OBJECTIVE To elucidate the mechanisms responsible for the inhibitory effects of limonene on the proliferation of non-small cell lung cancer(NSCLC) by non-targeted metabolomics and additional approaches. METHODS The CCK-8 assay was utilized to evaluate the inhibitory effects of limonene on NSCLC A549 cell viability and to ascertain the IC50. In vitro experiments, encompassing colony formation, flow cytometry, iron content assessment, and mitochondrial staining, were conducted to assess the anti-lung cancer and iron-induced cell death effects of limonene. Metabolomic analysis was employed to identify potential pathways influenced by limonene, and Western blotting was carried out to validate pivotal proteins within these pathways. RESULTS In comparison to the control group, the limonene-treated group demonstrated a significant, dose-dependent reduction in A549 cell proliferation and colony formation. Optical microscopy revealed cellular detachment and pronounced changes in cellular morphology following exposure to limonene. Limonene induced apoptosis in A549 cells and arrested them in the G0-G1 phase of the cell cycle. Confocal microscopy unveiled diminished mitochondrial fluorescence and an augmented intracellular iron content, indicative of the classical phenomenon of ferroptosis. Metabolomic investigations unveiled divergent metabolic pathways, including glutathione(GSH) metabolism, arginine biosynthesis, D-glutamine and D-glutamate metabolism, as well as cysteine and methionine metabolism, with many of them intricately linked to intracellular GSH synthesis. Western blotting experiments underscored a marked reduction in the levels of SLC40A1, SLC7A11(xCT), and GPX4 proteins within the cells post-limonene treatment. CONCLUSION Limonene may induce ferroptosis in lung cancer cells by reducing GSH synthesis and increasing Fe2+ levels.

Objective:To compare and evaluate the quality of wild and different cultivation methods of Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai through analysis on UPLC characteristic atlas and multi-component content determination results. Methods:UPLC was used to establish the characteristic chromatogram and multi-component content determination method of Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai, and clustering analysis, orthogonal partial least squares - discriminant analysis method were used for chemical pattern recognition analysis. Results:The results showed that there were 10 common peaks in 18 batches of Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai. Five components were identified, erythrothioneine(peak 1), protocatechuic acid (peak 2), protocatechualdehyde (peak 3), caffeic acid (peak 4) and Hispidin (peak 5). HCA and OPLS-DA could distinguish Sanghuang porus vaninii (Ljub.) with different cultivation methods. Conclusion:Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai in wood is closer to wild Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai than in substitute cultivation. The UPLC characteristic atlas and multi-component content determination method established in this study can provide reference for the quality evaluation of Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai.

Objective·To analyze the differences and classify hypertrophic cardiomyopathy(HCM)patients and healthy controls(HC)using short-axis cine cardiac magnetic resonance(CMR)images-derived radiomics features.Methods·One hundred HCM subjects were included,and fifty HC were randomly selected at 2∶1 ratio during January 2018 to December 2021 in the Department of Cardiology,Renji Hospital,Shanghai Jiao Tong University School of Medicine.The CMR examinations were performed by experienced radiologists on these subjects.CVI 42 post-processing software was used to obtain left ventricular morphology and function measurements,including left ventricular ejection fraction(LVEF),left ventricular end-diastolic volume(LVEDV)and left ventricular end-diastolic mass(LVEDM).The 3D radiomic features of the end-diastolic myocardial region were extracted from short-axis images CMR cine.The distribution of the radiomic features in the two groups was analysed and machine learning models were constructed to classify the two groups.Results·One hundred and seven 3D radiomic features were selected and extracted.After exclusion of highly correlated features,least absolute shrinkage and selection operator(LASSO)was used,and a 5-fold cross-validation was performed.There were still 11 characteristics with non-zero coefficients.The K-best method was used to decide the top 8 features for subsequent analysis.Among them,four features were significantly different between the two groups(all P<0.05).Support vector machine(SVM)and random forest(RF)models were constructed to discriminate the two groups.The results showed that the maximum area under the curve(AUC)for the single-feature model(first order grayscale:entropy)was 0.833(95%CI 0.685?0.968)and the maximum accuracy for the multi-feature model was 83.3%with an AUC of 0.882(95%CI 0.705?0.980).Conclusion·There are significant differences in both left ventricular function and left ventricular morphology between HCM and HC.The 3D myocardial radiomic features of the two groups are also significantly different.Although single feature is able to distinguish the two groups,the combination of multi-features show better classification performance.

