Plasma Reactive Oxygen Species Research Articles

Effect of fumigation height and time on cryopreservation of ram semen

The cooling rate is a crucial factor in the process of freezing semen, influencing the overall freezing effectiveness. The height and time of fumigation can significantly impact the rate of cooling. Appropriate cooling rates can help minimize the formation of ice crystals in spermatozoa and reduce potential damage to them. Therefore, the aim of this study was to evaluate the effect of different fumigation heights and time for the cryopreservation of Hu ram semen. Experiments I–IV assessed the effect of semen cryopreservation by testing the post-thawed spermatozoa total motility (TM), progressive motility (PM) and kinetic parameters fumigated at distances of 2, 4, 6 and 8 cm for durations of 5, 10, 15 and 20 min, respectively. Based on the results of experiments I to IV, experiment V evaluated the effect of semen cryopreservation by testing the post-thawed spermatozoa TM, PM, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level fumigated at distances of 2, 4, 6 and 8 cm for duration of 20 min. The results indicated that fumigation at 2 cm for 20 min significantly (P < 0.05) improved spermatozoa TM, PM, mean angular displacement (MAD), plasma membrane integrity and acrosome integrity compared to other groups. Additionally, it significantly (P < 0.05) reduced spermatozoa ROS level compared to the 6 and 8 cm groups. In conclusion, fumigation for 20 min at a distance of 2 cm from the liquid nitrogen surface is the most suitable cooling method for the cryopreservation of Hu ram semen.

Effect of Different Dilution Methods and Ratios of Ram Semen on Sperm Parameters after Cryopreservation.

The dilution method and ratio were tested to assess their effects on the Hu ram semen after cryopreservation. Experiment I aimed to explore the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I (Tris-based and egg yolk) under the condition of 1:1 dilution of diluent II (diluent I and glycerol) on the Hu ram semen preserved in liquid nitrogen regarding spermatozoa motility and kinetic parameters. Experiment II aimed to investigate the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I under the condition of 1:2 dilution of diluent II to the Hu ram semen for cryopreservation on spermatozoa motility and kinetic parameters. The purpose of experiment III is to assess the effect of various dilution methods and ratios on the cryopreservation of Hu ram semen by detecting spermatozoa motility, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level. Experiment III includes four groups: one-step dilution method and two-step dilution method. The two-step dilution method includes two groups: 1:2, 1:1 and 1:3, 1:2, and the one-step dilution method includes two groups: 1:5 and 1:11. The results indicated that the post-thawed spermatozoa total motility (TM), progressive motility (PM) and average motion degree (MAD) were highest in the 1:2 group and significantly higher (p < 0.05) than those in the 1:1 and 1:4 groups under the condition of 1:1 dilution of diluent II. The post-thawed spermatozoa TM and PM of the 1:3 group were significantly higher (p < 0.05) than those of the other groups under the condition of 1:2 dilution of diluent II. The post-thawed spermatozoa TM, PM, plasma membrane integrity and acrosome integrity of the two-step group (1:3, 1:2) were the highest and significantly higher (p < 0.05) than those in the other groups. Additionally, the post-thawed spermatozoa ROS level of the two-step group (1:3, 1:2) was significantly lower (p < 0.05) than that in the one-step groups (1:5 and 1:11). Therefore, a two-step dilution (1:3, 1:2) was found to be the most suitable method and ratio for diluting the Hu ram semen after cryopreservation.

Anti-COVID Drugs (MMV COVID Box) as Leishmanicidal Agents: Unveiling New Therapeutic Horizons.

Leishmaniasis, a neglected tropical disease, poses a significant global health challenge, necessitating the urgent development of innovative therapies. In this study, we aimed to identify compounds from the COVID Box with potential efficacy against two Leishmania species, laying the foundation for future chemical development. Four promising molecules were discovered, demonstrating notable inhibitory effects against L. amazonensis and L. donovani. Our study revealed that bortezomib, almitrine, and terconazole induced a significant decrease in mitochondrial membrane potential, while the above compounds and ABT239 induced plasma permeability alterations, chromatin condensation, and reactive oxygen species accumulation, indicating early apoptosis in Leishmania amazonensis promastigotes, preventing inflammatory responses and tissue damage, thereby improving patient outcomes. Furthermore, ADME predictions revealed favorable pharmacokinetic profiles for all compounds, with bortezomib and ABT239 standing out as potential candidates. These compounds exhibited intestinal absorption, blood-brain barrier penetration (excluding bortezomib), and good drug-likeness for bortezomib and ABT239. Toxicity predictions for CYP-inhibition enzymes favored bortezomib as the safest candidate. In conclusion, our study identifies bortezomib as a promising aspirant for leishmaniasis treatment, demonstrating potent antiparasitic activity, favorable pharmacokinetics, and low toxicity. These findings emphasize the potential repurposing of existing drugs for neglected diseases and highlight the importance of the COVID Box in drug discovery against tropical diseases.

