[Study on the inhibitory and pro-apoptotic effects of different concentrations of total tanshinone alone and in combination with tyrosine kinase inhibitors on human myeloid leukemia cell lines].

Objective: To explore the effect and investigate the molecular mechanism of different concentrations of total tanshinones alone and in combination with tyrosine kinase inhibitors (TKIs) on the proliferation inhibition and apoptosis of human myeloid leukemia cell lines. Methods: K562 and Kasumi-1 cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the TKIs-resistant strain K562/T315I cell line was constructed in Molecular Medicine Research Center, Beijing Lu Daopei Institute of Hematology. Logarithmic growth phase cells were taken and divided into intervention groups with total tanshinone of 0, 2.19, 4.38, 8.75, 17.50 and 35.00 µg/ml intervention groups, which were inoculated in 96-well plates at a density of 1×104 cells/well and exposed to the drug for 24 h, and a control group treated with dimethyl sulfoxide was also set up simultaneously. All experiments were repeated independently 3-5 times. The proliferative activity of the cells was assessed using the CCK-8 assay, the apoptotic rates were measured by flow cytometry, and the expression levels of apoptosis-regulating proteins Bcl-2 and Bax were analyzed by Western blotting. The cell lines treated and untreated with total tanshinone were subjected to transcriptome sequencing and gene set enrichment analysis to identify differentially expressed genes. Results: The half-inhibitory concentration (IC50) values of 8.75 µg/ml total tanshinone at 24 h for K562, K562/T315I and Kasumi-1 cells were (4.11±0.02), (4.95±0.04) and (3.98±0.01) µg/ml, respectively. When combined with 0.25 µmol/L imatinib, 8.75 µg/ml total tanshinone could enhance the induction of apoptosis effects on K562 and K562/T315I cell lines. After being treated with 4.38, 8.75, and 17.50 µg/ml of total tanshinone for 24 h, compared with the control group, total tanshinone upregulated the expression level of Bax protein, downregulated the expression level of Bcl-2 protein, and decreased the Bcl-2/Bax ratio (all P<0.05). Total tanshinone inhibited the proliferation-related signaling pathway and DNA damage repair pathway of myeloid leukemia cell lines, and activated the signaling pathway that induces apoptosis in leukemia cells. Conclusion: Different concentrations of total tanshinoneinhibites proliferation and promote apoptosis in K562, Kasumi-1 and TKIs-resistant K562/T315I cell lines, and further enhance the anti-leukemic effect when combined with TKIs.

[Establishment of a prognostic model for non-nephrotic membranous nephropathy based on unbalanced data].

Objective: To explore the construction of a machine learning model based on unbalanced data to predict the progression of non-nephrotic membranous nephropathy. Methods: The clinical and pathological data of patients diagnosed with non-nephrotic membranous nephropathy by renal biopsy in Shanxi People's Hospital from January 2018 to December 2021 were retrospectively analyzed.The prediction models were constructed based on logistic regression, support vector machine (SVM) and light gradient boosting machine (lightGBM), respectively. The mixed sampling technology was used to process the unbalanced data, and the area under the receiver operating characteristic curve (AUC) was used to evaluate the predictive performance of the models. Finally, Shapley additive explanation (SHAP) was used to interpret the results of the optimal prediction model. Results: A total of 148 patients were included in the study, including 84 males and 64 females, with a mean age of (47.2±12.5) years. The follow-up time [M(Q1, Q3)] was 14(7, 20) months. Twenty-three patients (15.5%) achieved the renal end-point event in the study. The SVM model had the highest AUC (0.868, 95%CI: 0.813-0.925), followed by logistic regression (AUC=0.865, 95%CI: 0.755-0.899) and lightGBM (AUC=0.791, 95%CI: 0.690-0.882). The feature recursive elimination cross validation (RFECV) method based on random forest (RF) and the SHAP plot based on the SVM model showed that immunohistochemistry IgG, total protein (TP), anti-phospholipase A2 receptor (anti-PLA2R), blood chloride and D-Dimer were risk factors affecting the progress of non-nephrotic membranous nephropathy. Moreover, patients with high immunohistochemistry IgG, anti-PLA2R and D-Dimer had an increased risk of achieving the renal end-point event. Conclusion: The SVM model established in this study can effectively predict the progress of non-nephrotic membranous nephropathy, and provide a new method for the early identification of high-risk patients and precision therapy.

