[Effect of adenovirus-mediated shRNA down-regulates SHP2 expression on the apoptosis of human hepatic stellate cells LX-2].

Objective: To investigate the effect of adenovirus-mediated short hairpin RNA (shRNA) downregulating SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) on the apoptosis of human hepatic stellate cells LX-2 cultured in vitro. Methods: The recombinant adenovirus Ad-shRNA/SHP2 carrying shRNA targeted SHP2 and expressing green fluorescent protein (GFP), and the empty control virus Ad-GFP expressing GFP were transfected into LX-2 cells cultured in vitro. Real-time fluorescence quantitative PCR was used to detect SHP2 mRNA expression in LX-2 cells. Western blot was used to detect the protein expressions of SHP2, Bax, and Bcl-2 in LX-2 cells. TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry were used to detect apoptosis in LX-2 cells. Experimental group: (1) Control group: LX-2 cells were transfected with DMEM instead of adenovirus; (2) Ad-GFP group: transfected with empty virus Ad-GFP; (3) Ad-shRNA/SHP2 group: transfected with recombinant adenovirus Ad-shRNA/SHP2. The means between multiple groups were compared using a one-way ANOVA and the LSD test was used for inter group comparisons. Results: shRNA-targeted SHP2 significantly down-regulated the expression of SHP2 protein and mRNA in LX-2 cells (P < 0.05). The TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry results showed that the apoptosis rate of LX-2 cells in the Ad-shRNA/SHP2 group (12.755%±1.606%, 19.340%±2.505%) (P < 0.05) was significantly higher compared to the control group (3.077%±0.731%, 9.438%±0.804%) and the Ad-GFP group (3.250%±0.851%, 8.893%±1.982%), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). Western blot analysis of Bax and Bcl-2 protein expression in LX-2 cells of each group revealed that the Bax protein expression was significantly higher in the Ad shRNA/SHP2 group (2.493 ± 0.203) (P < 0.05) compared to the control group and Ad-GFP group (1.989 ± 0.147, 1.999 ± 0.162), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05), while the Bcl-2 protein was significantly decreased in the Ad-shRNA/SHP2 group (1.042±0.148) compared with the control group and the Ad-GFP group (1.707±0.146, 1.521±0.142), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). Conclusions: SHP2 expression down-regulation induces apoptosis of human hepatic stellate cells LX-2 in vitro by reducing Bcl-2/Bax.

[Phenotypes and ATP7B gene variants in 316 children with Wilson disease].

Objectives: To summarize the clinical phenotypes and the variation spectrum of ATP7B gene in Chinese children with Wilson's disease (WD) and to investigate their significance for early diagnosis. Methods: Retrospective analysis was performed on the clinical data of 316 children diagnosed as WD in Guangzhou Women and Children's Medical Center during the period from January 2010 to June 2021. The general situations, clinical manifestations, lab test results, imaging examinations, and ATP7B gene variant characteristics were collected. The patients were divided into asymptomatic WD group and symptomatic WD group based on the presence or absence of clinical symptoms at the time that WD diagnosis was made. The χ2 test, t test or Mann-Whitney U test were used to compare the differences between groups. Results: Among the 316 children with WD, 199 were males and 117 were females, with the age of 5.4 (4.0, 7.6) years at diagnosis; 261 cases (82.6%) were asymptomatic with the age of 4.9 (3.9, 6.4) years; whereas 55 cases (17.4%) were symptomatic with the age of 9.6 (7.3, 12.0) years. The main symptoms invloved liver, kidney, nervous system, or skin damage. Of all the patients, 95.9% (303/316) had abnormal liver function at diagnosis; 98.1% (310/316) had the serum ceruloplasmin lever lower than 200 mg/L; 97.7% (302/309) had 24-hour urine copper content exceeding 40 µg; only 7.4% (23/310) had positive corneal K-F rings, 8.2% (23/281) had abnormal MRI signals in the lenticular nucleus, and all of them had symptoms of damage in liver, kidney or nervous system. Compared with the group of symptomatic WD, asymptomatic group had higher levels of serum alanine aminotransferase and lower levels ceruloplasmin and 24-hour urine copper [(208±137) vs. (72±78) U/L, (55±47) vs. (69±48) mg/L, 103 (72, 153) vs. 492 (230, 1 432) µg; t=9.98, -1.98, Z=-4.89, all P<0.001]. Among the 314 patients completing genetic sequencing, a total of 107 mutations in ATP7B gene were detected, of which 10 are novel variants, and 3 cases (1.0%) had large heterozygous deletion (exons 10 to exon 11) in ATP7B gene. The percentage of missense mutation in asymptomatic WD children was significantly higher than that in symptomatic WD (81.5% (422/518) vs. 69.1% (76/110), χ²=8.47, P<0.05). WD patients carrying homozygous variant of c.2 333G>T had significantly low levels of ceruloplasmin than those not carrying this variant ((23±5) vs. (61±48) mg/L, t=-2.34, P<0.001). Conclusions: The elevation of serum ALT is an important clue for early diagnosis of WD in children, while serum ceruloplasmin and 24-hour urine copper content are specific markers for early diagnosis of WD. In order to confirm the diagnosis of WD, it is necessary to combine the Sanger sequencing with multiplex ligation-dependent probe amplification or other testing technologies.

