A widespread inversion polymorphism conserved among Saccharomyces species is caused by recurrent homogenization of a sporulation gene family.

Saccharomyces genomes are highly collinear and show relatively little structural variation, both within and between species of this yeast genus. We investigated the only common inversion polymorphism known in S. cerevisiae, which affects a 24-kb 'flip/flop' region containing 15 genes near the centromere of chromosome XIV. The region exists in two orientations, called reference (REF) and inverted (INV). Meiotic recombination in this region is suppressed in crosses between REF and INV orientation strains such as the BY x RM cross. We find that the inversion polymorphism is at least 17 million years old because it is conserved across the genus Saccharomyces. However, the REF and INV isomers are not ancient alleles but are continually being re-created by re-inversion of the region within each species. Inversion occurs due to continual homogenization of two almost identical 4-kb sequences that form an inverted repeat (IR) at the ends of the flip/flop region. The IR consists of two pairs of genes that are specifically and strongly expressed during the late stages of sporulation. We show that one of these gene pairs, YNL018C/YNL034W, codes for a protein that is essential for spore formation. YNL018C and YNL034W are the founder members of a gene family, Centroid, whose members in other Saccharomycetaceae species evolve fast, duplicate frequently, and are preferentially located close to centromeres. We tested the hypothesis that Centroid genes are a meiotic drive system, but found no support for this idea.

Carbon Dioxide Sensing by Immune Cells Occurs through Carbonic Anhydrase 2-Dependent Changes in Intracellular pH.

CO2, the primary gaseous product of respiration, is a major physiologic gas, the biology of which is poorly understood. Elevated CO2 is a feature of the microenvironment in multiple inflammatory diseases that suppresses immune cell activity. However, little is known about the CO2-sensing mechanisms and downstream pathways involved. We found that elevated CO2 correlates with reduced monocyte and macrophage migration in patients undergoing gastrointestinal surgery and that elevated CO2 reduces migration in vitro. Mechanistically, CO2 reduces autocrine inflammatory gene expression, thereby inhibiting macrophage activation in a manner dependent on decreased intracellular pH. Pharmacologic or genetic inhibition of carbonic anhydrases (CAs) uncouples a CO2-elicited intracellular pH response and attenuates CO2 sensitivity in immune cells. Conversely, CRISPR-driven upregulation of the isoenzyme CA2 confers CO2 sensitivity in nonimmune cells. Of interest, we found that patients with chronic lung diseases associated with elevated systemic CO2 (hypercapnia) display a greater risk of developing anastomotic leakage following gastrointestinal surgery, indicating impaired wound healing. Furthermore, low intraoperative pH levels in these patients correlate with reduced intestinal macrophage infiltration. In conclusion, CO2 is an immunomodulatory gas sensed by immune cells through a CA2-coupled change in intracellular pH.

Alterations in immune cell phenotype and cytotoxic capacity in HER2+ breast cancer patients receiving HER2-targeted neo-adjuvant therapy.

BACKGROUND: The phase II neo-adjuvant clinical trial ICORG10-05 (NCT01485926) compared chemotherapy in combination with trastuzumab, lapatinib or both in patients with HER2+ breast cancer. We studied circulating immune cells looking for alterations in phenotype, genotype and cytotoxic capacity (direct and antibody-dependent cell-mediated cytotoxicity (ADCC)) in the context of treatment response. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from pre- (n = 41) and post- (n = 25) neo-adjuvant treatment blood samples. Direct/trastuzumab-ADCC cytotoxicity of patient-derived PBMCs against K562/SKBR3 cell lines was determined ex vivo. Pembrolizumab was interrogated in 21 pre-treatment PBMC ADCC assays. Thirty-nine pre-treatment and 21 post-treatment PBMC samples were immunophenotyped. Fc receptor genotype, tumour infiltrating lymphocyte (TIL) levels and oestrogen receptor (ER) status were quantified. RESULTS: Treatment attenuated the cytotoxicity/ADCC of PBMCs. CD3+/CD4+/CD8+ T cells increased following therapy, while CD56+ NK cells/CD14+ monocytes/CD19+ B cells decreased with significant post-treatment immune cell changes confined to patients with residual disease. Pembrolizumab-augmented ex vivo PBMC ADCC activity was associated with residual disease, but not pathological complete response. Pembrolizumab-responsive PBMCs were associated with lower baseline TIL levels and ER+ tumours. CONCLUSIONS: PBMCs display altered phenotype and function following completion of neo-adjuvant treatment. Anti-PD-1-responsive PBMCs in ex vivo ADCC assays may be a biomarker of treatment response.

