Ryegrass tilled and returned to the field increased soil organic matter content by 6.89–7.92%, increased rhizosphere soil available phosphorus content by 2.22–17.96%, and converted the soil non-exchangeable potassium into potassium that was available for plant absorption and utilization. Ryegrass tilling and returning to the field increased the potassium content of middle leaves of flue-cured tobacco by 7.69–10.07%, the increased potassium content in flue-cured tobacco was accompanied by increased total sugar, reducing sugar, and the ratio of reducing sugar to nicotine, which facilitated the harmonization of the chemical composition of cured tobacco leaves. Moreover, the increased number of markedly improved operational taxonomic units enhanced the complexity of the soil bacterial community and its compactness after ryegrass tillage and their return to the field. The available potassium, available phosphorus, total potassium content, pH, and sampling period of the rhizosphere soil had considerable effects on the rhizosphere microbial. Ryegrass tilling and returning to the field changed the soil microbiome, which increased the abundance of bulk soil Proteobacteria, rhizosphere soil Fibrobacterota, and microbes with anti-pathogen activity ; and reduced the abundance of pathogenic fungi Neocosmospore genus in the soil. In brief, ryegrass returned to the field, improved soil microecology and restored soil nutrients, and established a new dynamic balance of soil ecology, thereby improving the quality of cultivated land and the quality of flue-cured tobacco.The widespread and continuous cultivation of tobacco has led to soil degradation and reduced crop yields and quality. Green manure is an essential organic fertilizer that alleviates obstacles to continuous cultivation. However, the plant–soil microecological effects of green manure on flue-cured tobacco cultivation remain unclear. Thus, a positioning trail including two treatments, chemical fertilizer application only (treatment NPK) and chemical fertilizer application with turning ryegrass (treatment NPKG) was conducted, and the effect of ryegrass returning on the soil physicochemical properties, soil microbiome, crop yield, and quality of flue-cured tobacco in continuous cropping soil were investigated. Results showed that returning ryegrass to the field increased the thickness of soil humus layer from 13 cm to 15 cm, reduced the humus layer soil bulk density to 1.29 cm Green manure has attracted the interest of many researchers due to its ability to accomplish both food safety and green farming development  and chemical fertilizer application with turning ryegrass (treatment NPKG) was conducted, and the effect of ryegrass returning on the growth and development of flue-cured tobacco, soil properties, and soil microbiome in continuous cropping soil were investigated. Further, the relationship between soil physicochemical properties and soil microbiome was analyzed. This research provides fundamental knowledge that mitigates the detrimental effects of continuous cultivation and enhances the quality and yield of flue-cured tobacco to promote sustainable agricultural production.2.2.1.2 plots representing the overall growth of ryegrass in the plot were selected. The ryegrass was dug out with the roots, and the amount of tillage in each plot was counted after removing the soil and impurities. The ryegrass tilled in 2019, 2020, and 2021 were 6297.58, 7807.68, and 7496.53 kg/hm2 (on dry a weight basis), respectively. Ryegrass was irrigated, tilled, and not fertilized during the planting period. The test soil was sandy loam with the following basic physicochemical properties: pH 7.57, organic matter (OM) 16.76 g/kg, total nitrogen (TN) 0.69 g/kg, alkaline nitrogen (AN) 32.40 mg/kg, available phosphorus (AP) 29.19 mg/kg, and available potassium (AK) 135.47 mg/kg.The investigation was conducted in the Science and Education Park of Henan Agricultural University , Zhengzhou, China. It has a north temperate continental monsoon climate, with an average annual temperature of 14°C, an average annual rainfall of about 640 mm, a frost-free period of 220 days, and 2,400 h of annual sunlight. Ryegrass was planted for three seasons before the start of the positioning trial to ensure homogeneous soil fertility. The first ryegrass was sown on April 28, 2017, and was removed from the field on September 5, 2017. On September 22, 2017, the ryegrass was sown for the second time, and on May 10, it was removed from the field. Ryegrass was sown for the third time on May 26, and removed from the field on August 31, 2018. After homogenizing the fields, ryegrass was planted on October 27, 2018, and turned over on March 28, 2019. We started growing flue-cured tobacco in 2019 and returned the entire amount of ryegrass to the tilling plots planted with ryegrass. Before tilling, two 1 m2 (12 m × 5.5 m) was arranged in randomized groups. Cement slabs  were set around the plot to prevent fertilizer and water from crossing the irrigation. The dosage of N was 52.5 kg/hm2, N: P2O5: K2O were 1: 2: 3. Special compound fertilizers (N: P2O5: K2O = 8: 12: 20), potassium sulfate (K2O 52%), and calcium magnesium phosphate (P2O5 16%) were used as base fertilizers, which were applied by band application before transplanting. Potassium nitrate (N: P2O5: K2O = 13.5: 0: 44.5) was applied as topdressing and dissolved in water for hole application.Two treatments were tested: no ryegrass, chemical fertilizer application only (treatment NPK) and chemical fertilizer application with turning ryegrass (treatment NPKG). Each treatment was replicated three times for six plots, and each plot with an area of 66 mThe tobacco variety tested was Zhongyan 100, with 110 cm row spacing and 50 cm plant spacing. Seedling transplantation was performed on April 30, 2020, and April 29, 2021, and harvesting was completed on September 15, 2020, and September 6, 2021. Other management measures were implemented in accordance with local management standards for quality tobacco leaves.2.2.2.2.1.Bulk and rhizosphere soils were collected 45 and 75 days after transplanting the tobacco seedlings, respectively. Bulk soil collection: The middle position of two tobacco plants was collected at 0–20 cm using a soil auger. Five soil samples were collected from each plot and mixed as one sample. Rhizosphere soil collection: Sampling was carried out using destructive methods. The complete tobacco root system was dug and tapped to allow the loose soil combined with the roots to fall naturally. Then, the soil that was tightly combined with the root system (within 2 mm) was placed into the sampling bag, and the rhizosphere soil samples were collected. Three tobacco plants were selected from each plot to collect the rhizosphere soil, and the rhizosphere soil from the same treatment was mixed as one sample. Each soil sample was divided into two parts: one part was stored in a refrigerator at −80°C for high-throughput sequencing of soil microbrome, and the other was dried in the shade and screened for soil physicochemical indicators. After the flue-cured tobacco was mature and harvested, the soil profile was observed, and soil samples from different soil layers were collected to determine their bulk density.2.2.2.The ring-knife method was used to determine bulk soil density and soil pH was determined using a pH meter (soil: water = 1: 2.5) . AN and 2.2.3.Before harvesting the flue-cured tobacco in 2020 and 2021, three representative tobacco plants were selected for each plot. The height, stem circumference, maximum leaf length, and maximum leaf width of each tobacco plant were determined according to the standard method of YC/T 142–2010. The selected tobacco plant was then centered on the stem base, and the surrounding 30 cm root belt soil was removed. The roots, stems, and leaves of the tobacco were separated and washed with tap water and deionized water. The tobacco plant was placed in an oven for 15 min at 105°C and then dried at 65°C until constant weight.A continuous flow analyzer  was used to determine the total sugar, reducing sugar, nicotine, chlorine, and potassium contents of flue-cured tobacco leaves according to the standards YC/T159-2002, YC/T160-2002, YC/T217-2007, and YC/T162-2011, the ratio of potassium to chloride, the ratio of reducing sugar to nicotine, and the ratio of reducing sugar to total sugar.2.2.4.DNA was extracted from the soil samples using the MagPure Soil DNA KF Kit. Subsequently, the purity and concentration of DNA were detected by agarose gel electrophoresis, and PCR amplification was performed using specific primers with barcodes and high-efficiency, high-quality fidelity enzymes. Bacterial amplification was performed using 343F 5’-TACGGRAGGCAGCAG-3′ and 798R 5’-AGGGTATCTAATCCT-3′. The internal transcribed spacer (ITS) segments of fungal amplification, ITS1F 5’-CTTGGtcatttagaggaagtaa-3′ and ITS2 5’-GCTGCGTTCTTCATCGATGC-3′. Shanghai Ouyi Biomedical Technology was commissioned to perform the sequencing and biological information analysis using a MiSeq sequencer (Illumina). The raw tags were trimmed, merged, and assigned using the split library software (version 1.8.0) in QIIME to remove sequences with more than eight single-base repeats and sequences with lengths less than 200 bp to obtain clean tag sequences. The quality sequence valid tags obtained from the quality control were classified into operational taxonomic units (OTUs) based on 97% similarity using the Vsearch (version 2.4.2) software, and the most abundant sequence in each OTU was selected as the typical sequence of that OTU.2.3.p < 0.05). The microbial co-occurrence networks for the different treatments were graphed using Gephi software (version 0.9.2), and the topological properties of the networks were calculated. Principal component analysis (PCoA) based on the Bray-Curtis distance matrix was used to analyze the differences in the beta diversity of the microbial community. Redundancy analysis (RDA) was performed using the “vegan” package in R (4.2.2) to assess the relationship between soil physicochemical factors and soil microbial communities. Linear discriminant analysis effect size (LEfSe) was used to search for biomarkers that differed significantly between treatments, with a linear discriminant analysis score of two.The least significant difference (LSD) test in one-way analysis of variance was used to analyze the differences and significance of soil physicochemical properties, flue-cured tobacco agronomic traits, dry matter accumulation, and soil microbial diversity among the different treatments. Co-occurrence network analysis was performed on OTUs with a total abundance in the top 200 to characterize the complex links between microbial communities. OTUs that were statistically significantly correlated were calculated using the “psych” package in R  data was obtained with a distribution of 65,558–72,622. The fungal ITS rRNA was sequenced, and the data volume of valid tags obtained by removing the chimeras of clean tags was distributed in 38,028–64,929 . There wCo-occurrence network analysis of the soil microbial community was conducted at the OTU level to further explore the complexity of the microbial community relationships among the different treatments. As shown in The response of the soil microbial