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 explore the effect of enhanced CT and enhanced MRI image fusion technique in making treatment decisions for primary liver cancer (PLC). Methods Clinical data of 55 patients with PLC who were treated in the Third Affiliated Hospital of Sun Yat-sen University between January 2013 and January 2015 were analyzed retrospectively. There were 42 males and 13 females, aged from 18-84 and with a median age of 52 years old. The informed consents of all patients were obtained and the local ethical committee approval was received. All the patients underwent enhanced CT and gadolinium ethoxybenzyl-diethylenetriaminepentaacetic acid (Gd-EOB-DTPA) enhanced MRI. CT and MRI images were fused by using flexible registration method based on finite element. Treatment decisions for these patients were discussed and made by HCC multidisciplinary consultation group. Discussion was conducted twice for each patient. The first discussion was based on enhanced CT images and the second was based on fused images. Changes of treatment decisions were observed and analyzed. Results Treatment decisions based on enhanced CT images included radical treatment in 8 cases, palliative surgical treatment in 35 cases and systemic medicine treatment in 12 cases. Treatment decisions based on fused images included radical treatment in 4 cases, palliative surgical treatment in 36 cases and systemic medicine treatment in 15 cases. Compared with those based on enhanced CT images, the conversion rate of radical treatment, palliative surgical treatment and systemic medicine treatment based on fused images was respectively 50%(4/8), 3%(1/35) and 25%(3/12). Conclusions Enhanced CT and Gd-EOB-DTPA enhanced MRI image fusion can change the treatment decisions for some patients with HCC, and it is of certain significance in optimizing the treatment protocols.

Objective:To evaluate the efficacy and safety of transcatheter arterial chemoembolization (TACE) combined with tislelizumab and tyrosine kinase inhibitor (TKI) as a conversion therapy in patients with initially unresectable hepatocellular carcinoma (HCC).Methods:The clinical data of 51 initially unresectable HCC patients admitted to the Department of Hepato-Pancreato-Biliary Surgery, the Eighth Affiliated Hospital of Guangxi Medical University from March 2022 to November 2023 were prospectively collected, including 46 males and 5 females, aged (53±11) years old. All patients received TACE combined with tislelizumab and TKI. For initially unresectable HCC patients who have successfully undergone conversion therapy, surgical resection was performed sequentially. Follow-up was conducted through regular outpatient visits or hospitalization combined with telephone contact, and the surgical conversion, relapse-free survival and adverse reactions of patients were recorded.Results:Among the 51 patients with initial unresectable HCC, there were 12 cases of stage Ib, 14 cases of stage IIa, 10 cases of stage IIb, and 15 cases of stage IIIa in Chinese liver cancer staging. The 51 patients were evaluated according to the modified solid tumor response evaluation criteria, and 15 patients had complete response, 26 had partial response, 5 had stable disease, and 5 had disease progression. The objective response rate was 80.4% (41/51), and the disease control rate was 90.2% (46/51). The conversion success rate was 49.0% (25/51), 2 patients gave up surgery, and the actual conversion rate was 45.1% (23/51). Among the 23 patients who underwent surgical resection, irregular hepatectomy was performed in 11 cases, lobectomy in 8 cases, and hemihepatectomy in 4 cases. Common treatment-related adverse events were hand-foot syndrome, hypertension, gingival bleeding, etc. Most of the drug-related adverse events were grade 1 to grade 2. A total of 10 patients (19.6%, 10/51) had grade 3 drug-related adverse events, and no grade 4 or above adverse events occurred, and no treatment-related deaths occurred. The cumulative recurrence free survival rates of 23 patients with initial unresectable HCC at 6 and 12 months after sequential hepatectomy were 100% and 94.7% respectively.Conclusion:The triple combination therapy of TACE combined with tislelizumab and TKI in the treatment of initial unresectable HCC has good clinical efficacy, and the adverse reactions are safe and controllable.

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.