The aphid Acyrthosiphon pisum exhibits a greater survival after a heat shock when parasitized by the wasp Aphidius ervi.

The pea aphid Acyrthosiphon pisum is a common pest of many species of legumes and its parasitoid Aphidius ervi is regarded as a successful biocontrol agent. In this study, we report a greater survival rate of parasitized aphids compared with unparasitized ones, after exposure to a very high temperature (39°C for 30min). After the heat shock, the survival of unparasitized aphids decreases according to their age at the heat shock treatment, suggesting a different adaptation of the aphid life stage to the different microclimatic conditions they experience. Survival of parasitized aphids does not change according to the time of the heat shock treatment, but it is always significantly higher compared with the unparasitized ones. Parasitized aphids are very quickly subjected to a wide range of physiological modifications and the observed increased survival could be a consequence of these modifications before the heat shock treatment. The possible explanations as well as the possible adaptive nature of the observed phenomenon are discussed.

Spleen contributes significantly to increased circulating levels of fibroblast growth factor 23 in response to lipopolysaccharide-induced inflammation.

Background: Circulating levels of fibroblast growth factor 23 (FGF23) increase progressively and correlate with systemic inflammation in chronic kidney disease (CKD). The aim of this study was to identify and characterize the causal relationship between FGF23 and inflammation in CKD. Methods: Circulating FGF23 and inflammatory cytokines were correlated in healthy subjects and patients with varying levels of CKD. In addition, FGF23 expression in blood and solid organs was measured in normal mice that were exposed acutely (one time) or chronically (2-week) to low-dose lipopolysaccharide (LPS); chronic exposure being either sustained (subcutaneous pellets), intermittent (daily injections) or combined sustained plus acute (subcutaneous pellets plus acute injection on the day of sacrifice). Blood was analyzed for both terminal (cFGF23) and intact (iFGF23) FGF23 levels. Solid tissues were investigated with immunohistochemistry, enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. Results: FGF23 levels correlated significantly with neutrophil gelatinase-associated lipocalin ( r = 0.72, P < 0.001), C-reactive protein ( r = 0.38, P < 0.001), tumor necrosis factor-α ( r = 0.32, P = 0.001) and interleukin-6 ( r = 0.48, P < 0.001). Acute LPS administration increased tissue FGF23 mRNA and plasma levels of cFGF23 but not iFGF23. Neither chronic sustained nor chronic pulsatile LPS increased the tissue or circulating levels of FGF23. However, acute on chronic LPS raised tissue FGF23 mRNA and both circulating cFG23 and iFGF23. Interestingly, the spleen was the major source of FGF23. Conclusion: Acute on chronic exposure to LPS stimulates FGF23 production in a normal mouse model of inflammation. We provide the first evidence that the spleen, under these conditions, contributes substantially to elevated circulating FGF23 levels.

Tumor induced osteomalacia: associated with elevated circulating levels of fibroblast growth factor-7 in addition to fibroblast growth factor-23.

Tumor induced osteomalacia (TIO) is a rare paraneoplastic syndrome characterized by renal phosphate wasting, hypophosphatemia, and osteomalacia. Fibroblast growth factor (FGF)-23, a phosphatonin i.e., phosphaturia-promoting hormone, is commonly implicated in the pathogenesis of TIO. However, very limited information is available about the circulating levels and clinical significance of other phosphatonins that are expressed by TIO-associated tumors. In addition, identification of the primary tumor constitutes a frequent major challenge in the management of TIO. Here, we report a patient with the clinical diagnosis of TIO with elevated blood levels of the phosphatonins FGF-23 and FGF-7; and extensive but unrewarding radiological search for the primary tumor. In selective venous sampling, both FGF-23 and FGF-7 displayed highest concentrations in the left femoral and iliac veins; although lateralization was much more pronounced for FGF-7 than FGF-23. This laboratory finding allowed us to focus on the left lower extremity as the likely location of the primary tumor. Our case is the first to show that FGF-7 can be analyzed in the circulation and used to assist in the diagnosis and localization of TIO-associated tumors.

Ergocalciferol treatment does Not improve erythropoietin utilization and hospitalization rate in hemodialysis patients.

