Protocol for CRISPR-Cas12a genome editing of protein tyrosine phosphatases in human pluripotent stem cells and functional ß-like cell generation.

Gene editing of human pluripotent stem cells is a promising approach for developing targeted gene therapies for metabolic diseases. Here, we present a protocol for generating a CRISPR-Cas12a gene knockout of protein tyrosine phosphatases in human embryonic stem cells. We describe steps for differentiating the edited clones into pancreatic islet-like spheroids rich in ß-like cells. We then detail procedures for implanting these spheroids under the murine kidney capsule for in vivo maturation.

Punicalagin increases follicular activation, development and activity of superoxide dismutase 1, catalase, and glutathione peroxidase 1 in cultured bovine ovarian tissues.

Context The overproduction of reactive oxygen species (ROS) during in vitro culture of ovarian tissues impairs follicular development and survival. Aims To evaluate the effects of punicalagin on the development and survival of primordial follicles, stromal cell and collagen fibres, as well as on the levels of mRNA for nuclear factor erythroid 2-related factor 2 (NRF2 ), superoxide dismutase 1 (SOD1 ), catalase (CAT ), glutathione peroxidase 1 (GPX1 ) and perirredoxin 6 (PRDX6 ), and activity of antioxidant enzymes in cultured bovine ovarian tissues. Methods Bovine ovarian cortical tissues were cultured for 6days in α-MEM+ alone or with 1.0, 10.0, or 100.0µM punicalagin at 38.5°C with 5% CO2 . Follicle morphology and growth, stromal cell density, and collagen fibres were evaluated by classical histology, while the expression of mRNA was evaluated by real-time PCR. The activity of enzymes was analysed by the Bradford method. Key results Punicalagin improved follicle survival and development, reduced mRNA expression for SOD1 and CAT , but did not influence stromal cells or collagen fibres. Punicalagin (10.0µM) increased the levels of thiol and activity of SOD1, CAT , and GPX1 enzymes. Conclusions Punicalagin (10.0µM) promotes follicle survival and development and activates SOD1, CAT , and GPX1 enzymes in bovine ovarian tissues. Implications Punicalagin improves follicle development and survival in cultured ovarian tissues.

Pericardial Fluid Accumulates microRNAs That Regulate Heart Fibrosis after Myocardial Infarction.

Pericardial fluid (PF) has been suggested as a reservoir of molecular targets that can be modulated for efficient repair after myocardial infarction (MI). Here, we set out to address the content of this biofluid after MI, namely in terms of microRNAs (miRs) that are important modulators of the cardiac pathological response. PF was collected during coronary artery bypass grafting (CABG) from two MI cohorts, patients with non-ST-segment elevation MI (NSTEMI) and patients with ST-segment elevation MI (STEMI), and a control group composed of patients with stable angina and without previous history of MI. The PF miR content was analyzed by small RNA sequencing, and its biological effect was assessed on human cardiac fibroblasts. PF accumulates fibrotic and inflammatory molecules in STEMI patients, namely causing the soluble suppression of tumorigenicity 2 (ST-2), which inversely correlates with the left ventricle ejection fraction. Although the PF of the three patient groups induce similar levels of fibroblast-to-myofibroblast activation in vitro, RNA sequencing revealed that PF from STEMI patients is particularly enriched not only in pro-fibrotic miRs but also anti-fibrotic miRs. Among those, miR-22-3p was herein found to inhibit TGF-ß-induced human cardiac fibroblast activation in vitro. PF constitutes an attractive source for screening diagnostic/prognostic miRs and for unveiling novel therapeutic targets in cardiac fibrosis.

Cardiac implantable electronic device infection awareness - A European survey amongst implanting physicians.

BACKGROUND AND AIMS: Cardiac Implantable Electronic Device (CIED) infections pose significant mortality and morbidity despite optimal treatment. This survey aimed to understand whether and how the risk of CIED infection is assessed and mitigated in clinical practice in Europe, and to detect gaps with respect to EHRA recommendations. METHODS: An Expert Group of 8 European cardiologists with specific expertise across CIED therapy designed and distributed electronically a survey to a number of European Cardiologists. RESULTS: 302 physicians from 18 European countries responded to the survey. 288/302 (95%) physicians agreed that CIED-related infections represent a burden on healthcare resources and are associated with significant morbidity and mortality. 285/302 respondents (94%) primarily assess the risk of CIED infections by only evaluating the patient's clinical profile (137/302, 46%) or with the support of a risk score (148/302, 49%). Intravenous antibiotic prophylaxis is used by 282/302 (93%), followed by the implantation of the lowest number of leads possible (182/302, 60%), and by the use of an antibacterial envelope (173/302, 57%). 230/302 respondents (76%) declared that there is need for clear and concise guidelines and more sensitive risk-scores for CIED infection, to maximize the chances of preventative strategies. CONCLUSIONS: This survey demonstrates a high level of awareness about the multifaceted issue of CIED infection, however, it also highlights an incomplete penetration of scoring systems for risk stratification owing to their perceived limitations, and detects a strong commitment to increase the effectiveness of preventative strategies.

