Optimisation of a fast DMS sensor (FDS) for real time quantification of dimethyl sulfide production by algae

Production of dimethyl sulfide (DMS) from marine samples is often quantified using gas chromatography techniques. Typically, these are labour intensive and have a slow sample turnover rate. Here we demonstrate the use of a portable fast DMS sensor (FDS) that utilises the chemiluminescent reaction of DMS and ozone to measure DMS production in aqueous samples, with a maximum frequency of 10?Hz. We have developed a protocol for quantifying DMS production that removes potential signal interference from other biogenic trace gases such as isoprene (2-methyl-1,3-butadiene) and hydrogen sulfide. The detection limit was 0.89?pM (0.02?ppbv) when using a DMS standard gas mixture. The lowest DMS production rates quantified with the FDS and verified using conventional gas chromatography with flame photometric detection (GC-FPD) were around 0.01?nmol?min^?1. There was a strong correlation in DMS production when comparing the FDS and GC-FPD techniques with a range of marine samples (e.g., r ² ?=?0.94 for Emiliania huxleyi). However, the combined dataset showed the FDS measured 22% higher DMS production than the GC-FPD, with the differences in rates likely due to interfering gases, for example hydrogen sulfide and isoprene. This possible overestimation of DMS production is smaller than the two-fold difference in DMS production between day and night samples from a culture of E. huxleyi. The response time of the instrument to changes in DMS production is method dependent (e.g., geometry of incubation vessel, bubble size) and was approximately 4?min under our conditions when using a culture of E. huxleyi (800?ml) with aeration at 100?ml?min^?1. We suggest the FDS can reduce sample handling, is suitable for short- and long-term measurements of DMS production in algal cultures, and will widen the range of DMS research in marine environments.     

Control of malodorous hydrogen sulfide compounds using microbial fuel cell

In this study, a microbial fuel cell (MFC) was used to control malodorous hydrogen sulfide compounds generated from domestic wastewaters. The electricity production demonstrated a distinct pattern of a two-step increase during 170 h of system run: the first maximum current density was 118.6 ± 7.2 mA m^?2 followed by a rebound of current density increase, reaching the second maximum of 176.8 ± 9.4 mA m^?2. The behaviors of the redox potential and the sulfate level in the anode compartment indicated that the microbial production of hydrogen sulfide compounds was suppressed in the first stage, and the hydrogen sulfide compounds generated from the system were removed effectively as a result of their electrochemical oxidation, which contributed to the additional electricity production in the second stage. This was also directly supported by sulfur deposits formed on the anode surface, which was confirmed by analyses on those solids using a scanning electron microscope equipped with energy dispersive X-ray spectroscopy as well as an elemental analyzer. To this end, the overall reduction efficiencies for HS^? and H₂S_(g) were as high as 67.5 and 96.4 %, respectively. The correlations among current density, redox potential, and sulfate level supported the idea that the electricity signal generated in the MFC can be utilized as a potential indicator of malodor control for the domestic wastewater system.     

Total plasma homocysteine determination by liquid chromatography before and after methionine loading: Results in cerebrovascular disease

Elevated homocysteine (HCY) levels in tissues and blood are associated with premature occlusive diseases. A number of techniques have been developed to assay HCY, including high-performance liquid chromatography (HPLC) with fluorimetric or electrochemical detection, and radioenzymatic methods. The present study evaluated the adaptation of a liquid chromatographic, ion-exchange technique with postcolumn derivatization using ninhydrin. Fasting and moreover post-methionine load total plasma HCY were assayed in 50 patients three months after a stroke and in 20 age-matched controls. Ion-exchange liquid chromatography was performed on an amino acid analyzer using a modified procedure to improve methionine and HCY separation. HCY values in the fasting state were moderately but significantly increased (P<0.05) in the patients compared to the controls: 10.5±3.4 versus 9.3±2.3 μmol/l. The difference between the two groups was amplified in post-load HCY results, which were significantly increased (P<0.05) in the patients: 41.6±17.8 versus 29.2±5.5 μmol/l in controls. The relationship between cerebrovascular disease and impaired HCY metabolism has previously been emphasized by other investigators. Our findings suggest that certain inherited and/or acquired HCY disorders observed in the fasting state (14%) and especially in post-methionine load conditions (32%) may occur during acute disease, and that total plasma HCY can be determined by ion-exchange chromatography even after oral methionine loading.     