BACKGROUND: Vitamin D (25-hydroxyvitamin D; 25[OH]D) deficiency (VDD) is highly prevalent in chronic kidney disease. The aim of this study was to evaluate the effect of oral ergocalciferol supplementation on requirement of erythropoietin (EPO) and active vitamin D analogues, and hospitalization rate in maintenance hemodialysis (HD) patients. METHODS: This retrospective cohort study included 186 patients who were on HD for 3 months and had 25(OH)D levels < 30 ng/ml. Over 1-year period, 107 patients were supplemented with protocol-based ergocalciferol (D2 group) and 79 were not (control). Parameters of erythropoiesis and bone-mineral metabolism, and monthly doses of EPO and paricalcitol were assessed at 6- and 12- months of ergocalciferol supplementation. Total hospitalizations were recorded for the same year. RESULTS: Baseline characteristics were similar across two arms except higher serum ferritin, transferrin saturation and prevalence of stroke in D2 and higher coronary artery disease in control group with overall mean ± SD 25(OH)D level of 16.8 ± 7 ng/ml. At 12 months, 25(OH)D levels increased significantly in D2 group compared to control (30.5 ± 11.7 vs. 14.2 ± 9.3 ng/ml; p < 0.001). The EPO dose remained same with no difference in hemoglobin values between the two groups during the intervention period. On multivariate regression which included above variables there was no effect of ergocalciferol treatment on EPO dose (p = ns). Hospitalization rate was similar in two arms; however, ergocalciferol treatment inversely associated with paricalcitol dose (ß ± SE = -10.4 ± 2.8; p < 0.001). CONCLUSIONS: One-year of ergocalciferol supplementation was not associated with reduction in EPO requirement or hospitalization rate in HD patients with VDD. Further studies are warranted to determine definitive role of nutritional vitamin D in these patients.

Targeting NADPH oxidase with a novel dual Nox1/Nox4 inhibitor attenuates renal pathology in type 1 diabetes.

Reactive oxygen species (ROS) generated by Nox NADPH oxidases may play a critical role in the pathogenesis of diabetic nephropathy (DN). The efficacy of the Nox1/Nox4 inhibitor GKT137831 on the manifestations of DN was studied in OVE26 mice, a model of type 1 diabetes. Starting at 4-5 mo of age, OVE26 mice were treated with GKT137831 at 10 or 40 mg/kg, once-a-day for 4 wk. At both doses, GKT137831 inhibited NADPH oxidase activity, superoxide generation, and hydrogen peroxide production in the renal cortex from diabetic mice without affecting Nox1 or Nox4 protein expression. The increased expression of fibronectin and type IV collagen was reduced in the renal cortex, including glomeruli, of diabetic mice treated with GKT137831. GKT137831 significantly reduced glomerular hypertrophy, mesangial matrix expansion, urinary albumin excretion, and podocyte loss in OVE26 mice. GKT137831 also attenuated macrophage infiltration in glomeruli and tubulointerstitium. Collectively, our data indicate that pharmacological inhibition of Nox1/4 affords broad renoprotection in mice with preexisting diabetes and established kidney disease. This study validates the relevance of targeting Nox4 and identifies GKT137831 as a promising compound for the treatment of DN in type 1 diabetes.

Ferric pyrophosphate citrate (Triferic™) administration via the dialysate maintains hemoglobin and iron balance in chronic hemodialysis patients.

