samples was extracted using the cetyltrimethylammonium bromide (CTAB) method, and the purity and concentration of DNA were tested by agarose gel electrophoresis. The DNA sample was subsequently diluted to 1 ng/μL with sterile water in a centrifugal tube. The 16S rRNA genes in different regions were amplified using a barcode-specific primer. The same volume of IX loading buffer (containing SYB green) was mixed with polymerase chain reaction (PCR) products. PCR products were detected by electrophoresis with 2% agarose gel. The qualified PCR products were purified using magnetic beads, quantified by enzyme standard, and mixed in equal quantities according to the concentration of PCR products. After full mixing, the PCR products were detected by 2% agarose gel electrophoresis, and the target bands were recovered using gel recovery kits provided by QIAGEN. TruSeq® DNA PCR-free Sample Preparation Kit was used for library construction. The constructed library was quantified by Qubit and Q-PCR. The samples were then sent for sequencing using NovaSeq6000.Quality control was carried out in accordance with QIIME version 1.9.1  to obtaihttp://www.genome.jp/kegg/), HMDB (http://www.hmdb.ca/), and Lipidmaps database (http://www.lipid maps.org/). Quality control through the QC sample overlap diagram and the correlation analysis were performed. After data conversion using metaX [P < 0.05 and FC > 1.2 or FC < 0.833. Ipath was used to analyze whether the intestinal microbiota were associated with metabolic pathways. The KEGG database was used to analyze the function and metabolic pathway of metabolites between the normal control and low calcium control group, and low calcium control group and Chondroitin sulfate group, and calcium carbonate control group and Chondroitin sulfate group. All graphs and plots were generated using ggplot2 package in R.Novogene provides 16S RNA sequencing and metabolomics services.Target-like metabolomics is based on a SCIEX QTRAP® 6500 + mass spectrometer with triple quadrupole-linear ion trap and Multiple Reaction Monitoring (MRM) to accurately characterize and quantify metabolites in biological samples. The experimental process mainly includes sample collection, metabolite extraction, mass spectrometry detection and data analysis. Qualitative analysis of parent ion (Q1), daughter ion (Q3), retention time (RT), cluster potential (DP) and collision energy (CE) was carried out. The SCIEX OS version 1.4 was used to open the mass spectrum file, and the chromatographic peak integration and correction work was performed. The metabolites were interpreted by KEGG . On the other hand, The calcium content of femur, bone mineral density at the midpoint of femur and distal femur in group C were significantly lower than those in groups N, Ca and CS . . The calcium content of femur, bone mineral density of midpoint of femur and bone mineral density of distal femur in CS group were not lower than those in Ca group. HE staining showed that compared with group C, bone mineral density was significantly increased in group CS and Ca  were found in the N group and the C group, with 838 and 1233 OTUs specific to the two groups, respectively  analysis was used to identify potential biomarkers for intestinal flora in the N group and the C group, and a t-test was used to further examine and visualize the results , delete mismatched entries and count the number of DEL that accompany each category, the most highly ranked category is Fatty Acyls (FA). The levels of plasma testosterone and stearic content in normal feeding rats were significantly higher than those in the other two groups , 107 lipids were detected, among which 93 lipids were different, we obtained lipid classification by matching screened DEL with Lipidmaps database  and Bacteroidetes (B) were the dominant bacteria in the three groups in this study. Previous studies have also suggested that changes in gut microbiota composition are involved in the development of osteoporosis .In this study, a low-calcium diet significantly reduced Firmicutes and Lactobacilli, and increased Actinobacteriota and Prevotella at the phylum and genus levels. Firmicutes are positively correlated with calcium absorption . They acPrevious studies have analyzed changes in microbial composition during calcium supplementation, dietary calcium acted in a prebiotic manner by promoting significant increases in two species analyzed from Bacteroidetes and Actinobacteria phyla (Bacteroides/Prevotella and Bifidobacterium) . In addiIn our study, a low-calcium diet was found to affect vitamin B6 metabolism, lysosome, pantothenic acid, and CoA biosynthesis, and the AMPK signaling pathway in the fecal metabolome. It also affected lysine degradation in the plasma metabolome. Bacteria-derived compounds, such as vitamins, may enter the bloodstream and directly affect bone cell activity . VitaminWhen calcium carbonate was administered to the low-calcium diet group, it affected the folate resistance of fecal metabolites, olfactory transduction, production and secretion of cortisol, Cushing's syndrome, and the cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling pathway, as well as the longevity regulation pathways of plasma metabolites—worm nicotinamide and platelet-activated ADP. Folic acid is a water-soluble vitamin that helps maintain bone density by maintaining optimal nitric oxide synthase activity in bone cells . It alsoHowever, compared to the calcium carbonate group, under CS intervention, the difference in fecal and plasma lipid profiles of metabolites was particularly significant, and the difference in fecal metabolism was concentrated in linoleic acid metabolism. Linoleic acid may indirectly affect bone mass by stimulating calcium absorption, which has a beneficial effect on bone loss . We alsoThere are several mechanisms that can cause osteoporosis. But chronic low-grade inflammation has been identified as the root cause of many diseases, including osteoporosis . We idenAdditional file 1: Table S1. MRPP analysis showed significant differences in microbial community structure between group N and group C and between group C and group CS and between group CS and group Ca.Additional file 2: Table S2. The differential metabolites of fecal metabolomics between the N group and the C group. The differential metabolites of fecal metabolomics between the C group and the Ca group. The differential metabolites of fecal metabolomics between the C group and the CS group. The differential metabolites of fecal metabolomics between the CS group and the Ca group.Additional file 3: Table S3. The differential metabolites of plasma metabolomics between the N group and the C group. The differential metabolites of plasma metabolomics between the C group and the Ca group. The differential metabolites of plasma metabolomics between the C group and the CS group. The differential metabolites of plasma metabolomics between the CS group and the Ca group.


Subacute combined degeneration of the spinal cord (SCD) is mainly caused by deficiency of Vitamin B12 and characterized by deep hypoesthesia, sensory ataxia and spasmodic paralysis of lower limbs. SCD often accompanies with megaloblastic anemia. Psychiatric symptoms could be the initial manifestations of SCD by lack of Vitamin B12, but are rarely considered secondary to physical discomfort and psychological factors in SCD. Additionally, treatment experience for psychiatric symptoms in SCD remains little reported.We presented a case of a 37-year-old female who complained of being persecuted and controlled for one week and thus was admitted to the psychiatry department. Before that, she had went through persistent paresthesia and numbness of her lower extremities for two-month. Low Vitamin B12 level and hemoglobin concentration, neurologic symptoms and bone marrow smear results supported the clinical diagnosis of SCD and megaloblastic anemia. With supplementation of Vitamin B12 and blood transfusion and short-term prescription of antipsychotics and antidepressants, physical symptoms were improved and psychological symptoms disappeared within 2 weeks.Psychiatric symptoms of SCD could be generated from lack of Vitamin B12, anemia and neurologic symptoms, where short-term use of antipsychotics and antidepressants may be effective. Subacute combined degeneration of the spinal cord (SCD) is the most common neurological disorder caused by Vitamin B12 deficiency, which often accompanied with megaloblastic or pernicious anemia . Complete blood count (CBC) revealed HGB 47 g/L [reference range 115-150 g/L]). MRI of the brain and whole spine showed abnormal signals of the cervical thoracic cord below the level of the spine centrum No. 6 and small circular hyper-intensity in the pituitary , RBC 1.91 × 10The primary diagnosis of this patient was SCD. We excluded the possibility of multiple sclerosis, compressive myelopathy, autoimmune and gastrointestinal diseases. The secondary diagnoses included organic mental disorder, megaloblastic anemia and Vitamin B12 deficiency.The patient received intramuscular injection of Vitamin B12 500 mg per day. Folic acid was supplemented 10 mg orally 3 times per day. Vitamin B12-rich diet was recommended. We administered blood transfusion (3.5 units of suspended RBC) when found HBG of 47 g/L. Duloxetine 60 mg per day was used to improve depressive symptoms, with a combination of Olanzapine 10 mg per day which was administered for her psychiatric symptoms. Three days after caring in psychiatric quarantine ward, the patient increased inappetence. And her fear and suspicion worsened with persisting physical inability and maladaptation. Her vital signs remained stable. Her HGB increased to 84 g/L. A week later, her abnormal sensations, weakness, and numbness began to resolve, with fear and suspicion improving simultaneously. She achieved complete remission of her neurologic and psychiatric symptoms after 14 days of treatment. Eighteen days after admission, she was discharged with suggestions of supplementation of Vitamin B12, folic acid and other nutrients.SCD is a reversible neurodegenerative disease manifested as peripheral neuropathy damage, paresthesia, weakness, ataxia, deep sensitivity disturbances, and very rarely, optic atrophy and psychiatric symptoms . HoweverFirstly, based on principle of monism diagnosis, both SCD and psychiatric symptoms are likely to be directly caused by Vitamin B12 deficiency instead of being irrelevant comorbidity. Her vegetarian diet for 8 years implied long-term influence from Vitamin B12 deficiency. Hallucinations, irritability, catatonia, delirium and apathy were commonly reported psychiatric symptoms caused by Vitamin B12 deficiency . In addiAnother possibility to consider is the effects from anemia. The patient's HGB was already at a low level when the psychiatric symptoms appeared. The anemia may cause reduced oxygen-carrying capacity and insufficient energy supply to interfere brain metabolism , 8. AnemThirdly, the persecutory delusions and delusions of being controlled were more likely to be secondary to abnormal body experiences like numbness and weakness. The psychiatric symptoms came after physical discomfort, which is time coherent. With undefined diagnosis and possible future of disability, it’s reasonable that the patient occurred psychological experience resembling that from trauma and stress. Moreover, with inability to take care of herself, decreased social contact, being always surrounded by her husband in daily life and medical staff after admission, plus our closed inpatient environment and still worsening physical condition, her fear was undoubtedly aggravated, leading to paranoid suspicion of everyone around her. Nonetheless, this supposition utilized a dualism perspective, where the physical mechanism is neglected.Taking the 3 possible mechanisms in together, we guess it’s time to take a comprehensive thinking. These direct physiological effects of Vitamin B12 deficiency and anemia might have increased neurological and psychological vulnerability. Combined with severe physical manifestations on the lower extremities, we highlighted the possibility of psychiatric symptoms being caused by a combination of psychological and organic factors, where exacerbation of neurologic symptoms and rapid change of living conditions appeared to accelerate the development of psychiatric symptoms in this vulnerable individual. After physical recovery, the vulnerability increased, so the patient didn’t relapse at one-month follow-up while we already discontinued antipsychotics and antidepressants at discharge. The vulnerability perspective seems to be reasonable.Our case also highlights the importance of evaluating Vitamin B12 levels in psychiatric patients, especially among high-risk populations such as vegetarians, the elderly, and patients with gastrointestinal disorders , 11. TheTo the best of our knowledge, we are the first to raise an argument that psychiatric symptoms in SCD patients could not only be generated from direct physiological effects of vitaminB12 deficiency but also associated with physical discomfort and environmental factors. We emphasized the importance of interdisciplinary consultation to get early detection and timely intervention of specialty diseases, which could effectively prevent treatment delay of primary disease and even the emergence of psychiatric symptoms. We reviewed the process of the patient's visit and found that although the medical workup did provide clues for diagnosis, the patient did not come for a scheduled follow-up visit and therefore did not receive systematic treatment until hospitalization. Confronting psychiatric symptoms combined with SCD, the use of antipsychotics and antidepressants is an additional key to improving the prognosis. Thus, a further systematical study is needed to confirm the effectiveness of different antipsychotics and antidepressants for treatments of SCD patients with psychiatric symptoms.In conclusion, the psychiatric symptoms of SCD patient are attributable to a combination of Vitamin B12 deficiency, anemia, worsening physical condition, and personal and environmental disruption. Testing Vitamin B12 levels in patients with psychiatric symptoms, especially for whom with anemia, should not be neglected. The efficacy of antipsychotics and antidepressants in patients with SCD warrants further investigation.


