[Effects of immune responses mediated by topological structures of three-dimensional bioprinted scaffolds on hair follicle cycle in mice].

Objective: To explore the effects of the immune responses mediated by topological structures of three-dimensional bioprinted scaffolds on hair follicle cycle in mice. Methods: The study was an experimental research. The alginate-gelatin composite hydrogels were printed into scaffolds using a three-dimensional bioprinter and named T45 scaffolds, T60 scaffolds, and T90 scaffolds according to the 3 topological structures of the scaffolds (the rotation angles of the printhead during printing were 45°, 60°, and 90°, respectively), and the morphology of the three scaffolds was observed after cross-linking by naked eyes. Nine 8-week-old female C57BL/6J mice were divided into T45 group, T60 group, and T90 group, according to the random number table, with three mice in each group, and the T45, T60, and T90 scaffolds were subcutaneously implanted on the back of mice, respectively. On post implantation day (PID) 7, the hair growth in the dorsal depilated area of mice was observed, the thickness of the fiber capsule around the scaffolds was observed by hematoxylin-eosin staining, and the expression levels of CD68, bone morphogenetic protein-2 (BMP-2), and tumor necrosis factor (TNF) protein in the tissue surrounding the scaffolds were observed by immunofluorescence staining. The samples of the above experiments were all 3. Results: The topological structures of the three scaffolds were all clear with high fidelity after cross-linking. On PID 7, the hair growth was obvious in the dorsal depilated area of mice in T45 group and T90 group, while hair growth was slow in the scaffold implantation area of mice in T60 group, which was significantly different from that of the unimplanted area. On PID 7, compared with (18±4) µm in T90 group, the thickness of both the fiber capsule around the scaffolds ((39±4) and (55±8) µm) of mice in T45 group and T60 group was significantly increased (P<0.05); the thickness of the fiber capsule around the scaffolds of mice in T60 group was also significantly increased compared with that in T45 group (P<0.05). On PID 7, the expression level of CD68 protein in the tissue surrounding the scaffolds of mice in T60 group was significantly higher than the levels in T45 group and T90 group (with both P values <0.05). The expression level of BMP-2 protein in the tissue surrounding the scaffolds of mice in T60 group was significantly higher than the levels in T45 group and T90 group (with both P values <0.05), and the expression level of BMP-2 protein in the tissue surrounding the scaffolds of mice in T45 group was significantly higher than that in T90 group (P<0.05). The expression level of TNF protein in the tissue surrounding the scaffolds of mice in T60 group was significantly lower than the levels in T45 group and T90 group (with both P values <0.05). Conclusions: Three-dimensional bioprinted scaffolds with different topological structures mediate different degrees of immune responses after being implanted in mice. A moderate immune response promotes hair growth in depilated area of mice, while an excessive immune response results inhibits the hair follicle entering into the anagen phase.

[Long term follow-up evaluation of combined surgery for congenital tibial pseudarthrosis in children].

Objective: To explore the long-term effect of combined surgery for the treatment of congenital tibial pseudarthrosis in children. Methods: The clinical data of 44 children with congenital tibial pseudarthrosis who underwent combined surgery (tibial pseudarthrosis tissue resection, intramedullary rod fixation, Ilizarov external fixator fixation, wrapped autologous iliac bone graft) from August 2007 to October 2011 at the Department of Pediatric Orthopedics, Hunan Children's Hospital were collected retrospectively. There were 33 males and 11 females. The age at the time of surgery was (3.7±2.2)years (range:0.6 to 12.4 years), including 25 cases under 3 years old and 19 cases above 3 years old.Among them, 37 cases were complicated with neurofibromatosis type 1.The operation status, postoperative complications and follow-up results were recorded. Results: The follow-up time after surgery was (10.9±0.7)years (range:10 to 11 years).Thirty-nine out of 44 patients (88.6%) achieved initial healing of tibial pseudarthrosis, with an average healing time of (4.3±1.1)months (range:3 to 10months).In the last follow-up, 36 cases (81.8%) had unequal tibial length, 20 cases (45.4%) had refractures, 18 cases (40.9%) had ankle valgus, 9 cases (20.4%) had proximal tibial valgus, and 11 cases (25.0%) had high arched feet.Nine cases (20.4%) developed distal tibial epiphyseal plate bridging.17 cases (38.6%) had abnormal tibial mechanical axis.Seven cases (15.9%) developed needle infection, and one case (2.3%) developed tibial osteomyelitis. 21 patients (47.7%) had excessive growth of the affected femur.Five patients (11.3%) had ankle stiffness, and 34 patients (77.2%) had intramedullary rod displacement that was not in the center of the tibial medullary cavity.Among them, 8 cases (18.1%) protruded the tibial bone cortex and underwent intramedullary rod removal.18 children have reached skeletal maturity, while 26 children have not been followed up until skeletal maturity. Conclusion: Combined surgery for the treatment of congenital pseudarthrosis of the tibia in children has a high initial healing rate, but complications such as unequal tibia length, refracture, and ankle valgus occur during long-term follow-up, requiring multiple surgical treatments.

