[Circular RNA expression profiles and circRNA-miRNA-mRNA crosstalk in pre-eclamptic placenta].

Objective: To identify the expression profile of circular RNA (circRNA) in placenta of pre-eclampsia (PE) pregnant women by high-throughput sequencing, and to construct the circRNA-microRNA (miRNA)-messenger RNA (mRNA) interaction network, so as to reveal the related pathways and regulatory mechanisms of PE. Methods: The clinical data and placentas of 42 women with PE (PE group) and 30 normal pregnant women (control group) who delivered in West China Second University Hospital from November 2019 to June 2021 were collected. (1) High-throughput sequencing was used to establish the differentially expressed circRNA profiles in placental tissues of 5 pairs of PE group and the control group. (2) Real-time quantitative PCR (qRT-PCR) was used to verify the expression levels of 6 differentially expressed circRNAs in placental tissues of PE group and control group. (3) Bioinformatics analysis was used to predict the target miRNA and analyze the co-expressed mRNA to construct a competitive endogenous RNA (ceRNA) network. The differentially expressed circRNAs were analyzed by Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. (4) Logistic regression analysis, Pearson correlation and Kendall's tau-b correlation analysis were used to test the correlation between the three differentially expressed circRNAs and the risk of PE and clinical characteristics. (5) circRNA_05393 was selected for subsequent functional study. Small interfering RNA (siRNA) and overexpression plasmid were used to knock down or increase the expression level of circRNA_05393 in trophoblast cell line HTR-8/SVneo cells, respectively. Transwell assay was used to detect the migration and invasion ability of the trophoblasts in vitro. Cell counting kit-8 assay was used to detect the proliferation ability of the trophoblasts. Results: (1) Seventy-two differentially expressed circRNAs were identified by high-throughput sequencing, of which 35 were up-regulated and 37 were down-regulated. (2) qRT-PCR showed that compared with the control group, circRNA_00673 (1.306±0.168 vs 2.059±0.242; t=2.356, P=0.021) and circRNA_07796 (1.275±0.232 vs 1.954±0.230; t=2.018, P=0.047) were significantly increased, while circRNA_05393 (1.846±0.377 vs 0.790±0.094; t=3.138, P=0.002) was significantly decreased. (3) The circRNA-miRNA-mRNA interaction network contained 3 circRNAs, 8 miRNAs and 53 mRNAs. GO functional annotation analysis showed that the biological process was mainly enriched in iron ion homeostasis, membrane depolarization during action potential and neuronal action potential. In terms of cellular components, they were mainly enriched in cytoskeleton and membrane components. In terms of molecular function, they were mainly enriched in the activity of voltage-gated sodium channel and basic amino acid transmembrane transporter. KEGG pathway enrichment analysis showed that mRNAs in the interaction network were mainly enriched in complement and coagulation cascade, glycine, serine and threonine metabolism, p53 signaling pathway and peroxisome proliferators-activated receptors (PPAR) signaling pathway. (4) Logistic regression analysis showed that down-regulation of circRNA_05393 expression was a risk factor for PE (OR=0.044, 95%CI: 0.003-0.596; P=0.019). Correlation analysis showed that circRNA_05393 was significantly correlated with systolic blood pressure and diastolic blood pressure in PE pregnant women (both P<0.05). (5) Knock down or overexpression of circRNA_05393 significantly reduced or increased the migration and invasion abilities of HTR-8/SVneo cells (all P<0.05), but had no significant effect on the ability of tube formation and proliferation (all P>0.05). Conclusions: The construction of circRNA expression profile in placenta and the exploration of circRNA-miRNA-mRNA interaction network provide the possibility to reveal the regulatory mechanism of specific circRNA involved in PE. Inhibition of circRNA_05393 may induce the progression of PE by reducing the migration and invasion of trophoblasts.

Increased expression of high mobility group box 1 (HMGB1) in the cytoplasm of placental syncytiotrophoblast from preeclamptic placentae.