Croton grewioides essential oil and anethole reduce oxidative stress and improve growth of bovine primordial follicles during culture of ovarian tissue.

OBJECTIVES: This study aims to evaluate the effects of Croton grewioides essential oil (CGEO) and anethole on follicle survival, growth, and oxidative stress in cultured bovine ovarian tissues. METHODS: Ovarian tissues were cultured for 6 days in a medium supplemented with different concentrations (1, 10, 100, or 1000 µg mL-1) of CGEO or anethole and then, follicular survival and growth, collagen content, and stromal cell density in ovarian tissues cultured in vitro were evaluated by histology. The mRNA levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1), peroxirredoxin 6 (PRDX6), and nuclear factor erythroid 2-related factor 2 (NRF2) were evaluated by real-time PCR. The activity of SOD, CAT, glutathione peroxidase (GPx), and thiol concentrations were investigated. KEY FINDINGS: Ovarian tissues cultured with 1 µg mL-1 CGEO or anethole had a higher percentage of healthy follicles than those cultured in a control medium (P < .05). The 1 µg mL-1 CGEO also increased the number of stromal cells, collagen fibers, and thiol levels. Anethole (1 µg mL-1) increased CAT activity and reduced that of GPx. The activity of SOD was reduced by CGEO. In contrast, 1 µg mL-1 anethole reduced mRNA for CAT, PRDX1, and NRF2 (P < .05). In addition, 1 µg mL-1 CGEO reduced mRNA for CAT, PRDX6, and GPx1 (P < .05). CONCLUSIONS: The presence of 1 µg mL-1 anethole or CGEO in a culture medium promotes follicle survival and regulates oxidative stress and the expression of mRNA and activity of antioxidant enzymes in cultured bovine ovarian tissues.

Thymol increases primordial follicle activation, protects stromal cells, collagen fibers and down-regulates expression of mRNA for superoxide dismutase 1, catalase and periredoxin 6 in cultured bovine ovarian tissues.

This study aims to investigate the influence of thymol on primordial follicle growth and survival, as well as on collagen fibers and stromal cells density in bovine ovarian tissues cultured in vitro. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), the thiol levels and the expression of mRNAs for SOD1, CAT, periredoxin 6 (PRDX6) and GPX1 were also investigated. Ovarian cortical tissues were cultured in α-MEM+ alone or with thymol (400, 800, 1600 or 3200 µg/mL) for six days. Before and after culture, the tissues were processed for histological analysis to evaluate follicular activation, growth, morphology, ovarian stromal cell density and collagen fibers. The levels of mRNA for SOD1, CAT, GPX1 and PRDX6 were evaluated by real-time PCR. The results show that tissues cultured with thymol (400 and 800 µg/mL) had increased percentages of normal follicles, when compared to tissues cultured in other treatments. At concentrations of 400 and 800 µg/mL, thymol maintained the rate of normal follicles similar to the uncultured control. In addition, 400 µg/mL thymol increased follicle activation, collagen fibers and stromal cell density of when compared to tissues cultured in control medium. The presence of 800 µg/mL thymol in culture medium increased CAT activity, while 400 or 800 µg/mL thymol reduced mRNA levels for SOD1, CAT and PRDX6, but did not alter GPX1 expression. In conclusion, 400 µg/mL thymol increases primordial follicle activation, preserves stromal cells, collagen fibers, and down-regulates expression of mRNA for SOD1, CAT and PRDX6 in cultured bovine ovarian tissues.

α-Pinene Improves Follicle Morphology and Increases the Expression of mRNA for Nuclear Factor Erythroid 2-Related Factor 2 and Peroxiredoxin 6 in Bovine Ovarian Tissues Cultured In Vitro.