Single nucleotide polymorphisms in the Dicer gene modify the risk of systemic lupus erythematosus

Objective MicroRNA-related single-nucleotide polymorphisms (miR-SNPs) can alter microRNA (miRNA) expression profiles, thereby influencing the risk of rheumatic diseases. Herrin a case control study, six miR-SNPs in miRNA processing machinery genes, namely RAN (rs14035), XPO5 (rs11077), Dicer (rs3742330), GEMIN3 (rs197412), GEMIN4 (rs2740348), and TNRC6B (rs9623117), were genotyped to assess their correlation with the risk of systemic lupus erythematosus (SLE). Methods We included 119 patients with SLE and 130 healthy controls. The genotypes of the six miR-SNPs were determined using polymerase chain reaction (PCR). Serum cytokine levels were assessed using a cytometric bead array, and fluorescent probe technology was used to determine plasma reactive oxygen species (ROS) levels. Results The AA genotype of Dicer was correlated with a 0.566-fold decreased risk of SLE compared with that of the AG + GG genotype (odds ratio, 0.566; 95% CI, 0.342–0.935; p = .026), and the rs3742330 A allele was associated with a significantly decreased risk of SLE ( p = .035) compared with that of the rs3742330G allele. Additionally, AA genotype carriers exhibited lower levels of interleukin-6 (IL-6) in the blood ( p = .013). Subsequent analysis revealed increased ROS production in patients with SLE than that in the controls (621.042 ± 425.285 vs 499.966 ± 302.273, p = .011). Conclusion Our findings suggest that ROS generation participates in SLE pathogenesis. The identification of Dicer gene SNP rs3742330 as a potential modifier of SLE risk via mediating IL-6 overproduction suggests a potential avenue for targeted interventions to manage SLE and its associated immune dysregulation.

Bactericidal effect of low-temperature atmospheric plasma against the Shigella flexneri

BackgroundShigella flexneri (S. flexneri) is a common intestinal pathogenic bacteria that mainly causes bacillary dysentery, especially in low socioeconomic countries. This study aimed to apply cold atmospheric plasma (CAP) on S. flexneri directly to achieve rapid, efficient and environmentally friendly sterilization.MethodsThe operating parameters of the equipment were determined by plasma diagnostics. The plate count and transmission electron microscope were employed to calculate bacterial mortality rates and observe the morphological damage of bacterial cells. Measurement of intracellular reactive oxygen species (ROS) and superoxide anions were detected by 2,7-dichlorodihydrofluorescein (DCFH) and Dihydroethidium fluorescence probes, respectively. The fluorescence intensity (a. u.) reflects the relative contents. Additionally, the experiment about the single effect of temperature, ultraviolet (UV), and ROS on bacteria was conducted.ResultsThe peak discharge voltage and current during plasma operation were 3.92kV and 66mA. After discharge, the bacterial mortality rate of 10, 20, 30 and 40 s of plasma treatment was 60.71%, 74.02%, 88.11% and 98.76%, respectively. It was shown that the intracellular ROS content was proportional to the plasma treatment time and ROS was the major contributor to bacterial death.ConclusionIn summary, our results illustrated that the plasma treatment could inactivate S. flexneri efficiently, and the ROS produced by plasma is the leading cause of bacterial mortality. This highly efficient sterilization method renders plasma a highly promising solution for hospitals, clinics, and daily life.

Hydroxyurea does not reverse functional alterations of the nitric oxide-cGMP pathway associated with priapism phenotype in corpus cavernosum from sickle cell mouse.