community after ryegrass tillage and return to the field at the OTU level was specifically analyzed using volcano plots. As shown in To further clarify the differences in the microbial community composition among the samples, the soil bacterial and fungal communities were analyzed by PCoA based on the Bray-Curtis algorithm, as shown in p < 0.05). The results indicated that ryegrass tilling and returning to the field for 3 years significantly changed the soil microbiome.PCoA was performed on the soil microbial communities of the NPK and NPKG treatments for the same period to further explore the timing effect on the soil microbial community after ryegrass tillage and return. As shown in 3.5.The relative abundance and variance analyses of soil microbiome at the phylum level are shown in Prevotella, Sphingomonas, AKAU4049, MND1, Muribaculaceae, S0134_terrestrial_group, Lysobacter, Nitrospira, Subgroup_7, Dongia. The LEfSe results showed that tilling ryegrass significantly increased Sphingomonas, Lysobacter, and Longimicrobiaceae in bulk soil and Sphingomonas and Pseudarthrobacter in rhizosphere soil. The AKAU4049 and S0134_terrestrial_group genera showed notable or extremely significant reductions in the bulk and rhizosphere soils.To further explore the changes in soil microbial communities, changes in soil microbial communities at the genus level were analyzed . At the Mortierella, Chaetomium, Coprinellus, Neocosmospora, Minimedusa, Paurocotylis, Stachybotrys, and Preussia. The soil fungi Chaetomium, Minimedusa, and Paracylindrocarpon increased significantly, whereas the bulk and rhizosphere soil fungi Neocosmospora markedly decreased after ryegrass tillage and its return to the field.At the fungal genus level, the dominant genera shared by the bulk and rhizosphere soils were 3.6.p values for all four models were less than 0.05, indicating that all soil environmental factors significantly affected soil microbiome. To investigate the effects of soil environmental factors on the soil microbiome, RDA analyses were performed at the OTU level, and correlation heatmap analyses were conducted between the soil physicochemical properties and soil microbial community composition at the phylum level. As shown in 4.4.1.Generally, the soil profile is a comprehensive measure of the internal and external morphologies of soil. In this study, returning ryegrass to the field increased the thickness of the soil humus layer, and large soil particles were produced in the deposition layer. Returning ryegrass to the field also reduced the bulk density of soil humus and leaching layers. On the one hand, the well-developed and dense root system of ryegrass is deeply rooted in the soil to produce a drilling effect , which rThe soil pH decreased after returning the ryegrass to the field, which was due to the ability of the ryegrass root system to secrete small molecule organic acids  and the 4.2.The stem circumference, plant height, and dry matter weight of flue-cured tobacco were significantly higher after turning ryegrass, mainly because tilling ryegrass back into the field improved the soil structure and fertility. Ryegrass tillage increases the content of the rapidly AN, AP, AK, and OM in the soil, tillage of ryegrass reduces the soil bulk, loosens the soil, promotes root development, and enhances the ability of tobacco plants to absorb nutrients and water from the soil, thus facilitating the development and reproduction of tobacco plants. The content and coordination of the conventional chemical components in flue-cured tobacco leaves are important for their quality of tobacco leaves. Total sugar, reducing sugar, nicotine, chlorine, and potassium contents in tobacco measure the intrinsic quality, aroma, and taste of tobacco. In this study, ryegrass tillage increased the potassium content, which was closely related to the increase in soil AK content. Potassium in flue-cured tobacco is mainly derived from the soil system and is then absorbed into the body through the root system. The ratio of reducing sugar to nicotine is an important indicator for controlling the quality of cigarettes, which not only reflects the balance between acidic and alkaline substances in tobacco but also characterizes the coordination of the chemical composition of tobacco. In this study, returning ryegrass reduced the nicotine content, increased the potassium, total sugar, and reducing sugar contents, and reduced the sugar-to-nicotine ratio of flue-cured tobacco, indicating that returning ryegrass promotes harmonization of the chemical composition of flue-cured tobacco.4.3.S0134_terrestrial_group genus and AKAU4049 genus) in the soil after returning ryegrass also showed that the soil entropy of the returning ryegrass was suitable and rich in nutrients. Both Lysobacter and Sphingomonas genera were significantly higher in bulk soil with ryegrass than in soil without ryegrass. Lysobacter produces a variety of extracellular enzymes and metabolites with antagonistic activities against various soil-borne diseases. Sphingomonas also has the ability to degrade a wide range of organic pollutants (Chaetomium and Minimedusa increased significantly, whereas Neocosmospore decreased in both the rhizosphere and bulk soil after returning ryegrass. Chaetomium not only promotes the degradation of OM, such as cellulose, cellulose disaccharides, and lignin but also has biocontrol effects on various plant pathogens. Minimedusa also has been shown to be effective against fungal diseases and has a good control effect (Neocosmospore is pathogenic genera that cause rot in the stems, roots, and tubers of Solanaceae crops (An increase in soil fertility is inseparable from the contribution of the soil microbiome. In this study, the dominant phylum Proteobacteria increased after returning ryegrass, which is closely related to the organic carbon and OM produced by the decomposition of ryegrass in the soil. The strong positive correlation between rhizosphere soil OM content and Proteobacteria after ryegrass tillage and return to the field confirmed the response of Proteobacteria to soil OM, which is consistent with the findings of llutants . An incrl effect . Neocosmae crops . These r5.Ryegrass tilling and returning to the field provides an important carbon source for soil microbiome and promotes microbiome reproduction. The large number of microbiome in turn promoted the decomposition of ryegrass, increased the thickness of the soil humus layer, and reduced the soil bulk density of the plow layer. Returning ryegrass into the field also provides a favorable environment for the reproduction of microbiome with antibacterial function that inhibits the propagation of pathogenic microorganisms and enhances the soil microorganism community toward the direction of making good use of soil nutrients, activating soil nutrients while establishing a new dynamic balance of soil nutrients, thereby improving the quality of cultivated land and raising the quality of flue-cured tobacco. This study provides a technical basis for using the winter idle time of continuous tobacco fields to plant and return ryegrass on limited arable land to alleviate the strain on arable land resources and improve tobacco yield and quality.The data presented in the study are deposited in the NCBI repository, under accession numbers PRJNA990362 and PRJNA994851.HZ: Formal analysis, Writing – original draft. MZ: Investigation, Writing – original draft. JY: Data curation, Writing – review & editing. JW: Writing – review & editing. YC: Writing – review & editing. XY: Funding acquisition, Writing – review & editing, Conceptualization.


Rhabdomyolysis (RM) refers to a clinical syndrome in which muscle cells are damaged by various causes and the clinical manifestations are mainly muscle pain, weakness, and dark urine. Acute kidney injury (AKI) is a serious complication of RM with complex mechanisms and high mortality. Therefore, understanding the pathogenesis and clinical manifestations, early diagnosis and treatment of RM are crucial to improve its prognosis. P < 0.05 indicates a statistically significant difference between the groups.  Analysis of medical records of RM patients admitted to Tianjin Medical University General Hospital from October 2019 to October 2022. Statistical software SPSS 25.0 was used to analyze the data. The risk factors of RM-complicated AKI were analyzed by logistic regression. The receiver operating characteristic (ROC) curve was plotted, the area under the curve (AUC) was calculated, and the optimal cutoff value was determined by the Youden index.  Among the 71 patients, the median age of the patients was 53.0  years and was 2.5 times higher in men than in women. Infection was the most common etiology. History of alcohol consumption, CK, and creatinine were independent influencing factors for AKI due to RM. Logistic regression analysis showed that CK combined with creatinine had a better predictive value than the single index.  Our study revealed the clinical and laboratory characteristics of RM in the population attending the Tianjin Medical University General Hospital in the last three years, which is a reference for future multicenter, prospective studies. Rhabdomyolysis (RM) is a clinical syndrome in which skeletal muscle is destroyed due to physiological, traumatic, genetic, toxic, or other causes, and muscle cytotoxic substances such as myoglobin and creatine kinase are released into the circulatory system, causing metabolic disorders and organ dysfunction . ClinicaThis retrospective study analyzed the medical records of RM patients, who were diagnosed and treated at the Tianjin Medical University General Hospital from October 2019 to October 2022. This study was approved by the Medical Ethics Committee of Tianjin Medical University General Hospital. Informed consent was not required due to the retrospective nature of the study.n = 71) were divided into AKI or non-AKI groups for comparison (The inclusion criteria for this study were as follows: (1) the presence of causes and symptoms of muscle damage, such as muscle pain, weakness, and dark urine; (2) an increase in the serum creatine kinase (CK) level over five times the normal level; and (3) significant increase in the levels of myoglobin (MYO) in blood and urine. A total of 90 patients were eligible for inclusion. The exclusion criteria were as follows: (1) a history of chronic kidney disease; (2) a history of muscle and nervous system myopathy; (3) acute myocardial infarction; (4) a history of hypertension or diabetes mellitus for over 10 years with elevated serum creatinine on admission, which did not decrease significantly even after treatment; (5) patients with end-stage tumors or major diseases affecting survival; and (6) discharge or abandonment of treatment within 24 hours with incomplete data. All subjects (mparison .