Gonadal intersex in teleosts: Mechanisms, molecular biomarkers and diagnostic assays

Natural and synthetic estrogenic and androgenic compounds are continuously released into aquatic ecosystems. Exposure of teleost fishes to these contaminants can negatively impact sex differentiation and reproductive output. Specifically, development of gonadal intersex in gonochoristic (fixed sex) fish species has been studied extensively in relation to exposure to this class of compounds. The main objectives of this dissertation were to: 1) conduct field and laboratory studies to investigate the molecular signaling pathways behind the development of gonadal intersex; and 2) establish molecular biomarkers and assays for testing the ability of environmental pollutants to develop this condition using a battery of molecular, cellular and organ-level tools. First, we conducted a literature review to summarize all available articles reporting gonadal intersex in wild populations of gonochoristic fish (Chapter 1). We also included the limited information available on this topic in amphibians. We analyzed studies from across the globe, identified families and species with reported cases of gonadal intersex, and highlighted the contaminants often linked with this condition. In addition, we discussed the current knowledge of molecular signaling pathways behind the development of gonadal intersex and summarized molecular biomarkers tested and others that require further investigation. We then conducted a field study investigating the prevalence of testicular oocytes (TOs), the most prevalent form of gonadal intersex, in a sentinel freshwater fish species, the smallmouth bass Micropterus dolomieu, inhabiting the St. Joseph River and its tributaries in northern Indiana (Chapter 2). This constitutes the first study of this nature. Sites on this river were previously identified as having medium to high intersex induction potential based on contaminant quantification and estrogen equivalence estimations. We reported prevalence and severity of gonadal intersex reaching 100% in some sites, and significant decreases in prevalence and increases in severity of TOs occurrence after the spawning season. We evaluated changes in the transcription levels of several genes involved in sex differentiation and gonadal development. Significantly higher levels of vitellogenin ( vtg) transcripts were found in livers of males with TOs, but only when sampled in the spawning season. Further, we quantified contaminant levels in surface water to identify possible correlations between contamination levels and the observed prevalence of gonadal intersex. Multiple sites had detectable levels of endocrine disruptors, but no correlations with the prevalence or severity of TOs was recorded. In order to develop molecular biomarkers and assays that test contaminants’ ability to develop gonadal intersex, short-term laboratory exposures were performed using Japanese medaka Oryzias latipes, a fish model with a well-understood sex determination mechanism and high sensitivity to exogenous hormone exposure (Chapter 3). First, we identified a gene, ovary structure protein 1 (osp1), with strong female-specific expression during gonadal differentiation and observed a significant downregulation in its expression in females following short-term (10 d) exposure to a potent synthetic androgen, 17β-trenbolone. Importantly, this decrease in osp1expression was correlated with changes in ovarian phenotype, namely ovarian intersex, later in life. We decided to further utilize this promising molecular biomarker by incorporating it in a visual in vivo reporter assay for rapid detection of contaminants with estrogenic/androgenic potential (Chapter 4). For this purpose, we built a pOSP1-AcGFP (promoterOSP1-Aequorea coerulescens green florescence protein) Japanese medaka transgenic line with osp1 promoter region driving the expression of a reporter protein, AcGFP. After establishing this line, we tested its use in an in vivo visual reporter system for identifying estrogenic contaminants. Significant upregulation in fluorescence intensity was recorded in 30 d post hatch females following a 24 h exposure to 500 ng/L of a synthetic potent estrogen, ethinyl estradiol. Overall, our results support earlier findings suggesting that gonadal intersex is highly prevalent in impacted rivers across the US and that smallmouth bass are highly sensitive to developing this condition. We also conclude that hepatic and plasma VTG are promising biomarkers for diagnosing gonadal intersex, but only in males sampled during the spawning season. Our findings also support the hypothesis that molecular biomarkers, such as osp1 , are sensitive tools that can be used for early detection of the effects of contaminants with estrogenic and androgenic activity on fish and are ideal endpoints in wide-scale contaminant screening assays