Paclitaxel-resistant related lncRNA DBH-AS1 promotes the proliferation and invasion of esophageal cancer.

OBJECTIVE: Chemoresistance is one of the main obstacles in the clinical treatment of cancer. However, secondary resistance to paclitaxel poses new challenges for cancer treatment. Long noncoding RNAs regulate cellular functions at different levels and mechanisms and play an important role in the biological behavior of tumors. MATERIALS AND METHODS: LncRNA microarrays were used to detect lncRNAs in Paclitaxel-resistant cells and corresponding parental cells. Cell counting kit 8 and Transwell analysis were used to test the effect of lncRNA on function. RESULTS: The expression of lncRNA DBH-AS1 in TE-4 TAX-R cells was significantly higher than that in TE-4 cells. Transwell analysis showed that the overexpression of lncRNA DBH-AS1 increased the invasion of Eca cells. Cell scratches and Transwell analysis showed that the overexpression of lncRNA DBH-AS1 in Eca cell culture supernatants promoted the migration and invasion of HUVEC. In addition, lncRNA DBH-AS1 relies on miR-21 to regulate the expression of YOD1. CONCLUSIONS: Paclitaxel-resistant lncRNA DBH-AS1 appears to promote ECa cell proliferation and invasion by acting as a ceRNA and regulating miR-21-5p /YOD1 signaling pathway.

[Safety and feasibility of stereotactic radiation therapy on porcine ventricular septum: a preliminary study].

Objective: To explore the safety and feasibility of stereotactic radiation therapy (SBRT) strategy for irradiating porcine ventricular septum, see if can provide a preliminary experimental evidence for clinical SBRT in patients with hypertrophic obstructive cardiomyopathy (HOCM). Methods: Five male pigs (39-49 kg, 6 months old) were used in this study. Pigs were irradiated at doses of 25 Gy (n=2) or 40 Gy (n=3). Delineation of the target volume was achieved under the guidance of 3-dimensional CT image reconstruction, and SBRT was then performed on defined target volume of porcine ventricular septum. Blood biomarkers, electrocardiogram and echocardiography parameters were monitored before and after SBRT. Pathological examination (HE staining, Masson staining) was performed on the target and non-target myocardium at 6 months post SBRT. Results: SBRT was successful and all animals survived to the designed study endpoint (6 months) after SBRT. Serum cardiac troponin T (cTnT) level was significantly higher than the baseline level at 1 day post SBRT, and reduced at 1 week after SBRT, but was still higher than the baseline level(P<0.05). Serum N-terminal pro-B type natriuretic peptide (NT-proBNP) was also significantly increased at 1 day post SBRT (P<0.05) and returned to baseline level at 1 week post SBRT. The serum NT-proBNP level was (249±78), (594±37) and (234±46) pg/ml, respectively, and the cTnT was (14±7), (240±40) and (46±34) pg/ml, respectively at baseline, 1 day and 1 week after SBRT in the 40 Gy dose group. The serum NT-proBNP level was (184±20), (451±49) and (209±36) pg/ml, respectively, the cTnT values ​​were (9±1), (176±29) and (89±27) pg/ml, respectively at baseline, 1 day and 1 week after SBRT in the 25 Gy dose group. Both NT-proBNP and cTnT values tended to be higher post SBRT in the 40 Gy dose group as compared with the 25 Gy dose group, but the difference was not statistically significant (P>0.05). The left ventricular ejection fraction and the left ventricular end-diastolic diameter remained unchanged before and after SBRT (P>0.05). The interventricular septum thickness showed a decreasing trend at 6 months after SBRT, but the difference was not statistically significant ((9.54±0.24) mm vs. (9.82±8.00) mm, P>0.05). The flow velocity of the left ventricular outflow tract, and the valve function and morphology were not affected by SBRT. At 6 months after SBRT, HE staining revealed necrosis in the irradiated target area of ​​the myocardium in the 40 Gy dose group and the 25 Gy dose group, and the degree of necrosis in the irradiated interventricular septum was more obvious in the 40 Gy dose group as compared with the 25 Gy group. The combined histological analysis of the two groups showed that the necrotic area of ​​the irradiated target area accounted for (26±9)% of the entire interventricular septum area, which was higher than that of the non-irradiated area (0) (P<0.05). There was no damage or necrosis of myocardial tissue outside the target irradiation area in both groups. The results of Masson staining showed that the percentage area of myocardial fibrosis was significantly higher in the irradiated target area than non-irradiated area ((12.6±5.3)% vs. (2.5±0.8)%, P<0.05). Conclusion: SBRT is safe and feasible for irradiating porcine ventricular septum.

