Triclosan administration to humanized UDP-glucuronosyltransferase 1 neonatal mice induces UGT1A1 through a dependence on PPARα and ATF4.

Triclosan (TCS) is an antimicrobial toxicant found in a myriad of consumer products and has been detected in human tissues, including breastmilk. We have evaluated the impact of lactational TCS on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) neonatal mice. In hUGT1 mice, expression of the hepatic UGT1A1 gene is developmentally delayed resulting in elevated total serum bilirubin (TSB) levels. We found that newborn hUGT1 mice breastfed or orally treated with TCS presented lower TSB levels along with induction of hepatic UGT1A1. Lactational and oral treatment by gavage with TCS leads to the activation of hepatic nuclear receptors constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor alpha (PPARα), and stress sensor, activating transcription factor 4 (ATF4). When CAR-deficient hUGT1 mice (hUGT1/Car-/-) were treated with TCS, TSB levels were reduced with a robust induction of hepatic UGT1A1, leaving us to conclude that CAR is not tied to UGT1A1 induction. Alternatively, when PPARα-deficient hUGT1 mice (hUGT1/Pparα-/-) were treated with TCS, hepatic UGT1A1 was not induced. Additionally, we had previously demonstrated that TCS is a potent inducer of ATF4, a transcriptional factor linked to the integrated stress response. When ATF4 was deleted in liver of hUGT1 mice (hUGT1/Atf4ΔHep) and these mice treated with TCS, we observed superinduction of hepatic UGT1A1. Oxidative stress genes in livers of hUGT1/Atf4ΔHep treated with TCS were increased, suggesting that ATF4 protects liver from excessive oxidative stress. The increase oxidative stress may be associated with superinduction of UGT1A1. The expression of ATF4 in neonatal hUGT1 hepatic tissue may play a role in the developmental repression of UGT1A1.

Oral arsenic administration to humanizedUDP-glucuronosyltransferase1 neonatal mice induces UGT1A1 through a dependence on Nrf2 and PXR.

Inorganic arsenic (iAs) is an environmental toxicant that can lead to severe health consequences, which can be exacerbated if exposure occurs early in development. Here, we evaluated the impact of oral iAs treatment on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) mice. We found that oral administration of iAs to neonatal hUGT1 mice that display severe neonatal hyperbilirubinemia leads to induction of intestinal UGT1A1 and a reduction in total serum bilirubin values. Oral iAs administration accelerates neonatal intestinal maturation, an event that is directly associated with UGT1A1 induction. As a reactive oxygen species producer, oral iAs treatment activated the Keap-Nrf2 pathway in the intestinal tract and liver. When Nrf2-deficient hUGT1 mice (hUGT1/Nrf2-/-) were treated with iAs, it was shown that activated Nrf2 contributed significantly toward intestinal maturation and UGT1A1 induction. However, hepatic UGT1A1 was not induced upon iAs exposure. We previously demonstrated that the nuclear receptor PXR represses liver UGT1A1 in neonatal hUGT1 mice. When PXR was deleted in hUGT1 mice (hUGT1/Pxr-/-), derepression of UGT1A1 was evident in both liver and intestinal tissue in neonates. Furthermore, when neonatal hUGT1/Pxr-/- mice were treated with iAs, UGT1A1 was superinduced in both tissues, confirming PXR release derepressed key regulatory elements on the gene that could be activated by iAs exposure. With iAs capable of generating reactive oxygen species in both liver and intestinal tissue, we conclude that PXR deficiency in neonatal hUGT1/Pxr-/- mice allows greater access of activated transcriptional modifiers such as Nrf2 leading to superinduction of UGT1A1.

Single-Molecule Fluorescence Lifetime Imaging Using Wide-Field and Confocal-Laser Scanning Microscopy: A Comparative Analysis.