How Knowledge Management Implementation Affects the Performance of Egyptian Construction Companies

This study examines the effectiveness of knowledge management systems within the construction industry in Egypt from the perspective of knowledge infrastructure capability (KIC), knowledge process capability (KPC) and their impact on business performance (BP) from the financial, consumer, learning and growth, supplier and internal perspectives. The sample consists of 75 first class Egyptian construction companies. The authors used a questionnaire that was modified from the questionnaire previously used by Gold et al., (2001) and Smith (2006). The authors used one-way ANOVA, t-tests and OLS regressions. The results indicated that both knowledge infrastructure capability (KIC) and knowledge process capability (KPC) have a positive effect on business performance (BP). The results also indicate that organizations with well-developed training and development plans have significantly higher KIC and KPC scores compared to those that do not have such plans

Modulation of macrophage functions by sheeppox virus provides clues to understand interaction of the virus with host immune system

BACKGROUND: Poxviruses encode a range of immunomodulatory genes to subvert or evade the challenges posed by the innate and adaptive immune responses. However, the inactivated poxviruses possessed immunostimulating capacity and were used as a prophylactic or metaphylactic application that efficiently reduced susceptibility to infectious diseases in different species. This fact is intensively studied in different genera of poxviruses. However, little is known about the basic mechanisms adopted by sheeppox virus (SPPV). SPPV causes an acute disease of sheep that recently, has been observed to reinfect its host in spite of vaccination. RESULTS: By injecting inactivated or attenuated sheeppox virus SPPV vaccine in adult male Swiss mice, SPPV was found to reduce macrophages' functions in a local event that occurs at the site of application 12 h after vaccine administration as indicated by increased level of IL-10 and decreased level of SOD from cultured peritoneal macrophages. In contrast increased levels of IL-12, and SOD activity from cultured splenic macrophages, lymphocyte response to PHA-P, and in-vivo response to T-dependant Ag were detected. These effects were observed in both attenuated and inactivated SPPV, but more prominent in attenuated one. CONCLUSION: The results of this study help to elucidate, the phenomenon of existence natural SPPV infections in sheep instead of vaccination and the basic mechanisms responsible for the immunostimulating capacity of sheeppox virus. Locally, SPPV shows evidence for an immune escape mechanism that alleviates the host's immune response. Later and systemically, the virus protects the host from any fatal consequences of the immune system suppression

Influence of using Straight and Twisted Elliptical Section Heater Tubes on Stirling Engine Performance

The heat transfer area of the heater tubes is a significant factor that deeply affects net output power and thermal efficiency in Stirling engines. It is greatly affected by the input heat transfer rate, heater tube geometries, and heat transfer removal rate. The alpha Stirling engine heater is our concern in this study. An ordinary circular and straight elliptical section heater tubes having different twisting ratios with a rectangular section-connecting duct are used to study the different heater tube configurations and twisting effect on the heat transfer characteristics and working fluid motion inside the engine. Three twisting ratios of two, three, and four with each section of the heater tube are used in this study. The 3D simulation model using the SST K-ω model using ANSYS FLUENT-16 is used for simulating airflow through the hot cylinder, heater tubes, regenerator, cooler, and cold cylinder of the Stirling engine, during a complete engine cycle. The results showed that increasing the twisting value increases the net output power and the thermal efficiency. The maximum net power output occurs at the elliptical section heater tube with a two-twist ratio with a value of 1249.26 W by an increase of 86.90 W with respect to the ordinary circular heater. In addition, the maximum thermal efficiency occurs at the elliptical section heater tube with a two-twist ratio with a value of 29.55% by an increase of 1.07% with respect to the ordinary circular heater

Impact of Hydroxychloroquine on Fructose-induced Metabolic Syndrome in Rats: Promising Protective Effect