Kinase Suppressor of RAS 1 (KSR1) Maintains the Transformed Phenotype of BRAFV600E Mutant Human Melanoma Cells.

Kinase Suppressor of RAS 1 (KSR1) is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS-mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E-transformed melanoma cell line. KSR1 loss produced a complex phenotype characterised by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation.

Hydroxylase Inhibition Selectively Induces Cell Death in Monocytes.

Hypoxia is a common and prominent feature of the microenvironment at sites of bacteria-associated inflammation in inflammatory bowel disease. The prolyl-hydroxylases (PHD1/2/3) and the asparaginyl-hydroxylase factor-inhibiting HIF are oxygen-sensing enzymes that regulate adaptive responses to hypoxia through controlling the activity of HIF and NF-κB-dependent transcriptional pathways. Previous studies have demonstrated that the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) is effective in the alleviation of inflammation in preclinical models of inflammatory bowel disease, at least in part, through suppression of IL-1ß-induced NF-κB activity. TLR-dependent signaling in immune cells, such as monocytes, which is important in bacteria-driven inflammation, shares a signaling pathway with IL-1ß. In studies into the effect of pharmacologic hydroxylase inhibition on TLR-induced inflammation in monocytes, we found that DMOG selectively triggers cell death in cultured THP-1 cells and primary human monocytes at concentrations well tolerated in other cell types. DMOG-induced apoptosis was independent of increased caspase-3/7 activity but was accompanied by reduced expression of the inhibitor of apoptosis protein 1 (cIAP1). Based on these data, we hypothesize that pharmacologic inhibition of the HIF-hydroxylases selectively targets monocytes for cell death and that this may contribute to the anti-inflammatory activity of HIF-hydroxylase inhibitors.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Fasciola hepatica products can alter the response of bovine immune cells to Mycobacterium avium subsp. paratuberculosis.

BACKGROUND: Fasciola hepatica causes economically important disease in livestock worldwide. The relevance of this parasitic infection extends beyond its direct consequences due to its immunoregulatory properties. OBJECTIVES: Given the importance of the T helper 1 (Th1) immune response in controlling infections with Mycobacterium avium subspecies paratuberculosis (MAP) in cattle, we aimed to establish the immunological consequences that co-infection with F. hepatica might have on the course of Johne's disease (JD). METHODS: This study compared the in vitro response of bovine immune cells to infection with MAP or exposure to MAP antigens following F. hepatica infection or stimulation with F. hepatica products. RESULTS: We found a decreased proliferation of peripheral blood mononuclear cells (PBMCs) after infection with F. hepatica. This reduction was inversely correlated with fluke burden. Pre-stimulation with F. hepatica molecules produced a significant reduction of ileocaecal lymph node leucocyte proliferation in response to MAP antigens. Additionally,F. hepatica products reduced expression of the CD14 receptor by macrophages and increased levels of apoptosis and bacterial (MAP) uptake. CONCLUSIONS: Overall, F. hepatica infection had little impact on the in vitro response of immune cells to MAP, whereas in vitro co-stimulation with F. hepatica molecules had a measurable effect. Whether this is likely to affect JD progression during in vivo chronic conditions remains unclear.

Intramyocardial bone marrow mononuclear cells versus bone marrow-derived and adipose mesenchymal cells in a rat model of dilated cardiomyopathy.