A recent addition to the toolbox of super-resolution microscopy methods is fluorescence-lifetime single-molecule localization microscopy (FL-SMLM). The synergy of SMLM and fluorescence-lifetime imaging microscopy (FLIM) combines superior image resolution with lifetime information and can be realized using two complementary experimental approaches: confocal-laser scanning microscopy (CLSM) or wide-field microscopy. Here, we systematically and comprehensively compare these two novel FL-SMLM approaches in different spectral regions. For wide-field FL-SMLM, we use a commercial lifetime camera, and for CLSM-based FL-SMLM we employ a home-built system equipped with a rapid scan unit and a single-photon detector. We characterize the performances of the two systems in localizing single emitters in 3D by combining FL-SMLM with metal-induced energy transfer (MIET) for localization along the third dimension and in the lifetime-based multiplexed bioimaging using DNA-PAINT. Finally, we discuss advantages and disadvantages of wide-field and confocal FL-SMLM and provide practical advice on rational FL-SMLM experiment design.

Technological Pathways to Produce Compressed and Highly Pure Hydrogen from Solar Power.

Hydrogen (H2 ) produced from renewables will have a growing impact on the global energy dynamics towards sustainable and carbon-neutral standards. The share of green H2 is still too low to meet the net-zero target, while the demand for high-quality hydrogen continues to rise. These factors amplify the need for economically viable H2 generation technologies. The present article aims at evaluating the existing technologies for high-quality H2 production based on solar energy. Technologies such as water electrolysis, photoelectrochemical and solar thermochemical water splitting, liquid metal reactors and plasma conversion utilize solar power directly or indirectly (as carbon-neutral electrons) and are reviewed from the perspective of their current development level, technical limitations and future potential.

The metabolite p-cresol impairs dendritic development, synaptogenesis, and synapse function in hippocampal neurons: Implications for autism spectrum disorder.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopment disorder resulting from different etiological factors, both genetic and/or environmental. These factors can lead to abnormal neuronal development on dendrite and synaptic function at the central nervous system. Recent studies have shown that a subset of ASD patients display increased circulation levels of the tyrosine metabolite, p-cresol, related to chronic intestinal disorders because of dysbiosis of the intestinal microbiota. In particular, abnormal presence of intestinal Clostridium sp. has been linked to high levels of p-cresol in ASD children younger than 8 years. However, the role of p-cresol during development of the central nervous system is unknown. Here, we evaluated in vitro the effect of p-cresol on neurite outgrowth in N2a and PC12 cell lines and dendritic morphology, synaptic density, neuronal activity, and calcium responses in primary rat hippocampal neurons. p-cresol inhibits neural differentiation and neurites outgrowth in N2a and PC12 neuronal cell lines. In hippocampal neuronal cultures, Sholl's analysis shows a decrease in the dendritic arborization of neurons treated with p-cresol. Synaptic density analyzed with the synaptic markers Piccolo and Shank2 is diminished in hippocampal neurons treated with p-cresol. Electrically evoked intracellular calcium rise was drastically, but reversely, blocked by p-cresol, whereas that spontaneous neuronal activity was severely affected by early addition of the metabolite. These findings show that p-cresol alters dendrite development, synaptogenesis, and synapse function of neurons in culture, therefore, neuronal alterations occurring in ASD children may be related to this metabolite and dysbiosis of the intestinal microbiota.

Intersexual Twins due to Tetragametic Chimerism.

Disorders of or differences in sexual development (DSD) are defined by congenital conditions in which development of chromosomal, gonadal, or anatomic sex are atypical. Here, we report on monochorionic diamniotic twins delivered by caesarean section in the 36th week of pregnancy. Monochorionic twins are usually monozygous and thus should have the same sexual differentiation. In this case, one twin had female external genitalia, while the other showed ambiguous genitalia. At first, a diagnosis of mixed gonadal dysgenesis was proposed because of the obvious sexual discrepancy between the supposedly monozygous twins. Cytogenetic analyses were performed to assure the sex chromosome status for both children. Male and female cells were found subsequently in both children. While hematopoietic chimerism of monochorionic dizygous twins as a result of twin-to-twin blood transfusion is a rare but already well-documented phenomenon, to our knowledge this is the first case description of tetragametic chimerism that led to intersexuality.

