P–111 Development of a flow cytometric assay for membrane lipid oxidation in human sperm

Study question Is a commercially available lipid peroxidation assay sensitive enough to detect sperm lipid membrane damage and thus provide a novel indicator of male fertility status? Summary answer Provisional results demonstrate the novelty of creating a protocol to identify and quantify sperm lipid membrane damage and indicate possible insight into individual male fertility. What is known already Cytotoxic lipid aldehydes such as 4-hydroxynonenal (4HNE) created by the damaging effects of reactive oxygen species (ROS) have been studied extensively in sperm, as an indicator of male fertility. This is due to their connection with detrimental effects on sperm function such as morphology, acrosome reactions, motility and fertilization of the oocyte. Although literature states the mechanisms of damage caused to the lipid membrane of the sperm cell, there is no evidence of its quantification or usage as a commercial fertility indicator for human males. Study design, size, duration Since the assay is still being developed, there is no formal study size or duration. The goal of this pilot study is to determine whether a commercial lipid peroxidation assay can detect the difference between sperm with high levels of oxidative damage and control sperm cells. We used the remains of sperm samples initially collected for standard semen analysis, which were flash-frozen and then assayed with / without hydrogen peroxide treatment to induce oxidative damage. Participants/materials, setting, methods Frozen sperm from consenting donors (n = 21) were washed, optionally treated with hydrogen peroxide to induce oxidative damage, stained with a commercially available lipid peroxidation sensor (LPS, Abcam ab243377), and the resulting fluorescence quantitated by flow cytometry. Assay optimization varied the numbers of sperm input to the protocol, the concentration of the peroxidation sensor, the amount and duration of hydrogen peroxide treatment and the effect of paraformaldehyde (PFA) fixation of samples before or after staining. Main results and the role of chance Successful detection of lipid damage in control samples We observed a significant difference at a p-value < 0.05 between untreated samples and all positive controls with hydrogen peroxide concentrations stronger than 500uM (p < 0.038) . This indicates that we can detect sperm bearing oxidative damage, and establishes the conditions required to make a positive control sample. Establishment of assay parameters Results indicate the concentration of sperm input to the protocol is not a significant factor for concentrations below 5 million/ml. Low concentration samples thus do not require further dilution before testing. Correlation with DNA damage A significant direct strong positive Pearson correlation coefficient (R = 0.93, p < 0.023) was found between samples with low DNA fragmentation index (DFI (%), measured by flow cytometric staining with acridine orange) and the LPS flow cytometric data (%). Limitations, reasons for caution As yet our data only addresses high level lipid damage induced by peroxide treatment. It remains to be established whether it is possible to detect endogenous LPO damage due to oxidative stress in semen. Future work will correlate our data with motility information and oxidative stress data (measured by MiOXSYS). Wider implications of the findings: If we are able to develop a direct assay for sperm LPO, this will allow an additional avenue for testing patients with unexplained male infertility, which could in turn affect treatment choices and ART methodology. Improved diagnosis and treatment will potentially improve the lives of families with their fertility matters. Trial registration number Not applicable

1,2-benzenedicarboxylic acid, bis (2-methyl propyl) ester isolated from Onosma bracteata Wall. inhibits MG-63 cells proliferation via Akt-p53-cyclin pathway

Onosma bracteata Wall. (Boraginaceae family) is one of the important constituents of Ayurvedic drugs which enhance immunity. Among all the fractions isolated from O. bracteata, ethyl acetate fraction (Obea) showed good antioxidant activity in Superoxide radical scavenging assay and Lipid peroxidation assay with EC50 value of 95.12 and 80.67 µg/ml, respectively. Silica gel column chromatography of Obea yielded ObD1 fraction which was characterized as Di-isobutyl phthalate (DIBP) using NMR, FTIR and HRMS spectroscopic techniques. DIBP showed antiproliferative activity in human osteosarcoma MG-63, human neuroblastoma IMR-32 and A549 cell lines with GI50 value of 37.53, 56.05 and 47.12 µM, respectively, in MTT assay. In Flow cytometric studies, DIBP has shown disruption of mitochondrial membrane potential (MMP) and enhancement of ROS, indicating the apoptosis induction. The cells were found to be delayed at G0/G1 phase which might be due to the downregulation of Cyclin E and CDK2 as shown in RT-PCR studies. Western blotting analysis revealed an increased expression of p53, caspase 3 and caspase 9 and downregulation of p-NF-kB, p-Akt and Bcl-xl. Molecular docking studies also displayed the interaction of DIBP with p53 (− 151.13 kcal/mol) and CDK1 (− 133.96 kcal/mol). Thus, DIBP has exhibited great potential as chemopreventive/chemotherapeutic agent against osteosarcoma.