[Effects of adenovirus-mediated shRNA down-regulates PTEN expression on fibril-binding proteins vinculin, filamin A and cortactin in activated hepatic stellate cells].

Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.

MiR-193 promotes cell proliferation and invasion by ING5/PI3K/AKT pathway of triple-negative breast cancer.

OBJECTIVE: Triple-negative breast cancers (TNBC) are a subtype of breast cancer lacking of estrogen receptor (ER), progesterone receptor (PR), and human EGF-like receptor 2 (HER2). MiR-193 always acted as an oncogene and promoted toxic aldehyde accumulation and tyrosine hydroxylase dysfunction. The purpose of this study is to explore the function of miR-193 in triple-negative breast cancer. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the mRNA level of miR-193 expression in 50 cases of TNBC tissues and para-cancerous specimens. Also, the relation between miR-193 level and the overall survival of TNBC patient was analyzed. MiR-193 mimic and miR-193 inhibitor oligos, as well as the corresponding negative control, were synthesized from RiboBio (Guangzhou, China). RESULTS: MiR-193 expression was higher in triple-negative breast cancer tissues and cell lines than the corresponding adjacent non-tumor tissues and normal cell lines. Upregulation of miR-193 predicted poor prognosis of TNBC patients. Overexpression of miR-193 promoted cell proliferation and invasion, while that was suppressed by the knockdown of miR-193. MiR-193 binds to the 3'-UTR of an inhibitor of growth family member 5 (ING5) mRNA to mediate the expression of ING5 in TNBC cells. The knockdown of miR-193 inhibited cell invasion-mediated epithelial-mesenchymal transition (EMT). Furthermore, the knockdown of miR-193 suppressed cell proliferation through the ING5/phosphatidylinositol 3-hydroxy kinase/protein kinase B (PI3K/AKT) signal pathway. CONCLUSIONS: MiR-193 enhanced cell invasion-mediated EMT and improved cell proliferation through the ING5/PI3K/AKT signal pathway in triple-negative breast cancer. The newly identified miR-193/ING5/PI3K/AKT axis provides novel insight into the pathogenesis of triple-negative breast cancer.

[Performance of combined liquid based cytology and HPV nucleic acid test for detecting cervical precancer among women attending screening].

Objective: To evaluate the clinical performance of HPV genotyping with cytology for detecting cervical precancer among women attending co-testing. Methods: A total of 2 883 females who participated in cervical cancer screening program were recruited from Erdos in 2016. All the participants were tested by cytology and HPV genotyping. In 2017, women with abnormal cytology results or HPV positive were followed up. Pathological cervical intraepithelial neoplasia (CIN) 2+ was the study end-point. Clinical performance indexes were calculated, including sensitivity, specificity, positive predictive value, negative predictive value, referral rate and missed cases. Results: INNO-LiPA resulted in a detection rate of 18.87%(544/2 883) for the 14-type high risk HPV. HPV16 was the most common infectious genotype (4.06%), followed by HPV52 (3.61%), HPV51 (2.50%), HPV58 (1.98%), and HPV18 (1.56%). With more HPV genotypes added into the group, sensitivity increased and the specificity decreased. Addition of HPV16, 58, 33, 39, 52, 18, 31 for detection lead to the maximun value of area under the curve (AUC)=0.913 (95%CI: 0.882-0.944). Compared with traditional screening method by cytology, cotesting decreased the number of missed diagnosis. Meanwhile, the fifth method (co-testing: triage of women with HPV16/18+ , cytological minor abnormalities and HPV58, 33, 39, 52, 31+ or cytological high grade abnormalities) did not increase referral rate (8.99% vs. 8.71%, P=0.525), with five cases of missed diagnosis (sensitivity of 92.1% and specificity of 93.2%). Conclusions: Co-testing with triage of women with HPV16/18+ , cytological minor abnormalities and HPV58, 33, 39, 52, 31+ or cytological high grade abnormalities would provide better clinical performance. In co-testing, triage of HPV16/18 was used in women with normal cytology; triage of HPV58, 33, 39, 52 and 31 was used in women with low-grade abnormal cytology; referral colposcopy was used in women with high-grade abnormal cytology, which would provide better clinical performance.