Sickle cell disease (SCD) is a genetic disorder that has been associated with priapism. The role of hydroxyurea, a common SCD therapy, in influencing the nitric oxide (NO)-cGMP pathway and its effect on priapism is unclear. To investigate the effect of hydroxyurea treatment on smooth muscle relaxation of corpus cavernosum induced by stimulation of the NO-cGMP pathway in SCD transgenic mice and endothelial NO synthase gene-deficient (eNOS-/-) mice, which are used as model of priapism associated with the low bioavailability of endothelial NO. Four-month-old wild-type (WT, C57BL/6), SCD transgenic, and eNOS-/- male mice were treated with hydroxyurea (100 mg/Kg/day) or its vehicle (saline) daily for three weeks via intraperitoneal injections. Concentration-response curves for acetylcholine (ACh), sodium nitroprusside (SNP), and electrical field stimulation (EFS) were generated using strips of mice corpus cavernosum. The SCD mice demonstrated an amplified CC relaxation response triggered by ACh, EFS, and SNP. The corpus cavernosum relaxation responses to SNP and EFS were found to be heightened in the eNOS-/- group. However, the hydroxyurea treatment did not alter these escalated relaxation responses to ACh, EFS, and SNP in the corpus cavernosum of the SCD group, nor the relaxation responses to EFS and SNP in the eNOS-/- group. In conclusion, hydroxyurea is not effective in treating priapism associated with SCD. It is likely that excess plasma hemoglobin and reactive oxygen species, which are reported in SCD, are reacting with NO before it binds to GCs in the smooth muscle of the corpus cavernosum, thus preventing the restoration of baseline NO/cGMP levels. Furthermore, the downregulation of eNOS in the penis may impair the pharmacological action of hydroxyurea at the endothelial level in SCD mice. This study emphasize the urgency for exploring alternative therapeutic avenues for priapism in SCD that are not hindered by high plasma hemoglobin and ROS levels.

Mitochondrial displacement loop region single nucleotide polymorphisms and mitochondrial DNA copy number associated with risk of ankylosing spondylitis.

The pathogenesis of ankylosing spondylitis (AS) seems to be associated with genetics, the environment, heredity, and oxidative stress. Single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) region of mitochondrial DNA (mtDNA) and mtDNA copy number were investigated for their correlation with AS patients. This study included 83 AS patients and 100 healthy controls from the Second Hospital of Hebei Medical University. DNAs were extracted from blood samples for polymerase chain reaction analysis and quantitative real-time polymerase chain reaction analysis. Plasma reactive oxygen species (ROS) levels were measured by fluorescent probe technology. The distribution frequencies of the minor alleles of nucleotides 16304C (p = .037), 16311C (p = .027), and 152C (p = .034) were remarkably higher in AS patients than in healthy controls, which indicated that the16304C, 16311C, and 152C alleles were correlated with an increased risk of AS. Simultaneously, mtDNA copy number was statistically higher in patients with AS compared with controls (1.450 ± 0.876 versus 0.835 ± 0.626, p < .001). We also observed an increased ROS generation in AS patients compared with controls (27 066.169 ± 18 364.819 versus 14 758.330 ± 5854.946, p < .001) subsequently. In addition, the AS susceptible SNP 16311C is associated with high ROS levels (35 065.177 ± 26 999.934 vs. 25 005.818 ± 14 999.495, p = .043). Our study demonstrated that SNPs in the mtDNA D-loop could be AS risk biomarkers with the potential to promote oxidative stress levels; mtDNA copy number-induced mitochondrial dysfunction may also be involved in AS pathogenesis.

Galectin-3 Mediates Vascular Dysfunction in Obesity by Regulating NADPH Oxidase 1.

Obesity is associated with increased risk of cardiovascular disease, but underlying mechanisms remain elusive. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor, but how glucose impacts vascular function is unclear. GAL3 (galectin-3) is a sugar-binding lectin upregulated by hyperglycemia, but its role as a causative mechanism of cardiovascular disease remains poorly understood. Therefore, the objective of this study was to determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity. GAL3 was measured and found to be markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate causative mechanisms in cardiovascular disease, mice deficient in GAL3 were bred with obese db/db mice to generate lean, lean GAL3 knockout, obese, and obese GAL3 knockout genotypes. Endothelial cell-specific GAL3 knockout mice with novel AAV-induced obesity recapitulated whole-body knockout studies to confirm cell specificity. Deletion of GAL3 did not alter body mass, adiposity, or plasma indices of glycemia and lipidemia, but levels of plasma reactive oxygen species as assessed by plasma thiobarbituric acid reactive substances were normalized in obese GAL3 knockout mice. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells from obese mice had increased expression of NOX1 (nicotinamide adenine dinucleotide phosphate oxidase 1), which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, which was normalized in microvascular endothelium from mice lacking GAL3. Cell-specific deletion confirmed that endothelial GAL3 regulates obesity-induced NOX1 overexpression and subsequent microvascular function. Furthermore, improvement of metabolic syndrome by increasing muscle mass, improving insulin signaling, or treating with metformin decreased microvascular GAL3, and thereby NOX1, expression levels. Deletion of GAL3 normalizes microvascular endothelial function in obese db/db mice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3, and in turn NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.