µmol/L) within 48 h or SCr ≥ 1.5 times the basal value and is known or presumed to occur within 7 days, or the continuous urine output in 6 h is <0.5 mL/(kg·h). All laboratory tests were conducted on the day before admission or before performing any treatment after admission.The medical records of patients were organized to record the gender, age, height, weight, symptoms, underlying disease, etiology and complications of rhabdomyolysis, vital signs, biochemical parameters , treatment methods, and outcomes. According to the AKI clinical practice guidelines of the Kidney Disease Improvement Global Outcomes (KDIGO) organization , AKI is t-test or Mann–Whitney U test, while categorical variables were compared using the chi-square test or Fisher's exact test. The statistical software SPSS 25.0 was used for the analysis. The normally distributed measurement data were represented as P value <0.05 indicated statistically significant differences between the groups.The continuous variables between groups were compared using the Student 71 patients were included in the study according to the inclusion and exclusion criteria.P < 0.05). In addition, 50.0% of the patients with AKI had drinking history, 50.0% had oliguria or no uria symptoms, and 62.5% were accompanied by gastrointestinal symptoms of varying degrees , which were statistically different from those without AKI  years. There were 2.5 times more men than women (51 : 20). 2 (2.8%) patients were recurrent cases of rhabdomyolysis. 3 (4.2%) patients died. The median length of hospital days was 18.5  days in the AKI group and 10.0  days in the non-AKI group, with a statistically significant difference .In the results of the univariate analysis , ALT, ASP < 0.01). As mentioned above, drinking history (P < 0.01) also had significant differences. Therefore, a binary logistic regression analysis was performed using whether AKI occurred as the dependent variable  and the above significantly different variables as independent variables. The results suggested that history of alcohol consumption, CK, and SCr were independent influencing factors for AKI in RM  (Only 18.3% of the 71 patients had no complications. 16 (22.5%) had AKI, 48 (67.6%) had varying degrees of hepatic impairment, and 32 (45.1%) had electrolyte metabolism disorders. The proportion of electrolyte disorders was higher in the AKI group, which was statistically different from the non-AKI group ( < 0.05) . 3  patients received aggressive rehydration therapy, 41 (57.8%) patients received alkalinization of urine, and 10 (14.1%) patients received continuous renal replacement therapy (CRRT). Of the patients treated with CRRT, 4 died or abandoned treatment, and the remaining 6 had improved renal function after a mean of 9 ± 4.1 days of hemodialysis, and all renal function indexes returned to normal at discharge.In this study, among the total population with RM, males were 2.5 times more likely than females, which may be related to higher alcohol intake and smoking status in males, especially in RM due to strenuous exercise, where adolescent males accounted for 83.3%, revealing a male gender advantage. A survey of 235 patients with COVID-19 showed that  males anIn the analysis of underlying diseases, 32.4% of patients had previous neurological disorders, such as epilepsy, Parkinson's disease, and old cerebral infarction. On the one hand, epilepsy itself can be the cause of RM. Certain antiepileptic drugs (carbamazepine and levetiracetam) and lipid-lowering drugs (atorvastatin and rosuvastatin) taken orally in patients with epilepsy and old cerebral infarction have shown to be associated with the development of RM . In a stStreptococcus pneumoniae, and salmonella are common pathogens that cause RM [Streptococcus pneumoniae, should be alerted to the development of RM.Infection was the primary cause of RM in our study. 45.1% of patients had infection, and in the AKI group, 63.5% of patients had infection of varying degrees. Bacterial infections were predominant, with the most common foci of infection being the lung, followed by the intestinal, intracranial, and urinary tracts. Pathogenic bacteria can directly invade myofibers and produce bacterial toxins that can directly cause damage to myocytes. However, the specific pathogens of infection were not statistically analyzed in this study. According to the literature, influenza virus, legionella, cause RM , 9. TherAmong the 20 cases of RM due to drugs and poisons, 17 cases had a history of statins' use (mainly rosuvastatin and atorvastatin), 2 cases of alcoholism, and 1 case of excessive consumption of freshwater crayfish. RM caused by lipid-lowering drugs occurred mostly in patients with renal insufficiency, hypothyroidism, and infections and was induced by regular doses . This maStrenuous exercise was also a major etiology. 15 (21.1%) patients were admitted with varying degrees of myalgia, malaise, and deepening of urine color after strenuous exercise. This group of patients contained mainly young adults, and the types of exercise were mainly long-distance running, swimming, and frog jumping. This is almost consistent with previous exercise-related rhabdomyolysis studies, in which patients are typically young exercisers who engage in high-intensity exercise, especially in hot or humid conditions, without adequate rest and hydration , 13. 1 cIn addition to the abovementioned causes, endocrine or metabolic diseases are also causes of RM. Among the 12 cases of RM due to endocrine or metabolic diseases, there were 6 cases of hypokalemia, 3 cases of acute complications of diabetes mellitus with diabetic ketoacidosis, 2 cases of primary hypothyroidism, and 1 case of hyperthyroidism. The presentation of these patients was usually atypical, with only 2 patients presenting with the classical triad. Therefore, these patients are more likely to be missed and misdiagnosed and need to be highly alerted by clinicians.α (TNF-α) [AKI is a serious complication of RM. It has been reported in the literature that the incidence of AKI in RM is 13%–50% . 16 (22. (TNF-α) , as well (TNF-α) , thereby (TNF-α) , which i (TNF-α) . Under n (TNF-α)   patients received aggressive rehydration therapy after admission to the hospital. In addition, AKI caused by RM is associated with a decrease in urinary pH. Therefore, sodium bicarbonate can be used to alkalinize the urine to prevent nephrotoxic metabolites from myoglobin catabolism. However, it is important to note that large doses of bicarbonate may aggravate hypocalcemia and deposition of calcium salts in tissues. In this study, more than half of the patients (57.8%) were given alkalinizing urine treatment. Mannitol is an osmotic diuretic originally thought to promote increased renal blood flow, reduce tubular cast, and act as a free radical scavenger. However, there is no randomized controlled trial evidence that sodium bicarbonate or mannitol is more effective than aggressive fluid resuscitation alone in reducing acute kidney injury, the need for dialysis, or death. According to the available literature, mannitol, sodium bicarbonate, and furamide should not be routinely used for rhabdomyolysis. In this study, the population in the non-AKI group showed significant relief of muscle soreness in 2-3 d after treatment with removal of etiology, active rehydration, and maintenance of electrolyte balance. According to the degree of muscle injury in different patients, although the recovery time of CK, CK-MB, MYO, AST, SCr, and other laboratory test indicators are different, the prognosis is good. Although the above treatment is preferred, continuous renal replacement therapy (CRRT) should be considered for acute renal failure with severe hyperkalemia, metabolic acidosis, and oliguria . In thisSince this study was a retrospective single-center study, there were some limitations. First, only patients in the past three years were included in the study, and the sample size was relatively small. Second, some data on the etiology, history, and laboratory indicators were missing, leading to the exclusion of some cases. Third, AKI and oliguria may be underestimated or overestimated due to issues in the methodology, involving the measurement of baseline SCr levels and urine output. Fourth, the analytical methods and equipment used by different sections of the laboratory may vary, which may cause errors in the statistical results. Our study revealed the clinical and laboratory characteristics of RM among patients at the Tianjin Medical University General Hospital in the past three years, explored the characteristics and distribution of RM caused by different etiologies, described the incidence and characteristics of AKI concomitant with RM, and identified the factors associated with its occurrence. These findings will contribute to a wider understanding of RM, facilitate the early diagnosis and treatment of RM, prevent the occurrence of serious complications, and provide a reference for future multicenter and prospective studies.


The cell of origin (a stem cell) of primary myelofibrosis (PMF) is still partially unknown. We used next generation sequencing to explore this aspect of PMF pathobiology deeper. We found evidence that higher progenitors  harbor genetic lesions found in PMF cells. However, genetic events can also occur in later stages of differential contribution to disease development and progression. This new finding sheds light on the complexity of the transformation of hematopoietic pluripotent precursors in the genesis of PMF.JAK2V617F), indicating that most genetic events likely to contribute to disease pathogenesis occurred in a non-commissioned precursor. In contrast, only 9/27 InDels were similar, suggesting that this type of lesion contributed instead to disease progression, occurring at more differentiated stages, or maybe just represented “passenger” lesions, not contributing at all to disease pathogenesis. In conclusion, we showed for the first time that genetic lesions characteristic of PMF occur at an early stage of hematopoietic stem cell differentiation, this being in line with the possible transformation of the disease in either myeloid or lymphoid acute leukemia.Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm (MPN) characterized by progressive bone marrow sclerosis, extra-medullary hematopoiesis, and possible transformation to acute leukemia. In the last decade, the molecular pathogenesis of the disease has been largely uncovered. Particularly, genetic and genomic studies have provided evidence of deregulated oncogenes in PMF as well as in other MPNs. However, the mechanisms through which transformation to either the myeloid or lymphoid blastic phase remain obscure. Particularly, it is still debated whether the disease has origins in a multi-potent hematopoietic stem cells or instead in a commissioned myeloid progenitor. In this study, we aimed to shed light upon this issue by using next generation sequencing (NGS) to study both myeloid and lymphoid cells as well as matched non-neoplastic DNA of PMF patients. Whole exome sequencing revealed that most somatic mutations were the same between myeloid and lymphoid cells, such findings being confirmed by Sanger sequencing. Particularly, we found 126/146 SNVs to be the e same (including  The classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are a group of chronic hematological diseases which include three major entities: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), all of which are characterized by the clonal expansion of one or several myeloid cell lineages  and bone marrow fibrosis ; howeverPMF is characterized by a stepwise evolution. There is an initial prefibrotic/early stage, distinguished by hypercellular bone marrow. Subsequently, an overt fibrotic stage with marked fibrosis in the bone marrow occurs. The fibrotic stage of PMF is clinically characterized by leukoerythroblastosis in the blood, hepatomegaly, and splenomegaly ,12,13.JAK2 gene  are the most frequent (around 50–60%) genetic aberrations in PMF patients, which lead to the constitutive activation of the JAK/STAT pathway. However, patients lacking this mutation often have alternative mutations [CALR mutations are found in about one fourth of PMF cases, and MPL mutations are found in less than 10% of cases. MPL and CALR also activate the JAK/STAT pathway [JAK2, CALR, and MPL [Genetic studies have revealed that mutations in the utations ,15. CALR pathway ,16. In t and MPL ,16. Alth and MPL ,19.JAK2, MPL, and CALR. Non-driver mutations, identified in these patients, involved TET2, ASXL1, SRSF2, IDH2, SF3B1, and EZH2 [The application of next generation sequencing (NGS) later allowed the further definition of the genetic landscape of MPN. Garcia-Gisbert et al. detected molecular alterations on cell-free DNA (cfDNA) and paired granulocyte DNA in plasma samples of a series of patients with MPNs. cfDNA and granulocyte DNA showed an equivalent mutational profile. The 14 PMF patients, included in the study cohort, exhibited mutations in driver genes such as and EZH2 . JAK2V617F mutation in both myeloid and lymphoid cells in myeloproliferative neoplasms, has resulted in the hypothesis that PMF originates from a multipotent hematopoietic stem/progenitor cell (HSPC) [The cell of origin of PMF in the hematological cell development hierarchy has been a matter of contradiction. Some evidence, such as clonal myelopoiesis and the detection of l (HSPC) ,21. In tl (HSPC) ,23. Anotl (HSPC) . The most comprehensive study aiming to define the clonal evolution of myeloproliferative neoplasms on a molecular level was presented by Levine and colleagues . In thisIn this study, we aimed to further investigate the cell of origin of PMF. To address this issue, we applied whole exome sequencing to both myeloid and lymphoid cells from PMF patients and confirmed the sparsely available evidence concerning the derivation of PMF from multipotent hematopoietic stem cells. Three PMF samples were collected at the Hematology and Stem Transplantation Unit, Azienda Ospedaliera Marche Nord, Pesaro, Italy. The pathological diagnosis was confirmed by at least 2 expert hematopathologists by a bone marrow trephine biopsy. The final diagnosis was then established based on clinical and pathological features. An additional amount of nine cases was included in the validation cohort studied by Sanger sequencing. The details on these patients had been previously reported . A summaPeripheral blood cells and saliva were collected, serving as neoplastic and non-neoplastic cells, respectively (see below), before treatment initiation. A detailed description of the three patients studied by WES is presented in In order to obtain granulocytes (as the myeloid cells) and lymphocytes, we followed a procedure that has been previously described . First, Total DNA was extracted from 9 samples  with QIAamp DNA mini kit Qiagen according to the manufacturer’s instructions . A Covaris instrument was used to shear one microgram of DNA into 100–500 bp fragments, following fragmentation quality control using a DNA-7500 kit . Further, based on Illumina’s TruSeq DNA Sample Preparation v2 Guide, DNA libraries were pre-enriched. Briefly, after end repair, specific adapters (adenylate 3’ends and ligate) were used in a PCR reaction to selectively enrich the DNA fragments with adaptor molecules on both ends. After purifying the product of the PCR libraries using AmpureXP beads , we implanted Illumina’s TruSeq Exome Enrichment Guide  to perform exome enrichment. Two hybridizations of 20 h using biotinylated bait-based were performed, and each was followed with a step of streptavidin magnetic bead binding, the product of which was eluted after washing. After the second elution, a PCR reaction of 10 cycles was performed for enrichment, followed by a quality control analysis of the enriched libraries using a DNA-1000 kit . Quant-it PicoGreen dsDNA Assay Kit was used for the quantification, according to the manufacturer’s protocol . http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 8 January 2014)) was used for quality control, and the reads were mapped to Human GRCh37 Genome Assembly using Burrows-Wheeler Aligner version 0.6.1 [Using Illumina HiScan SQ , we sequenced the paired-end libraries (2 × 100 base pair), following the manufacturer’s instructions. On average, about 17 million 100 bp paired-ends raw reads were generated, and the theoretical coverage was calculated based on the hg19 RefSeq non-redundant exome length, which ranged from 21X to 60X. The FastQC V0.10.0 tool (on 0.6.1 . In ordeon 0.6.1 . Mappingon 0.6.1 . Single-http://www.ensembl.org/Homo_sapiens/Info/Index (accessed on 8 January 2014)). To reduce the presence of germline mutations, variants already present in saliva samples were excluded. Saliva was macroscopically not contaminated by blood. However, in order to exclude the possible puzzling effects of scattered contaminating blood cells, SNVs/InDels with allele frequencies below 10% were excluded.All the mutations detected were filtered using thresholds based on the quality, coverage, and strand of the mapped reads and according to the variants already present in public databases  (http://www.openbioinformatics.org/annovar/ (accessed on 8 January 2014)) was used for functional annotation of variants, including exonic functions and aminoacid changes and only non-synonymous variants, including stop-gain SNVs, splicings, and frameshift InDels were selected for further analysis.The Annovar tool with the 20 May 2013 update ) for the analysis of the gene ontology (GO) molecular function of the genes with novel SNVs. For the investigation of the molecular interaction between the mutated genes and the possible drug candidates, and thus for defining actionable genes, the Cognoscente database was utilized (http://vanburenlab.tamhsc.edu/cognoscente.html (accessed on 8 January 2014)) [Furthermore, we used Molecular Signature Database ) .To confirm the efficiency of WES, we performed Sanger sequencing on a selected array of the genes found to be mutated. For this aim, DNA was extracted from the granulocytes and lymphocytes using QIAamp DNA mini kit Qiagen  according to the manufacturer’s procedure  . PrimersBy means of exome capture and high-throughput sequencing, we analyzed granulocytes and lymphocytes in three PMF patients and filtered the results with non-malignant cells obtained from saliva. The number of mate pair reads for patients 1, 2, and 3 was 16 M, 8 M and 8 M, respectively, with more than 97% of total mapped reads in all cases. The mean coverage for the patients was 27X, 14X, and 13.3X, respectively, with more than 86% of the exomes covering at least 1X . The discovered genetic aberrations included both SNVs and InDels, . In totaIFI16) [ERBB4) and fibroblast growth factor receptor 2 (FGFR2) [AMFR) and ubiquitin-like modifier activating enzyme 2 (UBA2) [Among the mutated genes, several genes with notable roles in cancer emerged, including several members of the zinc finger family of transcription factors  and otheIFI16) , importa (FGFR2) ,35, and 2 (UBA2) ,37. HLA-B, VSIG10L, and ZNF717 , 6  and 7  novel frameshift InDels A. In totd ZNF717 B,C. The d ZNF717 .Details on novel SNVs are reported in In order to clarify the probable stage(s) during hematopoiesis in which the mutations occurred, we compared the discovered mutations in granulocytes and lymphocytes. Very interestingly, this comparison revealed that a major part of the SNVs were common between the two cell lineages. In fact, 126 out of the 136 (93%) SNVs in each cell type were found to be common between them A. In theFurthermore, to gain insights into the biological functions of mutated genes, we analyzed the GO function of the genes found to be mutated both in granulocytes and lymphocytes. The enriched functions turned out to be related to important signaling functions such as those related to free nucleotides and kinase activity, plus transcription-related functions such as RNA plolymerase II . To idenADAMTSL3, TET1, EFCAB4B, KIR2DL1, IKBKE, TMEM216, and JAK2. These genes were chosen based on their biological relevance and known role in cancer. The studied cases included the ones used for the WES plus nine additional ones  investigation.The possible multipotent cell of origin of PMF has already been suggested. A study by Xu et al. evaluated the presence of a high frequency of CD34+ HSPC in the peripheral blood of PMF patients and suggested that PMF could arise from multipotent CD34+ HSPC . In addiJAK2 and non-driver genes such as ASXL1, IDH1/2, SRSF2, EZH1 [Farina et al. also identified molecular alterations in CD34 + HSPCs in PMF patients by next-generation sequencing. These findings appear to confirm the origin of PMF as multipotent CD34+ HPSCs. The most frequent mutations concern driver genes such as F2, EZH1 . FurtherF2, EZH1 .JAK2V617F. Very importantly, a study reported the carriage of this mutation in lymphoid cells as well, which further confirms our hypothesis regarding the cell of origin for of [CALR nor MPL mutations were observed in the initial cohort of three patients. This makes our series quite unique as triple negative cases are not common. In this regard, it should be noted how the evolution of liquid biopsy and liquid biopsy-based biomarkers , might turn out to be useful in PMF and especially in triple negative cases to differentiate subtypes and secondary causes of erythrocytosis, thrombocytosis, and myelofibrosis, as well as to predict the development of thrombosis [One of the most common mutations reported in PMF patients is n for of . Indeed,rombosis . MED25, TAOK2, RERE, ZNF84, and NLRP6 which play significant roles in cell signaling, chromatin modification, and apoptosis. These mutations might explain the lymphoblastic leukemia transformation in PMF, which is associated with the advent of new clonal expansions in the disease. Since PMF shows a pure myeloproliferative phenotype, the risk of myeloid transformation in this disease is significantly high; however, PMF also shows a rare association with lymphoid transformation, which happens for CML ,45,46,47The main limitations of our analysis were represented by the small sample size and the fact that the study design was simpler than that which was presented by Levine and colleagues . Of noteIn addition, since the patient population comprised elderly patients, we cannot exclude the fact that some mutations did just occur due to aging in hematopoietic stem cells. Further testing on younger patients (though it is not so common) would certainly help in clarifying this issue.In conclusion, we found that most genetic lesions found in PMF patients share a myeloid and lymphoid lineage. This implies their occurrence in an early, uncommissioned hematopietic precursor, at least in a proportion of PMF cases, and provides a rationale for the possible occurrence of either myeloid or lymphoid blastic transformation. Further studies are recommended to analyze these results in a larger cohort of patients and to evaluate the role of distinct mutations in the pathogenesis of PMF and its transformation.