BACKGROUND: Preeclampsia is a pregnancy-specific disorder characterised by an inappropriate maternal inflammatory response during pregnancy. High mobility group box 1 (HMGB1) was originally characterised as a nuclear protein but when released into the extracellular environment following necrotic cell death, it is proinflammatory. HMGB1 is expressed in the syncytiotrophoblast of human placenta. Higher levels of uric acid are reported in preeclampsia. The aim of this study was to investigate whether the expression of HMGB1differed between early onset and late onset preeclampsia or severe and mild preeclampsia and whether its expression correlated with the levels of uric acid. METHODS: 74 preeclamptic placentae and 110 normotensive placentae were included in this study. The levels of uric acid in women with preeclampsia were measured. The expression of HMGB1 in preeclamptic placentae or in first trimester and term placentae that had been treated with uric acid was measured. RESULTS: HMGB1 was expressed predominantly in the syncytiotrophoblast of the placenta and the expression of HMGB1 in the cytoplasm of the syncytiotrophoblast was significantly increased in both severe preeclampsia and early onset preeclampsia compared to normotensive pregnancies. The circulating levels of uric acid were significantly increased in preeclampsia and correlated with the expression of HMGB1. Increased levels of HMGB1 were significantly correlated with the severity and the time of onset of preeclampsia, but pathologic levels of uric acid did not increase the expression of HMGB1. CONCLUSION: Our data provides a better understanding of the function of HMGB1, a danger molecule in the pathogenesis of preeclampsia.

Heat shock protein 27: a potential biomarker of peritoneal metastasis in epithelial ovarian cancer?

Ovarian cancer is the major gynaecologic malignancy and the leading cause of death in gynaecological cancer. Heat shock proteins (HSPs) are highly expressed in many malignant cancers and involved in metastasis including ovarian cancer. The early detection of peritoneal metastases in epithelial ovarian cancer may be more important in clinical care. HSP27, a small heat shock protein, is correlated with peritoneal metastases in epithelial ovarian cancer tissues. In this study, we investigated whether the levels of total HSP27 were detectable in serum and whether it could be a predictive biomarker for peritoneal metastases in epithelial ovarian cancer. Serum samples from 48 patients with epithelial ovarian cancer, 35 patients with benign ovarian tumours and 24 healthy women were included in this study. The serum levels of total HSP27 were measured by enzyme-linked immunosorbent assay (ELISA). There was no difference in the serum levels of total HSP27 between women with benign ovarian tumours and healthy women. However, the serum levels of total HSP27 were significantly increased in patients with epithelial ovarian cancer. The increased serum levels of total HSP27 were only seen in patients with peritoneal metastases. Furthermore, increased serum levels of total HSP27 were significantly reduced after the combination chemotherapies in patients with peritoneal metastases. These data suggest that circulating HSP27 levels were increased in epithelial ovarian cancer and correlated with peritoneal metastases. The measurement of serum HSP27 levels may be used as a potential additional indicator for peritoneal metastases in epithelial ovarian cancer and response to treatment.

Cloning and expression analysis of heat-shock transcription factor gene CaHsfA2 from pepper (Capsicum annuum L.).

The heat-shock transcription factor (Hsf) gene CaHsfA2 (GenBank accession No. JX402923) was cloned from the Capsicum annuum thermotolerant line R9 by combining the techniques electron cloning and rapid amplification of cDNA ends. The gene, which is 1436 bp in length, had an open reading frame of 1089 bp that encoded 362 amino acids. There was an 831-bp intron between positions 321 and 322 of the cDNA. The deduced amino acid sequence of CaHsfA2 contained the conserved domains of Hsf, including DNA binding domain, adjacent domain with heptad hydrophobic repeats (A/B), activator motifs, nuclear localization signal, and nuclear export signal, and it had the highest E value of hypothesized annotation of HsfA2. CaHsfA2 had the nearest phylogenetic relationship with HsfA2 from Lycopersicon peruvianum and Mimulus guttatus, which was consistent with its botanical classification. After heat-shock treatment at 40°C for 2 h, the expression of CaHsfA2 was observed in different tissues of thermotolerant cultivar R9 and thermosensitive line B6; however, the expression levels of the CaHsfA2 gene were significantly different as follows: expression in B6 leaf > stem > flower > root, and expression in R9 flower > leaf > stem ≈ root.

