Fat area and lipid droplet morphology of porcine oocytes during in vitro maturation with trans-10, cis-12 conjugated linoleic acid and forskolin

Lipid droplets (LD) in porcine oocytes form a dark mass reaching almost all cytoplasm. Herein we investigated changes in fat areas, cytoplasmic tone and LD morphology during in vitro maturation (IVM) of porcine oocytes cultured with 100mM trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA) or 10mM forskolin at different time periods. Four groups were constituted: control, excipient, t10,c12 CLA and forskolin, with drugs being supplemented during 44 to 48h and the initial 22 to 24h in Experiments 1 and 2, respectively. In Experiment 3, forskolin was supplemented for the first 2 h. Matured oocytes were inseminated with frozen-thawed boar semen and cleavage rate recorded. Before and during IVM, samples of oocytes were evaluated for LD, total and fat areas and fat gray value or for meiotic progression. Results showed that forskolin supplementation during 44 to 48 h or 22 to 24 h inhibits oocyte maturation (exp. 1: forskolin = 5.1±8.0%, control = 72.6±5.0%; exp. 2: forskolin =24.3±7.4%, control =71.6±5.6%) and cleavage (exp. 1: forskolin=0.0±0.0%, control=55.4±4.1%; exp. 2: forskolin=8.3±3.3%, control=54.5±3.0%). Forskolin also reduced oocyte and fat areas. In Experiment 3, forskolin negative effect on oocyte maturation and cleavage disappeared, although minor (P<0.03) LD and oocyte fat areas were identified at 22 to 24 h of IVM. Oocytes supplemented with t10,c12 CLA during 44 to 48h presented a lighter (P<0.04) colour tone cytoplasm than those of control and forskolin. In conclusion, t10,c12 CLA and forskolin were capable of modifying the distribution and morphology of cytoplasmic LD during porcine oocyte maturation, thus reducing its lipid content in a time-dependent manner

Efeitos de Anestésico Local no Tempo Entre Bólus e na Duração do Trabalho de Parto em Patient-Controlled Epidural Analgesia: Estudo Prospectivo de Dois Regimes de Doses Ultra-Baixas com Ropivacaina e Sufentanil

BACKGROUND: Patient-controlled epidural analgesia with low concentrations of anesthetics is effective in reducing labor pain. The aim of this study was to assess and compare two ultra-low dose regimens of ropivacaine and sufentanil (0.1% ropivacaine plus 0.5 μg.ml-1 sufentanil vs. 0.06% ropivacaine plus 0.5 μg.ml-1 sufentanil) on the intervals between boluses and the duration of labor. MATERIAL AND METHODS: In this non-randomized prospective study, conducted between January and July 2010, two groups of parturients received patient-controlled epidural analgesia: Group I (n = 58; 1 mg.ml-1 ropivacaine + 0.5 μg.ml-1 sufentanil) and Group II (n = 57; 0.6 mg.ml-1 ropivacaine + 0.5 μg.ml-1 sufentanil). Rescue doses of ropivacaine at the concentration of the assigned group without sufentanil were administered as necessary. Pain, local anesthetic requirements, neuraxial blockade characteristics, labor and neonatal outcomes, and maternal satisfaction were recorded. RESULTS: The ropivacaine dose was greater in Group I (9.5 [7.7-12.7] mg.h-1 vs. 6.1 [5.1-9.8 mg.h-1], p < 0.001). A time increase between each bolus was observed in Group I (beta = 32.61 min, 95% CI [25.39; 39.82], p < 0.001), whereas a time decrease was observed in Group II (beta = -1.40 min, 95% CI [-2.44; -0.36], p = 0.009). The duration of the second stage of labor in Group I was significantly longer than that in Group II (78 min vs. 65 min, p < 0.001). CONCLUSIONS: Parturients receiving 0.06% ropivacaine exhibited less evidence of cumulative effects and exhibited faster second stage progression than those who received 0.1% ropivacaine

Engineering modular half-antibody conjugated nanoparticles for targeting CD44v6-expressing cancer cells