Investigation of the binding characteristics between ligands and epidermal growth factor receptor by cell membrane chromatography

The binding property between a ligand and its receptor is very important for numerous biological processes. In this study, we developed a high epidermal growth factor receptor (EGFR)‐expression cell membrane chromatography (CMC) method to investigate the binding characteristics between EGFR and the ligands gefitinib, erlotinib, canertinib, afatinib, and vandetanib. Competitive binding analysis using gefitinib as the marker was used to investigate the interactions that occurred at specific binding sites on EGFR. The ability of displacement was measured from the HEK293‐EGFR/CMC column on the binding sites occupied by gefitinib for these ligands, which revealed the following order: gefitinib (K_D, 8.49 ± 0.11 × 10^?7 M) > erlotinib (K_D, 1.07 ± 0.02 × 10^?6 M) > canertinib (K_D, 1.41 ± 0.07 × 10^?6 M) > afatinib (K_D, 1.80 ± 0.12 × 10^?6 M) > vandetanib (K_D, 1.99 ± 0.03 × 10^?6 M). This order corresponded with the values estimated by frontal displacement analysis and the scores obtained with molecular docking. Furthermore, thermodynamic analysis indicated that the hydrogen bond or Van der Waals force was the main interaction force in the process of EGFR binding to all 5 ligands. Overall, these results demonstrate that a CMC method could be an effective tool to investigate the binding characteristics between ligands and receptors.     

Digital twin of a continuous chromatography process for mAb purification: Design and model-based control

Model-based design of integrated continuous train coupled with online process analytical technology (PAT) tool can be a potent facilitator for monitoring and control of Critical Quality Attributes (CQAs) in real time. Charge variants are product related variants and are often regarded as CQAs as they may impact potency and efficacy of drug. Robust pooling decision is required for achieving uniform charge variant composition for mAbs as baseline separation between closely related variants is rarely achieved in process scale chromatography. In this study, we propose a digital twin of a continuous chromatography process, integrated with an online HPLC-PAT tool for delivering real time pooling decisions to achieve uniform charge variant composition. The integrated downstream process comprised continuous multicolumn capture protein A chromatography, viral inactivation in coiled flow inverter reactor (CFIR), and multicolumn CEX polishing step. An online HPLC was connected to the harvest tank before protein A chromatography. Both empirical and mechanistic modeling have been considered. The model states were updated in real time using online HPLC charge variant data for prediction of the initial and final cut point for CEX eluate, according to which the process chromatography was directed to switch from collection to waste to achieve the desired charge variant composition in the CEX pool. Two case studies were carried out to demonstrate this control strategy. In the first case study, the continuous train was run for initially 14 h for harvest of fixed charge variant composition as feed. In the second case study, charge variant composition was dynamically changed by introducing forced perturbation to mimic the deviations that may be encountered during perfusion cell culture. The control strategy was successfully implemented for more than ±5% variability in the acidic variants of the feed with its composition in the range of acidic (13%–17%), main (18%–23%), and basic (59%–68%) variants. Both the case studies yielded CEX pool of uniform distribution of acidic, main and basic profiles in the range of 15 ± 0.8, 31 ± 0.3, and 53 ± 0.5%, respectively, in the case of empirical modeling and 15 ± 0.5, 31 ± 0.3, and 53 ± 0.3%, respectively, in the case of mechanistic modeling. In both cases, process yield for main species was >85% and the use of online HPLC early in the purification train helped in making quicker decision for pooling of CEX eluate. The results thus successfully demonstrate the technical feasibility of creating digital twins of bioprocess operations and their utility for process control.     