Oxidative stress during in vitro of ovarian tissues has adverse effects on follicle survival. α-pinene is a monoterpenoid molecule with antioxidant activity that has great potential to maintain cell survival in vitro. This study investigated the effect of α-pinene (1.25, 2.5, 5.0, 10.0, or 20.0 µg/mL) on primordial follicle growth and morphology, as well as on stromal cells and collagen fibers in bovine ovarian slices cultured for six days. The effect of α-pinene on transcripts of catalase (CAT), superoxide dismutase (SOD), peroxiredoxin 6 (PRDX6), glutathione peroxidase (GPX1), and nuclear factor erythroid 2-related factor 2 (NRF2) was investigated by real-time PCR. The tissues were processed for histological analysis to evaluate follicular growth, morphology, stromal cell density, and collagen fibers. The results showed that 2.5, 5.0, or 10.0 µg/mL α-pinene increased the percentages of normal follicles but did not influence follicular growth. The α-pinene (10.0 µg/mL) kept the stromal cell density and collagen levels in cultured bovine ovarian tissue like uncultured tissues. Ovarian tissues cultured in control medium had reduced expression of mRNA for NRF2, SOD, CAT, GPX1, and PRDX6, but α-pinene (10.0 µg/mL) increased mRNA levels for NRF2 and PRDX6. In conclusion, 10.0 µg/mL α-pinene improves the follicular survival, preserves stromal cell density and collagen levels, and increases transcripts of NRF2 and PRDX6 after in vitro culture of bovine ovarian tissue.

Coronary atherosclerotic burden in veteran athletes: The relationship between cardiovascular risk and volume of exercise.

INTRODUCTION AND OBJECTIVES: The association between exercise and coronary atherosclerosis still remains unclarified. We aimed to analyze the prevalence of high coronary atherosclerotic burden in veteran athletes, considering cardiovascular (CV) risk and volume of exercise. METHODS: A total of 105 asymptomatic male veteran athletes (48±5.6 years old) were studied. A high coronary atherosclerotic burden was defined as one of the following characteristics in coronary computed tomography angiography: calcium score >100, >75th percentile, obstructive plaques, involving left main, three-vessels or two-vessels including proximal anterior descending artery, segment involvement score >5 or CT-adapted Leaman score ≥5. CV risk was stratified by SCORE2 and volume of exercise by metabolic equivalent task score. RESULTS: Most athletes (n=88) were engaged in endurance sports for 17.1±9.8 years, with a median exercise volume of 66 [IQR 44-103] metabolic equivalent of tasks/hour/week. The mean Systematic Coronary Risk Evaluation 2 was 2.8±1.5%; 76.9% of athletes had a low-moderate risk and none a very high risk. High coronary atherosclerotic burden was present in 25.7% athletes. Athletes with high cardiovascular risk and high exercise volume (above the median) showed significantly high coronary atherosclerotic burden compared to those with low-moderate risk and high volume (50.0% vs. 15.6%; p=0.017). Among athletes with low to moderate risk, a high volume of exercise tended to be protective, while in those with low volume, there was similar rate of high coronary atherosclerotic burden, regardless of CV risk. CONCLUSIONS: A combination of higher volume of exercise and high cardiovascular risk revealed the worst association with coronary atherosclerosis in veteran athletes. The relationship between these variables is controversial, but integrating exercise characteristics and risk assessment into preparticipation evaluation is essential.

Field comparison of broad-spectrum white LED-baited traps with narrow-spectrum green LED-baited traps in the capture of Anopheles mosquitoes (Diptera: Culicidae).

Light-Emitting Diodes (LEDs) have been effective light sources in attracting Anopheles mosquitoes, but the broad-spectrum white light, even with a wide-ranging application in lighting, have not been evaluated yet. In this study, the white light was field evaluated against the green one in the light trapping of anopheline mosquitoes by using two non-suction Silva traps and two CDC-type suction light traps. Anopheline mosquitoes were captured for two 21-night periods of collecting (2022 and 2023). In the first period, two LEDs were used per Silva trap, but three were used in the second one to increase the luminance/illuminance at traps. A CDC-type suction light trap equipped with an incandescent lamp was used in 2022 and a CDC-type suction light trap equipped with a 6 V-white light (higher luminance/illuminance) in 2023. A total of eight species and 3,289 specimens were captured in both periods. The most frequent species were Anopheles triannulatus s.l., An. goeldii, An. evansae and An. argyritarsis. In 2022, white LEDs were less attractive to anopheline mosquitoes than the other light sources, but without statistical difference among treatments (F = 2.703; P = 0.0752; df = 2). In 2023, even with an increased luminance/illuminance at traps, no statistical difference was found between the two LED-baited Silva traps (F = 6.690; P = 0.0024; df = 2), but rather between the 6 V-white-baited CDC-type suction light trap and green-baited Silva traps. Due to some drawbacks and the lower abundance of individuals caught by using white LEDs, the narrow-banded green LEDs is preferable to white ones for attracting anophelines.