Genetic determinants of the defense response of resistant and susceptible pepper (Capsicum annuum) cultivars infected with Phytophthora capsici (Oomycetes; Pythiaceae).

Based on culture isolation and morphological observation blight-infected pepper plants in Shaanxi Province, China, we identified the pathogen causing pepper phytophthora blight as Phytophthora capsici. Varieties that differed in resistance (CM334, PBC602, and B27) were inoculated with this pathogen. The root activity of resistant CM334 variety was the highest while that of susceptible B27 variety was the lowest. Also, significant differences in the activity of POD, PAL, and ß-1,3-glucanase were found; there was a positive correlation between disease resistance and activity of these three enzymes. We inhibited mycelial growth and sporangia formation of P. capsici using crude ß-1,3-glucanase and PAL enzymes isolated from the resistant variety CM334 after it had been inoculated with P. capsici. These two enzymes had a synergistic effect on inhibition of P. capsici mycelial growth and sporangia formation. Expression of the defensive genes CaPO1, CaBGLU, CaBPR1, and CaRGA in the three varieties was higher in the leaves than in the roots. All three genes were upregulated in infected leaves and roots of the pepper plants, always expressing at higher levels in the resistant cultivar than in the susceptible cultivar, suggesting that the differences in resistance among the pepper genotypes involve differences in the timing and magnitude of the defense response.

The increased maternal serum levels of IL-6 are associated with the severity and onset of preeclampsia.

Preeclampsia is a complex disease of pregnancy with both feto-placental and maternal factors contributing to its pathogenesis. Although the cause of this disease is uncertain, imbalance between pro-and anti-inflammatory cytokines has been implicated in the pathogenesis of preeclampsia. Increased levels of the inflammatory cytokine IL-6 has been postulated to be involved in some ways in the pathogenesis of preeclampsia. However studies investigating whether levels of IL-6 in the maternal circulation differ between the disease severities or between times of onset of preeclampsia, or between preeclamptic pregnancies that are or are not complicated by fetal growth restriction (FGR) are limited. 104 women with preeclampsia and 75 health pregnant women were included into this study. The levels of IL-6 in maternal circulation were measured by enzyme-linked immunosorbent assay (ELISA). The levels of IL-6 in serum were significantly increased in women with preeclampsia in early onset and late onset preeclampsia compared to gestation matched health pregnant women. In addition, the levels of IL-6 were significantly increased in women with severe preeclampsia, but not with mild preeclampsia compared to gestation matched health pregnant women. Furthermore there was no correlation in IL-6 levels between preeclamptic with or without FGR. Our data shows increased level of circulating IL-6 levels in both women with early onset or late onset preeclampsia and in women with severe preeclampsia. These results suggest the excessive maternal inflammatory response in preeclampsia.

Increased expression of Rab25 in breast cancer correlates with lymphatic metastasis.

Breast cancer is the most common cancer in women worldwide. Studies have suggested that Ras-related protein 25 (Rab25), a member of Rab small GTPase family, is involved in the pathogenesis of breast cancer. In this study, we investigated whether the expression of Rab25 correlated with lymphatic metastasis in breast cancer and whether the expression of Rab25 was positively correlated with oestrogen receptor (ER) and progesterone receptor (PR) expression in breast cancer. Breast cancer tissues from 42 invasive ductal breast cancer patients with or without lymphatic metastasis were collected and the levels of Rab25 mRNA and protein measured by quantitative real-time PCR and immunohistochemistry, respectively. The mRNA level of Rab25 was significantly increased in invasive ductal breast cancer with lymphatic metastasis compared to that in invasive ductal breast cancer without lymphatic metastasis. Immunohistochemical analysis demonstrated that Rab25 and vascular endothelial growth factor (VEGF) were highly expressed in invasive ductal breast cancer with lymphatic metastasis regardless of whether the cancer is ER and PR positive or negative. Higher expression of Rab25 positively correlated with VEGF expression. However, the expressions of Rab25 in ER and PR-positive cancers were much higher than ER and PR-negative cancers regardless of whether lymphatic metastasis occurred. These data suggest that higher level of Rab25 was associated with lymphatic metastasis, specifically in ER and PR-positive breast cancer. The better understanding of the mechanism of Rab25 may provide a basis for the development of a novel therapeutic target in breast cancer.

