[Single-cell transcriptomic sequencing coupled with Mendelian randomization analysis elucidates the pivotal role of CTSC in chronic rhinosinusitis].

Objective: To investigate the molecular mechanisms of chronic rhinosinusitis (CRS), to identify key cell subgroups and genes, to construct effective diagnostic models, and to screen for potential therapeutic drugs. Methods: Key cell subgroups in CRS were identified through single-cell transcriptomic sequencing data. Essential genes associated with CRS were selected and diagnostic models were constructed by hdWGCNA (high dimensional weighted gene co-expression network analysis) and various machine learning algorithms. Causal inference analysis was performed using Mendelian randomization and colocalization analysis. Potential therapeutic drugs were identified using molecular docking technology, and the results of bioinformatics analysis were validated by immunofluorescence staining. Graphpad Prism, R, Python, and Adobe Illustrator software were used for data and image processing. Results: An increased proportion of basal and suprabasal cells was observed in CRS, especially in eosinophilic CRS with nasal polyps (ECRSwNP), with P=0.001. hdWGCNA revealed that the "yellow module" was closely related to basal and suprabasal cells in CRS. Univariate logistic regression and LASSO algorithm selected 13 key genes (CTSC, LAMB3, CYP2S1, TRPV4, ARHGAP21, PTHLH, CDH26, MRPS6, TENM4, FAM110C, NCKAP5, SAMD3, and PTCHD4). Based on these 13 genes, an effective CRS diagnostic model was developed using various machine learning algorithms (AUC=0.958). Mendelian randomization analysis indicated a causal relationship between CTSC and CRS (inverse variance weighted: OR=1.06, P=0.006), and colocalization analysis confirmed shared genetic variants between CTSC and CRS (PPH4/PPH3>2). Molecular docking results showed that acetaminophen binded well with CTSC (binding energy:-5.638 kcal/mol). Immunofluorescence staining experiments indicated an increase in CTSC+cells in CRS. Conclusion: This study integrates various bioinformatics methods to identify key cell types and genes in CRS, constructs an effective diagnostic model, underscores the critical role of the CTSC gene in CRS pathogenesis, and provides new targets for the treatment of CRS.

[Clinical features and prognostic analysis of testicular relapse in pediatric acute lymphoblastic leukemia].

Objective: To investigate the clinical features and prognosis of testicular relapse in pediatric acute lymphoblastic leukemia (ALL). Methods: Clinical data including the age, time from initial diagnosis to recurrence, relapse site, and therapeutic effect of 37 pediatric ALL with testicular relapse and treated in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences between November 2011 and December 2022 were analyzed retrospectively. Patients were grouped according to different clinical data. Kaplan-Meier analysis was used to evaluate the overall survival (OS) rate and event free survival (EFS) rate for univariate analysis, and Cox proportional-hazards regression model was used to evaluate the influencing factors of OS rate and EFS rate for multivariate analysis. Results: The age at initial diagnosis of 37 pediatric testicular relapse patients was (5±3) years and the time from initial diagnosis to testicular recurrence was (37±15) months. The follow-up time was 43 (22, 56) months. Twenty-three patients (62%) were isolated testis relapse. The 5-year OS rate and EFS rate of the 37 relapsed children were (60±9) % and (50±9) % respectively. Univariate analysis showed that the 2-year EFS rate in the group of patients with time from initial diagnosis to testicular recurrence >28 months was significantly higher than those ≤28 months ((69±10)% vs. (11±11)%, P<0.05), 2-year EFS rate of the isolated testicular relapse group was significantly higher than combined relapse group ((66±11)% vs. (20±13) %, P<0.05), 2-year EFS rate of chimeric antigen receptor T (CAR-T) cell treatment after relapse group was significantly higher than without CAR-T cell treatment after relapse group ((78±10)% vs. (15±10)%, P<0.05). ETV6-RUNX1 was the most common genetic aberration in testicular relapsed ALL (38%, 14/37). The 4-year OS and EFS rate of patients with ETV6-RUNX1 positive were (80±13) % and (64±15) %, respectively. Multivariate analysis identified relapse occurred≤28 months after first diagnosis (HR=3.09, 95%CI 1.10-8.72), combined relapse (HR=4.26, 95%CI 1.34-13.52) and CAR-T cell therapy after relapse (HR=0.15,95%CI 0.05-0.51) were independent prognostic factors for 2-year EFS rate (all P<0.05). Conclusions: The outcome of testicular relapse in pediatric ALL was poor. They mainly occurred 3 years after initial diagnosis. ETV6-RUNX1 is the most common abnormal gene.Patients with ETV6-RUNX1 positive often have a favorable outcome. Early relapse and combined relapse indicate unfavorable prognosis, while CAR-T cell therapy could significantly improve the survival rate of children with testicular recurrence.

[Clinical characteristics of epileptic seizure in neurofibromatosis type 1 in 15 cases].