The three-dimensional (3D) structure of human SFRP4 was predicted through comparative modeling techniques and evaluated by various online bioinformatics tools. The molecular docking and MD simulation investigations were carried out against phytochemicals with anti-diabetic and anti-obesity properties to shortlist the best SFRP4 inhibitor. Hesperetin, Curcumin, Isorhamnetin, Embelin, Epicatechin, and Methyl Eugenol interacted strongly with SFRP4 by displaying zero RMSD and binding affinities of -6.5, -6.4, -6.3, -5.3, -6.3 and -5.8 kcal/mol respectively. Additionally, the root mean square fluctuation and root mean square deviation graphs from the MD simulation results demonstrated that hesperetin has good variations throughout the simulation period as compared to others. This dynamic stability and control behavior of hesperetin, when it interacts with SFRP4, shows that it has the potential to modulate the function and activity of the protein. Therefore, hesperetin is identified as an effective and top drug candidate through this analysis for preserving beta-cell function and treating diabesity by targeting SFRP4. The findings of this study could be useful in the design and development of diabesity drugs.The simultaneous coexistence of complicated metabolic conditions like obesity and diabetes within an individual is known as diabesity. Obesity is the key factor for many chronic diseases, including insulin resistance and type 2 diabetes (T2D). Metabolic stress due to nutrient overload releases different inflammatory mediators. Secreted frizzled-related protein 4 (SFRP4) is also an inflammatory mediator that impairs insulin secretion. SFRP4 acts as an early biomarker for diabesity expressed with interleukin-1 beta (IL-1β) in the adipose tissues that hinder the exocytosis of insulin-secreting granules from the pancreatic β-cells and is a potential target for preserving β-cell dysfunction and the diabesity treatment. The current study aimed to screen potential bioactive compounds targeting and inhibiting the diabesity-linked SFRP4 protein through an  Then thsy.org/) , PDBsum-sy.org/) , ERRAT, sy.org/) .https://pubchem.ncbi.nlm.nih.gov/). The retrieved structure was saved in .sdf format. The compounds downloaded from the database were loaded onto PyRx docking software with the help of the Open Babel tool existing in the software. The energy minimization and force field optimization was done using uff geometry, and the optimization algorithm was set to conjugate gradients at a total 200 number of steps. The resulting optimized ligands were then changed and saved into the PDBQT format, a suitable input for docking by auto dock vina [Plants with potential anti-diabetic and anti-obesity properties were chosen from already existing literature and the active phytochemicals were listed. A total of 300+ phytochemicals were retrieved from the Chemical Entities of Biological Interest (ChEBI)  and the ock vina .The active pocket of the SFRP4 protein was predicted using the pocket analysis and detection tool DoGSiteScorer . After mDocking between SFRP4 protein and several phytochemical ligands was performed by virtual screening software PyRx using the AutoDock Vina run option . The actAfter docking, each phytochemical generated different poses of results. The pose with the maximum negative binding energy and zero RMSD value was selected . The proThe selected hit compounds after docking were screened based on Lipinski’s rule of five (RO5)  and ADMEin-silico technique known as molecular dynamics (MD) simulation. Molecular docking studies in static situations may forecast a ligand’s binding state and give a static view of the molecule’s binding posture at the protein’s active site. Whereas, MD simulations utilize Newton’s classical equation of motion to track the movement of atoms across time and provide a dynamic perspective of molecular behavior. The Desmond/ Maestro (2022.1 version) was used to conduct simulation studies [The dynamic behavior and stability of the protein-ligand or docked complex under various conditions are frequently studied using the  studies , 46. In  studies –49.The atomic composition and physio-chemical analyses were done using ProtParam. The atomic composition of SFRP4 showed the presence of 1752 carbon, 2832 hydrogen, 502 nitrogen, 494 oxygen, and 32 sulfur atoms and a formula mass of C1752H2832N502O494S32. The estimated molecular weight of the SFRP4 protein is 39858.75, and 9.12 is its theoretical isoelectric pI. The total integer of negatively charged residues (Asp + Glu) is 37, and the total integer of positively charged residues (Arg + Lys) is 53. The total integer of atoms is 5612, and the atomic composition of all the atoms is given in The crystal structure of the SFRP4 protein (Accession no. CAG46532.1) with 346 amino acids was predicted by using the template of secreted frizzled-related protein 3 (1ijx). The selected template showed 73.98% identity with the SFRP4 protein sequence. The QMEAN score of our predicted structure is 0.85 . The preThe active site determined by the online server DoGSiteScorer based on calculated shape, size, and chemical content was confirmed through literature mining. The active pocket residues include Glu60, Tyr61, Glu63, Leu64, Val67, Leu111, Met112, Tyr115, His117, Ser 118, Try119, and Pro 120 , 51.After molecular docking of 300+ phytochemicals, the top six phytochemicals were chosen based on their best binding energies, lower RMSD values, their capability to bind well in the pose contrast investigation, and their interaction with active site residues. The lead compound binding energies are shown in 2 permeability and in terms of distribution, not all of them are permeable to cross the blood-brain barrier. The ligands were also auspicious towards cytochrome enzymes P450 having shown few possible inhibitions to CYP1A2, and CYP2C19. Renal and total clearance were also low for all the lead ligands. Finally, in terms of toxicity prediction results of selected lead ligands passed the AMES test for toxicity. However, the overall results of these lead ligand ADMET drug screenings were acceptable, and these leads might be considered effective therapeutic candidates against SFRP4.The top six ligands based on good binding affinities against SFRP4 showed zero violation of the Lipinski rule as given in All the docking results showed a good binding affinity with SFRP4 then to check the stability of binding MD simulation was performed by Desmond at 100ns. The conformational changes, stability, and major binding events of the selected ligands within the receptor’s binding pocket were assessed. After performing the simulation analysis results of the simulation trajectories were used for several dynamics analyses, including root mean square fluctuation (RMSF), root mean square deviation (RMSD), protein secondary structure elements, and Protein-ligand interactions Figs –8. The cProtein RMSD of the Hesperetin-SFRP4 complex attained stability at 10 ns and maintained it until the end of the 100 ns MD simulation. P-RMSD of the Hesperetin system lies in the range of ∼2.8–3.0 Å. Similarly, the ligand RMSD of the Hesperetin system stabilizes from 10 ns to 58 ns. After 58 ns, L-RMSD slightly decreases but maintains stability. At 80 ns, hesperetin experiences a peak due to five rotatable bonds and again stabilizes over the simulation . The P-RThe RMSF (root mean square fluctuation) of the receptor in contact with ligands was calculated to assess the mobility of each residue. There are some higher peaks, and the residues of the higher peaks represent loops, coils, and turns. These peaks represent more flexibility than other regions of the protein. In this respect, it is worth noting that the residues with greater peaks are generally found in loop regions of the protein’s N and C termini. As well, the N and C terminal regions depict more variations. These results are based on an examination of MD trajectories. The P-RMSF of the hesperetin complex fluctuates within ∼2.4 Å, with four smaller peaks that lie in an acceptable range and one higher peak at the C terminal . The curSecondary structure elements (SSE) exhibit structural stability, the distribution of different structural elements over time, and ligand-receptor interactions during simulation. In the case of hesperetin, it was found that 28.07% of the secondary structure of this complex was made up of helixes. This shows that some parts of the protein have an alpha-helical shape. Strands, on the other hand, accounted for 1.68% of the total, reflecting areas with a beta-strand or beta-sheet conformation. The total composition consisting of secondary structural components within the hesperetin complex was 29.75% . In the By using the MD simulation, various intermolecular interactions, including hydrogen bonds, water bridges, hydrophobic interactions, and ionic interactions, were thoroughly examined to critically evaluate their significance. Protein-ligand interactions are summarized in the form of a plot. Interaction types are categorized with different colors during MD simulation. The noteworthy hydrogen bonds (Gln60 and Glu63) were identified in the initial docked configuration of the hesperetin compound they remained unchanged during the simulation period. Hydrophobic interactions were noticed with residues Tyr61, Leu64, Tyr115, and His117. Water bridges were most significantly observed with the residues Gln60, Glu63, Asp66, and Tyr 115 . The curDiabesity refers to the simultaneous coexistence of challenging diseases like obesity and diabetes within one individual. Over the world, the prevalence of T2DM and obesity is rising quickly . T2DM isIt was found that T2DM patients had increased blood concentrations of SFRP-4 even before the development of hyperglycemia . SFRP-4 in silico approaches. The phytochemicals identified in the current study demonstrated very promising effects, indicating that dietary photochemical intake could be a promising strategy for obesity and diabetes prevention [Natural products are gaining more and more attention for their potential applications in the prevention and treatment of type 2 diabetes mellitus and obesity. These products include plant extracts, phytochemicals, and microbial metabolites. The development of drugs from natural sources is a significant and quick-creating zone. Thousands of year’s plants have been used to cure a range of diseases including diabetes and obesity . Severalevention .in vitro and in vivo experiments. It can efficiently block the proliferation and migration of malignant cells. Embelin has been utilized as an anthelminthic treatment and is successful in treating rats with acetic acid-induced ulcerative colitis. Additionally, it reduced the total cholesterol and triglycerides, heart rate, systolic blood pressure, and systolic blood pressure in diabetic rats [Our docking results of six reported leads showed the strongest interaction against SFRP4 protein with the closest RMSD values and the most active site residues of amino acids participating in the interaction. Lead one Hesperetin was found to be the utmost potential inhibitor based on the number of interactions with amino acids in an active pocket. It is a trihydroxy flavanone with three hydroxy groups, and it possesses antioxidant, anti-allergic, anti-inflammatory, Vaso-protective, and hypolipidemic characteristics. The natural sources of Hesperetin are citrus fruits, which are also utilized to decrease cholesterol levels . Flavanotic rats . Lead fitic rats . Lead sitic rats .in vitro and in vivo investigations are required to confirm the antidiabesity properties of these phytochemicals.All these leads support its additional role and increased secretion of insulin by modulating and inhibiting the activity of SFRP4, following the results of Yasmin et al., 2020; Hassan et al., 2018; and Bukhari et al., 2014. In the interaction studies, the SFRP4 protein’s His 117, Ser 118, Tyr 61, Ser 122, Ser 118, Gln 60, and Pro 120 amino acid residues were the most reactive, and they participated in interactions more frequently than other amino acid residues, in accordance to results by Yasmin et al., 2020, Hassan et al., 2018 and Bukhari et al., 2014. Then these leads were subjected to MD simulation, a standard method used in molecular modeling, to further validate or examine our results of docking. To determine the stability of the docked protein-ligand complexes, a 100 ns MD simulation was run using the Schrodinger module Desmond, and certain parameters, such as RMSD, were used to examine the stability. The simulation findings demonstrated that the phytochemicals Hesperetin and Epicatechin, respectively, exhibited good stability with SFRP4 during the simulation time, and these results supported their inhibitory potential in accordance with the results of Yasmin et al., 2020; Hassan et al., 2018; Bukhari et al., 2014 , 51. HowSFRP4 is an inflammatory mediator and a potential target for preserving β-cell function and acts as an early biomarker for the treatment of obesity-induced diabetes (diabesity). In this current study, the CADD approaches are used to identify promising phytochemical candidates for modulating SFRP4—a potential avenue for treating obesity-induced diabetes. The three-dimensional structure of SFRP4 was predicted, and molecular docking was performed to investigate its interactions with phytochemical inhibitors. In molecular docking, six phytochemicals, including hesperetin, curcumin, isorhamnetin, embelin, epicatechin, and methyl eugenol, showed strong interactions with the active site residues Gln60, Glu63, Tyr61, Leu64, Met112, Tyr115, His117, and Pro120 of SFRP4. Based on binding interactions, binding affinities, and ADMET analysis all six docked complexes were utilized in 100ns MD simulations to check the good dynamics and stability within the SFRP4 active site. The top six phytochemicals found in our study have the ability to bind with the SFRP4 protein and might inhibit the activity of the protein. However, predicted RMSD and RMSF graphs justified the finding that hesperetin had a better potential to inhibit the SFRP4 protein as compared to other compounds. These phytochemicals can be used as an antidiabesity compound directly from the plant extracts to control obesity-induced diabetes (diabesity). In short, the comprehensive evaluation of all tested drug-like phytochemicals offers a promising avenue in the quest for discovering a novel treatment against diabesity.S1 File(DOCX)Click here for additional data file.