Gastric cancer (GC) remains a major cause of death worldwide mainly because of the late detection in advanced stage. Recently, we proposed CD44v6 as a relevant marker for early detection of GC, opening new avenues for GC-targeted theranostics. Here, we designed a modular nanoscale system that selectively targets CD44v6-expressing GC cells by the site-oriented conjugation of a new-engineered CD44v6 half-antibody fragment to maleimide-modified polystyrene nanoparticles (PNPs) via an efficient bioorthogonal thiol-Michael addition click chemistry. PNPs with optimal particle size (200 nm) for crossing a developed biomimetic CD44v6-associated GC stromal model were further modified with a heterobifunctional maleimide crosslinker and click conjugated to the novel CD44v6 half-antibody fragment, obtained by chemical reduction of full antibody, without affecting its bioactivity. Collectively, our results confirmed the specific targeting ability of CD44v6-PNPs to CD44v6-expressing cells (1.65-fold higher than controls), highlighting the potential of CD44v6 half-antibody conjugated nanoparticles as promising and clinically relevant tools for the early diagnosis and therapy of GC. Additionally, the rational design of our nanoscale system may be explored for the development of several other nanotechnology-based disease-targeted approaches.This work was supported by Norte Portugal Regional Operational Programme (NORTE2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF) projects Norte-01-0145-FEDER-000012 and NORTE-07-0124-FEDER-000029, through COMPETE 2020-Operational Programme for Competitiveness and Internationalization (POCI) Portugal 2020 and Portuguese Foundation for Science and Technology (FCT) in the framework of the projects POCI-01-0145-FEDER-007274, POCI-01-0145-FEDER-016390, and PTDC/CTMNAN/120958/2010, B.N.L. doctoral grant (SFRH/BD/87400/2012) and postdoctoral grant (PTDC/MEC-GIN/29232/2017). R.F.P. was supported by Institute of Network Bioengineering for Healthy Aging (0245_IBEROS_1_E)

Eating Habits During Pregnancy of Women Giving Birth Very Prematurely: An Exploratory Analysis

Introduction: Preterm birth is increasing worldwide, representing a major cause of death and long-term loss of human potential among survivors. Some morbidities during pregnancy are well-known risk factors for preterm labor, but it is not yet known whether deviations from adequate dietary patterns are associated with preterm delivery. Diet may be an important modulator of chronic inflammation, and pro-inflammatory diets during pregnancy were reported to be associated with preterm birth. The aim of this study was to assess the food consumption during pregnancy of Portuguese women giving birth very prematurely and the association between the food consumption and the major maternal morbidities during pregnancy related with preterm delivery. Material and Methods: A single-center cross-sectional observational study including consecutive Portuguese women giving birth before 33 weeks of gestation was conducted. Recall of eating habits during pregnancy was obtained within the first week after delivery, using a semi quantitative food frequency questionnaire validated for Portuguese pregnant women. Results: Sixty women with a median age of 36.0 years were included. Of these, 35% were obese or overweight at the beginning of pregnancy, 41.7% and 25.0% gained excessive or insufficient weight during pregnancy, respectively. Pregnancy-induced hypertension was present in 21.7% of cases, gestational diabetes in 18.3%, chronic hypertension in 6.7%, and type 2 diabetes mellitus in 5.0%. Pregnancy-induced hypertension was significantly associated with increased daily consumption of pastry products (31.2 vs 10.0 g, p=0.022), fast food (39.6 vs 29.7 g, p=0.028), bread (90.0 vs 50.0 g, p=0.005), pasta, rice and potatoes (225.7 vs 154.3 g, p=0.012). In a multivariate analysis, only bread consumption maintained a significant, albeit weak, association (OR=1.021; 1.003 - 1.038, p=0.022). Conclusion: Pregnancy-induced hypertension was associated with increased consumption of pastry products, fast food, bread, pasta, rice, and potatoes, although only bread consumption had a weak but statistically significant association with pregnancy-induced hypertension in a multivariate analysis

Hábitos Alimentares Durante a Gravidez em Mulheres com Parto Muito Pré-Termo: Uma Análise Exploradora