The aim of this study was to analyze subjective appetite assessments, dietary intake, and appetite hormones in relationship to these polymorphisms. Subjects (n = 132) with normal BMIs were enrolled. Dietary intake was analyzed with 3-day diet records. Subjective appetite was measured by visual analogue scales. Biochemical parameters were measured after 12 h of fasting and 120′ following ingestion of a test meal. Ghrelin and leptin levels were measured by ELISA assay (enzyme-linked immunosorbent assay) and insulin by chemiluminescence assay. The polymorphisms were determined by allelic discrimination using TaqMan® probes. Fasting ghrelin levels differed significantly between men and women. The consumption of fruit and bread/starch with added sugar servings, as indicated by dietary records, and measured ghrelin levels were higher in carriers of Leu72Met/Met72Met compared to Leu72Leu carriers; total sugar intake was higher in Gln223Gln carriers than in Gln223Arg/Arg223Arg carriers. In conclusion, the Leu72Met and Gln223Arg polymorphism in ghrelin and LEPR may contribute to differential responses to a standardized meal as evidenced by higher postprandial levels of ghrelin and may also contribute to a higher dietary sugar intake.Appetite regulation has been recognized as a promising target for the prevention of obesity, which has become a worldwide health issue. Polymorphisms in the genes of hormones or receptors including Leu72Met for  Obesity is a worldwide health issue that has proven to be hard to prevent and treat. A leading cause of obesity is the absence of energy homeostasis characterized by an imbalance between the intake and expenditure of energy, which depends on mechanisms that modulate the amount of food consumed and the frequency of meals ,2. ThereAppetite can be evaluated through visual analogue scales (VAS) for aspects of appetite such as hunger, fullness, and desire to eat. In addition, the quantification of peptides that influence gastrointestinal motility, satiety, and hunger provide a biochemical insight into appetite . A key pGhrelin is a 28-aminoacid peptide produced and secreted by the P/D1 type cells of the stomach. Upon interaction with its receptor, signals are sent to the ventral tegmental area (VTA) to stimulate food-seeking behavior and the ingestion of palatable food . In turnIn addition to environmental and physiological responses, some genetic factors may be related to an imbalance in appetite control and food intake, which can increase the susceptibility to being overweight or obese ,12. The ghrelin (GHRL), which codes for ghrelin, contains the rs696217 polymorphism (Leu72Met) that has been associated with early onset of obesity [LEPR) has been focused mainly on obesity risk with controversial results [The gene  obesity , higher  obesity , alcohol obesity , and die obesity , dietary obesity , or eati obesity . On the  results ,23,24, wGHRL (Leu72Met) and LEPR (Gln223Arg) polymorphisms.Hence, there is a relative paucity of studies describing how these two genetic variants affect appetite responses. Therefore, this study aimed to analyze subjective appetite, dietary intake, and appetite hormones according to the 2) and having the habit of breakfast. Exclusion criteria were subjects who were vegans or vegetarians, with food allergies, elite-athletes, medical or in nutritional treatment for weight loss, appetite-altering medications, smokers, having respiratory symptoms, aversion to breakfast ingredients, and women who were pregnant, breastfeeding, or using hormonal contraceptives. Finally, only subjects who signed a written informed consent and complete all the sessions were included  , 132 unrincluded . This stThe subjects were summoned twice in a state of fasting for 12 h. In the first session, we obtained the anthropometric measurements and gathered the clinical history. In the second session, we took vital signs and a peripheral blood sample, and asked them to fill a VAS. Then, they consumed an isocaloric breakfast (see breakfast design section). At the end of breakfast, postprandial VAS at 30’, 60’, 90’, and 120’ were applied. Finally, a blood sample was taken at 120’ postprandial. Details are shown in ® stadiometer, SECA GMBH & Co., Hamburg, Germany; model 213). Body composition was analyzed by electrical bioimpedance . Waist circumference was measured in the narrowest diameter between the last rib and the iliac crest using a Lufkin Rosscraft® tape . Blood pressure was measured with an Omron Automatic arm digital blood pressure monitor  after 15 min of rest, for which subjects were instructed to sit with their backs touching the back of the chair, to rest their arms on a horizontal surface, and to keep their legs without crossing. Anthropometric measurements were taken after 12 h of fasting in all participants. Measurements were performed in light clothes and without shoes. Height was determined using a stadiometer with a precision of 0.1 cm and a measuring range up to 205 cm  cholesterol was measured with a dry chemistry analyzer Vitros 350 Chemistry . Low-density lipoprotein cholesterol (LDL-c) was calculated with the Friedewald formula, except when triglycerides levels were higher than 400 mg/dL . Very-lo®, model Human GHRL/Ghrelin ELISA Kit catalog number ELH-GHRL . The serum leptin levels were determined using ALPCO® Leptin ELISA (Ultrasensitive) catalog number 22-LEPHUU-E01 . The serum insulin levels were determined by an assay based on the chemiluminescence (CLIA) principle in a LIAISON® analyzer according to the manufacturer’s recommendations using the LIAISON® Insulin kit (REF 310360) DiaSorin S.p.A  Italy). Ghrelin and leptin levels were measured by ELISA assay (Enzyme-Linked Immuno-Sorbent Assay) according to the manufacturer’s recommendations. The serum ghrelin levels were determined using RayBioGHRL gene and Gln223Arg (rs1137101) of the LEPR gene was performed by allelic discrimination using TaqMan® probes. . The amplification reaction was carried out in a Light Cycler® 96 Real-Time PCR System . The amplification protocol consisted of a pre-incubation stage at 95 °C for 10 min, then 40 cycles of 15 s each at 95 °C, and 1 min at 60 °C. A 10 μL mixture was prepared for the reaction with 5 μL of FastStart probe master 2×, 1 μL of TaqMan® probe 20×, 2.5 μL of gDNA at 20 ng/μL, and 1.5 μL of molecular biology grade water. gDNA at a final concentration of 50 ng was used. A total of 30% of the samples were analyzed by duplicated to avoid genotyping errors.We used 200 µL of peripheral blood sample for the extraction of genomic deoxyribonucleic acid (gDNA). The assay was performed with the High Pure Polymerase Chain Reaction (PCR) Template Preparation Kit . The quantification and purity of gDNA were analyzed with the Multiskan™ SkyHigh Microplate Spectrophotometer . Genotyping of the Leu72Met (rs696217) of the ® Wisconsin, Fort Atkinson, WI, USA). The energy intake, and macro- and micronutrients composition were analyzed with the Nutritionist Pro™ version 8.1 software .We assessed the dietary intake with a 3-day dietary food record questionnaire . All subjects were instructed to provide the quantity and correct food description of their habitual dietary intake using a report of food consumption, for which participants were shown food scale models , Hass avocado (58 g), natural turkey breast ham (48 g), tomato (20 g), grated carrot (60 g), 1 leaf of Italian lettuce (20 g), almonds (8 g), no sugar-added plain yogurt (114 g), Cantaloupe melon (160 g), and 236 mL of simple water at 22 °C. The ingredients used in the preparation of breakfast were of well-known commercial brands, carefully prepared, measured, and weighed by the staff of CUCSINE Food Service Management Laboratory . The energy composition of the breakfast was 526.5 kilocalories . Energy and macronutrient content were analyzed with the Nutritionist Pro™ version 8.1 software . Appetite was evaluated through visual analogue scales (VAS). The VAS are composed of a straight horizontal line of 100 mm with the words “None” or “Not at all” located at the left end and the words “Extreme” or “As much as I have never felt” at the right end. This instrument is frequently employed in appetite evaluation due to its reproducibility and simplicity . Particit-test or the Mann–Whitney U test. The repeated measures analysis of variance (ANOVA) was used to analyze the scores of the VAS at different times and genotypes. Analysis of covariance (ANCOVA) test was used to analyze diet variables or hormone levels according to genotypes, adjusted by covariates and using Bonferroni correction for multiple comparisons. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS v28.0 software . Figures were made with GraphPad Prism v9.0  and with BioRender.com.For the descriptive statistics, quantitative variables were expressed as mean ± standard deviation (SD) or median and interquartile range (IQRs); the qualitative variables were expressed as frequencies or percentages. The Shapiro–Wilk test was used to analyze the normality distribution of quantitative variables. Variables without normal distribution were log-transformed and their normality was checked again; the variables that maintained a non-normally distribution were analyzed with non-parametric statistical tests. The comparative analysis between two independent groups was analyzed with the Student’s A total of 132 participants (77.3% women) were enrolled with a mean age of 22.0 ± 2.0 years. All anthropometric variables were different between women and men; however, no differences were found concerning the biochemical parameters, except in ghrelin levels, which were higher in men. In addition, women consumed more carbohydrates and less fat than men the night previous to the day of the intervention. Other variables such as dinner kilocalories the day before the intervention or the time of the last meal before intervention showed no differences between women and men .All polymorphisms in this study were in Hardy–Weinberg Equilibrium (HWE). The genotype and allelic frequencies are shown in GHRL or LEPR genes in energy intake or macronutrient intake  were compared between genotypes of Leu72Met of re found .LEPR gene, the leptin basal levels were higher in carriers of wild-type genotype compared to those with the polymorphic allele , and no differences were found between the Leu72Met genotypes. However, regarding the Gln223Arg polymorphism in the c allele . Becausec allele , as wellc allele . Other vGHRL (Leu72Met) and LEPR (Gln223Arg) and dietary sugar intake, which provides a perspective that had not been previously reported. Additionally, we analyzed aspects of appetite, dietary intake, and appetite hormones according to genotypes of GHRL and LEPR. We found that Leu72Met and Gln223Arg polymorphism contribute to a differential response to the standardized