Exposure to pesticides changes the microbial community structure in contaminated agricultural fields. To analyze the changes in the native microbial composition qRT-PCR, a metagenomic study was conducted. The qRT-PCR results exhibited that the uncontaminated soil has a higher copy number of 16S rDNA relative to the soil contaminated with pesticide. Metagenome analysis interprets that uncontaminated soil is enriched with proteobacteria in comparison with pesticide-contaminated soil. However, the presence of Actinobacteria, Firmicutes, and Bacteroides was found to be dominant in the pesticide-spiked soil. Additionally, the presence of new phyla such as Chloroflexi, Planctomycetes, and Verrucomicrobia was noted in the pesticide-spiked soil, while Acidobacteria and Crenarchaeota were observed to be extinct. These findings highlight that exposure to pesticides on soil significantly impacts the biological composition of the soil. The abundance of microbial composition under pesticide stress could be of better use for the treatment of biodegradation and bioremediation of pesticides in contaminated environments. The application of agrochemicals including fertilizers and pesticides has become an essential component of agriculture  was found to be dominant. Pesticide contaminates the metagenomic analysis of soil sample, revealing that as the concentration of pesticide increases in the soil, the expression and number of pesticide-biodegrading genes also increases and are mostly peroxidase, monooxydase, and cytochrome P450 , the researchers preferably work on targeted and non-targeted genes to explore more information using some advanced techniques such as metagenomics and metatranscriptomics. These molecular tools have enough potential to extract the entire microbial composition and their metabolic potential without having any prior knowledge , Uttarakhand, India. One field was contaminated with pesticides, and the other was uncontaminated. The rice crop was grown in both fields during the Kharif season. The soil sample was collected using stainless steel auger. The 10 cm of soil at the top was discarded, and the next 5 cm of soil was collected from both the contaminated and uncontaminated sites. Both the soil samples were labeled and stored in a deep freezer at −20°C. The soil sample taken from uncontaminated sites acts as a control for this study. Pesticide residues such as chlorpyrifos, cypermethrin fipronil, and imidacloprid were majorly found in the pesticide-contaminated soil. Soil DNA was extracted from both soil samples (500 mg) using the HiPurATM Soil DNA Purification Kit. The purity was checked using a NanoDrop spectrophotometer at wavelengths of 260 and 280 nm with a concentration of 50 ng/L  of the 16SrRNA gene in the soil bacterial community was targeted using the Illumina MiSeq platform. The primers used for amplification were V3: 341 F (5′ CCTACGGGAGGCAGCAG 3′) and V4: 806 R (5′ GGACTACHVGGGTWTCTAAT 3′). Paired-end reads obtained from sequencing were subjected to several quality control checks, including score distribution, base quality, average base content, and GC distribution. The FLASH program  and SYBR green dye , subsequently followed by Nocardioides (3.41%), Bellilinea (3.14%), Anaerolinea (2.75%), Longilinea (2.48%), Caldilinea (2.33%), and Phycicoccus (2.21%). In 2GC soil sample, the second most abundant genus was Candidatus Koribacter (5.89%), subsequently followed by Bacillus (5.06%), Candidatus Solibacter (5.00%), Clostridium (3.74%), Conexibacter (1.94%), Streptomyces (1.90%), and Edaphobacter (1.87%)  and non-contaminated (2GC) soil samples were 562,416 and 873,083, respectively. Furthermore, the reads were classified at the phylum and genus levels, i.e., for contaminated soil, the reads were 562,416 and 716, while for non-contaminated soil, the reads were 873,083 and 725. During the study, only the top 8 dominant phyla and genera were selected for comparative analysis. At the genus level, the unclassified category comprised of most abundant genera in both the 2G (20.35%) and 2GC (18.52%) soil samples. In the 2G soil sample, the second most dominant genus was  (1.87%) .Candida tuskoribacter and Candida tussolibacter), followed by Firmicutes (Bacillus and Clostridium), Actinobacteria (Conexibacter and Streptomyces), and Proteobacteria (Edaphobacter). The phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Planctomycetes were consistently present in both soil samples. Two unique phyla, Chloroflexi and Nitrospira, were found exclusively in the 2G soil sample. With the exception of Clostridium, the genus-level composition of the microbial communities exhibited variations between the two soil samples.Additionally, the comparative study at the phylum level exhibited the prominent existence of Firmicutes , Actinobacteria (Nocardioides and Phycicoccus), and Chloroflexi  in the 2G soil sample . HoweverThe genus-level relative abundance of the top 25 classified operational taxonomic units (OTUs) was investigated. In the 2G soil sample, the Shannon species diversity index was high (3.198). Genotypically, a total of 1,627 species were identified, whereas in the 2GC soil sample (control), the Shannon species diversity index was 2.739, and 1,850 species were identified genotypically .Clostridium was larger, whereas in the 2GC soil sample, the population of Bacillus was dominant. Unclassified bacteria at the genus level were predominantly found in both soils, but their abundance decreased in the 2GC soil sample. Genera from the phyla Actinobacteria (Nocardioides) and Firmicutes (Clostridium and Oenococcus) were evenly distributed in both soil samples. Abundant genera in the 2G soil sample included Clostridium (10.6%), Nocardioides (4.3%), Bellilinea (4.0%), Anaerolinea (3.5%), Longilinea (3.2%), and Phycicoccus (2.8%). The remaining microbial population in the 2G soil sample was distinct from that in the 2GC soil sample.In the 2G soil sample, the population of The heat map generated by hierarchical clustering exhibited the number of OTUs per sample. The color intensity on the heat map corresponds to the relative abundance of an OTU within a sample . In the Bacillus cereus 2D from the same sampling sites, i.e., Gularbhoj, and found a highly efficient strain to tolerate higher concentrations of pesticides and a high rate of degradation, and they evaluated the expression of protein profiling under pesticide stress. The decrease in the copy number of 16S rDNA genes in the pesticide-contaminated soil indicated exposure of the pesticide to the native microbial flora, leading to inhibition of metabolic activity and growth. Application of real-time PCR was also employed by several researchers for the investigation of gene expression, microbial abundance, and functional and taxonomic gene expressions  was maximum for the 2G soil sample, while in the 2GC soil sample, the Shannon species diversity index was minimum (2.739). This indicates that more the Shannon index value, the more the diversity in species. Hence, it could be analyzed easily that the more diversity in the system, the more the system stable. Although the total number of species  present in 2GC soil is more as compared with the 2G , in the context of species diversity, 2G soil sample is more dynamic. The possible reason is that pesticides create selective pressure on the microbial community to adapt and acclimatize under stress.In the hierarchical map, the different classes of microbial communities were analyzed in a comparative manner between 2G and 2GC soil samples. More intense color signifies the dominance of the native microbial class. A hierarchical map was used by Parks et al.  and JeffThe application of indigenous microorganisms present in contaminated environments is a more reliable approach for the biodegradation of agricultural pesticides. Under pesticide stress, the bacterial communities altered their genetic pool and developed as efficient mutant strains for the utilization of pesticides as a source of carbon and energy. Monitoring of microbial communities is important because they play a crucial role in the regulation of biogeochemical cycles and soil health, enhancing crop productivity, and maintaining a sustainable environment. Additionally, by conducting metagenomic studies of contaminated soil samples, it becomes possible to identify various types of microbial populations present and the interaction between them. This knowledge is indispensable for comprehending the natural biodegradation of pesticides in environmental settings. Looking ahead, exploring the activity of bacterial isolates against other xenobiotic compounds and delving into the mechanisms of gene regulation involved would be valuable avenues for research and potential advancements in the field.SAMN37722849 and SAMN37722850.The original contributions presented in the study are publicly available. This data can be found in the NCBI repository under accession numbers: SG: Conceptualization, Data curation, Formal analysis, Writing—original draft. SJ: Conceptualization, Formal analysis, Writing—review and editing. GB: Formal analysis, Investigation, Methodology, Writing—review and editing. GP: Writing—review and editing, Formal analysis. KP: Formal analysis, Validation, Writing—review and editing. NB: Formal analysis, Validation, Writing—review and editing. RR: Validation, Writing—review and editing. AS: Formal analysis, Supervision, Validation, Writing—review and editing.


