Objective: To investigate the pyroptosis-inducing effects of celastrol on tumor cells and to explore the potential mechanisms involved, specifically focusing on the role of the caspase-3/gasdermin E (GSDME) signaling pathway and the impact of endoplasmic reticulum (ER) stress and autophagy. Methods: Necrostatin-1 (Nec-1), lactate dehydrogenase release (LDH) assay, and Hoechst/propidium iodide (PI) double staining were employed to validate the mode of cell death. Western blot was used to detect the cleavage of GSDME and the expression of light chain 3 (LC3) and BIP. Results: Celastrol induced cell swelling with large bubbles, which is consistent with the pyroptotic phenotype. Moreover, treatment with celastrol induced GSDME cleavage, indicating the activation of GSDME-mediated pyroptosis. GSDME knockout via CRISPR/Cas9 blocked the pyroptotic morphology of celastrol in HeLa cells. In addition, cleavage of GSDME was attenuated by a specific caspase-3 inhibitor in celastrol-treated cells, suggesting that GSDME activation was induced by caspase-3. Mechanistically, celastrol induced endoplasmic reticulum (ER) stress and autophagy in HeLa cells, and other ER stress inducers produced effects consistent with those of celastrol. Conclusion These findings suggest that celastrol triggers caspase-3/GSDME-dependent pyroptosis via activation of ER stress, which may shed light on the potential antitumor clinical applications of celastrol.

Objective:To compare the differences in important parameters between the articulation assessment and training system of intelligently extracted speech with those from the Praat acoustic software and those manually extracted.Methods:The speech of thirty-two normal subjects was captured using the intelligent articulation assessment and training system and using Praat acoustic software. The former analyzed the mean fundamental frequencies (mF0s), the first formant peaks (F1s) and the second formant peak (F2s) of the sustained vowels /a/, /i/ and /u/. The speech parameters collected by the traditional Praat software were extracted and analyzed by professionals. The two tools′ consistency in terms of these important acoustic parameters was analyzed.Results:The results with all 32 subjects when retested returned ICC values above 0.9 with all three vowels with the exception of mF0 for /u/ (ICC=0.75), indicating excellent retest reliability for the articulation assessment and training system. The ICC values also indicated excellent consistency between the two kinds of software in analyzing mF0, F1 and F2 of the three vowels. The mF0, F1, F2, FCR, VAI, tongue spacing, VSA, and mandibular spacing of all three vowels were mostly distributed within the 95% confidence interval of the data points in Bland-Altman plots, indicating the high accuracy of both acoustic analysis systems in speech measurement. The mean fundamental frequency values of the male long vowels /a/, /i/ and /u/ were all significantly lower than for the female long versions.Conclusions:The retest reliability of the articulation assessment and training system was good, and the results of the articulation check in the natural state were in good consistency compared to the Praat check and were interchangeable in the articulation check.

Via an insufficient coat protein complex I (COPI) retrieval signal, the majority of SARS-CoV-2 spike (S) is resident in host early secretory organelles and a tiny amount is leaked out in cell surface. Only surface-exposed S can be recognized by B cell receptor (BCR) or anti-S therapeutic monoclonal antibodies (mAbs) that is the trigger step for B cell activation after S mRNA vaccination or infected cell clearance by S mAbs. Now, a drug strategy to promote S host surface exposure is absent. Here, we first combined structural and biochemical analysis to characterize S COPI sorting signals. A potent S COPI sorting inhibitor was then invented, evidently capable of promoting S surface exposure and facilitating infected cell clearance by S antibody-dependent cellular cytotoxicity (ADCC). Importantly, with the inhibitor as a probe, we revealed Omicron BA.1 S is less cell surface exposed than prototypes because of a constellation of S folding mutations, possibly corresponding to its ER chaperone association. Our findings not only suggest COPI is a druggable target against COVID-19, but also highlight SARS-CoV-2 evolution mechanism driven by S folding and trafficking mutations.