Mycotoxins degradation by reactive oxygen species in plasma: Reactive molecular dynamics insights

The food industry is plagued by mycotoxin contamination, and obtaining mycotoxin-free products has become a great challenge. With the advancement of cold atmospheric plasma (CAP) technology, new ideas for mycotoxin decontamination have emerged. The reactive species in plasma can act on the toxic structural groups through various reaction pathways, which are not yet completely clear. In this paper, we investigated the reaction mechanisms of six mycotoxins upon the impact of reactive oxygen species (ROS) employing reactive molecular dynamics (RMD) simulation. The degradation pathways of mycotoxins were revealed, and the reaction types of main toxic groups were summarized, including addition, oxidation, reduction, ring opening, side chain shedding, and even skeleton structure breaking. The degradation patterns of the double C=C bond and lactone ring were analyzed in detail. Besides the addition reaction, the double C=C bond can also be reduced by bond breaking or hydrogen atom abstraction reaction. The degradation processes of the lactone ring mainly include ring-opening reaction and reduction of lactone carbonyl group. The reaction processes can contribute to an improved understanding of the mechanisms of mycotoxins degradation by plasma and are also valuable for the degradation of other organic pollutants with the same structural groups.

Comparative study of touchable plasma devices on transdermal drug delivery

AbstractLow‐temperature plasma is a new transdermal drug delivery method that is different from the traditional physical and chemical methods. In this study, three touchable plasma jet devices are selected, such as direct current (DC) air plasma jet, DC helium plasma jet, and pulsed helium plasma jet. The patent blue V 1159.43 Da has been chosen for percutaneous penetration studies. The results show that 1 min DC air and helium plasma treatment significantly enhanced transdermal drug delivery by 9 and 26 times, respectively. The enhancement of pulsed plasma jet is the weakest. The skin histological analysis and reactive oxygen species (ROS) measurement shows that nonuniform distribution of ROS in DC helium and DC air plasma may cause perforation on the whole epidermis and allow more drug permeation.

Relationship among seminal antigenicity, antioxidant status and metabolically active sperm from Holstein-Friesian (Bos taurus) bulls

Sperm antigenicity has been implicated as a regulatory factor for acquiring fertilizing competence in the female reproductive tract. Overt immune response against the sperm proteins leads to idiopathic infertility. Hence, the aim of the study was to evaluate the influence of the auto-antigenic potential of sperm on the antioxidant status, metabolic activities and reactive oxygen species (ROS) in bovine. Semen from Holstein-Friesian bulls (n = 15) was collected and classified into higher (HA, n = 8) and lower (LA, n = 7) antigenic groups based on micro-titer agglutination assay. The neat semen was subjected to the evaluation of bacterial load, leukocyte count, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lipid peroxidation (LPO) levels. Antioxidant activities in seminal plasma and intracellular ROS levels in the post-thawed sperm were estimated. The number of leukocytes was lower (p < .05) in the HA than the LA semen. The percentage of metabolically active sperm was higher (p < .05) in HA than the LA group. The activities of total non-enzymatic antioxidant, superoxide dismutase (SOD) and catalase (CAT) were higher (p < .05) while glutathione peroxidase activity was lower (p < .05) in the seminal plasma of LA group. The LPO levels of neat sperm and the percentage sperm positive for intracellular ROS in the cryopreserved sample were lower (p < .05) in the HA group. Auto-antigenic levels were positively correlated with the percentage of metabolically active sperm (r = 0.73, p < .01). However, the seminal auto-antigenicity was negatively (p < .05) correlated with the levels of SOD (r=–0.66), CAT (r=–0.72), LPO (r=–0.602) and intracellular ROS (r=–0.835). The findings were represented in graphical abstract. It is inferred that the higher auto-antigenic levels protect the quality of bovine semen by promoting sperm metabolism and lowering ROS and LPO levels.

Alkaline semen diluent combined with R848 for separation and enrichment of dairy goat X-sperm.