Outcomes of ventricular tachycardia ablation in patients with ischemic and non-ischemic cardiomyopathy: A propensity-score matched analysis.

INTRODUCTION AND OBJECTIVES: Catheter ablation (CA) is effective in the treatment of ventricular tachycardia (VT). Although some observational data suggest patients with non-ischemic cardiomyopathy (NICM) have less favorable outcomes when compared to those with an ischemic etiology (ICM), direct comparisons are rarely reported. We aimed to compare the outcomes of VT ablation in a propensity-score matched population of ICM or NICM patients. METHODS: Single-center retrospective study of consecutive patients undergoing VT ablation from 2012 to 2023. A propensity score (PS) was used to match ICM and NICM patients in a 1:1 fashion according to age, sex, left ventricular ejection fraction (LVEF), NYHA class, electrical storm (ES) at presentation, and previous endocardial ablation. The outcomes of interest were VT-free survival and all-cause mortality. RESULTS: The PS yielded two groups of 71 patients each (mean age 63±10 years, 92% male, mean LVEF 35±10%, 36% with ES at presentation, and 23% with previous ablation), well matched for baseline characteristics. During a median follow-up of 2.3 (interquartile range IQR 1.3-3.8) years, patients with NICM had a significantly lower VT-free survival (53.5% vs. 69.0%, log-rank p=0.037), although there were no differences regarding all-cause mortality (22.5% vs. 16.9%, log-rank p=0.245). Multivariate analysis identified NICM (HR 2.34 [95% CI 1.32-4.14], p=0.004), NYHA class III/IV (HR 2.11 [95% CI 1.11-4.04], p=0.024), and chronic kidney disease (HR 2.23 [95% CI 1.25-3.96], p=0.006), as independent predictors of VT recurrence. CONCLUSION: Non-ischemic cardiomyopathy patients were at increased risk of VT recurrence after ablation, although long-term mortality did not differ.

Melatonin improves the viability and ultrastructure of bovine oocyte-granulosa complexes of in vitro cultured early antral follicles.

This study aims to investigate the effects of melatonin on follicular growth, viability and ultrastructure, as well as on the levels of mRNA for antioxidant enzymes, reactive oxygen species (ROS) and meiotic progression in oocytes from in vitro cultured bovine early antral follicles. To this end, isolated early antral follicles (500-600 µm) were cultured in TCM-199+ alone or supplemented with 10-6 , 10-7 or 10-8 M melatonin at 38.5°C with 5% CO2 for 8 days. Follicle diameters were evaluated at days 0, 4 and 8 of culture. At the end of culture, ultrastructure, chromatin configuration, viability (calcein-AM and ethidium homodimer-1 staining), and the levels of ROS and mRNA for catalase (CAT), superoxide dismutase (SOD) and peroxiredoxin 6 (PRDX6) and glutathione peroxidase (GPx) were investigated in oocyte-granulosa cell complexes (OGCs). The results showed that early antral follicles cultured with 10-6 and 10-8 M melatonin had a progressive and significant increase in their diameters throughout the culture period (p < .05). Additionally, oocytes from follicles cultured with 10-7 or 10-8 M melatonin had increased fluorescence for calcein-AM, while those cultured with 10-6 or 10-7 M had reduced fluorescence for ethidium homodimer-1. Different from follicles cultured in other treatments, those cultured with 10-8 M melatonin had well-preserved ultrastructure of oocyte and granulosa cells. Melatonin, however, did not influence the levels of ROS, the mitochondrial activity, oocyte meiotic resumption and expression mRNA for SOD, CAT, GPX1 and PRDX6. In conclusion, the presence of 10-8 M melatonin in culture medium improves viability and preserves the ultrastructure of oocyte and granulosa cells of early antral follicles cultured in vitro.