Introduction: Preterm birth is increasing worldwide, representing a major cause of death and long-term loss of human potential among survivors. Some morbidities during pregnancy are well-known risk factors for preterm labor, but it is not yet known whether deviations from adequate dietary patterns are associated with preterm delivery. Diet may be an important modulator of chronic inflammation, and pro-inflammatory diets during pregnancy were reported to be associated with preterm birth. The aim of this study was to assess the food consumption during pregnancy of Portuguese women giving birth very prematurely and the association between the food consumption and the major maternal morbidities during pregnancy related with preterm delivery. Material and methods: A single-center cross-sectional observational study including consecutive Portuguese women giving birth before 33 weeks of gestation was conducted. Recall of eating habits during pregnancy was obtained within the first week after delivery, using a semi quantitative food frequency questionnaire validated for Portuguese pregnant women. Results: Sixty women with a median age of 36.0 years were included. Of these, 35% were obese or overweight at the beginning of pregnancy, 41.7% and 25.0% gained excessive or insufficient weight during pregnancy, respectively. Pregnancy-induced hypertension was present in 21.7% of cases, gestational diabetes in 18.3%, chronic hypertension in 6.7%, and type 2 diabetes mellitus in 5.0%. Pregnancy-induced hypertension was significantly associated with increased daily consumption of pastry products (31.2 vs 10.0 g, p = 0.022), fast food (39.6 vs 29.7 g, p = 0.028), bread (90.0 vs 50.0 g, p = 0.005), pasta, rice and potatoes (225.7 vs 154.3 g, p = 0.012). In a multivariate analysis, only bread consumption maintained a significant, albeit weak, association (OR = 1.021; 1.003 - 1.038, p = 0.022). Conclusion: Pregnancy-induced hypertension was associated with increased consumption of pastry products, fast food, bread, pasta, rice, and potatoes, although only bread consumption had a weak but statistically significant association with pregnancy-induced hypertension in a multivariate analysis.info:eu-repo/semantics/publishedVersio

Biomarkers of Thermal Adaptation: New Tools in Sustainable Livestock Production under Climate Change

Climate changes have been identified as one of the greatest environmental, social and economic threats to the planet and humanity. The increase of extreme weather events, such as prolonged droughts, extreme ambient temperatures or periods with high and intensive precipitation has effects on animal production systems. Crops, and consequently forage productivity and availability are compromised, the risk of new diseases increase, and animal production is impaired (growth, reproductive performance, metabolic and health status, and immune response can be affected). In this way the development of resilient and robust animal production systems, together with an improvement in the knowledge of the environmental impact in animal production and welfare are crucial to enhance innovation, sustainability and productivity in the animal sector. Ambient temperature and its abrupt extreme events have a major impact on the energy metabolism of livestock. This implies that animals presenting more physiological versatility can be best adapted, and therefore less susceptible to thermal stress and more productive. To achieve a production system where the detrimental effect of the climate change can be the minimum is necessary to improve the ability of the animal to cope with environmental stress by management and selection. The existence of biomarkers that allow to identify the levels of thermal stress and/or acclimation are valuable in the process of selecting the best well suited animals for each environmental condition, to propose selection programs based on that and for the herds management. Ideally biomarkers should be obtained from readily accessible samples, preferably non-invasively or minimally invasive, such as saliva, sweat and milk, hair and feces. Nowadays, the most commonly fluid used in biomarkers studies is the blood/plasma, but with growing tendency for being replaced. Blood cortisol has been one of the parameters more frequently used for assessing stressful conditions such as thermal stress. Nevertheless, it does not allow a full understanding of heat stress, due to its circadian cycle and because the confounding with other types of stress. Moreover, and taking the advantage of saliva as a non-invasive source of this corticosteroid, salivary cortisol has been also referred as potentially interesting. However, some of the limitations in taking conclusions from salivary cortisol results are the same reported for blood cortisol. Consequently, new and better non-invasive methods, than allow assessing stress, are necessary. The aim of this chapter is to present the state of the art on stress responses (climate, housing) and the principal effects of great temperature amplitudes in livestock production and the existing means to evaluate heat stress and acclimation capacity. Focus will be put on the importance of new reliable biomarkers. Saliva, hair, milk and feces will be discussed as potential sources of such non-invasive biomarkers