Effects of sodium fluoride and Ocimum sanctum extract on the lifespan and climbing ability of Drosophila melanogaster

Background Fluoride may induce oxidative stress and apoptosis. It may also lead to neurobehavioural defects including neuromuscular damage. The present study aimed to explore the effects of sub lethal concentrations of sodium fluoride (NaF) on the lifespan and climbing ability of Drosophila melanogaster. In total, 0.6 mg/L and 0.8 mg/L of NaF were selected as sublethal concentrations of NaF for the study. Lifespan was measured and climbing activity assay was performed. Results The study showed significant decrease in lifespan of flies treated with fluoride. With increasing age, significant reduction in climbing activity was observed in flies treated with sodium fluoride as compared to normal (control) flies. Flies treated with tulsi (Ocimum sanctum) and NaF showed increase in lifespan and climbing activity as compared to those treated with NaF only. Lipid peroxidation assay showed significant increase in malondialdehyde (MDA) values in the flies treated with NaF as compared to control. The MDA values decreased significantly in flies treated with tulsi mixed with NaF. Conclusions The results indicate that exposure to sub lethal concentration of NaF may cause oxidative stress and affect the lifespan and climbing activity of D. melanogaster. Tulsi extract may help in reducing the impact of oxidative stress and toxicity caused by NaF.

Systematic prompting the new physiological and morphological, biological responses of modified metabolic induction process of Calendula officinalis L under heat stress with couple of supplementary nutritional applications as safety of floriculture crops

Purpose the objective of study to modified the metabolism of Calendula officinalis L. under heat stress couple with N and P application in two years of study. Methods the trail was conducted during peak summer season (months of May, June, and July) couple with different levels of N & P application to monitor the quality and quantity characterization of marigold (Calendula officinalis L.). The traits of three doses of N and P (0.6 g, 0.9 g, and 0.8 g, 1− 1 g) given to Calendula officinalis L.) to display the vegetative, reproductive, physiological parameters such as Malondialdehyde (MDA), Chlorophyll contents, Lipid Peroxidation Assay in leaves, Li and K contents, phytochemicals and nitrogen and phosphorus used efficacy Results showed that maximum values of Malondialdehyde (MDA), Chlorophylls, Lipid Peroxidation Assay in leaves observed followed by the alteration in Li and K measured in the month of May, June with slight differences July. The phytochemicals like total phenolic contents (84.41 mg GAE/g), total antioxidants (36.3% DPPH), total carotenoids and total flavonoids contents (16.2 and 0.9 mg/100 g) were measured by 0.9 g of Nitrogen application and followed by 1 g of Phosphorus in both years of study. The respirational changes were observed in the higher rate of P levels. The liner changes of N and P rates showed some fluctuation in heat stress months. Conclusions It was concluded that the higher doses of both N and P fertilizers were effective in controlling the heat stress and mentioned the quality of florets by various induction and biological active process.