[The bacterial flora and drug sensitivity of chronic tonsillitis in different age groups].

Objective:To investigate the pathogenic bacteria and drug sensitivity tests in patients with chronic tonsillitis.Method：We chose a group of patients who were diagnosed chronic tonsillitis as the research object. According to age, they were divided into the children group, the adolescents group and the adults group. Collect secretions of tonsil in the operation, then summarize and analyze the secretions.Result: The detection rate of gram-negative bacteria in adult group was significantly higher than that of children and adolescents groups.Conclusion: Broad-spectrum antibiotics should be preferred in adult patients. While others should choose the antibiotic that is sensitive to gram positive bacterium first, before the pathogenic bacteria and drug sensitivity tests. The multi-drug resistant bacterium infection can not be neglected.

[Association between high risk human papillomavirus DNA load and cervical lesions in different infection status].

Objective: To explore the relationship between high risk HPV (HR-HPV) DNA load and cervical lesions in HR-HPV single/ multiple infections. Methods: Two thousand six hundred and forty-six women from Shanxi, Henan and Xinjiang were recruited into a cervical cancer screening program. Cervical exfoliated cell specimens collected from all of the participants were detected by hybrid capture Ⅱ (HC2), cytological diagnosis was performed according to the Bethesda System, and pathological diagnosis was interpreted using cervical intraepithelial neoplasia (CIN) terminology.Totally 571 cervical specimens were selected and retested to ascertain the HPV types and single/ multiple infections by liner array, a PCR-based method. Semi-quantitative result of HR-HPV DNA load (pg/ml) was estimated by HR HC2.According to the taxonomy of "International Human Papillomavirus Reference Center" , 13 HR-HPVs, including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, which could be detected by HR HC2 were divided into 4 subgroups. Results: The positive rate of HR-HPV in normal cervix (436 cases), CIN1 (88 cases), CIN2+ (47 cases) group were 29.82%, 85.23% and 100%, respectively. The overall prevalence and median viral load increased coordinating with the pathological degree of cervical lesions (P<0.001). The positive rate and viral load of single infection with HR-HPV belongs to α9 species increased coordinating with the pathological degree of cervical lesions (P<0.05). The viral load of single infection with HR-HPV belongs to α7 species in CIN1 group was higher than those of normal group and CIN2+ group, but without statistical significance (P=0.130). The viral load of multiple infections in CIN1 group was 559.13 pg/ml, significantly higher than 37.73 pg/ml of normal histology (P=0.025), but without significant difference of 332.91 pg/ml of CIN2+ group (P=0.790). The median viral load of HPV single infection in CIN1 group was 167.93 pg/ml, significantly lower than 559.73 pg/ml of multiple infections (P=0.044). The incidence of co-infection with HR-HPVs belong to α9 species was 80.56%, dominated in all patterns of multiple infections and their median viral load increased coordinating with the pathological degree of cervical lesions, but without significant difference (P>0.05). The incidence of co-infection with HR-HPVs belong to α7 species was 66.67%, their median viral load in CIN1 group was higher than that of CIN2+ group, but without statistical significance (P>0.05). Conclusions: Viral loads of single/ multiple infections with HR-HPVs belong to different species show different tendencies coordinating with the pathological degree of cervical lesions. Women with high grade of cervical lesion were dominantly infected with high viral load of HR-HPVs belong to α9 species, and the viral load of multiple infections is higher than that of single infection in low grade of cervical lesion.

