Enhanced Nitrite-Mediated Relaxation of Placental Blood Vessels Exposed to Hypoxia Is Preserved in Pregnancies Complicated by Fetal Growth Restriction

Nitric oxide (NO) is essential in the control of fetoplacental vascular tone, maintaining a high flow−low resistance circulation that favors oxygen and nutrient delivery to the fetus. Reduced fetoplacental blood flow is associated with pregnancy complications and is one of the major causes of fetal growth restriction (FGR). The reduction of dietary nitrate to nitrite and subsequently NO may provide an alternative source of NO in vivo. We have previously shown that nitrite induces vasorelaxation in placental blood vessels from normal pregnancies, and that this effect is enhanced under conditions of hypoxia. Herein, we aimed to determine whether nitrite could also act as a vasodilator in FGR. Using wire myography, vasorelaxant effects of nitrite were assessed on pre-constricted chorionic plate arteries (CPAs) and veins (CPVs) from normal and FGR pregnancies under normoxic and hypoxic conditions. Responses to the NO donor, sodium nitroprusside (SNP), were assessed in parallel. Nitrate and nitrite concentrations were measured in fetal plasma. Hypoxia significantly enhanced vasorelaxation to nitrite in FGR CPAs (p < 0.001), and in both normal (p < 0.001) and FGR (p < 0.01) CPVs. Vasorelaxation to SNP was also potentiated by hypoxia in both normal (p < 0.0001) and FGR (p < 0.01) CPVs. However, compared to vessels from normal pregnancies, CPVs from FGR pregnancies showed significantly lower reactivity to SNP (p < 0.01). Fetal plasma concentrations of nitrate and nitrite were not different between normal and FGR pregnancies. Together, these data show that nitrite-mediated vasorelaxation is preserved in FGR, suggesting that interventions targeting this pathway have the potential to improve fetoplacental blood flow in FGR pregnancies.     

Tadalafil Treatment of Mice with Fetal Growth Restriction and Preeclampsia Improves Placental mTOR Signaling

Fetal growth restriction (FGR) is a major cause of poor perinatal outcomes. Although several studies have been conducted to improve the prognosis of FGR in infants, no effective intrauterine treatment method has been established. This study aimed to use tadalafil, a phosphodiesterase 5 inhibitor (PDE5) inhibitor, as a novel intrauterine treatment and conducted several basic and clinical studies. The study investigated the effects of tadalafil on placental mTOR signaling. Tadalafil was administered to mice with L-NG-nitroarginine methyl ester (L-NAME)-induced FGR and associated preeclampsia (PE). Placental phosphorylated mTOR (p-mTOR) signaling was assessed by fluorescent immunohistochemical staining and Western blotting. The expression of p-mTOR was significantly decreased in mice with FGR on 13 days post coitum (d.p.c.) but recovered to the same level as that of the control on 17 d.p.c. following tadalafil treatment. The results were similar for 4E-binding protein 1 (4E-BP1) and S6 ribosomal (S6R) protein, which act downstream in the mTOR signaling pathway. We demonstrate that the tadalafil treatment of FGR in mice improved placental mTOR signaling to facilitate fetal growth. Our study provides the key mechanistic detail about the mode of action of tadalafil and thus would be helpful for future clinical studies on FGR.     

YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.     

A Novel Homozygous Variant in DYSF Gene Is Associated with Autosomal Recessive Limb Girdle Muscular Dystrophy R2/2B