Intestinal UDP-Glucuronosyltransferase 1A1 and Protection against Irinotecan-Induced Toxicity in a Novel UDP-Glucuronosyltransferase 1A1 Tissue-Specific Humanized Mouse Model.

The human UDP-glucuronosyltransferases (UGTs) represent an important family of drug-metabolizing enzymes, with UGT1A1 targeting the conjugation and detoxification of many exogenous substances, including pharmaceutical drugs. In this study we generated humanized UGT1A1 mice expressing the human UGT1A1 gene in either liver (hUGT1A1HEP ) or intestine (hUGT1A1GI ), enabling experiments to examine tissue-specific properties of UGT1A1-specific glucuronidation. Hepatic and intestinal tissue-specific expression and function of UGT1A1 were demonstrated. Although the liver is considered a major organ for detoxification, intestinal UGT1A1 is an important contributor for drug clearance. Mice were challenged with irinotecan (CPT-11), a prodrug hydrolyzed by carboxylesterases to form the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) and detoxified by UGT1A1. Humanized UGT1A1HEP mice that have no intestinal UGT1A1 displayed a greater lethality rate when exposed to CPT-11 than hUGT1A1GI mice. When exposed to a low dose of CPT-11 (10 mg/kg), hUGT1A1HEP mice displayed greater intestinal inflammatory (IL-1ß and IL-6) insult in addition to p53-triggered apoptotic responses. In vitro studies with intestinal crypt organoids exposed to CPT-11 confirmed the results observed in vivo and indicated that CPT-11 impacts stemness, apoptosis, and endoplasmic reticulum (ER) stress in organoids deficient in UGT1A1. When we examined the induction of ER stress in organoids with thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase, apoptosis and the caspase surge that occurred in hUGT1A1HEP mice were blocked in hUGT1A1GI organoids. This study reveals the importance of intestinal UGT1A1 in preventing inflammation, apoptosis, and loss of stemness capacity upon systemic challenge with an important chemotherapeutic agent. SIGNIFICANCE STATEMENT: Hepatic and intestinal UGT1A1 play a key role in the metabolism and detoxification of endogenous and exogenous compounds. The use of tissue-specific humanized models expressing UGT1A1 in liver or intestine has confirmed the relevance of the intestinal tract in the detoxification of irinotecan. Mechanistic studies using intestinal organoids highlighted the importance of UGT1A1 in reducing inflammation, apoptosis, and loss of stemness. These new models provide valuable tools for studying tissue-specific glucuronidation of substances that are metabolized by human UGT1A1.

Scale up of Transmembrane NADH Oxidation in Synthetic Giant Vesicles.

The transfer of electrons across and along biological membranes drives the cellular energetics. In the context of artificial cells, it can be mimicked by minimal means, while using synthetic alternatives of the phospholipid bilayer and the electron-transducing proteins. Furthermore, the scaling up to biologically relevant and optically accessible dimensions may provide further insight and allow assessment of individual events but has been rarely attempted so far. Here, we visualized the mediated transmembrane oxidation of encapsulated NADH in giant unilamellar vesicles via confocal laser scanning and time-correlated single photon counting wide-field microscopy. To this end, we first augmented phospholipid membranes with an amphiphilic copolymer in order to check its influence on the oxidation kinetics spectrophotometrically. Then, we scaled up the compartments and followed the process microscopically.

Regulation of Intestinal UDP-Glucuronosyltransferase 1A1 by the Farnesoid X Receptor Agonist Obeticholic Acid Is Controlled by Constitutive Androstane Receptor through Intestinal Maturation.