meal by elevation of postprandial levels of ghrelin. When we applied the VAS for subjective appetite, we did not find differences related to the genotypes of GHRL and LEPR.Our study shows a novel association related to the genetic variants One interesting finding in this study was the higher fasting ghrelin concentrations in men compared to women. Similar results were found by Espinoza-García et al., who reported that in a group of young adults with normal weight, men presented higher concentration of ghrelin compared to women . In contGHRL Leu72Met in our study were similar to those reported in a Mexican-American population of Los Angeles [LEPR gene were similar to those reported in the Mexican-American population of Los Angeles [The genotypic and allelic frequencies of the genotypes of  Angeles . Neverth Angeles . Moreove Angeles , as well Angeles ,35,36.We compared dietary variables between genotypes, and we found differences in the intake of carbohydrates and sugars. To our knowledge, only the study of Takezawa et al. analyzed diet intake according to the Leu72Met polymorphism. They did not find significant differences in either the consumption of total energy, fat, or sugar in subjects with the Leu72Leu genotype, but the intake of dairy products was higher in subjects with the wild-type genotype . SeveralGHRL in the Mexican population. Indeed, the higher postprandial ghrelin levels we found in the subjects with the Leu72Met polymorphism could be derived from the low-carbohydrate breakfast, as these individuals did not achieve a postprandial suppressive effect on ghrelin since, as mentioned above, they would need a higher carbohydrate intake to decrease the ghrelin levels to levels similar to those of Leu72Leu genotype. However, these results must be replicated in other populations that include more subjects with the Met allele and with different degrees of BMI. Ukkola et al. measured fasting ghrelin in a Swedish population with obesity and found that Met72Met carries have higher levels than subjects with the Leu72Leu or Leu72Met . AdditioLEPR gene and dietary intake, especially related to the servings of food groups. Dominguez-Reyes et al. observed that the consumption of carbohydrates was very similar between genotypes [LEPR in the Japanese adult population, in which no difference was reported regarding the Gln223Arg polymorphism [There are few studies about the Gln223Arg polymorphism in the enotypes . In addimorphism . Studiesmorphism ,45. Howemorphism . Human smorphism . The LEPmorphism ,49, and Concerning the subjective appetite assessment, we did not find previous studies that assess the association between these aspects of appetite and the two genetic variants of this study. Nevertheless, we did not find differences among genotypes, probably because the aspects of appetite do not always correlate with physiological variables. For example, ghrelin levels do not always correlate with hunger or leptin with satiety ,51. It iLimitations of this research include a small number of studied individuals and a different proportion of female and male participants. In addition, the lack of a control group during the intervention does not allow us to know how subjects with different genotypes respond to meals with different nutritional content. Finally, the application of eating behavior questionnaires and measuring short-term satiety hormones such as GLP-1 and PYY is recommendable.GHRL and Gln223Arg of LEPR could affect sugar intake in this population, since subjects with different genotypes of Leu72Met polymorphism responded differently to a standardized meal as evidenced by higher postprandial levels of ghrelin. This could lead to consuming more carbohydrates in the short- or medium-term (as we observed in the dietary intake between genotypes) and therefore being more susceptible to obesity at earlier ages. More studies of nutritional interventions considering this genetic variant are necessary.The results of our study suggest that the genetic variables Leu72 Met of


BRCA1 and BRCA2 high-risk genes. Recently, the BRCA1-associated ring domain (BARD1), that partners BRCA1 in DNA repair, has been confirmed as a moderate-risk breast cancer susceptibility gene. Taking advantage of next-generation sequencing techniques, and with the purpose of defining the whole spectrum of possible pathogenic variants (PVs) in this gene, here we have performed a comprehensive mutational analysis of BARD1 in a cohort of 1946 Spanish patients who fulfilled criteria to be tested for germline pathogenic mutations in BRCA1 and BRCA2. We identified 22 different rare germline variants, being 5 of them clearly pathogenic or likely pathogenic large deletions, which account for 0.26% of the patients tested. Our results show that the prevalence and spectrum of mutations in the BARD1 gene might vary between different regions of Spain and expose the relevance to test for copy number variations.Only up to 25% of the cases in which there is a familial aggregation of breast and/or ovarian cancer are explained by germline mutations in the well-known  BRCA1 and BRCA2 are the main high-risk susceptibility genes responsible for Hereditary Breast and Ovarian Cancer (HBOC), however their germline mutations only explain up to 25% of the cases in which a familial aggregation of these diseases is observed. Other moderate-high susceptibility genes have been identified in recent years, whose association with breast and/or ovarian cancer susceptiblity has been accurately established, thanks to advances in next-generation sequencing (NGS) technologies3. Recently, the two largest BC case–control studies published so far, have confirmed those genes that have an actual association with the disease, and therefore should be included in diagnostic testing, in addition to BRCA1/2. These genes are ATM, BARD1, CDH1, CHEK2, PALB2, RAD51C, RAD51D and TP535. All of those were already considered clinically relevant8, except BARD1, whose role in BC susceptibility had not been so clearly established until now.Breast cancer (BC) is the most common malignancy in women and the second cause of death due to cancer in this sex in the worldBARD1) gene encodes a 777 amino acid protein which was discovered in 1996 to directly interact with BRCA1, through their homologous N-terminal RING domains, to form a stable complex9. BARD1 also contains two tandem conserved C-terminal BRCT domains shared with BRCA1, and four tandem ankyrin repeats. The tumour suppressor complex BRCA1-BARD1 interacts with DNA and other DNA damage response factors in order to repair double-strand breaks, including damaged replication forks, via homologous recombination. Besides, the heterodimer acts as an efficient E3 ubiquitin ligase, targeting proteins involved in cell-cycle, genome stability and hormone signaling13. BARD1 has several BRCA1-independent functions, including the inhibition of mRNA maturation and the induction of apoptosis by means of p53 stabilization14. Furthermore, BARD1 spliced isoforms lacking regions of functional domains, have been found overexpressed in some breast and ovarian tumors in comparison with the full-length protein, and have shown oncogenic properties15.The BRCA1-associated ring domain . These studies also report a stronger association of pathogenic variants (PVs) in BARD1 with ER-negative tumors and, more remarkably, with triple-negative breast cancer (TNBC).The two studies by Dorling et al. and Hu et al. have definitely confirmed the role of BARD1 gene in different cohorts16. However, to our knowledge, none of them has taken in account the whole spectrum of possible pathogenic variants (PV) in the gene. Regarding the Spanish population, only one study has investigated the prevalence of BARD1 PVs in an important number of patients, focusing on the region of Catalonia17. With the purpose of continuing to deepen our knowledge of the gene in breast and ovarian cancer predisposition, here we report a comprehensive mutational analysis of the BARD1 gene in a cohort of 1946 patients who fulfilled criteria to be tested for pathogenic mutations in the BRCA1/2 genes18 from different regions of Spain.Numerous studies have been performed in the last years aiming to define the prevalence of mutations in the BARD1 rare germline variants, 5 Copy Number Variants (CNV), 11 missenses and 6 synonymous, distributed in 49 patients . Strickingly, we did not find any frameshift, nonsense nor splicing variants. The position across the protein of the detected variants is represented in Fig. p = 0.318 and 0.492 respectively), suggesting that these variants are not potentially associated with BC risk.Among the 1946 patients screened using targeted NGS, we identified 22 different https://ncbi.nlm.nih.gov/clinvar/), literature search and Franklin Genoox platform  variant interpretation.All variants found were classified according to their pathogenicity based on ACMG guidelines and taking into consideration ClinVar database  database, from which along 9884 noncancer controls, the only deletion reported (once) comprised exons 4–11 with an overall frequency of 0.0001012 in contrast to our 0.00257 prevalence of large deletions in the cases studied. As local controls, we included 1119 DNA samples from Spanish individuals provided by Spanish National Biobank (BNADN), that had been genotyped using Illumina Global Screening Array v1.0 + MD containing 700.078 SNPs. We made use of these data to analyse CNVs in the BARD1 gene, and no CNVs were detected.Only the five CNVs detected in our series were considered as pathogenic or likely pathogenic following ACMG guidelines. CNVs were detected from NGS data, based on the depth of coverage which is correlated with the copy number of the regionein Fig. . Each on22. In this study, we report for the first time the deletion of exons 10–11, deletion of exon 9 and deletion of exons 2–11.Of note, out of the five CNVs detected, only deletion of exon 1 and deletion of the entire gene have been previously describedBARD1 PVs found in each group are shown in Table Carriers of these PVs were women diagnosed with BC, whose genotype and phenotype data are summarized in Table Most of the synonymous variants detected in our population were previously described with conflicting interpretations of pathogenicity in ClinVar  motifs causing the deletion of exons 2 to 9 and resulting in a frame-shift which created a premature stop codon (p.Cys53_Trp635delinsfsX12)BARD1 missense variants is performed. Although we did not conduct functional assays, we performed an exhaustive review of the literature to try to add any piece of information that could contribute to their classification  alteration28. Of our detected missense variants, p.Asp612Val, p.Thr343Ile and p.Leu239Gln were described as functional, although for these two last variants a significantly