[Analysis of short-term efficacy of overlapping delta-shaped anastomosis in totally laparoscopic left hemicolectomy for digestive tract reconstruction].

Objective: At present, though the laparoscopic delta-shaped anastomosis and overlapping delta-shaped anastomosis have been gradually applied to complete laparoscopic radical resection of left hemicolon cancer, the comparative evaluation of their efficacy has not been mentioned in the published literatures. This study aims to explore the safety, feasibility and short-term efficacy of overlapping delta-shaped anastomosis (ODA) in totally laparoscopic left hemicolectomy. Methods: A retrospective cohort study was performed. The clinical and pathological data of patients who underwent totally laparoscopic left hemicolectomy at Department of Colorectal Surgery, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College from May 2017 to October 2020 were retrospectively analyzed. The case inclusion criteria were as follows: (1) age of 18-75 years; (2) body mass index (BMI) of 18.5-30 kg/m(2); (3) descending colonic and proximal sigmoid colonic adenocarcinoma was confirmed by preoperative colonoscopy and pathology. The exclusion criteria: (1) multiple primary colorectal cancers; (2) uncontrolled or poorly controlled diabetes mellitus, immune system diseases, or hematological diseases; (3) severe intestinal obstruction; (4) left transverse colonic or splenic flexure colonic adenocarcinoma; (5) distant metastasis of liver, lung and other viscera determined by enhanced computed tomography in the chest, abdomen and pelvis. According to the above criteria, a total of 115 patients with left hemicolon cancer were enrolled. All the patients underwent totally laparoscopic left hemicolectomy. Patients who underwent laparoscopic traditional delta-shaped anastomosis were selected as the control group. Patients who underwent laparoscopic ODA were selected as the ODA group. Effects of these two laparoscopic reconstruction methods on postoperative recovery and perioperative complications were analyzed and compared. Results: A total of 60 patients were enrolled in the ODA group, including 32 males and 28 females, with mean age of (57.3±10.4) years and body mass index (BMI) of (25.0±3.1) kg/m(2). While mean 55 patients were enrolled in the control group, including 31 males and 24 females, with mean age of (56.7±9.9) years and BMI of (24.4±2.9) kg/m(2). There was no statistically significant differences between the two groups in gender, age, BMI, American Society of Anesthesiologist (ASA) classification, TNM staging, preoperative abdominal surgery history, neoadjuvant chemotherapy and nutritional status (levels of hemoglobin, lymphocyte count, prealbumin, and albumin) (all P>0.05). All the patients in both groups received R0 resection without conversion to open laparotomy or conversion to extra-abdominal anastomosis. The digestive tract reconstruction time of the ODA group was significantly shorter than that of the control group [(15.1±1.7) minutes vs. (15.9±2.4) minutes, t=-2.053, P=0.042]. There were no statistically significant differences in the total operation time, intraoperative blood loss, length of skin incision, tumor size, proximal and distal margins, harvested lymph nodes, postoperative first ambulatory time, and postoperative hospital stay (all P>0.05). However, the time to the first flatus and the first defecation in the ODA group was significantly shorter as compared to control group [(1.5±0.5) days vs. (1.7±0.5) days, t=-2.028, P=0.045; (3.1±0.6) days vs. (3.4±0.7) days, t=-2.095, P=0.039], indicating faster intestinal function recovery in patients with ODA. The morbidity of postoperative complication was 6.7% (4/60) in the ODA group and 7.3% (4/55) in the control group and no significant difference was found (χ(2)=0.016, P=0.898). Two cases of incision infection, 1 case of lung infection, and 1 case of intra-abdominal infection occurred in the ODA group, while 3 cases of lung infection and 1 case of intra-abdominal infection occurred in the control group. All these complications were resolved after conservative treatment, and no secondary operation was performed due to complications. Conclusion: Compared with the traditional delta-shaped anastomosis, ODA is associated with a faster recovery of postoperative intestinal function without increasing the morbidity of postoperative complications, and has the satisfactory short-term efficacy.

[Transcriptome sequencing-based classification and quantification of IKZF1 transcript isoforms in B-cell acute lymphoblastic leukemia].