A large number of female goats are needed for the dairy goat industry; therefore, the development of a method to ensure the birth of more females than males in a single pregnancy will lead to economic benefits. Increasing the number of X-sperm would be an effective way to increase the proportion of female offspring. In this study, goat semen was incubated at pH 7.4 in alkaline diluent combined with resiquimod (R848) and the number of X-sperm was enriched by the swim-up method. The percentage of X-sperm was determined using the double TaqMan qPCR method. Sperm total motility, progressive motility, average path velocity, straight-line velocity, and curvilinear velocity were measured using a computer-aided sperm analysis system, and the functional parameters of the sperm plasma membrane, the acrosome, mitochondrial activity, ATP content, and reactive oxygen species levels were also measured. Lastly, the ratio of female embryos was determined by in vitro fertilization, and the number of female kids and the pregnancy rate of does was assessed by artificial insemination. The results showed that dilution of semen in an alkaline buffer containing R848 could enrich the number of X-sperm to 85.57% ± 3.27%. The progressive motility, average path velocity, straight-line velocity, curvilinear velocity, mitochondrial activity, and ATP level of the collected X-sperm-enriched semen were significantly reduced, but its total motility, plasma membrane, and acrosome were not affected. The in vitro fertilization experiments showed that the rate of female embryo production using X-sperm-rich seminal fluid could reach 83.25% (174/209), which was significantly higher than the proportion of female embryos in the control group, 47.71% ± 1.80% (104/218). As determined by artificial insemination, the number of female kids in the test group increased by 62.79% (243/387), which was significantly higher than that in the control group (47.65%, 193/405). There was no significant difference in pregnancy rate between the test group and the control group (71.71% vs. 78.48%). Therefore, this study demonstrated that use of a pH 7.4 diluent containing R848 is a simple and effective method of X-sperm enrichment for dairy goat production. Its application would allow does to produce more female offspring for herd expansion and milk production.

Effects of prenatal dietary rumen-protected choline supplementation during late gestation on calf growth, metabolism, and vaccine response.

The objective of this study was to examine the effects of prenatal supplementation and dose of rumen-protected choline (RPC) on neonatal calf growth, metabolism, and vaccine response. Parous Holstein cows were blocked by calving month and randomly assigned within block to receive 45 g/d of RPC [20.4 g/d of choline ions (CHOL45), n = 19], 30 g/d of RPC [13.6 g/d of choline ions (CHOL30), n = 22], or no RPC (CON, n = 19) as a top-dress, starting 24 d before expected calving. Calf body weights were recorded for the first 3 wk of life. All calves were fed colostrum replacer (300 g of IgG) at birth, and apparent efficiency of IgG absorption was calculated. On d 1, 7, 14, and 21, blood samples were taken to quantify plasma reactive oxygen and nitrogen species, antioxidant potential, haptoglobin, nonesterified fatty acids (NEFA), β-hydroxybutyrate, and glucose. Calves received an intranasal vaccine at birth, and nasal secretions were collected on d 0, 7, 10, 14, and 21 to quantify bovine respiratory syncytial virus-specific IgA. Data were analyzed using linear mixed models including the fixed effects of treatment, time (when applicable), calf sex, and prepartum dam data (-24 d) along with interactions. Treatment did not affect calf body weight, β-hydroxybutyrate, or glucose concentrations. For apparent efficiency of IgG absorption, treatment interacted with the dam's prepartum body condition score. Where the dam's body condition score was ≤3.25, IgG absorption was reduced in calves born from CHOL45 dams as compared with calves from either CHOL30 or CON dams. Calves from CHOL30 dams had a lesser oxidative stress index (OSi; reactive oxygen and nitrogen species/antioxidant potential) than calves from CON dams. Haptoglobin concentrations were less in heifer calves from CHOL45 dams as compared with heifers from CON dams. The dam's prepartum NEFA concentration interacted with treatment. When dam NEFA was minimal, calves from CHOL45 and CHOL30 dams had greater or tended to have greater NEFA, respectively. Conversely, when dam NEFA was greater, calves from CHOL30 and CHOL45 dams had lesser or tended to have lesser NEFA than calves from CON dams, respectively. For vaccine response, treatment interacted with the dam's prepartum OSi. Among calves born from dams with a greater OSi, calves from CHOL45 and CHOL30 dams had lesser bovine respiratory syncytial virus-specific IgA concentrations in nasal secretions as compared with CON. Prenatal RPC supplementation during late gestation affected IgG absorption, neonatal calf metabolism, and vaccine response with some effects dependent on the dam's prepartum parameters.