increased loss of heterozygosity (LOH) has been reported. In addition, they recognized p.Thr716Ala as functional, while other studies had described this variant as deleterious as mentioned before. This suggests that HDR assay, although informative, might not be sufficient for classifying BARD1 variants.A total of 105 29. In this context, BARD1 has been recently confirmed as a moderate-risk BC gene by two large case–control studies, sorting out the uncertainity about its association with the disease5. With the aim of establishing the mutational spectrum of BARD1 in the Spanish population in order to improve genetic diagnosis, we screened the gene for PVs by a multigene panel analysis in a series of 1946 Spanish patients who fulfilled criteria to be tested for germline mutations in the BRCA1/2 genes. To our knowledge, this is the first study that comprehensively reports all types of rare variants in the BARD1 gene, subsequently discovering 5 large deletions, 11 missense and 6 synonymous from which only the large deletions were finally regarded as pathogenic or likely pathogenic. Surprisingly, no frameshift, nonsense nor splicing variants were found, which could be due to the limited size of our cohort or to populations or even regional differences in the spectrum of BARD1 mutations.Familial BC is a polygenic disease that can be partially explained, by the effect of PVs distributed over several susceptibility genes with different levels of riskBARD1 gene in the Spanish population17. Among 4015 patients with clinical suspicion of HBOC, they identified 19 PVs: 8 truncating, 1 splicing and 2 large deletion, ending up with a PV prevalence of 0.47% which approximately doubles the 0.22% reported by Couch et al. and the 0.25% described by Suszynska and Kozlowski30, and triples the 0.13% of protein-truncating variants reported by Dorling et al. and the 0.15% identified by Hu et al.5. In our cohort, we found a prevalence of 0.26%, which is similar to previous studies, but lower than that reported in the other Spanish cohort. This could be explained by the lower number of patients studied here, or differences in the ascertainment of the cases. These differences in the frequency of mutations, could also be explained by the possibility of variation in the mutational spectrum of BARD1 between different geographical areas of Spain, as most of our patients proceeded from Madrid and Castilla la Mancha regions, while those reported in Rofes et al. proceeded mainly from Catalonia. These regional differences have been already observed for mutations in the BRCA1/2 genes, and detecting them is very relevant to improve genetic diagnosis31, however, larger studies performed in Spanish population would be necessary to confirm this hypothesis.Previously, another study investigated the presence of germline PVs within the 32. Only a few studies have characterized the CNV landscape within the BARD1 gene, having reported a total of 8 different large deletions so far in BC or OC patients35. In our study, we have detected the deletion of exon 1 and the deletion of the entire gene previously reported21 and, what is more, we have identified for the first time exons 2–11, exon 9 and exons 10–11 deletions present in three BC cases. It is very important to highlight that any deletion can not be assumed to be pathogenic, and special caution should be taken with the ones that affect one exon and/or do not produce a frameshift. In our case, all the large deletions were predicted to cause an out of frame RNA, and classified as pathogenic or likely pathogenic by following the ACMG guidelines adaptation for single-gene copy number variants36.Germline CNVs are recognized as the genetic cause of diverse hereditary pathologies, even though their detection via NGS is still challenging28. Unfortunately, we were not able to classify most of the BARD1 missense variants we identified, but we think it is relevant to include them in further screenings and perfom functional assays, as otherwise, the percentage of PVs could be underestimated. Regarding synonymous variants, it is important to carefully analyze the effect of apparently silent changes, taking into consideration that this type of variants are usually classified as (likely) benign, obviating their possible effect in splicing, which could turn out in a missclassification. For all the rare synonymous variants in our population, damaging effect in splicing was experimentally ruled out, which corresponds with BS3 criteria of ACMG guidelines that allowed us to classify them all as definitively benign. These includes the c.1977A > G variant, that had been previously suggested to be deleterious by altering splicing and causing the partial deletion of exons 2–9 of the gene23. In our hands, no alteration in splicing was seen for this variant . Taking this in account, the variant would meet the BS1 and BP7 criteria and, even in the absence of BS3, could be classified as likely benign.In addition to clearly PVs, to our knowledge, this is the first study in which a comprehensive analysis of all missense and synonymous rare variants is performed. There are already several functional studies reporting that some of these rare variants could be pathogenicBARD1 with high risk of TNBC compared to other BC subtypes39. This assumption turns out to be of great importance, given that these patients often show a more aggressive disease course, with higher rates of metastases, earlier age at diagnosis and recurrence, and worse survival rates40. In our study, only one out of the 5 BC patients who carried a PV, was diagnosed with TNBC, representing 0.4% of the 243 patients that were diagnosed with TNBC in our series, regardless of fulfilling any other criteria. While doubling the overall rate, the reduced size of the TNBC subset does not allow us to drive any conclusion in this regard.Finally, many independent studies have associated germline PVs in BARD1 performed in Spanish population, and the first one in which all possible rare variants are comprehensively reported. Our results show that the prevalence and spectrum of mutations might vary between different regions in Spain, and highlights the large deletions in BARD1 as important contributors to BC susceptibility in our population. Larger studies and incorporation of independent functional assays are needed in order to properly classify each variant by its clinical impact, which is essential to determine the degree of susceptibility to the disease for each carrier.In summary, here we report the second largest study on BRCA1/2 genes were recruited from the Spanish National Cancer Center (CNIO), Hospital General La Mancha Centro, Complejo Hospitalario Universitario de Albacete and Hospital General Universitario de Ciudad Real. Families fulfilled one or more of the following criteria: (i) one BC diagnosed at < 40 years; (ii) ≥ 2 first-degree relatives diagnosed with BC at least one of them at < 50 years; (iii) one TNBC diagnosed at < 60 years; (iv) ≥ 3 first-degree BC relatives; (v) ≥ 1 male BC case; (vi) ≥ 1 ovarian cancer case; (vii) BC not fulfilling i-vi criteria but being bilateral or having any antecedent of pancreatic or prostate cancer  and 2094 Spanish unrelated individuals from the Collaborative Spanish Variant Server (http://csvs.clinbioinfosspa.es/).For frequency comparison of single nucleotide change variants detected in our patients, we used data from 125,748 exome sequences and 15,708 whole-genome sequences from unrelated individuals from gnomAD (https://whi.color.com/). We also included a total of 1119 DNA samples from Spanish individuals provided by Spanish National Biobank (BNADN). DNA samples were genotyped using Illumina Global Screening Array v1.0 + MD containing 700.078 SNPs according to the manufacturer's protocols and scanned using a iScan system. GenomeStudio software v2.0.4 (Illumina) was used for genotype calling. Before CNV analysis, BARD1 region was carefully inspected to ensure enough probe coverage of the gene, resulting in 59 probes thorugh all the gene. CNVs were detected and called using PennCNV v1.0.5 software from genotype, Log R Ratio (LRR) and B Allele Frequency (BAF) data values41. BARD1 gene locus  and an additional 100 KB up and downstream, were inspected to identify CNVs in this region.For CNV classification, we used data from more than 21,000 alleles from gnomAD SVs v2.1 dataset, 9884 noncancer controls from Fabulous Ladies Over Seventy  according to the manufacturer’s protocol. Purified DNA was quantitated making use of Quant-iT PicoGreen dsDNA reagent (Invitrogen).BARD1 was analyzed within a larger NGS panel, Onco-GeneSGKit (Sistemas Genómicos). Libraries were prepared employing capture-based target enrichment covering 111 genes involved in familial cancer . Briefly, 50 ng of total gDNA from cases was tagmented and amplified. Quality and quantity assessment of the resulting libraries was performed on a 2100 Bioanalyzer system (Agilent). Libraries were hybridized with capture probes to targeted regions, followed by hybrid capture with streptavidin-coated magnetic beads. Finally, libraries were indexed by PCR and combined in a single run before sequencing on a MiSeq platform (Illumina). The average read depth per sample was 200X, and, on average, 99.9% of the tardeted regions had a coverage of at least 20X.BARD1 variant nomenclature following the Human Genome Variation Society (HGVS) guidelines (http://varnomen.hgvs.org/). All coding exons and at least + /− 25 intronic nucleotides flanking each exon were analysed. Variants showing a minor allele frequency (MAF) < 0.01 in every subpopulation of the Genome Aggregation Database v2.1.1  were considered as “rare” and selected for further analysis.FastQ files generated were uploaded in GeneSystems platform (Sistemas Genómicos) to perform variant annotation. GenBank reference sequence NM_000465.3, with numbering starting at the A of the first ATG, was used for 42. In the case of large deletions, the adaptation of this classification to single-gene copy number variants was applied36.Sequence variants identified in this work were grouped by their clinical significance according to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines as pathogenic, likely pathogenic, uncertain significance (VUS), benign, or likely benignBARD1 gene were predicted by the CNid method developed by Sistemas Genómicos based on a read depth (RD) approach. Briefly, this approach is based on the comparison of the tested sample with a pool of normal samples, under the hypothesis that depth of coverage in a genomic region is correlated with the copy number of the region19. Identified CNVs were confirmed by Multiplex Ligation-dependent Probe Amplification (MLPA). The assay was performed using the MLPA Probemix P489 BARD1 (MRC-Holland) according to manufacturer’s instructions. MLPA results were analyzed with Peak Scanner Software (Thermo Fisher Scientific). For MLPA analysis, each sample harbouring a candidate CNVs in the BARD1 gene, was compared with five reference samples. Data were analysed and visualized using the Coffalyser.net software (MRC-Holland) . For the sample harbouring the single exon 9 deletion, sanger sequencing of the genomic region close to the ligation site of the half-probes (underlined in red) specific for exon 9, was performed to confirm the absence of single nucleotide polymorphisms that could have caused a decrease of the fluorescence signal . PCR products were sequenced with the Sanger method using the Big Dye Terminator Cycle sequencing kit on an ABI 3730 Sequencer (Applied Biosystems). Results were visualized in FinchTV software.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