BACKGROUND:The pedicle navigation template has many advantages,but there are still some problems.For example,poor soft tissue dissection leads to poor adhesion of the pedicle navigation template,resulting in screw path deviation;careful dissection of soft tissue to fit the pedicle navigation template leads to prolonged surgery time and increased bleeding;the design of the pedicle navigation template cannot predict the vertebral rotation and the impact of body position changes,resulting in the poor fitting. OBJECTIVE:To explore the utility of a new 5-point positioning point-contact pedicle navigation template in the case of scoliosis and complex pedicle. METHODS:A total of 20 patients with scoliosis and complicated pedicle admitted to the Department of Spinal Surgery,Guizhou Hospital,Beijing Jishuitan Hospital from February 2020 to February 2023 were selected for scoliosis orthopedics.During the operation,the 5-point positioning point-contact pedicle navigation template was used to guide the screws.According to the inclusion and exclusion criteria,34 cases were matched as the empirical nail placement group,and conventional barehanded nail placement was performed.The time of placement,the amount of bleeding,the number of fluoroscopies,the number of manual diversions,the level and accuracy of pedicle screws,the complications of placement,and the rate of correction of main curvature were compared between the two groups. RESULTS AND CONCLUSION:(1)There were no significant differences in sex,age,coronal Cobb's angle of the main curvature,bending Cobb's angle of the main curvature,pedicle variation,apex rotation,fusion segment,number of screws,level of screws,accuracy of screws,and rate of correction of main curvature between the navigation template group and the empirical nail placement group(P>0.05).(2)Compared with the empirical nail placement group,the navigation template group had more advantages in time of placement(P=0.034),amount of bleeding(P=0.036),number of fluoroscopies(P=0.000)and number of manual diversions(P=0.021).(3)There were 0 cases of screw-related complications in both groups.(4)In conclusion,the 5-point positioning point-contact 3D printing pedicle navigation template has a claw-like structure.It can firmly adapt to various deformities of the lamina articular process,avoid drift,and accurately place the screws.It has a point-like contact lamina structure to avoid extensive and complete dissection of the posterior structure,and reduce bleeding,operation time,and trauma.Pre-designed screw entry points and directions can reduce the number of fluoroscopy and operation time.Segmental design can avoid discomfort due to changes in anesthesia position.The operation is simple and the accuracy of screw placement is high.

BACKGROUND:Molecular mechanisms targeting the miRNA/mRNA axis to regulate osteoarthritis disease process have been studied.We identified the mRNA:phospholipase C delta 3(PLCD3)and its target miRNA(miR-34a-5p)with clinical predictive value through previous bioinformatics studies,while experiments to verify their specific roles and mechanisms in regulating osteoarthritis are still lacking. OBJECTIVE:To investigate the regulatory role and mechanism of miR-34a-5p/PLCD3 axis on osteoarthritis progression. METHODS:The synovium of 15 patients with knee osteoarthritis was selected as the osteoarthritis group,and the synovium of 15 young patients with internal fixation of patellar fracture caused by trauma during the same period was selected as the control group.The expression of PLCD3 and miR-34a-5p in the synovium was detected by real-time PCR.Human fibroblast like synovial cells-osteoarthritis(HFLS-OA)cells were treated by cell transfection and divided into miR-34a-5p mimic group,pCDH-PLCD3 group,miR-34a-5p mimic+pCDH-PLCD3 group,miR-34a-5p inhibitor group,si-PLCD3 group,and miR-34a-5p inhibitor+si-PLCD3 group.The relationship between PLCD3 and miR-34a-5p expression was detected by real-time PCR.The effects of HFLS-OA cell viability and cell migration in each group were detected by CCK-8 assay and cell scratch test.Western blot assay was used to detect the expression level of apoptosis marker protein.The expression of inflammatory factors was detected by ELISA. RESULTS AND CONCLUSION:(1)PLCD3 was a direct target of miR-34a-5p,and the expression levels of PLCD3 and miR-34a-5p were negatively correlated.(2)Upregulation of PLCD3 promoted proliferation of HFLS-OA cells and inhibited cell migration.The up-regulation of miR-34a-5p significantly inhibited the activity of HFLS-OA cells and enhanced cell migration.Overexpression of miR-34a-5p significantly increased the levels of Casp3 and Casp9 proteins in HFLS-OA cells,while overexpression of PLCD3 showed the opposite trend.(3)PLCD3 overexpression significantly increased the expression of interleukin 6 and tumor necrosis factor alpha in HFLS-OA cells,while miR-34a-5p mimics showed protective activity.(4)The miR-34a-5p/PLCD3 axis may affect the progression of osteoarthritis by regulating the inflammatory process or apoptosis of synovial cells.

