OBJECTIVE: To explore the potential mechanism of resistance to axitinib in clear cell renal cell carcinoma (ccRCC), with a view to expanding the understanding of axitinib resistance, facilitating the design of more specific treatment options, and improving the treatment effectiveness and survival prognosis of patients. METHODS: By exploring the half maximum inhibitory concentration (IC₅₀) of axitinib on ccRCC cell lines 786-O and Caki-1, cell lines resistant to axitinib were constructed by repeatedly stimulated with axitinib at this concentration for 30 cycles in vitro. Cell lines that were not treated by axitinib were sensitive cell lines. The phenotypic differences of cell proliferation and apoptosis levels between drug resistant and sensitive lines were tested. Genes that might be involved in the drug resistance process were screened from the differentially expressed genes that were co-upregulated in the two drug resistant lines by transcriptome sequencing. The expression level of the target gene in the drug resistant lines was verified by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB). The expression differences of the target gene in ccRCC tumor tissues and adjacent tissues were analyzed in the Gene Expression Profiling Interactive Analysis (GEPIA) public database, and the impact of the target gene on the prognosis of ccRCC patients was analyzed in the Kaplan-Meier Plotter (K-M Plotter) database. After knocking down the target gene in the drug resistant lines using RNA interference by lentivirus vector, the phenotypic differences of the cell lines were tested again. WB was used to detect the levels of apoptosis-related proteins in the different treated cell lines to find molecular pathways that might lead to drug resistance. RESULTS: Cell lines 786-O-R and Caki-1-R resistant to axitinib were successfully constructed in vitro, and their IC₅₀ were significantly higher than those of the sensitive cell lines (10.99 μmol/L, P < 0.01; 11.96 μmol/L, P < 0.01, respectively). Cell counting kit-8 (CCK-8) assay, colony formation, and 5-ethynyl-2 '-deoxyuridine (EdU) assay showed that compared with the sensitive lines, the proliferative ability of the resistant lines decreased, but apoptosis staining showed a significant decrease in the level of cell apoptosis of the resistant lines (P < 0.01). Although resistant to axitinib, the resistant lines had no obvious new replicated cells in the environment of 20 μmol/L axitinib. Nuclear protein 1 (NUPR1) gene was screened by transcriptome sequencing, and its RNA (P < 0.0001) and protein expression levels significantly increased in the resistant lines. Database analysis showed that NUPR1 was significantly overexpressed in ccRCC tumor tissue (P < 0.05); the ccRCC patients with higher expression ofNUPR1had a worse survival prognosis (P < 0.001). Apoptosis staining results showed that knockdown ofNUPR1inhibited the anti-apoptotic ability of the resistant lines to axitinib (786-O, P < 0.01; Caki-1, P < 0.05). WB results showed that knocking downNUPR1decreased the protein level of B-cell lymphoma-2 (BCL2), increased the protein level of BCL2-associated X protein (BAX), decreased the protein level of pro-caspase3, and increased the level of cleaved-caspase3 in the resistant lines after being treated with axitinib. CONCLUSION ccRCC cell lines reduce apoptosis through theNUPR1 -BAX/ BCL2 -caspase3 pathway, which is involved in the process of resistance to axitinib.
Humans ; Carcinoma, Renal Cell/metabolism* ; Axitinib/pharmacology* ; Kidney Neoplasms/metabolism* ; bcl-2-Associated X Protein ; Nuclear Proteins ; Cell Line, Tumor ; Apoptosis ; Cell Proliferation

