OBJECTIVE: To elucidate the role and mechanism of microRNA-145-5p (miR-145-5p) in hypoxia-induced pyroptosis of human alveolar epithelial cells. METHODS: In vitro, human alveolar epithelial cell line BEAS-2B was cultured. Cells in the logarithmic growth phase were cultured to 80% confluence and then used for the experiment. (1) BEAS-2B cells were cultured under 1% O₂ hypoxic condition, with a normoxic control group. Western blotting was employed to detect the expressions of pyroptosis marker proteins [NOD-like receptor protein 3 (NLRP3), Gasdermin D N-terminal domain (GSDMD-N), and caspase-1] in cells cultured for 24 hours. Real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-145-5p in cells cultured for 6 hours and 12 hours. (2) Cells were transfected with 30 nmol/L miR-145-5p mimic to overexpress miR-145-5p expression under normoxic condition or 30 nmol/L miR-145-5p inhibitor to suppress miR-145-5p expression under hypoxic condition. Control group and negative control group were respectively set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of pyroptosis marker proteins and nuclear factor-E2-related factor 2 (Nrf2) in cells. Flow cytometry was applied to detect the level of reactive oxygen species (ROS) in cells. The target genes of miR-145-5p were predicted by miR target gene prediction software miRWalk and verified by Western blotting. (3) Under hypoxic condition, cells were transfected with 6.94 ng/μL silent information regulator 5 (Sirt5) overexpression plasmid or pretreated with 12.5 mmol/L N-acetyl-L-cysteine (NAC) as an ROS inhibitor. The empty plasmid group and control group were set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of Sirt5, Nrf2, and pyroptosis marker proteins in cells. Flow cytometry was used to detect the level of ROS in cells. RESULTS: (1) Compared with the normoxic control group, the expression levels of pyroptosis marker proteins in the 24-hour hypoxia group was significantly increased, indicating that hypoxia could induce pyroptosis in BEAS-2B cells. The expression level of miR-145-5p in cells gradually increased with the extension of hypoxia induction time, indicating that hypoxia could cause the increase of miR-145-5p expression level. (2) The expression levels of pyroptosis marker proteins in cells of miR-145-5p mimic group significantly increased under normoxic condition as compared with the control and negative control groups [NLRP3 protein (NLRP3/β-actin): 1.58±0.07 vs. 1.00±0.01, 0.98±0.07, GSDMD-N protein (GSDMD-N/β-actin): 1.71±0.03 vs. 1.01±0.01, 0.85±0.03, caspase-1 protein (caspase-1/β-actin): 2.33±0.04 vs. 1.01±0.01, 1.05±0.04, all P < 0.05], Nrf2 protein expression level was significantly decreased (Nrf2/β-actin: 0.79±0.03 vs. 1.00±0.01, 1.03±0.04, both P < 0.05), ROS level was significantly up-regulated (fluorescence intensity: 1.74±0.03 vs. 1.00±0.01, 0.92±0.03, both P < 0.05). Under hypoxia condition, compared with control group and negative control group, the expression levels of pyroptosis marker proteins in miR-145-5p inhibitor group were significantly decreased [NLRP3 protein (NLRP3/β-actin): 0.21±0.04 vs. 1.70±0.02, 1.63±0.04; GSDMD-N protein (GSDMD-N/β-actin): 1.32±0.02 vs. 2.51±0.02, 2.72±0.03; caspase-1 protein (caspase-1/β-actin): 0.56±0.01 vs. 2.77±0.02, 3.12±0.03; all P < 0.05], Nrf2 protein expression level was significantly increased (Nrf2/β-actin: 1.57±0.04 vs. 1.22±0.01, 1.28±0.04, both P < 0.05), ROS level was significantly down-regulated (fluorescence intensity: 0.64±0.05 vs. 1.87±0.04, 1.70±0.07, both P < 0.05). The results indicated that miR-145-5p could promote cell pyrodeath. The predictive result of miRWalk showed that the 3' untranslated region (3'UTR) of Sirt5 had complementary base binding sites with miR-145-5p. The expression level of Sirt5 protein in cells of miR-145-5p mimic group was significantly lower than that of control group and negative control group under normoxic condition (Sirt5/β-actin: 0.59±0.03 vs. 1.00±0.01, 1.01±0.03, both P < 0.05), which verified that Sirt5 was the target gene of miR-145-5p. (3) The occurrence of pyrodeath could be partially reversed by transfection with Sirt5 overexpression plasmid or adding ROS inhibitor NAC into cells, and Sirt5 overexpression could also up-regulate Nrf2 expression and eliminate intracellular ROS. CONCLUSION In human alveolar epithelial cells, miR-145-5p can down-regulate Nrf2 by targeting Sirt5, thereby increasing ROS expression and inducing pyrodeath.
Humans ; MicroRNAs ; Pyroptosis ; Cell Hypoxia ; Alveolar Epithelial Cells/cytology* ; Cell Line ; NLR Family, Pyrin Domain-Containing 3 Protein ; Caspase 1/metabolism* ; Epithelial Cells/metabolism* ; Gasdermins ; Phosphate-Binding Proteins