Exogenous abscisic acid increases antioxidant enzymes and related gene expression in pepper (Capsicum annuum) leaves subjected to chilling stress.

To elucidate how physiological and biochemical mechanisms of chilling stress are regulated by abscisic acid (ABA) pretreatment, pepper variety (cv. 'P70') seedlings were pretreated with 0.57 mM ABA for 72 h and then subjected to chilling stress at 10°/6°C (day/night). Chilling stress caused severe necrotic lesions on the leaves and increased malondialdehyde and H(2)O(2) levels. Activities of monodehydroascorbate reductase (DHAR), dehydroascorbate reductase, glutathione reductase, guaiacol peroxidase, ascorbate peroxidase, ascorbate, and glutathione increased due to chilling stress during the 72 h, while superoxide dismutase and catalase activities decreased during 24 h, suggesting that chilling stress activates the AsA-GSH cycle under catalase deactivation in pepper leaves. ABA pretreatment induced significant increases in the above-mentioned enzyme activities and progressive decreases in ascorbate and glutathione levels. On the other hand, ABA-pretreated seedlings under chilling stress increased superoxide dismutase and guaiacol peroxidase activities and lowered concentrations of other antioxidants compared with untreated chilling-stressed plants. These seedlings showed concomitant decreases in foliage damage symptoms, and levels of malondialdehyde and H(2)O(2). Induction of Mn-SOD and POD was observed in chilling-stressed plants treated with ABA. The expression of DHAR1 and DHAR2 was altered by chilling stress, but it was higher in the presence than in the absence of ABA at 24 h. Overall, the results indicate that exogenous application of ABA increases tolerance of plants to chilling-induced oxidative damage, mainly by enhancing superoxide dismutase and guaiacol peroxidase activities and related gene expression.

Construction of the intermediate vector pVBG2307 by incorporating vital elements of expression vectors pBI121 and pBI221.

Molecular chaperones of plasmid pBI121 carrying CaMV35S promoter and a nucleotide sequence of plasmid pBI221 were inserted into plasmid pCAMBIA2300 to construct an intermediate vector: pVBG2307. This novel vector pVBG2307 contains a greatly expanded multiple cloning site with an adjacent imported CaMV35S promoter sequence. This vector allows controlled transformation of DNA in both Escherichia coli and Agrobacterium tumefaciens. Cloned PG, orf456, ipt genes and E8, a fruiting promoter, were amplified by PCR of cDNA libraries of Capsicum annum and Lycopersicon esculentum and were then transferred into vector pVBG2307. The viability of this vector was demonstrated, as it regulated PG, orf456, ipt and E8 genes in E. coli and could be transferred into Agrobacterium strain EHA105-4.

Activity and expression of polygalacturonase vary at different fruit ripening stages of sweet pepper cultivars.

Activity and expression of polygalacturonase (PG), a hydrolytic enzyme involved in ultrastructural changes in the pericarp of sweet pepper (Capsicum annaum), were investigated at different ripening stages of the pepper cultivars Mandi and Talanduo. Molecular cloning of CaPG was carried out by constructing a cDNA library from three stages of fruit ripening. Morphological determination, PG assay, RT-PCR, and ultrastructural studies were used to quantify changes in CaPG gene expression in the pericarp from green, color change and fully ripened stages. We found that CaPG gene expression, PG activity and striking changes in the structure of the cell wall occurred with the transition of ripening stages. CaPG gene expression was high (obvious PCR products) in mature and ripened stages of both cultivars; however, the CaPG gene was not expressed in preclimacteric fruits or vegetative tissues. We conclude that developmental regulation of CaPG gene expression is instrumental for sweet pepper fruit ripening; its expression during development leads to dissolution of middle lamella and eventually disruption of the fully ripened cell wall.

Research Note: Effects of polysaccharide-enriched Acanthopanax senticosus extract on growth performance, immune function, antioxidation, and ileal microbial populations in broiler chickens.

