[Efficiency of CNV-seq in detecting fetal DMD gene deletion or duplication in prenatal diagnosis].

Objective: To evaluate the diagnostic efficiency of copy number variation sequencing (CNV-seq) to detect the deletion or duplication of DMD gene in prenatal diagnosis. Methods: A retrospective analysis was carried out on the CNV-seq results of 34 544 fetuses diagnosed in the First People's Hospital of Yunnan Province from January 2018 to July 2023. A total of 156 cases of fetuses were collected, including Group 1:125 cases with family history of Duchenne muscular dystrophy or Becker muscular dystrophy (DMD/BMD), and Group 2:31 cases with no family history but a DMD gene deletion or duplication was detected unexpectedly by CNV-seq. Multiplex ligation-dependent probe amplification (MLPA) was used as a standard method to detect the deletion or duplication. Consistency test was carried out basing on the results of CNV-seq and MLPA of all 156 cases. Results: Comparing to MLPA, CNV-seq had a coincidence rate of 92.3% (144/156) for DMD gene deletion or duplication, with a sensitivity and positive predictive value of 88.2%, with a specificity and negative predictive value of 94.3%, a missed detection rate of 3.8%, and a Kappa value of 0.839. CNV-seq missed 4 cases with deletions and 2 with duplications due to involved fragments less than 100 Kb, among 20 cases of deletions and 6 cases of duplications detected by MLPA in Group 1. In Group 2, the deletions and duplications detected by CNV-seq were 42% (13/31) and 58% (18/31), respectively, in which the percentage of duplication was higher than that in Group 1. Among those 18 cases with duplications, 3 cases with duplication locating in exon 42~67 were likely pathogenic; while 9 cases with duplication covering the 5' or 3' end of the DMD gene, containing exon 1 or 79 and with only one breakpoint within the gene, along with the last 6 cases with duplications locating at chrX: 32650635_32910000 detected only by CNV-seq, which might be judged as variants of uncertain significance. Conclusions: CNV-seq has a good efficiency to detect fetal DMD gene deletion or duplication in prenatal diagnosis, while a further verification test by MLPA is recommended. The duplications on chrX: 32650635_32910000, 5' or 3' end of DMD gene detected by CNV-seq should be carefully verified and assessed because those variants appear to be nonpathogenic polymorphisms.

[Study on the predictive significance of PLR, SII and RPR in ovarian endometriotic cyst].

Objective: To investigate the predictive value of platelet-to-lymphocyte ratio (PLR), red blood cell distribution width to platelet count ratio (RPR) and systemic immune inflammation index (SII) in the staging and postoperative recurrence of ovarian endometrial cysts. Methods: Retrospective analysis was made on the clinical data of patients who underwent laparoscopic surgery for ovarian cysts in the Affiliated Hospital of Qingdao University from January 2018 to January 2020. The patients with ovarian endometriosis cyst confirmed by pathology after surgery were the observation group (n=350), and the patients with other benign ovarian cyst were the control group (n=150). The preoperative platelet count, platelet distribution width, absolute number of neutrophils, lymphocyte absolute number, absolute number of monocytes, red blood cell distribution width, and serum cancer antigen 125 (CA125) of the patients in two groups were recorded, and PLR, neutrophil-to-lymphocyte ratio (NLR), RPR, SII, and systemic inflammation response index (SIRI) were calculated and analyzed. The general data of all patients and the follow-up data within 2 years after the operation of the observation group were statistically recorded to evaluate the diagnostic value of PLR, RPR and SII for ovarian endometrial cyst, and the predictive value of staging and recurrence within 2 years after the operation. Results: PLR, NLR, SII (median: 147.53, 1.86, and 488.70 respectively) and CA125 (median: 59.41 kU/L) in the observation group were significantly higher than those in the control group, while RPR (median: 0.16) was lower than that in the control group, with significant differences (all P<0.01). There was no significant difference in SIRI between the two groups (P>0.05). The PLR and SII (median: 122.73, 345.00) of the observation group at stage Ⅲ and Ⅳ were higher than those of patients at stage Ⅰ and Ⅱ, and the RPR was lower than that of patients with stage Ⅰ and Ⅱ, with significant differences (all P<0.001). The PLR, NLR, SII, SIRI (median: 179.63, 2.75, 762.96, and 1.06 respectively) and CA125 (median: 108.83 kU/L) in patients with recurrence were significantly higher than those in patients without recurrence 2 years after the operation, and the differences were statistically significant (all P<0.001). The area under curve (AUC) of CA125 in the diagnosis of ovarian endometriosis cyst was 0.951, the sensitivity was 85.7%, and the specificity was 93.0%, which were higher than those of PLR and SII; the AUC of PLR+SII+CA125 in the diagnosis of ovarian endometriosis cyst was 0.952. The AUC of RPR predicting the stage of ovarian endometriosis cyst was 0.713, higher than PLR and SII, lower than CA125; the AUC of RPR+SII+CA125 in predicting the stage of ovarian endometriotic cyst was 0.825, with sensitivity of 68.7% and specificity of 85.7%. The AUC predicted by SII for recurrence of ovarian endometriotic cyst within 2 years after the operation was 0.803, higher than NLR, PLR, SIRI and CA125; the AUC of PLR+SII+CA125, sensitivity, specificity was 0.813, 81.5% and 73.0%, higher than SII. Conclusion: PLR, RPR and SII are related to the staging of ovarian endometriotic cyst, and SII has a certain predictive value for the recurrence of ovarian endometriotic cyst after surgery.