Objective: To summarize the clinical characteristics of epileptic seizure associated with neurofibromatosis type 1 (NF1). Methods: From January 2017 to July 2023 at Children's Hospital Capital Institute of Pediatrics, medical records of patients with both NF1 and epileptic seizure were reviewed in this case series study. The clinical characteristics, treatment and prognosis were analyzed retrospectively. Results: A total of 15 patients(12 boys and 3 girls) were collected. Café-au-lait macules were observed in all 15 patients. There were 6 patients with neurodevelopmental disorders and the main manifestations were intellectual disability or developmental delay. The age at the first epileptic seizure was 2.5 (1.2, 5.5) years. There were various seizure types, including generalized tonic-clonic seizures in 8 patients, focal motor seizures in 6 patients, epileptic spasm in 4 patients, tonic seizures in 1 patient, absence in 1 patient, generalized myoclonic seizure in 1 patient and focal to bilateral tonic-clonic seizure in 1 patient. Among 14 patients whose brain magnetic resonance imaging results were available, there were abnormal signals in corpus callosum, basal ganglia, thalamus or cerebellum in 6 patients, dilated ventricles of different degrees in 3 patients, blurred gray and white matter boundary in 2 patients, agenesis of corpus callosum in 1 patient and no obvious abnormalities in the other patients. Among 13 epilepsy patients, 8 were seizure-free with 1 or 2 antiseizure medications(ASM), 1 with drug resistant epilepsy was seizure-free after left temporal lobectomy, and the other 4 patients who have received 2 to 9 ASM had persistent seizures. One patient with complex febrile convulsion achieved seizure freedom after oral administration of diazepam on demand. One patient had only 1 unprovoked epileptic seizure and did not have another seizure without taking any ASM. Conclusions: The first epileptic seizure in NF1 patients usually occurs in infancy and early childhood, with the main seizure type of generalized tonic-clonic seizure and focal motor seizure. Some patients have intellectual disability or developmental delay. Most epilepsy patients achieve seizure freedom with ASM.

[Genomics of next generation sequencing in pediatric B-acute lymphoblastic leukemia and its impact on minimal residual disease].

Objective: To describe the gene mutation profile of newly diagnosed pediatric B-acute lymphoblastic leukemia (B-ALL) and analyze its effect on minimal residual disease (MRD). Methods: A total of 506 newly diagnosed B-ALL children treated in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences from September 2018 to July 2021 were enrolled in this retrospective cohort study. The enrolled children were divided into MRD ≥1.00% group and <1.00% group according to MRD results on the 19th day since chemotherapy, and MRD ≥0.01% group and <0.01% group according to MRD results on the 46th day. Clinical characteristics and gene mutations of two groups were compared. Comparisons between groups were performed with chi-square test or Fisher's exact test. Independent risk factors of MRD results on the 19th day and the 46th day were analyzed by Logistic regression model. Results: Among all 506 patients, there were 318 males and 188 females. On the 19th day, there were 114 patients in the MRD ≥1.00% group and 392 patients in the MRD <1.00% group. On the 46th day, there were 76 patients in the MRD ≥0.01% group and 430 patients in the MRD <0.01% group. A total of 187 gene mutations were detected in 487 (96.2%) of 506 children. The most common gene mutations were signal transduction-related KRAS gene mutations in 111 cases (22.8%) and NRAS gene mutations in 99 cases (20.3%). Multivariate analysis showed that PTPN11 (OR=1.92, 95%CI 1.00-3.63), KMT2A (OR=3.51, 95%CI 1.07-11.50) gene mutations and TEL-AML1 (OR=0.48, 95%CI 0.27-0.87), BCR-ABL1 (OR=0.27, 95%CI 0.08-0.92) fusion genes and age >10 years (OR=1.91, 95%CI 1.12-3.24) were independent influencing factors for MRD ≥1.00% on the 19th day. BCORL1 (OR=2.96, 95%CI 1.18-7.44), JAK2 (OR=2.99, 95%CI 1.07-8.42) and JAK3 (OR=4.83, 95%CI 1.50-15.60) gene mutations and TEL-AML1 (OR=0.43, 95%CI 0.21-0.87) fusion gene were independent influencing factors for MRD ≥0.01% on the 46th day. Conclusions: Children with B-ALL are prone to genetic mutations, with abnormalities in the RAS signaling pathway being the most common. Signal transduction related PTPN11, JAK2 and JAK3 gene mutations, epigenetic related KMT2A gene mutation and transcription factor related BCORL1 gene mutation are independent risk factors for MRD.

Inhibition of the myeloid differentiation primary response protein 88 reducres neuron injury in the early stages of subarachnoid hemorrhage in an in vitro experimental model.

Subarachnoid hemorrhage (SAH) is a life-threatening cerebrovascular disease with high rates of morbidity and mortality and a paucity of effective therapies. The development of early brain injury (EBI) is closely related to prognosis in SAH, and inflammation plays an important role in its pathophysiology. A previous experiment showed that ST2825, a selective inhibitor of MyD88, could alleviate EBI in vivo. However, this protective effect in vivo is affected by a variety of pathophysiology processes making the result to some extent uncertain. whether there is a coincident result in vitro ruling out the effect of other factors remains unknown, and further investigation using cultured neurons is necessary. Primary neuronal cells were cultured to construct an in vitro model of SAH. The cells were cultured and then divided into three groups: (1) a blank control group, (2) an oxygenated hemoglobin + vehicle group, and (3) an oxygenated hemoglobin + ST2825 group. In each group, apoptosis of neuronal cells along with changes in the expression of proteins including MyD88, p-JNK, p-Erk, p-p38, NFκB, Bcl-2, and P53 were measured. Results showed that after stimulating neurons with oxygenated hemoglobin, the expression of the MyD88 protein in the vehicle group increased significantly. The quantity of p-JNK, p-p38, and p-Erk also increased significantly, as did the quantity of p65 in the nucleus. Expression of the anti-apoptotic protein Bcl-2 was markedly reduced, while that of the cleaved caspase-3 protein was significantly increased. In addition, in this group, the apoptosis rate of neurons was significantly increased. In the ST2825 group, the expression of p-JNK, p-p38, p-Erk, cleaved caspase-3, and p65 in the nucleus was significantly decreased, the expression of Bcl-2 was significantly increased, and the apoptosis rate of neurons was significantly reduced. The results of this study suggest that in an experimental in vitro SAH model, ST2825, a selective inhibitor of MyD88, can have a neuroprotective effect by inhibiting neuronal apoptosis mediated by the MAPK and NFκB signaling pathways, and this has a certain protective effect on EBI after SAH.