Yunüjian （Jing Yue's Collected Works）， composed of Gypsum Fibrosum， Rehmanniae Radix Praeparata， Anemarrhenae Rhizoma， Ophiopogonis Radix， and Achyranthis Bidentatae Radix， is used for the treatment of "headache， toothache， consumptive thirst， and other diseases caused by heat and Yin deficiency in the stomach". In modern clinical settings， it has definite hypoglycemic efficacy， as it improves insulin resistance， inhibits oxidative stress and inflammatory response， and down-regulates estrogen level. It is often directly used， modified before use， combined with other formulae （Liuwei Dihuangtang， Shashen Maidongtang， Shenqi Sanjingtang， Huangqi Guizhi Wuwutang， Zengyetang， Zengye Baihutang， etc.）， or used together with western medicine （insulin， metformin， pioglitazone， glurenorm， etc.）. Modified Yunüjian can be applied for the treatment of type 2 diabetes mellitus， gestational diabetes， polycystic ovary syndrome with insulin resistance， pruritus induced by diabetes mellitus， diabetic peripheral neuropathy， diabetic nephropathy， diabetic ketoacidosis， diabetic retinopathy， and diabetic periodontitis. According to previous experiment， the protection for pancreatic islet β-cells is an important way of Yunüjian to improve diabetes mellitus. The formula can remove damaged proteins and organelles by increasing the expression of autophagy-related 2B （Atg2B）， ubiquitin-like-conjugating enzyme （Atg3）， γ-aminobutyric acid type A receptor-associated protein （GABARAP）， selective autophagy receptor （p62/SQSTM1）， etc. Moreover， it plays an active role in islet cell proliferation and apoptosis by regulating the expression of mammalian target of rapamycin （mTOR） and reducing the expression of islet β-cell autophagy gene （Beclin）. These are pivotal for maintaining the quantitative structure and functions of islet cells. The modulatory effect of Yunüjian on growth hormone-releasing peptide （ghrelin）， gastrin， and growth hormone secretagogue receptor （GHSR） mRNA expression is also the underlying mechanism for the formula to protect pancreatic β-cells. In this study， we summarized the clinical and experimental studies on the therapeutic effect of this formula on diabetes mellitus， discussed the mechanisms， and proposed suggestions on the problems， hoping to provide a reference for the clinical application and further development of Yunüjian. This study is of practical significance for the scientific interpretation of the modern connotation of Yunüjian and the expansion of its clinical application.

Objective To evaluate Aptima HPV E6 and E7 mRNA assay (Aptima HPV) combined with Aptima HPV 16 and 18 or 45 (18/45) genotype assay (Aptima HPV-GT) as a means of cervical cancer opportunistic screening. Methods From October 2016 to October 2017, a total of 23 258 women aged 25-65 years were enrolled in the physical examination center and gynecological clinic of Huzhou Maternity and Child Health Care Hospital. All the women had Aptima HPV tested, further Aptima HPV-GT testing for positive women and liquid-based thin layer cytology Thinprep cytologic test (TCT). Women with Aptima HPV (+) or ≥low-grade squamous intraepithelial lesion (LSIL) or obvious clinical symptoms (including vaginal bleeding after intercourse and watery, bloody vaginal discharge) were referred for colposcopy and further biopsy with or without endocervical curettage (ECC) if indicated. Expression of Aptima HPV, HPV 16 and HPV 18/45 with different cytological diagnostic groups and histological diagnosis groups were compared respectively. Sensitivity, specificity, positive predictive value and negative predictive value of Aptima HPV detection and TCT in identifying histological diagnosis of high-grade squamous intraepithelial lesion (HSIL) or worse (HSIL+) were compared. Results (1) The positive rates of Aptima HPV, HPV 16 and HPV 18/45 were 14.00% (3 257/23 258), 1.85% (430/23 258) and 0.86% (199/23 258) respectively.The positive rates of Aptima HPV, HPV 16 and HPV 18/45 increased with cytology grading in squamous epithelium [negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of undetermined significance (ASCUS), LSIL, atypical squamous cell cannot exclude HSIL (ASC-H), HSIL and squamous cell carcinoma (SCC), all P=0.000)]. According to histology results, the positive rates of Aptima HPV, HPV 16 and HPV 18/45 increased with histology grading in squamous epithelium (normal cervical tissue, LSIL, HSIL and SCC, all P=0.000). The positive rate of Aptima HPV was significantly higher in HSIL+group than that in the LSIL or better (LSIL-) group [98.11% (311/317) vs 12.84% (2 946/22 941), P=0.000]. The positive rate of Aptima HPV-GT was significantly higher in HSIL+group than that in LSIL-group [58.36% (185/317) vs 1.91% (439/22 941), P=0.000]. (2) Compared with cytology, Aptima HPV resulted in significant higher sensitivity (98.11% vs 59.62%, P=0.000) and negative predictive value (99.97% vs 99.42%, P=0.000), significant lower specificity (87.16% vs 95.37%, P=0.000) and positive predictive value (9.55% vs 15.10%, P=0.000) when identified HSIL+. Conclusions Women with Aptima HPV positive, especially those with Aptima HPV-GT positive, are more likely to have histological diagnosis of HSIL+. Aptima HPV combined with Aptima HPV-GT is feasible as a means of cervical cancer opportunistic screening in tertiary hospitals.