UDP-glucuronosyltransferase (UGT) 1A1 is the only transferase capable of conjugating serum bilirubin. However, temporal delay in the development of the UGT1A1 gene leads to an accumulation of serum bilirubin in newborn children. Neonatal humanized UGT1 (hUGT1) mice, which accumulate severe levels of total serum bilirubin (TSB), were treated by oral gavage with obeticholic acid (OCA), a potent FXR agonist. OCA treatment led to dramatic reduction in TSB levels. Analysis of UGT1A1 expression confirmed that OCA induced intestinal and not hepatic UGT1A1. Interestingly, Cyp2b10, a target gene of the nuclear receptor CAR, was also induced by OCA in intestinal tissue. In neonatal hUGT1/Car -/- mice, OCA was unable to induce CYP2B10 and UGT1A1, confirming that CAR and not FXR is involved in the induction of intestinal UGT1A1. However, OCA did induce FXR target genes, such as Shp, in both intestines and liver with induction of Fgf15 in intestinal tissue. Circulating FGF15 activates hepatic FXR and, together with hepatic Shp, blocks Cyp7a1 and Cyp7b1 gene expression, key enzymes in bile acid metabolism. Importantly, the administration of OCA in neonatal hUGT1 mice accelerates intestinal epithelial cell maturation, which directly impacts on induction of the UGT1A1 gene and the reduction in TSB levels. Accelerated intestinal maturation is directly controlled by CAR, since induction of enterocyte marker genes sucrase-isomaltase, alkaline phosphatase 3, and keratin 20 by OCA does not occur in hUGT1/Car -/- mice. Thus, new findings link an important role for CAR in intestinal UGT1A1 induction and its role in the intestinal maturation pathway. SIGNIFICANCE STATEMENT: Obeticholic acid (OCA) activates FXR target genes in both liver and intestinal tissues while inducing intestinal UGT1A1, which leads to the elimination of serum bilirubin in humanized UGT1 mice. However, the induction of intestinal UGT1A1 and the elimination of bilirubin by OCA is driven entirely by activation of intestinal CAR and not FXR. The elimination of serum bilirubin is based on a CAR-dependent mechanism that facilitates the acceleration of intestinal epithelium cell differentiation, an event that underlies the induction of intestinal UGT1A1.

Biomarker responses induced by bisphenol A on spermatogenesis in a Neotropical teleost fish are temperature-dependent.

Bisphenol A (BPA) is an organic synthetic compound used in the plastic industry with endocrine disrupting activity. Although it is frequently found in surface waters, few studies have investigated its impact on fish gametogenesis, particularly when associated with natural stressors. In this regard, the present study evaluated BPA toxicity on spermatogenesis in the lambari Astyanax bimaculatus under controlled conditions and its interactive effects with water temperature. Adult specimens were exposed in duplicate to 40 µg/L and 400 µg/L BPA at 23 °C and 28 °C for 21 days; the control group did not receive BPA. Testicular samples were collected and analyzed using different cellular and molecular techniques. The results showed a significant reduction in the gonadosomatic index in the BPA-treated groups at both temperatures. A decrease in the testicular levels of 11-ketotestosterone was observed in the 400 µg/L BPA group at 23 °C, 17ß-estradiol increased significantly in the treated groups at 28 °C, and vitellogenin showed no difference between the treatments. The morphometric analysis of spermatogenesis revealed a significant increase in the proportion of spermatogonia, spermatocytes, and Sertoli cells in the treated groups, with a higher proportion at 23 °C than at 28 °C. Otherwise, the proportion of spermatozoa was significantly lower in the BPA-treated groups, with a greater reduction at 23 °C. In addition, BPA also stimulated spermatogonial proliferation in the treated groups, but apoptosis was significantly increased in spermatids at 23 °C. Testis-ova, cell degeneration, and chromatin alterations in spermatids and Sertoli cells were observed in the germinal epithelium of the BPA-treated groups. The integrated biomarker response (IBR) index revealed that the analyzed endpoints are suitable for assessing estrogenic contamination. Taken together, our results indicate that the interactive effects of BPA and temperature contribute to the impairment of spermatogenesis in A. bimaculatus with more severe effects observed on sperm production at 23 °C than at 28 °C.

Evaluation of the oestrogenic potential of oestrone and bisphenol-A on the reproduction of Astyanax bimaculatus males after subacute exposure.