Mutations in the DYSF gene, encoding dysferlin, are responsible for Limb Girdle Muscular Dystrophy type R2/2B (LGMDR2/2B), Miyoshi myopathy (MM), and Distal Myopathy with Anterior Tibialis onset (MDAT). The size of the gene and the reported inter and intra familial phenotypic variability make early diagnosis difficult. Genetic analysis was conducted using Next Gene Sequencing (NGS), with a panel of 40 Muscular Dystrophies associated genes we designed. In the present study, we report a new missense variant c.5033G>A, p.Cys1678Tyr ({"type":"entrez-nucleotide","attrs":{"text":"NM_003494","term_id":"1675063869"}}NM_003494) in the exon 45 of DYSF gene related to Limb Girdle Muscular Dystrophy type R2/2B in a 57-year-old patient affected with LGMD from a consanguineous family of south Italy. Both healthy parents carried this variant in heterozygosity. Genetic analysis extended to two moderately affected sisters of the proband, showed the presence of the variant c.5033G>A in both in homozygosity. These data indicate a probable pathological role of the variant c.5033G>A never reported before in the onset of LGMDR2/2B, pointing at the NGS as powerful tool for identifying LGMD subtypes. Moreover, the collection and the networking of genetic data will increase power of genetic-molecular investigation, the management of at-risk individuals, the development of new therapeutic targets and a personalized medicine.     

Transforming Growth Factorβ1 Overexpression Is Associated with Insulin Resistance and Rapidly Progressive Kidney Fibrosis under Diabetic Conditions

Diabetes mellitus is a global health problem. Diabetic nephropathy is a common complication of diabetes mellitus and the leading cause of end-stage renal disease. The clinical course, response to therapy, and prognosis of nephropathy are worse in diabetic than in non-diabetic patients. The role of transforming growth factorβ1 in kidney fibrosis is undebatable. This study assessed whether the overexpression of transforming growth factorβ1 is associated with insulin resistance and the rapid progression of transforming growth factorβ1-mediated nephropathy under diabetic conditions. Diabetes mellitus was induced with streptozotocin in wild-type mice and transgenic mice with the kidney-specific overexpression of human transforming growth factorβ1. Mice treated with saline were the controls. Glucose tolerance and kidney fibrosis were evaluated. The blood glucose levels, the values of the homeostasis model assessment for insulin resistance, and the area of kidney fibrosis were significantly increased, and the renal function was significantly impaired in the diabetic transforming growth factorβ1 transgenic mice compared to the non-diabetic transgenic mice, diabetic wild-type mice, and non-diabetic mice. Transforming growth factorβ1 impaired the regulatory effect of insulin on glucose in the hepatocyte and skeletal muscle cell lines. This study shows that transforming growth factorβ1 overexpression is associated with insulin resistance and rapidly progressive kidney fibrosis under diabetic conditions in mice.     

Dysregulation of PAK1 Is Associated with DNA Damage and Is of Prognostic Importance in Primary Esophageal Small Cell Carcinoma

Primary esophageal small cell carcinoma (PESCC) is a rare, but fatal subtype of esophageal carcinoma. No effective therapeutic regimen for it. P21-activated kinase 1 (PAK1) is known to function as an integrator and an indispensable node of major growth factor signaling and the molecular therapy targeting PAK1 has been clinical in pipeline. We thus set to examine the expression and clinical impact of PAK1 in PESCC. The expression of PAK1 was detected in a semi-quantitative manner by performing immunohistochemistry. PAK1 was overexpressed in 22 of 34 PESCC tumors, but in only 2 of 18 adjacent non-cancerous tissues. Overexpression of PAK1 was significantly associated with tumor location (p = 0.011), lymph node metastasis (p = 0.026) and patient survival (p = 0.032). We also investigated the association of PAK1 with DNA damage, a driven cause for malignancy progression. γH2AX, a DNA damage marker, was detectable in 18 of 24 (75.0%) cases, and PAK1 expression was associated with γH2AX (p = 0.027). Together, PAK1 is important in metastasis and progression of PESCC. The contribution of PAK1 to clinical outcomes may be involved in its regulating DNA damage pathway. Further studies are worth determining the potentials of PAK1 as prognostic indicator and therapeutic target for PESCC.     