The FG1-treated bees showed extensive cytoarchitecture changes in the fat body and pericardial cells, inducing cell death. Bees from the FG2 group showed disarranged oenocytes, peripheral vacuolization, and pyknotic nuclei of pericardial cells, but the cytoarchitecture was not compromised as observed in FG1. Additionally, immune system cells were observed through the fat body in the FG1 group. Bees exposed to FG3 demonstrated only oenocytes vacuolization. A significant decrease in the oenocyte’s surface area for bees exposed to all pyraclostrobin concentrations was observed compared to the CTL and CAC groups. The bees from the FG1 and FG2 treatment groups presented a reduced surface area of pericardial cells compared to the controls and the FG3 group. This study highlighted the harmful effects of fungicide pyraclostrobin concentrations at the individual bee cellular level, potentially harming the colony level on continuous exposure.Managed honey bees are daily exposed in agricultural settings or wild environments to multiple stressors. Currently, fungicide residues are increasingly present in bees’ pollen and nectar and can harm colonies’ production and survival. Therefore, our study aimed to evaluate the effects of the fungicide pyraclostrobin on the fat body and pericardial cells of Africanized honey bees. The foragers were divided into three experimental treatment groups and two controls: pyraclostrobin 0.125 ng/µL (FG1), 0.025 ng/µL (FG2), 0.005 ng/µL (FG3), untreated control (CTL), and acetone control (CAC). After five days of oral exposure ( Apis mellifera Linnaeus, 1758) is a managed species worldwide. It is recognized for its economic importance due to its pollination in agricultural landscapes used for human consumption and its high honey production [The honey bee  with standardized strength and visually healthy, free of symptomatic diseases and pests. All these colonies were installed in the apiary of São Paulo State University “Júlio de Mesquita Filho” , which is located in the municipality of Rio Claro, in the countryside of the state of São Paulo, Brazil. These colonies were kept in an urban area in order to avoid the bees collected being exposed to pesticides by the local farmers. The ecotoxicological bioassay was performed at the Laboratório de Ecotoxicologia e Conservação de Abelhas (LECA), located at the Centro de Estudos de Insetos Sociais (CEIS), from the same aforementioned institution .w/w) and added to all bee cages at the LECA.Foraging bee collection consisted of inserting bee cages of 250 mL  at the entrance of three different colonies at the selected foraging time (7:00–9:00 a.m.) and with temperatures from 20 °C during the summer season of 2018 in Brazil. These cages were previously prepared with small holes using a needle (1.20 × 40 mm) around them for air circulation, such as filter paper at the bottom of the plastic pots for better hygiene of the place, a larger hole in the lid for the removal of dead bees and a microtube in the lid, with four small holes (0.70 × 30 mm), for the food introduction. Immediately after foraging bee collection, polypropylene microtubes  were filled with syrup  was purchased from Sigma-Aldrich . A stock solution (1000 ng a.i./mL) was prepared using acetone (100% purity) and autoclaved distilled water in the proportion of 40–60%, respectively. After that, serial dilutions were performed to obtain working concentrations for the oral exposure to Africanized honey bees. These are based on previous research conducted in our group with A. mellifera [Melipona scutellaris  [The fungicide pyraclostrobin Pestanalellifera  and Melie, 1811) , as welle, 1811) ,40. ad libitum) as five days, based on Domingues et al. [After the foraging honey bee collection  for 24 h at 4 °C. Following the fixation period, these organs were immersed in PBS (pH 7.2–7.6 at 25 °C) for one hour and gradually dehydrated in ethanol, according to Silva-Zacarin et al. [For the histological procedure, five foragers bees were randomly collected from each experimental group and anesthetized at a cold temperature (4 °C) for one minute. Then, the bees’ parietal fat body was dissected with a digital stereo microscope (LEICA EZ4 HD) and immersed in a fixative solution , 0.1 mol Ln et al. . Upon thn et al. , and fixThe analysis and photo documentation of slides was performed using bright-field light microscopy (Olympus BX51) by the program DP Manager Software (Olympus). In order to define the morphological pattern of each experimental group and guarantee a high-accuracy histopathological diagnosis, six slides each with 12 nonsequential histological sections at different depths were made for each individual (n = 5 per group), which resulted in 72 analyses per individual and 360 for each experimental group . On aver2. In the end, a total of 2.500 pericardial cells and 1.250 oenocytes were evaluated in each experimental group through the ImageJ software bundled with 64-bit Java 1.8.0_172. From this point of view, were measured 12.5 times more pericardial cells and 6.25 times more oenocytes than suggested by Balsamo et al. [The morphometry of parietal fat body oenocytes and pericardial cells was performed by analyzing histological sections in slides stained using the HE technique  . The graphs presented the results as mean ± standard error, and the significance level adopted was The morphological pattern of fat body oenocytes and trophocytes from all experimental groups is summarized in p < 0.0001, Kruskal–Wallis statistic = 125.7), as shown in p > 0.9999) in the oenocytes surface area between the groups exposed to pyraclostrobin concentrations . Bees from the control groups had similar oenocytes surface areas (p = 0.3750).The morphometric results showed a highly significant decrease in the oenocytes surface area for bees exposed to all pyraclostrobin concentrations compared to the CTL and CAC groups  and FG2 (p < 0.001) groups presented a reduced surface area of pericardial cells  compared to the controls, being that FG1 showed a smaller size among all experimental groups (p = 0.3262), as well as FG3 and the control groups . The control groups had similar pericardial cells surface areas (p > 0.9999).The bees from the FG1  located in the lumen of the dorsal vessel A, betweeOur study revealed that oral exposure to the fungicide pyraclostrobin, even at residual concentrations, causes histopathological and morphometric injuries to the fat body and the pericardial cells of foragers of Africanized honey bees. These findings are relevant as they contribute to reducing the knowledge gap on the effects of fungicides on bees in undeveloped regions, such as South America (Brazil), with extensive beekeeping practice on one side and high use of pesticides in agriculture on the other side , along tConsidering that the effects observed here in forager bees were obtained in a laboratory with exposure-controlled conditions, maintaining the colony balance tends to be threatened in a realistic field scenario. According to Domingues et al. , foragerMelipona scutellaris (Latreille 1811) [Tetrapedia diversipes (Klug 1810) [In accordance with recent literature, other sublethal studies have also shown adverse effects of pyraclostrobin on different bees and organs such as the midgut, mandibular and hypopharyngeal glands of Africanized honey bee ,47, midgle 1811)  and on tug 1810) . Our datug 1810) , highligRegarding the fat body, this tissue can be located below the integument  or between the organs  and is mainly composed of two cell types: trophocytes of mesodermal origin and oenocytes of ectodermal origin ,49. It iThe morphological changes observed in oenocytes and trophocytes in bees exposed to the highest concentration of pyraclostrobin suggest a process of cell death due to the stress involved by the fungicide, since it noticed strongly condensed chromatin, a fragmented nucleus, intense vacuolization, and cytoarchitecture disarrangement. However, these effects were not observed by Domingues et al.  after orA. mellifera after exposure to the insecticide thiamethoxam, Nosema ceranae (microsporidian) inoculation [Oenocytes are responsible for bee detoxification (enzymes) and homeostasis . Accordiculation , and theculation . The troculation ,48,53, wculation , which dIn addition to the earlier changes, the pericardial cells were also injured in the FG1 and FG2 groups compared to the control groups. These cells have a mesodermal origin and are responsible for capturing, filtering, and eliminating toxic components in the hemolymph ,54. Acco®  in Africanized honey bee larvae induced midgut cytotoxicity, confirmed by immunohistochemical techniques (DNA fragmentation and HSP70). In the same way, Carneiro et al. [A. mellifera) after exposure to Rovral® SC (iprodione 500 g/L active ingredient).Additionally, it is important to highlight that the outcomes obtained in this present work come from exposure to the active principle alone—pyraclostrobin. However, commercial fungicide formulation may also impact the bees and the ecosystem “in situ”. Tadei et al.  highligho et al.  showed sOverall, studies of the sublethal effects of pesticides on bees still represent a low amount, and even so, most of the research focuses only on insecticides, leaving a worrying gap of 84% of fungicides with no data on sublethal effects (lowest observable adverse effect level (LOAEL)) . From thThe data presented here highlighted the harmful effects of fungicide pyraclostrobin, even in residual concentrations, on individual foragers’ fitness, with potentially harmful effects at the colony level. The used biomarkers show high sensitivity at the cellular level and are suitable parameters to be used in future toxicological studies. In conclusion, the present study revealed that the fungicide potentially affected cells involved in the bee’s intermediary metabolism (fat body) and excretion of toxicants , which contributed to reducing knowledge gaps about the sublethal effects of fungicides on bees.


