Uncovering microplastics contamination in canned seafood.

There is limited research on the occurrence of microplastics (MPs) in canned seafood. All types of canned seafood investigated in the present study were contaminated. After sample digestion in 30 % hydrogen peroxide, a total of 40 MPs were recovered. Fibers were the most common type, blue was the dominant colour, and Fourier Transform Infrared Spectroscopy (FTIR) identified polyester as the most common polymer. Considering all samples, an average of 3.5 ± 5.2 MPs/can was obtained, with octopus in tomato sauce and tuna in olive oil presenting the highest contamination (5.2 ± 7.5 MPs/can and 5.2 ± 5.1 MPs/can, respectively). Also, significant differences between the number of MPs in the seafood tissues and immersion liquids were verified. The present study demonstrates MPs occurrence in canned seafood, a potential contamination pathway for humans. More research on the different stages of the canning processing is vital for understanding MPs contamination in cans.

Optimization of an Analytical Protocol for the Extraction of Microplastics from Seafood Samples with Different Levels of Fat.

Marine organisms are affected by the ubiquitous occurrence of microplastics (MPs) in the environment. Several protocols have been described to extract and quantify MPs in seafood, although their complex matrices, with high level of fat, can compromise the efficiency of MPs extraction. To solve this issue, the present study aimed to develop a detailed methodology suitable to process seafood samples with different levels of fat, namely fish and molluscs, from fresh and canned sources, including the immersive liquids from the cans. Sample digestion was tested using different solutions (10% KOH, 30% H2O2), temperatures (40 °C, 65 °C) and incubation times (24, 48, 72 h). For fat removal, three detergents (two laboratory surfactants and a commercial dish detergent) and 96% ethanol were tested, as well as the manual separation of fat. The methodology optimized in this study combined a digestion with 30% H2O2 at 65 °C, during 24 to 48 h, with a manual separation of the fat remaining after the digestion. All steps from the present methodology were tested in six types of polymers (PE-LD, PET, PE, AC, PS, and lycra), to investigate if these procedures altered the integrity of MPs. Results showed that the optimized methodology will allow for the efficient processing of complex seafood samples with different fat levels, without compromising MPs integrity (recoveries rate higher than 89% for all the polymers tested).

Dimethylformamide Preserves the Integrity of Cryopreserved Goat Semen in a Soybean Lecithin-Based Extender.

This study investigated the cryoprotectant effects of dimethylformamide (DMF), ethylene glycol (EG), and dimethyl sulfoxide (DMSO) as substitutes for glycerol (GLY) in a soybean lecithin (SL)-based extender in the cryopreservation of buck sperm. In this study, the semen of three Saanen bucks was individually extended in SL supplemented with 5% GLY (control), DMF, EG, or DMSO. After this, the extended semen was cryopreserved and two straws from each group were thawed (37°C for 30 seconds), pooled, and analyzed for sperm motion parameters, plasma membrane integrity (PMI), acrosomal integrity (ACI), and high mitochondrial membrane potential (HMMP). Samples were analyzed after 15 minutes (T0) and after 2 hours of incubation at 37°C (T2). The results revealed higher values of motility (total and progressive) and sperm motion parameters for DMF than the other cryoprotectants (p < 0.0001). PMI and HMMP did not differ (p > 0.05) between GLY and DMF, but ACI was higher (p < 0.01) for DMF compared with GLY. Based on these results, DMF and GLY samples were used in heterologous in vitro fertilization assays by using bovine oocytes (n = 337) obtained from a slaughterhouse. No differences (p > 0.05) were observed between GLY and DMF for unfertilized (GLY: 38.8%; DMF: 25.33%), pronucleus (GLY: 25.68%; DMF: 27.92%), and cleavage rates (GLY: 35.52%; DMF: 46.75%). Based on these results, it is concluded that DMF preserves sperm motion characteristics and ACI better than GLY, EG, and DMSO, and it is the penetrating cryoprotectant of choice for the cryopreservation of buck sperm in SL extender.

Targeting transglutaminase 2 partially restores extracellular matrix structure but not alveolar architecture in experimental bronchopulmonary dysplasia.