Toll-like Receptor 2 Is Associated with the Immune Response,Apoptosis, and Angiogenesis in the Mammary Glands of Dairy Cows with Clinical Mastitis

Toll-like receptor 2 (TLR2) plays a crucial role in bacterial recognition and the host immune response during infection. However, its function and downstream biological processes (BPs) in the mammary glands (MGs) of Holstein cows with clinical mastitis (CM) are not fully understood. This study aimed to comprehensively identify the BPs and differentially expressed proteins (DEPs) associated with the bacterial response and TLR2 using data-independent acquisition (DIA) proteomic data. A possible mechanism for the action of TLR2 was proposed, and the results suggested that the expression levels of TLR2 and caspase 8 (CASP8) were positively correlated with the apoptosis of MGs. The expression patterns of TLR2 and TEK receptor tyrosine kinase 2 (Tie2) were negatively correlated with angiogenesis. These results indicated that TLR2 might promote apoptosis in mammary epithelial cells (MECs) and vascular endothelial cells (VECs) via upregulation of CASP8 expression, and inhibition of angiogenesis in VECs via downregulation of Tie2 expression in dairy cows with CM. In conclusion, TLR2 is associated with inflammation, apoptosis, and angiogenesis in the MGs of dairy cows with bacteria-induced mastitis. These results contribute to a deeper understanding of the pathogenic mechanisms and provide the knowledge needed for developing the prevention and treatment of dairy mastitis.     

Mesothelin Expression Is Not Associated with the Presence of Cancer Stem Cell Markers SOX2 and ALDH1 in Ovarian Cancer

Mesothelin (MSLN) overexpression (OE) is a frequent finding in ovarian carcinomas and increases cell survival and tumor aggressiveness. Since cancer stem cells (CSCs) contribute to pathogenesis, chemoresistance and malignant behavior in ovarian cancer (OC), we hypothesized that MSLN expression could be creating a favorable environment that nurtures CSCs. In this study, we analyzed the expression of MSLN and CSC markers SOX2 and ALDH1 by immunohistochemistry (IHC) in different model systems: primary high-grade serous carcinomas (HGSCs) and OC cell lines, including cell lines that were genetically engineered for MSLN expression by either CRISPR-Cas9-mediated knockout (Δ) or lentivirus-mediated OE. Cell lines, wild type and genetically engineered, were evaluated in 2D and 3D culture conditions and xenografted in nude mice. We observed that MSLN was widely expressed in HGSC, and restricted expression was observed in OC cell lines. In contrast, SOX2 and ALDH1 expression was limited in all tissue and cell models. Most importantly, the expression of CSC markers was independent of MSLN expression, and manipulation of MSLN expression did not affect CSC markers. In conclusion, MSLN expression is not involved in driving the CSC phenotype.     

miR-218-5p/RUNX2 Axis Positively Regulates Proliferation and Is Associated with Poor Prognosis in Cervical Cancer

The overexpression of miR-218-5p in cervical cancer (CC) cell lines decreases migration, invasion and proliferation. The objective was to identify target genes of miR-218-5p and the signaling pathways and cellular processes that they regulate. The relationship between the expression of miR-218-5p and RUNX2 and overall survival in CC as well as the effect of the exogenous overexpression of miR-218-5p on the level of RUNX2 were analyzed. The target gene prediction of miR-218-5p was performed in TargetScan, miRTarBase and miRDB. Predicted target genes were subjected to gene ontology (GO) and pathway enrichment analysis using the Kyoto Encyclopaedia of Genes and Genomes (KEGG). The miR-218-5p mimetic was transfected into C-33A and CaSki cells, and the miR-218-5p and RUNX2 levels were determined by RT–qPCR. Of the 118 predicted targets for miR-218-5p, 86 are involved in protein binding, and 10, including RUNX2, are involved in the upregulation of proliferation. Low miR-218-5p expression and a high level of RUNX2 are related to poor prognosis in CC. miR-218-5p overexpression is related to decreased RUNX2 expression in C-33A and CaSki cells. miR-218-5p may regulate RUNX2, and both molecules may be prognostic markers in CC.     