[The value of high-definition intelligent endoscopy combined with stroboscopy in the diagnosis and treatment of vocal cord leukoplakia].

Objective: To evaluate the clinical value of high-definition intelligent endoscopy (iSCAN) combined with stroboscopy in identifying vocal cord leukoplakia. Methods: Seventy-nine patients with vocal cord leukoplakia who underwent CO2 laser laryngeal microsurgery and diagnosed by histopathology were recruited between October 2020 to August 2021. The morphological features, microvascular morphology and mucosal waves were observed by stroboscope; SPSS 20.0 software was used for statistical analysis. Results: There were 79 patients with a total of 119 lesions (56 on left and 63 on right).Pathological examination showed that 51 sides of the vocal cords were malignant lesions (severe dysplasia, carcinoma in situ and invasive carcinoma), and 68 sides were benign lesions.Under stroboscopy, 69 sides of mucosal wave were normal or slightly decreased, and 50 sides were severely decreased or disappeared.The decrease degree of mucosal wave was positively correlated with malignant lesions (ρ=0.687, P<0.001).Under iSCAN endoscopy, there was a positive correlation between the morphological changes of microvessels at the lesion site (vertical) and the malignant lesion (ρ=0.687, P<0.001).Univariate analysis showed that lesion size, thickness, uneven color, granular elevation, peripheral erythema and asymmetry were positively correlated with malignant lesions (ρ=0.530, 0.401, 0.538, 0.315, 0.497, 0.281, P<0.05).Logistic regression analysis showed that the risk of pathological malignancy with large lesions was 5.437 times higher than those of small lesions, the vertical vascular changes under iSCAN were 8.711 times higher than that of normal vascular morphology, and the severe reduction or disappearance of mucosal waves was 9.12 times higher than that of normal or mild reduction of mucosal waves. Conclusion: ISCAN can be combined with staphyloscopy to comprehensively observe and evaluate the changes of vocal cord morphology, submucosal microvessels and mucosal wave of vocal cord in patients with vocal cord leukoplosis, thus improving the ability to distinguish benign and malignant lesions.

[Secondhand smoke exposure and its influencing factors among Chinese people aged 15 years and above in 2010 and 2018].

Objective: To investigate secondhand smoke (SHS) exposure among non-current smokers aged 15 and over and its influencing factors in China in 2010 and 2018. Methods: The 2010 and 2018 China Adult Tobacco Surveys used multistage stratified cluster random sampling methods to obtain national representative samples across 31 provinces (autonomous regions and municipalities) in China. This study selected non-current smokers aged 15 and over as the research subjects to describe the general demographic characteristics, perceptions of SHS hazards, attitudes towards smoking bans in indoor areas in public places, and SHS exposure and the smoking restriction regulations in different places. The Rao-Scott χ2 test was used to compare the rates, and the unconditional logistic regression was used to analyze the influencing factors of SHS exposure. All the subjects in the analysis were weighted based on a complex sampling design. Results: Among non-current smokers aged 15 and over, from 2010 to 2018, the percentage of indoor smokers had decreased from 84.7% to 71.9%. The rates of people who saw smoking in various places declined with a statistically significant difference (P<0.001). The exposure to SHS for females, people aged 45-64, teachers, medical staff, workers in enterprises, businesses, waiters, and people who live in rural and central areas declined. The differences were statistically significant (P<0.05). In 2018, the percentage of SHS exposure for those who reported a comprehensive smoking ban in households or allowed smoking in certain areas was lower than for those who reported allowed or without a smoking ban. Among those aged 25-44, SHS exposure for those who reported complete smoking prohibition in workplaces was lower than those who reported allowed or without smoking prohibition (OR=0.65, 95%CI: 0.49-0.87). The proportion of SHS exposure for those who believe that smoking should not be allowed in indoor areas of 0-5 public places is higher than that smoking should not be allowed in the indoor spaces of 8 public places (OR=2.13, 95%CI: 1.35-3.36). Among people aged 45-64, the proportions of SHS exposure for teachers (OR=0.37, 95%CI: 0.17-0.78) and medical staff (OR=0.35, 95%CI: 0.16-0.76) and staff working in agriculture, forestry, animal husbandry, fishery, and water conservancy were lower than governmental staff (OR=0.49, 95%CI: 0.29-0.84). Conclusions: Data from the decreased exposure of Chinese non-current smokers aged 15 and over to SHS suggests the initial achievement through constructing a smoke-free environment, but continued efforts are needed. It is necessary to actively advocate for smoke-free families, strengthen publicity and education on the hazards of tobacco and SHS, and improve the skills of non-smokers in rejecting SHS exposure.

[Analysis of clinical features and etiological diagnostic indices of reproductive age women with hyperandrogenism].