A meta-analysis of effects and safety of Tripterygium wilfordii polyglycoside in the treatment of IgA nephropathy.

OBJECTIVE: To evaluate the effects and safety of Tripterygium wilfordii polyglycoside (TWP) in the treatment of immunoglobulin A (IgA) nephropathy. SUBJECTS AND METHODS: A computer-assisted study search of Chinese Biomedical Database (CBM), Chinese Journal Full-text Database (CNKI), Wanfang Database, Chinese Scientific Journal Database (VIP), PubMed, Medline, EMBASE and Cochrane Library was performed, with the time range of retrieval set between the establishment of the database to December 31, 2019. Articles of randomized controlled trials on the treatment of IgA nephropathy by Tripterygium wilfordii polyglycoside were collected, and then screened according to the inclusion and exclusion criteria. Next, the quality of the papers was assessed, effective data were extracted, and a meta-analysis of the included studies was conducted using the Review Manager 5.3 software provided by the Cochrane Collaboration. RESULTS: Thirty randomized controlled trials (RCT) were included ultimately, and the meta-analysis showed that 1) Single (Sgl) TWP group was superior to angiotensin-converting enzyme inhibitor/angiotension receptor blocker (ACEI/ARB) group in terms of complete remission [odds ratio (OR) = 4.74, p-value < 0.00001], total remission (OR = 3.90, p-value < 0.0001), 24-hour proteinuria [mean difference (MD) = 1.18, p-value < 0.00001], and serum albumin (MD = - 8.23, p-value < 0.00001), and no significant difference in serum creatinine (MD = 2.09, p-value = 0.08) was found between Sgl TWP and control groups; TWP + ACEI/ARB group was superior in complete remission (OR = 2.57, p-value < 0.00001), total remission (OR = 4.36, p-value < 0.00001), serum albumin [standardized mean difference (SMD) = -0.68, p-value = 0.0005], 24-hour proteinuria (SMD = 1.24, p-value < 0.00001) and serum creatinine (SMD = 0.48, p-value = 0.006); 2) TWP group was superior to glucocorticoid group in complete remission (OR = 1.93, p-value < 0.0010), total remission (OR = 3.71, p-value < 0.00001), serum albumin (MD = -3.50, p-value = 0.002), 24-hour proteinuria (SMD = 0.93, p-value < 0.0001) and serum creatinine (SMD = 0.88, p-value = 0.006); 3) TWP group was better than mycophenolate mofetil (MMF) group in complete remission (OR = 2.05, p = 0.005), total remission (OR = 3.30, p-value = 0.002), 24-hour proteinuria (MD = 2.61, p-value < 0.0001), and serum albumin (MD = -6.43, p-value < 0.00001), but the differences in serum creatinine (MD = 1.28, p-value = 0.89) between TWP and control groups were not significant. Besides, TWP + ACEI/ARB group had a higher adverse reaction rate than the control group (OR = 2.21, p-value = 0.04), but there was no significant difference in the adverse reaction rate between other control and experimental groups (p-value > 0.05). CONCLUSIONS: The present evidence shows that Tripterygium wilfordii polyglycoside can effectively improve the remission rate, reduce proteinuria, and protect kidney function of IgA nephropathy patients, and also has good safety. However, limited by the quality of the included studies, the effects and safety of Tripterygium wilfordii polyglycoside in the treatment of IgA nephropathy need to be verified by more high-quality, large-scale, multi-center RCTs.

[Interpretation of the management of type 1 diabetes in adults: a consensus report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) in 2021].