Actinobacteria  and lower absolute levels of Proteobacteria, often linked to gut inflammation, in comparison with WD-fed mice. Furthermore, an increased abundance of the typically rare genus Atopobium was observed. These changes may indicate the possible anti-inflammatory effects of the KD. However, since the overall changes in the microbiota seem low, the KD effects might be linked to the differential abundance of only a few key genera in mice.The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that has been reported to have neuroprotective effects. The health effects of KD might be linked to an altered gut microbiome, which plays a major role in host health, leading to neuroprotective effects via the gut-brain axis. However, results from different studies, most often based on the 16S rRNA gene and metagenome sequencing, have been inconsistent. In this study, we assessed the effect of a 4-week KD compared to a western diet (WD) on the colonic microbiome of female C57Bl/6J mice by analyzing fecal samples using fluorescence in situ hybridization. Our results showed distinct changes in the total number of gut bacteria following the 4-week KD, in addition to changes in the composition of the microbiome. KD-fed mice showed higher absolute numbers of  The ketogenic diet (KD) is characterized by high-fat and low-carbohydrate dietary intake, leading to a state of ketosis . It has Bacteroidetes to Firmicutes [Interestingly, the therapeutic potential of KD to improve seizure activity has been linked to its effect on the gut microbiota and related metabolites, although KD has been reported to reduce gut microbial diversity ,13. The rmicutes . Howeverrmicutes .Most studies on KD have used 16S rRNA gene sequencing or metagenome sequencing to assess the relative abundance of bacteria, and the results from different studies, most often based on the 16S rRNA gene and metagenome sequencing, have been inconsistent. This difference is due to the fact that metagenomics is able to detect less abundant taxa, and sometimes, these less abundant genera could be more biologically meaningful than the highly abundant ones that are only detected by 16S rRNA sequencing . For chan = 10/group) were maintained under standard conditions in a specific pathogen-free environment at 12 h day-night cycles according to the FELASA recommendations with food and water ad libitum. Following a one-week acclimatization period, female mice were divided randomly into homogeneous groups according to their weight and age (10–12 weeks of age) and fed an ad libitum Western or ketogenic diet for four weeks. The Western diet contained 18% protein, 59% carbohydrates, and 23% fat in terms of energy content. The ketogenic diet contained 15% protein, 1.4% carbohydrates, and 83.6% fat. Health status, such as the observation of behavior, physical assessment, and body weight, was monitored twice per week throughout the intervention. At the end of the dietary intervention, fecal samples from individual mice were fixed in modified Carnoy’s solution  for at least 24 h at room temperature [C57BL/6J mice  and performed according to the guidelines for the care and use of laboratory animals adopted by the U.S. National Institutes of Health, and the ARRIVE guidelines.The Fecal microbiota was investigated using FISH analysis with ribosomal RNA-derived probes. Hybridization was performed on 4 µM thick sections of Carnoy-fixed and paraffin-embedded stool samples . The samThe bacteria were quantified using group-specific C3 probes. The FITC-marked universal probe was used in each hybridization to evaluate the number of all bacteria, and C5 marked probes with a different specificity to C3 probes were used to determine the spatial relationship between different bacterial groups. Only signals that hybridized with a specific FISH probe and the universal FISH probe that did not hybridize with specific FISH probes from unrelated bacterial groups were evaluated .9 bacteria/mL, which was equivalent to the formula used previously [Bacterial concentrations in homogeneous populations were enumerated visually in one of the 10 × 10 fields of the ocular raster, corresponding to 10 µm × 10 µm of the section surface at a magnification of ×1000. This number was assigned to a concentration of ×10eviously .In the case of uneven distribution of bacteria over the microscopic field, the positive signals were enumerated in 10 fields of the ocular raster along the gradient of distribution, and an average was used after dividing by 10.https://probebase.csb.univie.ac.at (accessed on 16 February 2022)) [A total of 48 bacterial FISH probes were applied. Seven of these probes  were excluded from the analysis because the bacteria detected by these probes were not specific to the mouse intestine. The names of the FISH probes are listed according to abbreviations of the probeBase online resource (y 2022)) . DetailsThe probes in p < 0.05. The data are expressed as individual values ± SEM.Statistical analyses were performed using the GraphPad Prism 8.1.2 software . Since some data were not normally distributed, statistical significance was determined using the non-parametric Mann-Whitney U test. Differences were considered statistically significant at Mice were fed for 4 weeks with a WD in the control group and with KD in the treatment group. As expected, a significant reduction in blood glucose concentration and a significant increase in ketone levels were observed in the KD-fed group compared with those in the WD-fed group a,b. HoweSubstantial bacterial groups were found in at least 20% of the fecal samples at concentrations higher than 109 bacteria/mL. Marginal bacterial groups showed low occurrence as well as marginal concentrations. They occurred in less than 20% of fecal samples at concentrations below 109 bacteria/mL and, in most cases, lower than 108 bacteria/mL. According to this classification, 19 of the FISH probes represented substantial bacterial groups, and 22 of the probes presented marginal bacterial groups , CFB560 , and MIB661  probes were consistently present in the fecal samples of mice fed the WD and KD at concentrations between 10 × 109 and 25 × 109 bacteria/mL, contributing to approximately half of the colonic microbiota in each mouse. The invariability and predominance of these three bacterial groups in C57BL/6J mice contributed to a major part of the microbiome and were designated as highly conserved bacteria. All other substantial bacterial groups were present only in a subset of the mice and were designated as individual bacteria. As their concentrations reached 1010 bacteria/mL, they contributed substantially to the fecal biomass, comprising approximately 50% of the biomass.The bacteria were subdivided into highly conserved and individual bacterial groups. Three bacteria detected using CF319a , followed by the Eubacterium hallii group , and Bacteroides putredinis (Bputre698 probe), and the numbers of these three bacterial groups were lower in KD-fed mice (Sphingomonas/Erythrobacter (SPH492 probe) was lower in the KD group (Bacteroides distasonis (Bdis656 probe), Eubacterium cylindroides (Ecyl387 probe), and Bacteroides spp. (Bac303 probe) (Atopobium cluster (Ato291 probe), Bifidobacterium spp. (Bif662 probe), and the Eubacterium cylindroides group (Ecyl387 probe)  . Three o7 probe) .As mentioned before, differences in single marginal bacterial groups were not statistically evaluated between WD- and KD-fed mice.Bacteroidetes (62.8%), Proteobacteria (17.8%), and Firmicutes (17.3%), whereas Verrucomicrobia (1.5%) and Actinobacteria (0.6%) were only present in a minor proportion in the stool samples of mice consuming the WD (Actinobacteria (9.1%) and a lower abundance of Proteobacteria (8.5%)  and the KD-fed groups (0.220 ± 0.091).The three most abundant phyla in the stool of WD-fed mice were g the WD b. In cona (8.5%) b. These bacteria c. IntereDiet plays a major role in causing 50% of gut microbial variations in mice and 20% in humans, making it a potential strategy for disease management via modulation of the gut microbiota ,33,34. S5 per ml. Nevertheless, Swidsinski et al. explained that even though the information provided by sequence analysis on the physical abundance and contribution of bacteria to bio-fermentation is substandard, their abundance within the fecal matter directly expresses their bio-fermenting power [In our study, we also used the FISH technique, but we could not have a final conclusion on the whole biodiversity or composition of the microbiota, as FISH only detects bacteria at concentrations higher than 10ng power .Actinobacteria. Numerous reports have shown that members of Actinobacteria, such as Bifidobacterium spp., play a protective role against colitis and are able to reduce the overall level of systemic inflammation [Actinobacteria, and in particular Bifidobacterium spp., which have previously been reported to have lower blood glucose and act as an anti-diabetic agent [Bifidobacterium spp. after KD may indicate that KD can have a possible beneficial effect on the inflammatory status, as well as on glucose homeostasis.Our data quantifying bacterial participants demonstrated a clear difference in bacterial concentration and composition after KD compared with WD in C57BL/6J mice. For instance, the total number of bacteria in the fecal samples of mice consuming KD was reduced compared to those consuming WD, confirming previous data . Feedingammation ,43,44,45ic agent . This siProteobacteria. Members of the Proteobacteria phylotype have been shown to play a role in the onset and progression of ulcerative colitis and inflammatory diseases, such as Crohn’s disease [Proteobacteria represent a minor part of the healthy gut microbiota, but their disproportionate increase leads to inflammation [Proteobacteria have been considered as a microbial signature of intestinal dysbiosis [Proteobacteria in mice with DSS-induced colitis [Proteobacteria, leading to their increase [Proteobacteria [On the other hand, in our study, KD in mice led to a decrease in the number of  disease ,48,49,50ammation ; as suchysbiosis . Similar colitis . This maincrease . Howeverbacteria .Verrucomicrobia between WD- and KD-fed mice. This is an important finding as this taxon contains members such as Akkermansia muciniphila, whose presence has been associated with gut health and reversal of experimental colitis [A. muciniphila after a KD in mice [A. muciniphila has been reported to have anti-diabetic effects, leading to an improvement in insulin secretion and glucose homeostasis [A. muciniphila numbers after KD [A. muciniphila numbers in BTBR mice following a KD compared to a chow diet [A. muciniphila [A. muciniphila was significantly increased after a 16-week ketogenic diet [A. muciniphila.Interestingly, we found no differences in the abundance of  colitis ,55. Our  in mice ,21,36. Aeostasis . This waafter KD ,21,36. Hhow diet , possibliniphila . This efiniphila . Lookingnic diet , suggestAtopobium cluster in the KD group compared to the WD group, which has been implicated in the pathogenesis of several diseases such as type 2 diabetes mellitus [Sphingomonas and Erythrobacter, in the KD group. Looking more closely at the individual bacteria detected by the FISH probes, we found a significant increase in the mellitus , and ulcmellitus . HoweverBacteroidetes were overrepresented and Firmicutes were underrepresented in comparison to previous studies based on 16S rRNA sequencing [Firmicutes in the murine gut. Therefore, claims based on the Firmicutes/Bacteroides ratio should be carefully considered. Here, the 4-week KD in C57Bl/6J mice did not have a significant effect on the Firmicutes to Bacteroidetes ratio compared to the WD. As Firmicutes and Bacteroidetes represent more than 90% of the gut microbial community, the ratio of Firmicutes to Bacteroidetes is considered important for microbial balance and gut health [Furthermore, our results showed that quencing  or metagt health . This cot health .Proteobacteria leading to weight loss and favouring Firmicutes, Ruminococcaceae, and