Natural infection of free-ranging capybaras (Hydrochoerus hydrochaeris) with Anaplasmataecea and Rickettsiaceae bacteria in the Iberá wetlands ecoregion, Argentina.

The current work assessed the infection with Ehrlichia and Anaplasma species, and exposure to Rickettsia spp. in free-ranging capybaras in the Iberá wetlands ecoregion in Argentina. By indirect immunofluorescence assay, 37 out of 51 (73%) capybara sera were seropositive to Rickettsia spp., with 23.5% and 4% samples considered homologous to Rickettsia parkeri and Rickettsia bellii, respectively (or very closely related serotypes). Anaplasmataceae DNA was found to be highly prevalent in capybaras, with 33 out of 62 samples positive for Anaplasma sp. with Ct values of 28.64 ± 0.35 (average ± standard error), and 12 samples positive for Ehrlichia sp. with Ct values of 31.74 ± 0.87. Anaplasma sp. from capybaras was closely related to Anaplasma sp. reported to infect Amblyomma dubitatum in Iberá wetlands and to Anaplasma odocoilei, while the detected Ehrlichia sp. was closely related to "Candidatus Ehrlichia hydrochoerus" previously reported to infect capybaras in Brazil and A. dubitatum in Iberá wetlands. Structures compatible with Anaplasma morulae were observed in the cytoplasm of platelets from Anaplasma-positive capybaras. Our findings show that capybaras from the Iberá wetlands were exposed to Rickettsia species related to R. bellii and to the pathogen R. parkeri, and were infected with "Ca. Ehrlichia hydrochoerus" and a novel Anaplasma species, herein named "Candidatus Anaplasma capybara".

Quality indicators in atrial fibrillation ablation (RIQAFA). A national registry from the Portuguese Association of Arrhythmology, Pacing and Electrophysiology (APAPE).

INTRODUCTION AND OBJECTIVES: Atrial fibrillation (AF) is the most common sustained arrhythmia, with significant burden for patients. Catheter ablation is safe and superior for symptom improvement. The purpose of this work was to assess how clinical practice compares with current scientific evidence and quality indicators for AF ablation. METHODS: The Portuguese Association of Arrhythmology, Pacing and Electrophysiology conducted a prospective registry among Portuguese centers to assess clinical practice regarding management of patients referred for ablation and the methodology used in the procedures and related outcomes. RESULTS: A total of 337 patients were referred for ablation, 102 (37.91%) female, age 65 (56-70.8) years. The median CHADS2-VaSC2 thromboembolic risk score was 2 (1-3), and 308 (92.49%) were on anticoagulants. AF was mainly paroxysmal (224, 66.97%) and symptomatic (mEHRA score 3; 2-3). Before ablation most patients (273, 81.49%) underwent cardiac computed tomography and only 24 (7.36%) procedures were performed with uninterrupted anticoagulation. For ablation, Carto® (194; 59.15%) and Ensite® (55; 16.77%) were mainly used, and the preferential strategy was pulmonary vein isolation (316; 94.61%). Acute complications occurred in five (1.49%) patients, while most had symptom improvement at one month (200; 86.21%), sustained at one year. There were 40 (12.6%) relapses within 30 days and 19 (26.39%) at one year. CONCLUSIONS: In a population of patients with AF referred for ablation in Portuguese centers, patient management is provided according to the best scientific evidence and there is a high standard of practice with respect to the quality of AF ablation practice.

Field evaluation of a new suction light trap for the capture of phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae), vectors of leishmaniasis.

Phlebotomine sand flies are crepuscular and nocturnal small dipteran insects in the family Psychodidae. Several disease agents, including Leishmania parasites, are transmitted to humans and other vertebrate hosts by the bite of an infected female sand fly. As part of leishmaniasis surveillance programs, light traps have been routinely used in sand fly collections. In this context, new trapping devices are always being required to improve vector monitoring. Here, the efficiency of a new suction light trap, named Silva suction trap or SS trap, was field evaluated in collecting sand flies. Two SS traps, one with green (520 nm, 15,000 mcd) and the other with white (wide spectrum, 18,000 mcd) LEDs, and one CDC-type trap were deployed in a rural forested environment. A total of 4686 phlebotomine sand flies were captured. The most frequent species were females of the Ps. Chagasi series (77.8%) followed by males of Ps. wellcomei (11.6%), Nyssomyia whitmani (3.3%), and Bichromomyia flaviscutellata (2.4%). The CDC-type light trap collected 101.9 ± 20.89 sand flies and 14 species, followed by the white-baited SS trap (87.78 ± 16.36, 14), and the green-baited SS trap (70.61 ± 14.75, 15), but there were no statistically significant differences among traps. A discussion on the considerable advantages of the use of SS traps over CDC traps is included. In this study, the Silva suction trap proved to be efficient and can be an alternative to CDC traps for monitoring adult phlebotomine sand fly populations.