In the last decades, oestrogenic compounds have often been reported in environmentally relevant concentrations in aquatic environments around the world. Most laboratory studies of oestrogens try to understand the effects of a single contaminant, but in natural environments, the effects may be quite different due to interactions with other compounds. The present study aimed to compare the action of oestrone (E1) and bisphenol-A (BPA), acting singularly and in combination, on the spermatogenesis of Astyanax bimaculatus. After exposure to 100 ng/L of E1, BPA and a mixture of the two for 15 days, our results showed that E1 and the E1 + BPA mixture significantly altered the number of spermatogenic cells. BPA presented high cytotoxicity when compared to other treatments. Analysis of the two oestrogenic compounds suggests that the E1 + BPA mixture has no additive or synergistic effects. Together, the results of the present study indicate that endocrine-disrupting chemicals (EDCs) analysed alone may behave differently than when administered with other substances.

Impact of Bone Cement Augmentation on the Fixation Strength of TFNA Blades and Screws.

Background and Objectives: Hip fractures constitute the most debilitating complication of osteoporosis with steadily increasing incidences in the aging population. Their intramedullary nailing can be challenging because of poor anchorage in the osteoporotic femoral head. Cement augmentation of Proximal Femoral Nail Antirotation (PFNA) blades demonstrated promising results by enhancing cut-out resistance in proximal femoral fractures. The aim of this study was to assess the impact of augmentation on the fixation strength of TFN-ADVANCEDTM Proximal Femoral Nailing System (TFNA) blades and screws within the femoral head and compare its effect when they are implanted in centre or anteroposterior off-centre position. Materials and Methods: Eight groups were formed out of 96 polyurethane low-density foam specimens simulating isolated femoral heads with poor bone quality. The specimens in each group were implanted with either non-augmented or cement-augmented TFNA blades or screws in centre or anteroposterior off-centre positions, 7 mm anterior or posterior. Mechanical testing was performed under progressively increasing cyclic loading until failure, in setup simulating an unstable pertrochanteric fracture with a lack of posteromedial support and load sharing at the fracture gap. Varus-valgus and head rotation angles were monitored. A varus collapse of 5° or 10° head rotation was defined as a clinically relevant failure. Results: Failure load (N) for specimens with augmented TFNA head elements (screw/blade centre: 3799 ± 326/3228 ± 478; screw/blade off-centre: 2680 ± 182/2591 ± 244) was significantly higher compared with respective non-augmented specimens (screw/blade centre: 1593 ± 120/1489 ± 41; screw/blade off-centre: 515 ± 73/1018 ± 48), p < 0.001. For both non-augmented and augmented specimens failure load in the centre position was significantly higher compared with the respective off-centre positions, regardless of the head element type, p < 0.001. Augmented off-centre TFNA head elements had significantly higher failure load compared with non-augmented centrally placed implants, p < 0.001. Conclusions: Cement augmentation clearly enhances the fixation stability of TFNA blades and screws. Non-augmented blades outperformed screws in the anteroposterior off-centre position. Positioning of TFNA blades in the femoral head is more forgiving than TFNA screws in terms of failure load.

Wide-Field Fluorescence Lifetime Imaging of Single Molecules.

Fluorescence lifetime imaging (FLIM) has become an important microscopy technique in bioimaging. The two most important of its applications are lifetime-multiplexing for imaging many different structures in parallel, and lifetime-based measurements of Förster resonance energy transfer. There are two principal FLIM techniques, one based on confocal-laser scanning microscopy (CLSM) and time-correlated single-photon counting (TCSPC) and the other based on wide-field microscopy and phase fluorometry. Although the first approach (CLSM-TCSPC) assures high sensitivity and allows one to detect single molecules, it is slow and has a small photon yield. The second allows, in principal, high frame rates (by 2-3 orders of magnitude faster than CLSM), but it suffers from low sensitivity, which precludes its application for single-molecule imaging. Here, we demonstrate that a novel wide-field TCSPC camera (LINCam25, Photonscore GmbH) can be successfully used for single-molecule FLIM, although its quantum yield of detection in the red spectral region is only ∼5%. This is due to the virtually absent background and readout noise of the camera, assuring high signal-to-noise ratio even at low detection efficiency. We performed single-molecule FLIM of different red fluorophores, and we use the lifetime information for successfully distinguishing between different molecular species. Finally, we demonstrate single-molecule metal-induced energy transfer (MIET) imaging which is a first step for three-dimensional single-molecule localization microscopy (SMLM) with nanometer resolution.