BACKGROUND: Administration of ferric pyrophosphate citrate (FPC, Triferic™) via hemodialysate may allow replacement of ongoing uremic and hemodialysis-related iron losses. FPC donates iron directly to transferrin, bypassing the reticuloendothelial system and avoiding iron sequestration. METHODS: Two identical Phase 3, randomized, placebo-controlled trials (CRUISE 1 and 2) were conducted in 599 iron-replete chronic hemodialysis patients. Patients were dialyzed with dialysate containing 2 µM FPC-iron or standard dialysate (placebo) for up to 48 weeks. Oral or intravenous iron supplementation was prohibited, and doses of erythropoiesis-stimulating agents were held constant. The primary efficacy end point was the change in hemoglobin (Hgb) concentration from baseline to end of treatment (EoT). Secondary end points included reticulocyte hemoglobin content (CHr) and serum ferritin. RESULTS: In both trials, Hgb concentration was maintained from baseline to EoT in the FPC group but decreased by 0.4 g/dL in the placebo group (P < 0.001, combined results; 95% confidence interval [CI] 0.2-0.6). Placebo treatment resulted in significantly larger mean decreases from baseline in CHr (-0.9 pg versus -0.4 pg, P < 0.001) and serum ferritin (-133.1 µg/L versus -69.7 µg/L, P < 0.001) than FPC treatment. The proportions of patients with adverse and serious adverse events were similar in both treatment groups. CONCLUSIONS: FPC delivered via dialysate during hemodialysis replaces iron losses, maintains Hgb concentrations, does not increase iron stores and exhibits a safety profile similar to placebo. FPC administered by hemodialysis via dialysate represents a paradigm shift in delivering maintenance iron therapy to hemodialysis patients.

Dietary Intake of Proteins and Calories Is Inversely Associated With The Oxidation State of Plasma Thiols in End-Stage Renal Disease Patients.

OBJECTIVES: Oxidative stress contributes to the pathogenesis of protein-energy wasting in maintenance hemodialysis (MHD) patients, but knowledge of specific effectors and mechanisms remains fragmented. Aim of the study was to define whether and how food intake is involved in the causal relationship between oxidative stress and protein-energy wasting. METHODS: Seventy-one adult MHD patients and 24 healthy subjects (control) were studied cross-sectionally with analyses of diet record and of oxidative stress, as measured by a battery of plasma thiols including the protein sulfhydryl (-SH) group (PSH) levels (a marker of total protein-SH reducing capacity), the protein thiolation index (PTI, the ratio between disulfide, i.e., oxidized and reduced -SH groups in proteins), low molecular mass (LMM) thiols, LMM disulfides, and mixed LMM-protein disulfides. In addition, interleukin-6 (IL-6), albumin, C-reactive protein, and neutrophil gelatinase-associated lipocalin (NGAL) were measured as markers of inflammation. RESULTS: The patients showed low energy (22.0 ± 8.4 kcal/kg/day) and adequate protein (1.0 ± 0.4 g/kg/day) intakes, high levels of cystine (CySS; patients vs. CONTROL: 113.5 [90.9-132.8] vs. 68.2 [56.2-75.7] µM), cysteinylated proteins (CySSP; 216.0 [182.8-254.0] vs. 163.5 [150.0-195.5] µM), and high PTI (0.76 [0.61-0.88] vs. 0.43 [0.40-0.54]; P < .001 in all comparisons). In patients, variation of CySSP was explained by a standard regression model (R = 0.775; P = .00001) that included significant contributions of protein intake (ß = -0.361), NGAL (ß = 0.387), age (ß = 0.295), and albumin (ß = 0.457). In the same model, variation of PTI (R = 0.624; P = .01) was explained by protein intake (ß = -0.384) and age (ß = 0.326) and NGAL (ß = 0.311). However, when PSH was entered as dependent variable (R = 0.730; P = .0001), only serum albumin (ß = 0.495) and age (ß = -0.280), but not dietary intake or NGAL, contributed to the model. CONCLUSIONS: In MHD, markers of thiol oxidation including CySSP and PTI show independent association with dietary intake and NGAL, whereas PSH, a marker of thiol-reducing capacity, did not associate with these same variables. The mechanism(s) responsible for inverse association between oxidative stress and food intake in MHD remain undefined.

Transgenerational plasticity in aphids reared in a poor-resource environment.

The changing environmental conditions can affect insect biology over multiple generations and phenotypic plasticity is important for coping with these changes. Transgenerational plasticity occurs when the environment in which the parents developed influences the plastic response of the offspring phenotype. In the present study, the plastic effects of resource limitation on important life history traits such as body size, fecundity, survival, and resistance to starvation of the pea aphid Acyrthosiphon pisum were investigated over two generations. This study focused on understanding how resource limitation can determine an adaptive expression of maternal effects and transgenerational plasticity in fitness-related traits. Aphids showed phenotypic plasticity for the life history traits investigated, as they performed better when grown in an optimal environment than in a resource-poor one. Also, aphids had a poorer performance if their mothers were raised in a resource-poor environment. The effects of transgenerational plasticity were observed only in response to resistance to starvation, through increased survival in the offspring of the mother reared in a resource-poor environment, suggesting an evolutionary bet-hedging strategy. The results of this study showed that the effects of adaptive transgenerational plasticity may be partially masked in stressful environments, where developmental problems instead predominate. More information on the transgenerational response to resource limitation across generations can contribute to a better understanding of aphid biology.