Three-dimensional bioprinting of artificial ovaries by an extrusion-based method using gelatin-methacryloyl bioink.

PURPOSE: The aim of this study was to design and fabricate a three-dimensional (3D) printed artificial ovary. METHODS: We first compared the printability of gelatin-methacryloyl (GelMA), alginate and GelMA-alginate bioinks, of which GelMA was selected for further investigation. The swelling properties, degradation kinetics and shape fidelity of GelMA scaffolds were characterized by equilibrium swelling/lyophilization, collagenase processing and micro-computed tomography evaluation. Commercial ovarian tumor cell lines (COV434, KGN, ID8) and primary culture ovarian somatic cells were utilized to perform cell-laden 3D printing, and the results were evaluated by live/dead assays and TUNEL detection. Murine ovarian follicles were seeded in the ovarian scaffold and their diameters were recorded every day. Finally, in vitro maturation was performed, and the ovulated oocytes were collected and observed. RESULTS: Our results indicated that GelMA was suitable for 3D printing fabrication. Its scaffolds performed well in terms of hygroscopicity, degradation kinetics and shape fidelity. The viability of ovarian somatic cells was lower than that of commercial cell lines, suggesting that extrusion-based 3D culture fabrication is not suitable for primary ovarian cells. Nevertheless, the GelMA-based 3D printing system provided an appropriate microenvironment for ovarian follicles, which successfully grew and ovulated in the scaffolds. Metaphase II oocytes were also observed after in vitro maturation. CONCLUSIONS: The GelMA-based 3D printing culture system is a viable alternative option for follicular growth, development and transfer. Accordingly, it shows promise for clinical application in the treatment of female endocrine and reproductive conditions.

Docetaxel and bevacizumab with or without trastuzumab as first-line treatment for patients with metastatic breast cancer: a meta-analysis.

OBJECTIVE: Although bevacizumab and trastuzumab have been widely added to the standard regimen for metastatic breast cancer, the clinical outcomes remain controversial. The purpose of this study was to conduct meta-analysis to verify the clinical efficacy and safety of docetaxel and bevacizumab with or without trastuzumab as first-line treatment for patients with metastatic breast cancer (MBC). MATERIALS AND METHODS: All available literature of clinical trials about docetaxel, bevacizumab, trastuzumab and metastatic breast cancer was pooled from PubMed, Embase and Cochrane library database. The meta-analysis combined the progression free survival (PFS), overall response rate (ORR) and incidence of all grades adverse events in MBC patients. RESULTS: Seven clinical trials were included by two reviewers. Docetaxel and bevacizumab with trastuzumab show the pooled PFS was 16.53 months (95% CI: 13.95-19.11 months), the pooled ORR was 0.75 (95% CI: 0.69-0.80) in HER2-positive MBC patients. Docetaxel and bevacizumab show that the pooled PFS was 8.49 months (95% CI: 7.80-9.18 months), the pooled ORR was 0.51(95% CI: 0.47-0.55) in HER2-negative MBC patients. CONCLUSIONS: Both for patients with HER2-positive and negative metastatic breast cancer, docetaxel and bevacizumab with or without trastuzumab as first-line treatment resulted in long survival, especially in terms of progression-free survival. Although the overall response rates are also significantly improved, it is still controversial based on the current evidence.

[Hypoxic stimulation leads to the changes of inflammatory factors in mucosal epithelial cells of nasal polyps].

Objective: To investigate the roles of hypoxic stimulation in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) by comparing the variation and differences of inflammatory factors secreted from epithelial cells of nasal polyps and normal nasal mucosa under hypoxic stimulation. Methods: Sixty-eight patients who were diagnosed with CRSwNP from June 2015 to January 2018 at China-Japan Union Hospital of Jilin University were analyzed, including 36 males and 32 females, aged (45.2±12.5) years. Nasal polyps mucosa was included in CRS-NP group and inferior turbinate mucosa was included in CRS-IT group. According to the degree of eosinophil infiltration in histopathologic results, each of these two groups was further divided into eosinophil infiltration and non-eosinophil infiltration as Eos-NP group (n=34), Non-Eos-NP group (n=34), Eos-IT group (n=20) and Non-Eos-IT group (n=20). The inferior turbinate mucosa of twenty-five patients who were diagnosed with cyst of paranasal sinus or deviation of nasal septum was classified as control group (n=25), including 14 males and 11 females, aged (42.8±10.2) years. The expression of interleukin 17A (IL-17A), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and hypoxia-inducible factor 1α (HIF-1α) in each group was analyzed by immunohistochemical staining. After 0, 24 and 48 h hypoxic stimulation, the secretion of IL-17A, IFN-γ, TNF-α in primary nasal mucosa epithelial cells of each group was tested by enzyme-linked immune sorbent assay (ELISA) experiment; the expression of HIF-1α was tested by immunofluorescent staining and imaging and Western blot. SPSS 17.0 software and two-way ANOVA were used for statistical analysis. Results: Immunohistochemical staining showed that the expression of IL-17A and TNF-α was much higher in control group (optical density (OD) value was 0.37±0.03, 0.53±0.02, respectively) and the expression of IFN-γ and HIF-1α was much higher in Eos-IT group (OD value was 0.47±0.03, 0.39±0.02, respectively). The secretion of IL-17A and TNF-α was much lower in control group than that in other groups under normal condition. After 48 h hypoxic stimulation, the secretion of IL-17A and TNF-α was much higher in control group compared with other groups. The secretion of IFN-γ in Eos-NP group was much higher than that in control group under normal condition ((13.7±1.3) pg/ml vs (11.1±1.6) pg/ml, P<0.05). After 48 h hypoxic stimulation, there was no difference of IFN-γ between control group and Eos-NP group. The expression of HIF-1α decreased in Eos-NP group and Non-Eos-NP group while increased in CRS-IT group and control group upon prolonged exposure to hypoxia. HIF-1α was mostly located at cytoplasm of epithelial cells in control and CRS-IT group while mainly located at nucleus of epithelial cells in CRS-NP group. Conclusions: The secretion of IL-17A, TNF-α, IFN-γ and the expression of HIF-1α show significant difference between normal nasal mucosa, polyps and inferior turbinate of CRSwNP under hypoxic stimulation, presenting different subcellular localization. This illustrates the proteins above are involved in transcription and regulation of the gene responsible for the pathogenesis of CRSwNP.

