Estrogen therapy increases the risk of ovarian cancer and exogenous estradiol accelerates the onset of ovarian cancer in mouse models. Both in vivo and in vitro, ovarian surface epithelial (OSE) ...cells exposed to estradiol develop a subpopulation that loses cell polarity, contact inhibition, and forms multi-layered foci of dysplastic cells with increased susceptibility to transformation. Here, we use single-cell RNA-sequencing to characterize this dysplastic subpopulation and identify the transcriptional dynamics involved in its emergence. Estradiol-treated cells were characterized by up-regulation of genes associated with proliferation, metabolism, and survival pathways. Pseudotemporal ordering revealed that OSE cells occupy a largely linear phenotypic spectrum that, in estradiol-treated cells, diverges towards cell state consistent with the dysplastic population. This divergence is characterized by the activation of various cancer-associated pathways including an increase in Greb1 which was validated in fallopian tube epithelium and human ovarian cancers. Taken together, this work reveals possible mechanisms by which estradiol increases epithelial cell susceptibility to tumour initiation.
A cost and time effective inspection strategy for in-service health monitoring of composites is demonstrated using the fundamental anti-symmetric A
0 Lamb mode at frequencies of 15–20 kHz. In ...principle, this method involves analysis of the transmitted and/or reflected wave generated by a piezoelectric device after interacting with the test-piece boundaries or discontinuities (defects). In the present work, the applicability of the technique to inspect sandwich and repaired composite laminated carbon fibre reinforced structures is explored.
Women who have experienced childhood sexual abuse (CSA) have an increased risk of alcoholism and antisocial personality disorder (ASPD). Among male subjects, a functional polymorphism (MAOA-LPR, ...monoamine oxidase A linked polymorphic region) in the promoter region of the monoamine oxidase A gene (MAOA) appears to moderate the effect of childhood maltreatment on antisocial behavior. Our aim was to test whether MAOA-LPR influences the impact of CSA on alcoholism and ASPD in a sample of 291 women, 50% of whom have experienced CSA; we also tested whether haplotypes covering the region where both MAOA and monoamine oxidase B (MAOB) genes are located predict risk of alcoholism and ASPD better than the MAOA-LPR locus alone. Participants included 168 alcoholics (39 with ASPD (antisocial alcoholics) and 123 controls (no alcoholics, no ASPD). Antisocial behavior was also modeled as a continuous trait: ASPD symptoms count. The MAOA-LPR low activity allele was associated with alcoholism (P=0.005), particularly antisocial alcoholism (P=0.00009), only among sexually abused subjects. Sexually abused women who were homozygous for the low activity allele had higher rates of alcoholism and ASPD, and more ASPD symptoms, than abused women homozygous for the high activity allele. Heterozygous women displayed an intermediate risk pattern. In contrast, there was no relationship between alcoholism/antisocial behavior and MAOA-LPR genotype among non-abused women. The MAOA-LPR low activity allele was found on three different haplotypes. The most abundant MAOA haplotype containing the MAOA-LPR low activity allele was found in excess among alcoholics (P=0.008) and antisocial alcoholics (P=0.001). Finally, a MAOB haplotype, which we termed haplotype C, was significantly associated with alcoholism (P=0.006), and to a lesser extent with antisocial alcoholism (P=0.03). In conclusions, MAOA seems to moderate the impact of childhood trauma on adult psychopathology in female subjects in the same way as previously shown among male subjects. The MAOA-LPR low activity allele appears to confer increased vulnerability to the adverse psychosocial consequences of CSA. Haplotype-based analysis of the MAOA gene appeared to strengthen the association, as compared to the MAOA-LPR locus alone. A MAOB haplotype was associated with alcoholism independently from ASPD.
Producing animal protein while reducing the animal's impact on the environment, e.g., through improved feed efficiency and lowered methane emissions, has gained interest in recent years. Genetic ...selection is one possible path to reduce the environmental impact of livestock production, but these traits are difficult and expensive to measure on many animals. The rumen microbiome may serve as a proxy for these traits due to its role in feed digestion. Restriction enzyme-reduced representation sequencing (RE-RRS) is a high-throughput and cost-effective approach to rumen metagenome profiling, but the systematic (e.g., sequencing) and biological factors influencing the resulting reference based (RB) and reference free (RF) profiles need to be explored before widespread industry adoption is possible. Metagenome profiles were generated by RE-RRS of 4,479 rumen samples collected from 1,708 sheep, and assigned to eight groups based on diet, age, time off feed, and country (New Zealand or Australia) at the time of sample collection. Systematic effects were found to have minimal influence on metagenome profiles. Diet was a major driver of differences between samples, followed by time off feed, then age of the sheep. The RF approach resulted in more reads being assigned per sample and afforded greater resolution when distinguishing between groups than the RB approach. Normalizing relative abundances within the sampling Cohort abolished structures related to age, diet, and time off feed, allowing a clear signal based on methane emissions to be elucidated. Genus-level abundances of rumen microbes showed low-to-moderate heritability and repeatability and were consistent between diets. Variation in rumen metagenomic profiles was influenced by diet, age, time off feed and genetics. Not accounting for environmental factors may limit the ability to associate the profile with traits of interest. However, these differences can be accounted for by adjusting for Cohort effects, revealing robust biological signals. The abundances of some genera were consistently heritable and repeatable across different environments, suggesting that metagenomic profiles could be used to predict an individual's future performance, or performance of its offspring, in a range of environments. These results highlight the potential of using rumen metagenomic profiles for selection purposes in a practical, agricultural setting.