Balcinrenone plus dapagliflozin in patients with heart failure and chronic kidney disease: Results from the phase 2b MIRACLE trial.

AIMS: Many patients with heart failure (HF) have chronic kidney disease (CKD) and may not tolerate mineralocorticoid receptor antagonists. We investigated the efficacy and safety of the novel mineralocorticoid receptor modulator balcinrenone in combination with dapagliflozin in a phase 2b study. METHODS AND RESULTS: From January 2021 to October 2023, we randomized 133 adults with symptomatic HF, ejection fraction <60%, estimated glomerular filtration rate (eGFR) ≥30 to ≤60 ml/min/1.73 m2 and urinary albumin-to-creatinine ratio (UACR) ≥30 to <3000 mg/g, to receive balcinrenone 15, 50 or 150 mg/day plus dapagliflozin 10 mg/day, or dapagliflozin 10 mg/day plus placebo, for 12 weeks. Enrolment was stopped early because of slow recruitment. Relative reductions in UACR from baseline to week 12 (primary endpoint) were not significantly different between the balcinrenone plus dapagliflozin groups versus dapagliflozin plus placebo. There was no clear balcinrenone dose-response relationship. There were possible dose-dependent increases in serum potassium levels, reduced eGFR in the highest dose group, and non-significant trends towards reduced N-terminal pro-B-type natriuretic peptide levels. Hyperkalaemia adverse events led to discontinuation in two participants receiving balcinrenone plus dapagliflozin and none in those receiving dapagliflozin plus placebo. CONCLUSION: While the smaller than planned sample size limits interpretation, we did not see significant reduction in UACR in patients treated with balcinrenone plus dapagliflozin compared with dapagliflozin plus placebo.

Pioglitazone improves glucose metabolism and modulates skeletal muscle TIMP-3-TACE dyad in type 2 diabetes mellitus: a randomised, double-blind, placebo-controlled, mechanistic study.

AIMS/HYPOTHESIS: Pioglitazone (PIO) is a peroxisome proliferator-activated receptor (PPAR)γ agonist insulin-sensitiser with anti-inflammatory and anti-atherosclerotic effects. Our objective was to evaluate the effect of low-dose PIO (15 mg/day) on glucose metabolism and inflammatory state in obese individuals with type 2 diabetes. METHODS: A randomised, double-blind, placebo-controlled, mechanistic trial was conducted on 29 patients with type 2 diabetes treated with metformin and/or sulfonylurea. They were randomised to receive PIO or placebo (PLC) for 6 months, in a 1:1 ratio. Participants were allocated to interventions by central office. All study participants, investigators and personnel performing measurements were blinded to group assignment. At baseline and after 6 months patients underwent: (1) OGTT; (2) muscle biopsy to evaluate expression of TNF-α, tissue inhibitor of metalloproteases 3 (TIMP-3) levels, TNF-α converting enzyme (TACE) expression and enzymatic activity; (3) euglycaemic-hyperinsulinaemic clamp; (4) measurement of plasma high-sensitivity C-reactive protein (hsCRP), plasminogen activator inhibitor type-1 (PAI-1), TNF-α, IL-6, monocyte chemotactic protein-1 (MCP-1), adiponectin and fractalkine (FRK). The interventions were PIO 15 mg/day vs placebo and the main outcomes measured were absolute changes in whole-body insulin sensitivity, insulin secretion and inflammatory state. RESULTS: Fifteen participants were randomized to receive PIO and 14 participants were randomized to receive PLC. Eleven participants completed the study in the PIO group and nine participants completed the study in the PLC group and were analysed. Fasting plasma glucose and HbA1c decreased modestly (p < 0.05) after PIO and did not change after PLC. M/I (insulin-stimulated whole-body glucose disposal), adipose tissue insulin resistance (IR) index, insulin secretion/IR (disposition) index and insulinogenic index improved significantly after PIO, but not after PLC. Circulating MCP-1, IL-6, FRK, hsCRP and PAI-1 levels decreased in PIO- as compared with PLC-treated patients, while TNF-α did not change. TNF-α protein expression and TACE enzymatic activity in muscle were significantly reduced by PIO but not PLC. Adiponectin levels increased significantly after PIO as compared with PLC treatment. Given that the mean TACE enzymatic activity level at baseline in the PIO group was 0.29 ± 0.07 (fluorescence units [FU]), and at end of study decreased to 0.05 vs 0.14 in the PLC group, the power to reject the null hypothesis that the population means of the PIO and PLC groups are equal after 6 months is greater than 0.80. Given that M/I was 2.41 ± 0.35 µmol kg(-1) min(-1) (pmol/l)(-1) at baseline and increased by 0.55 in the PIO and 0.17 in the PLC groups, the power to reject the null hypothesis that the population means of the PIO and PLC groups are equal after 6 months is greater than 0.85. The type I error probability associated with this test of this null hypothesis is 0.05. No serious adverse events occurred in either group. CONCLUSIONS/INTERPRETATION: Low-dose PIO (15 mg/day) improves glycaemic control, beta cell function and inflammatory state in obese patients with type 2 diabetes. TRIAL REGISTRATION: Clinical.Trial.gov NCT01223196. FUNDING: This study was funded by TAKEDA.