BACKGROUND: Effects of cell therapy on dilated cardiomyopathy (DCM) have been investigated in pre-clinical models using distinct cellular types in each study. A single study that compares the effectiveness of different cells is lacking. METHODS: We have compared the effects of intramyocardial injection (IMI) of bone marrow (BM)-derived mononuclear cells (MNCs), BM and adipose tissue (AT) mesenchymal stromal cells (BM-MSCs and AT-MSCs) on heart function, histological changes and myocardial ultrastructure in a rat model of DCM. Isogenic Wistar rats were used to isolate the different cell types and to induce DCM by autoimmune myocarditis. Animals were randomly assigned to receive BM-MNCs, BM-MSCs, AT-MSCs or placebo at day 42 by IMI. Serial echocardiography was used to assess cardiac function and hearts obtained after sacrifice at day 70, were used for histological and ultrastructural analysis. Serum levels of type B-natriuretic peptide (BNP) and vascular endothelial growth-factor (VEGF) were determined at different time points. RESULTS: BM-MSC treatment induced significant improvement in ejection fraction (EF), fractional shortening (FS), left ventricular systolic diameter (LVESD) and systolic volume (LVESV). In contrast, changes in echocardiographic parameters with respect to pre-treatment values in animals receiving placebo, AT-MSCs or BM-MNCs were not statistically significant. EF and FS in animals receiving AT-MSCs were superior to those receiving placebo. BM-MSC transplantation induced also improvement in cardiac fibers organization and capillary density, fibrotic tissue reduction, increase in final VEGF concentration and BNP decrease. DISCUSSION: IMI of BM or AT-MSCs improves LV function and induces more angiogenesis processes than BM-MNCs. In addition, BM-MSCs showed more anti-fibrotic effects and more ability to reorganize myocardial tissue compared with the other cell types.

Metabolic risk management, physical exercise and lifestyle counselling in low-active adults: controlled randomized trial (BELLUGAT).

BACKGROUND: The primary aim of this study is to evaluate the effectiveness of different doses (intensity) of supervised exercise training - concomitant with lifestyle counselling - as a primary care intervention tool for the management of metabolic syndrome risk factors in low-active adults with one or more such factors (programme name in Catalan: Bellugat de CAP a peus). METHODS/DESIGN: Three-arm, randomized controlled clinical trial implemented in the primary care setting, with a duration of 40 weeks (16 weeks intervention and 24-week follow-up). Adults aged 30 to 55 years with metabolic risk factors will be randomized into three intervention groups: 1) aerobic interval training (16 supervised training lessons) plus a healthy lifestyle counselling programme (6 group and 3 individual meetings); 2) low-to-moderate intensity continuous training (16 supervised training lessons) plus the same counselling programme; or 3) the counselling- programme without any supervised physical exercise. The main output variables assessed will be risk factors for metabolic syndrome (waist circumference, blood pressure, and levels of plasma triglycerides, high-density lipoproteins and glucose), systemic inflammation, cardiorespiratory fitness, physical activity and sedentary behaviour, dietary habits, health-related quality of life, self-efficacy and empowerment. Economic factors will also be analysed in order to determine the cost-effectiveness of the programme. These variables will be assessed three times during the study: at baseline, at the end of the intervention, and at follow-up. We estimate to recruit 35 participants per group. DISCUSSION: The results of this study will provide insight into the immediate and medium-term effects on metabolic risk and lifestyle of a combined approach involving aerobic interval training and a multidisciplinary behavioural intervention. If effective, the proposed intervention would provide both researchers and practitioners in this field with a platform on which to develop similar intervention programmes for tackling the repercussions of an unhealthy lifestyle. TRIAL REGISTRATION: Clinical trials.gov. NTC02832453 . Registered 6 July 2016 (retrospectively registered).

Dietary l-carnitine prevents histopathological changes in tilapia (Oreochromis Niloticus) exposed to cylindrospermopsin.