Objective: To classify and quantify IKZF1 mutant transcripts in B-cell acute lymphoblastic leukemia (B-ALL) by RNA sequencing (RNA-seq) and bioinformatics analysis. Methods: A cohort of 263 B-ALL cases was enrolled at Hebei Yanda Ludaopei Hospital from September 2018 to September 2020. An integrated bioinformatics pipeline was developed to adapt the classification and quantification of IKZF1 transcripts from RNA-seq and was applied to sequencing data of these cases. The IKZF1 mutant transcripts classified by RNA-seq analysis were compared with the qualitative reverse transcription PCR (RT-PCR). Results: IKZF1 mutant transcripts were identified in 53 B-ALL patients by RT-PCR and Sanger sequencing, among which IK6 and IK10 transcripts accounted for 67.9% (36/53) and 28.3% (15/53) respectively. Additionally, 2 patients were double positive for IK6 and IK10. RNA-seq analysis identified 51 patients with IKZF1 mutant transcripts. Compared with the RT-PCR result, the detection sensitivity and specificity of RNA-seq analysis reached 94.3% (50/53) and 99.5% (209/210), respectively. Among the 50 patients with IKZF1 mutant transcripts both in RNA-seq and RT-PCR analysis, the ratio of mutant transcripts to total IKZF1 transcripts in 6 patients was 0.14 (0.11, 0.35), which was significantly lower than that of the other 44 patients [0.88 (0.35, 0.97), Z=-3.945,P<0.001]. IKZF1 mutations mostly occurred in Ph+and Ph-like B-ALL, characterized by abnormal JAK-STAT pathway, and B-ALL with PAX5 translocation. Conclusions: Through the optimized bioinformatics analysis process, RNA-seq data can be used to classify and quantitatively analyze IKZF1 transcripts in B-ALL. Furthermore, the relative expression of mutant IKZF1 transcripts was found to cluster into two groups, and IKZF1 mutation was found often accompanied with PAX5 translocations.

[ABO gene subtypes and gene expression analysis in three cases of hematological malignancies patients].

Objective: To explore the application and discovery of genotyping, gene sequencing, and gene expression analysis in the determination of ABO blood group subtypes and antigen expression abnormalities in hematological malignancies patients. Methods: From June 2019 to May 2020, three clinical cases were found with forward and reverse ABO typing discrepancy or atypical serologic agglutination pattern in the laboratory and blood transfusion department of Hebei Yanda Ludaopei Hospital were selected. Sequence-specific primer PCR (PCR-SSP) and Sanger sequencing of ABO gene coding regions were performed to determine the ABO genotypes, and whole transcriptome sequencing was used to analyze ABO and FUT1 gene expression levels. Results: A 12-year-old female acute lymphoblastic leukemia patient was determined as O.01.02 and BA.04 sub-genotype, corresponding to the serological B(A) subtype, and her ABO gene expression was normal (354.80). A 41-year-old female acute myeloid leukemia patient was determined as A1.02 and B.01 genotype, corresponding to the serological A(1)B phenotype, and her ABO gene expression was significantly reduced (45.70). A 42-year-old male with myelodysplastic syndrome and myelofibrosis was determined as A1.02 and A2.05 sub-genotype, corresponding to the serological A(1) and A(2) phenotype, respectively, and his ABO expression was negative. FUT1 expression was in the normal range in all three cases. The clinical blood product infusion strategy was formulated according to the genotype and the corresponding immunological subtype, and no significant transfusion-related adverse reactions occurred. Conclusion: Blood group sub-genotypes or aberrant gene expression can lead to ambiguities in serological blood group determination in hematological malignancies patients. ABO genotyping and gene expression analysis can help in this scenario and escort blood product infusion safety.

A QM protein from Bombyx mori negatively regulates prophenoloxidase activation and melanization by interacting with Jun protein.