The generation, maturation and remodelling of the extracellular matrix (ECM) are essential for the formation of alveoli during lung development. Alveoli formation is disturbed in preterm infants that develop bronchopulmonary dysplasia (BPD), where collagen fibres are malformed, and perturbations to lung ECM structures may underlie BPD pathogenesis. Malformed ECM structures might result from abnormal protein cross-linking, in part attributable to the increased expression and activity of transglutaminase 2 (TGM2) that have been noted in affected patient lungs, as well as in hyperoxia-based BPD animal models. The objective of the present study was to assess whether TGM2 plays a causal role in normal and aberrant lung alveolarization. Targeted deletion of Tgm2 in C57BL/6J mice increased septal thickness and reduced gas-exchange surface area in otherwise normally developing lungs. During aberrant lung alveolarization that occurred under hyperoxic conditions, collagen structures in Tgm2-/- mice were partially protected from the impact of hyperoxia, where normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance was restored; however, the lung alveolar architecture remained abnormal. Inhibition of transglutaminases (including TGM2) with cysteamine appreciably reduced transglutaminase activity in vivo, as assessed by Nε -(γ-l-glutamyl)-l-lysine abundance and TGM catalytic activity, and restored normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance under pathological conditions. Furthermore, a moderate improvement in alveoli size and gas-exchange surface density was noted in cysteamine-treated mouse lungs in which BPD was modelled. These data indicate that TGM2 plays a role in normal lung alveolarization, and contributes to the formation of aberrant ECM structures during disordered lung alveolarization.

Influência da neoplasia mamária na concentração sérica de hormônios e na expressão de receptores de estrógeno e progesterona em cadelas / Influence of mammary neoplasm on the serum concentration of hormones and on the expression of estrogen and progesterone receptors in bitches

O objetivo desse estudo foi avaliar as concentrações séricas de estradiol, progesterona e prolactina, bem como a expressão gênica dos receptores de estrógeno α e β e de progesterona em cadelas com neoplasias mamárias. Foram utilizadas 60 cadelas adultas, sem raça definida que foram distribuídas em dois grupos. O Grupo I constituído por 30 cadelas portadoras de neoplasias mamárias e o Grupo II constituído por 30 cadelas saudáveis, não portadoras de neoplasia. Para os tutores, foram aplicados questionários sobre fatores epidemiológicos da doença. Após avaliação dos exames pré-operatórios, as cadelas com neoplasia mamária foram submetidas à mastectomia, coletaram-se fragmentos das neoplasias e linfonodos regionais, os quais foram processados para análise histopatológica. Para as dosagens hormonais de estradiol, progesterona e prolactina foram colhidas amostras de sangue em tubos sem anticoagulante e os soros foram submetidos à técnica de eletroquimioluminescência. A expressão gênica dos receptores hormonais foi realizada por meio da técnica de Real-time PCR e para isso foram coletados fragmentos das neoplasias mamárias e extraído o RNA para obtenção do cDNA. A expressão do mRNA para os REα, REβ e RP foi avaliada a partir da amplificação desses genes utilizando primers específicos. Verificaram-se maiores níveis séricos de estradiol (média de 38,98±13,68pg/mL) em cadelas portadoras de neoplasias mamárias malignas quando comparadas as cadelas do grupo controle (p<0,05). Já os níveis séricos de prolactina foram maiores (média de 0,231±0,201ng/mL) nas cadelas que não possuíam neoplasias mamárias quando comparadas ao Grupo I (p<0,05). Para os níveis de progesterona não foram observadas diferença entre os diferentes grupos (p>0,05). Tanto os tumores malignos como os benignos expressaram REα, REβ e RP, não havendo diferença (p>0,05) na expressão entre tumores malignos ou benignos ou relacionada aos outros fatores prognósticos investigados (estadiamento clínico, presença de ulceração, vascularização e tempo de evolução do processo). Os níveis séricos de estradiol aumentaram significativamente com o estadiamento clínico da doença (p<0,05). Verificou-se moderada correlação negativa entre os níveis séricos de estradiol e prolactina. Dessa forma, conclui-se que as dosagens séricas de estradiol e PRL foram influenciadas pela malignidade do tumor e pelo estadiamento clínico das neoplasias. Os receptores hormonais foram expressos pelas neoplasias, independentemente do tipo tumoral e não estão associados aos outros fatores prognóstico clássicos, como presença de ulceração, vascularização ou estadiamento clínico.(AU)