A utilização de sémen fresco na fertilização in vitro de embriões ovinos melhora a qualidade dos blastocistos na raça portuguesa Merino

A produção de embriões em ovinos é uma tarefa difícil, exigindo experiência e condições onerosas, principalmente na produção de embriões in vivo. A recolha sistemática de oócitos em animais de matadouro ou em animais vivos por ovum pick-up, permite a produção in vitro de embriões (IVP), em larga escala e menos dispendiosa, nos pequenos ruminantes. Esta possibilidade é importante não só como fonte de embriões mas também de oócitos e zigotos para fins comerciais ou de investigação, facilitando a sua disponibilidade em tecnologias emergentes tais como a clonagem ou a transgénese. Para IVP foram desenvolvidos vários protocolos de maturação, utilizando fertilização in vitro (IVF) com sémen fresco ou congelado. Em Portugal, a produção de embriões in vitro foi somente realizada com sémen congelado dada a sua disponibilidade em condições de rotina. Contudo, o sémen fresco poderá melhorar a produção de embriões frescos ou criopreservados. Este trabalho teve como objectivo comparar a eficiência da IVP em ovinos usando diferentes protocolos de maturação de oócitos e IVF com sémen fresco ou congelado. Oócitos (n=1768) recolhidos em matadouro foram maturados em meio TCM199 com 100 μM cisteamine, 10 ng mL-1 EGF, 10 μg mL-1 E2 e gentamicina (mat A, n=692) ou suplementada com 10 μg mL-1 FSH e 0,3 mM piruvato de sódio (mat B, n=707) a 39 ºC e 5% CO2 durante 22h. O sémen fresco (FS) e congelado/descongelado (TS) de carneiros de raça Merino Branco (n=3) foi lavado ou submetido a swim-up, respectivamente. Após a fertilização (18h p.i.), os presumíveis zigotos foram cultivados em meio de fluido sintético do oviducto (SOF) enriquecido com aminoácidos e BSA a 38,5 ºC, em atmosfera humidificada com 5% O2, 5% CO2 e 90% N2 até ao estadio de 2-4-8 células. Após clivagem, o desenvolvimento embrionário prosseguiu até ao estadio de blastocisto em meio SOF, BSA e 10% FCS. A qualidade foi avaliada no dia 6-7, classificando-se como bons, médios e maus, baseado nos parâmetros IETS. Os dados das taxas de produção embrionária foram analisados utilizando ANOVA. Foi utilizado o teste de Mann-Whitney U para avaliação da qualidade dos embriões. Os diferentes protocolos de maturação não interferiram (p>0,05) quer com as taxas de maturação quer com as taxas de produção de embriões. A qualidade embrionária foi superior (p=0,004) na fertilização com sémen fresco (bom: FS=40,1±8,0% vs TS=32,9±5,6%; média: FS=20,1±4,7% vs TS=35,7±5,8%; má: FS=39,8±9,8% vs TS=31,4±7,6%). Em conclusão, estes resultados preliminares mostram que o sémen fresco de carneiro pode ser facilmente utilizado para fertilização in vitro e melhora a qualidade dos embriões produzidos.#Embryo production in sheep is a difficult task demanding experience and expensive facilities, particularly when dealing with in vivo embryo production. Easy ways to obtain ovine embryos consist of collecting oocytes at slaughterhouses or systematically pick them up from live animals, allowing a large scale and cheaper in vitro embryo production (IVP) for small ruminants. Those are important sources of embryos, oocytes and zygotes for commercial, laboratorial and research proposes, making easier the availability of resources for emerging techniques like cloning or transgenesis. For IVP, several oocyte maturation protocols have been developed using fertilization (IVF) either with fresh or frozen-thawed semen. In Portugal, IVP has been done through IVF using cryopreserved semen because it is easily available for routine use. However, the use of fresh semen could improve embryo production and cryopreservation results. The aim of this work was to compare the efficiency of in vitro embryo production in ovine using different oocyte maturation protocols and fresh or frozen semen for IVF. Abattoir-derived oocytes (n=1768) were matured in TCM199, 10 μM cysteamine, 10 ng mL-1 EGF, 10 μg mL-1 E2 and gentamicin (mat A, n=692) or plus 10 μg mL-1 FSH and 0.3 mM sodium piruvate (mat B, n=707) at 39 ºC and 5% CO2 for 22h. Prior to fertilization, either fresh (FS) or frozen/thawed (TS) semen from Merino rams (n=3) was washed or submitted to swim-up respectively. Presumptive zygotes (18h p.i.) were cultured in synthetic oviductal fluid (SOF) enriched with aminoacids and 6 mg mL-1 BSA at 38.5 ºC, under 5% O2, 5% CO2 and 90% N2 in an humidified atmosphere until the stage of 2-4-8 cell embryos. After assessing cleavage, embryo development proceeded until the blastocyst stage in SOF+BSA and 10% FCS. Quality was evaluated on D6-7 by scoring embryos as good, fair and bad based on IETS guidelines. Data from embryo production rates were analysed using ANOVA. Mann-Whitney U test was used for embryo quality evaluation. Different maturation protocols did not interfere (P>0.05) either on maturation or on embryo quality or production rates. Embryo quality was higher (P=0.004) when fertilization was accomplished with fresh semen (good: FS=40.1±8.0% vs TS=32.9±5.6%; fair: FS=20.1±4.7% vs TS=35.7±5.8%; bad: FS=39.8±9.8% vs TS=31.4±7.6%). Preliminar results show that ram fresh semen can be easily used for in vitro fertilization and improves the quality of produced embryos