Traumatic spinal cord injury (TSCI) causes neurological dysfunction below the injured segment of the spinal cord, which significantly impacts the quality of life in affected patients. The phosphoinositide 3kinase/serine‐threonine kinase (PI3K/AKT) signaling pathway offers a potential therapeutic target for the inhibition of secondary TSCI. This review summarizes updates concerning the role of the PI3K/AKT pathway in TSCI.By searching articles related to the TSCI field and the PI3K/AKT signaling pathway, we summarized the mechanisms of secondary TSCI and the PI3K/AKT signaling pathway; we also discuss current and potential future treatment methods for TSCI based on the PI3K/AKT signaling pathway.Early apoptosis and autophagy after TSCI protect the body against injury; a prolonged inflammatory response leads to the accumulation of pro‐inflammatory factors and excessive apoptosis, as well as excessive autophagy in the surrounding normal nerve cells, thus aggravating TSCI in the subacute stage of secondary injury. Initial glial scar formation in the subacute phase is a protective mechanism for TSCI, which limits the spread of damage and inflammation. However, mature scar tissue in the chronic phase hinders axon regeneration and prevents the recovery of nerve function. Activation of PI3K/AKT signaling pathway can inhibit the inflammatory response and apoptosis in the subacute phase after secondary TSCI; inhibiting this pathway in the chronic phase can reduce the formation of glial scar.The PI3K/AKT signaling pathway has an important role in the recovery of spinal cord function after secondary injury. Inducing the activation of PI3K/AKT signaling pathway in the subacute phase of secondary injury and inhibiting this pathway in the chronic phase may be one of the potential strategies for the treatment of TSCI. During secondary injury after spinal cord injury (SCI), recovery is impaired by inflammation, cell death and glial scar formation. Therefore, these pathological processes may be potential treatment targets for SCI. The phosphoinositide 3‐kinase/serine‐threonine kinase (PI3K/AKT) signalling pathway has a key role in the secondary injury after SCI. PI3K/AKT signalling pathway can inhibit the inflammation and cell death after SCI, and can also promote the formation of glial scar. PI3K regulates cell proliferation, differentiation, programmed death and migration by activating the downstream target protein kinase B (known as PKB or AKT).2Secondary SCI begins within a few minutes after the primary injury and can last for several weeks or months, leading to progressive damage to the lesion and surrounding spinal cord tissue.2.1Inflammation protects against tissue damage caused by trauma, ischemia, infection or toxins.Microglia are resident immune cells (a type of macrophage) in the central nervous system that respond to early central nervous system damage.In addition to the changes in microglial morphology and the release of chemokines and inflammatory factors, peripheral neutrophils and macrophages are recruited to the injured spinal cord.After SCI, neutrophil infiltration decreases within 1 week, while there is increasing infiltration of macrophages derived from monocytes.Activated immune cells remove cell debris, prevent the spread of damage and secrete anti‐inflammatory factors, which control secondary injury. However, prolonged inflammation leads to the accumulation of proinflammatory factors, which damages normal nerve cells and aggravates SCI. Thus, there is a need to identify the favourable and harmful time points of the inflammatory response, suppress the activation of immune cells and inhibit the release of inflammatory factors during the harmful time period, and promote the polarization of macrophages and astrocytes to the anti‐inflammatory phenotype (M2/A2 phenotype).2.2Cell death is an important cause of secondary injury to tissues surrounding the primary SCI. Cell death is the lysis of cells in an uncontrolled manner , which causes cell contents to flow into the surrounding tissues or in a programmed manner through a series of biochemical and molecular pathways.Apoptosis  of neurons and oligodendrocytes after SCI causes secondary injury.Necrosis was originally regarded as unprogrammed cell death. When necrosis is stimulated by hypoxia or external physical/chemical damage, the permeability of the necrotic cell membrane increases, causing cell swelling, organelle enlargement or deformation and finally cell rupture. The cell contents overflow into the surrounding area, which triggers inflammation and tissue damage.Autophagy is a self‐protective mechanism of cells, a cytoprotective response activated by cells to cope with stress.Various direct and indirect interactions exist between autophagy and apoptosis, suggesting that a coordinated balance between autophagy and apoptosis regulatory proteins determines the cell fate.2.3PTPRS gene, which encodes PTPR σ, significantly enhances axon regeneration after SCI.After the primary injury, inflammatory cells infiltrate into the injured tissue and release inflammatory factors, chemokines and enzymes. In addition, astrocytes, oligodendrocyte precursor cells and fibroblasts are activated, which causes them to undergo hypertrophy, thickening and migration to the centre of the lesion.Based on the aforementioned evidence, glial scars formed at different time points after SCI may have protective or harmful roles in the clinical recovery. In the acute and subacute phases, glial scars limit the expansion of the damage and protect the remaining spinal cord tissue; however, in the chronic phase, mature glial scars form physical and chemical barriers, which hinder axon regeneration and prevent the recovery of nerve function. Therefore, the removal of glial scars in the chronic phase, rather than the acute or subacute phase, may offer a treatment strategy for spinal cord injury.3The PI3K family is divided into three categories (classes I–III) based on differences in primary structure and specificity for lipid substrates. Class I PI3Ks have been studied extensively. In mammals, class I PI3Ks exist in all cell types and are activated by receptor tyrosine kinases, G protein‐coupled receptors and Ras family proteins on the cell membrane.Class II PI3Ks were identified on the basis of sequences homologous to classes I and III PI3Ks, instead of their functions. The mechanism of action of class II PI3Ks is poorly understood and unclear. Class II PI3Ks are mainly present in the intracellular membrane and may be expressed in the nucleus, but they are only present at low levels in the cytoplasm.Class III PI3Ks  were first discovered in yeast. The function of class III PI3Ks is highly conserved from yeasts to mammals; these proteins have key roles in diverse cellular processes.4Many studies have evaluated the roles of the class I PI3K/AKT signalling pathway in secondary SCI, such as the regulation of cell growth and the control of anti‐inflammatory and anti‐apoptosis effects. Here, we discuss the complex roles of the class I PI3K/AKT signalling pathway in secondary injury, mainly related to inflammation, cell death and glial scar formation.4.1The PI3K/AKT signalling pathway has an important role in regulating the inflammatory response after SCI. After SCI, damage‐associated molecular patterns  activate TLRs on inflammatory cells, thus triggering inflammation.The aforementioned studies have shown that the PI3K/AKT signalling pathway plays an important role in regulating the inflammatory response in SCI. PI3K/AKT pathway activation inhibits the activation of immune cells, as well as the release of proinflammatory cytokines. In addition, PI3K/AKT pathway activation may decrease the expression of proinflammatory cytokines by inhibiting the transcription factor NF‐κB. This pathway also reduces the conversion of macrophages to the inflammatory phenotype and limits the secretion of anti‐inflammatory factors. Therefore, activation of the PI3K/AKT signalling pathway via inflammation inhibition offers a potential treatment strategy for SCI.4.2The PI3K/AKT pathway has an important role in regulating programmed cell death. PI3K/AKT pathway activation during necroptosis inhibits MLKL activation and necroptosis.PI3K/AKT is a key signal pathway involved in the regulation of apoptosis. PI3K/AKT signalling pathway activation reduces the expression levels of caspase‐9 and ‐3; it inhibits apoptosis after SCI in rats.As mentioned previously, PI3K/AKT signalling pathway activation inhibits apoptosis. In theory, PI3K/AKT signalling pathway activation promotes autophagy. However, multiple studies have shown contradictory results. The autophagy inhibitor mTORC1 is activated by phosphorylated AKT; therefore, inhibition of the PI3K/AKT/mTOR pathway may promote autophagy.Inhibition of mTORC1, a downstream target of the PI3K/AKT signalling pathway, promotes autophagy and inhibits apoptosis. However, activation of the PI3K/AKT signalling pathway may have anti‐apoptotic effects. Further research is needed to determine whether activation or inhibition of the PI3K/AKT signalling pathway has anti‐apoptotic effects. To produce rigorous results, future research should use models to modify the doses of activators or inhibitors of the PI3K/AKT signalling pathway, effects on other signalling pathways, durations of action, time points to detect the impact on apoptosis or autophagy and cell types.4.3PI3K/AKT signalling pathway activation is the main determinant of glial scar formation5PI3K/AKT, a classical signalling pathway, has an important role in secondary SCI and is therefore a potential therapeutic target. Numerous studies have shown that activators or inhibitors of the PI3K/AKT signalling pathway can be used to treat SCI. Here, we discuss the roles of these factors in the treatment of SCI through regulation of the PI3K/AKT signalling pathway, including non‐coding RNA, biomolecules and drugs.5.1According to the Human Genome Project, less than 2% of the RNA sequences generated by human DNA can be translated into proteins. Most of the remaining RNA sequences cannot be translated and are known as non‐coding RNAs (ncRNAs). Although ncRNAs cannot be transcribed into proteins, they have indispensable roles in the growth and development of organisms through their effects on transcription and translation. Abnormal expression of ncRNAs after SCI has been shown to affect the development of secondary injury.