A method for improving the accuracy of non-invasive prenatal screening by cell-free foetal DNA size selection.

Background Non-invasive prenatal screening (NIPS) using cell-free foetal DNA (cfDNA) has been widely used for identifying common foetal aneuploidies (e.g. trisomy 21 (T21), trisomy (T18) and trisomy 13 (T13)) in clinical practice. The sensitivity and specificity of NIPS exceeds 99%, but the positive prediction value (PPV) is approximately 70% (combined T21, T18 and T13). Thus, some 30% of pregnant women who have positive NIPS results are eventually identified as normal by amniocentesis. These women therefore must undertake needless invasive tests and risk miscarrying healthy babies because of false positive NIPS results. Methods In order to achieve higher accuracy, we amended the standard NIPS (s-NIPS) protocol with an additional cfDNA size selecting step in agarose-electrophoresis. The advantage of the new method (named e-NIPS) was validated by comparing the results of e-NIPS and s-NIPS using 114 retrospective cases selected from 15,930 cases. Results Our results showed that the foetal cfDNA fraction can be enriched significantly by a size selection step. With this modification, all 98 negative cases and 9 of 11 false positive cases of s-NIPS were correctly identified by e-NIPS, resulting in an increased PPV from 71% to 77%. Additionally, a simulation test showed that e-NIPS is more reliable than s-NIPS, especially when the foetal cfDNA concentration and sequencing coverage are low. Conclusion cfDNA size selection is an important step in improving the accuracy of non-invasive prenatal screening for chromosomal abnormalities.

Expression of nestin in ovarian serous cancer and its clinicopathologic significance.

OBJECTIVE: Nestin has been found to be overexpressed in several human malignancies. The primary goal of this research was to investigate whether nestin expression correlates with the occurrence and development of ovarian cancer. PATIENTS AND METHODS: Expression of nestin was evaluated by immunohistochemistry in 315 cases of various epithelial ovarian lesions as well as 52 cases of normal ovarian epithelia. The association between nestin expression and various clinical pathological parameters was analysed. RESULTS: Immunohistochemical results showed that the positive correlation of nestin expression gradually increased from benign and borderline to malignant ovarian tumours. Nestin overexpression was associated with more advanced International Federation of Gynaecology and Obstetrics stage and higher histological grade in serous adenocarcinoma. Nestin expression was significantly associated with chemoresistance. However, there was no significant correlation between nestin expression and the age and level of CA125 in patients. CONCLUSIONS: We propose that nestin expression might contribute to the initiation, promotion, and progression of serous ovarian carcinoma; nestin expression is associated with poor chemoresponse. It could be a potential therapeutic target in patients with epithelial ovarian carcinoma.

Early adjunctive diagnostic value of contrast-enhanced ultrasound-related quantitative parameter and its relationship with micro-perfusion of nontraumatic necrosis of femoral head.

OBJECTIVE: The aim of this study is to explore the early diagnostic value of contrast-enhanced ultrasound (CEUS)-related quantitative parameter and its relationship with the micro-perfusion of nontraumatic necrosis of the femoral head. PATIENTS AND METHODS: According to the random and double-blind method, the patients with non-traumatic femoral head necrosis diagnosed and treated in our hospital from July 2019 to January 2022 were selected as the subjects (the research group). According to the staging of the International Society of Bone Circulation for Femoral Head Necrosis, 89 patients with stage Ⅱ and Ⅲ A were included (39 patients with stage Ⅱ and 50 patients with stage Ⅲ A). 25 patients who conducted physical examination in our hospital during the same time were taken as the control group. Quantitative parameters of CEUS were analyzed. The content of serum vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) were evaluated. The relationship among the quantitative parameters of CEUS, the expression of VEGF and BMP-2 in serum and the patient's condition, and the value for assisting the early diagnosis of nontraumatic femoral head necrosis were analyzed. RESULTS: The body mass, body mass index (BMI), blood lipid, and cholesterol levels were much higher in the research group than in the control group (p < 0.05). The research group had a markedly higher slope of ascending branch (AS), strength enhancement index (EI), and VEGF and obviously lower decay slope (DS), mean transit time (MTT), and time to peak (TTP) than the control group (p < 0.05). In the research group, compared to stage Ⅱ, the levels of AS, EI, and VEGF in stage Ⅲ A patients were memorably higher, and the levels of DS, MTT, TTP and BMP-2 were dramatically lower (p < 0.05). Pearson's correlation test showed that AS, EI, and VEGF were positively correlated with the patients' condition, while DS, MTT, TTP and BMP-2 were negatively correlated with the patients' condition (p < 0.05). The receiver operating characteristic (ROC) curve analysis showed that the diagnostic area under the curve (AUC) of quantitative parameters of CEUS was 0.961, with sensitivity and specificity of 88.0% and 97.4%, respectively. The AUC of the combined detection of VEGF and BMP-2 was 0.945 with sensitivity and specificity of 82.3% and 87.5%, respectively, and the combined detection had a high diagnostic value (p < 0.05). CONCLUSIONS: The quantitative parameters of CEUS were of great value in the early diagnosis of nontraumatic necrosis of the femoral head with microvascular perfusion and the patients' condition, and provided a reference for the clinical treatment of non-traumatic necrosis of the femoral head. These parameters were expected to be useful indicators for judging the efficacy before and after treatment.