Objective To investigate the distribution differences of human papillomavirus(HPV) genotypes in the patients with cervical intraepithelial neoplasia grade Ⅱ/Ⅲ and the patients with cervical cancer. Methods 260 cases of cervical cancer and 280 cases of cervical intraepithelial neoplasia grade Ⅱ /Ⅲ patients who were admitted to our hospital from January 2010 to October 2017 were selected and divided into cancer group and neoplasia group respectively. The HPV infection of patients was detected and typing identification was performed. The distribution differences of HPV genotypes in cancer group and neoplasia group were compared. The differences of genotypes of HPV infection in different types of tissues in cancer group were also compared. Results The negative constituent ratio of HPV infection in cancer group was lower than that in neoplasia group(P＜0. 05), and the constituent ratio of all the high-risk types in single infection was significantly higher than that in the neoplasia group(P＜0. 05). The constituent ratio of all the low-risk types in single infection was significantly similar to that in the neoplasia group(P＞0. 05). The constituent ratio of multiple infections in cancer group was significantly higher than that in neoplasia group(P＜0. 05); the constituent ratios of negative HPV infections and 18 types of single infections in cervical squamous cell carcinoma was much lower than that of cervical adenocarcinoma(P＜0. 05). The constituent ratios of 16 types of HPV and multiple infections in cervical squamous cell carcinoma were much higher than that of cervical adenocarcinoma(P＜0. 05). Conclusion The risk of HPV infection in cervical cancer patients is significantly higher than that of cervical intraepithelial neoplasia grade Ⅱ /Ⅲ, the infection rate of cervical cancer high-risk type HPV is higher, and the risk of multiple infections is also higher. The tissue types of cervical cancer can be determined according to HPV typing.

Objective To examine the application of On-Board Imaging (OBI) system-based image-guided radiotherapy (IGRT) in the improvement of the precision of intensity-modulated radiotherapy (IMRT) for nasopharyngeal carcinoma.Methods Ten patients with nasopharyngeal carcinoma were treated with IMRT using the OBI system. The IGRT images after positioning, position adjustment, and treatment were observed and recorded to investigate the image difference between CT simulation and IGRT. Results The deviations in the x (lateral), y (cranial-caudal), and z (ventral-dorsal) directions between CT simulation and IGRT images were 0.22±1.00 mm,-0.37±1.28 mm, and 0.04±1.36 mm, respectively, after positioning, 0.29±0.76 mm,-0.04±0.78 mm, and -0.01±0.92 mm, respectively, after position adjustment, and 0.20±0.78 mm, 0.16±0.80 mm, and 0.05±0.92 mm, respectively, after treatment. The probabilities of a ≤1 mm deviation in the x, y, and z directions were 81.0%, 77.6%, and 88.2%, respectively, after positioning, 92.5%, 96.4%, and 96.4%, respectively, after position adjustment, and 91.7%, 94.9%, and 96.8%, respectively, after treatment. Conclusions The application of OBI system-based IGRT is very important in the improvement of the precision of fractionated IMRT for patients with nasopharyngeal carcinoma. The position of the patient should be adjusted based on the IGRT image after positioning in order to correct set-up error and effectively increase the precision of fractionated IMRT.