[Mechanism of lncRNA-SRLR induced invasion and metastasis in U2OS osteosarcoma cells].

Objective: To explore the potential mechanism of sorafenib resistance associated long non-coding RNA (lncRNA-SRLR) promoted invasion and metastasis in U2OS osteosarcoma cells. Methods: We transfected U2OS cells with negative control lentivirus (LV-NC) or lncRNA-SRLR overexpressed lentivirus (LV-over/SRLR) particles. LV-NC and LV-over/SRLR stable transfected cells (U20S/NC and U20S/SRLR) were selected by primary cell culture medium containing puromycin. The mRNA expressions of lncRNA-SRLR and procollagen-lysine, procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of lncRNA-SRLR on the invasion of U2OS cells were determined by wound-healing assay and Transwell migration assay. The effect of SRLR on the interleukin-6 (IL-6) secretion of U2OS cells was evaluated by enzyme-linked immunosorbent assay (ELISA) analysis. The subcellular distribution of SRLR in U2OS cells was detected by fluorescence in situ hybridization (FISH) analysis.The expression of PLOD2 in cells was detected by immunofluorescence (IF). The expressions of PLOD2 and focal adhesion kinase (FAK)/signal transducer and activator of transcription 3 (STAT3) signal pathway related proteins in U2OS/NC and U2OS/SRLR cells were detected by western blotting. Results: qRT-PCR assay showed that mRNA expressions of lncRNA-SRLR and PLOD2 in U2OS/SRLR cells were (3 964.97±0.05) and (2.77±0.11), respectively, significantly higher than those in U2OS/NC cells (P<0.001 or P<0.01). The results of wound-healing and Transwell migration assay showed that over-expression of SRLR markedly promoted the invasion ability of U2OS cells (P<0.05). The result of ELISA analysis showed that the IL-6 secretions in U2OS/NC or U2OS/SRLR cells were (125.38±11.22) pg/ml or (119.97±13.43) pg/ml, without statistical significance (P>0.05). The subcellular distribution assay revealed that lncRNA-SRLR is predominately located in the nucleus. The result of IF showed that compared with U2OS/NC cells, the expression of PLOD2 was up-regulated in U2OS/SRLR cells. The result of western blotting showed that over-expression of SRLR significantly increased the expression levels of PLOD2, phosphorylation (p)-FAK and p-STAT3 in U2OS cells (P<0.01). Conclusion: lncRNA-SRLR promotes invasion and metastasis of osteosarcoma by activating PLOD2-FAK/STAT3 signal axis.

[Genetic characteristics and clinical outcomes of pediatric acute myeloid leukemia with NUP98-NSD1 fusion gene].

Objective: To investigate the genetic characteristics and clinical outcomes of pediatric acute myeloid leukemia patients with NUP98-NSD1 fusion gene. Methods: A total of 80 pediatric AML patients were enrolled in this study, and bone marrow specimens were collected at initial diagnosis and relapse. NUP98-NSD1 was screened by fluorescence in situ hybridization (FISH) and PCR. Other laboratory test results and clinical outcomes were further analyzed for the NUP98-NSD1 positive cases. Results: A total of eight patients (10.0%) were positive for NUP98-NSD1, which were all fusions of NUP98 exon12 and NSD1 exon 6. There were two M2, three M4, and three M5 cases according to the French-American-British classification. Seven patients had karyotype results at the time of initial diagnosis, and none of them had complicated karyotype abnormalities. Among these patients, two cases had normal karyotype, three cases had trisomy 8, one case had trisomy 6, and two cases had anomalies involving 9q13 or 9q21. Additional karyotypic abnormalities and clonal evolutions were observed during disease progression or relapse, five cases had 9q13 or 9q32 abnormalities. Five cases (62.5%) were positive with FLT3-ITD mutation. Patients were treated with DAE/NAE/HAE/IA chemotherapy. Three cases did not achieve remission after several courses of chemotherapy, and five cases achieved remission but relapsed in 1 to 19 months. Five cases underwent salvage allogeneic hematopoietic stem cell transplantation (allo-HSCT). Among whom, four died in 40 days to 4 months after transplantation, and one survived 8.5 months till the last follow-up. Conclusions: NUP98-NSD1 is a recurrent genetic abnormality with significant clinical prognostic significance, and this group of disease has unique clinical and genetic characteristics. NUP98-NSD1 should be screened by FISH or PCR for children with AML who are newly diagnosed or refractory and relapsed to identify the high-risk genetic marker.