The QM gene that encodes for the ribosomal protein L10 was firstly identified from human tumour cells as a tumour suppressor. In this study, a QM gene was identified in silkworm Bombyx mori (BmQM) and its immunomodulatory function was explored. BmQM messenger RNA (mRNA) and protein were highly expressed in the silk gland and fat body, and expressed in all stages of silkworm growth. After challenged with four different microorganisms, the expression levels of BmQM mRNA in fat body or haemocytes were significantly upregulated compared with the control. After knock-down of BmQM gene, the expressions of some immune genes (PGRPS6, Gloverin0, Lysozyme and Moricin) were affected, and the transcripts of prophenoloxidase1 and prophenoloxidase2 have different degrees of change. The phenoloxidase activity was significantly reduced when the purified recombinant BmQM protein was injected. Recombinant BmQM protein inhibited systemic melanization and suppressed prophenoloxidase activation stimulated by Micrococcus luteus, but it did not affect phenoloxidase activity. Far-western blotting assays showed that the BmQM protein interacted with silkworm BmJun protein, which negatively regulates AP-1 expression. Our results indicated that BmQM protein could affect some immune gene expression and negatively regulate the prophenoloxidase-activating system, and it may play an important role in regulation of the innate immunity in insects.

Quercetin induces protective autophagy and apoptosis through ER stress via the p-STAT3/Bcl-2 axis in ovarian cancer.

Quercetin (3,3',4',5,7-pentahydroxyflavone, Qu) is a promising cancer chemo-preventive agent for various cancers because it inhibits disease progression and promotes apoptotic cell death. In our previous study, we demonstrated that Qu could evoke ER stress to enhance drug cytotoxicity in ovarian cancer (OC). However, Qu-induced ER stress in OC is still poorly understood. Here, we demonstrated that Qu evoked ER stress to involve in mitochondria apoptosis pathway via the p-STAT3/Bcl-2 axis in OC cell lines and in primary OC cells. Unexpectedly, inhibition of ER stress did not reverse Qu-induced cell death. Further functional studies revealed that Qu-induced ER stress could activate protective autophagy concomitantly by activating the p-STAT3/Bcl-2 axis in this process. Moreover, the autophagy scavenger 3-MA was shown to enhance Qu's anticancer effects in an ovarian cancer mice xenograft model. These findings revealed a novel role of ER stress as a "double edge sword" participating in Qu-induced apoptosis of OC and might provide a new angle to consider in clinical studies of biological modifiers that may circumvent drug resistance in patients by targeting protective autophagy pathways.

Bone Morphogenetic Protein-2 regulates in vitro osteogenic differentiation of mouse adipose derived stem cells.

OBJECTIVE: The aim of this study is to investigate the feasibility and efficiency of Bone Morphogenetic Protein-2 (BMP-2) in regulating in vitro osteogenic differentiation of mouse adipose derived stem cells (ADSCs). MATERIALS AND METHODS: Mouse ADSCs were isolated from adipose tissues of C57/BL6 mice (age of 4-6 w) and cultured. Surface antigens of passage 3 (P3) ADSCs, including CD31, CD34, CD90, CD105 and CD133, were analyzed using flow cytometry. Overexpression of BMP-2 was achieved through gene transfection of ADSCs. In vitro osteogenic differentiation of transfected and non-transfected ADSCs cultured in specific induction media was evaluated by Alizarin Red staining. In addition, expression of osteoblast-specific gene, Runx2, was analyzed by quantitative RT-PCR (qRT-PCR). RESULTS: Abundant ADSCs could be isolated from adipose tissue. P3 ADSCs expressed stem cell-specific molecular markers, CD90 and CD105 but did not express CD31, CD34 or CD133. BMP-2 could efficiently transfect mouse ADSCs. Alizarin Red staining revealed that more calcified nodules were formed in BMP-2 transfected ADSCs. qRT-PCR further confirmed higher level of Runx2 expression in BMP-2 transfected ADSCs (p < 0.05). CONCLUSIONS: BMP-2 can promote in vitro osteogenic differentiation of mouse adipose stem cells.

Effect of gender on the development of hypocapnic apnea/hypopnea during NREM sleep.