Cylindrospermopsin (CYN) is a cytotoxin highly water-soluble, which is easily taken up by several aquatic organisms. CYN acts as a potent protein and glutathione synthesis inhibitor, as well as inducing genotoxicity, oxidative stress, and histopathological alterations. This is the first study reporting the protective effect of a l-carnitine (LC) pretreatment (400 or 880 mg LC/kg bw fish/day, for 21 days) on the histopathological alterations induced by pure CYN or Aphanizomenon ovalisporum lyophilized cells (400 µg CYN/kg bw fish) in liver, kidney, heart, intestines, and gills of tilapia (Oreochromis niloticus) acutely exposed to the toxin by oral route. The main histopathological changes induced by CYN were disorganized parenchyma with presence of glycogen and lipids in the cytoplasm (liver), glomerulonephritis, glomerular atrophy, and dilatation of Bowman's capsule (kidney), myofibrolysis, loss of myofibrils, with edema and hemorrhage (heart), intestinal villi with necrotic enterocytes and partial loss of microvilli (gastrointestinal tract), and hyperemia and hemorrhage (gills). LC pretreatment was able to totally prevent those CYN-induced alterations from 400 mg LC/kg bw fish/day in almost all organs, except in the heart, where 880 mg LC/kg bw fish/day were needed. In addition, the morphometric study indicated that LC managed to recover totally the affectation in the cross sections of the proximal and distal convoluted tubules in CYN-exposed fish. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 241-254, 2017.

Effects of depuration on histopathological changes in tilapia (Oreochromis niloticus) after exposure to cylindrospermopsin.

Cylindrospermopsin (CYN) is a highly water-soluble cytotoxin produced by several species of freshwater cyanobacteria and it is considered the second most studied cyanotoxin worldwide. CYN acts as a potent protein and glutathione synthesis inhibitor, as well as inducing genotoxicity, oxidative stress and histopathological alterations. Studies concerning the depuration of cyanobacterial toxins in aquatic organisms, especially in fish, are of great interest for fish economy and public health, but are scarce in the case of CYN. This is the first study reporting the ability of depuration (3 - 7 days) in reversing or ameliorating the histopathological lesions induced in liver, kidney, heart, intestines, and gills of tilapia (Oreochromis niloticus) due to exposure by immersion to repeated doses of a CYN-containing culture of A. ovalisporum for 14 days. The main histopathological changes induced by CYN were glucogenic degeneration and loss of the normal hepatic cord-structure (liver), hyperemia, dilated Bowman's capsule and cellular tumefaction (kidney), myofibrolysis, hemorrhages and edema (heart), necrosis and partial loss of microvilli (gastrointestinal tract), and hyperemia and inflammatory cells infiltrates (gills). After 3 days of depuration, gills were totally recovered, while the liver, kidney, and gastrointestinal tract required 7 days, and longer depuration periods may be needed for a full recovery of the heart. In addition, the morphometric study indicated that depuration managed to reverse the affectation in the hepatocytes nuclear diameters and cross sections of the proximal and distal convoluted tubules induced in CYN-exposed fish. In general, these results validate depuration as an effective practice for detoxification of fish contaminated with CYN. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1318-1332, 2017.

A method for isolation of cone photoreceptors from adult zebrafish retinae.

BACKGROUND: Cone photoreceptors are specialised sensory retinal neurons responsible for photopic vision, colour perception and visual acuity. Retinal degenerative diseases are a heterogeneous group of eye diseases in which the most severe vision loss typically arises from cone photoreceptor dysfunction or degeneration. Establishing a method to purify cone photoreceptors from retinal tissue can accelerate the identification of key molecular determinants that underlie cone photoreceptor development, survival and function. The work herein describes a new method to purify enhanced green fluorescent protein (EGFP)-labelled cone photoreceptors from adult retina of Tg(3.2gnat2:EGFP) zebrafish. RESULTS: Methods for dissecting adult zebrafish retinae, cell dissociation, cell sorting, RNA isolation and RNA quality control were optimised. The dissociation protocol, carried out with ~30 retinae from adult zebrafish, yielded approximately 6 × 106 cells. Flow cytometry cell sorting subsequently distinguished 1 × 106 EGFP+ cells and 4 × 106 EGFP- cells. Electropherograms confirmed downstream isolation of high-quality RNA with RNA integrity number (RIN) >7.6 and RNA concentration >5.7 ng/µl obtained from both populations. Reverse Transcriptase-PCR confirmed that the EGFP-positive cell populations express known genetic markers of cone photoreceptors that were not expressed in the EGFP-negative cell population whereas a rod opsin amplicon was only detected in the EGFP-negative retinal cell population. CONCLUSIONS: This work describes a valuable adult zebrafish cone photoreceptor isolation methodology enabling future identification of cone photoreceptor-enriched genes, proteins and signalling networks responsible for their development, survival and function. In addition, this advancement facilitates the identification of novel candidate genes for inherited human blindness.