Mogibacteriaceae [Proteobacteria in the KD-fed mice. However, we did not observe a corresponding increase in the other genera, which could be due to the short-term period of the diet followed in our study. Furthermore, the proven anti-inflammatory effects of KD reported in different studies make it a potential strategy for the prevention or treatment of CVD. Another anti-inflammatory factor in KD is the elimination of pro-inflammatory simple sugars, which has been reflected in CVD improvement because the restriction of carbohydrate content has shown anti-inflammatory benefits in the case of cardiometabolic health. As such, these studies recommend a high-fat, well-composed KD that is rich in omega-3 polyunsaturated fatty acids due to its anti-inflammatory and cardioprotective effects [Evidence from different studies has shown a clear effect of diet on the gut microbiome and a clear relationship between the state of the gut microbiome and chronic diseases such as type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) . For inseriaceae . In our  effects .In conclusion, KD led to a lower total number of bacteria in the guts of mice. A limitation to consider is the duration of the dietary intervention, as longer durations have been shown to have a stronger effect on shaping the gut microbiota. Our study was also limited by the fact that the fecal microbiota analysis was performed using the FISH technique. This technique does not allow an in-depth study of all the major species involved in health and disease; therefore, we still lack data to make a definitive conclusion about the effect of KD at the species level. Another limitation of the study is that the effect of KD on the fecal microbiota was only investigated in female mice, and the estrous cycle was not determined, as recent studies have shown sex differences in response to KD ,65. Ther


Stigma is common in patients with breast cancer after surgery, which has a negative impact on the quality of life (QOL). This study aimed to investigate the QOL of breast cancer patients after surgery and to analyze the multiple chains mediating effects of self-disclosure and social support between stigma and QOL.A total 292 patients of breast cancer patients after operation were recruited in this study. A questionnaire survey was conducted using the general information questionnaire, the consumer experiences of stigma questionnaire (CESQ), the distress disclosure index(DDI), the perceived social support scale(PSSS), and the functional assessment of cancer therapy-breast(FACT-B). Path analysis was conducted to test the hypothesized serial multiple mediation model.The total scores of stigma, self-disclosure, social support and QOL were 15 (10 ~ 22), 39 (31 ~ 46), 58 (50 ~ 67) and 88 (74 ~ 104) respectively. QOL of breast cancer patients after the operation was negatively correlated with stigma (p < 0.01), and positively correlated with self-disclosure and social support (p < 0.01). Self-disclosure and social support played a complete mediating effect between stigma and QOL, and the total mediating effect value was 85. 87%.Self-disclosure and social support play a complete intermediary role between stigma and QOL. In order to improve the quality of life of patients, medical staff should pay attention to the assessment of stigma, encourage patients to express their emotions, and encourage their families and friends to respond to their expression and needs of patients. In Chin22.1A total of 292 breast cancer patients from 5 hospitals in Guangdong Province  from March 2021 to March 2022 were selected. To increase the sample size and combine with clinical practice, a convenient sampling method is adopted, and the number of participants in each hospital was evenly distributed as far as possible Figure 1Inclusion criteria: (1) pathological diagnosis of breast cancer ; (2) patients accepted mastectomy, breast-conserving surgery, or breast reconstruction surgery;(3)age ≥ 18 years old; (4) voluntarily participate in this study on the premise of informed consent; (5)clear awareness, reading comprehension and expression ability; (6)awareness of their condition. Exclusion criteria: (1)combined with other malignant tumors or recurrence of breast cancer; (2)mental disorders, unable to cooperate; (3)critical condition, unable to understand or answer questions clearly; (4)combined with myocardial infarction, heart failure and other serious diseases affecting the QOL. Sample size calculation: Combined with popular rules-of-thumb and Monte Carlo analysis method, the minimum sample size is 265 –18.2.22.2.1Basic information such as gender, age, marital status, educational level, and religious belief was self-reported. Disease staging, pathological classification, and surgical methods were extracted from medical records.2.2.2The questionnaire was developed by Wahl et al. in 2013 to provide a tool for assessing stigma in breast cancer patients. This study adopted the Chinese version of the CESQ , which m2.2.3DDI was used , which c2.2.4PSSS was a 12-item scale developed by Blumenthal  in 1987.2.2.5The 36-item of Functional Assessment of Cancer Therapy-Breast scale  was used2.3This study was a cross-sectional design and had been approved by the ethics committee of the hospital. Before the study, researchers were trained and assessed in a unified way, and the researchers were required to use unified guidelines in the survey process. The subjects formally conducted a questionnaire survey after oral or signed informed consent. The completed questionnaire was checked and taken back by the researchers on the spot. Questionnaire exclusion criteria: questionnaire blank ≥ 15%; the answer was single and the content was contradictory. A total of 300 questionnaires were distributed, excluding 8 invalid questionnaires, 292 valid questionnaires were recovered, and the effective recovery rate was 97.3%. Descriptive statistics and correlations were performed in SPSS 26.0. The descriptive data were presented as mean ± SD for variables obeying normal distribution, Md  for variables not obeying normal distribution. The enumeration data were expressed by frequency and constituent ratio. In this study, the scores of stigma, social support, self-disclosure, and quality of life do not obey normal distribution, however, the scores of demographic characteristics are expressed by Md , and the correlation analysis is conducted by Spearman rank correlation analysis. Amos 26. 0 was used to construct the structural equation model, the bootstrap was used to evaluate the direct and indirect effects, and the effects of each path were tested. All tests were performed two-sided, and a p-value of less than 0.05 was considered a significant level.33.13.2p<0.01), and positively correlated with self-disclosure and social support. Stigma was negatively correlated with self-disclosure and social support; Self-disclosure was positively correlated with social support.The total score of CESQ was 15 (10~22), the total score of DDI was 39 (31~46), the total score of PSSS was 58 (50~67); The total score of FACT-B was 88 (74~104), and the 5 dimensions were: physiological status, 21 (19~23); social/family status, 17 (11~22); emotional status, 16 (13~19); functional status, 13 (8~17); additional concerns, 24 (21~26), respectively Table 2.3.32/df=X2.512, goodness of fit(GFI)=0.963, adjusted goodness of fit (AGFI)=0.906, root mean square error of approximation(RMSEA)=0.072, normed fit index(NFI)=0.958, comparative fit index(CFI)=0.974, which indicated that the fitting degree of the model was good , and stigma can indirectly affect the QOL through self-disclosure and social support. Through a large number of literature research and correlation analysis results, it is hypothesized that QOL can be directly affected by stigma and indirectly affected by self-disclosure and social support. The maximum likelihood method was used to fit the model structure, and the model was corrected according to the correction index. The fitting results of the model: was good Table 4.44.1This research has shown that the median total score of QOL was 88 points (range 0-144 points), which is above average. Compared with the research of Criscitiello C et al. , the QOL4.2This research explores the relationship between stigma and QOL and its influence path in patients with breast cancer after surgery. Among them, the stigma of patients is above average, which is similar to most studies, indicating that patients with breast cancer after surgery generally experience stigma , 9. The 4.3In this survey, self-disclosure and social support of patients after breast cancer surgery are negatively correlated with stigma and positively correlated with QOL Table 3.4.3.1The results of this study show that the indirect effect value of stigma → social support →QOL is -0.014, accounting for 3.73% of the total indirect effect. The stigma experience can improve the QOL through the increase of social support. The breast cancer patients who have finished surgery often have social support needs , when pa4.3.2The results of this study show that the indirect effect value of stigma → self-disclosure →QOL was -0.271, accounting for 84.16% of the total indirect effect, ranking first among the three mediating effect paths. Chinese breast cancer patients believe that coping with disease and misfortune is a private matter, and they are reluctant to disclose their diagnosis, treatment, and disease-related thoughts and feelings to others . Women wTo sum up, the QOL of breast cancer patients after surgery still needs to be improved, which can be affected by stigma, self-disclosure, and social support. Stigma can affect QOL through multiple mediating effects of self-disclosure and social support. To improve the QOL of patients, we should encourage patients and their families to express themselves and carry out relevant psychological counseling activities.4.4There are some limitations to this study. First, this study uses a cross-sectional design, only the independent time point data are collected, and it can not assess patients at different times. A longitudinal study design can be introduced in the later research to explore the trajectory of patients’ QOL at different times. Secondly, this study adopted a convenient sampling method to collect data in five hospitals, and the representativeness of the sample needs to be improved. Although we used convenient sampling, we tried our best to achieve “stratified sampling” by hospital and operation. In the future, we will continue to expand the sample size, increase the hospitals included in the study, adopt a random sampling method, and include more influencing factors for analysis to obtain more accurate conclusions.5The QOL of patients with breast cancer after surgery is at the upper middle level, which is higher than before, but it can still be improved compared with other countries. Stigma, self-disclosure, social support, and QOL are correlated with each other, and self-disclosure and social support play a multiple chain mediation effects between stigma and QOL. To improve the QOL of patients with breast cancer after surgery, medical staff should not only pay attention to the physical condition of the patients, but also pay attention to the evaluation of their stigma experience, encourage patients to express their emotions, and also encourage their families and friends to respond to the expression and needs of the patients.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.All methods were performed in accordance with the relevant guidelines and regulations or declaration of Helsinki. The study was approved by the ethics committee of Shantou University Medical College . Informed consent was obtained from all individual participants and legal guardian. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by LB, SZ and GF. The first draft of the manuscript was written by LB and all authors commented on previous versions of the manuscript. GF ultimately modified the manuscript. All authors read and approved the final manuscript.