Objective: To investigate the clinical features and the value of different diagnostic indices for etiology in reproductive age women with hyperandrogenism. Methods: The medical records of 96 reproductive age women with hyperandrogenism in the multi-disciplinary team of Peking University First Hospital from January 2020 to April 2021 were collected. The patients were divided into four groups based on final diagnosis: congenital adrenal hyperplasia (CAH) (n=8), polycystic ovary syndrome (PCOS) (n=67), idiopathic hyperandrogenism (n=13) and other specific diseases (n=8), respectively. The indices related to androgens in different groups were compared, and then their efficiency for diagnosis of CAH and PCOS were analyzed with receiver operator characteristic curve (ROC curve). Results: A total of 96 patients with hyperandrogenism were recruited, with the age of 19-45 (29±6) years old. Overall, 4.2% (4/96) of the patients were with single clinical hyperandrogenism, 56.3% (54/96) were with single laboratory hyperandrogenaemia and 39.6% (38/96) were with both. The breakdown into laboratory hyperandrogenaemia subtypes was as follows: only T elevation 22.8% (21/92), only A2 elevation 7.6% (7/92), none DHEAS elevation, only FAI elevation 5.4% (5/92) and elevation of more than one of the androgen indices mentioned above accounted for 64.1% (59/92). In the reasons of consultation, simple irregular menstruation (36.0%, 32/89) or accompanied by clinical hyperandrogenism with or without infertility (36.0%, 32/89) were the most common. As for primary visiting departments, Obstetrics and Gynecology accounted for 53.2% (51/96), and then Endocrinology as 39.5% (38/96). The 17-OHP level of CAH, PCOS and idiopathic hyperandrogenism group was 20.0 (8.2, 33.1), 1.1 (0.8, 1.4), 0.9 (0.8, 1.3) ng/ml, respectively. The androstenedione level in these groups was 6.3 (4.6, 8.7), 3.8 (2.9, 4.8) and 3.2 (2.7, 3.7) ng/ml, respectively. The 17-OHP and androstenedione levels of CAH group were significantly higher than that in PCOS or idiopathic hyperandrogenism group (all P<0.05). The ratio of LH and FSH in these three groups was 0.8(0.5, 1.0), 1.3(0.6, 1.9) and 0.6(0.3, 0.7), respectively. The ratio of LH and FSH was significantly higher in PCOS than that in idiopathic hyperandrogenism group (P=0.024), but yet there was no significant difference compared with CAH group (P>0.05). The AUC of ROC curve of 17-OHP for CAH diagnosis was 0.94, followed by androstenedione 0.83, whereas LH/FSH for PCOS diagnosis was only 0.63. Conclusions: Among the reasons of consultation in reproductive age women who visited our multi-disciplinary team for female hyperandrogenism, simple irregular menstruation or accompanied by clinical hyperandrogenism with or without infertility are the most common. PCOS accounts for the majority of different androgen excess disorders. 17-OHP is the most valuable parameter for the diagnosis of CAH and secondly androstenedione.

[Effects of anti-liver fibrosis treatment on the occurrence of liver cancer in patients with hepatitis B-related liver cirrhosis].

Objective: To investigate the effect of anti-liver fibrosis treatment on the occurrence of liver cancer in patients with hepatitis B-related liver cirrhosis within three years. Methods: 1,049 cases with hepatitis B-related liver cirrhosis who were hospitalized in Beijing Ditan Hospital affiliated to Capital Medical University from October 2008 to August 2016 were enrolled. Clinical data were collected, and COX regression analysis was used to find the independent influencing factors for the occurrence of liver cancer in patients with hepatitis B-related liver cirrhosis within three years. According to whether the patients had received anti-liver fibrosis treatment for ≥ 6 months, they were divided into combination and antiviral group. There were 388 cases in combination group and 661 cases in antiviral group. In addition, the combination group received anti-liver fibrosis therapy with Chinese patent medicine on the basis of antivirus, and the antiviral group received antiviral treatment. The incidence of liver cancer within three years were compared between the two groups, and the incidence of liver cancer in patients with different Child-Pugh grades and mPAGE-B risks was further analyzed. The independent samples t-test, Mann Whitney U test, χ2 test or Fisher's exact probability method were used for data comparison. Results: Anti-liver fibrosis treatment was an independent protective factor to prevent liver cancer in patients with hepatitis B-related liver cirrhosis within 3 years (P < 0.05). The incidence of liver cancer in the combination group was lower than antiviral group within 3 years (10.3% vs. 15.4%, χ (2) = 5.480, P < 0.05). Child-Pugh stratified analysis showed that the risk of liver cancer was significantly reduced in Child-Pugh grade A patients (6.7% vs. 12.6%, χ (2) = 2.857, P = 0.040). Among high-risk patients with mPAGE-B, the incidence of liver cancer was significantly lower in combination group than control group (13.7% vs. 19.9%, χ (2) = 6.671, P = 0.031). Conclusion: Compared to antiviral therapy alone, combined anti-liver fibrosis and antiviral therapy can reduce the liver cancer occurrence risk in patients with hepatitis B-related liver cirrhosis for 3 years. Patients with Child-Pugh grade A and high-risk group by mPAGE-B scores are the dominant population to receive treatment.