[Efficacy and peripheral immunity analysis of allogeneic natural killer cells therapy in patients with hepatocellular carcinoma].

OBJECTIVE: To evaluate the safety and efficacy of allogeneic natural killer (NK) cells in the treatment of primary hepatocellular carcinoma (HCC), and to elucidate the mechanism of NK cells therapy. METHODS: Twenty-one patients with primary HCC treated with allogeneic NK cells at the Fifth Medical Center of the PLA General Hospital were followed up for 1 year. Peripheral blood mononuclear cells (PBMCs) were isolated from patient-related donors and cultured in vitro for 15 days and infused to the patients in two consecutive days. Clinical data and laboratory data were collected and analyzed, including survival, clinical features, imaging changes, hematology, immunology, and biochemical indicators to evaluate the safety and efficacy of allogeneic NK cell therapy. The changes of peripheral blood lymphocyte subsets after treatment were also analyzed to explore the possible anti-tumor mechanisms. RESULTS: (1) Of the 21 patients with primary HCC, 11 patients were treated once, 5 patients were treated twice, and 5 patients were treated 3 times. After allogeneic NK cells infusion, 10 patients had fever, 1 patient had slight hepatalgia and 1 patient had slight headache, no other adverse events occurred including acute and chronic graft-versus-host disease (GVHD). They resolved spontaneously within 8 hours without other treatment. (2) The total disease control rate was 76.2% during one-year follow-up. Among them, the patients with Barcelona clinic liver cancer (BCLC) stage A had a disease control rate of 100%, stable disease (SD) in 10 cases; BCLC stage B patients had a disease control rate of 60%, partial response (PR) in 1 case, and SD 2 in cases; BCLC stage C patients had a disease control rate of 50%, complete response (CR) in 1 case, and 2 cases of PR. (3) The frequencies of NK cells and CD8+ T cells in peripheral blood were significantly lower than that before at 24 hours after treatment, and the frequencies of CD4+ T cells and CD4/CD8 were significantly higher than the baseline. CONCLUSION: Allogeneic NK cells have good safety and efficacy in the treatment of primary HCC. The anti-tumor effect of the allogeneic NK cells may play an important role in the activation of the patient's natural immune system and delay disease progression, suggesting that allogeneic NK cells combined with sorafenib may be a very effective treatment for advanced HCC, and further large-sample multicenter randomized controlled clinical trials are needed to validate this result.

[Mechanism of MALAT1 induced osimertinib resistance in HCC827 lung cancer cells].

Objective: To test the effect of metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) and/or osimertinib on the proliferation and apoptosis of HCC827 cells, and explore the potential mechanism of MALAT1 induced resistance to osimertinib. Methods: We transfected HCC827 cells with LV-vector or LV-over/MALAT1. Stable transfected cells (HCC827/Vector, HCC827/MALAT1) were selected by adding puromycin. HCC827/MALAT1 cells were further transfected with the shRNA-negative control (NC) or shRNA-human epidermal growth factor receptor 3 (ERBB3) plasmid. The effects of overexpression of MALAT1, knockdown of ERBB3 and/or osimertinib on the proliferation of HCC827 cells were evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. Cell apoptosis induced by MALAT1 overexpression, knockdown of ERBB3 and/or osimertinib treatment were analyzed by flow cytometry analysis. The expressions of EGFR and ERBB3 signal pathway related proteins in HCC827 cells treated with overexpression of MALAT1, knockdown of ERBB3 and/or osimertinib treatment were detected by western blot. Results: The MTT assay showed that sensitivity to osimertinib of HCC827/MALAT1 cells were significantly repressed. The 50% inhibitive concentration (IC(50)) of osimertinib >4 000 nmol/L in HCC827/MALAT1 cells. However, knockdown of ERBB3 facilitated the anti-proliferation effect of osimertinib, and the IC(50) of osimertinib in shRNA-ERBB3 cells was (17.27±3.21) nmol/L. The results of flow cytometry analysis showed that the apoptotic rate of HCC827/MALAT1 cells induced by 10 nmol/L osimertinib was (8.38±0.92)%, significantly lower than (27.17±5.83)% of knockdown of ERBB3 (P<0.01). Western blotting showed that the expression of p-ERBB3, p-AKT and p-extracellular regulated protein kinases (ERK) in HCC827/MALAT1 cells was markedly up-regulated, while the expression of p-epithelial growth factor receptor (EGFR) was inhibited. The expressions of p-ERBB3, p-AKT and p-ERK were marginally affected by osimertinb. However, osimertinib downregulated the expressions of p-EGFR, p-ERBB3, p-AKT and p-ERK in ERBB3 deleted cells. Conclusions: MALAT1 confers resistance to osimertinb in HCC827 cells by activating of the ERBB3/PI3K/AKT and ERBB3/MAPK/ERK signaling pathways.

[Relationship between urinary cadmium and type 2 diabetes mellitus in adults].

Objective: To explore the relationship between environmental factors as urinary cadmium and type 2 diabetes mellitus (DM) in adults. Methods: Case-control study was adopted, including 166 cases and 427 controls. General characteristics of the subjects were collected by a structured questionnaire. FPG, biochemical indexes and urinary cadmium (UCd) were detected respectively, while UCd was corrected with creatinine. Unconditioned logistic regression model was applied to analyze the relationship between UCd and DM. Results: Levels of UCd appeared higher in cases with the following characteristics as: having primary school education (P=0.016), being female (P=0.013), being non-smokers (P=0.014) or non-alcoholic (P=0.025), and with BMI>25.00 kg/m(2) (P=0.040, P=0.025) than those appeared in the control group. Same results were shown in the 60-69 years (P=0.024) old group. Data from the unconditional logistic regression analysis showed that family history of DM (OR=3.19, 95%CI: 1.45-7.03), education status (OR=1.50,95%CI: 1.08-2.08) and UCd (OR=1.61, 95%CI: 1.08-2.41) were influencing factors on DM. Conclusion: A close association between UCd and DM was noticed. UCd appeared a risk factor on DM that called for setting up related prevention program to reduce the exposure of Cd and to control the risk on DM.