Objective: To construct growth standards for Chinese children under 7 years of age. Methods: Cross-sectional study design based on national representative data on children's growth and development in 2015 was used. Stratified cluster sampling method was used. A total of 83 628 healthy children aged 0-<7 years from 9 cities, including Beijing, Harbin, Xi'an, Shanghai, Nanjing, Wuhan, Guangzhou, Fuzhou, and Kunming, were investigated from June to November 2015, excluding those with adverse conditions that may impact the establishment of the growth standards. Weight, length (height) and head circumference were measured using unified measurement tools and measurement methods. The Lambda-Mu-Sigma method was employed to establish percentile and standard deviation score reference values of weight-for-age, length (height)-for-age, head circumference-for-age, weight-for-length (height) and body mass index (BMI)-for-age of the study population. The standard deviation score curves of the new-established growth standards were compared with the 2009 reference standards. Results: Reference values of percentile (P₃, P₁₀, P₂₅, P₅₀, P₇₅, P₉₀, P₉₇) and standard deviation score (-3, -2, -1, 0,+1,+2,+3) of weight-for-age, length (height)-for-age, head circumference-for-age, weight-for-length (height) and BMI-for-age were obtained. Compared with the 2009 growth standards, the difference of weight at P₅₀ was -0.1-0.4 kg, the difference of length (height) at P₅₀ was 0.1-1.3 cm, the difference of head circumference at P₅₀ was -0.2-0.2 cm, the difference of weight for length (height) at P₅₀ was -0.2-0.5 kg, and the difference of BMI at P₅₀ was -0.2-0.2 kg/m². The main differences were as follows: weight for girls aged 5.0-<7.0 years was 0.4-0.6 kg higher at +2 s, height for boys and girls aged 2.0-<7.0 years was 0.4-1.4 cm higher at -2 s, and BMI for boys and girls aged 5.0-<7.0 years was 0.1-0.3 kg/m² higher at +2 s than the 2009 reference standards. Conclusion: The newly established growth standards for Chinese children under 7 years of age that have achieved a minor revision to the 2009 reference standards, are recommended for nationwide use in growth monitoring and nutritional assessment.
Male ; Female ; Child ; Humans ; Infant ; Child Development ; Body Weight ; Cross-Sectional Studies ; East Asian People ; Body Height ; China/epidemiology* ; Reference Standards ; Reference Values ; Body Mass Index

Objective: To establish the reference values and growth curves of length for weight and head circumference for weight among Chinese newborns in order to provide a reference for the assessment of body proportionality at birth. Methods: A cross-sectional design was applied. A total of 24 375 singleton live birth newborns with gestational ages at birth of 24⁺⁰ to 42⁺⁶ weeks were recruited from June 2015 to November 2018 from 13 cities including Beijing, Harbin, Xi'an, Shanghai, Nanjing, Wuhan, Guangzhou, Fuzhou, Kunming, Tianjin, Shenyang, Changsha, and Shenzhen, excluding those with maternal or newborn conditions that may impact the establishment of the reference values. The generalized additive model for location, scale, and shape was employed to establish reference values in terms of percentile and growth curves of length for weight and head circumference for weight for male and female newborns. The random forest machine learning method was employed to analyze the importance of variables between the established reference values in this study and the previous published weight/length, body mass index (BMI), ponderal index (PI), weight/head circumference, length/head circumference in the assessment of symmetrical and asymmetrical small for gestational age (SGA) newborns. Results: A total of 24 375 newborns with 13 197 male infants (preterm birth 7 042 infants and term birth 6 155 infants) and 11 178 female infants (preterm birth 5 222 infants and term birth 5 956 infants) were included in this study. The reference values in terms of percentile (P₃, P₁₀, P₂₅, P₅₀, P₇₅, P₉₀, P₉₇) and growth curves of length for weight and head circumference for weight were obtained for male and female newborns with gestational ages of 24⁺⁰ to 42⁺⁶ weeks. The median birth lengths corresponding to the birth weights of 1 500, 2 500, 3 000, and 4 000 g were 40.4, 47.0, 49.3 and 52.1 cm for males and 40.4, 47.0, 49.2, and 51.8 cm for females, respectively; the median birth head circumferences were 28.4, 32.0, 33.2 and 35.2 cm for males and 28.4, 32.0, 33.1, and 35.1 cm for females, respectively. The differences of length for weight between males and females were minimum, with the difference range of -0.3 to 0.3 cm at P₅₀; the differences of head circumference for weight between males and females were minimum, with the difference range of 0 to 0.2 cm at P₅₀. Based on the match between birth length and birth weight for classifying symmetrical and asymmetrical SGA, length for weight and PI contributed the most, accounting for 0.32 and 0.25, respectively; based on the match between birth head circumference and birth weight, head circumference for weight and weight/head circumference contributed the most, accounting for 0.55 and 0.12, respectively; based on the match between birth length or head circumference with birth weight, head circumference for weight and length for weight contributed the most, accounting for 0.26 and 0.21, respectively. Conclusion: The establishment of the new standardized growth reference values and growth curves of length for weight and head circumference for weight among Chinese newborns are useful for clinical practice and scientific research.
Female ; Humans ; Infant, Newborn ; Male ; Birth Weight ; Body Height ; China ; Cross-Sectional Studies ; East Asian People ; Fetal Growth Retardation ; Gestational Age ; Premature Birth ; Reference Values