Objective To explore the effect of open reading frame 66(C12ORF66)located at chromosome 12 on the viability of MYCN amplified NB cell lines.Methods DDatasets GSE16476 and GSE49710 in R2 database were analyzed for expression level of C12ORF66 in MYCN amplified and MYCN non-amplified NB cells and its potential correlation with the prognosis of pediatric patients.C12ORF66 mRNA expression level in normal tissue immortalized cell lines,MYCN amplified and MYCN non-amplified cell lines were detected by RT-qRCR.Transient or stable knockdown of C12ORF66 cell lines were constructed to compare the difference in real time cellular analysis(RTCA),colony formation,Ki67 positive cells between the control group and the C12ORF66 knockdown group.Results By analyzing R2 datasets,C12ORF66 level in MYCN amplified samples was significantly higher than that in MYCN non-amplified samples,and the expression of C12ORF66 was negatively correlated with the prognosis of pediatric patients(P＜0.05).C12ORF66 highly expressed in MYCN-amplified BE(2)-C and SK-N-BE(2)cell lines than in MYCN non-amplified CHLA-255 and SH-SY5Y cell lines(P＜0.001).Transient or stable knockdown of C12ORF66 resulted in significant slow down of proliferation of MYCN amplified NB cells(P＜0.001),the colony formation ability was significantly reduced(P＜0.001),and the proportion of Ki67 positive cells was significantly decreased(P＜0.05).Conclusions C12ORF66 was highly expressed in MYCN amplified clinical NB samples and cell lines which is believed to be correlated with poor prognosis of pediatric patients.C12ORF66 knockdown signifi-cantly inhibits cell viability of NB cells.

Objective To evaluate the potential risk of transmission of angiostrongyliasis by common freshwater snails in Dali Bai Autonomous Prefecture, Yunnan Province, so as to provide insights into local surveillance of angiostrongyliasis. Methods Common freshwater snails were collected from Dali Bai Autonomous Prefecture, Yunnan Province from March to April, 2020, and identified and bred in laboratory. SD rats were infected with third-stage larvae of Angiostrongylus cantonensis that were isolated from commercially available Pomacea canaliculata snails in Dali Bai Autonomous Prefecture, and freshwater snails were infected with the first-stage larvae of A. cantonensis that were isolated from the feces of SD rats 39 days post-infection at room temperature. The developmental process and morphological characteristics of worms in hosts were observed, and the percentages of A. cantonensis infections in different species of freshwater snails were calculated. Then, SD rats were infected with the third-stage larvae of A. cantonensis that were isolated from A. cantonensis-infected freshwater snails, and the larval development and reproduction was observed. Results More than 3 000 freshwater snail samples were collected from farmlands, ditches and wetlands around Erhai Lake in Dali Bai Autonomous Prefecture, and Cipangopaludina chinensis, P. canaliculata, Parafossarulus striatulus, Oncomelania hupensis robertsoni, Galba pervia, Physa acuta, Radix swinhoei, Assiminea spp., Tricula spp. and Bellamya spp. were morphologically identified. A total of 105 commercially available P. canaliculata snails were tested for A. cantonensis infections, and 2 P. canaliculata snails were found to be infected with A. cantonensis, in which the third-stage larvae of A. cantonensis were isolated. Ten species of freshwater snails were artificially infected with the third-stage larvae of A. cantonensis, and all 10 species of freshwater snails were found to be infected with A. cantonensis, with the highest positive rate of A. cantonensis infections in Bellamya spp. (62.3%, 137/204), and the lowest in C. chinensis (35.5%, 11/31). After SD rats were infected with the third-stage larvae of A. cantonensis isolated from different species of freshwater snails, mature adult worms of A. cantonensis were yielded. Conclusions Multiple species of freshwater snails may serve as intermediate hosts of A. cantonensis under laboratory conditions in Dali Bai Autonomous Prefecture of Yunnan Province. Further investigations on natural infection of A. cantonensis in wild snails in Dali Bai Autonomous Prefecture seem justified.