[Correlation between clinical manifestation, prognosis and antibody in anti-muscle-specific tyrosine kinase antibody positive myasthenia gravis].

Objective: To determine the correlation between the titer of anti-muscle-specific tyrosine kinase (anti-MuSK) antibodies (Abs) and the severity and prognosis of Musk-associated myasthenia gravis (Musk-MG). Methods: A total of 33 MuSK-MG patients diagnosed at Department of Neurology, Peking Union Medical College Hospital from May 2018 to June 2020 were prospectively included. Patients were divided into different groups according to immune state, and the immune naive patients were further divided by the Myasthenia Gravis Foundation of America (MGFA) classification. There were 28 Musk-MG patients who completed the follow-up and subdivided into different groups according to post-intervention status (PIS). Twenty-five patients who received immunotherapy were divided into corticosteroid monotherapy group (n=17) and corticosteroid combined with immunosuppressant group (n=8). The comparison of Ab titers between different MGFA groups and PIS groups was determined by Kruskal-Wallis method, and the comparison of Ab titers between different time points was analyzed by Mann-Whitney U method. Results: There were 11 males and 22 females included in the study, with an onset age of 48 (18, 73) years, of which 16 cases were immune naive and 17 cases were treated with corticosteroids or immunosuppressant at least once. In immune naive population, a significant difference of Ab titers among different MGFA phenotypes was detected (P=0.04). Ab titers were reduced by immunosuppression therapy (the median value decreased from 1.20 to 0.87, P=0.01). Twenty-four (85.7%) MuSK-MG patients achieved a good prognosis (PIS-PR/MM), 1 (3.6%) case achieved improvement (PIS-I), and 3 (10.7%) patients' condition worsened (PIS-W), there was no significant difference of Ab titers among the three groups (P=0.21). Meanwhile, there was no significant difference of Ab titers between different treatment groups (P=0.95). Conclusions: In the immune naive state, the concentration of MuSK-Ab is consistent with the severity of the disease, and the Ab titers decrease after immunotherapy. Change of Ab titers is related to the daily dosage of corticosteroid and is not consistent with PIS grades.

[Effect of autoflation on the prognosis of otitis media with effusion in children].

Objective: To investigate the effect of autoinflation on the prognosis of OME in children. Methods: Total of 325 pediatric patients, age ranged from 3 to 8 years, with OME(486 ears)diagnosed in our department from January 2019 to January 2020 were collected. Among them, 177 were males, 148 were females. Disease course ranged from 25 to 86 days. According to watchful waiting and autoinflation application during follow-up, these children were divided into two groups including 183 cases(271 ears) and 142 cases(215 ears), respectively. The average auditory threshold and tympanogram curve type in two groups were analyzed in the period of 3 months follow-up, and the recovery of OME was evaluated. Results: At the end of 1- and 2- month follow-up, the auditory threshold of patients in autoinflation group was significantly lower than that in watchful waiting group (t=2.139 5 and 2.680 6, P<0.05). However, at the end of 3- month follow-up, there was no significant difference between two groups (t=1.158 5, P>0.05). At the end of 1-, 2- and 3- month follow-up, 89 (33%, 89/271), 200 (74%, 200/271), 220 (81%, 220/271) and 176 (82%, 176/215), 178 (83%, 178/215), 183 (85%, 183/215) ears in watchful waiting group and autoinflation group had a hearing threshold <20 dB HL, respectively, in which ears with auditory threshold<20 dB HL in watchful waiting group were significantly less than those in autoinflation group at the end of 1 and 2 month follow-up (P<0.05), However, ears with auditory threshold<20 dB HL in watchful waiting group were not significantly different from that in the autoflation group at the end of 3- month follow-up (P>0.05). The proportion of ears with type A tympanogram curve was 74%(159/215), 79%(170/215), and 85%(183/215) at the end of 1-, 2- and 3- month follow-up in autoinflation group and 36%(98/271), 71%(192/271) and 76%(206/271) in watchful waiting group, respectively. Proportion of ears with type A tympanogram curve in autoflation group was significantly higher than that in watchful waiting group (P<0.05). Conclusion: Autoinflation can improve the hearing of children with OME in early stage, restore normal middle ear pressure, increase recovery rate, and reduce the choice of surgical treatment of OME.

Diagnostic and prognostic values of KLK11 in nasopharyngeal carcinoma.