Insulin-like growth factors 1 and 2 are associated with testicular germ cell proliferation and apoptosis during fish reproduction.

To support sperm production, fish testes undergo intense tissue remodelling, with endocrine, paracrine and autocrine signals regulating gonad physiology. The aim of this study was to investigate the testicular expression of insulin-like growth factor (Igf) 1 and Igf2 during spermatogenesis, and their relationship with cell proliferation and apoptosis throughout the reproductive cycle. The study was performed in male Hypostomus garmani, a catfish living in headwater rivers of the São Francisco River basin, Brazil. Spermatogenesis was analysed using histology, morphometry, immunohistochemistry and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) analysis at different maturity stages. The results showed the proliferation of spermatogonia throughout the reproductive cycle, with a higher rate during the ripe stage. Germ and Sertoli cells expressed Igf1 at all stages of testicular maturity, Igf2 was predominant at the ripe stage and both Igf1 and Igf2 occurred at the spent stage. Caspase-3 and TUNEL analysis revealed a higher rate of apoptosis at the spent stage associated with reduced expression of Igf1 and Igf2. Sertoli cell proliferation was associated with spermatogonia and spermatocyte cysts at different stages of the reproductive cycle. Together, the data support a proliferative role for Igf1 and Igf2 in regulating testicular apoptosis in H. garmani, with cyclical variation in their expression during gonad maturation.

Effects of metal contamination on liver in two fish species from a highly impacted neotropical river: A case study of the Fundão dam, Brazil.

Environmental disasters such as the rupturing of mine tailings dams are a major concern worldwide. In the present study, we assess the effects of the release of mine waste due to the rupture of the Fundão dam on two native fish species (Hoplias intermedius and Hypostomus affinis) from the Doce River basin. Two sampling sites were chosen: S1, a reference site, and S2, contaminated by mining waste. Water and sediment were collected to evaluate metals concentration. Adult fish were caught to analyse biological parameters, hepatic histopathology, and biomarkers of metal contamination. Compared to site S1, the concentration of manganese was statistically higher in water while lead, nickel, and arsenic were statistically higher in the sediment from site S2, and iron had no significant difference between sites. At site S1, fish of both species presented hepatic tissue with normal architecture. At site S2, hepatic alterations, such as cytoplasmic vacuolization and necrosis were frequently found in both species. Regarding the histopathological index, higher values were found in both species from site S2. The positive antibody reactions for cytochrome P450 1A (CYP1A) and metallothionein (MT) were statistically greater in site S2 for both species. The oxidative stress biomarkers, superoxide dismutase (SOD) and catalase (CAT) were statistically higher in H. intermedius from site S2, but only CAT was statistically greater in H. affinis at site S2. These results demonstrate that the release of mineral residues from the rupture of the Samarco mine dam is provoking hepatic damage in the fish from the Doce River besides inducing the expression of proteins and enzymes related to metal contamination.

Environmental exposure to oestrogenic endocrine disruptors mixtures reflecting on gonadal sex steroids and gametogenesis of the neotropical fishAstyanax rivularis.