[Cellular damage of low-dose combined exposure to mercury, lead and cadmium on hippocampal neurons in rats].

Objective: To observe the cellular damage of low-dose combined exposure to Hg, Pb and Cd on hippocampal neurons in rat. Methods: SH-SY5Y cells were randomly divided into 8 groups by 2×2×2 factorial design: control group, Pb exposure group, Hg exposure group, Pb+Hg exposure group, Pb+Cd exposure group, Hg+Cd exposure group and Pb+Cd+Hg exposure group. And the cell viabilities were measured. On this basis, an animal model was established. Twenty eight-week-old SD pregnant rats were randomly divided into four groups by random number table, and five in each group: the control group(distilled water), 1-fold metal mixture exposure group (1×MM, poisoning solution containing mercury chloride 0.15 mg/L, lead acetate trihydrate 25 mg/L, cadmium chloride 7.5 mg/L), 5-fold metal mixture exposure group (5×MM, poisoning solution containing mercury chloride 0.75 mg/L, lead acetate trihydrate 125.00 mg/L, cadmium chloride 37.50 mg/L), 10-fold metal mixture exposure group (10×MM, poisoning solution containing mercury chloride 1.50 mg/L, lead acetate trihydrate 250.00 mg/L, cadmium chloride 75.00 mg/L). Pregnant rats drank water until delivery. Twenty male pups were selected and exposed to these metals through breast milk until weaned. The heavy metals dose of poisoning water was adjusted, and then the weaned rats were exposed to heavy metals via drinking poisoning water until adulthood (postnatal day 83). The blood samples and brain hippocampus samples were collected to observe the ultrastructural changes of hippocampus, and to determine the levels of Hg, Pb and Cd in blood. In addition, apoptosis rate and fluorescence intensity of reactive oxygen species and intracellular free calcium concentration ([Ca(2+)](i)) in hippocampal neurons were measured. Results: Cellular factorial design analysis showed that Hg+Pb+Cd (at no observed adverse effect level, 1.0, 0.5 and 0.1 µmol/L, respectively)had a interaction on cell viability after 48 or 72 hours of combined exposure (P<0.05). The results of ultrastructure showed that mitochondria decreased, ridges and matrixes gradually dissolved in rat hippocampal neurons of 5×MM group; nuclear chromatin aggregated, more ridges and matrixes dissolved and the mitochondria also decreased in rat hippocampal neurons of 10×MM group. The concentration of Hg, Pb and Cd in the blood of 1×MM group, 5×MM group and 10×MM group were higher than those in the control group, and the differences were statistically significant (P<0.001). There was no significant difference in apoptosis rate between the 1×MM group and the control group. The apoptosis rate of 5×MM group and 10×MM group was higher than that in the control group, and the differences were statistically significant (P<0.001). There was no statistically significant difference in the fluorescence intensity of reactive oxygen species in hippocampal neurons of the 1×MM group and the control group. The fluorescence intensity of reactive oxygen species in the 5×MM group and the 10×MM group was higher than that in the control group, the difference was statistically significant (P<0.05). There was no significant difference in the fluorescence intensity of [Ca(2+)](i) between the 1×MM group and the control group. The fluorescence intensity values of [Ca(2+)](i) in the 5×MM group and the 10×MM group were higher than the control group, the differences were statistically significant (P<0.001). Conclusion: Low-level combined exposure to Hg, Pb, and Cd caused synergistic neurotoxic damage, and the process may be related to the changes of neuronal apoptosis, reactive oxide species, and [Ca(2+)](i) levels.

[Mechanism of miR-221 contributes to gefitinib resistance in PC-9 cells].

Objective: To investigate the effect of microRNA-221 (miR-221) overexpression on gefitinib resistance in PC-9 cells and study its underlying mechanisms. Methods: PC-9 cells were transfected with LV-NC and LV-miR-221 to establish cell stabilizing strains (PC-9/NC and PC-9/miR-221), then they were used to detect the relative expression of miR-221 and apoptotic protease activating factor-1 (APAF-1) mRNA by real-time fluorescence quantitative PCR (qRT-PCR). The effects of gefitinib (0-4 µmol/L) on the growth and proliferation of cell stabilizing strains were detected by CCK-8 Assay. After gefitinib treatment, cell apoptosis was detected by Flow Cytometry Assays. The expression of epidermal growth factor receptor (EGFR), phosphorylated epidermal growth factor receptor (p-EGFR), APAF-1 and cleaved cysteinyl aspartate specific proteinase-3 (Cleaved-caspase-3) were detected by Western blot. Dual-Luciferase Reporter Assay was used to evaluate the relationship between APAF-1 and miR-221. Results: The relative expression of miR-221 in PC-9/NC cells was significantly lower than that in PC-9/miR-221 cells[(1.00±0.082) vs (40.24±0.017)](P<0.01). The half maximal inhibitory concentration (IC(50)) in PC-9/NC cells was significantly lower than that in PC-9/miR-221 cells[(IC(50)=0.1 µmol/L) vs (IC(50)>4 µmol/L)](P<0.05). Apoptosis rate of PC-9/NC cell was significantly higher than PC-9/miR-221[(33.42±4.28)% vs (10.27±1.12)%](P<0.05); APAF-1 mRNA expression was significantly higher in PC-9/NC cells than PC-9/miR-221[(1.000+ 0.069) vs (0.701±0.072)](P<0.05), and the expression of APAF-1 protein in PC-9/NC cells was significantly higher than that of PC-9/miR-221 cells. The dual luciferase reporter gene results showed that miR-221a inhibited luciferase activity significantly stronger than transfected miRNA negative control group after co-transfection of luciferase plasmids pmir-REPORT-APAF-1-wt and miR-221a mimics (P<0.01). p-EGFR was down-regulated in both PC-9/NC and PC-9/miR-221 cells after treatment with gefitinib. APAF-1 and Cleaved-caspase-3 proteins were significantly down-regulated in PC-9/miR-221 cells compared with PC-9/NC cells, while APAF-1 and Cleaved-caspase-3 proteins were up-regulated in PC-9/NC cells treated with gefitinib compared with PC-9/miR-221 cells (P<0.05). Conclusion: miR-221 induces resistance to gefitinib in PC-9 cells by downregulating APAF-1 expression.