A Literature Review on the Use of Aortic Allografts in Modern Cardiac Surgery for the Treatment of Infective Endocarditis: Is There Clear Evidence or Is It Merely a Perception?

Infective valve endocarditis is caused by different pathogens and 60% of those involve the aortic valve with valve failure. Although S. aureus is recognized as the most frequently isolated causative bacterium associated with IE in high-income countries, Gram-positive cocci nevertheless play a crucial role in promoting infection in relation to their adhesive matrix molecules. The presence of pili on the surface of Gram-positive bacteria such as in different strains of Enterococcus faecalis and Streptococcus spp., grants these causative pathogens a great offensive capacity due to the formation of biofilms and resistance to antibiotics. The indications and timing of surgery in endocarditis are debated as well as the choice of the ideal valve substitute to replace the diseased valve(s) when repair is not possible. We reviewed the literature and elaborated a systematic approach to endocarditis management based on clinical, microbiological, and anatomopathological variables known to affect postoperative outcomes with the aim to stratify the patients and orient decision making. From this review emerges significant findings on the risk of infection in the allograft used in patients with endocarditis and no endocarditis etiology suggesting that the use of allografts has proved safety and effectiveness in patients with both pathologies.

Effects of cyclic adenosine monophosphate modulating agents during oocyte pre-maturation and the role of melatonin on in vitro maturation of bovine cumulus-oocyte complexes.

This study investigated the effects of cyclic adenosine monophosphate modulating during cumulus-oocyte complexes (COCs) pre-maturation and the role of melatonin on in vitro maturation (IVM) of bovine COCs. In experiment one, COCs were pre-matured for 8 h in control medium or with 3-isobutyl-1-methylxanthine (IBMX) and forskolin, IBMX and C-type natriuretic peptide, c-type natriuretic peptide and forskolin or IBMX, forskolin and c-type natriuretic peptide. Then, meiotic progression was evaluated. In experiment two, COCs were pre-matured, followed by IVM in control medium alone or with 10-6, 10-7 or 10-8 M melatonin. After IVM, chromatin configuration, transzonal projections (TZPs), reactive oxygen species, mitochondrial distribution, ultrastructure and mRNA expression for antioxidant enzymes were evaluated. In experiment 1, COCs pre-matured with both C-type natriuretic peptide and forskolin or C-type natriuretic peptide, forskolin and IBMX had lower meiotic resumption rate when compared to control. Considering that IBMX had not an additional effect to potentiate inhibition of meiotic resumption, a combination of C-type natriuretic peptide and forskolin was chosen. In experiment 2, COCs matured with 10-8 M melatonin had greater rates of meiotic resumption when compared to the other treatments (P < 0.05). The COCs matured with 10-7 or 10-8 M melatonin had greater mitochondrial activity (P < 0.05), while those matured with 10-6 or 10-8 M of melatonin had greater levels of TZPs. Ultrastructure of oocyte and cumulus cells after IVM with melatonin was relatively well preserved. COCs matured with 10-8 M melatonin increased mRNA expression for superoxide dismutase (SOD) and catalase (CAT) (P < 0.05), when compared to non-cultured and pre-matured COCs, respectively. In conclusion, bovine COC pre-maturation with C-type natriuretic peptide and forskolin, followed by IVM with 10-8 M melatonin improves meiotic resumption rates, TZPs, mitochondrial distribution and mRNA expression for SOD and CAT.

Injectable hydrogels for the delivery of nanomaterials for cancer combinatorial photothermal therapy.

Progress in the nanotechnology field has led to the development of a new class of materials capable of producing a temperature increase triggered by near infrared light. These photothermal nanostructures have been extensively explored in the ablation of cancer cells. Nevertheless, the available data in the literature have exposed that systemically administered nanomaterials have a poor tumor-homing capacity, hindering their full therapeutic potential. This paradigm shift has propelled the development of new injectable hydrogels for the local delivery of nanomaterials aimed at cancer photothermal therapy. These hydrogels can be assembled at the tumor site after injection (in situ forming) or can undergo a gel-sol-gel transition during injection (shear-thinning/self-healing). Besides incorporating photothermal nanostructures, these injectable hydrogels can also incorporate or be combined with other agents, paving the way for an improved therapeutic outcome. This review analyses the application of injectable hydrogels for the local delivery of nanomaterials aimed at cancer photothermal therapy as well as their combination with photodynamic-, chemo-, immuno- and radio-therapies.