Rice glutamate receptor-like channel OsGLR3.4 modulates the root tropism growth towards amino acids via plasma membrane depolarization and ROS generation

Understanding how plants determine root directional growth towards nutrient sources is important to reveal optimal strategies during plant growth. GLUTAMATE RECEPTOR-LIKE (GLR) channels play essential roles in various Ca2+-mediated plant-specific physiological processes, but how GLRs are involved in the root tropism growth towards surrounding nutrients remains largely unknown. Here, we report that OsGLR3.4, a rice Ca2+-permeable channel, specifically modulates the root growth and tropism towards Glycine (Gly) via Ca2+-mediated electrical activity and reactive oxygen species (ROS) production. Mutation of OsGLR3.4 significantly reduced the inhibitory effect of Gly on the primary root growth, coupled with the decrease of Gly-induced membrane depolarization and ROS levels at the elongation zone in the root tip. More importantly, our results showed that OsGLR3.4 was required for root nutritropism growth towards Gly. Mechanistic study showed that, when grown in the agar with unequal Gly application, the bilaterally asymmetrical distribution of membrane depolarization and ROS evoked by OsGLR3.4-mediated [Ca2+]cyt fluctuation across the root resulted in the differential cell elongation at two sides of the root tip, which therefore led to the root nutritropism growth. Nevertheless, addition of Ca2+ channel blocker eliminated the Gly-induced asymmetrical distribution of depolarization and ROS across the root tip. Notably, OsGLR3.4 promoted the nitrate uptake in the root by enhancing expression of nitrate transporter genes in response to Gly treatment, suggesting its potential roles in crop yield. Our findings established a mechanism by which OsGLR3.4 modulates rice root growth and tropism towards nutrient sources, and shed light on the root behavioral property used by plants to acquire nutrients efficiently.

Risk-associated single nucleotide polymorphisms of mitochondrial D-loop mediate imbalance of cytokines and redox in rheumatoid arthritis.

We have identified rheumatoid arthritis (RA) risk-associated single nucleotide polymorphisms (SNPs) in the mitochondrial displacement loop (D-loop) including the major alleles of nucleotides 195T/C, 16260C/T, and 16519C/T as well as the minor alleles of nucleotides 146T/C and 150C/T previously. We evaluated the potential relationships of these SNPs with status for oxidative stress and inflammation cytokines. The DNA was extracted from blood samples of RA patients, and the SNPs of DNA D-loop were verified by polymerase chain reaction amplification and sequence analysis. Serum levels of inflammatory cytokines including interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-6, IL-10, and tumor necrosis factor-α (TNF-α) were determined by cytometric bead array. Plasma reactive oxygen species (ROS) levels were measured by fluorescent probe technology. The RA risk-related allele 16519C was significantly associated with high IFN-γ levels (100.576 ± 11.769 vs 64.268 ± 8.199, 95% confidence interval [CI] -66.317 to -6.299, P=0.018). This allele also associated with ROS at borderline statistics level (619.295 ± 36.687 vs 526.979 ± 25.896, 95% CI -186.145 to -1.513, P=0.054). The subsequent analysis also showed that the ROS levels were positively correlated with IFN-γ levels (R=0.291, P=0.002). Further analysis showed that RA patients with high C-reactive protein levels displayed a higher ROS level (P=0.001). Our results imply that the 16519C allele of the mtDNA D-loop might promote ROS and IFN-γ levels by altering the replication and transcription of mtDNA, thereby modifying RA development. The potential relationships of RA-associated SNPs in the mitochondrial D-loop with status for oxidative stress and inflammation were evaluated. The 16519C allele of the mtDNA D-loop might promote ROS and IFN-γ levels by altering the replication and transcription of mtDNA to modify RA development.