OBJECTIVE: Clinical characteristics and outcome in COVID-19 with brucellosis patients has not been well demonstrated, we tried to analyze clinical outcome in local and literature COVID-19 cases with brucellosis before and after recovery. METHODS: We retrospectively collected hospitalization data of comorbid patients and prospectively followed up after discharge in Heilongjiang Infectious Disease Hospital from January 15, 2020 to April 29, 2022. Demographics, epidemiological, clinical symptoms, radiological and laboratory data, treatment medicines and outcomes, and follow up were analyzed, and findings of a systematic review were demonstrated. RESULTS: A total of four COVID-19 with brucellosis patients were included. One patient had active brucellosis before covid and 3 patients had nonactive brucellosis before brucellosis. The median age was 54.5 years, and all were males (100.0%). Two cases (50.0%) were moderate, and one was mild and asymptomatic, respectively. Three cases (75.0%) had at least one comorbidity (brucellosis excluded). All 4 patients were found in COVID-19 nucleic acid screening. Case C and D had only headache and fever on admission, respectively. Four cases were treated with Traditional Chinese medicine, western medicines for three cases, no adverse reaction occurred during hospitalization. All patients were cured and discharged. Moreover, one case (25.0%) had still active brucellosis without re-positive COVID-19, and other three cases (75.0%) have no symptoms of discomfort except one case fell fatigue and anxious during the follow-up period after recovery. Conducting the literature review, two similar cases have been reported in two case reports, and were both recovered, whereas, no data of follow up after recovery. CONCLUSION These cases indicate that COVID-19 patients with brucellosis had favorable outcome before and after recovery. More clinical studies should be conducted to confirm our findings.
Female ; Humans ; Male ; Middle Aged ; Brucellosis ; COVID-19 ; Retrospective Studies ; SARS-CoV-2 ; Treatment Outcome ; Case Reports as Topic

Interleukin 6 (IL-6), an important component of cardiac microenvironment, favors cardiac repair by improving cardiomyocyte regeneration in different models. This study aimed to investigate the effects of IL-6 on stemness maintenances and cardiac differentiation of mouse embryonic stem cells (mESCs). The mESCs were treated with IL-6 for two days, and then subjected to CCK-8 essay for proliferation analysis and quantitative real-time PCR (qPCR) to evaluate the mRNA expression of genes related to stemness and germinal layers differentiation. Phosphorylation levels of stem cell-related signal pathways were detected by Western blot. siRNA was used to interfere the function of STAT3 phosphorylation. Cardiac differentiation was investigated by the percentage of beating embryoid bodies (EBs) and qPCR analysis of cardiac progenitor markers and cardiac ion channels. IL-6 neutralization antibody was applied to block the endogenous IL-6 effects since the onset of cardiac differentiation (embryonic day of 0, EB0). The EBs were collected on EB7, EB10 and EB15 to investigate the cardiac differentiation by qPCR. On EB15, Western blot was applied to investigate the phosphorylation of several signaling pathways, and immunochemistry staining was adopted to trace the cardiomyocytes. IL-6 antibody was administered for two days (short term) on EB4, EB7, EB10 or EB15, and percentages of beating EBs at late developmental stage were recorded. The results showed that exogenous IL-6 promoted mESCs proliferation and favored maintenances of pluripotency, evidenced by up-regulated mRNA expression of oncogenes (c-fos, c-jun) and stemness markers (oct4, nanog), down-regulated mRNA expression of germ layer genes (branchyury, FLK-1, pecam, ncam, sox17), and increased phosphorylation of ERK1/2 and STAT3. siRNA targeting JAK/STAT3 partially attenuated the effects of IL-6 on cell proliferation and mRNA expression of c-fos and c-jun. During differentiation, long term IL-6 neutralization antibody application decreased the percentage of beating EBs, down-regulated mRNA expression of ISL1, GATA4, α-MHC, cTnT, kir2.1, cav1.2, and declined the fluorescence intensity of cardiac α actinin in EBs and single cell. Long term IL-6 antibody treatment decreased the phosphorylation of STAT3. In addition, short term (2 d) IL-6 antibody treatment starting from EB4 significantly reduced the percentage of beating EBs in late development stage, while short term IL-6 antibody treatment starting from EB10 significantly increased the percentage of beating EBs on EB16. These results suggest that exogenous IL-6 promotes mESCs proliferation and favors stemness maintenance. Endogenous IL-6 regulates mESC cardiac differentiation in a development-dependent manner. These findings provide important basis for the study of microenvironment on cell replacement therapy, as well as a new perspective for understanding the pathophysiology of heart diseases.
Animals ; Mice ; Interleukin-6 ; Mouse Embryonic Stem Cells ; Cell Differentiation ; Proto-Oncogene Proteins c-fos ; RNA, Messenger