[Dynamic investigation of nutritional risk in patients with malignant tumor during hospitalization].

Objective: To prospectively investigate the changes in nutritional status of patients with malignant tumors during hospitalization by using nutritional risk screening (NRS2002), and to analyze the correlation between the nutritional status and clinical outcomes . Methods: This was a prospective and parallel research done by multi-center collaboration from 34 hospitals in China from June to September 2014.Hospitalized patients with malignant tumors inthese departments (Department of Gastroenterology, respiratory medicine, oncology, general surgery, thoracic surgery and geriatrics)were investigated. Only the patients with age≥ 18 years and hospitalization time between 7-30 days were included. During hospitalization, the physical indexes of human bodywere measured, and the NRS 2002 scores, and monitored the nutritional support at the time points of admission and 24 hours before discharge were recorded.And whether there was a nutritional risk in hospitalized patients and its association with clinical outcomes were investigated. Results: A total of 2 402 patients with malignancies were enrolled in this study. Seventy fourpatients who did not complete NRS2002 were eliminated, and 2 328 patients were included. The number of the main diseases was the top five, including 587 cases of colorectal cancer, 567 cases of lung cancer, 564 cases of gastric cancer, 146 cases of esophageal cancer, and 119 cases of liver tumor. At the time of discharge, compared with admission, the BMI, body weight, grip and calf circumferences of patients with malignant tumor were significantly decreased (P<0.05). The total protein, albumin, prealbumin and hemoglobin were significantly lower than those at admission (P<0.05). In 2 328 patients who were completed nutritional risk screening, the rate of malnutrition at admission was 11.1% (BMI =18.5, 258/2 328) and the rate of malnutrition at discharge was 10.9% (BMI =18.5, 254/2 328), there were no significant differences (χ(2)=0.019 7, P=0.888). There were 1 204 patients with nutritional risk at admission (51.7%, NRS2002 score≥3)and 1 352 patients with nutritional risk at discharge (58.1%, NRS2002 score≥3), with significant differences (χ(2)=49.9, P<0.001). The incidence of nutritional risk in patients with colorectal, stomach, and lung tumors at discharge was significantly higher than that at admission (P<0.05). The infective complications and other complications of patients with nutritional risk were significantly greater than those without nutritional risk at admission and at discharge.ICU hospitalization stay of patients with nutritional risk was increased significantly than those without nutritional risk at admission(P=0.042). Hospitalization expenses of patients with nutritional risk was increased significantly than those of patients without nutritional risk at discharge(P<0.01). Conclusion: The patients with malignant tumor have a higher incidence rate of malnutrition at both admission and discharge and malnutritionhas correlation with adverse clinical outcomes.The aboveindicators did not improve significantly at discharge.Doctors should pay more attention to the nutritional status (screening and evaluation)of patients before discharge and use appropriate and adequate nutrition support in order to prevent the weight loss and improve the life quality of patients.

[Clinical significance of four quadrant localization in the diagnosis and treatment of metastatic carcinoma of the neck with unknown primary].

Objective:The aim of this study is to investigate the clinical significance of four quadrant localization in the diagnosis and treatment of unknown primary cervical metastases. Method:The clinical data with unknown primary cervical metastases, were analyzed retrospectively. All the patients have not been found the original site in the initial treatment. There are four quadrants in the neck, the neck line as the longitudinal axis, and edge of cricoid cartilage as the horizontal axis. When cervical metastasis occurred in the left and right upper quadrant, the primary tumor site and radiotherapy from the skull base to the root of the neck; when appear in left and right lower quadrant, the primary investigation site and radiotherapy from neck to thoracic mediastinum, left lower abdomen also includes following primary search. At the same time, bilateral cervical metastasis cancers, focusing on the central line near the primary focus. Specific treatment strategies include ipsilateral total neck dissection and radical radiotherapy of the above radiotherapy site. Result:Left upper neck in 4 cases, right upper neck in 5 cases, left lower neck in 7 cases, lower right neck in 8 cases and mixed area in 6 cases. Only 10 of 30 patients (33.3%) with primary sites were found in the follow up period. In accordance with the four quadrant localization, the median time was 6 months. Conclusion:Four quadrant localization to locate the primary site is accurate, and individualized comprehensive treatment is the key to improve the curative effect.