Objective:To investigate the prevalence of adult skeletal fluorosis caused by drinking tea-type endemic fluorosis in Yushu Tibetan Autonomous Prefecture (hereinafter referred to as Yushu Prefecture), Qinghai Province, and provide scientific basis for prevention and control of the disease.Methods:In August 2021, one village was selected as a survey site in six counties (cities) in Yushu Prefecture, including Nangqian, Chindu, Yushu, Zadoi, Qumarlêb, and Zhiduo. Drinking water samples and 10 brick tea samples were collected from each village to determine the fluoride content in water and brick tea; at least 100 permanent residents aged ≥ 25, who had a habit of drinking brick tea and had lived in the local area for more than 5 years, were selected for X-ray imaging to examine the prevalence of adult skeletal fluorosis.Results:A total of 75 samples of residential drinking water were collected, with a fluoride content of (0.21 ± 0.05) mg/L, ranging from 0.11 to 0.34 mg/L; 60 samples of brick tea, with a fluoride content of (626.70 ± 157.27) mg/kg, ranging from 324.00 to 2 102.00 mg/kg. A total of 1 136 adults were examined, and 318 cases of skeletal fluorosis were diagnosed, with a detection rate of 27.99%. Among them, the detection rates of mild, moderate, and severe skeletal fluorosis were 20.95% (238/1 136), 6.07% (69/1 136), and 0.97% (11/1 136), respectively, with mild symptoms being the main. The detection rates of skeletal fluorosis in males and females were 29.09% (121/416) and 27.36% (197/720), respectively, with no statistically significant difference between the gender (χ 2 = 0.39, P = 0.533). Comparison of the skeletal fluorosis in different gender, the differences were statistically significant (χ 2 = 22.31, P < 0.001). The detection rates of skeletal fluorosis in the age groups of 25 - 35, 36 - 45, 46 - 55, 56 - 65, 66 - 75, and ≥76 years old were 6.86% (7/102), 22.37% (51/228), 24.02% (92/383), 37.44% (73/195), 43.48% (70/161), and 37.31% (25/67), respectively. The differences between the groups were statistically significant (χ 2 = 59.84, P < 0.001). Moreover, there was a statistically significant difference in the composition of skeletal fluorosis among different age groups ( H = 37.66, P < 0.001). The Spearman correlation analysis results showed that the severity of adult skeletal fluorosis was positively correlated with age ( r = 0.34, P < 0.001). Conclusions:There is a certain degree of prevalence of adult skeletal fluorosis in Yushu Prefecture. And as age increases, the condition of skeletal fluorosis becomes more severe.

Objective The purpose of this study was to investigate the bacterial communities of biting midges and ticks collected from three sites in the Poyang Lake area,namely,Qunlu Practice Base,Peach Blossom Garden,and Huangtong Animal Husbandry,and whether vectors carry any bacterial pathogens that may cause diseases to humans,to provide scientific basis for prospective pathogen discovery and disease prevention and control. Methods Using a metataxonomics approach in concert with full-length 16S rRNA gene sequencing and operational phylogenetic unit(OPU)analysis,we characterized the species-level microbial community structure of two important vector species,biting midges and ticks,including 33 arthropod samples comprising 3,885 individuals,collected around Poyang Lake. Results A total of 662 OPUs were classified in biting midges,including 195 known species and 373 potentially new species,and 618 OPUs were classified in ticks,including 217 known species and 326 potentially new species.Surprisingly,OPUs with potentially pathogenicity were detected in both arthropod vectors,with 66 known species of biting midges reported to carry potential pathogens,including Asaia lannensis and Rickettsia bellii,compared to 50 in ticks,such as Acinetobacter lwoffii and Staphylococcus sciuri.We found that Proteobacteria was the most dominant group in both midges and ticks.Furthermore,the outcomes demonstrated that the microbiota of midges and ticks tend to be governed by a few highly abundant bacteria.Pantoea sp7 was predominant in biting midges,while Coxiella sp1 was enriched in ticks.Meanwhile,Coxiella spp.,which may be essential for the survival of Haemaphysalis longicornis Neumann,were detected in all tick samples.The identification of dominant species and pathogens of biting midges and ticks in this study serves to broaden our knowledge associated to microbes of arthropod vectors. Conclusion Biting midges and ticks carry large numbers of known and potentially novel bacteria,and carry a wide range of potentially pathogenic bacteria,which may pose a risk of infection to humans and animals.The microbial communities of midges and ticks tend to be dominated by a few highly abundant bacteria.