OBJECTIVE: This study was aimed at investigating the carrier rate of, and molecular variation in, α- and β-globin gene mutations in Hunan Province. METHODS: We recruited 25,946 individuals attending premarital screening from 42 districts and counties in all 14 cities of Hunan Province. Hematological screening was performed, and molecular parameters were assessed. RESULTS: The overall carrier rate of thalassemia was 7.1%, including 4.83% for α-thalassemia, 2.15% for β-thalassemia, and 0.12% for both α- and β-thalassemia. The highest carrier rate of thalassemia was in Yongzhou (14.57%). The most abundant genotype of α-thalassemia and β-thalassemia was -α ^3.7/αα (50.23%) and β ^IVS-II-654/β ^N (28.23%), respectively. Four α-globin mutations [CD108 (ACC>AAC), CAP +29 (G>C), Hb Agrinio and Hb Cervantes] and six β-globin mutations [CAP +8 (C>T), IVS-II-848 (C>T), -56 (G>C), beta nt-77 (G>C), codon 20/21 (-TGGA) and Hb Knossos] had not previously been identified in China. Furthermore, this study provides the first report of the carrier rates of abnormal hemoglobin variants and α-globin triplication in Hunan Province, which were 0.49% and 1.99%, respectively. CONCLUSION Our study demonstrates the high complexity and diversity of thalassemia gene mutations in the Hunan population. The results should facilitate genetic counselling and the prevention of severe thalassemia in this region.
Humans ; beta-Thalassemia/genetics* ; alpha-Thalassemia/genetics* ; Hemoglobinopathies/genetics* ; China/epidemiology* ; High-Throughput Nucleotide Sequencing

AIM To observe the protective effects of Qigu Capsule-medicated serum on C2C12 myotube cell atrophy induced by dexamethasone.METHODS The Qigu Capsule-medicated serum was prepared.C2C12 cells cultured in vitro were divided into the normal control group,the dexamethasone group,the 10%blank serum group and the 10%Qigu Capsule-medicated serum group for 48 h,corresponding drug treatment,followed by 24 h culture with 10 μmol/L dexamethasone except the control group,and had cell viability detection by CCK-8 method.With their myotube cells intervened accordingly by the aforementioned regime following 6-7 days differentiation with 2%horse serum induction,the C2C12 cells had their myotube cells diameter measured;and the expressions of proteins related to MyHC,MuRF-1,Atrogin-1 and PI3K/Akt signaling pathway detected by Western blot.The impact of PI3K inhibitor LY294002 on the diameter of myotube cells and the expression of MuRF-1,Atorgin-1 and PI3K/Akt signaling pathway related proteins were detected as well.RESULTS Compared with the normal control group,the dexamethasone group and the blank serum group were observed with decreased viability of C2C12 cells(P<0.01),decreased diameter of myotube cells(P<0.01),increased protein expressions of MuRF-1 and Atrogin-1(P<0.01),and decreased expression of MyHC protein and phosphorylation levels of PI3K,Akt,mTOR and FoxO3a(P<0.01).Compared with the dexamethasone group,the Qigu Capsule-medicated serum group showed increased viability of C2C12 cells(P<0.01);increased diameter of myotube cells(P<0.01);decreased protein expressions of MuRF-1 and Atrogin-1(P<0.01);and increased expression of MyHC protein and phosphorylation levels of PI3K,Akt,mTOR and FoxO3a(P<0.05,P<0.01).The intervention of LY294002 upon the Qigu Capsule-medicated serum group offset the anti-muscular tube atrophy effect(P<0.01),increased the protein expressions of MuRF-1 and Atorgin-1(P<0.01),and decreased the phosphorylation levels of PI3K,Akt,mTOR and FoxO3a(P<0.05,P<0.01).CONCLUSION The Qigu Capsule-medicated serum can alleviate the atrophy of C2C12 myotubes induced by dexamethasone,and its mechanism may be related to the activation of PI3K/Akt signaling pathway.

