Objective To construct expression vectors of calmodulin(CaM)mutants N2 and C2,and to express,purify,and identify the mutant proteins,in order to study the interactions between CaM and calcium channels. Methods The cDNA of N?lobe and C?lobe of CaM were used to prepare the cDNA of N2 and C2. Next,the recombinant cDNAs were cloned into a pGEX?6p?3 plasmid,and the recombinant plasmids were trans?ferred into E.coli BL21 cells. The transfected BL21 cells were stimulated with IPTG. The fusion proteins were extracted by ultrasonication and puri?fied by using GS?4B beads. Finally,protein activity was identified by the pull?down assay. Results Both the restriction digestion map and the DNA sequence identification results confirmed that the recombinant plasmids were successfully constructed. SDS?PAGE results showed high purity and concentration of N2 and C2 proteins. Their activities and binding abilities with the calcium channel fragment were confirmed by the pull?down assay.Conclusion In this study,expression vectors of N2 and C2 are successfully constructed,and physiologically active N2 and C2 CaM mutant proteins are obtained.

Objective To explore the association between moxibustion sensation and therapeutic efficacy during moxibustion treatment. Methods By applying Taiyi moxa stick to Shiqizhui (EX-B 8) to treat primary dysmenorrhea, the association between the change of Visual Analogue Scale (VAS) score and the topical moxibustion sensation and transmission types during the 30 min moxibustion treatment was observed. Besides, the occurrence time of transmission, and the transmission distance, width, depth, and direction were recorded. Results Superficial moxibustion sensation occurred in forty patients, of whom, the VAS score changed by (38.50±14.38) mm; heat-penetrating moxibustion sensation occurred in 18 patients, and the VAS score changed by (38.89±12.43) mm; heat-expanding sensation occurred in 6 patients, and the VAS score changed by (45.00±14.10) mm; distant transmission happened in 13 patients, and the VAS score changed by (41.54±13.90) mm. Patients with 4 types of moxibustion sensation had the highest VAS scores both before and after treatment, followed by 3 types, 2 types, and 1 type moxibustion sensation. In comparing the changes of VAS score between 10 min and 20 min treatment and between 20 min and 30 min treatment, the patients with 4 types of moxibustion sensation had the most significant change. Conclusions Different moxibustion sensations occur at different frequencies, and the occurrence of moxibustion sensation is related to the severity of disease condition. The number of moxibustion sensation type can affect the remission process of disease, but can merely influence the treatment result. The single moxibustion sensation (superficial heat only) works faster, usually taking 0~10 min; while the other forms of moxibustion sensation (heat penetrating, heat-expanding, and distant transmission) works slower, usually taking over 20 min.

Objective: To compare the therapeutic effects of two different moxibustion methods both with tai-yi moxa stick in treating primary dysmenorrhea. ＜br> Methods: Forty-three patients were randomized into two groups by the random number table according to their treatment orders. The causalgic group was intervened by causalgic stimulation with tai-yi moxa stick while the tepid group was treated by mild thermal stimulation with tai-yi moxa stick. Shiqizhui (EX-B 8) was selected for both groups. Visual analogue scale (VAS) was used for observation before and during the treatment by every 10 min to compare the clinical efficacies between the two groups. ＜br> Results: Before treatment, there was no statistically significant difference in pain intensity between the two groups (P>0.05). After treatment, both groups achieved significant improvements in pain intensity (P＜0.05), but the inter-group difference in pain intensity was still statistically insignificant (P>0.05), but the difference was enlarged comparing with that before treatment. The pain relief during the first 10 min of treatment was slower in the causalgic group than that in the tepid group. However, during the later 20 min, the pain relief in the calsalgia group gradually outpaced that in the tepid group. ＜br> Conclusion: The two moxibustion methods with tai-yi moxa stick both have a good instant analgesic effect in treating primary dysmenorrhea. For patients with primary dysmenorrhea, if 30 min is regarded as the treatment time, mild stimulation was suggested to be used for the first 10 min, and causalgic stimulation for the later 20 min to achieve a better curative effect.

Testicular aging is mainly characterized by a gradual decline in the capability of testosterone synthesis and spermatogenesis, which not only affects male fertility, but also correlates with aging-related chronic diseases intimately. Therefore, delaying testicular aging plays a significant role in improving the health and quality of life of middle-aged and elderly men. Stem cells are a cell group with potent self-renewal capability and multi-directional differentiation potential. In recent years, the research of stem cells in basic and clinical application has been carried out in-depth, which has accelerated the development of cell therapy. Currently, stem cell transplantation has been employed to treat multiple diseases, which has captivated widespread attention in the field of aging and regenerative medicine. Stem cell transplantation has demonstrated promising prospects in the treatment of testicular aging. In this article, research profile and progress of stem cell transplantation in the treatment of testicular aging were reviewed, and bottleneck issues encountered in clinical translation and strategies for optimizing clinical efficacy were discussed, aiming to provide novel ideas for the research and development and clinical translation of stem cell therapy for testicular aging.