ECM-enriched alginate hydrogels for bioartificial pancreas: an ideal niche to improve insulin secretion and diabetic glucose profile

Introduction: The success of a bioartificial pancreas crucially depends on ameliorating encapsulated beta cells survival and function. By mimicking the cellular in vivo niche, the aim of this study was to develop a novel model for beta cells encapsulation capable of establishing an appropriate microenvironment that supports interactions between cells and extracellular matrix (ECM) components. Methods: ECM components (Arg-Gly-Asp, abbreviated as RGD) were chemically incorporated in alginate hydrogels (alginate-RGD). After encapsulation, INS-1E beta cells outcome was analyzed in vitro and after their implantation in an animal model of diabetes. Results: Our alginate-RGD model demonstrated to be a good in vitro niche for supporting beta cells viability, proliferation, and activity, namely by improving the key feature of insulin secretion. RGD peptides promoted cell–matrix interactions, enhanced endogenous ECM components expression, and favored the assembly of individual cells into multicellular spheroids, an essential configuration for proper beta cell functioning. In vivo, our pivotal model for diabetes treatment exhibited an improved glycemic profile of type 2 diabetic rats, where insulin secreted from encapsulated cells was more efficiently used. Conclusions: We were able to successfully introduce a novel valuable function in an old ally in biomedical applications, the alginate. The proposed alginate-RGD model stands out as a promising approach to improve beta cells survival and function, increasing the success of this therapeutic strategy, which might greatly improve the quality of life of an increasing number of diabetic patients worldwide.The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by FCT/MEC through National Funds and co-financed by FEDER through the PT2020 Partnership Agreement under the 4293 Unit I&D, FCT Strategic Project PEst-C/SAU/UI3282/2011-2013 and UID/NEU/04539/2013, FCT in the framework of project UID/BIM/04293/2013, FCT in the framework of project IF/00939/2013/CP1179/CT0001, FCT for Joana Crisóstomo (grant number SFRH/BD/72964/2010), FCT for Sílvia J Bidarra (grant number SFRH/BPD/80571/2011), and FCT and POPH/ESF (EC) for Cristina C Barrias research position FCT Investigator (IF2013)