Tomato below ground-above ground interactions: Trichoderma longibrachiatum affects the performance of Macrosiphum euphorbiae and its natural antagonists.

Below ground and above ground plant-insect-microorganism interactions are complex and regulate most of the developmental responses of important crop plants such as tomato. We investigated the influence of root colonization by a nonmycorrhizal plant-growth-promoting fungus on direct and indirect defenses of tomato plant against aphids. The multitrophic system included the plant Solanum lycopersicum ('San Marzano nano'), the root-associated biocontrol fungus Trichoderma longibrachiatum strain MK1, the aphid Macrosiphum euphorbiae (a tomato pest), the aphid parasitoid Aphidius ervi, and the aphid predator Macrolophus pygmaeus. Laboratory bioassays were performed to assess the effect of T. longibrachiatum MK1, interacting with the tomato plant, on quantity and quality of volatile organic compounds (VOC) released by tomato plant, aphid development and reproduction, parasitoid behavior, and predator behavior and development. When compared with the uncolonized controls, plants whose roots were colonized by T. longibrachiatum MK1 showed quantitative differences in the release of specific VOC, better aphid population growth indices, a higher attractiveness toward the aphid parasitoid and the aphid predator, and a quicker development of aphid predator. These findings support the development of novel strategies of integrated control of aphid pests. The species-specific or strain-specific characteristics of these below ground-above ground interactions remain to be assessed.

The antioxidant silybin prevents high glucose-induced oxidative stress and podocyte injury in vitro and in vivo.

Podocyte injury, a major contributor to the pathogenesis of diabetic nephropathy, is caused at least in part by the excessive generation of reactive oxygen species (ROS). Overproduction of superoxide by the NADPH oxidase isoform Nox4 plays an important role in podocyte injury. The plant extract silymarin is attributed antioxidant and antiproteinuric effects in humans and in animal models of diabetic nephropathy. We investigated the effect of silybin, the active constituent of silymarin, in cultures of mouse podocytes and in the OVE26 mouse, a model of type 1 diabetes mellitus and diabetic nephropathy. Exposure of podocytes to high glucose (HG) increased 60% the intracellular superoxide production, 90% the NADPH oxidase activity, 100% the Nox4 expression, and 150% the number of apoptotic cells, effects that were completely blocked by 10 µM silybin. These in vitro observations were confirmed by similar in vivo findings. The kidney cortex of vehicle-treated control OVE26 mice displayed greater Nox4 expression and twice as much superoxide production than cortex of silybin-treated mice. The glomeruli of control OVE26 mice displayed 35% podocyte drop out that was not present in the silybin-treated mice. Finally, the OVE26 mice experienced 54% more pronounced albuminuria than the silybin-treated animals. In conclusion, this study demonstrates a protective effect of silybin against HG-induced podocyte injury and extends this finding to an animal model of diabetic nephropathy.