+‐dependent deacetylase with important roles in the regulation of apoptosis and inflammation. The expression of miR‐138‐5p increases after SCI; miR‐138‐5p can directly bind to the 3′‐UTR of sirtuin 1. miR‐138‐5p inhibition has been shown to reduce apoptosis and inflammation by upregulating sirtuin 1, thereby decreasing PTEN expression and activating AKT.miRNAs are highly conserved ncRNAs, approximately 22 nucleotides in length, which bind to the 3′‐untranslated regions (3′‐UTRs) of mRNAs through complementary base pairing. Such interactions inhibit protein synthesis through mRNA degradation (caused by complete base pairing) or transient translation arrest (caused by incomplete base pairing). A single miRNA can target multiple mRNAs and bind to multiple miRNAs.lncRNAs are ncRNAs with >200‐nucleotide transcripts; they can participate in the regulation of protein‐coding genes at multiple levels, including epigenetic regulation, transcription regulation and post‐transcriptional regulation. In addition, some lncRNAs bind to miRNAs, thus acting as miRNA ‘sponges’ by sharing a common binding site with miRNAs and preventing miRNAs from binding to their target mRNAs.circRNAs are single‐stranded transcripts with a covalent closed loop structure. The lengths of circRNAs range from tens to thousands of base pairs. Compared with linear RNAs, circRNAs have a more stable structure. The circRNAs regulate gene transcription, participate in protein translation and regulate RNA‐binding proteins. In addition, similar to lncRNAs, circRNAs act as ‘sponges’ for miRNAs, thus inhibiting miRNA functions.5.2In addition to the receptor tyrosine kinase, G protein‐coupled receptor and Ras families, various cellular molecules can regulate the PI3K/AKT signalling pathway after SCI Table . Nerve gFibroblast growth factor (FGF) is a family of cell signalling molecules released by various tissues; it has a wide range of biochemical and biological properties. FGF10, a member of the FGF family, has important roles in the regulation of several biological functions, such as morphogenesis, proliferation and the inhibition of apoptosis.IGF‐1 is an important neurotrophic factor that regulates neuron growth, dendritic branching and synapse formation.After SCI, cell necrosis leads to increased levels of extracellular glutamate, which promotes inflammation, glial scar formation and neuronal cell death. Blood glutamate scavengers, such as the blood resident enzyme glutamate‐oxaloacetate transaminase 1 and its co‐substrate oxaloacetate, remove excess glutamate from the spinal cord tissue to the systemic blood circulation; this process facilitates the neutralization of glutamate and its transamination into 2‐ketoglutarate. In blood glutamate scavenger‐treated SCI mice, the blood glutamate level was significantly reduced; moreover, p‐AKT protein expression was increased, the expression levels of proinflammatory factors  were significantly decreased, and the expression level of caspase‐3 in nerve cells at the lesion site was reduced.Oestrogen is a class of steroid compounds with a wide range of biological activities. Because of its strong antioxidant, anti‐inflammatory and anti‐apoptotic properties, oestrogen has been widely studied to investigate its potential therapeutic role for nerve injury after SCI. Oestrogen and its agonists can reduce PTEN expression and activate AKT to inhibit apoptosis after SCI.In addition to the previously mentioned biomolecules, many other biomolecules can promote recovery after SCI by activating the PI3K/AKT signalling pathway. Neuromodulin‐1β1,5.3Several drugs have been found to exhibit neuroprotective effects in SCI Table . MetformFTY720 is a functional antagonist of sphingosine 1‐phosphate receptor‐1; it is widely used as an immunosuppressant in various diseases.The polyunsaturated fatty acid docosahexaenoic acid is a highly unsaturated fatty acid that is essential for brain nutrition. The use of docosahexaenoic acid prevents the deposition of cholesterol on the blood vessel wall and reduces atherosclerosis and coronary heart disease.In addition, various natural compounds extracted from plants activate the PI3K/AKT signalling pathway and participate in the treatment of SCI. In both in vitro and in vivo models of SCI, baicalin,6Secondary injury after SCI is a global problem. There is a need to understand the pathological processes involved in secondary SCI, then identify potential therapeutic targets to prevent the progression of secondary injury. This review summarized the pathological processes and mechanisms underlying inflammation, cell death and glial scar formation in secondary injury. Early apoptosis and autophagy after SCI protect the body against injury; a prolonged inflammatory response leads to the accumulation of proinflammatory factors and excessive apoptosis, as well as autophagy in the surrounding normal nerve cells, thus aggravating SCI in the subacute stage of secondary injury. Initial glial scar formation in the subacute phase is a protective mechanism for SCI, which limits the spread of damage and inflammation. However, mature scar tissue in the chronic phase hinders axon regeneration and prevents the recovery of nerve function. Therefore, in the acute phase of SCI, surgical intervention and suppression of inflammatory responses may be needed. In the subacute phase, apoptosis, disruption of autophagic flow and excessive autophagy should be suppressed. In the chronic phase, glial scars can be ablated to promote axon regeneration. These strategies may be feasible for the treatment of SCI, but additional studies are necessary to compare the onset timings of acute, subacute and chronic phases.The PI3K/AKT signalling pathway has an important role in the recovery of spinal cord function after secondary injury. Activation of the PI3K/AKT signalling pathway can inhibit the activation of immune cells and block the release of proinflammatory cytokines. In addition, this pathway can promote the polarization of macrophages to the anti‐inflammatory type and lead to increased secretion of anti‐inflammatory factors. The PI3K/AKT signalling pathway can also regulate the programmed death pathway. Furthermore, activation of this pathway inhibits apoptosis after SCI through effects on the extrinsic or intrinsic apoptosis pathway; it also inhibits excessive autophagy and cell death. However, inhibition of this pathway can promote autophagy, which leads to inhibition of apoptosis. This contradictory biological function may be explained by differences in time points, activators or inhibitors and cell types used in vitro experiments. Many studies have explored the biological effects of the activation or inhibition of the pathway at different time periods by knocking out or knocking in key molecules of the pathway. In addition, PI3K/AKT signalling pathway activation can promote the proliferation of spinal cord astrocytes, whereas inhibition of this pathway can reduce the formation of glial scars and promote recovery after SCI. Therefore, PI3K/AKT signalling pathway activation is involved in the subacute phase of secondary injury; inhibition of this pathway in the chronic phase may offer a strategy for the treatment of SCI.In summary, this review discussed the pathological processes of inflammation, cell death and glial scar formation in secondary SCI, as well as the potential therapeutic effects of the PI3K/AKT signalling pathway. Additional studies are needed to improve the general understanding of the pathogenesis of secondary SCI and the complex mechanism of action of the PI3K/AKT pathway, thus establishing a comprehensive theoretical basis for the treatment of SCI based on the PI3K/AKT pathway.Xuegang He, Yong Yang and Xuewen Kang conceived and designed the review. Xuegang He drafted the manuscript; Ying Li and Bo Deng assisted in the preparation of the charts; Aixin Lin, Guangzhi Zhang and Miao Ma edited and revised the manuscript; all authors read and approved the final version of the manuscript.The authors declare no competing interests.