Objective:To investigate the surgical treatment effect and prognostic factors of hilar cholangiocarcinoma.Methods:This is an ambispective cohort study. From August 2005 to December 2022,data of 510 patients who diagnosed with hilar cholangiocarcinoma and underwent surgical resection at the Hepatobiliary Center of the First Affiliated Hospital of Nanjing Medical University were retrospectively collected. In the cohort,there were 324 males and 186 females,with an age of ( M (IQR)) 63(13)years (range:25 to 85 years). The liver function at admission was Child-Pugh A (343 cases,67.3%) and Child-Pugh B (167 cases,32.7%). Three hundred and seventy-two(72.9%) patients had jaundice symptoms and the median total bilirubin was 126.3(197.6) μmol/L(range: 5.4 to 722.8 μmol/L) at admission. Two hundred and fourty-seven cases (48.4%) were treated with percutaneous transhepatic cholangial drainage or endoscopic nasobiliary drainage before operation. The median bilirubin level in the drainage group decreased from 186.4 μmol/L to 85.5 μmol/L before operation. Multivariate Logistic regression was used to identify the influencing factors for R0 resection,and Cox regression was used to construct multivariate prediction models for overall survival(OS) and disease-free survival(DFS). Results:Among 510 patients who underwent surgical resection,Bismuth-Corlett type Ⅲ-Ⅳ patients accounted for 71.8%,among which 86.1% (315/366) underwent hemi-hepatectomy,while 81.9% (118/144) underwent extrahepatic biliary duct resection alone in Bismuch-Corlett type Ⅰ-Ⅱ patients. The median OS time was 22.8 months, and the OS rates at 1-,3-,5-and 10-year were 72.2%,35.6%,24.8% and 11.0%,respectively. The median DFS time was 15.2 months,and the DFS rates was 66.0%,32.4%,20.9% and 11.0%,respectively. The R0 resection rate was 64.5% (329/510), and the OS rates of patients with R0 resection at 1-,3-,5-and 10-year were 82.5%, 48.6%, 34.4%, 15.2%,respectively. The morbidity of Clavien-Dindo grade Ⅲ-Ⅴ complications was 26.1%(133/510) and the 30-day mortality was 4.3% (22/510). Multivariate Logistic regression indicated that Bismuth-Corlett type Ⅰ-Ⅲ ( P=0.009), hemi-hepatectomy and extended resection ( P=0.001),T1 and T2 patients without vascular invasion (T2 vs. T1: OR=1.43 (0.61-3.35), P=0.413;T3 vs. T1: OR=2.57 (1.03-6.41), P=0.010;T4 vs. T1, OR=3.77 (1.37-10.38), P<0.01) were more likely to obtain R0 resection. Preoperative bilirubin,Child-Pugh grade,tumor size,surgical margin,T stage,N stage,nerve infiltration and Edmondson grade were independent prognostic factors for OS and DFS of hilar cholangiocarcinoma patients without distant metastasis. Conclusions:Radical surgical resection is necessary to prolong the long-term survival of hilar cholangiocarcinoma patients. Hemi-hepatectomy and extended resection,regional lymph node dissection and combined vascular resection if necessary,can improve R0 resection rate.