BACKGROUND: Hydroxychloroquine (HCQ) is used in the treatment of malaria and rheumatoid arthritis for a long time. Its effects on inflammation and immune modulation were noted. AIM: This study aims to investigate the effects of HCQ in fructose-induced metabolic syndrome and to explore its possible mechanisms. METHODS AND MATERIALS: Sixty male Sprague-Dawley rats were divided into Group I (negative control), Group II fed on high-fructose diet, and Group III fed on high fructose and subdivided into Group III-a (HCQ 50 mg/kg), Group III-b (HCQ 100 mg/kg), Group III-c (HCQ 200 mg/kg), and Group III-d (metformin 100 mg/kg). Body weight, blood glucose, liver enzymes, and lipid profile were measured. Insulin level, homeostatic model assessment (HOMA), soluble-intercellular adhesion molecule, and vascular cell adhesion molecule were assayed. Tumor necrosis factor (TNF)-α, adipokines (leptin, resistin, and adiponectin), and histological examination of pancreas were assessed. RESULTS: HCQ induces good effects on lipid profile and improves significantly HOMA, endothelial stress markers, and adiponectin, and reduces leptin and TNF-α levels. In addition, significant improvement in structural changes was noted in pancreas with different doses of HCQ. CONCLUSION: Favorable effects of HCQ in fructose-induced metabolic syndrome are promising and can be used early in those at risk of diabetes

A ring-connected dual active bridge based DC-DC multiport converter for EV fast-charging stations

This paper proposes a multiport DC-DC converter for EV fast-charging stations. The proposed converter is comprised of Ring-Connected Dual Active Bridge (RCDAB) DC-DC converters, where the connection point between every two adjacent DABs provides a DC port. Bypass switches are added to each DAB to eliminate unnecessary power processing stages in the event of idle ports (no EVs) (open circuit ports). The nature of the ring connection of the RCDAB theoretically allows infinite internal power flow solutions within the ring to satisfy a certain power flow scenario at the DC ports, hence, the optimal power flow solution can be selected to minimize total RMS current and losses. Single-phase shift control is applied to this optimization problem to make it simple. A novel closed-loop control scheme using Bisection optimization is developed to minimize the total RMS current. A control-hardware-in-the-loop (CHiL) validation is carried out for a 5-port network of the proposed topology to investigate the converter efficiency and fault tolerance/availability characteristics. Also, an experimental hardware validation is implemented for a 3-port network where different scenarios for power flow and faults are performed. Finally, a comparative discussion between the proposed topology and other multiport topologies in literature is presented revealing the superior performance of the RCDAB topology