Predicting neurological outcomes after cardiac arrest remains a major issue. This study aimed to identify novel biomarkers capable of predicting neurological prognosis after cardiac arrest. Expression profiles of GSE29540 and GSE92696 were downloaded from the Gene Expression Omnibus (GEO) database to obtain differentially expressed genes (DEGs) between high and low brain performance category (CPC) scoring subgroups. Weighted gene co-expression network analysis (WGCNA) was used to screen key gene modules and crossover genes in these datasets. The protein-protein interaction (PPI) network of crossover genes was constructed from the STRING database. Based on the PPI network, the most important hub genes were identified by the cytoHubba plugin of Cytoscape software. Eight hub genes  were finally screened and validated, which were downregulated in the group with poor neurological prognosis. In addition, GSEA identified critical pathways associated with these genes. Finally, a Pearson correlation analysis showed that the mRNA expression of hub genes EEF1B2, PSMD14, RPFDN5, RBX1, and SNRPD2 were significantly and positively correlated with NDS scores in rats. Our work could provide comprehensive insights into understanding pathogenesis and potential new biomarkers for predicting neurological outcomes after cardiac arrest. Cardiac arrest (CA) remains a major health burden around the globe, most often occurring in the community with a worldwide incidence of approximately 96 cases per 100,000 people per year ,3. Post-Among all the serum markers used to assess the neurological prognosis of comatose survivors, neuron-specific enolase (NSE) is the most widely studied measure for predicting neurological outcomes after CA. Research has shown the importance of NSE in outcome prediction, and it has been recommended for use in clinical practice ,14. In aThe use of high-throughput sequencing/gene chips to mine new biomarkers is expected to meet these new stringent requirements and could elucidate the molecular mechanisms underlying poor prognosis. In a previous study, Iwahana et al. used RNA-sequencing to analyze genetic markers that can predict LV reverse remodeling in advanced non-ischemic heart failure and showed that the novel myocardial markers GADD45G and NDUFS5 can play this predictive role . In addiHerein, we identified differentially expressed genes (DEGs) between the high and low cerebral performance category (CPC) score groups by analyzing the mRNA expression profiles and clinical information of GSE29540 and GSE92696. Then, an enrichment analysis of the DEGs was conducted to reveal the potential molecular mechanisms associated with neurological outcomes after CA, and WGCNA analysis was applied to explore the relationship between DEGs and clinical parameters. The significant gene modules associated with a high CPC score were also selected. Next, PPI network analysis was performed by Cytoscape’s cytoHubba plug-in to screen out eight hub genes, and co-expressed genes and potential pathways of the eight hub genes were also investigated. These central genes were further experimentally validated by animal models and correlation analysis was performed to analyze the correlation between hub genes and NDS scores in rats. Our work may provide new and in-depth insights for predicting neurological prognosis and exploring potential molecular mechanisms.The mRNA expression profiles and clinical information of GSE29540 and GSE92696 were acquired from the GEO Datasets. GSE29540 was based on the GPL13218 platform with 84 blood samples with low CPC scores (CPC 1–2) and 56 blood samples with high CPC scores (CPC 3–5). GSE92696 was also based on the platform GPL10558 with 10 blood samples with low CPC scores (CPC 1–2) and 12 blood samples with high CPC scores (CPC 3–5). CPC scores 3–5 was defined as poor prognosis of neurological function, while CPC score 1–2 was defined as good neurological prognosis function.p-value < 0.05. Genes that were simultaneously upregulated or downregulated in both two datasets were selected as DEGs for subsequent analysis. The R package “ggplot2” and “pheatmap” were used for volcano plots and heatmaps respectively.Before performing differential expression analysis, the data were homogenized using the R (version 4.1.1) package “preprocessCore”. After pre-processing, DEGs were screened using the “limma” package with a threshold of Functional enrichment analysis of DEGs was carried out using the R package “clusterProfiler”, including Gene Ontology (GO) terms and the KEGG pathways. Gene set enrichment analysis (GSEA) was performed using the R package “clusterProfiler” to explore the function of the hub genes.The co-expression network of DEGs was investigated using the R package “WGCNA”, and the gene modules associated with high CPC scores were screened using GSE92696. The gene modules most relevant to high CPC scores were selected for further PPI construction.https://string-db.org/, accessed on 10 April 2022) database to build a protein–protein interaction (PPI) network. Cytoscape (V3.7.2) was used to visualize the PPI networks. The cytoHubba plugin in Cytoscape was used to identify hub genes. Three algorithms, including Degree, Stress, and Radiality were used to identify hub genes in the PPI network. The top 12 ranked hub genes of the three algorithms were selected and intersected. Key genes from WGCNA were inputted into the STRING  and model group (n = 14). The 10-min asphyxial cardiac arrest (CA)model was performed in our study [A total of 21 male adult Wistar rats (350–400 g) were obtained from the Animal Experimental Center of Southern Medical University and randomly divided into sham-operated group  and paraffin-embedded for TUNEL staining, and the rats were euthanized 24 h after the operation. Then the hippocampal region of each brain (3.2 to 4.2 mm from the bregma) was cut into sections of 10 μm sections, and two sections from each sample were randomly selected for in situ cell death detection using a Roche In Situ Cell Death Detection Kit  according to the manufacturer’s protocol for paraffin-embedded tissues.Paraffin-embedded hippocampal sections were heated at +60 °C followed by washing in xylene and rehydrating through a graded series of ethanol and double-distilled water. Then the sections were placed in a plastic jar containing 200 mL 0.1 M citrate buffer and 300 W microwave irradiation was applied for 5 min. After rinsing twice with PBS, the sections were incubated with the TUNEL reaction mixture in a dark humidified chamber at 37 °C for 1 h. Then, the sections were washed with PBS and incubated for 30 min. After washing with PBS, the sections were stained with 4′-6-diamidino-2-phenylindole (DAPI) for 15 min. The number of neurons in the pyramidal layer (200 μm in length) of the CA1 region of the Hippocampal was analyzed using a fluorescence microscope equipped with the Image-pro Plus System . The number of apoptotic neurons induced by ischemia–reperfusion was determined by comparing the number of TUNEL-positive cells with the number of DAPI-positive cells.Rats were euthanized within 24 h after the cardiopulmonary resuscitation, and three rats in each group were assigned to H&E staining and Nissl staining to determine morphological changes in the CA1 region of the hippocampus. Brain tissue was routinely embedded in paraffin and cut into 10-μm coronal sections and brain tissue sections were stained with H&E or Nissls for routine histological examinations and the morphological changes were observed under a light microscope .The total RNA of rat hippocampal tissue was extracted with a trizol reagent, and the RNA concentration was measured using a nanodrop 2000 instrument, followed by conversion of RNA to cDNA using Takara RT Kit (RR047a). TB Green™ Premix Ex Taq™ II(Tli RNaseH Plus) (RR820Q) was performed on the CFX96 Real-Time PCR Detection System  to evaluate relative mRNA levels. The 2-ΔΔCt method was employed to measure the relative mRNA expression , and all experiments were carried out three times. The primers used in this study were produced by Servicebio , and the sequences are shown in Four animals per group were used to assess the protein levels of RPL26, PSMD14, HINT1, RBX1, EEF1B2, RPL27, SNRPD2, and PFDN5. The hippocampus of the rat was rapidly removed and frozen at −80 °C after decapitation. Samples were homogenized in a standard lysis buffer  and protease inhibitor solution  and centrifuged at 12,000 rpm for 20 min. The lowry method was selected to determine the protein concentration. The proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membranes. The membranes were blocked with 5% BSA in tris-buffered saline and then incubated at 4 °C overnight with respective primary antibodies  deviation and the count data were expressed using M . In comparison, a student’s T-test was used between two groups. For comparisons between three groups, one-way ANOVA was used. While, for comparisons between the 3 groups, an analysis of variance was used. In addition, a Pearson correlation analysis was used to analyze the correlation between gene expression levels and protein expression levels of the 8 hub genes and NDS scores in rats. RBefore performing differential expression analysis, the mRNA expression data was homogenized to get more accurate results . The difFunctional enrichment of these DEGs was performed to identify possible biological functions. For the results of KEGG, pathways of neurodegeneration-multiple diseases, Parkinson’s disease, Prion’s disease, and Alzheimer’s disease pathways were enriched E. GO anaWe further conducted WGCNA in GSE92696 and used DEGs to screen for key gene modules associated with high CPC scores. A PPI network of 175 DEGs was constructed using the STRING database A. Using The ssGSEA function of the R package “GSVA” was used to evaluate the infiltration level of 23 immune cells. We found that the infiltration level of activated B cells and immature B cells was higher in the high CPC score group A. The coThe correlation between hub genes and all genes was analyzed in GSE92696. The heatmaps of the top 50 genes with positive correlations are displayed, respectively, in We further constructed a rat CA model. The neurological function of rats after CA was evaluated according to the international NDS score. The rats were divided into the sham group, the NDS high group, (NDS > 60) and the NDS low group (NDS < 60). The representative pictures of H&E staining, Nissl staining, and Tunel staining were displayed in Furthermore, the correlation between gene expression levels and protein expression levels of the eight hub genes and NDS scores in rats was also analyzed. Our results showed a positive correlation between the protein expression of PSMD14, RPL26, PFDN5, RBX1, SNRPD2, and the NDS scores of rats, whereas there was no correlation between the protein expression levels of EEF1B2, RPL27, HINT1, and the NDS score of rats A. In addCardiac arrest is common, and the consequences of it can be devastating. The cause of cardiac arrest is most often cardiogenic disease, the others including respiratory insufficiency, neurological causes, and so on ,25. HypoRecently, research showed that transcriptomics plays an essential role in assessing neurological prognosis with brain injury. Meng et al. found that genomic information has the potential in predicting trauma brain injury (TBI) pathogenesis, and hippocampal transcriptome signatures were assosiated with behavior phenotypes . Osier eTo date, our study is the first work performed to analyze the gene expression profiles of GSE29540 and GSE92696 and reveal important prognostic markers. The PPI network of 175 DEGs was constructed using the STRING database using three algorithms . Finally, eight overlapping genes were identified, including RPL27, EEF1B2, PFDN5, RBX1, PSMD14, HINT1, SNRPD2, and RPL26, which were downregulated in the high CPC score group. To the best of our knowledge, all these genes have not ever been studied in CA. We further explored the correlation of the eight hub genes and revealed that these genes were positively correlated with each other. Then, we analyzed the potential function of hub genes to predict the outcome of patients with CA. EEF1B2, PSMD14, HINT1, and SNRPD2 were proved to be eligible predictors (AUC > 0.7). It was reported that a novel biallelic loss of EEF1B2 function was reported to be associated with autosomal recessive intellectual disability . In addiIn the early stages of post-cardiac arrest syndrome, the immune cell reactivity could explain the potent role of rapid hypothermia . We founWe further constructed a rat CA model to validate the expression of the eight hub genes. The neurological function of rats after CA was evaluated according to the international NDS score. We examined the mRNA and protein levels of the eight hub genes and found that the mRNA and protein levels of the eight hub genes were all lowest in the low NDS group and highest in the control group, which was consistent with our analysis results. In addition, we performed a Pearson correlation analysis to investigate the correlation of gene expression profiles with NDS scores and prognosis. The results showed a positive correlation between the protein expression of PSMD14, RPL26, PFDN5, RBX1, SNRPD2 and the NDS score of rats, whereas there was no correlation between the protein expression level of EEF1B2,HINT1,RPL27 and the NDS score of rats. In addition, the mRNA levels of all these eight genes were significantly and positively correlated with the NDS score. This is the first study is to correlate gene expression profiles with NDS scores in this field. Previously, Osier et al. reported the associations between genetic polymorphisms related to blood-based biomarkers and neurological outcomes after TBI; in their study, the variation in S100B was found to significantly predict the outcome . HoweverThere are limitations to this study, and more future research is needed. The ideal biomarker should be easy to detect, stable, and sensitive. Biomarkers in peripheral blood are often available . In the In summary, our study identified eight potential biomarkers predicting neurological outcomes after cardiac arrest and investigated their potential pathways and the correlation of these gene expression profiles with NDS scores. Our work provides a new direction for future research.