The American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) convened a writing group to develop a consensus report on the management of type 1 diabetes in adults in September 2021. The writing group has proposed principles of the diagnosis and management of adult patients with type 1 diabetes, and has made suggestions for glycemic control with individualized glycemic targets to avoid hypoglycemia. They have also emphasized the importance of education and support for the self-management of diabetes in the management of type 1 diabetes.

Low expression of CircRNA HIPK3 promotes osteoarthritis chondrocyte apoptosis by serving as a sponge of miR-124 to regulate SOX8.

OBJECTIVE: CircRNA, a type of circular RNA, has recently been shown to be a potential target for osteoarthritis (OA). Circular RNA HIPK3 (CircHIPK3) is reported to be abnormally expressed in various disease tissues and affects the occurrence and development of the disease. However, the role and underlying mechanism of CircRNA HIPK3 in osteoarthritis are still unclear. The purpose of this study is to explore the effect of CircRNA HIPK3 on osteoarthritis and analyze its regulatory mechanism. PATIENTS AND METHODS: We took human OA tissues, normal knee cartilage, human OA chondrocytes and normal chondrocytes as the research objects. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was used to detect CircHIPK3 expression level, its target gene miRNA 124 (miR-124) and downstream target molecule SOX8. Flow cytometry analysis was applied to discover the apoptosis of CircHIPK3 and miR-124 on OA cartilage in different transfection situations. Moreover, Western blot and RT-qPCR were used to detect the expression of caspase-3 in OA chondrocytes. The binding site of CircRNA HIPK3 and miR-124, miR-124, and SOX8 were verified by using Dual-Luciferase assay. RESULTS: High expressed CircHIPK3 and low expressed miR-124 were found in OA tissues and OA chondrocytes. In addition, the Dual-Luciferase report showed CircHIPK3 acted as a sponge of miR-124 in OA chondrocytes. CircHIPK3 and miR-124 expression in OA tissue were confirmed to be negatively correlated. To our surprise, knocking down CircHIPK3 and transfected miR-124 mimics both inhibited the apoptosis of OA chondrocytes. Further experiments verified that the downstream target molecule of miR-124 was SOX8 in OA chondrocytes. Besides, miR-124 inhibitors reversed the knockdown of CircHIPK3 while si-SOX8 reversed the miR-124 inhibitors effect of apoptosis on OA chondrocytes. CONCLUSIONS: Our results demonstrated that CircHIPK3 was significantly upregulated in OA cartilage tissue and cells. Low expression of CircHIPK3 promoted the apoptosis of OA chondrocytes by promoting miR-124 to suppress SOX8 expression. The molecular mechanism of CircHIPK3 in present study is expected to provide new ideas for the treatment of osteoarthritis.

MicroRNA-124 regulates lactate transportation in the muscle of largemouth bass (micropterus salmoides) under hypoxia by targeting MCT1.

Carbohydrate metabolism switches from aerobic to anaerobic (glycolysis) to supply energy in response to acute hypoxic stress. Acute hypoxic stress with dissolved oxygen (DO) levels of 1.2 ±â€¯0.1 mg/L for 24 h and 12 h re-oxygenation was used to investigate the response of the anaerobic glycolytic pathway in Micropterus salmoides muscle. The results showed that the glucose concentration was significantly lower in muscle, while the lactic acid and pyruvic acid concentrations tended to increase during hypoxic stress. No significant difference was observed in muscle glycogen, and ATP content fluctuated significantly. The activities of gluconeogenesis-related enzymes were slightly elevated, such as phosphoenolpyruvate carboxykinase (PEPCK). The activities of the glycolytic enzymes increased after the induction of hypoxia, such as hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH). Curiously, phosphofructokinase (PFK) activity was significantly down-regulated within 4 h during hypoxia, although these effects were transient, and most indices returned to control levels after 12 h of re-oxygenation. Upregulated hif-1α, ampkα, hk, glut1, and ldh mRNA expression suggested that carbohydrate metabolism was reprogrammed under hypoxia. Lactate transport was regulated by miR-124-5p according to quantitative polymerase chain reaction and dual luciferase reporter assays. Our findings provide new insight into the molecular regulatory mechanism of hypoxia in Micropterus salmoides muscle.

Pyruvate kinase genes in grass carp Ctenopharyngodon idella: molecular characterization, expression patterns, and effects of dietary carbohydrate levels.