Objective:To investigate the surgical treatment effect and prognostic factors of hilar cholangiocarcinoma.Methods:This is an ambispective cohort study. From August 2005 to December 2022,data of 510 patients who diagnosed with hilar cholangiocarcinoma and underwent surgical resection at the Hepatobiliary Center of the First Affiliated Hospital of Nanjing Medical University were retrospectively collected. In the cohort,there were 324 males and 186 females,with an age of ( M (IQR)) 63(13)years (range:25 to 85 years). The liver function at admission was Child-Pugh A (343 cases,67.3%) and Child-Pugh B (167 cases,32.7%). Three hundred and seventy-two(72.9%) patients had jaundice symptoms and the median total bilirubin was 126.3(197.6) μmol/L(range: 5.4 to 722.8 μmol/L) at admission. Two hundred and fourty-seven cases (48.4%) were treated with percutaneous transhepatic cholangial drainage or endoscopic nasobiliary drainage before operation. The median bilirubin level in the drainage group decreased from 186.4 μmol/L to 85.5 μmol/L before operation. Multivariate Logistic regression was used to identify the influencing factors for R0 resection,and Cox regression was used to construct multivariate prediction models for overall survival(OS) and disease-free survival(DFS). Results:Among 510 patients who underwent surgical resection,Bismuth-Corlett type Ⅲ-Ⅳ patients accounted for 71.8%,among which 86.1% (315/366) underwent hemi-hepatectomy,while 81.9% (118/144) underwent extrahepatic biliary duct resection alone in Bismuch-Corlett type Ⅰ-Ⅱ patients. The median OS time was 22.8 months, and the OS rates at 1-,3-,5-and 10-year were 72.2%,35.6%,24.8% and 11.0%,respectively. The median DFS time was 15.2 months,and the DFS rates was 66.0%,32.4%,20.9% and 11.0%,respectively. The R0 resection rate was 64.5% (329/510), and the OS rates of patients with R0 resection at 1-,3-,5-and 10-year were 82.5%, 48.6%, 34.4%, 15.2%,respectively. The morbidity of Clavien-Dindo grade Ⅲ-Ⅴ complications was 26.1%(133/510) and the 30-day mortality was 4.3% (22/510). Multivariate Logistic regression indicated that Bismuth-Corlett type Ⅰ-Ⅲ ( P=0.009), hemi-hepatectomy and extended resection ( P=0.001),T1 and T2 patients without vascular invasion (T2 vs. T1: OR=1.43 (0.61-3.35), P=0.413;T3 vs. T1: OR=2.57 (1.03-6.41), P=0.010;T4 vs. T1, OR=3.77 (1.37-10.38), P<0.01) were more likely to obtain R0 resection. Preoperative bilirubin,Child-Pugh grade,tumor size,surgical margin,T stage,N stage,nerve infiltration and Edmondson grade were independent prognostic factors for OS and DFS of hilar cholangiocarcinoma patients without distant metastasis. Conclusions:Radical surgical resection is necessary to prolong the long-term survival of hilar cholangiocarcinoma patients. Hemi-hepatectomy and extended resection,regional lymph node dissection and combined vascular resection if necessary,can improve R0 resection rate.

Objective To investigate whether microRNA(miR)-199a-5p regulates blood-brain barrier(BBB)integrity through PI3K/Akt pathway after cerebral ischemia.Methods A permanent middle cerebral artery occlusion(MCAO)model was established in SPF adult male SD rats.Totally 48 rats were randomly divided into sham group(n=12),model group(n=12),MCAO+miR-199a-5p group(n=12),and MCAO+miR-199a-5p negative control group(n=12).The Ludmila Bellayev 12 point score was used to evaluate the neurobehavioral performance of rats;The integrity of the BBB after ischemia stroke was detected through Evans blue staining;Immunofluorescent staining was used to determine apoptosis after cerebral ischemia;Western blotting technology was used to detect the protein expression of claudin-5,phosphatidylinositol-3 kinase regulatory subunit 2(PIK3R2),p-Akt,Akt,and vascular endothelial growth factor(VEGF)-A;Real-time PCR was used to investigate the expression levels of miR-199a-5p,claudin-5,and VEGF-A in the ischemic penumbra and infarcted area of the brain.Results The result showed that miR-199a-5p mimic intervention improved proprioception and motor ability in MCAO rats.MiR-199a-5p mimic reduced the expression of PIK3R2 following ischemia stroke,activated the Akt signaling pathway,and increased the expression of claudin-5 and VEGF-A in the ischemic penumbra.In addition,miR-199a-5p alleviated inflammation after cerebral ischemia.MiR-199a-5p mimic reduced BBB permeability and reduced neuronal apoptosis after cerebral ischemia.Conclusion MiR-199a-5p can reduce the expression of PIK3R2 following ischemic stroke,activate the Akt signaling pathway,reduce the expression of inflammatory cytokines,and alleviate the damage to the blood-brain barrier.