Vitamin D enhances reactive oxygen intermediates production in phagocytic cells in term and preterm infants.

BACKGROUND: Newborn infants are endotoxin tolerant which may be responsible for their increased susceptibility to bacterial sepsis. Vitamin D has an immunomodulatory effect and newborn infants are at risk of vitamin D deficiency. We examined the in vitro effect of 1, 25-dihydroxyvitamin D (1,25OHD) on whole blood phagocytic toll-like receptor 4 (TLR4), CD11b, and reactive oxygen intermediates (ROIs) in newborn infants during sepsis. METHODS: Whole blood from preterm infants <32-wk gestation, control term neonates, and adults were sampled for phagocytic expression of ROI, TLR4, CD11b in response to lipopolysaccharide (LPS), and 1,25OHD using flow cytometer. RESULTS: ROI production from newborn phagocytes incubated with LPS alone was decreased. Pretreatment with 1,25OHD demonstrated increased (P = 0.001) phagocytic ROI production in newborns but not in adults. 1,25OHD did not have any effect on TLR4 and CD11b in both newborns and adults. Pretreatment with ROI inhibitors (apocynin (APO) and diphenyleneiodonium), phosphoinositide 3-kinase (PI3K) inhibitor, and p38 inhibitor blocked neutrophil ROI production. CONCLUSION: Neonatal phagocytic cells had diminished ROI production in the presence of LPS, however, pretreatment with 1,25OHD reversed this hyporesponsiveness. This action by 1,25OHD was mediated by activation of nicotinamide adenine dinucleotide phosphate oxidase system through PI3K signaling enzymes.

Vitamin E pretreatment prevents histopathological effects in tilapia (Oreochromis niloticus) acutely exposed to cylindrospermopsin.

Cylindrospermopsin (CYN) is a cyanotoxin frequently involved in blooms with a predominantly extracellular availability, which makes it easily taken up by a variety of aquatic organisms. CYN is a potent protein and glutathione synthesis inhibitor, and also induces genotoxicity, oxidative stress and several histopathological lesions. The present study investigates the protective role of a vitamin E pretreatment (700 mg vit E/kg fish bw/day, for 7 days) on the histopathological alterations induced in different organs of tilapia (Oreochromis niloticus) acutely exposed to a single oral dose of 400 µg pure CYN/kg bw fish. The major histological changes observed were degenerative glucogenic process and loss of the hepatic structure in the liver, glomerulopathy and tubular tumefaction in the kidney, myofibrolysis and edema in the heart, catarrhal enteritis and necrosis in the gastrointestinal tract, hyperemic processes in the gill lamellae, and high basophilia, degeneration and tumefaction of granular neurons in the brain. Vitamin E pretreatment was effective in preventing or ameliorating the abovementioned alterations induced by CYN. In addition, a morphometric study indicated that the average nuclear diameter of hepatocytes, and cross-sections of proximal and distal convoluted tubules, together with the cardiac fiber and capillaries diameters represent a useful tool to evaluate the damage induced by CYN. This is the first study reporting vitamin E prevention of histopathological damage in tissues (liver, kidney, heart, gastrointestinal tract, gills and brain) of fish intoxicated with CYN. Therefore, vitamin E can be considered a useful chemoprotectant in the treatment of histopathological changes induced in CYN-intoxicated fish. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1469-1485, 2016.

Energy expenditure in low active overweight and obese children at varying treadmill grades.