Effects of Δ(9)-tetrahydrocannabinol (THC) on human amniotic epithelial cell proliferation and migration.

BACKGROUND: The deleterious effects of cannabis consumption for fertility and pregnancy outcome are recognized for years. The main psychoactive molecule of cannabis, Δ(9)-tetrahydrocannabinol (THC) is able to cross the placenta barrier and cause alterations in fetal growth, low birth weight and preterm labor. However, the effects of THC on the human placenta amnion are still unknown. METHODS: The distributions of CB1R and CB2R in human amnion tissues were observed by immunohistochemistry (IHC). Human amniotic epithelial cell proliferation and migration in response to THC treatment were measured by MTS and transwell assays, respectively. The PCR array was performed to study the key regulators involved in the cell migration. The protein levels of CB1R, CB2R in amnion tissues and MMP2, MMP9 in cells were detected by western blotting. Small interfering RNAs (siRNAs) were used to knockdown MMP2 and MMP9 in WISH cells. RESULTS: Our results indicated that both CB1R and CB2R primarily identified in the epithelial layer of human placental amnion tissue. The CB1R expression in the amnion tissue was higher in the preterm group than normal control. High-dose of THC (30uM, but not 20 and 10uM) significantly inhibited (p<0.01) human amniotic epithelial cell lines (WISH) proliferation. Meanwhile, THC at both 10uM and 20uM (p<0.05) significantly suppressed cells migration in both WISH and primary human amniotic epithelial cells. The PCR array data and siRNA experiments demonstrated that MMP2/9 were tightly involved in the regulation of THC-inhibited cell migration in WISH cells. CONCLUSION: These results suggested that THC inhibited the migration of human amniotic epithelial cell through the regulation of MMP2 and MMP9, which in turn altered the development of the amnion during the gestation and partially resulted in preterm labor and other adverse pregnancy outcomes.

Hsa-miR-206 represses the proliferation and invasion of breast cancer cells by targeting Cx43.

OBJECTIVE: Hsa-miR-206, a microRNA, was found to be able to switch subtypes by targeting ER-α in breast cancer. However, there are few studies addressing the role of miR-206 in triple-negative breast cancer (TNBC). The purpose of this study was to evaluate the metastatic-regulatory ability of miR-206 in TNBC. MATERIALS AND METHODS: We treated two TNBC lines (MDA-MB-231 and MDA-MB-436) with miR-206 mimics, inhibitors and paired controls and examined the in vitro and in vivo functions of miR-206 via the degradation of Connexin43 (Cx43). A luciferase reporter assay was used to identify the binding site of GJA1 (gap junctional intercellular communication) (Cx43) and miR-206. Furthermore, quantitative RT-PCR was used to evaluate miR-206 expression in 77 breast cancer samples to determine the association with lymph node status and Cx43 expression. RESULTS: Up-regulation of miR-206 in TNBC contributed to a decreasing metastatic potential, as demonstrated by a reduction of cell viability and proliferation, decreased cell migration and invasion, lower expression levels of matrix metalloproteinase (MMP)-2, MMP-9 and a higher expression level of breast cancer metastatic suppressor (BRMS)-1. In vitro dual luciferase assays showed GJA1 (Cx43) is a target of miR-206. Quantitative RT-PCR was conducted to evaluate miR-206 expression in 77 breast cancer samples to determine the associations between miR-206 levels and both lymph node status and Cx43 expression. Restoring Cx43 expression positively regulated cell adhesion and GJA1, which may facilitate metastasis. MiR-206 significantly attenuated the proliferation and metastatic potential of cancer cells but did not inhibit tumor onset in a mouse xenograft model because of the dual function of Cx43. CONCLUSIONS: Our results suggest hsa-miR-206 may repress the tumor proliferation and invasion in breast cancer by targeting Cx43.

Effect of morphine and electro-acupuncture (EA) on apoptosis of thymocytes.