Oxidative Stress Mediates Anxiety-Like Behavior Induced by High Caffeine Intake in Zebrafish: Protective Effect of Alpha-Tocopherol

Anxiety is a common symptom associated with high caffeine intake. Although the neurochemical mechanisms of caffeine-induced anxiety remain unclear, there are some evidences suggesting participation of oxidative stress. Based on these evidences, the current study is aimed at evaluating the possible protective effect of alpha-tocopherol (TPH) against anxiety-like behavior induced by caffeine (CAF) in zebrafish. Adult animals were treated with CAF (100 mg/kg) or TPH (1 mg/kg)+CAF before behavioral and biochemical evaluations. Oxidative stress in the zebrafish brain was evaluated by a lipid peroxidation assay, and anxiety-like behavior was monitored using light/dark preference and novel tank diving test. Caffeine treatment evoked significant elevation of brain MDA levels in the zebrafish brain, and TPH treatment prevented this increase. Caffeine treatment also induced anxiety-like behavior, while this effect was not observed in the TPH+CAF group. Taken together, the current study suggests that TPH treatment is able to inhibit oxidative stress and anxiety-like behavior evoked by caffeine.

Antioxidant-mediated neuro-protective and GABAergic calming effect of Stephania japonica

Stephania japonica, a tropically-habituated plant is widely distributed in Bangladesh. Traditionally, it has been used in the treatment of inflammation, asthma, fever, sleep disturbance, painful conditions, and rheumatism. However, scientific evidence of its biological activities are very limited. This study evaluated neuroprotctivity along with a possible anxiolytic-like effect of the methanol leaf extract of S. japonica (MSJ). The antioxidant test was performed by using the scavenging capacity of hydroxyl (●OH) and nitric oxide (●OH) radicals; and an inhibition of lipid peroxidation assay. In vitro, egg albumin denaturation (IELD) and anti-acetylcholinesterase (anti-AChE) tests were performed to evaluate the anti-inflammatory and anti-cholinesterase activity, respectively. Additionally, a possible anxiolytic action of the MSJ was investigated in Swiss mice by taking diazepam (DZP) and flumazenil (FLU) as gamma amino butyric acid (GABA) receptor agonist and antagonist, respectively. The MSJ concentration (50-200 µg/mL)/dose (50-200 mg/kg, oral)-dependently exhibited antioxidant, anti-inflammatory, anti-AChE and anxiolytic-like effects. The MSJ also increased the activities of the standards used in antioxidant and anti-AChE (trolox), anti-inflammatory (acetyl salicylic acid), and anxiolytic (DZP: agonist) test. The MSJ exhibited antioxidant and anti-inflammatory activities. It also potentiated the action of DZP, while antagonizing the effect of the FLU, suggesting a possible GABAergic, DZP-agonistic anxiolytic-like action in experimental animals. S. japonica may be one of the good sources of neuroprotective phytochemicals.

Phytochemical and pharmacological investigation of Teucrium muscatense

<p>The effect of enzyme inhibition, anticancer, antifungal, antioxidants activities of the extracts of <em>Teucrium muscatense </em>obtained with different organic solvents was investigated. Chloroform fraction exhibited promising inhibition (64%) with IC₅₀value (390 ± 2.0 μg/mL) against urease, while other fractions displayed moderate activity. In case of α-glucosidase and acetyl cholinesterase enzyme inhibition assays, all fractions were found inactive at a concentration of 1.0 mg/mL. The <em>n</em>-hexane fraction of <em>Teucrium muscatense </em>exhibited highest anticancer activity against breast cancer cell (MDAMB231) line at high (100 µg/mL) concentration and also inhibited the growth of <em>F. oxysporum </em>up to 64.3% in case of antifungal activity. EtOAc fraction showed highiest DPPH (70.6%) and ABTS (76.7%) radical scavenging activity at highest concentration (1000 μg/mL). Bioassay guided isolation of EtOAc fraction afforded two flavonoids (<strong>1</strong> and <strong>2</strong>). Both compounds showed highiest ABTS activity and could be a significant markers in the EtOAc fraction. Anti-lipid peroxidation assay was also performed in which aqueous fraction showed highest percentage of 73.2% at higher concentration (1000 μg/mL) followed by n-BuOH (64.1%) and EtOAc (51.4%) fractions. GC/MS analysis of the essential oil of <em>T. mascatense</em> showed higher percentage of linalool (34.18%), limonene (13.45%), linalyl acetate (10.04%), and β-eudesmol (9.21%). Proximate composition of <em>Teucrium muscatense </em>showed that it contained high amount of ash (19.6%), protein (10.3%), and fiber (17.5%).</p>