UNLABELLED: This study aimed to evaluate differences between low active overweight and obese children in terms of energy expenditure (EE), ventilation (VE), and cardiac response during graded submaximal treadmill testing at constant speed. METHODS: We categorized 20 children into two weight groups according to the International Obesity Task Force criteria: overweight (n = 10; age = 9.7 ±1.34 years) and obese (n = 10; age = 10.4 ± 1.4 years). Children performed treadmill testing at a constant speed (1.53 m·s-1) and increasing grade (0%, 4%, and 8%). every 3 min. RESULTS: The EE across all grades was significantly higher (p < .001) in obese than in overweight children. Differences at each grade disappeared when EE was adjusted by body mass; however, several differences remained when the EE was adjusted by fat-free mass or body surface area. The increase in EE with increasing grade was greater in obese children (effect size between 0% and 8% for EE was 1.17). BMI z-score and fat mass (kg) were the main predictors of EE (Kcal·min-1) and contributed to explaining 66%, 70%, and 83.4% of the variance in EE at 0%, 4% and 8% gradients respectively. CONCLUSION: We suggest that when assessing EE response to exercise, the degree of obesity should be taken into consideration.

Cyanobacterium producing cylindrospermopsin cause histopathological changes at environmentally relevant concentrations in subchronically exposed tilapia (Oreochromis niloticus).

The acute toxicity of cylindrospermopsin (CYN) has been established in rodents, based on diverse intraperitoneal an oral exposure studies and more recently in fish. But no data have been reported in fish after subchronic exposure to cyanobacterial cells containing this cyanotoxin, so far. In this work, tilapia (Oreochromis niloticus) were exposed by immersion to lyophilized Aphanizomenon ovalisporum cells added to the aquaria using two concentration levels of CYN (10 or 100 µg CYN L(-1)) and deoxy-cylindrospermopsin (deoxy-CYN) (0.46 or 4.6 µg deoxy-CYN L(-1)), during two different exposure times: 7 or 14 d. This is the first study showing damage in the liver, kidney, hearth, intestines, and gills of tilapia after subchronic exposure to cyanobacterial cells at environmental relevant concentrations. The major histological changes observed were degenerative processes and steatosis in the liver, membranous glomerulopathy in the kidney, myofibrolysis and edema in the heart, necrotic enteritis in the gastrointestinal tract, and hyperemic processes in gill lamellae and microhemorrhages. Moreover, these histopathological findings confirm that the extent of damage is related to the CYN concentration and length of exposure. Results from the morphometric study indicated that the average of nuclear diameter of hepatocytes and cross-sections of proximal and distal convoluted tubules are useful to evaluate the damage induced by CYN in the main targets of toxicity.

In vivo toxicity evaluation of the migration extract of an organomodified clay-poly(lactic) acid nanocomposite.

The food packaging industry is in continuous development in order to obtain more secure and stable food and beverages. The incorporation of inorganic and organic materials with plastic polymers leads to polymer composites. Among the inorganic compounds, clays such as montmorillonite (MTT) and its derivatives are of great interest due to their advantageous properties. The Technological Institute of Packaging, Transport,and Logistics (ITENE) developed a novel nanocomposite based on a poly(lactic) acid (PLA) polymer using an MMT derivative, named Clay1, as filler, to be used in the beverage industry. The improvement of the technological properties of this new material was demonstrated, but safety issues are also of concern. In the present study, a histopathological examination by optical and electron microscopy of organs from Wistar rats exposed for 90 d to a migration extract of PLA-Clay1 nanocomposite was carried out. Moreover, different clinical biochemistry, inflammation,and oxidative stress biomarkers were determined. Results showed no apparent evidence of damage, indicating that this nanocomposite has a good profile to be used in the food packaging industry, although further research is still needed.

Acute exposure to pure cylindrospermopsin results in oxidative stress and pathological alterations in tilapia (Oreochromis niloticus).

Cylindrospermopsin (CYN) is increasingly recognized as a potential threat to drinking water safety, due to its ubiquity. This cyanotoxin has been found to cause toxic effects in mammals, and although fish could be in contact with this toxin, acute toxicity studies on fish are nonexistent. This is the first study showing that single doses of CYN pure standard (200 or 400 µg CYN/kg fish bw) by oral route (gavage) generate histopathological effects in fish (Tilapia-Oreochromis niloticus) exposed to the toxin under laboratory condition. Among the morphological changes, disorganized parenchymal architecture in the liver, dilated Bowman's space in the kidney, fibrolysis in the heart, necrotic enteritis in the intestines, and hemorrhages in the gills, were observed. Moreover, some oxidative stress biomarkers in the liver and kidney of tilapias were altered. Thus, CYN exposure induced increased protein oxidation products in both organs, NADPH oxidase activity was significantly increased with the kidney being the most affected organ, and decreased GSH contents were also detected in both organs, at the higher dose assayed.