Objective To investigate the reliability and feasibility of spectral entropy in evaluate the effectiveness of analgesic sedation in patients with ischemic stroke.Methods A total of 64 patients with acute ischemic stroke admitted to Hongqi Hospital Affiliated to Mudanjiang Medical University from July 2021 to November 2022 were selected as study objects,and the included patients were divided into control group and experimental group according to random number table method,with 32 cases in each group.Patients in control group adjusted analgesia and sedation regimen according to Richmond agitation-sedation scale(RASS)score and critical-care pain observation tool(CPOT)score.Patients in experimental group adjusted the analgesic and sedation regimen according to the results of spectral entropy.The vital signs,C-reactive protein(CRP),dose of sedative and analgesic drugs and incidence of adverse reactions were compared between two groups.The correlation between spectral entropy and RASS score and CPOT score was used Spearman correlation analysis.Results The spectral entropy values were positively correlated with the RASS score and CPOT score,respectively(r=0.719,0.556,P<0.001).There were no significant differences in mean arterial pressure and percutaneous arterial oxygen saturation between two groups at different time points(P>0.05).The heart rate at T3 in experimental group was significantly lower than that in control group(P<0.05).At T1,T2 and T3,CRP levels in experimental group were significantly lower than those in control group(P<0.05).The dosage of sufentanil and midazolam in experimental group were significantly lower than those in control group(P<0.05).The incidence of adverse reactions in experimental group was significantly lower than that in control group(12.50%vs.34.38%,χ2=4.267,P=0.039).Conclusion Spectral entropy can be used as an objective method to monitor the depth of analgesia and sedation in patients with ischemic stroke,and has a good correlation with RASS score and CPOT score,which can reduce the incidence of adverse reactions,effectively avoid stress reactions,and reduce the application of analgesia and sedation drugs.

Angiostrongyliasis cantonensis is an emerging infectious disease in China. Snails are intermediate hosts of Angiostrongylus cantonensis and play a critical role in the transmission of angiostrongyliasis cantonensis. Detection of A. cantonensis in snails is an important part of epidemiological surveys. Currently, the rapid developments in the techniques for detection of A. cantonensis in snails facilitate the surveillance of angiostrongyliasis cantonensis and provide an important support for angiostrongyliasis cantonensis prevention and control. This review summarizes the advances in the techniques for detection of A. cantonensis in snails.

Objective:To construct the scientific nursing standards for extracorporeal membrane oxygenation combined with intra-aortic balloon pumping after heart transplantation that meets clinical needs.Methods:Through literature retrieval, case review, and expert interviews, a preliminary letter questionnaire was established for nursing standards of extracorporeal membrane oxygenation combined with intra-aortic balloon pumping after heart transplantation. From February to June 2018, we selected 21 medical and nursing experts in anesthesia, cardiopulmonary bypass, critical care medicine for two rounds of Delphi consultation.Results:Through two rounds of expert consultation, the nursing standards for extracorporeal membrane oxygenation combined with intra-aortic balloon pumping after heart transplantation were determined. The standards included 4 first-level indicators, 10 second-level indicators and 33 third-level indicators.Conclusions:The nursing standards for extracorporeal membrane oxygenation combined with intra-aortic balloon pumping after heart transplantation are scientific and reliable, which provides a reference for the nursing of extracorporeal membrane oxygenation combined with intra-aortic balloon pumping after heart transplantation.

OBJECTIVES: To investigate the genetic causes of hearing loss with enlarged vestibular aqueduct (EVA) in two children from unrelated two Chinese families. METHODS: Sanger sequencing of all coding exons in SLC26A4 (encoding Pendrin protein) was performed on the two patients, their sibling and parents respectively. To predict and visualize the potential functional outcome of the novel variant, model building, structure analysis, and in silico analysis were further conducted. RESULTS: The results showed that the proband from family I harbored a compound heterozygote of SLC26A4 c.1174A>T (p.N392Y) mutation and c.1181delTCT (p.F394del) variant in exon 10, potentially altering Pendrin protein structure. In family II, the proband was identified in compound heterozygosity with a known mutation of c.919-2A>G in the splice site of intron 7 and a novel mutation of c.1023insC in exon 9, which results in a frameshift and translational termination, consequently leading to truncated Pendrin protein. Sequence homology analysis indicated that all the mutations localized at high conservation sites, which emphasized the significance of these mutations on Pendrin spatial organization and function. CONCLUSION: In summary, this study revealed two compound heterozygous mutations (c.1174A>T/c.1181delTCT; c.919- 2A>G/c.1023insC) in Pendrin protein, which might account for the deafness of the two probands clinically diagnosed with EVA. Thus this study contributes to improve understanding of the causes of hearing loss associated with EVA and develop a more scientific screening strategy for deafness.
Asian Continental Ancestry Group ; Child ; Clinical Coding ; Computer Simulation ; Deafness ; Exons ; Extravehicular Activity ; Frameshift Mutation ; Hearing Loss ; Heterozygote ; Humans ; Introns ; Mass Screening ; Parents ; Sequence Homology ; Siblings ; Vestibular Aqueduct