Objective To explore the methods of preparing whole blood control of seven trace elements (magnesium,manganese,iron,copper,zinc,lead,calcium) in laboratory and evaluate its performance.Methods Heparin sodium anticoagulant calf whole blood was used as substrateMetal salt or standard solution with target concentration of each element was added.And whole blood control product was made after process of anticorrosion,mixing and sub-packaging.Antibacterial effect was observed,uniformity and stabilitywasevaluatedaccording to CNAS-GL03 and matrix effects was evaluatedaccording to CLSI EP14.SDI (standard deviation index) and detection coefficient of variation (CV)were calculated to evaluateapplication effectiveness.Results Laboratory preparation of whole blood control reached target concentration,sterility tests was qualified,results of uniformity and stability indicated that the substrate was even and stable at least for one year.Besides,matrix effects of other six elements can be ignored except lead.Historical and inter-laboratory comparisons had shown that laboratory preparation of whole blood control has no obvious difference with commercial ones in performance.Conclusion The formulation and evaluation scheme of whole blood control of seven trace elements (magnesium,manganese,iron,copper,zinc,lead,calcium) was feasible and can be used as commercial ones for elementary tests in medical laboratory.

Objective The aim of the current study is to determine the clinical value of incidental internal mammary lymph node biopsy in free abdominal flap breast reconstruction using internal mammary vessels as recipient vessels and to investigate the risk factors of internal mammary lymph nodes metastasis. Methods The clinical data of all patients who underwent free abdominal flap breast reconstruction using internal mammary vessels as recipient vessels from November 2006 to December 2015 in the Department of Breast Surgery, Fudan University Shanghai Cancer Center were reviewed in the study. The incidence of internal mammary lymph node biopsy and the rate of metastasis were analyzed. Statistical analysis was conducted to evaluate the risk factors of internal mammary lymph node metastasis. Results A total of 113 patients met the inclusion criteria, 53 (46.9%) of whom had internal mammary lymph nodes harvested. Four of these were positive for metastatic disease, all in immediate breast reconstructions. The incidence of metastasis was 7.5% in patients who had successful internal mammary lymph node biopsies.The multi?variate Logistic regression analysis showed that invasive tumor size, tumor location and axillary lymph node metastasis were not risk factors for internal mammary lymph node metastasis ( P>0. 05 ) . Conclusions Internal mammary lymph nodes found incidentally during recipient vessel exposure may provide important information about internal mammary lymph node metastasis in free flap breast reconstruction. This approach for internal mammary lymph node biopsy reveals an appreciable success rate and is convenient in clinical practice. The size of invasive tumor and the axillary lymph node metastasis are probably associated with internal mammary lymph node positivity.

Objective The aim of the current study is to determine the clinical value of incidental internal mammary lymph node biopsy in free abdominal flap breast reconstruction using internal mammary vessels as recipient vessels and to investigate the risk factors of internal mammary lymph nodes metastasis. Methods The clinical data of all patients who underwent free abdominal flap breast reconstruction using internal mammary vessels as recipient vessels from November 2006 to December 2015 in the Department of Breast Surgery, Fudan University Shanghai Cancer Center were reviewed in the study. The incidence of internal mammary lymph node biopsy and the rate of metastasis were analyzed. Statistical analysis was conducted to evaluate the risk factors of internal mammary lymph node metastasis. Results A total of 113 patients met the inclusion criteria, 53 (46.9%) of whom had internal mammary lymph nodes harvested. Four of these were positive for metastatic disease, all in immediate breast reconstructions. The incidence of metastasis was 7.5% in patients who had successful internal mammary lymph node biopsies.The multi?variate Logistic regression analysis showed that invasive tumor size, tumor location and axillary lymph node metastasis were not risk factors for internal mammary lymph node metastasis ( P>0. 05 ) . Conclusions Internal mammary lymph nodes found incidentally during recipient vessel exposure may provide important information about internal mammary lymph node metastasis in free flap breast reconstruction. This approach for internal mammary lymph node biopsy reveals an appreciable success rate and is convenient in clinical practice. The size of invasive tumor and the axillary lymph node metastasis are probably associated with internal mammary lymph node positivity.