Reliability of two behavioral tools to assess pain in preterm neonates

CONTEXT: One of the main difficulties in adequately treating the pain of neonatal patients is the scarcity of validated pain evaluation methods for this population. OBJECTIVE: To analyze the reliability of two behavioral pain scales in neonates. TYPE OF STUDY: Cross-sectional. SETTING: University hospital neonatal intensive care unit. PARTICIPANTS: 22 preterm neonates were studied, with gestational age of 34 ± 2 weeks, birth weight of 1804 ± 584 g, 68% female, 30 ± 12 hours of life, and 30% intubated. PROCEDURES: Two neonatologists (A and B) observed the patients at the bedside and on video films for 10 minutes. The Neonatal Facial Coding System and the Clinical Scoring System were scored at 1, 5, and 10 minutes. The final score was the median of the three values for each observer and scale. A and B were blinded to each other. Video assessments were made three months after bedside evaluations. MAIN MEASUREMENTS: End scores were compared between the observers using the intraclass correlation coefficient and bias analysis (paired t test and signal test). RESULTS: For the Neonatal Facial Coding System, at the bedside and on video, A and B showed a significant correlation of scores (intraclass correlation score: 0.62), without bias between them (t test and signal test: p > 0.05). For the Clinical Scoring System bedside assessment, A and B showed correlation of scores (intraclass correlation score: 0.55), but bias was also detected between them: A scored on average two points higher than B (paired t test and signal test: p 0,05). Para a Escala de Conforto Clínico à beira do leito, os escores obtidos por A e B mostraram uma correlação significante (0,55), foi detectado: o escore obtido por A foi, em média, dois pontos superior ao de B (teste t e do sinal: p < 0,05). Para a mesma escala aplicada em vídeo, os escores obtidos por A e B não mostraram correlação (0,25) e detectou-se viés (teste t e do sinal: p < 0,05). CONCLUSÃO: Os resultados reforçam a confiabilidade do Sistema de Codificação da Atividade Facial Neonatal aplicado à beira do leito para a avaliação da dor no recém-nascido pré-termo.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Neonatal DivisionUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Department of EpidemiologyUNIFESP, EPM, Neonatal DivisionUNIFESP, EPM, Department of EpidemiologySciEL

Evaluation of urinary cysteinyl leukotrienes as biomarkers of severity and putative therapeutic targets in COVID-19 patients

Background Cysteinyl leukotrienes (CysLT) are potent inflammation-promoting mediators, but remain scarcely explored in COVID-19. We evaluated urinary CysLT (U-CysLT) relationship with disease severity and their usefulness for prognostication in hospitalized COVID-19 patients. The impact on U-CysLT of veno-venous extracorporeal membrane oxygenation (VV-ECMO) and of comorbidities such as hypertension and obesity was also assessed. Methods Blood and spot urine were collected in severe (n = 26), critically ill (n = 17) and critically ill on VV-ECMO (n = 17) patients with COVID-19 at days 1-2 (admission), 3-4, 5-8 and weekly thereafter, and in controls (n = 23) at a single time point. U-CysLT were measured by ELISA. Routine markers, prognostic scores and outcomes were also evaluated. Results U-CysLT did not differ between groups at admission, but significantly increased along hospitalization only in critical groups, being markedly higher in VV-ECMO patients, especially in hypertensives. U-CysLT values during the first week were positively associated with ICU and total hospital length of stay in critical groups and showed acceptable area under curve (AUC) for prediction of 30-day mortality (AUC: 0.734, p = 0.001) among all patients. Conclusions U-CysLT increase during hospitalization in critical COVID-19 patients, especially in hypertensives on VV-ECMO. U-CysLT association with severe outcomes suggests their usefulness for prognostication and as therapeutic targets.This work was supported by a RESEARCH 4 COVID-19 grant (project 519, reference number: 613690173) from FCT-Fundacao para a Ciencia e a Tecnologia (special support for rapid implementation projects for innovative response solutions to COVID-9 pandemic). CS-P is a recipient of a Ph.D. fellowship from FCT and MedInUP (UI/BD/150816/2020). P-PT was supported by a research contract within the scope of the RIFF-HEART project funded by FEDER via COMPETE, Portugal 2020-Operational Programme for Competitiveness and Internationalization (POCI) (POCI-01-0145-FEDER-032188) and by FCT (PTDC/MEC-CAR/32188/2017). Open access funding provided by FCT|FCCN (b-on)