Previous studies showed that liver transplant rejection in the Piebald Virol Glaxo (PVG)-to-Lewis combination was associated with more intragraft interleukin (IL)-4 mRNA expression than in ...spontaneously tolerant grafts in the PVG-to-Dark Agouti (DA) combination. There was also immunoglobulin (Ig) G1 antibody deposition, suggesting an IL-4-induced IgG class switch in rejection. The aim of this study was to investigate whether IL-4 treatment converts PVG-->DA liver transplant tolerance to rejection.
DA (RT1a) rats were recipients of orthotopic PVG (RT1c) liver transplants and DA liver transplants were syngeneic controls. Supernatant from IL-4-transfected Chinese hamster ovary cells (0.5 mL, 30,000 U) or from untransfected cells was injected intraperitoneally on days 3 through 7. Samples were taken for immunohistochemical staining of frozen tissue sections to analyze cellular infiltrate and antibody deposition.
IL-4 treatment significantly reduced survival of liver allografts from greater than 100 days in untreated animals to 9 days (P=0.004). Pathologic analysis of IL-4-treated animals showed that death was caused by liver transplant rejection, with a heavy infiltrate of mononuclear cells, disruption of portal tract areas, and infarction. Immunohistochemistry revealed an extensive infiltrate of T cells, CD25-expressing cells, and B cells that was similar to the level in PVG--> Lewis liver allograft recipients that reject the liver. There was also a more extensive monocyte-macrophage infiltrate and more major histocompatibility complex class II expression in IL-4-treated animals compared with untreated animals. There was moderate increase of IgM, little IgG1, and no IgE or IgG2a antibody deposition.
IL-4, a T-helper type 2 cytokine that has previously been shown to inhibit delayed-type hypersensitivity responses such as rejection, was found to promote rejection of liver allografts. There was only slight evidence of a graft-specific antibody response, showing that IL-4 induces liver allograft rejection in association with some, but not all, of the changes accompanying rejection in the PVG-->Lewis strain combination.
Exogenous 17β‐estradiol (E2) accelerates the progression of ovarian cancer in the transgenic tgCAG‐LS‐TAg mouse model of the disease. We hypothesized that E2 has direct effects on ovarian cancer ...cells and this study was designed to determine the molecular mechanisms by which E2 accelerates ovarian tumor progression. Mouse ovarian cancer ascites (MAS) cell lines were derived from tgCAG‐LS‐TAg mice. Following intraperitoneal engraftment of two MAS cell lines, MASC1 and MASE2, into SCID mice, exogenous E2 significantly decreased the survival time and increased the tumor burden. Microarray analysis performed on MASE2‐derived tumors treated with E2 or placebo showed that E2 treatment caused the upregulation of 197 genes and the downregulation of 55 genes. The expression of gene regulated by estrogen in breast cancer 1 (Greb1) was upregulated in mouse tumors treated with E2 and was overexpressed in human ovarian cancers relative to human ovarian surface epithelium, suggesting a role for GREB1 in human ovarian tumor progression. RNA interference‐mediated knockdown of GREB1 in MASE2 cells decreased their proliferation rate in vitro and increased survival time in mice engrafted with the cells. These results emphasize the importance of E2 in ovarian tumor progression and identify Greb1 as a novel gene target for therapeutic intervention.
What's new?
Post‐menopausal estrogen‐only hormone replacement therapy is associated with an increased risk of epithelial ovarian cancer, the development of which may be influenced by the actions of 17β‐estradiol (E2) on ovarian surface epithelium (OSE). Here, in an orthotopic mouse model of ovarian cancer, E2 was found to accelerate tumor progression. Microarray analysis identified 197 E2‐upregulated and 55 E2‐downregulated genes. Among E2‐upregulated genes was Greb1. GREB1 protein was highly upregulated in human tumors relative to normal human OSE, suggesting that it may be a tumor‐promoting factor and potential mediator of E2‐stimulated tumor growth.