PSXI-18 Evaluation of Plasma Oxidation Products in Beef Calves Challenged with Lipopolysaccharide (LPS) Following Methionine Supplementation

Abstract This study evaluated the plasma oxidative products of beef calves challenged with LPS following methionine supplementation (L0 = Control; L1 = 10 g/hd · d; L2 = 20 g/hd · d). Following an initial 40-d feeding period, steers (n = 32; 379kg±30.7) were challenged i.v. with LPS (0.25 µg/kg body weight) on d 41. Blood was collected at -2, 0, 2, 4, 6, 8, 10, 12, 18, 24, 36 and 48 h relative to the LPS challenge at 0 h. Samples were processed to isolate plasma for indicators of oxidative stress with colorimetric assays determining malondialdehyde (MDA) and reactive oxygen species (ROS) concentrations. Data were analyzed as repeated measures using the GLIMMIX procedure of SAS. Concentrations of MDA varied by time (P &amp;lt; 0.001), showing a large increase (P &amp;lt; 0.05) 2 h post-LPS, then decreasing again at 4 h. MDA values decreased (P &amp;lt; 0.05) from h 6 to 18 where values returned to initial concentrations (P &amp;lt; 0.05). A treatment × time interaction (P &amp;lt; 0.001) occurred for ROS concentrations. Concentrations for all treatments were less than the detectable limit at -2, 0, 10, 12, 24, 36, and 48h (P &amp;gt; 0.05). At 2 h post-LPS, ROS was greatest in L0 cattle, least in L2, and intermediate in L1 (P &amp;lt; 0.05), but declined at 4 h in all treatments. Values peaked at 6 h for L1 and L2 cattle, followed by a decline at 8 h (P &amp;lt; 0.05). Control cattle were similar from 4 to 6 h (P &amp;gt; 0.05) but increased again at 8 h (P &amp;lt; 0.05). These data show that oxidative stress from an LPS challenge contributes to increased plasma ROS and secondary lipid oxidation products but may be mitigated by the inclusion of methionine supplements.

Regulation of the molecular repertoires of oxidative stress response in the gills and olfactory organ of Atlantic salmon following infection and treatment of the parasite Neoparameoba perurans

The present study investigated the involvement of key molecular regulators of oxidative stress in amoebic gill disease (AGD), a parasitic infestation in Atlantic salmon. In addition, the study evaluated how these molecular biomarkers responded when AGD-affected fish were exposed to a candidate chemotherapeutic peracetic acid (PAA). Atlantic salmon were experimentally infected with the parasite Neoparameoba perurans, the causative agent of AGD, by bath exposure and after 2 weeks, the fish were treated with three commercial PAA products (i.e., Perfectoxid, AquaDes and ADDIAqua) at a dose of 5 ppm. Two exposure durations were evaluated – 30 min and 60 min. Sampling was performed 24 h and 2 weeks after PAA treatment (equivalent to 2- and 4-weeks post infection). At each sampling point, the following parameters were evaluated: gross gill pathology, gill parasitic load, plasma reactive oxygen species (ROS) and total antioxidant capacity (TAC), histopathology and gene expression profiling of genes with key involvement in oxidative stress in the gills and olfactory organ. AGD did not result in systemic oxidative stress as ROS and TAC levels remained unchanged. There were no clear patterns of AGD-mediated regulation of the oxidative stress biomarkers in both the gills and olfactory organ; significant changes in the expression were mostly related to time rather than infection status. However, the expression profiles of the oxidative stress biomarkers in AGD-affected salmon, following treatment with PAA, revealed that gills and olfactory organ responded differently – upregulation was prominent in the gills while downregulation was more frequent in the olfactory organ. The expression of catalase, glutathione S-transferase and thioredoxin reductase 2 was significantly affected by the treatments, both in the gills and olfactory organ, and these alterations were influenced by the duration of exposure and PAA product type. Parasitic load in the gills did significantly increase after treatment regardless of the product and exposure duration; the parasite was undetectable in some fish treated with AquaDes for 30 mins. However, PAA treated groups for 30 min showed lower macroscopic gill scores than the infected-untreated fish. Histology disclosed the classic pathological findings such as multifocal hyperplasia and increased number of mucous cells in AGD-affected fish. Microscopic scoring of gill injuries showed that AGD-infected-PAA-treated fish had lower scores, however, an overall trend could not be established. The morphology and structural integrity of the olfactory organ were not significantly altered by parasitism or PAA treatment. Collectively, the results indicate that AGD did not affect the systemic and mucosal oxidative status of Atlantic salmon. However, such a striking profile was changed when AGD-affected fish were exposed to oxidative chemotherapeutics. Moreover, the gills and olfactory organ demonstrated distinct patterns of gene expression of oxidative stress biomarkers in AGD-infected-PAA-treated fish. Lastly, PAA treatment did not fully resolve the infection, but appeared not to worsen the mucosal health either.

Plasma ascorbic acid, antioxidant capacity, and reactive oxygen species in healthy foals.