OBJECTIVE: This study aims to clarify potential diagnostic and prognostic values of KLK11 in nasopharyngeal carcinoma (NPC). PATIENTS AND METHODS: KLK11 levels in 81 primary NPC tissues, 24 recurrent NPC tissues, and 60 nasopharyngeal tissues with chronic mucosal inflammation were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Then, receiver operating characteristic (ROC) curves were depicted for assessing the diagnostic value of KLK11 in primary and recurrent NPC. Next, correlation between KLK11 level and pathological indexes of NPC patients was analyzed by Chi-square test. Enrolled NPC patients were followed up for 5 years, and the follow-up data were recorded to determine the potential influence of KLK11 on overall survival by Kaplan-Meier method. In addition, Cox regression model was applied for assessing factors that could affect prognosis of NPC patients. RESULTS: It was found that KLK11 level was higher in primary NPC tissues than that in nasopharyngeal tissues with chronic mucosal inflammation. In recurrent NPC tissues, KLK11 was upregulated relative to primary ones. In addition, ROC curves revealed a certain diagnostic value of KLK11 in NPC. Overall survival was worse in primary and recurrent NPC patients expressing a high level of KLK11. By analyzing the pathological indexes of NPC patients, KLK11 level was found to be correlated with age, T stage, and clinical stage of NPC patients. Furthermore, KLK11 level was found to be the risk factor influencing the survival of NPC patients. CONCLUSIONS: KLK11 is upregulated in NPC tissues, and unfavorable to the prognosis of NPC. Besides, it can be utilized as a potential hallmark for diagnosing NPC.

Both Caspase and Calpain are Involved in Endoplasmic Reticulum-Targeted BNIP3-Induced Cell Death.

Bcl-2/E1B-19K-interacting protein 3 (BNIP3) is a member of the apoptotic B-cell lymphoma-2 family that regulates cell death. Although BNIP3 targeted normally to the mitochondrial outer membrane by its transmembrane domain was originally considered to be essential for its pro-apoptotic activity, accumulating evidence has shown that BNIP3 is localized to endoplasmic reticulum at physiological conditions and that forced expression of BNIP3 can initiate cell death via multiple pathways depending on the subcellular compartment it targets. Targeting BNIP3 to endoplasmic reticulum has been shown to participate in cell death during endoplasmic reticulum stress. However, the molecular events responsible for BNIP3-induced cell death in the endoplasmic reticulum remain poorly understood. In the present study, the transmembrane domain of BNIP3 was replaced with a segment of cytochrome b5 that targets BNIP3 into endoplasmic reticulum, which induced cell death as effectively as its wild-type molecule in the SW480 cell line (colon carcinoma). Furthermore, a pan-caspase inhibitor, z-VAD-fmk, and PD150606, a specific calpain inhibitor, both significantly suppressed the endoplasmic reticulum-targeted BNIP3-induced cell death. These results suggest that endoplasmic reticulum-targeted BNIP3 induced a mixed mode of cell death requiring both caspases and calpains.

STYK1 promotes the malignant progression of laryngeal squamous cell carcinoma through targeting TGF-ß1.

OBJECTIVE: The aim of this study was to uncover the expression characteristic and biological function of STYK1 in the progression of laryngeal squamous cell carcinoma (LSCC), and to explore the underlying mechanism. PATIENTS AND METHODS: Expression level of STYK1 in 44 paired LSCC and adjacent normal tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between STYK1 level and clinical parameters of LSCC patients was analyzed. Subsequently, the regulatory effect of STYK1 on the proliferative ability of AMC-HN-8 and Hep-2 cells was evaluated by cell counting kit-8 (CCK-8) assay and colony formation assay. Dual-Luciferase reporter gene assay and rescue experiments were conducted to uncover the role of STYK1/TGF-ß1 axis in regulating the progression of LSCC. RESULTS: STYK1 was significantly up-regulated in LSCC tissues than that of adjacent normal tissues (p<0.05). LSCC patients with high expression level of STYK1 exhibited significantly higher clinical stage and lower survival rate (p<0.05). Knockdown of STYK1 remarkably attenuated viability and clonality in Hep-2 cells, while overexpression of STYK1 achieved the opposite trends in AMC-HN-8 cells (p<0.05). TGF-ß1 was confirmed to be the direct target binding STYK1, whose expression level was negatively regulated by STYK1. TGF-ß1 was significantly down-regulated in LSCC tissues  (p<0.05). Meanwhile, its low expression predicted significantly poor prognosis of LSCC patients. In addition, TGF-ß1 was responsible for STYK1-regulated malignant progression of LSCC. CONCLUSIONS: STYK1 is upregulated in LSCC and is closely associated with T stage and poor prognosis. Furthermore, STYK1 promotes the proliferative ability of LSCC cells through targeting TGF-ß1, thus aggravating the malignant progression of LSCC.

Regulation of epithelial mesenchymal transition by the renin-angiotensin system: a role for klotho in renal tubular epithelial cells.