To explore features of carbohydrate metabolism and evolution of carbohydrate metabolism-associated genes in herbivorous fishes, the open reading frames (ORF) of PKL, PKMa, and PKMb genes of grass carp (Ctenopharyngodon idella) were obtained, encoding 538, 528, and 532 amino acids, respectively. Comparative genomic analysis showed that adjacent PK genes were highly conserved between fish and mammals. Gene expression profiles were quite different between the three PK genes in tissues and at developmental stages. PKL, PKMa, and PKMb had the highest expression levels in the liver, heart, and muscle, respectively. During embryogenesis, high expression levels of PKMa and PKMb were detected in unfertilized and fertilized eggs. Following a non-expression period, PKMa and PKMb exhibited high expressions again after the hatching stage. In contrast, PKL transcripts could not be detected in early developmental stages, and expression levels continued to increase from the hatching stage to 144 h post hatching. After the 8-week feeding trial with 18%, 30%, and 42% dietary carbohydrate levels, the concentrations of glucose and insulin in serum, pyruvate kinase enzymes, and gene expression levels in brain, muscle, and liver tissues all increased with the increase in carbohydrate levels in the diets. Furthermore, high carbohydrate levels (30% and 42% carbohydrate diets) had a greater effect on grass carp growth. This indicated that PKL, PKMa, and PKMb genes were not only very important in catalytic enzymes, which can be up-regulated by high carbohydrate dietary conditions, but also exhibited a complex and detailed division of labor in different tissues and developmental stages.

[Role and mechanism of the regulation of nuclear factor-κB by heparin binding-epidermal growth factor-like growth factor in the induction of paclitaxel resistance of ovarian cancer].

Objective: To investigate the role and mechanism of the regulation of nuclear factor-κB (NF-κB) by heparin binding-epidermal growth factor-like growth factor (HB-EGF) in paclitaxel resistance of ovarian cancer in vitro and in vivo. Methods: (1) The detection of NF-κB expression: parental (A2780) and paclitaxel-resistant (A2780/Taxol) ovarian carcinoma cells were divided into four groups, named A2780 group, A2780+cross-reacting material 197 (CRM197, HB-EGF inhibitor) group, A2780/Taxol group and A2780/Taxol+CRM197 group. Among four groups, the expression level HB-EGF and epidermal growth factor receptor (EGFR) were examined by immunofluorescence double staining on confocal microscopy. Western blot was used to detect the expression level of NF-κB. In vivo, A2780 and A2780/Taxol cells were injected intraperitoneally to nude mouse to determine the expression level of NF-κB of the tumors from these four groups by immunohistochemistry method. (2) The detection on the function of NF-κB: A2780/Taxol cells were divided into four groups, named transfected with empty vector+saline group, NF-κB small interference RNA (siRNA)+saline group, empty vector+CRM197 group and NF-κB siRNA+CRM197 group respectively. Among four groups, the 50% inhibitory concentrations (IC(50)) of A2780/Taxol cells to paclitaxel, the expression level of plasma membrane glycoprotein (P-gp) and the effect of intracellular rhodomine123 (Rh123) accumulation were detected. Results: (1) The detection of NF-κB expression: the expression scores of HB-EGF protein among four groups were 5.6±1.3, 2.1±1.2, 11.7±3.5 and 6.2±1.4; the expression scores of EGFR protein were 5.1±1.6, 2.8±0.6, 10.4±3.1 and 5.6±1.9, respectively. The expression levels of NF-κB protein in the cells of the group named A2780, A2780+CRM197, A2780/Taxol and A2780/Taxol+CRM197 group were 1.89±0.23, 0.74±0.12, 3.45±0.16 and 1.31±0.08, respectively; the expression scores of NF-κB protein in the tissue tumors from four groups were 3.3±1.1, 1.4±0.4, 8.7±2.3 and 3.6±1.2, respectively. The expression level of HB-EGF, EGFR and NF-κB protein between A2780 and A2780/Taxol groups in vivo and in vitro were higher than these in A2780+CRM197 and A2780/Taxol+CRM197 group, while the expression level of HB-EGF, EGFR and NF-κB protein in A2780 group were lower than those in A2780/Taxol groups in vivo and in vitro (P<0.05). (2) The examination of NF-κB function: the IC(50) of A2780/Taxol cells to paclitaxel in groups transfected with empty vector+saline, NF-κB siRNA+saline, empty vector+CRM197 and NF-κB siRNA+CRM197 group were respectively (39.4±0.8), (7.6±0.6), (6.7±0.5) and (4.2±0.4) µmol/L, while the expression levels of P-gp protein among four groups were respectively 3.11±0.23,1.45±0.16, 1.73±0.21 and 0.68±0.14, the cellular Rh123 accumulation among four groups were respectively 110±15, 246±19, 231±22 and 296±24. The expression levels of IC(50) and P-gp protein in groups transfected with NF-κB siRNA+saline, empty vector+CRM197 and NF-κB siRNA+CRM197 group were significantly higher than those in group transfected with empty vector+saline group (P<0.01), while the cellular Rh123 accumulation among three groups were significantly lower than that in group transfected with empty vector+saline (P<0.01). Conclusions: The expression of NF-κB may contributes to the paclitaxel resistance to ovarian cancer. HB-EGF may induce the paclitaxel resistance of ovarian cancer by the regulation of EGFR/NF-κB/P-gp pathway.