The influence of morphine and EA on the apoptosis of thymocytes were studied to investigate the posibility of its involvement in the mechanism of morphine-induced immunosuppression and the regulatory effect of EA on it. 1h after injecting 50 mg/kg morphine subcutaneously into 3-wk old Balb/c mice continually twice a day for 5 days, thymus was collected and the apoptotic cell was detected by a method of terminal deoxynucleotidyl transferase-meditaed dUTP nick end-labeling(TUNEL). The results showed that morphine significantly enhanced the percentage of TUNEL positive cells inside thymus with an appearing of apoptotic DNA ladder after 24 h incubation. Treating mice with EA of "Zusanli(St.36)" and "Lanwei(Ext.33)" for 1h after morphine administration decreased the percentage of TUNEL positive cells. EA also showed an regulatory effect on the increased the expression of CPP32 and decreased the expression of Bcl-2 by morphine. The significant enhancement of hypothalamic CRF and plasma ACTH level by morphine and the antagonize effect of EA on it suggested a possible role of Hypothalamus-pituitary-adrenal (HPA) axis played in the apoptosis of thymocytes by morphine and the regulatory effect of EA.

Effect of continued electroacupuncture on induction of interleukin-2 production of spleen lymphocytes from the injured rats.

The present study was to investigate the dynamic changes of the induction of interleukin-2 (IL-2) production of spleen lymphocytes from the rats after operative trauma stress and the regulatory effect of continued electroacupuncture (EA) stimulation of "Zusanli" (St. 36) and "Lanwei" (Extra 33) points on the induction of IL-3 production of spleen lymphocytes from the injured rats. The results showed that the induction of IL-2 production of the rat spleen lymphocytes was significantly decreased on day 1, 3, 5, 7 (P < 0.05), and the minimum level was on day 3 after operative trauma at both dilution 1:8 and dilution 1:16. The induction of IL-2 production was reserved to nearly normal on day 10. Continued stimulation by EA on "St. 36" and "Extra 33" points on day 3, 5, 7 increased the induction of IL-2 production in various degrees at the two dilutions (P < 0.01), or P < 0.05). The results mentioned above implied that the immune function could be depressed by trauma stress. Continued EA stimulation could improve the immunosuppression induced by trauma stress.

Effect of electroacupuncture on the activities of tyrosine protein kinase in subcellular fractions of activated T lymphocytes from the traumatized rats.

The present study was to observe the dynamic changes of tyrosine protein kinase (TPK) activity in subcellular fractions in the early stage of activation of T lymphocytes from normal and traumatized rats, and the regulatory effect of electroacupuncture (EA) stimulation on it. The results showed that the activities of TPK in membranous and cytosolic fractions of activated T lymphocytes were increased on second 5, and the peak was on scond 45 after ConA stimulation. Then it was decreased gradually. Comparing with the control group, the activities of TPK in membranous and cytosolic fractions of activated T lymphocytes from the traumatized rats were inhibited in various degrees especially in membrane. EA of "Zusanli"(ST-36) and "Lanwei"(Extra 33) points could enhance the activity of TPK in subcellular fractions of activated T lymphocytes from the traumatized rats. The results indicated that EA stimulation could prevent the inhibition of activation of TPK induced by trauma stress, and contribute to transmembrane signal transduction of T lymphocytes.

[Effects of tanshionone to bleomycin induced pulmonary fibrosis of rats on histological changes and production of lipid peroxides and hydroxyproline].

Study was designed to explore the effect of sodium tanshionone IIA sulfonate on bleomycin-induced pulmonary fibrosis in rat. The amount of lipid peroxides (LPO), hydroxyproline in rat lung tissue homogenate was determined at 3, 7, 14, 28 day after administration respectively. The results suggest that the amount of LPO, hydroxyproline of rat lung homogenate were significantly decreased in sodium tanshionone IIA sulfonate group. In addition, histological changes and width of alveolar septa and percentage area of pulmonary fibrosis scars was measured. The results show that sodium tanshionone IIA sulfonate could ameliorate bleomycin induced pulmonary fibrosis in rat. The mechanism that sodium tanshionone IIA sulfonate could prevent and treat bleomycin-induced pulmonary fibrosis in rat was discussed.

The nature of zinc in cytochrome c oxidase.

The zinc ion in bovine heart cytochrome c oxidase can be completely depleted from the enzyme with mercuric chloride without denaturing the protein. The metal atom stoichiometry of 5Cu/4Fe/0Zn/2Mg obtained for the enzyme following HgCl2 treatment indicates that this depletion is highly selective. Zinc depletion exposes one cysteine on subunit VIa and one cysteine on subunit VIb for N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine (1,5-I-AEDANS) labelling, suggesting that the zinc plays a structural role in the protein by providing a bridge between these two subunits. Although the treatment of cytochrome c oxidase with mercuric chloride inhibits the steady-state activity of the enzyme, subsequent removal of the Hg2+ bound to cysteine residues by 1,5-I-AEDANS significantly reverses the inhibition. This latter result indicates that the removal of the zinc itself does not alter the steady-state activity of the enzyme.