To describe ascorbic acid (AA) concentrations, plasma antioxidant capacity (PAC) and markers of oxidative stress, as measured by derivatives of reactive oxygen metabolites (dROMs), in healthy foals at birth and during the first month of life. Venous blood samples were collected from healthy Standardbred (n = 13) and Quarter Horse (n = 10) foals. Plasma AA, PAC, and dROMs were assessed at 3 to 12 hours, 3 days, and 1, 2, and 4 weeks of age. AA was measured via high-performance liquid chromatography. PAC and dROMs were measured with a free radical analytical system. Comparisons of AA, PAC, and dROMs at different time points were assessed. Mean ± standard deviation AA concentrations at 3 to 12 hours (44.7 ± 19.6 μmol/L; P ≤ .01), 1 (48.6 ± 22.5 μmol/L; P ≤ .001), and 2 weeks (41.8 ± 15.8 μmol/L; P ≤ .001) were higher than at 4 weeks of age (28.5 ± 12.7 μmol/L). Both PAC and dROMs significantly increased at different time points compared to 3 to 12 hours of age. Healthy foals have higher plasma AA concentrations shortly after birth, which then gradually decrease throughout the first month of life, suggesting that AA may represent a key antioxidant in the postnatal period. The concurrent increase in PAC and dROMs suggests that dynamic development of oxidative balance occurs after birth in foals. Development of AA, PAC, and dROM reference ranges in healthy foals could be used to guide therapeutic interventions and monitor during disease states characterized by increased oxidative stress.

Galectin‐3 Regulates Oxidant Stress in Obesity

ObjectiveInvestigate the mechanism by which overexpression of Galectin‐3 (GAL3) in the endothelium of obese mice contributes to excess reactive oxygen species (ROS) production and subsequent vascular dysfunction.Methodsdb/db mice, a well‐characterized model of human obesity, were crossed with mice lacking GAL3, a receptor for advanced glycation end products, to generate lean and obese mice with and without GAL3 deletion. Metabolic and cardiovascular endpoints were assessed by mRNA and protein expression in freshly isolated microvascular endothelial cells, microvascular reactivity assessed by pressure myography, and ROS production assessed by dihydroethidium (DHE) staining. Other previously characterized db/db colonies were utilized to further elucidate the mechanistic contributions of GAL3, including mice harboring deletions of NADPH Oxidase I (NOX1), Myostatin (MSTN), and Protein Tyrosine Phosphatase 1B (PTP1B).ResultsDeletion of GAL3 in obese mice had no observable effect on anatomical, metabolic, or plasma phenotype, except for normalizing levels of plasma ROS as evidenced by decrease in thiobarbituric acid reactive substances (TBARS). Obese mice exhibited profound endothelial dysfunction, which was rescued by deletion of GAL3. Our data demonstrate that NOX1, the predominant source of endothelial superoxide production, is significantly downregulated by GAL3 deletion, thereby restoring endothelial function and ameliorating endothelial ROS production in obesity. Furthermore, we demonstrate that GAL3‐mediated NOX1 overexpression is amenable to improvements in metabolic status, such as lowering blood glucose with metformin, improving glucose handling by augmenting muscle mass through deletion of MSTN, or improving insulin signaling through deletion of PTP1B.ConclusionTaken together, these data demonstrate that the overproduction of superoxide by endothelial NOX1 is regulated by the metabolic sensor GAL3 in obesity, leading to endothelial dysfunction and subsequent cardiovascular disease. This pathway presents an attractive target for therapeutic intervention to break the link between aberrant metabolism in obesity and its corresponding vascular pathologies.

Effect of lactoferrin on ram sperm motility after cryopreservation.

ObjectiveThe objective of this study was to analyse the differentially abundant proteins caused by freeze–thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro.MethodsSperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry. Among these proteins, lactoferrin (LTF) was selected to be added before cryopreservation. Next, sperm samples were diluted in Tris extender, with the addition of 0, 10, 100, 500, and 1,000 μg/mL of LTF. After thawing, sperm quality was evaluated by motility, plasma membrane integrity, mitochondrial activity and reactive oxygen species (ROS).ResultsCryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 μg/mL LTF compared to those in the control group. There was no significant difference in mitochondrial activity between the 0 μg/mL group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 μg/mL LTF led to decreased ROS levels compared with those in the control and other groups (p<0.05).ConclusionThe LTF is an important protein during cryopreservation, and the addition of 10 μg/mL LTF to a cryoprotective extender can significantly improve the function of frozen ram sperm.