Cystoisospora suis in Portugal: an observational study of prevalence, management, and risk factors.

BACKGROUND: Neonatal coccidiosis is a common and important disease of suckling piglets in modern farming caused by Cystoisospora suis. Prevalence rates are high, namely, in Portugal, although no recent data are available. The metaphylactic administration of a single dose of toltrazuril and hygienic measures are the backbone of control strategies on positive farms. However, several studies have shown that these programs are not always effective, underlining the need to revise the risk factors and control strategies currently applied. The present study evaluated C. suis prevalence on Portuguese farms and assessed the risk factors associated with facilities and farm rearing practices. RESULTS: From the 27 tested farms, 23 were positive to Cystoisopora suis (85.2%). In total, 258 litters were sampled (accounting for 516 samples-2 samples per litter), with an average of 59.7% of positive litters per positive farm. Faecal pools from litters, in which liquid faeces predominated had a higher probability of containing oocysts than litter pools with mainly solid (Odds Ratio: 9.87; p < 0.0001) or pasty faeces (OR: 7.05; p = 0.001), and samples obtained from younger animals had higher oocyst counts (coefficient: - 0.0720; 95% CI - 0.125; - 0.019). No significant effect of toltrazuril administration was observed on the positivity rate, and none of the tested farms used disinfectants with official claims against parasites and known anticoccidial effects (e.g., cresol-based products). CONCLUSIONS: The C. suis prevalence on Portuguese swine farms appears to be similar to the prevalence found in other European countries. Repeated sampling of the same litter and the use of autofluorescence microscopy after a modified Ritchie technique seems to have increased sensitivity and consequently the detection rate of positive litters. Finally, despite the common use of oral toltrazuril, C. suis control programs appear to not always be effective (based on the detection of oocysts in faecal samples), suggesting the need to revise the control strategies applied in the field, including management factors and choices of disinfectant products.

Enhanced apoptotic index in hepatocytes, Kupffer cells, and inflammatory infiltrate showed positive correlation with hepatic lesion intensity, parasite load, and clinical status in naturally Leishmania-infected dogs.

It is unknown if Leishmania amastigote infections affect hepatocytes and Kupffer cell apoptosis, and the role played by apoptosis in liver lesions in leishmaniasis is still unclear. Clinically affected and subclinically infected dogs with leishmaniosis and uninfected controls were assessed. Parasite load, biochemical markers for evaluation of liver damage, morphometry (area, perimeter, number of inflammatory focus, major and minor diameters), apoptosis in hepatic tissue (hepatocytes, Kupffer cells, and inflammatory infiltrates) and cellularity in inflammatory foci were quantified. The parasite load in clinically affected dogs proved to be higher than in the other groups. All morphometric parameters (area, perimeter, number of inflammatory focus, major and minor diameters) from clinically affected were higher than the values found in the subclinically infected and uninfected control dogs. Only clinically affected dogs presented high levels of ALT, FA, GGT and cholesterol in serum. Strong positive correlation was observed between biochemical markers for evaluation of liver damage (ALT, FA, GGT and cholesterol) and hepatic apoptosis (hepatocytes, Kupffer cells, and inflammation). Clinically affected dogs showed a more intense hepatic lesion. Hepatocytes showed a higher rate of apoptosis in Leishmania-infected dogs than in uninfected control dogs. The Kupffer cell apoptotic index and apoptosis within the inflammatory infiltrates were higher in clinically affected dogs. The apoptotic index evaluated in hepatocytes, Kupffer cells, and inflammatory infiltrates showed a positive correlation with the intensity of the hepatic lesion, parasite load, and clinical status. Apoptotic cells also showed positive immunostaining for TUNEL, Bcl2, and Bax. Our data showed that hepatic apoptosis was related to the severity of liver damage, the progression of infection, and the parasite load in leishmaniasis. Apoptotic regulated cell recruitment modulated the inflammatory response and favored the survival and dissemination of parasites, depending on the clinical status of the Leishmania-infected dogs.