Application of Specific Fragment Length Polymorphism of Algae rDNA in Identification of Drowning Cases.

OBJECTIVES: To identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death by detecting part of 5.8S sequence and second internal transcribed spacer （ITS2） （5.8S+ITS2） of diatom rDNA in water and organs. METHODS: Two cases identified by diatom examination, which received by Nanjing Municipal Public Security Bureau Forensic Center, were taken as the research objects. The difference of the population structure of algae in water and human tissue was analysed by length polymorphism of 5.8S+ITS2 marker. RESULTS: In case 1, similar species of diatom were detected from victim's lung and liver tissues and the water sample. Two kinds of DNA fragments with length of 330 bp and 376 bp were detected from victim's lung tissue and the water sample using 5.8S+ITS2 marker, which could confirm the victim was drowning before death. In case 2, there was no diatom found in victim's lung and liver tissues. Only one kind of DNA fragment with length of 331 bp and low relative fluorescence unit （RFU） was obtained from victim's lung tissue using 5.8S+ITS2 marker, thus the victim was thrown into the water after death. CONCLUSIONS: The experimental results of the two cases in present study are consistent with the actual facts and the result of the diatom microscopic examination. The difference of population structure of specific microorganism in water and human tissue can be detected by 5.8S+ITS2 marker, which can help to identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death.

[Correlation between Genetic Variants and Polymorphism of Caveolin and Sudden Unexplained Death].

OBJECTIVES: To explore the genetic variation sites of caveolin （CAV） and their correlation with sudden unexplained death （SUD）. METHODS: The blood samples were collected from SUD group （71 cases）, coronary artery disease （CAD） group （62 cases） and control group （60 cases）, respectively. The genome DNA were extracted and sequencing was performed directly by amplifying gene coding region and exon-intron splicing region of CAV1 and CAV3 using PCR. The type of heritable variation of CVA was confirmed and statistical analysis was performed. RESULTS: A total of 4 variation sites that maybe significative were identified in SUD group, and two were newfound which were CAV1： c.45C>T （T15T） and CAV1：c.512G>A （R171H）, and two were SNP loci which were CAV1：c.246C>T （rs35242077） and CAV3：c.99C>T （rs1008642） and had significant difference （P<0.05） in allele and genotype frequencies between SUD and control groups. Forementioned variation sites were not found in CAD group. CONCLUSIONS: The variants of CAV1 and CAV3 may be correlated with a part of SUD group.

Von Willebrand factor and ADAMTS13 plasma in older patients with high CHA2DS2-VASc Score with and without atrial fibrillation.

OBJECTIVE: Ischemic stroke risk rises with the increasing cardiovascular risk factors in patients with and without AF. How atrial fibrillation (AF) incrementally contributes to the risk for ischemic stroke with increasing age and multiple cardiovascular risk factors is unclear. Von Willebrand factor (vWF) is a biomarker of endothelial dysfunction. PATIENTS AND METHODS: We suggested that in older patients with high CHA2DS2-VASc Score, the vWF and ADAMTS13 would be comparable between patients with and without AF. Consecutive 196 old patients (≥ 60 years, 45.9% with concomitant AF) with and without non-valve atrial fibrillation were recruited from April 2014 to April 2016. Data on baseline clinical characteristics were recorded at study entry. Plasma ADAMTS13 levels and plasma vWF levels were determined. Statistical analyses were performed using SPSS19.0 statistical software package. RESULTS: There were significant correlations between plasma vWf levels, ADMATS13 and CHA2DS2- VASc Score in older patients with and without AF (with AF: Spearman, r = 0.215, p < 0.05; without AF: Spearman, r = 0.197, p < 0.05). Results of research indices in our older patients were as follows: vWf 180. 79 ± 28.27 IU/dL in AF and 153.5 ± 35.54 in non AF with p < 0.001, ADAMTS13 431.5 ± 160.33 IU/dL in AF and 536.7 ± 169.96 in non AF with p < 0.05. Results of research indices in our older patients (≥ 75 year) were as follows: vWf 181.4 ± 22.04 in AF and 174.1 ± 29.45 in non AF, and ADMATS-13 412.9 ± 130.76 IU/dL in AF and 451.7 ± 153.18 in non AF. There were no differences (p > 0.05). CHA2DS2-VASc Score can predict stroke risk in old patients without atrial fibrillation. At high CHA2DS2-VASc Score, the levels of vWF and ADAMTS13 have difference in old patients (60-74) with and without AF, but in such older patients, age (≥ 75 year), there were no differences. In elderly patients, atrial fibrillation has a limited effect on VWF, and the age is an important factor affecting the endothelial function. CONCLUSIONS: For elderly patients with a high incidence rate of stroke and thrombosis, we should pay more attention to the thrombotic events, and atrial fibrillation can be used as one of the risk factors involved and improving the risk scoring system of stroke.