The aim of this study was to evaluate the serum concentrations of estradiol, progesterone, prolactin, the gene expression of estrogen α and β and progesterone receptors in bitches with mammary neoplasms. Sixty adult crossbred bitches distributed in two groups were used. Group I consisted of 30 bitches with mammary neoplasms and Group II consisted of 30 healthy bitches without neoplasia. For the tutors, interviews were made about the disease epidemiology. After preoperative examinations, bitches with mammary neoplasia were submitted to mastectomy; fragments of the neoplasms and regional lymph nodes were collected and processed for histopathological analysis. Blood samples were collected in tubes without anticoagulant and the serum was analyzed by electrochemiluminescence to measure estradiol, progesterone and prolactin. The gene expression of the hormonal receptors was performed by means of the Real-time PCR technique, thus fragments of mammary neoplasms were collected and the RNA was extracted to obtain cDNA. Expression of the mRNA for ERα, ERβ and PR was assessed from the amplification of these genes using specific primers. Higher serum levels of estradiol (mean 38.98±13.68pg/mL) were observed in bitches with malignant neoplasms when compared to the control bitches (p<0.05). Serum prolactin levels were higher (mean of 0.231±0.201ng/mL) in bitches that did not have mammary neoplasms when compared to Group I (p<0.05). No difference was observed for related to the progesterone levels between the groups (p>0.05). Both malignant and benign tumors expressed ERα, ERβ and RP with no statistical difference (p>0.05) and there were no difference related to the other prognostic factors investigated (clinical staging, presence of ulceration, vascularization and aging of neoplasms). Serum estradiol levels increased significantly with the clinical staging of the disease (p<0.05). There was a moderate negative correlation between serum levels of estradiol and prolactin. It was concluded that serum levels of estradiol and PRL were influenced by tumor malignancy and clinical staging of neoplasms. Hormonal receptors were expressed by neoplasms, regardless of tumor type and are not associated with other classical prognostic factors, such as ulceration, vascularization or clinical staging.(AU)

High resolution mass spectrometry-based methodologies for identification of Etravirine bioactivation to reactive metabolites: In vitro and in vivo approaches.

Drug bioactivation to reactive metabolites capable of covalent adduct formation with bionucleophiles is a major cause of drug-induced adverse reactions. Therefore, elucidation of reactive metabolites is essential to unravel the toxicity mechanisms induced by drugs and thereby identify patient subgroups at higher risk. Etravirine (ETR) was the first second-generation Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) to be approved, as a therapeutic option for HIV-infected patients who developed resistance to the first-generation NNRTIs. Additionally, ETR came into market aiming to overcome some adverse effects associated with the previously used efavirenz (neurotoxicity) and nevirapine (hepatotoxicity) therapies. Nonetheless, post-marketing reports of severe ETR-induced skin rash and hypersensitivity reactions have prompted the U.S. FDA to issue a safety alert on ETR. Taking into consideration that ETR usage may increase in the near future, due to the possible use of the drug for coinfection with malaria and HIV, the development of reliable prognostic tools for early risk/benefit estimations is urgent. In the current study, high resolution mass spectrometry-based methodologies were integrated with MS3 experiments for the identification of reactive ETR metabolites/adducts: 1) in vitro incubation of the drug with human and rat liver S9 fractions in the presence of Phase I and II co-factors, including glutathione, as a trapping bionucleophile; and 2) in vivo, using urine samples from HIV-infected patients on ETR therapy. We obtained evidence for multiple bioactivation pathways leading to the formation of covalent adducts with glutathione and N-acetyl-L-cysteine. These results suggest that similar reactions may occur with cysteine residues of proteins, supporting a role for ETR bioactivation in the onset of the toxic effects elicited by the drug. Additionally, ETR metabolites stemming from amine oxidation, with potential toxicological significance, were identified in vitro and in vivo. Also noteworthy is the fact that new metabolic conjugation pathways of glucuronide metabolites were demonstrated for the first time, raising questions about their potential toxicological implications. In conclusion, these results represent not only a contribution towards the elucidation of new metabolic pathways of drugs in general but also an important step towards the elucidation of potentially toxic ETR pathways, whose understanding may be crucial for reliable risk/benefit estimations of ETR-based regimens.

Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in goat ovary and its effect on oocyte nuclear maturation in vitro.

Adiponectin is an adipokine secreted primarily by adipocytes and is involved in the control of male and female reproductive functions. Circulating levels of adiponectin are inversely correlated with body fat mass, and its biological effects are predominantly mediated through two receptors, AdipoR1 and AdipoR2. The aim of the present study was to verify the expression of the adiponectin system (adiponectin and its receptors, AdipoR1 and AdipoR2) in goat ovary using qPCR and immunohistochemistry analyses and further investigate the in vitro effects of recombinant adiponectin (5 µg/mL and 10 µg/mL) on goat oocyte nuclear maturation. We demonstrated that the mRNA and proteins of the adiponectin system are present in goat ovary. Gene and protein expression of AdipoR1 and AdipoR2 was detected in follicular cells (oocyte, cumulus, granulosa and theca) of small and large antral follicles, while adiponectin mRNA was not detected in oocytes from small and large follicles or in large follicle cumulus cells. Finally, addition of various concentrations of adiponectin in maturation medium affected the number of oocytes that reached metaphase II. In conclusion, in the present study, we detected expression of adiponectin and its receptors AdipoR1 and AdipoR2 in goat ovarian follicles. Furthermore, we demonstrated that recombinant adiponectin increases nuclear maturation of goat oocytes in vitro.

Standardisation of oxygen exposure in the development of mouse models for bronchopulmonary dysplasia.

Progress in developing new therapies for bronchopulmonary dysplasia (BPD) is sometimes complicated by the lack of a standardised animal model. Our objective was to develop a robust hyperoxia-based mouse model of BPD that recapitulated the pathological perturbations to lung structure noted in infants with BPD. Newborn mouse pups were exposed to a varying fraction of oxygen in the inspired air (FiO2) and a varying window of hyperoxia exposure, after which lung structure was assessed by design-based stereology with systemic uniform random sampling. The efficacy of a candidate therapeutic intervention using parenteral nutrition was evaluated to demonstrate the utility of the standardised BPD model for drug discovery. An FiO2 of 0.85 for the first 14 days of life decreased total alveoli number and concomitantly increased alveolar septal wall thickness, which are two key histopathological characteristics of BPD. A reduction in FiO2 to 0.60 or 0.40 also caused a decrease in the total alveoli number, but the septal wall thickness was not impacted. Neither a decreasing oxygen gradient (from FiO2 0.85 to 0.21 over the first 14 days of life) nor an oscillation in FiO2 (between 0.85 and 0.40 on a 24 h:24 h cycle) had an appreciable impact on lung development. The risk of missing beneficial effects of therapeutic interventions at FiO2 0.85, using parenteral nutrition as an intervention in the model, was also noted, highlighting the utility of lower FiO2 in selected studies, and underscoring the need to tailor the model employed to the experimental intervention. Thus, a state-of-the-art BPD animal model that recapitulates the two histopathological hallmark perturbations to lung architecture associated with BPD is described. The model presented here, where injurious stimuli have been systematically evaluated, provides a most promising approach for the development of new strategies to drive postnatal lung maturation in affected infants.

Identification and genetic characterization of classical swine fever virus isolates in Brazil: a new subgenotype.

The classical swine fever (CSF) is a highly contagious viral disease of pigs and wild boar. The CSF causes great economic losses for pork production and the occurrence of the disease is notifiable to the OIE. The objective of this work was to identify and characterize CSF virus isolates from Brazil. Seven viral isolates were obtained and the full-length E2 sequences were analyzed. Phylogenetic analysis revealed a different segregation pattern between Brazilian isolates and members of subgenotype 1.1, forming a separate group within genotype 1. Genetic distance analysis suggested the existence of two new subgenotypes, designated subgenotypes 1.5 and 1.6.