The 5-HT3 receptor is rapidly potentiated by ethanol and mediates fast excitatory serotonin (5-HT) transmission that modulates dopamine release in the reward circuitry. The 5-HT transporter regulates ...synaptic 5-HT availability. Functional polymorphisms in genes encoding the transporter and receptor may therefore influence addiction vulnerability. In this study, 360 treatment-seeking African American male patients with single and comorbid DSM-IV lifetime diagnoses of alcohol, cocaine and heroin dependence and 187 African American male controls were genotyped for the triallelic 5-HTTLPR functional polymorphism in the 5-HT transporter gene (SLC6A4) and 16 haplotype-tagging single-nucleotide polymorphisms (SNPs) across HTR3B (including the functional rs1176744 Tyr129Ser) and HTR3A, genes encoding 5-HT3 receptors. The HTR3B rs1176744 gain-of-function Ser129 allele predicted alcohol dependence (P=0.002) and low 5-HTTLPR activity predicted cocaine/heroin dependence (P=0.01). Both the HTR3B Ser129 allele (P=0.014, odds ratio (OR)=1.7 (1.1-2.6)) and low 5-HTTLPR activity (P=0.011, OR=2.5 (1.3-4.6)) were more common in men with alcohol+drug dependence compared with controls. Moreover, the HTR3B Ser129 allele and low 5-HTTLPR activity had an additive (but not an interactive) effect on alcohol+drug dependence (OR=6.0 (2.1-16.6)) that accounted for 13% of the variance. One possible explanation of our findings is that increased synaptic 5-HT coupled with increased 5-HT3 receptor responsiveness may result in enhanced dopamine transmission in the reward pathway, a predictor of increased risk for addiction. Our results may have pharmacogenetic implications for 5-HT3 therapeutic antagonists such as ondansetron.
Articular cartilage (AC) has a poor capacity to repair once damaged, with progressive degeneration often leading to osteoarthritis (OA). While biomaterials fabricated with extra cellular matrix (ECM) ...native to the AC have shown promise for repair of focal AC defects, several challenges must be overcome for the repair of larger load bearing defects due to poor scaffold mechanical properties and a lack of chondrogenic potential in diseased cells. Here, we develop a method to improve such biomaterials by incorporating a bioabsorbable 3D printed reinforcing framework and the delivery of pro-chondrogenic genes to infiltrating stem cells to enhance chondrogenesis and produce hyaline tissue that is more indicative of healthy AC. A bioabsorbable polycaprolactone (PCL) 3D printed framework was surface treated to improve its hydrophilicity and used to reinforce a collagen hyaluronic acid (CHyA) matrix. The mechanically reinforced scaffolds were then gene-activated (GA) with the chondrogenic transcription factor SOX9 which was complexed with non-viral nanoparticles (NPs) generated using the glycosaminoglycan-binding enhanced transduction (GET) system, before being seeded with human mesenchymal stromal cells (hMSC). After 28 days culture in chondrogenic media, hMSCs on the GA-scaffolds deposited an ECM more indicative of healthy hyaline cartilage compared to the gene free control. SOX9 mRNA expression on the GA scaffold was 2-orders of magnitude higher than on the control, with downstream chondrogenic targets of SOX9 (COL2α1, ACAN) also expressing significantly higher mRNA levels. Expression of pro-chondrogenic ECM proteins such as COL2, were 17.5 times greater (p = 0.0018) on the GA scaffold which also resulted in enhanced production and spatial distribution of sulphated glycosaminoglycans (sGAG), which are critical to the function of healthy AC. In summary, these findings provide evidence that functionalization of a 3D printed biomimetic pro-chondrogenic scaffold with SOX9 NPs enhances the quality of ECM produced by human stem cells on such mechanically reinforced scaffolds.
The aim of the present work is to develop a system of smart devices that could be permanently attached on the surface of the composite structure and monitor the interaction of low-frequency Lamb ...waves with defects. A linear array of transmitters would generate a relatively uniform wavefront allowing the inspection of large areas with a limited number of sensors. The asymmetric
A
0 Lamb mode is generated in carbon fibre reinforced plastic (CFRP) quasi-isotropic laminates using an array of thin piezoceramic transmitters operating in-phase. In this paper, the effect of damage size on the propagation of Lamb waves is presented. Experimental verification is also presented in multidirectional CFRP composite panels. Critical size impact damage is detected. Finally, the technique is applied to a stiffened panel. Damage on the skin or flange is detectable while damage on the web or cap is not detected with the current experimental set up.
Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic ...secretion machinery and a base that bridges both bacterial membranes, into which a hollow, external needle is embedded. When isolated, the latter two parts are termed the 'needle complex'. An incomplete understanding of the structure of the needle complex has hampered studies of T3SS function. To estimate the stoichiometry of its components, we measured the mass of its subdomains by scanning transmission electron microscopy (STEM). We determined subunit symmetries by analysis of top and side views within negatively stained samples in low-dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21-25-A resolution, three-dimensional reconstruction of the needle complex base, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component.
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