Objective:To discuss the causes and solutions for failed anterior ring fixation for unstable pelvic fractures.Methods:A retrospective analysis was conducted of the 84 patients who had been admitted to Department of Orthopedics and Joint Surgery, Jiangmen Central Hospital for unstable pelvic fractures from January 2009 to March 2019. They were 56 males and 28 females, aged from 19 to 64 years (mean, 42.5 years). By the Tile classification, 22 cases were type B1, 16 type B2, 10 type B3, 22 type C1, 6 type C2 and 8 type C3. Simple anterior ring fixation was performed for 21 cases and combined anterior and posterior ring fixation for 63 ones. The Matta's criteria were applied for the evaluation of fracture reduction. The cases of failed anterior ring fixation and their solutions were recorded and analyzed.Results:All patients were followed up for 6 to 36 months (average, 13.5 months). Failed anterior ring fixation was observed in 7 cases (8.3%) at 3 to 75 days after operation (average, 29.1 days). The failure was attributed to improper operation timing and unstable anterior ring fixation in 2 cases, mere unstable anterior ring fixation in one, wrong choice of anterior ring fixators and improper rehabilitation in 2 cases, poor intraoperative reduction in one and unstable posterior ring fixation in one. In the 2 failed cases that had been treated by external fixators, one underwent revision and the other conservative treatment. In the 5 cases that had been treated by plating, 4 underwent revision and one conservative treatment. By the Majeed criteria, the pelvic function was evaluated at the final follow-up as excellent in 2, good in 2 and fair in one in the 5 cases of revision who obtained follow-up from 11 to 24 months(average, 17.2 months) after revision; malunion was observed in the 2 cases of conservative treatment.Conclusions:Failed anterior ring fixation for unstable pelvic fracture may be caused by improper operation timing, wrong choice of anterior ring fixators, intraoperative malreduction, unstable pelvic ring fixation and improper rehabilitation. The key solution to failed anterior ring fixation is to find the specific causes. Plate revision may lead to fine therapeutic efficacy.

Objective:To investigate the application value of Metagenomic Next-Generation sequencing (mNGS) in infectious patients after allogeneic hematopoietic stem cell transplantation(allo-HSCT).Methods:Patients suspected with local or systemic infections were retrospectively included after allo-HSCT in our department from April 2019 to November 2020. Pathogenic microorganisms were tested by mNGS in samples from peripheral blood, cerebrospinal fluid, alveolar lavage Liquid, abscess, etc. Other diagnostic methods such as bacterial/fungal culture, viral PCR detection were simultaneously explored comparing with mNGS results.Results:A total of 112 samples in 83 patients were detected by mNGS, and 34 pathogenic microorganisms were determined. Among these positive samples, 11 strains of bacteria (17 times) with the most common Escherichia coli (4/17) were reported. There were 7 strains of fungi (10 times) detected with primary Candida albicans (7/29). Although arvovirus 30.2% (39/129) were predominantly detected, its diagnostic relevance with infections was not definite. Other pathogenic viruses including cytomegalovirus (CMV) 25.6% (33/129) and Epstein Barr virus (EBV) 14.0% (18/129)were of significance. Comparing with golden diagnostic criteria, the sensitivity of mNGS was 86.5%, and specificity was 45.0%. Regarding single pathogen infection, the consistency of mNGS and conventional methods was 82.9% (29/35), while it was 16/17 in combination infections.Conclusion:mNGS could be a potential method to determine pathogens in patients suspected with infections after allo-HSCT.

The effects of tanshinone IIA on the proliferation of the human non-small cell lung cancer cell line A549 and its possible mechanism on the VEGF/VEGFR signal pathway were investigated. The exploration of the interaction between tanshinone IIA and its target proteins provides a feasible platform for studying the anticancer mechanism of active components of herbs. The CCK-8 assay was used to evaluate the proliferative activity of A549 cells treated with tanshinone IIA (2.5-80 μmol/L) for 24, 48 and 72 h, respectively. Flow cytometry was used for the detection of cell apoptosis and cell cycle perturbation. VEGF and VEGFR2 expression were studied by Western blotting. The binding mode of tanshinone IIA within the crystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1. The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of tested group was higher than the vehicle control, and tanshinone IIA-treated cells accumulated at the S phase, which was higher than the vehicle control. Furthermore, the expression of VEGF and VEGFR2 was decreased in Western blot. Finally, molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π-π stacking interactions with Val848. In conclusion, tanshinone IIA may suppress A549 proliferation, induce apoptosis and cell cycle arrest at the S phase. This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2.

