Methionine and Tryptophan Play Different Modulatory Roles in the European Seabass (Dicentrarchus labrax) Innate Immune Response and Apoptosis Signaling—An In Vitro Study

The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. In vitro trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from Photobacterium damselae subsp. piscicida or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes’ immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher il1ß and nf-¿b expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including nf-¿b and p65. This study showed the ability of methionine surplus to improve leucocytes’ response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56.This work was partially supported by UIDB/04423/2020, UIDP/ 04423/2020 and INFLAMMAA (reference PTDC/CVT-CVT/ 32349/2017), financed by Portugal and the European Union through FEDER and COMPETE 2020, and national funds through Fundação para a Ciência e a Tecnologia (FCT, Portugal). MM and BC were supported by FCT, Portugal (SFRH/BD/108243/2015 and IF/00197/2015, respectively)

Selection of carbohydrate-active probiotics from the gut of carnivorous fish fed plant-based diets

Abstract The gastrointestinal microbiota plays a critical role on host health and metabolism. This is particularly important in teleost nutrition, because fish do not possess some of the necessary enzymes to cope with the dietary challenges of aquaculture production. A main difficulty within fish nutrition is its dependence on fish meal, an unsustainable commodity and a source of organic pollutants. The most obvious sustainable alternatives to fish meal are plant feedstuffs, but their nutritive value is limited by the presence of high levels of non-starch polysaccharides (NSP), which are not metabolized by fish. The composition of fish-gut microbial communities have been demonstrated to adapt when the host is fed different ingredients. Thus, we hypothesized that a selective pressure of plant-based diets on fish gut microbiota, could be a beneficial strategy for an enrichment of bacteria with a secretome able to mobilize dietary NSP. By targeting bacterial sporulating isolates with diverse carbohydrase activities from the gut of European sea bass, we have obtained isolates with high probiotic potential. By inferring the adaptive fitness to the fish gut and the amenability to industrial processing, we identified the best two candidates to become industrially valuable probiotics. This potential was confirmed in vivo, since one of the select isolates lead to a better growth and feed utilization efficiency in fish fed probiotic-supplemented plant-based diets, thus contributing for sustainable and more cost-effective aquaculture practices

Erectile dysfunction, physical activity and metabolic syndrome: differences in markers of atherosclerosis

<p>Abstract</p> <p>Background</p> <p>Erectile dysfunction (ED), impaired arterial elasticity, elevated resting heart rate as well as increased levels of oxidized LDL and fibrinogen associate with future cardiovascular events. Physical activity is crucial in the prevention of cardiovascular diseases (CVD), while metabolic syndrome (MetS) comprises an increased risk for CVD events. The aim of this study was to assess whether markers of subclinical atherosclerosis are associated with the presence of ED and MetS, and whether physical activity is protective of ED.</p> <p>Methods</p> <p>57 MetS (51.3 ± 8.0 years) and 48 physically active (PhA) (51.1 ± 8.1 years) subjects participated in the study. ED was assessed by the International Index of Erectile Function (IIEF) questionnaire, arterial elasticity by a radial artery tonometer (HDI/PulseWave™ CR-2000) and circulating oxLDL by a capture ELISA immunoassay. Fibrinogen and lipids were assessed by validated methods. The calculation of mean daily energy expenditure of physical exercise was based on a structured questionnaire.</p> <p>Results</p> <p>ED was more often present among MetS compared to PhA subjects, 63.2% and 27.1%, respectively (p < 0.001). Regular physical exercise at the level of > 400 kcal/day was protective of ED (OR 0.12, 95% CI 0.017-0.778, p = 0.027), whereas increased fibrinogen (OR 4.67, 95% CI 1.171-18.627, p = 0.029) and elevated resting heart rate (OR 1.07, 95% CI 1.003-1.138, p = 0.04) were independently associated with the presence of ED. In addition, large arterial elasticity (ml/mmHgx10) was lower among MetS compared to PhA subjects (16.6 ± 4.0 <it>vs</it>. 19.6 ± 4.2, p < 0.001), as well as among ED compared to non-ED subjects (16.7 ± 4.6 <it>vs</it>. 19.0 ± 3.9, p = 0.008). Fibrinogen and resting heart rate were highest and large arterial elasticity lowest among subjects with both MetS and ED.</p> <p>Conclusions</p> <p>Markers of subclinical atherosclerosis associated with the presence of ED and were most evident among subjects with both MetS and ED. Thus, especially MetS patients presenting with ED should be considered at high risk for CVD events. Physical activity, on its part, seems to be protective of ED.</p> <p>Trial registration</p> <p>ClinicalTrials.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT01119404">NCT01119404</a></p

The Role of the Proteinase Inhibitor Ovorubin in Apple Snail Eggs Resembles Plant Embryo Defense against Predation

BACKGROUND: Fieldwork has thoroughly established that most eggs are intensely predated. Among the few exceptions are the aerial egg clutches from the aquatic snail Pomacea canaliculata which have virtually no predators. Its defenses are advertised by the pigmented ovorubin perivitellin providing a conspicuous reddish coloration. The nature of the defense however, was not clear, except for a screening for defenses that identified a neurotoxic perivitellin with lethal effect on rodents. Ovorubin is a proteinase inhibitor (PI) whose role to protect against pathogens was taken for granted, according to the prevailing assumption. Through biochemical, biophysical and feeding experiments we studied the proteinase inhibitor function of ovorubin in egg defenses. METHODOLOGY/PRINCIPAL FINDINGS: Mass spectrometry sequencing indicated ovorubin belongs to the Kunitz-type serine proteinase inhibitor family. It specifically binds trypsin as determined by small angle X-ray scattering (SAXS) and cross-linking studies but, in contrast to the classical assumption, it does not prevent bacterial growth. Ovorubin was found extremely resistant to in vitro gastrointestinal proteolysis. Moreover feeding studies showed that ovorubin ingestion diminishes growth rate in rats indicating that this highly stable PI is capable of surviving passage through the gastrointestinal tract in a biologically active form. CONCLUSIONS: To our knowledge, this is the first direct evidence of the interaction of an egg PI with a digestive protease of potential predators, limiting predator's ability to digest egg nutrients. This role has not been reported in the animal kingdom but it is similar to plant defenses against herbivory. Further, this would be the only defense model with no trade-offs between conspicuousness and noxiousness by encoding into the same molecule both the aposematic warning signal and an antinutritive/antidigestive defense. These defenses, combined with a neurotoxin and probably unpalatable factors would explain the near absence of predators, opening new perspectives in the study of the evolution and ecology of egg defensive strategies

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research