Klotho is a putative aging suppressor gene that is primarily expressed in renal tubular epithelial cells. Its expression has been reported to protect against fibrosis in human chronic kidney disease. However, the roles of klotho in epithelial-mesenchymal transition (EMT) and renal fibrosis are yet to be elucidated. The present study aimed to investigate the putative roles of klotho in angiotensin (Ang) II-induced damage of renal tubular epithelial cells. NRK-52E rat cells were treated with various combinations of Ang II, the Ang-converting enzyme inhibitor fosinopril (Fos) and the Ang II receptor antagonist valsartan (Val). The levels of transforming growth factor (TGF)-ß1, soluble klotho, α-smooth muscle actin (α-SMA) and E-cadherin in NRK-52E culture supernatants were measured using enzyme-linked immunosorbent assays. Furthermore, the mRNA and protein expression of TGF-ß1, klotho, α-SMA and E-cadherin was detected using semiquantitative reverse transcription-polymerase chain reaction, immunocytochemistry and Western blot analysis. The results demonstrated that Ang II inhibited the expression of klotho and E-cadherin, while it upregulated the expression of TGF-ß1 and α-SMA, in NRK52E cells. Fos and/or Val were revealed to enhance klotho and E-cadherin expression, and suppress the expression of TGF-ß1 and α-SMA, compared with the Ang II-only group. Furthermore, a positive linear correlation was detected between the expression of klotho and E-cadherin, while negative linear correlations with klotho expression were detected for TGF-ß1 and α-SMA expression. In conclusion, the expression of klotho was demonstrated to be enhanced following treatment with Fos and Val in Ang II-treated NRK-52E cells. The present results indicate that klotho may be involved in the inhibition of Ang II-induced EMT in renal tubular epithelial cells. Therefore, klotho may serve as a protective factor in renal tubulointerstitial fibrosis and aid the treatment of chronic kidney disease (CKD) patients using precision therapy.

[Clinical phenotype and immunological features of a patient with A20 haploinsufficiency].

Objective: To explore the clinical phenotype, immunological features, pathogenesis and gene variation of a case with A20 haploinsufficiency (HA20). Methods: A patient diagnosed with tumor necrosis factor α-induced protein 3 (TNFAIP3) mutated HA20 was admitted into Shenzhen Children's Hospital in May,2019.The clinical data was analyzed. Flow cytometry was used to detect the patient's peripheral blood lymphocyte subsets, and also, the percentage of follicular helper T cell (TFH) cells in the patient and thirteen healthy controls. After the construction of empty vector, wild-type and mutant plasmid vectors, a wild-type or mutant overexpression system of the TNFAIP3 gene was established in 293T cells and Hela cells. Then, the expression level of A20 in 293T cells and the expression of inhibitor K binding α (IKBα) in green fluorescent protein (GFP)+Hela cells before and after tumor necrosis factor α (TNF-α) stimulation were measured, to verify the pathogenicity of this variation. Results: A 5 years and 11 months old boy, presented with recurrent oral ulcer, abdominal pain, joint swelling and arthralgia. Oral ulcer, chronic skin rashes, knee joint swelling were observed. The levels of inflammatory markers were increased. Colonoscopy showed congestion of mucosa and multiple ulcers in terminal ileum and ileocecus. The absolute number of naive B cells was 124×10(6) cells/L (reference range 147×10(6)-431×10(6) cells/L), accounting for 0.430 of the total B cells (reference range 0.484-0.758). Compared to healthy controls (0.016-0.071), the percentage of TFH cells in CD4(+)T cells was much lower (0.008).A heterozygous mutation of TNFAIP3 gene (c.909_913 del, p.L303fs) was identified by genetic analysis. In vitro study showed that truncated A20 protein was expressed in TNFAIP3 mutant overexpressed 293T cells, which verified the pathogenicity of this variation. Besides, after TNF-α stimulation, the degradation rate of IkBα protein in mutant overexpressed Hela cells (35%) was between the other two groups (15% in the wild-type group and 57% in the non-loaded group). Conclusions: This case with HA20 due to a de novo TNFAIP3 gene mutation presents with early onset Behcet-like autoinflammatory syndrome. This variation leads to expression of truncated A20 protein, enhanced degradation of IkBα, and further activation of nuclear factor κB signaling pathway.

[One case report of Mohr-Tranebjærg syndrome].

Summary A 4-year-old male patient was found poor development in hearing and speech, without family hereditary history. Hearing screening was failed at birth. From the age of 2, the patient showed poor response to sound and speech, but no audiological examination was carried out. After physical examination, no deformity was found in both ears, and the tympanic membranes were intact; the muscular tension was normal; and the visual acuity was normal. The acoustic immittance showed curve A; DPOAE showed that both ears passed; click ABR threshold was greater than 95 dB nHL, bone conduction was greater than 45 dB nHL; electrocochleogram was bilateral elicited. There were no malformations of cochlea and inner ear showed in temporal bone CT and internal auditory canal MRI. Gene detection indicated a mutation in TIMM8A gene of X chromosome. Combined with the patient's medical history, gene detection, audiological manifestations and imaging examination, the final diagnosis was Mohr-Tranebjærg syndrome, bilateral severe sensorineural hearing loss, and auditory neuropathy.