Role of CD133 in colorectal cancer

CD133 is a pentaspan transmembrane glycoprotein of ~120 kDa, which was initially used to identify haematopoietic stem cells and, later on, used for the isolation and study of cancer stem cells in many different types of solid tumour including colorectal cancer. Although CD133 expressing cells are thought to represent cancer stem cells, little is known about the exact role of CD133 and the molecular mechanisms underlying control of CD133 expression. This project sought to investigate these questions in colorectal cancer. Initially the expression of CD133 was tested by immunohistochemistry in a two tissue microarray (TMA) sets consisting of (a) 449 cases of primary colorectal cancer, and (b) 45 cases of primary and matched liver metastases. High CD133 expression was marginally associated with shorter overall survival (OS) (p=0.05, Log-rank test) but no difference in expression was found between primary tumours and corresponding metastases. Next, the functional activity of CD133 was evaluated in colorectal cancer (CRC) cell lines by knockdown in cell lines with high CD133 expression. In order to identify appropriate cell lines, the expression of CD133 was tested by quantitative RT -PCR in a series of 29 CRC cell lines and 10 samples of normal mucosa and, in selected cell lines, validated by testing for protein expression by flow cytometry. CD133 mRNA was expressed in 24/29 colorectal cancer cell lines with a heterogenous level of expression. 10 cell lines were chosen on the basis of CD133 mRNA expression level to assess the protein level. CD133 mRNA and protein expression were generally correlated (rs = 0.831, p= 0.003, spearman rank correlation coefficient test) although, interestingly, CD133 mRNA level was higher in normal samples compared with that in cancer cell lines and was significantly higher in cell lines derived from metastatic sites than those derived from site of primary tumour (p=0.009; Mann-whitney test). In addition, it was noted that many cell lines had a stable biphasic phenotype containing CD133+ and CD133- cell populations. This allowed functional analysis of CD133 by sorting the two populations. HT29 was identified as a high expresser of CD133 (95%) and was used for gene-knockdown studies, SW480 had a biphasic population consisting of 42% CD133+ cells and 58% CD133- cells and each population was isolated by cell sorting before functional analysis. Functional assays included proliferation, migration, colony formation and staurosporine induced apoptosis assays. These showed that CD133 expressing cells had greater cell motility (p= 0.04, and p = 0.03, unpaired t-test, for knocked down cells and sorted populations respectively) , enhanced colony forming abilities (p=0.0001, and p=0.003, unpaired t-test for 2D and 3D colony formation respectively using sorted populations only), and increased resistance to staurosporine induced apoptosis (p=0.01, and p=0.008, unpaired t-test, for knocked down and sorted populations respectively) than CD133 negative counterparts. In addition, sorted monophasic populations reverted to a biphasic state in both CD133+/- populations from SW480. Further studies demonstrated that CD133-induced cell motility was independent of E-cadherin, β-catenin, and suggestive of not being regulated by Cten or Wnt, but further work is warranted to verify these results. In addition, regulation of CD133 was partly dependent on STAT3 signalling and on CD133 promoter methylation. Levels of mRNA of some stem cell related genes such as KLF-4, Musashi-1, OCT4, Nanog, and Lgr5 were higher in CD 133 + compared to CD 133 negative cells (p=0.008, p=0.004, p=0.006, p=0.001, and p=0.11; unpaired t-test, respectively) In conclusion, in CRC, CD133 was found to be a significant prognostic factor which enhances cell motility and is associated with features of "stemness". It is a target of ST AT3 signalling and partly regulated by promoter methylation. More in depth studies are warranted to discover the downstream and upstream targets of CD133 before translating these preclinical and laboratory investigations into clinical management of colorectal cancer

S1 gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in Egypt

BACKGROUND: Infectious bronchitis is highly contagious and constitutes one of the most common and difficult poultry diseases to control. IBV is endemic in probably all countries that raise chickens. It exists as dozens of serotypes/genotypes. Only a few amino acid differences in the S1 protein of vaccine and challenge strains of IBV may result in poor protection. Tropism of IBV includes the respiratory tract tissues, proventriculus and caecal tonsils of the alimentary tract, the oviduct and the kidney. RESULTS: Infectious bronchitis virus (IBV) strain closely related to Massachusetts (Mass) serotype was isolated from broiler chickens suffering from severe renal and respiratory distresses. The isolate was serologically identified by Dot-ELISA and further characterized by RT-PCR then genotyped using S1 gene sequence analysis. Alignment of the S1 sequence of the isolate with 16 IBV strains revealed high homology to isolates related to Mass serotype. Inoculation with the strain reproduced the disease in experimental 1-day-old chickens and resulted in 20% mortality, severe renal and moderate respiratory distresses. Marked histopathological changes in both kidney and trachea were observed in experimentally infected chickens. A protection study using the H120 live attenuated vaccine showed low protection rate in spite of high S1 sequence homology (97%). Protection based criteria were: virus re-isolation attempts from trachea, tracheal and renal histopathology as well as IBV antigens detection by immunofluorescent antibody technique in kidney sections. CONCLUSION: Periodical evaluation of cross-protective capabilities of IBV vaccine(s) versus recently recovered field isolates should be performed to ensure optimum control of IBV