Revealing binding interaction between seven drugs and immobilized β2‐adrenoceptor by high‐performance affinity chromatography using frontal analysis

The development of new approaches to study the affinity between ligands and G‐protein‐coupled receptors proves to be of growing interest for pharmacologists, chemists, and biologists. The aim of this work was to determine the binding of seven drugs to β₂‐adrenoceptors by frontal analysis using immobilized receptor stationary phase. The dissociation constants (K_d) were determined to be (3.16 ± 0.09) × 10^?4 M for salbutamol, (4.29 ± 0.12) × 10^?4 M for terbutaline, (6.19 ± 0.16) × 10^?4 M for methoxyphenamine, (2.11 ± 0.07) × 10^?4 M for tulobuterol, (1.82 ± 0.11) × 10^?4 M for fenoterol, (9.75 ± 0.24) × 10^?6 M formoterol, and (9.84 ± 0.26) × 10^?5 M for clenbuterol. These results showed a good correlation with the data determined by radioligand binding assay. Further investigations revealed that the dissociation constant mainly attributed to the number of hydrogen bonds in the structures of ligands. This study indicates that affinity chromatography using immobilized receptor stationary phase can be used for the direct determination of drug‐receptor binding interactions and has the potential to become a reliable alternative for quantitative studies of ligand–receptor interactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Pharmacokinetic studies of amiloride and its analogs using reversed-phase high-performance liquid chromatography

We have studied the pharmacokinetics of amiloride and its analogs. A high-performance liquid chromatographic method has been adapted for the measurement of amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and 5-(N,N-hexamethylene)amiloride (HMA) in mouse plasma, kidney, liver and tumor tissues. The method uses a C₈ preparative solid-phase column, followed by separation using a reversed-phase C₁₈ column (250×4 mm I.D., 5 μm particle size) with detection by ultraviolet absorption at 365 nm. Reversed-phase separations were performed at ambient temperature using a non-linear gradient method with two different mobile phases: mobile phase A was 100% acetonitrile while mobile phase B was 0.15 M perchloric acid at pH 2.20 (flow-rate was 1.2 ml/min). The retention times for amiloride, benzamil (used as an internal standard), EIPA and HMA are 13.4, 19.5, 21.8 and 23.5 min, respectively. The calibration curves are linear over the range of 0.1–50 μM in plasma and in tissues. The half-lives of amiloride, EIPA and HMA (and their confidence intervals) in plasma after intraperitoneal injection of drugs into mice were 68.8±0.2, 31.2±2.5 and 39.3±7.9 min, respectively. Amiloride was detected as a metabolite of EIPA but not of HMA. When EIPA was injected at a dose of 10 μg/g body weight, it was cleared rapidly from liver, but concentrations > 1 μM were sustained for at least 2 h in murine kidney and in a transplantable tumor.     

Mechanistic modeling based process analytical technology implementation for pooling in hydrophobic interaction chromatography

A major challenge in chromatography purification of therapeutic proteins is batch-to-batch variability with respect to impurity levels and product concentration in the feed. Mechanistic model can enable process analytical technology (PAT) implementation by predicting impact of such variations and thereby improving the robustness of the resulting process and controls. This article presents one such application of mechanistic model of hydrophobic interaction chromatography (HIC) as a PAT tool for making robust pooling decisions to enable clearance of aggregates for a monoclonal antibody (mAb) therapeutic. Model predictions were performed before the actual chromatography experiments to facilitate feedforward control. The approach has been successfully demonstrated for four different feeds with varying aggregate levels (3.84%–5.54%) and feed concentration (0.6 mg/mL–1 mg/mL). The resulting pool consistently yielded a product with 1.32 ± 0.03% aggregate vs. a target of 1.5%. A comparison of the traditional approach involving column fractionation with the proposed approach indicates that the proposed approach results in achievement of satisfactory product purity (98.68 ± 0.03% for mechanistic model based PAT controlled pooling vs. 98.64 ± 0.16% for offline column fractionation based pooling). © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2758, 2019.     