We hypothesized that a decreased susceptibility to the development of hypocapnic central apnea during non-rapid eye movement (NREM) sleep in women compared with men could be an explanation for the gender difference in the sleep apnea/hypopnea syndrome. We studied eight men (age 25-35 yr) and eight women in the midluteal phase of the menstrual cycle (age 21-43 yr); we repeated studies in six women during the midfollicular phase. Hypocapnia was induced via nasal mechanical ventilation for 3 min, with respiratory frequency matched to eupneic frequency. Tidal volume (VT) was increased between 110 and 200% of eupneic control. Cessation of mechanical ventilation resulted in hypocapnic central apnea or hypopnea, depending on the magnitude of hypocapnia. Nadir minute ventilation in the recovery period was plotted against the change in end-tidal PCO(2) (PET(CO(2))) per trial; minute ventilation was given a value of 0 during central apnea. The apneic threshold was defined as the x-intercept of the linear regression line. In women, induction of a central apnea required an increase in VT to 155 +/- 29% (mean +/- SD) and a reduction of PET(CO(2)) by -4.72 +/- 0.57 Torr. In men, induction of a central apnea required an increase in VT to 142 +/- 13% and a reduction of PET(CO(2)) by -3.54 +/- 0.31 Torr (P = 0.002). There was no difference in the apneic threshold between the follicular and the luteal phase in women. Premenopausal women are less susceptible to hypocapnic disfacilitation during NREM sleep than men. This effect was not explained by progesterone. Preservation of ventilatory motor output during hypocapnia may explain the gender difference in sleep apnea.

Regenerative growth of respiratory bronchioles in dogs.

Loss of lung units due to pneumonectomy stimulates growth of the remaining lung. It is generally believed that regenerative lung growth involves only alveoli but not airways, a dissociated response termed "dysanaptic growth." We examined the structural response of respiratory bronchioles in immature dogs raised to maturity after right pneumonectomy. In another group of adult dogs, we also examined the effect of preventing mediastinal shift after right pneumonectomy on the response of respiratory bronchioles. In immature dogs after pneumonectomy, the volume of the remaining lung increased twofold, with no change in volume density, numerical density, or mean diameter of respiratory bronchiole, compared with that in the control lung. The number of respiratory bronchiole segments and branch points increased proportionally with lung volume. In adult dogs after pneumonectomy, prevention of mediastinal shift reduced lung strain at a given airway pressure, but lung expansion and regenerative growth of respiratory bronchiole were not eliminated. We conclude that postpneumonectomy lung growth is associated with proliferation of intra-acinar airways. The proportional growth of acinar airways and alveoli should optimize gas exchange of the regenerated lung by enhancing gas conductance and mixing efficiency within the acinus.

Plasma shock wave lithotripsy of gallstones in vitro. Animal experiments and clinical application.

It is sometimes very difficult to extract a huge impacted stone through the T-tube fistulous tract by conventional techniques with choledochoscope. To simplify the procedure, a lithotriptor PSW-G type using plasma shock wave to disintegrate the stone was designed. The efficacy to fragmentate stone was investigated both in vitro and in vivo. Stones can be shattered into pieces less than 3 mm in diameter in about 30 times of spark with lower energy ranging from 1.7-3.4 kV and 1-3 J. Animal experiments were carried out to prove the safety of the lithotriptor. There was neither interference with the cardiovascular and respiratory systems nor obvious damage to the adjacent tissue where plasma shock wave applied to break stones. Six patients with retained calculi impacted both in extra- and intra-hepatic duct were successfully treated by plasma shock wave lithotripsy. It appears very useful in dealing with a huge impacted stone in the biliary tract.

A retrospective study on diagnosis and treatment of severe acute pancreatitis.

Severe acute pancreatitis is highly controversial on its diagnostic criteria, the optimum time for surgery, the selection of surgical procedures, and the prevention and treatment of complications. We treated 40 patients with severe acute pancreatitis from July 1983 to July 1988. The comparison of clinical and laboratory data of severe acute pancreatitis and mild acute pancreatitis showed that in some patients neither Ranson's nor Bank's criteria are reliable in classifying or predicting the severity of the disease. The coexistence of acute peritonitis and bloody ascites with elevated amylase level is very helpful to identify the local conditions of pancreatic necrosis and hemorrhage. We suggest early operation (within 48 hours) be applied in severe acute pancreatitis. In our series, five types of surgical procedures were used. We consider that proper treatment of acute respiratory distress syndrome (ARDS) is most important in the management of severe pancreatitis.

[Value of endometrial aspiration cytology in assessing endometrial status].

Cytologic findings of endometrial aspiration from 135 women was correlated with pathologic diagnosis of endometrium. The results indicate that different endometrial changes, such as hyperplasia, carcinoma and other malignant endometrial lesions, different phases of normal menstrual cycle and senile atrophy can be distinguished by endometrial cytology. The author suggests that aspiration cytology may serve as a supplement to the biopsy or curettage in the assessment of the status of the endometrium.