[Reverse of the resistance to paclitaxel of the heparin binding-epidermal growth factor-like growth factor inhibitor in ovarian cancer].

Objective: To investigate the effect and mechanism of CRM197, the heparin binding-epidermal growth factor-like growth factor (HB-EGF) inhibitor, on the reverse of the resistance of ovarian cancer to paclitaxel. Methods: (1)The effect of CRM197 on the 50% inhibitory concentrations (IC(50)) of human ovarian carcinoma cell line A2780 and paclitaxel-resistant ovarian carcinoma cell line A2780/Taxol was tested by methyl thiazolyl tetrazolium (MTT) assay. Western blot was used to detect the effect of CRM197 on the expression of HB-EGF, epidermal growth factor receptor (EGFR) and plasma membrane glycoprotein (P-gp) protein in A2780 and A2780/Taxol cells. Real-time PCR was used to examine the MDR1 mRNA expression in these cells. (2) A2780/Taxol cells were divided into 4 groups, including the cells transfected with empty vector and saline treatment (empty vector group), MDR1 small interference RNA (siRNA) vector and saline treatment (MDR1 siRNA group), empty vector and CRM197 treatment (empty vector+CRM197 group) and MDR1 siRNA vector and CRM197 treatment (MDR1 siRNA+CRM197 group), respectively. Flow cytometry was used to detecte the effect of intracellular rhodomine 123 (Rh123) accumulation, and caspase-3 activity assay was used to test the effect of apoptosis in four groups of A2780/Taxol cells. (3) In experiments in vivo, A2780/Taxol cells were inoculated to nude mouse subcutaneously to determine the EGFR and P-gp protein expression following CRM197 treatment by immunohistochemistry. Results: (1) In vitro, MTT examination showed that the IC(50) of A2780/Taxol cells to paclitaxel in A2780/Taxol+CRM197 group [(6.4±0.3) µmol/L] was significantly lower than the IC(50) in A2780/Taxol group [ (34.1±0.5) µmol/L, P<0.01], and the reveral fold of CRM197 was 5.3. The expression level of HB-EGF protein in A2780/Taxol+CRM197 group (1.44±0.29) was significantly lower than HB-EGF protein in A2780/Taxol group (2.72±0.32), respectively (P<0.05). The expression level of EGFR protein (0.71±0.25) and P-gp protein (0.82±0.19) in A2780/Taxol+CRM197 group was significantly lower than EGFR protein (1.87±0.31) and P-gp protein (1.84±0.27) of A2780/Taxol group (P<0.05). Compared with A2780/Taxol group (1.78±0.27) , MDR1 mRNA was significantly down-regulated in A2780/Taxol+CRM197 group (0.79±0.13, P<0.05). (2) The fluorescence intensity of Rh123 of the A2780/Taxol cells in empty vector group, MDR1 siRNA group,empty vector+CRM197 group, MDR1 siRNA+CRM197 group was 33.4±1.6, 56.3±3.3, 43.5±3.1,100.4±7.4, and the pNA of the A2780/Taxol cells was (11.4±1.2) , (52.8±0.9) , (71.2±3.6) , (82.7±3.8) µmol/L. The expression levels in MDR1 siRNA+CRM197 group were both higher than the expression levels in empty vector+CRM197 group, and the expression levels in empty vector+CRM197 group, MDR1 siRNA group were both higher than the expression levels in empty vector group (P<0.05). (3) In vivo, the expression scores of EGFR protein in A2780/Taxol+CRM197 tumors (4.4±1.4) were lower than that in A2780/Taxol tumors (10.2±3.1, P<0.05). The expression scores of P-gp protein in A2780/Taxol+CRM197 tumors (3.8±1.1) were lower than that in A2780/Taxol tumors (8.8±2.7, P<0.05). Conclusion: CRM197 reverses the resistance of ovarian cancer to paclitaxel by increasing caspase-3 activity to advance apoptosis via EGFR/MDR1/P-gp pathway.