[Application of isothermal HPV DNA amplification test in cervical cancer screening].

Objective: To evaluate the feasibility and effectiveness of isothermal human papillomavirus (HPV) DNA amplification test as a primary screening test in the early detection of cervical cancer. Methods: From June to August 2016, 2, 774 women aged 30-64 years old from Inner Mongolia were recruited for cervical cancer screening. HPV DNA was detected by Isomega and cobas4800. INNO-LiPA HPV Genotyping Extra was served as a reference method for the cases whose results were inconsistent by using these two methods. Histological diagnosis was considered as a gold standard to estimate the effectiveness and accuracy of Isomega and cobas4800 for detecting CIN2 or greater. Results: The concordance of Isomega and cobas4800 was 94.84% (Kappa=0.82) for high risk HPV (HR-HPV), 99.68% (Kappa=0.95) for HPV16, 99.78% (Kappa=0.91) for HPV18 and 94.34% (Kappa=0.76) for other HR-HPV types. The concordances of Isomega and the reference were 99.71% (Kappa=0.96), 99.86% (Kappa=0.94) and 96.76% (Kappa=0.87) for HPV16, 18 and other HR-HPV, respectively, while the concordances of cobas4800 and the reference were 99.82% (Kappa=0.97), 99.86% (Kappa=0.94) and 97.51% (Kappa=0.90), respectively. The sensitivity and specificity of Isomega for detecting CIN2+ (including CIN2, CIN3 and squamous cell carcinoma) were 87.76% and 82.94%, respectively, while those of cobas4800 were 89.80% and 85.06%, respectively. Conclusions: The concordances of Isomega and cobas4800 is confident. These two methods can accurately detect the HPV16 and 18 genotyping, and have good sensitivity and specificity for clinical diagnosis and population screening of cervical cancer.

Respiratory signal prediction based on adaptive boosting and multi-layer perceptron neural network.

To improve the prediction accuracy of respiratory signals using adaptive boosting and multi-layer perceptron neural network (ADMLP-NN) for gated treatment of moving target in radiation therapy. The respiratory signals acquired using a real-time position management (RPM) device from 138 previous 4DCT scans were retrospectively used in this study. The ADMLP-NN was composed of several artificial neural networks (ANNs) which were used as weaker predictors to compose a stronger predictor. The respiratory signal was initially smoothed using a Savitzky-Golay finite impulse response smoothing filter (S-G filter). Then, several similar multi-layer perceptron neural networks (MLP-NNs) were configured to estimate future respiratory signal position from its previous positions. Finally, an adaptive boosting (Adaboost) decision algorithm was used to set weights for each MLP-NN based on the sample prediction error of each MLP-NN. Two prediction methods, MLP-NN and ADMLP-NN (MLP-NN plus adaptive boosting), were evaluated by calculating correlation coefficient and root-mean-square-error between true and predicted signals. For predicting 500 ms ahead of prediction, average correlation coefficients were improved from 0.83 (MLP-NN method) to 0.89 (ADMLP-NN method). The average of root-mean-square-error (relative unit) for 500 ms ahead of prediction using ADMLP-NN were reduced by 27.9%, compared to those using MLP-NN. The preliminary results demonstrate that the ADMLP-NN respiratory prediction method is more accurate than the MLP-NN method and can improve the respiration prediction accuracy.

Cargo-activated ATPase activity of kinesin.

We have measured the ATPase activity of squid optic lobe kinesin bound to polystyrene beads in the presence of microtubules. We find that there is a substantial increase (> 10-fold) in the microtubule-activated ATPase activity for bead-bound kinesin over free kinesin. We tentatively attribute such cargo-activated ATPase activity to the presence of a self-inhibited form of kinesin in solution, which becomes activated when bound to a bead in the presence of alpha-casein. Further experiments are underway to unravel this phenomenon and, in addition, to associate the traveling distance of beads with the observed ATPase rate to determine the average number of ATP consumed per kinesin-bead per micron of travel along microtubule.

Mechanics of myosin motor: force and step size.

How motor proteins induce mechanical movement at the molecular level has been a focus of biophysicists for a long time. While the whole picture is yet to be completely revealed, recent developments in looking at nanometer-scale movement with millisecond-time resolution driven by single motors have revealed important new details about the moving step size and amount of force generated per molecule.