Acanthopanax senticosus (AS) is a well-known, highly effective traditional Chinese herbal medicine. Polysaccharides extracted from AS (ASPS) have multiple pharmacologic and biological activities with potential use as additives in broiler chicken feed. This trial evaluated the effects of dietary ASPS on growth performance, immune function, antioxidation, and ileal microbial populations in broiler chickens. A total of 240 1-day-old Arbor Acres male broiler chicks were randomly divided into 4 groups, with 10 replicates of 6 chicks and fed a corn- and soybean-based diet supplemented with 0, 1, 2, or 4 g/kg ASPS. Compared with the control group, supplementation with 1 g/kg ASPS increased ADG and ADFI in the finisher and overall periods and decreased the feed conversion ratio in the finisher period (both P < 0.05). Serum IgA and IgM were significantly increased by supplementation with 1 and 2 g/kg of ASPS (P < 0.05). Superoxide dismutase and glutathione peroxidase activities were increased and malondialdehyde concentration was decreased in birds fed ASPS-supplemented diets compared with those in the control group (P < 0.05). Polysaccharides extracted from AS supplementation increased Lactobacillus and decreased Escherichia coli and Salmonella counts in the ileal contents compared with the control diet (both P < 0.05). The results show that dietary ASPS improved growth performance, immune status, and antioxidant capacity and stimulated the growth of beneficial gut bacteria in broiler chickens. In conclusion, ASPS was effective as a natural additive in broiler chicken feed; 1 g/kg can be considered as the optimum dosage.

[Clinical analysis of two cases of imported children Zika virus infection in China].

OBJECTIVE: To analyze the clinical characteristics, outcome and diagnosis of two cases of imported children Zika virus infection in China. METHOD: A retrospective analysis was performed on clinical characteristics, treatment and outcome of two cases of imported children with Zika virus infection in February 2016 in Enping People's Hospital of Guangdong. RESULT: Two cases of children with imported Zika virus infection resided in an affected area of Venezuela, 8-year-old girl and her 6 year-old brother. The main findings on physical examination included the following manifestations: fever, rash, and conjunctivitis. The rash was first limited to the abdomen, but extended to the torso, neck and face, and faded after 3-4 d. The total number of white blood cells was not high and liver function was normal. The diagnosis of two cases of Zika virus infection was confirmed by the expert group of Guangdong Provincial Center for Disease Control and Prevention, according to the epidemiological history, clinical manifestations and Zika virus nucleic acid detection results.Treatment of Zika virus infection involves supportive care. Two Zika virus infection children had a relatively benign outcome. CONCLUSION: At present, Zika virus infection in children is an imported disease in China. No specific therapy is available for this disease. Information on long-term outcomes among infants and children with Zika virus disease is limited, routine pediatric care is advised for these infants and children.

The reduction of circulating levels of IL-6 in pregnant women with preeclampsia by magnesium sulphate and nifedipine: In vitro evidence for potential mechanisms.

INTRODUCTION: Women with preeclampsia have elevated levels of inflammatory cytokines including IL-6. IL-6, which is known to activate endothelial cells and induce the production of necrotic trophoblastic debris from the placenta, may be important in the pathogenesis of preeclampsia. MgSO4 is a major therapy for the prevention of seizures in preeclampsia but it has been suggested to also have anti-inflammatory and vasodilatory properties. METHODS: 22 pregnant women with preeclampsia and 68 normotensive controls were recruited and circulating IL-6 levels in these women were measured before MgSO4 and nifedipine treatment and after delivery. In addition, endothelial cells were treated with IL-6 or necrotic trophoblastic debris, generated from first trimester placental explants in the presence or absence of MgSO4in vitro, and cell-surface ICAM-1 was measured by ELISA. The levels of IL-6 in the culture medium were also measured. Furthermore nitric oxide synthetase activity in endothelial cells that had been treated with IL-6 was measured using l-NAME. RESULTS: Circulating levels of IL-6 in preeclampsia were reduced significantly following administration of MgSO4. In vitro, MgSO4 reversed the activation of endothelial cells induced by IL-6 but not by necrotic trophoblastic debris. The effect of MgSO4 in reversing the IL-6 induced activation of endothelial cells was not dependent upon nitric oxide synthetase. Treating placental explants with MgSO4 prevented the production of necrotic trophoblastic debris induced by IL-6. DISCUSSION: we demonstrated that IL-6 levels drop following treatment with MgSO4 and nifedipine in vivo, and have identified several mechanisms by which this positive effect on IL-6 may occur in vitro.