[Abstract] Objective: To investigate the lung cancer-associated driver gene mutations in peripheral blood of patients with advanced non-small cell lung cancer (NSCLC) in Yunnan area, and to explore their association with clinical pathological features. Methods: Peripheral blood of 304 patients with stage Ⅳ NSCLC were collected from Molecular Diagnostic Center of Yunnan Cancer Hospital during January 2019 to December 2019. Next generation sequencing (NGS) technique was used to detect the mutation of NSCLC related driver genes, chi-square test was used to analyze the relationship between the major mutant genes and the clinicopathological features of patients, and Logistic regression was used to analyze the independent risk factors. Results: In the peripheral blood of 304 patients with stage Ⅳ NSCLC, there were 120 (39.47%) cases with EGFR mutations, 12 (3.95%) cases with ALK fusion, 36 (11.84%) case with other mutations such as KRAS, BRAF and RET. The main EGFR mutations were 19del and L858R (69.17%). The mutation rate of EGFR was higher in female, young, non-smoking, non-chemotherapy and lung adenocarcinoma patients (49.26% vs 31.55%, 45.39% vs 33.56%, 45.92% vs 27.78%, 45.07% vs 26.37%, 42.39% vs 10.71%, all P<0.05). Multivariate analysis showed that female, no history of chemotherapy and lung adenocarcinoma were independent risk factors for EGFR mutations (all P<0.05). Conclusion: Using NGS technology to detect the driver genes in peripheral blood of patients with advanced NSCLC in Yunnan area showed that the mutation rate of EGFR was higher in women and lung adenocarcinoma patients without chemotherapy history.

Objective To investigate the capillarization of liver sinusoidal endothelial cells (LSECs) and its association with hepatic fibrosis during the development of alveolar echinococcosis, so as to provide the basis for unraveling the mechanisms underlying the role of LSEC in the development and prognosis of hepatic injuries and hepatic fibrosis caused by alveolar echinococcosis. Methods Forty C57BL/6 mice at ages of 6 to 8 weeks were randomly divided into a control group and 1-, 2- and 4-week infection groups, of 10 mice in each group. Each mouse in the infection groups was intraperitoneally injected with 2 000 Echinococcus multilocularis protoscoleces, while each mouse in the control group was given an equal volume of phosphate-buffered saline using the same method. All mice were sacrificed 1, 2 and 4 weeks post-infection and mouse livers were collected. The pathological changes of livers were observed using hematoxylin-eosin (HE) staining, and hepatic fibrosis was evaluated through semi-quantitative analysis of Masson’s trichrome staining-positive areas. The activation of hepatic stellate cells (HSCs) and extracellular matrix (ECM) deposition were examined using immunohistochemical staining of α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1A1), and the fenestrations on the surface of LSECs were observed using scanning electron microscopy. Primary LSECs were isolated from mouse livers, and the mRNA expression of LSEC marker genes Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf was quantified using real-time fluorescence quantitative PCR (qPCR) assay. Results Destruction of local liver lobular structure was observed in mice 2 weeks post-infection with E. multilocularis protoscoleces, and hydatid cysts, which were surrounded by granulomatous tissues, were found in mouse livers 4 weeks post-infection. Semi-quantitative analysis of Masson’s trichrome staining showed a significant difference in the proportion of collagen fiber contents in mouse livers among the four groups (F = 26.060, P < 0.001), and a higher proportion of collagen fiber contents was detected in mouse livers in the 4-week infection group [(11.29 ± 2.58)%] than in the control group (P < 0.001). Immunohistochemical staining revealed activation of a few HSCs and ECM deposition in mouse livers 1 and 2 weeks post-infection, and abundant brown-yellow stained α-SMA and COL1A1 were deposited in the lesion areas in mouse livers 4 weeks post-infection, which spread to surrounding tissues. Semi-quantitative analysis revealed significant differences in α-SMA (F = 7.667, P < 0.05) and COL1A1 expression (F = 6.530, P < 0.05) in mouse levers among the four groups, with higher α-SMA [(7.13 ± 3.68)%] and COL1A1 expression [(13.18 ± 7.20)%] quantified in mouse livers in the 4-week infection group than in the control group (both P values < 0.05). Scanning electron microscopy revealed significant differences in the fenestration frequency (F = 37.730, P < 0.001) and porosity (F = 16.010, P < 0.001) on the surface of mouse LSECs among the four groups, and reduced fenestration frequency and porosity were observed in the 1-[(1.22 ± 0.48)/μm² and [(3.05 ± 0.91)%] and 2-week infection groups [(3.47 ± 0.10)/μm² and (7.57 ± 0.23)%] groups than in the control group (all P values < 0.001). There was a significant difference in the average fenestration diameter on the surface of mouse LSECs among the four groups (F = 15.330, P < 0.001), and larger average fenestration diameters were measured in the 1-[(180.80 ± 16.42) nm] and 2-week infection groups [(161.70 ± 3.85) nm] than in the control group (both P values < 0.05). In addition, there were significant differences among the four groups in terms of Stabilin-1 (F = 153.100, P < 0.001), Stabilin-2 (F = 57.010, P < 0.001), Ehd3 (F = 31.700, P < 0.001), CD209b (F = 177.400, P < 0.001), GATA4 (F = 17.740, P < 0.001), and Maf mRNA expression (F = 72.710, P < 0.001), and reduced mRNA expression of Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf genes was quantified in three infection groups than in the control group (all P values < 0.001). Conclusions E. multilocularis infections may induce capillarization of LSECs in mice, and result in a reduction in the expression of functional and phenotypic marker genes of LSECs, and capillarization of LSECs occurs earlier than activation of HSC and development of hepatic fibrosis.