[Prenatal diagnosis of chromosome abnormalities and nine microdeletion syndromes using both traditional karyotyping and BoBs].

OBJECTIVE: To evaluate a new prenatal diagnosis model of chromosomal abnormalities and nine microdeletion syndromes by using both traditional karyotyping and a newly-developed rapid prenatal diagnosis technology, BACs-on-Beads (BoBs) technique. METHODS: From June 2012 to December 2014, 807 pregnant women with high risk after screening or with other indicators, were performed amniocentesis. Traditional karyotyping and BoBs were employed simultaneously for prenatal diagnosis. RESULTS: Thirty-two cases with chromosome aneupoidies were successfully detected both by BoBs and karyotyping, including 18 cases of trisomy 21, 6 cases of trisomy 18, 1 case of trisomy 13, and 7 cases with sex chromosome abnormality. All 8 fetuses with chromosome structural abnormalities detected by karyotyping were missed by BoBs; while BoBs contributed more in detection of five microdeletion syndrome cases, including 3 cases of DiGeorge syndromes (two with microduplication and one with microdeletion), one case of Miller-Dieker syndrome, and one case of Wolf-Hirschhorn syndrome. CONCLUSION: Combined use of traditional karyotyping and BoBs, is a rapid and effective prenatal diagnosis model that may enlarge our horizon on chromosomal diseases and should be widely used in future clinical service.

[Outcomes of total deafness type of idiopathic suddendeafness in different ages].

Objective:This retrospective study was aimed to investigate the characteristics of hearing recovery in the complete deafness type of SSNHL(≥81 dBHL) in patients with different ages.Method:Clinical outcomes of 179 total deafness type of idiopathic sudden deafness were compared.Patients were divided into 5 groups according to age,they were,pediatric group(13 years or less),youthful group(14-44 years),middle-aged group(45-59 years),presenium group(60-74 years),senectitude group(75 years or higher).Patients were divided into 3 groups according to the initial degree of hearing loss: 81 dB group (81-89 dBHL),90 dB group(90- 99 dBHL),100 dB group(100 dBHL or higher).Routine comprehensive treatment including corticosteroids,the inner ear microcirculation improvement drugs,neurotrophic drugs,saturationoxygen and hyperbaric oxygen therapy,etc.was applied.Patients were treated in accordance with the age and body weight.Result:The percentage of youthful group(83/179,46.4%) was highest(P<0.05),middle-aged group(57/179,31.8%)followed(P<0.05),presenium group(26/179,14.5%)was lower(P<0.05),pediatric group(8/179,4.5%) and senectitude group(5/179,2.8%)were the lowest.No a complete recovery in either pediatric group or senectitude group.A complete recovery was rare in the other groups.Recovery rate of the different aged groups was similar(P>0.05).The percentage of 100 dB group(108/179,60.3%) was highest(P<0.05).The percentage of 81 dB group(39/179,21.8%)was similar to 90 dB group(32/179,17.9%)(P>0.05).Recovery rate was similar in 81 dB group(25/39,64.1%)and 90 dB group(18/32,56.2%)(P>0.05).Recovery rate of both 81 dB group and 90 dB group were greater than 100 dB group(24/108,22.2%)(P<0.05).The 100 dB group reduced the satisfactory recovery effects.There were no differences in the proportion of the patients with dizziness(95/179,53.1%)and without dizziness(84/179,46.9%)(P>0.05).Recovery rate of patients without dizziness(43/84,51.2%) was greater than with dizziness(24/95,25.3%)(P<0.05).The percentage of the patients without dizziness(31/39,79.5%)in 81 dB group was the highest(P<0.05),90 dB group(18/32,56.2%)followed(P<0.05).The percentage of the patients with dizziness in 100 dB group(73/108,67.6%)was highest(P<0.05).Recovery rate was similar in the patients without dizziness of 81 dB group(21/31,67.7%)and 90 dB group(11/18,61.1%)(P>0.05).Recovery rate of the above two groups was greater than that of 100 dB group(11/35,31.4%)(P<0.05).Conclusion:Recovery rate of the different aged groups was similar.The percentage of the patients with dizziness in 100 dB group was highest.Initial hearing threshold in excess of 100 dB reduced the satisfactory recovery in patients with total deafness type of SSNHL.Our results provided a good reference for other clinicians.

Retinoic acid receptor ß2 agonists restore glycaemic control in diabetes and reduce steatosis.