Objective Coronavirus disease 2019 (COVID-19) and tuberculosis (TB) are major public health and social issues worldwide. The long-term follow-up of COVID-19 with pulmonary TB (PTB) survivors after discharge is unclear. This study aimed to comprehensively describe clinical outcomes, including sequela and recurrence at 3, 12, and 24 months after discharge, among COVID-19 with PTB survivors. Methods From January 22, 2020 to May 6, 2022, with a follow-up by August 26, 2022, a prospective, multicenter follow-up study was conducted on COVID-19 with PTB survivors after discharge in 13hospitals from four provinces in China. Clinical outcomes, including sequela, recurrence of COVID-19, and PTB survivors, were collected via telephone and face-to-face interviews at 3, 12, and 24 months after discharge. Results Thirty-two COVID-19 with PTB survivors were included. The median age was 52 (45, 59) years, and 23 (71.9％) were men. Among them, nearly two-thirds (62.5％) of the survivors were moderate, three (9.4％) were severe, and more than half (59.4％) had at least one comorbidity (PTB excluded). The proportion of COVID-19 survivors with at least one sequela symptom decreased from 40.6％ at 3 months to 15.8％ at 24 months, with anxiety having a higher proportion over a follow-up. Cough and amnesia recovered at the 12-month follow-up, while anxiety, fatigue, and trouble sleeping remained after 24 months. Additionally, one (3.1％) case presented two recurrences of PTB and no re-positive COVID-19 during the follow-up period. Conclusion The proportion of long symptoms in COVID-19 with PTB survivors decreased over time, while nearly one in six still experience persistent symptoms with a higher proportion of anxiety. The recurrence of PTB and the psychological support of COVID-19 with PTB after discharge require more attention.

Objective To establish a predictive model using multiple layer perceptron (MLP) for short-term outcome after subacute ischemic stroke.Methods From January, 2019 to September, 2021, 60 readmission-inpatients in Department of Rehabilitation, Ruijin Hospital, Shanghai Jiaotong University School of Medicine were collected the clinical features of first admission (less than 30 days after attack), and the outcomes were assessed with modified Rankin Scale (MRS) three months after the first admission. The risk factors were screened with single factor analysis, and the short-term outcome predictive models were established with multi-factor Logistic regression and MLP. The predictive accuracy of both models was calculated, and the predictive effects were compared with Receiver Operating Characteristic (ROC) curve.Results For multi-factor Logistic regression, the predictive accuracy was 73.3%, and the area under ROC curve was 0.851. For MLP, the predictive accuracy was 88.9%, and the area under the ROC curve was 0.930.Conclusion The prediction of short-term outcome after subacute ischemic stroke can be done with MLP model.

Objective To investigate the morphological variation of Oncomelania hupensis shells in Yunnan Province, so as to provide insights into the understanding of O. hupensis genetic evolution and control. Methods According to the O. hupensis density, geographical location, altitude, water system and environmental type, 12 administrative villages were sampled from 10 schistosomiasis-endemic counties (districts) in 3 prefectures (cities) of Yunnan Province as snail collection sites. From December 2021 to January 2022, about 200 snails were collected from each collection site, among which thirty adult snails (6 to 7 spirals) were randomly selected from each site, and the 11 morphological indexes of snail shells were measured and subjected to cluster analysis and principal component analysis. Results Of O. hupensis snails from 12 localities of Yunnan Province, the longest shell (7.33 mm) was detected in snails from Yongle Village, Eryuan County, with the shortest (4.68 mm) in Dongyuan Village, Gucheng District, and the largest angle of apex (59.47°) was measured in snails from Caizhuang Village, Midu County, with the smallest (41.40°) in Qiandian Village, Eryuan County. The mean coefficient of variation was 9.075% among O. hupensis snails from 12 localities of Yunnan Province, with the largest coefficient of variation seen in the thickness of the labra brim (29.809%). Among O. hupensis snails from 12 localities of Yunnan Province, the mean Euclidean distance was 2.26, with the shortest Euclidean distance seen between O. hupensis snails from Qiandian Village of Eryuan County and Wuxing Village of Dali City (0.26), and the largest found between O. hupensis snails from Caizhuang Village of Midu County and Cangling Village of Chuxiong County (8.17). Cluster analysis and principal component analysis classified O. hupensis snails from 12 localities of Yunnan Province into three categories, including the O. hupensis snail samples from Caizhuang Village of Midu County, O. hupensis snail samples from Cangling Village of Chuxiong County, and O. hupensis snail samples from Qiandian Village of Eryuan County, Wuxing Village of Dali City, Yangwu Village of Yongsheng County, Xiaoqiao Village of Xiangyun County, Yongle Village of Eryuan County, Xiaocen Village of Dali City, Anding Village of Nanjian County, Dongyuan Village of Gucheng District, Lianyi Village of Heqing County, and Dianzhong Village of Weishan County. The variations in these three categories of snail samples were mainly measured in the principal component 2 related to the angle of apex and the thickness of the labra brim. Conclusions The variations in the Euclidean distance and morphological features of shells of O. hupensis from 12 localities of Yunnan Province gradually rise with the decrease in the latitude of the collection sites. The angle of apex is an indicator for the growth of O. hupensis whorl.