PKR-Mediated Phosphorylation of eIF2a and CHK1 Is Associated with Doxorubicin-Mediated Apoptosis in HCC1143 Triple-Negative Breast Cancer Cells

Triple-negative breast cancer is more aggressive than other types of breast cancer. Protein kinase R (PKR), which is activated by dsRNA, is known to play a role in doxorubicin-mediated apoptosis; however, its role in DNA damage-mediated apoptosis is not well understood. In this study, we investigated the roles of PKR and its downstream players in doxorubicin-treated HCC1143 triple-negative breast cancer cells. Doxorubicin treatment induces DNA damage and apoptosis. Interestingly, doxorubicin treatment induced the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) via PKR, whereas the inhibition of PKR with inhibitor C16 reduced eIF2α phosphorylation. Under these conditions, doxorubicin-mediated DNA fragmentation, cell death, and poly(ADP ribose) polymerase and caspase 7 levels were recovered. In addition, phosphorylation of checkpoint kinase 1 (CHK1), which is known to be involved in doxorubicin-mediated DNA damage, was increased by doxorubicin treatment, but blocked by PKR inhibition. Protein translation was downregulated by doxorubicin treatment and upregulated by blocking PKR phosphorylation. These results suggest that PKR activation induces apoptosis by increasing the phosphorylation of eIF2α and CHK1 and decreasing the global protein translation in doxorubicin-treated HCC1143 triple-negative breast cancer cells.     

填料与橡胶的化学和物理作用及其对橡胶的补强ⅢAl2O3、n-Al2O3和n-Al2O3/NDZ-101与NR

研究了Al2O3与NR的相互作用。发现Al2O3与NR相互作用的物理交联密度与其吸油值和比表面积呈正相关性，与其粒径和pH值呈负相关性，与其吸油值的相关性最大。Al2O3对硫化体系与NR的化学相互作用有屏蔽效应，使化学交联密度下降。其屏蔽效应与其比表面积和吸油值呈负相关性，与其粒径和pH值呈正相关性。Al2O3的粒径为4μm时对NR无补强作用，粒径为1μm时稍有补强作用，粒径小于25nm时补强作用很大，全部是物理健的贡献，添加偶联剂，强度只增加8．9％，但是，化学相互作用的贡献达89．8％，化学交联占总交联密度的77．5％。     

Altered Expression of ESR1, ESR2, PELP1 and c-SRC Genes Is Associated with Ovarian Cancer Manifestation

Ovarian cancer remains the leading cause of death due to gynecologic malignancy. Estrogen-related pathways genes, such as estrogen receptors (ESR1 and ESR2) and their coregulators, proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), and proto-oncogene tyrosine-protein kinase c-Src (SRC) are involved in ovarian cancer induction and development, still they require in-depth study. In our study, tissue samples were obtained from 52 females of Caucasian descent (control group without cancerous evidence (n = 27), including noncancerous benign changes (n = 15), and the ovarian carcinoma (n = 25)). Using quantitative analyses, we investigated ESRs, PELP1, and SRC mRNA expression association with ovarian tumorigenesis. Proteins’ presence and their location were determined by Western blot and immunohistochemistry. Results showed that PELP1 and SRC expression levels were found to differ in tissues of different sample types. The expression patterns were complex and differed in the case of ovarian cancer patients compared to controls. The most robust protein immunoreactivity was observed for PELP1 and the weakest for ESR1. The expression patterns of analyzed genes represent a potentially interesting target in ovarian cancer biology, especially PELP1. This study suggests that specific estrogen-mediated functions in the ovary and ovary-derived cancer might result from different local interactions of estrogen with their receptors and coregulators.     

The Quiescent Cellular State is Arf/p53-Dependent and Associated with H2AX Downregulation and Genome Stability

Cancer is a disease associated with genomic instability and mutations. Excluding some tumors with specific chromosomal translocations, most cancers that develop at an advanced age are characterized by either chromosomal or microsatellite instability. However, it is still unclear how genomic instability and mutations are generated during the process of cellular transformation and how the development of genomic instability contributes to cellular transformation. Recent studies of cellular regulation and tetraploidy development have provided insights into the factors triggering cellular transformation and the regulatory mechanisms that protect chromosomes from genomic instability.