[Fibronectin in gastrointestinal tumors and its relation to biological characteristics of tumor].

Through immunohistochemical technique, distribution of FN in normal mucosa, benign and malignant tumors of human gastrointestinal tract were studied. In normal and adenoma tissues, FN was found in both basement membrane (BN) and interstitial tissue. While in cancer tissue, there was a consistent decrease of BM FN content around the tumor nests particularly more apparently in cases of invading carcinoma. Statistical analysis showed that the reduction of BM FN was correlated with the degree of tumor dedifferentiation but not with the incidence of regional metastases. No association was noticed between the stroma FN and tumor behaviors. Since small blood vessels were usually delineated clearly by the staining for FN, FN might be considered as a marker in identifying the invasion of blood vessel wall by tumor cells. It is suggested that lack of BM FN in tumor tissues might be mainly due to decrease of FN synthesis by the tumor cells.

Increased expression of heat shock protein 27 correlates with peritoneal metastasis in epithelial ovarian cancer.

Ovarian cancer is the third most common gynecologic malignancy and the leading cause of death in gynecological cancer. Although the 5-year survival rate is increasing, peritoneal metastasis of ovarian cancer is still a problem because of no potential predictor. Heat shock proteins (HSPs) are a class of functionally related proteins that are highly expressed in many malignant cancers. Previous studies suggest high levels of HSP27 present in the serum of patients with ovarian cancer. In this study, we investigated whether the expression of HSP27 in epithelial ovarian cancer tissue was associated with peritoneal metastasis and whether HSP27 could be used as a potential predictor of peritoneal metastasis in epithelial ovarian cancer. Tissues from epithelial ovarian cancer with or without peritoneal metastasis were collected and the levels of HSP27 messenger RNA and protein determined by real-time polymerase chain reaction and Western blotting. Immunohistochemistry was used to determine the subcellular localization of HSP27. Immunohistochemistry images showed that HSP27 was highly expressed in the cytoplasm of epithelial cancer cells with peritoneal metastasis. Messenger RNA and protein levels of HSP27 were significantly increased in epithelial ovarian cancer with peritoneal metastasis compared with epithelial ovarian cancer without peritoneal metastasis. Higher expression of HSP27 correlated with poor clinical outcome. These data suggest that higher level of HSP27 was associated with peritoneal metastasis in epithelial ovarian cancer. Heat shock protein 27 may be a useful prognostic marker of poor survival and may provide a basis for the development of molecular therapeutics modulating this survival pathway.

A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration.

A CHO cell line with a single copy of the DHFR locus on chromosome Z2 was used to analyze the structure of the amplification target and products subsequent to the initial amplification event. Dramatic diversity in the number and cytogenetic characteristics of DHFR amplicons was observed as soon as eight to nine cell doublings following the initial event. Two amplicon classes were noted at this early time: Small extrachromosomal elements and closely spaced chromosomal amplicons were detected in 30-40% of metaphases in six of nine clones, whereas three of nine clones contained huge amplicons spanning greater than 50 megabases. In contrast, the incidence of metaphases containing extrachromosomal amplicons fell to 1-2% in cells analyzed at 30-35 cell doublings, and most amplicons localized to rearranged or broken derivatives of chromosome Z2 at this time. Breakage of the Z2 chromosome near the DHFR gene, and deletion of the DHFR gene and flanking DNA was also observed in cells that had undergone the amplification process. To account for these diverse cytogenetic and molecular consequences of gene amplification, we propose that chromosome breakage plays a central role in the amplification process by (1) generating intermediates that are initially acentric and lead to copy number increase primarily by unequal segregation, (2) creating atelomeric ends that are either incompletely replicated or resected by exonucleases to generate deletions, and (3) producing recombinogenic ends that provide preferred sites for amplicon relocalization.