Objective: To study the difference expression and diagnostic value of ribosomal protein L5 (RPL5) in papillary thyroid carcinoma (PTC) of children and adults. Methods: Realtime-PCR was performed to detect the expression of RPL5 in 22 PTC tissues and 13 pericarcinous tissues. Receiver operating characteristic (ROC) curve and Youden's index were used to evaluate the diagnostic value of RPL5 in PTC of children and adults. Results: The expression of RPL5 in PTC tissues was higher than in pericarcinous tissues. The area under curve (AUC) was 0.820 (P=0.001), and Youden′s index was 0.568. The expression of RPL5 in PTC of adults was higher than children (P<0.05). The AUC and Youden's index were respectively 0.721 (P=0.069) and 0.414 in children, whereas being respectively 0.896 (P=0.0005) and 0.709 in adults. RPL5 in diagnosis of PTC of adults was better than CK19, Galectin-3 and TPO, which are commonly used for the pathologic diagnosis of PTC. Conclusion The expression of RPL5 in PTC is higher than pericarcinous tissues, and its expression in PTC of adults is higher than children. Furthermore, PTC is a potential indicator for diagnosis of PTC.

ObjectiveTo check the isolated heart by coronary angiography to discover the location, na-ture and degree of the coronary artery lesions more accurately and increase the comprehensive evaluation ability of cardiovascular disease.MethodsTen fresh isolated hearts with different causes of death were extracted and injected with barium sulphate as contrast substance by ring injector, then developed under Xper FD20 angiography equipment. The obtained pictures and image data were handled by three-dimen-sional angiography images with the software attached to the angiography equipment. The coronary artery tissues were HE stained and observed by microscope. The HE staining results were compared with the angiographic results.ResultsThe imaging data obtained from the 10 cases for examination showed 8 cases without coronary artery stenosis and 2 cases with Ⅲ, Ⅳcoronary artery stenosis, which were consistent with HE staining results of coronary artery organization and the both results were confirmed. ConclusionIsolated coronary angiography has an unique advantage for accurate grading of classification of coronary artery stenosis, examination of vascular malformation and tiny lesions, which can provide reference for the localization of small lesions and basis during the autopsy for identification conclusion.

OBJECTIVE:To compare the efficacy,safety,vascular endothelial growth factor(VEGF)and matrix metallopro-teinase-2 (MMP-2) of docetaxel combined with carboplatin and paclitaxel combined with cisplatin (DDP) in the treatment of ad-vanced ovarian cancer. METHODS:120 patients with advanced ovarian cancer were randomly divided into docetaxel combined with carboplatin group(60 cases)and paclitaxel combined with DDP group(60 cases). Docetaxel combined with carboplatin group received 70 mg/m2 Docetaxel injection,intravenous infusion of 1 h,d1;50 mg/m2 carboplatin injection,intravenous infusion of 1 h,d2. Paclitaxel combined with DDP group received 135 mg/m2 Paclitaxel injection,intravenous infusion of 24 h,d1;30 mg/m2 DDP for injection,intravenous infusion,d3;60 mg/m2 Paclitaxel injection (a maximum of 2.0 m2) by intraperitoneal infusion,d8. 3-week was regarded as 1 treatment course,and it lasted 6 courses. Clinical efficacy,VEGF,MMP-2,progression-free survival, overall survival before and after treatment,mortality rate within 2 years of treatment and the incidence of adverse reactions in 2 groups were compared. RESULTS:There were no significant differences in the objective response rate,disease control rate,mortal-ity rate,incidence of adverse reactions between 2 groups(P>0.05). The progression-free survival in docetaxel combined with car-boplatin group was significantly longer than paclitaxel combined with DDP group,the difference was statistically significant (P<0.05). Before treatment,there were no significant differences in VEGF and MMP-2 level between 2 groups(P>0.05). After treat-ment,VEGF and MMP-2 level in 2 groups were significantly lower than before,and VEGF at different time points and MMP-2 level after 4 weeks,8 weeks and 12 weeks of treatment in docetaxel combined with carboplatin group were lower than paclitaxel combined with DDP group,the differences were statistically significant(P<0.05). CONCLUSIONS:Docetaxel combined with car-boplatin and paclitaxel combined with DDP shows similar efficacy and safety in the treatment of advanced ovarian cancer,but docetaxel carboplatin combined with is superior to paclitaxel combined with DDP in reducing VEGF and MMP-2 and improving pro-gression-free survival.