Discharge of municipal wastewater promotes the entry of diverse oestrogenic compounds into the water bodies. This complex mixture of substances interferes in the steroidogenic pathway, being able to promote severe reproductive impairment in freshwater fish populations. The purpose of the present study was to evaluate the effects of oestrogenic endocrine disruptors (EDCs) mixture on gonadal sex steroids (testosterone, T; 11-ketotestosterone, 11-KT; 17ß-oestradiol, E2; 17-hydroxyprogesterone, 17-OHP) in the peak of the reproductive season of Astyanax rivularis, correlating the results obtained with the proportion of germ cells and gonadal histopathology. Three sampling sites were chosen to conduct the study, one reference site (S1), without contamination by municipal wastewater and two sites (S2 and S3) receiving discharge of municipal wastewater. Males of A. rivularis presented higher concentrations of E2, lower androgens (T and 11-KT) in gonads when compared to males from site S1. Concentrations of 17-OHP did not present significant difference among sites. In sites S2 and S3, the proportion of early spermatocytes, spermatids and Leydig cells increased while spermatozoa decreased compared to fish from S1. The following gonadal histopathologies were detected in the male fishes: intersex gonads (28% in S3) and testicular degeneration with germinal epithelium exhibiting agglutinated germ cells masses and empty cysts (57% in S2 and 71% in S3). In females, concentrations of T, E2 and 17-OHP did not present significant difference among the sites, however higher 11-KT concentrations were detected in females from sites S2 and S3. A lower proportion of perinucleolar follicles and a higher incidence of vitellogenic follicles, besides, aged oocytes and the presence of eosinophilic proteinaceous fluid in the interstitial compartment were also found in females from impacted sites. These results indicate that the urbanization and consequent release of municipal wastewater containing oestrogenic compounds in the headwater creeks are altering the levels of sex hormones and gametogenesis of A. rivularis. Further studies should be performed to determine whether oestrogenic endocrine disrupters are disrupting the reproduction of A. rivularis.

Expression patterns and immunolocalisation of IGF-I and IGF-II in male and female gonads of the Neotropical characid fish Astyanax fasciatus.

The insulin-like growth factor (IGF) system plays important roles in fish reproduction, but the expression pattern and cellular location of IGF-I and IGF-II during gonadal maturation are uncertain. The present study reports a stage-specific assessment of gonadal expression levels and immunolocalisation of IGF-I and IGF-II in Astyanax fasciatus, a characid fish from South America. Adult fish in different maturity stages were caught in the Furnas Reservoir, Grande River, Brazil. Gonad samples were processed for histology, immunohistochemistry, and ELISA for IGF-I and IGF-II. Ovarian levels of IGF-I were low during ripening and ripe stages, higher in totally spent, and then decreased in resting. Levels of IGF-II increased during ovarian maturation, reaching significantly higher values at stage totally spent. In males, IGF-I levels followed gonadal maturation, with higher values in ripening and ripe stages, whereas IGF-II levels showed higher values in stage ripening and partially spent. A positive correlation was found between IGF-I and gonadosomatic index (GSI) for males (r = 0.59), while females showed a negative correlation (r = - 0.43), but IGF-II showed no correlation to GSI. IGF-I was expressed mainly in oogonia nests whereas IGF-II stained the follicular cells in the perinucleolar follicles, cortical vesicles in the previtellogenic follicles, and oogonia nests. In males, IGF-I was evident in spermatogonia and spermatocytes while IGF-II stained Sertoli cells surrounding spermatids cysts and spermatogonia in late stages. Together, these findings support a hypothesis that the balance between IGF-I and IGF-II levels is important in the regulation of gonad maturation in Astyanax fasciatus.

Establishment of a Human Immunocompetent 3D Tissue Model to Enable the Long-Term Examination of Biofilm-Tissue Interactions.