AIMS: To investigate the effects of specific retinoic acid receptor (RAR) agonists in diabetes and fatty liver disease. METHODS: Synthetic agonists for RARß2 were administered to wild-type (wt) mice in a model of high-fat-diet (HFD)-induced type 2 diabetes (T2D) and to ob/ob and db/db mice (genetic models of obesity-associated T2D). RESULTS: We show that administration of synthetic agonists for RARß2 to either wt mice in a model of HFD-induced T2D or to ob/ob and db/db mice reduces hyperglycaemia, peripheral insulin resistance and body weight. Furthermore, RARß2 agonists dramatically reduce steatosis, lipid peroxidation and oxidative stress in the liver, pancreas and kidneys of obese, diabetic mice. RARß2 agonists also lower levels of mRNAs involved in lipogenesis, such as sterol regulatory element-binding transcription factor 1 (SREBP1) and fatty acid synthase, and increase mRNAs that mediate mitochondrial fatty acid ß-oxidation, such as CPT1α, in these organs. RARß2 agonists lower triglyceride levels in these organs, and in muscle. CONCLUSIONS: Collectively, our data show that orally active, rapid-acting, high-affinity pharmacological agonists for RARß2 improve the diabetic phenotype while reducing lipid levels in key insulin target tissues. We suggest that RARß2 agonists should be useful drugs for T2D therapy and for treatment of hepatic steatosis.

Mitochondrial modulation is involved in the hepatoprotection of Limonium sinense extract against liver damage in mice.

AIM OF THE STUDY: Limonium sinense (Girard) Ktze is a Chinese folk medicine used to treat fever, hemorrhage, hepatitis, and other disorders. The present research focused on the protective effects of L. sinense extracts (LSE) against liver damage. MATERIALS AND METHODS: In this study the extract from the root of Limonium sinense was used. Aminotransferase activity detection, electron microscopy, mitochondrial function evaluation, RT-PCR and western blot were used to evaluate the hepatoprotection of LSE in LPS/d-GalN-intoxicated mice. RESULTS: Pretreatment with 100, 200 or 400mg/kg LSE significantly blocked the increase in both serum aspartate aminotransferase (sAST) and serum alanine aminotransferase (sALT) levels induced by treatment with LPS plus d-GalN (LPS/d-GalN). Ultrastructural observation by electron microscopy showed reduced hepatocyte nuclear condensation and less lipid deposition. The decrease in both the mitochondrial membrane potential (14.6%) and sensitivity to mitochondrial swelling induced by Ca(2+) (45.9%) observed in the liver of LPS/d-GalN-treated mice were prevented by pretreatment with LSE. In addition, different doses of LSE increased both the transcription and the translation of voltage-dependent anion channels (VDAC), which was down-regulated by LPS/d-GalN treatment. CONCLUSIONS: In summary, LSE protects livers against LPS/d-GalN-induced damage, possibly by mitochondrial mechanisms related to increased expression of VDAC.

A comparative approach to somatic cell nuclear transfer in the rhesus monkey.

BACKGROUND: Despite the potential utility of primate somatic cell nuclear transfer (SCNT) to biomedical research and to the production of autologous embryonic stem (ES) cells for cell- or tissue-based therapy, a reliable method for SCNT is not yet available. Employing the rhesus monkey as a clinically relevant animal model, we have compared a conventional electrofusion method for SCNT with a one-step micromanipulation (OSM) method. METHODS: A prospective, randomized trial was conducted using only oocytes that were mature [metaphase II (MII)] at collection and a fibroblast-like cell line as nuclear donor cells (fetal fibroblasts). The embryos produced were characterized for in vitro developmental potential, cell number, karyotype and expression of nuclear mitotic apparatus (NuMA) and OCT-4. RESULTS: An in vitro blastocyst development rate of 24.4% was achieved with the OSM method, significantly higher than the 12.2% obtained following electrofusion. SCNT-produced embryos expressed normal karyotypes, cell numbers and NuMA and OCT-4 proteins in most cases. SCNT with male nuclear donor cells resulted in the production of male, SCNT blastocysts, eliminating the possibility of a parthenogenetic origin. Of the four fibroblast cell lines tested as nuclear donor cells, two supported the routine production of blastocysts following SCNT. CONCLUSIONS: The application of a modified SCNT technique (OSM) followed by embryo culture in hamster embryo culture medium-10 (HECM-10) allows, for the first time, the routine production of SCNT blastocysts, most of which appear normal by immunochemical, cytochemical and in vitro developmental criteria. These embryos will provide a resource for isolating ES cells and for studies of nuclear reprogramming by monkey cytoplasts.