[Preoperative imaging and electrophysiological evaluation of cochlear implantation in children with cochlear nerve dysplasia].

Objective:The aim of this study is to investigate the use of CT, magnetic resonance imaging （MRI）, functional magnetic resonance （fMRI） and electrical evoked auditory brainstem response in children with cochlear nerve deficiency （CND） before cochlear implantation （CI） （electrically evoked auditory brain stem response, EABR） evaluated auditory pathway, auditory cortex function, and cochlear nerve function. Method:Of 6 children with bilateral profound sensorineural hearing loss, bilateral internal auditory meatus stenosis was diagnosed by CT as in 4 cases and unilateral internal auditory meatus stenosis in 1 case. In 3 cases, oblique sagittal MRI reconstruction of the internal auditory meatus showed only facial nerve and vestibular nerve existence. 6 cases were diagnosed as CND by imaging, among which underwent fMRI and EABR in 3 cases, respectively. All the children received unilateral CI and were followed up for at least 1 year after hearing and speech rehabilitation. Result:fMRI was examed in 3 cases with CND. It showed that the right auditory cortex was activated in 1 case after sound given to the left ear but no activation was found in the left cortex when sound given to the right ear. 1 case showed activation in bilateral transverse temporal gyrus while sound was given to the left, right and both ears at 2000Hz. Another case had no activation in both auditory cortex. EABR detection elicited V wave in 2 of 3 cases. 6 children received audiological and speech evaluation 1 month, 6 and 12 months after CI. Among them, the hearing level was improved within 6 months after surgery in 4 cases, and the threshold of sound field was （48.15±6.60） dB HL, MAIS and CAP scores were improved. However, SIR score improvement is limited in 3 cases. The hearing level was improved in 1 case, but speech rehabilitation was poor mainly due to the older age. Conclusion:Preoperative comprehensive application of imaging and electrophysiological evaluation of children with CND can more accurately assess the integrity of the auditory pathway and understand the function of the cochlear nerve. It has important reference significance for whether or not to perform surgery. There was a significant difference in auditory speech rehabilitation after CND in children with CND.

MicroRNA-138 inhibits proliferation and induces apoptosis of laryngeal carcinoma via targeting MAPK6.

OBJECTIVE: To explore whether microRNA-138 could regulate the incidence and progression of laryngeal carcinoma through modulating proliferation and apoptosis of laryngeal carcinoma cells via MAPK6. PATIENTS AND METHODS: MicroRNA-138 expression in laryngeal carcinoma tissues and paracancerous tissues were detected by qRT-PCR (Quantitative Real-Time Polymerase Chain Reaction). The regulatory effects of microRNA-138 on proliferation and apoptosis of laryngeal carcinoma cells were detected by colony formation assay and flow cytometry, respectively. Target gene of microRNA-138 was predicted by online software and verified by luciferase reporter gene assay. Corresponding plasmids of microRNA-138 and the target gene were constructed. Rescue experiments were conducted to explore the regulatory effect of microRNA-138 on the target gene. RESULTS: MicroRNA-138 was downregulated in laryngeal carcinoma tissues than that of paracancerous tissues. MicroRNA-138 knockdown resulted in increased proliferation and decreased apoptosis of laryngeal carcinoma cells. MAPK6 was predicted as the target gene of microRNA-138. Luciferase reporter gene assay further verified that MAPK6 could directly bind to microRNA-138. Both mRNA and protein levels of MAPK6 were downregulated after microRNA-13 overexpression in laryngeal carcinoma cells. Rescue experiment results indicated that increased proliferation and decreased apoptosis of laryngeal carcinoma cells resulted from microRNA-13 knockdown were partially reversed by MAPK6 overexpression. CONCLUSIONS: MicroRNA-138 is downregulated in laryngeal carcinoma patients. MicroRNA-138 knockdown promotes proliferation and inhibits apoptosis of laryngeal carcinoma cells via inhibiting MAPK6 expression.

[Strategy for minimally invasive cochlear implantation and residual hearing preservation].

In the past few decades, considerable development was achieved in the cochlear implantation following the emergence of innovative electrode array and advances in minimally invasive surgery. Minimally invasive technique led to a better preservation of residual low-frequency hearing. The loss of residual hearing was caused by complicated factors. According to previous studies, a slower and stable speed of electrode insertion and the use of perioperative steroids were demonstrated to have a positive impact on hearing preservation. The selection of electrode array or its insertion approaches didn't show any distinctive benefits in hearing preservation.