Different studies suggest an impact of biofilms on carcinogenic lesion formation in varying human tissues. However, the mechanisms of cancer formation are difficult to examine in vivo as well as in vitro. Cell culture approaches, in most cases, are unable to keep a bacterial steady state without any overgrowth. In our approach, we aimed to develop an immunocompetent 3D tissue model which can mitigate bacterial outgrowth. We established a three-dimensional (3D) co-culture of human primary fibroblasts with pre-differentiated THP-1-derived macrophages on an SIS-muc scaffold which was derived by decellularisation of a porcine intestine. After establishment, we exposed the tissue models to define the biofilms of the Pseudomonas spec. and Staphylococcus spec. cultivated on implant mesh material. After 3 days of incubation, the cell culture medium in models with M0 and M2 pre-differentiated macrophages presented a noticeable turbidity, while models with M1 macrophages presented no noticeable bacterial growth. These results were validated by optical density measurements and a streak test. Immunohistology and immunofluorescent staining of the tissue presented a positive impact of the M1 macrophages on the structural integrity of the tissue model. Furthermore, multiplex ELISA highlighted the increased release of inflammatory cytokines for all the three model types, suggesting the immunocompetence of the developed model. Overall, in this proof-of-principle study, we were able to mitigate bacterial overgrowth and prepared a first step for the development of more complex 3D tissue models to understand the impact of biofilms on carcinogenic lesion formation.

Oocyte adhesiveness and embryonic development of Astyanax bimaculatus (Linnaeus, 1758) (Pisces: Characidae).

This study shows for the first time the presence of a jelly coat on oocytes of neotropical Characiformes fish. This structure could be responsible for the adhesiveness of Astyanax bimaculatus oocytes, a species widely distributed in South America including in the São Francisco River basin in Brazil. Adult specimens of A. bimaculatus were submitted to artificial reproduction in order to analyse the egg morphology and embryonic development. The eggs were fertilised and kept in incubators with a water temperature of 24°C so that embryogenesis could be monitored. Ovulated and unfertilised oocytes were also collected and submitted to routine histological techniques. Astyanax bimaculatus oocytes were found to be spherical, yellowish, and covered by a thin jelly coat with a slightly adhesive surface. The mean oocyte diameter was 1.03 ± 0.03 mm, the perivitelline space was 0.21 ± 0.02 mm and the jelly coat's thickness was 0.04 ± 0.01 mm. Positive periodic acid-Schiff (PAS) stain and Alcian blue stain pH 2.5 indicated the presence of neutral glycoproteins, and carboxylated acid glycoconjugates on the jelly coat that formed mucosubstances that may be associated with egg adhesiveness. At a water temperature of 24°C, blastopore closure and hatching occurred at 5 h and 17 h after fertilisation, respectively. The results of this study provide essential information for phylogenetic studies and for a better understanding of the reproductive strategy of A. bimaculatus, currently included in the incertae sedis group of the Characidae family due to the lack of monophyly among the families of the group.

Chronic estrone exposure affects spermatogenesis and sperm quality in zebrafish (Danio rerio).

Estrone (E1) is a common environmental contaminant found in rivers and streams due to the farming of animals, such as swine and cattle. Our study evaluated the effects of chronic E1 exposure at environmentally relevant concentrations on spermatogenesis and the semen quality of zebrafish (Danio rerio). We exposed the fish to E1 at concentrations of 20, 200, and 2000 ng/L diluted in 0.001% ethanol (v/v) for 49 days. There were two control groups: one was exposed to water only and the other to ethanol at the same concentration used in the E1 groups. Following exposure, we analyzed the proportion of testicular cell types and other components (%), rate of cell proliferation and death, and sex steroid concentrations. Furthermore, we analyzed the expression of insulin-like growth factor 1 (IGF1), IGF2, IGF1 receptor (IGF1R), and inducible nitric oxide synthase and assessed the semen quality. E1 exposure increased spermatogonia, spermatids, Sertoli cells, Leydig cells, and the proportion of inflammatory infiltrate but decreased the spermatozoa amount. These changes were reflected by reductions in the gonadosomatic index and levels of 11-ketotestosterone in the testes. On the other hand, E1 exposure increased testicular estradiol, IGF1R expression, and nitric oxide production. After an evaluation using a computer-assisted sperm analysis (CASA) system, we observed reduced progressive motility, curvilinear velocity, and beat cross frequency of 20 and 2000 ng/L E1 groups. Our findings support that E1 causes deleterious effects on the testicular function and semen quality of D. rerio even at environmental concentrations. Thus, E1 concentrations should be monitored in surface waters for the purposes of fish conservation.