Freshwater microalgae Nannochloropsis limnetica for the production of ß-galactosidase from whey powder.

This study investigated the first-ever reported use of freshwater Nannochloropsis for the bioremediation of dairy processing side streams and co-generation of valuable products, such as ß-galactosidase enzyme. In this study, N. limnetica was found to grow rapidly on both autoclaved and non-autoclaved whey-powder media (referred to dairy processing by-product or DPBP) without the need of salinity adjustment or nutrient additions, achieving a biomass concentration of 1.05-1.36 g L-1 after 8 days. The species secreted extracellular ß-galactosidase (up to 40.84 ± 0.23 U L-1) in order to hydrolyse lactose in DPBP media into monosaccharides prior to absorption into biomass, demonstrating a mixotrophic pathway for lactose assimilation. The species was highly effective as a bioremediation agent, being able to remove > 80% of total nitrogen and phosphate in the DPBP medium within two days across all cultures. Population analysis using flow cytometry and multi-channel/multi-staining methods revealed that the culture grown on non-autoclaved medium contained a high initial bacterial load, comprising both contaminating bacteria in the medium and phycosphere bacteria associated with the microalgae. In both autoclaved and non-autoclaved DPBP media, Nannochloropsis cells were able to establish a stable microalgae-bacteria interaction, suppressing bacterial takeover and emerging as dominant population (53-80% of total cells) in the cultures. The extent of microalgal dominance, however, was less prominent in the non-autoclaved media. High initial bacterial loads in these cultures had mixed effects on microalgal performance, promoting ß-galactosidase synthesis on the one hand while competing for nutrients and retarding microalgal growth on the other. These results alluded to the need of effective pre-treatment step to manage bacterial population in microalgal cultures on DPBP. Overall, N. limnetica cultures displayed competitive ß-galactosidase productivity and propensity for efficient nutrient removal on DPBP medium, demonstrating their promising nature for use in the valorisation of dairy side streams.

The N-terminal domain is required for cell surface localisation of VapA, a member of the Vap family of Rhodococcus equi virulence proteins.

Rhodococcus equi pneumonia is an important cause of mortality in foals worldwide. Virulent equine isolates harbour an 80-85kb virulence plasmid encoding six virulence-associated proteins (Vaps). VapA, the main virulence factor of this intracellular pathogen, is known to be a cell surface protein that creates an intracellular niche for R. equi growth. In contrast, VapC, VapD and VapE are secreted into the intracellular milieu. Although these Vaps share very high degree of sequence identity in the C-terminal domain, the N-terminal domain (N-domain) of VapA is distinct. It has been proposed that this domain plays a role in VapA surface localization but no direct experimental data provides support to such hypothesis. In this work, we employed R. equi 103S harbouring an unmarked deletion of vapA (R. equi ΔvapA) as the genetic background to express C-terminal Strep-tagged Vap-derivatives integrated in the chromosome. The surface localization of these proteins was assessed by flow cytometry using the THE2122;-NWSHPQFEK Tag FITC-antibody. We show that VapA is the only cell surface Vap encoded in the virulence plasmid. We present compelling evidence for the role of the N-terminal domain of VapA on cell surface localization using fusion proteins in which the N-domain of VapD was exchanged with the N-terminus of VapA. Lastly, using an N-terminally Strep-tagged VapA, we found that the N-terminus of VapA is exposed to the extracellular environment. Given the lack of a lipobox in VapA and the exposure of the N-terminal Strep-tag, it is possible that VapA localization on the cell surface is mediated by interactions between the N-domain and components of the cell surface. We discuss the implications of this work on the light of the recent discovery that soluble recombinant VapA added to the extracellular medium functionally complement the loss of VapA.