Diabetic nephropathy among Mexican Americans.

The incidence of diabetic nephropathy (DN) is growing rapidly worldwide as a consequence of the rising prevalence of Type 2 diabetes mellitus (T2DM). Among U.S. ethnic groups, Mexican Americans have a disproportionately high incidence and prevalence of DN and associated end-stage renal disease (ESRD). In communities bordering Mexico, as many as 90% of Mexican American patients with ESRD also suffer from T2DM compared to only 50% of non-Hispanic Whites (NHW). Both socio-economic factors and genetic predisposition appear to have a strong influence on this association. In addition, certain pathogenetic and clinical features of T2DM and DN are different in Mexican Americans compared to NHW, raising questions as to whether the diagnostic and treatment strategies that are standard practice in the NHW patient population may not be applicable in Mexican Americans. This article reviews the epidemiology of DN in Mexican Americans, describes the pathophysiology and associated risk factors, and identifies gaps in our knowledge and understanding that needs to be addressed by future investigations.

Effects of Trichoderma harzianum Strain T22 on the Arthropod Community Associated with Tomato Plants and on the Crop Performance in an Experimental Field.

Fungi belonging to the genus Trichoderma have received much attention in recent years due to their beneficial effects on crop health and their use as pest control agents. Trichoderma activates direct plant defenses against phytophagous arthropods and reinforces indirect plant defense through the attraction of predators. Although the plant defenses against insect herbivores were demonstrated in laboratory experiments, little attention has been paid to the use of Trichoderma spp. in open field conditions. In the present study, we investigated the effects of the inoculation of the commercial Trichoderma harzianum strain T22 on the arthropod community associated with tomato plants and on the crop performance in an experimental field located in South Italy. Our results showed that inoculation with T. harzianum could alter the arthropod community and reduce the abundance of specific pests under field conditions with respect to the sampling period. The present study also confirmed the beneficial effect of T. harzianum against plant pathogens and on tomato fruit. The complex tomato-arthropod-microorganism interactions that occurred in the field are discussed to enrich our current information on the possibilities of using Trichoderma as a green alternative agent in agriculture.

Effects of Below-Ground Microbial Biostimulant Trichoderma harzianum on Diseases, Insect Community, and Plant Performance in Cucurbita pepo L. under Open Field Conditions.

Agrochemicals are generally used in agriculture to maximize yields and product quality, but their overuse can cause environmental pollution and human health problems. To reduce the off-farm input of chemicals, numerous biostimulant products based on beneficial symbiont plant fungi are receiving a great deal of attention. The evolution of plant diseases and the performance of insects are influenced by plant chemical defences, both of which are, in turn, influenced by below-ground symbionts. Direct and indirect plant defences mediated by belowground symbionts against plant diseases and insect herbivores were demonstrated in greenhouses experiments. However, little attention has been paid to the use of Trichoderma under open field conditions, and no data are available for zucchini (Cucurbita pepo L.) plants in the field. To determine the effects of a commercial Trichoderma harzianum strain T22 on plant viruses, powdery mildew, the arthropod community, and on the agronomic performance associated with zucchini plants, an experiment was conducted in 2022 under open field conditions in South Italy. Our results indicate that T. harzianum T22 makes zucchini plants more attractive to aphids and to Hymenoptera parasitoid but failed to control zucchini pathogens. The complex plant-disease-arthropod-microorganism interactions that occurred in the field during the entire plant cycle are discussed to enrich our current information on the possibilities of using these microorganisms as a green alternative in agriculture.

Role of Ovarian Proteins Secreted by Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) in the Early Suppression of Host Immune Response.

Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae). During oviposition, T. nigriceps injects into the host body, along with the egg, the venom, the calyx fluid, which contains a Polydnavirus (T. nigriceps BracoVirus: TnBV), and the Ovarian Proteins (OPs). Although viral gene expression in the host reaches detectable levels after a few hours, a precocious disruption of the host metabolism and immune system is observed right after parasitization. This alteration appears to be induced by female secretions including TnBV venom and OPs. OPs, originating from the ovarian calyx cells, are involved in the induction of precocious symptoms in the host immune system alteration. It is known that OPs in braconid and ichneumonid wasps can interfere with the cellular immune response before Polydnavirus infects and expresses its genes in the host tissues. Here we show that T. nigriceps OPs induce several alterations on host haemocytes that trigger cell death. The OP injection induces an extensive oxidative stress and a disorganization of actin cytoskeleton and these alterations can explain the high-level of haemocyte mortality, the loss of haemocyte functionality, and so the reduction in encapsulation ability by the host.

Evaluation of polymorphisms in paraoxonase 2 (PON2) gene and their association with cardiovascular-renal disease risk in Mexican Americans.

BACKGROUND/AIMS: Genetic polymorphisms in the paraoxonase 2 (PON2) gene are thought to alter its activity and contribute to the development of cardiovascular and renal disease risk. The purpose of this study is to determine whether the Arg148Gly, Cys311Ser and rs12794795 polymorphisms of PON2 examined previously by others, are associated with type 2 diabetes (T2DM), and subclinical measures of cardiovascular and renal disease risk in Mexican Americans. METHODS: Study participants (n = 848; 21 families) were genotyped for the three polymorphisms by TaqMan assay. Association between the genotypic and phenotypic data was performed by measured genotype approach as implemented in the variance component analytical tools. RESULTS: The Arg148Gly variant was found to be monomorphic in our dataset. Of the phenotypes examined for association, the A/C variant located in intron-1 (rs12794795) exhibited statistically significant association only with diastolic blood pressure (p = 0.018) after accounting for the trait-specific covariate effects. The Cys311Ser variant failed to show statistically significant association with any of the phenotypes examined. CONCLUSION: In conclusion, the variants examined at the PON2 locus in Mexican Americans do not appear to be a major contributor to T2DM, cardiovascular or renal disease risk, although they exhibited a small effect on the blood pressure values.

Immediate stabilization of human blood for delayed quantification of endogenous thiols and disulfides.

Endogenous thiols undergo rapid and reversible oxidation to disulfides when exposed to oxidants and are, therefore, suitable biomarkers of oxidative stress. However, accurate analysis of thiols in blood is frequently compromised by their artifactual oxidation during sample manipulation, which spuriously elevates the disulfide levels. Here, we describe a validated pre-analytical procedure that prevents both artifactual oxidation of thiols during sample manipulation and their oxidative decay for months in biosamples that are stored at -80°C. Addition of N-ethylmaleimide to blood samples from healthy donors was used to stabilize whole blood, red blood cells, platelets and plasma disulfides, whereas addition of citrate buffer followed by dilution of plasma with H2O was used to stabilize plasma thiols. The concentrations of thiols and disulfides were stable in all biosamples for at least 6 months when analyzed by UV/Vis HPLC at regular intervals. Only 3 ml of blood were needed to perform the analyses of thiols and disulfides in the different blood fractions. This pre-analytical procedure is reliable for use in both animal and human prospective studies. Its ease of implementation makes the method suitable for application to multicenter studies where blood samples are collected by different sites and personnel and are shipped to specific specialized laboratories.

Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room.

Glutathione (GSH) is the most abundant low-molecular-mass thiol within cells and one of the major antioxidant compounds in body fluids. Under pro-oxidant conditions, two GSH molecules donate one electron each and are converted into glutathione disulfide (GSSG). The GSH/GSSG molar ratio is considered a powerful index of oxidative stress and disease risk. Despite high interest in GSH/GSSG titration as measures of thiol redox balance, no broad agreement has yet been reached as to the best pre-analytical and analytical methods for the quantitation of these molecules in biological samples. Consequently, measured concentrations of GSH and GSSG and calculated GSH/GSSG molar ratios vary widely among laboratories. Here, we describe in detail the main analytical and pre-analytical problems related to the artificial oxidation of the sulfhydryl (SH) group of GSH that occur during sample manipulation. We underline how this aspect has been neglected for long time after its first description more than fifty years ago. Finally, selected reliable procedures and methods to measure GSH and GSSG in biological samples are discussed.