N-pentyl-nitrofurantoin induces apoptosis in HL-60 leukemia cell line by upregulating BAX and downregulating BCL-xL gene expression.

BACKGROUND: Nitrofurantoin is a nitroderivative antibiotic that has bactericidal activity against pathogens causing urinary tract infection. A few studies have reported that nitrofurantoin has cytotoxic activity against cancer cells; however, nitrofurans remain a poorly explored class of compounds with respect to their anticancer potential. The aim of this study was to investigate the anticancer effects of a nitrofurantoin derivative, n-pentyl-nitrofurantoin (NFP), on HL-60 leukemia cells. METHODS: Cytotoxicity was assayed by the MTT assay. Cell morphology and phosphatidylserine externalization were visualized after Giemsa-May-Grunwald and annexin V staining, respectively. DNA content and mitochondrial depolarization were measured by flow cytometry. BAX and BCL-xL expression was examined by RT-PCR. RESULTS: NFP was 3.8-fold more cytotoxic against HL-60 leukemia cells than against normal cells. NFP reduced the number of viable cells 24h after the treatment with a concomitant increase in the number of apoptotic cells indicated by the externalization of phosphatidylserine, DNA fragmentation, and mitochondrial depolarization. The mRNA levels of BAX increased, whereas the mRNA levels of BCL-xL decreased. CONCLUSION: The results indicate that NFP induces apoptosis in HL-60 cells by upregulating BAX and downregulating BCL-xL.

Expression of angiogenic factors and luteinizing hormone receptors in the corpus luteum of mares induced to ovulate with deslorelin acetate.

The effects of deslorelin acetate use in inducing ovulation need to be clarified to improve the results of equine embryo transfer. The mRNA abundance for angiogenic factors and LH receptor (LHR) in corpus luteum (CL) was studied in mares with natural (control group [CG]) and induced ovulation with deslorelin acetate (treatment group [TG]; follicles: ≥ 35 mm). Transrectal ultrasonography was used to verify the ovulation day, and on Days 4, 8, and 12 after ovulation (Day 0), CL samples were obtained through ultrasound-guided biopsy. The messenger RNA expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and LHR genes were analyzed by real-time polymerase chain reaction. A positive correlation was observed between VEGF and LHR (P < 0.00001, r = 0.78), and it was possible to detect higher LHR expression in the TG than in the CG on Day 4 (P < 0.05). Moreover, this expression was higher on Days 4 and 8 than on Day 12 in the TG. Basic fibroblast growth factor was also expressed in luteal tissue on all days for both groups; however, these differences were not significant. In conclusion, deslorelin acetate was effective for the induction of ovulation in mares, resulting in higher expression of LHR, especially on the fourth day after ovulation. In addition, VEGF expression was influenced by induced ovulation, with a lower level on Day 12, which is expected in nonpregnant mares.

Recent advances in the mechanisms of lung alveolarization and the pathogenesis of bronchopulmonary dysplasia.

Alveolarization is the process by which the alveoli, the principal gas exchange units of the lung, are formed. Along with the maturation of the pulmonary vasculature, alveolarization is the objective of late lung development. The terminal airspaces that were formed during early lung development are divided by the process of secondary septation, progressively generating an increasing number of alveoli that are of smaller size, which substantially increases the surface area over which gas exchange can take place. Disturbances to alveolarization occur in bronchopulmonary dysplasia (BPD), which can be complicated by perturbations to the pulmonary vasculature that are associated with the development of pulmonary hypertension. Disturbances to lung development may also occur in persistent pulmonary hypertension of the newborn in term newborn infants, as well as in patients with congenital diaphragmatic hernia. These disturbances can lead to the formation of lungs with fewer and larger alveoli and a dysmorphic pulmonary vasculature. Consequently, affected lungs exhibit a reduced capacity for gas exchange, with important implications for morbidity and mortality in the immediate postnatal period and respiratory health consequences that may persist into adulthood. It is the objective of this Perspectives article to update the reader about recent developments in our understanding of the molecular mechanisms of alveolarization and the pathogenesis of BPD.