Autophagy mediated by arginine depletion activation of the nutrient sensor GCN2 contributes to interferon-γ-induced malignant transformation of primary bovine mammary epithelial cells.

Autophagy has been linked to the regulation of both the prevention and progression of cancer. IFN-γ has been shown to induce autophagy in multiple cell lines in vitro. However, whether IFN-γ can induce autophagy and whether autophagy promotes malignant transformation in healthy lactating bovine mammary epithelial cells (BMECs) remain unclear. Here, we provide the first evidence of the correlation between IFN-γ treatment, autophagy and malignant transformation and of the mechanism underlying IFN-γ-induced autophagy and subsequent malignant transformation in primary BMECs. IFN-γ levels were significantly increased in cattle that received normal long-term dietary corn straw (CS) roughage supplementation. In addition, an increase in autophagy was clearly observed in the BMECs from the mammary tissue of cows expressing high levels of IFN-γ. In vitro, autophagy was clearly induced in primary BMECs by IFN-γ within 24 h. This induced autophagy could subsequently promote dramatic primary BMEC transformation. Furthermore, we found that IFN-γ promoted arginine depletion, activated the general control nonderepressible-2 kinase (GCN2) signalling pathway and resulted in an increase in autophagic flux and the amount of autophagy in BMECs. Overall, our findings are the first to demonstrate that arginine depletion and kinase GCN2 expression mediate IFN-γ-induced autophagy that may promote malignant progression and that immunometabolism, autophagy and cancer are strongly correlated. These results suggest new directions and paths for preventing and treating breast cancer in relation to diet.

[Cadmium burden and renal dysfunction among residents in cadmium-polluted areas: A 3-year follow-up study].

OBJECTIVE: To investigate dynamic change of cadmium body burden and renal dysfunction among residents living in cadmium-polluted areas. METHODS: From April to July of 2011, the cadmium-polluted areas of northern Guangdong province in China was chosen as the study site. Based on the levels of cadmium pollution in soil and rice, the survey areas were divided into low exposed group (average concentration of cadmium was 0.15-0.40 mg/kg, 0.5-1.0 mg/kg in rice and soil, respectively) and high exposed group (average concentration of cadmium was >0.40 mg/kg, >1.0 mg/kg in rice and soil, respectively). Stratified random sampling and cluster sampling method of epidemiological investigations were carried out among 414 local residents who lived in cadmium exposure areas for more than 15 years, aged above 40, and without occupational cadmium exposure, including 168 and 246 residents in low and high exposed group, respectively. From March to June of 2014, 305 respondents of those who participated in 2011 were successfully traced, including 116 and 189 respondents in low and high exposed group, respectively. We used health questionnaires to acquire their health status. Home-harvested rice and vegetable samples were collected using quartering method for detection of cadmium level, including 190 rice samples, 161 vegetable samples in 2011 and 190 rice samples, 153 vegetable samples in 2014. Urine specimens of residents were collected for the detection of urinary cadmium and creatinine as well as renal dysfunction biomarkers, namely, N-acetyl-beta-D-glucosamidase (NAG) and ß2-microglobulin (ß2-MG), respectively. In 2011 and 2014, Chi-square test was used to investigate the differences of abnormality of cadmium concentration in rice, vegetables and urinary cadmium, ß2-MG, and NAG that were expressed as odds ratio(OR) and 95% confidence intervals (95%CI). RESULTS: In 2011 and 2014, cadmium concentration P50 (P25-P75) in rice was 0.43 (0.17-1.10) mg/kg, and 0.42 (0.20-1.14) mg/kg, respectively (Z=-0.77,P=0.440). In 2011 and 2014, cadmium concentrations P50 (P25-P75) in vegetables were 0.13 (0.07-0.34) mg/kg, and 0.25(0.12-0.59) mg/kg, respectively, with abnormal rates of 38.5%(62/161) and 60.8%(93/153), respectively. In 2014, both average concentration and abnormal rate of cadmium in vegetables were higher than those in 2011 (Z=-4.69, P<0.001 and χ(2)=15.58,P<0.001). Concentrations of urinary cadmium P50 (P25-P75) in high exposed group were 7.90 (3.96-14.91) µg/g creatinine, 8.64 (4.56-17.60) µg/g creatinine in 2011 and 2014, respectively. Contrary to that in 2011, urinary cadmium of high exposed group was significantly increased in 2014 (Z=-2.80, P=0.005). In 2011 and 2014, concentrations of ß2-MG, NAG P50 (P25-P75) were 0.15(0.07-0.29) µg/g creatinine, 0.15 (0.07-0.45) µg/g creatinine, and 7.12 (5.05-10.65) U/g creatinine, 13.55(9.1-19.84) U/g creatinine, respectively, with abnormal rates of 7.5% (23/305), 15.1% (46/305), 8.2% (25/305) , and 33.8% (103/305), respectively. Compared with baseline in 2011, average concentrations of ß2-MG, NAG significantly increased in 2014 (Z=-2.263, P=0.024 and Z=-12.52, P<0.001), and abnormal rates of ß2-MG, NAG were also higher in 2014 (χ(2)=15.61 , P<0.001 and χ(2)=64.72, P<0.001), with odds ratio(OR) of 2.00 (95%CI:1.23-3.24) and 4.12 (95%CI:2.87-5.92). CONCLUSION: Environmental cadmium pollution of crops such as rice and vegetables in survey areas continued to remain high. Body burden of cadmium might kept at sustainably high levels and renal dysfunction was worsened after continuous, long-term cadmium exposure. Our results suggested that NAG might be more sensitive than ß2-MG to serve as an indicator for an individual's future tubular function.