Microemulsion and micellar electrokinetic chromatography of steroids

A mixture of ten steroids was separated by microemulsion and micellar (SDS and glycodeoxyholate) electrokinetic chromatography systems. Separations were done on a 50 cm (to the detector) × 50 μm I.D. fused-silica capillary. Complete separation of all the test compounds in the micellar mode was obtained with glycodeoxycholate (50 mM) in 25 mM borate buffer, pH 6.5, as the micelle-forming agent. The best results, however, were obtained using microemulsion electrokinetic chromatography in which higher aliphatic alcohols were used as the microemulsion-forming modifiers. The system consisted of n-hexanol (0.81%), SDS (3.31%) and n-butanol (6.61%) in 20 mM phosphate buffer, pH 10.0 (89.28%, w/w). In the microemulsion mode, linear calibration for steroid standards was obtained in the concentration range 3 × 10⁻⁴ − 3 × 10⁻⁵ mol l⁻¹ with a detection limit of 1 pmol. The method was validated and applied to an 11β-hydroxysteroid dehydrogenase assay in tissues.     

Comparison of ochratoxin A levels in edible pig tissues and in biological fluids after exposure to a contaminated diet

The aim of this study was to compare ochratoxin A (OTA) levels in pig tissues and biological fluids after animal exposure to contaminated diet (250 μg OTA/kg of feed) during 4 weeks of fattening. OTA concentrations were quantified using a validated immunoassay method (ELISA) and high-performance liquid chromatography with fluorescence detector (HPLC-FD). The highest mean OTA concentration in pig tissues was determined in kidneys of exposed animals (13.87?±?1.41 μg/kg), followed by lungs (10.47?±?1.97 μg/kg), liver (7.28?±?1.75 μg/kg), spleen (4.81?±?0.99 μg/kg), muscle tissue (4.72?±?0.86 μg/kg), fat tissue (4.11?±?0.88 μg/kg), heart (3.71?±?1.09 μg/kg), and brain (3.01?±?0.25 μg/kg). Furthermore, on the last day of exposure (day 28), significantly higher mean OTA levels were determined in urine (16.06?±?3.09 μg/L) in comparison to serum (4.77?±?1.57 μg/L) showing that OTA urine analysis could be a good marker to identify elevated levels of this contaminant in porcine tissues used for human consumption. This study gave guidelines for the most efficient OTA control in pig-derived biological materials that can be exercised at slaughterhouses.     

Removal of dimethyl sulfide,methyl mercaptan,and hydrogen sulfide by immobilized Thiobacillus thioparus TK-m

Cells of Thiobacillus thioparus TK-m were immobilized on cylindrical porous polypropylene pellets (5 mmφ × 5 mm) which were packed in an acrylic cylinder of 50 mm inner diameter up to the height of 800 mm. When a sulfur-containing malodorous gas was charged to this packed tower at the superficial velocity of 0.1 m/s, maximum loading capacity (mmol/l·d) for a malodorous gas to attain the removal rate of 95% or more was: 3.65 for dimethyl sulfide, 8.74 for methyl mercaptan, and 17.36 for hydrogen sulfide. At this time, the inlet concentration (μl/l) of the malodorous compound was: 7.44 for dimethyl sulfide, 17.8 for methyl mercaptan, and 35.4 for hydrogen sulfide. For every compound, higher loading resulted in greater removal quantities. The removal rate of dimethyl sulfide was not overly affected by the presence of a large amount of easily decomposable hydrogen sulfide.     

Phytochemical profiling of Azima tetracantha Lam. leaf methanol extract and elucidation of its potential as a chain-breaking antioxidant,anti-inflammatory and anti-proliferative agent

Azima tetracantha, a traditional medicinal plant included in the order Brassicales and family Salvadoraceae, is widely used as a dietary supplement in folklore medicines. The plant is also used for the treatment of rheumatism, diarrhea and other inflammatory disorders. The present investigation focused on the phytochemical composition, radical scavenging, reducing potential and anti-proliferative activities of the A. tetracantha leaves. Quantitative estimation of the polyphenols and flavonoids revealed significantly elevated levels in the methanol extract. Corroborating with this, methanol extract exhibited higher in vitro anti-radical scavenging effect against 2,2-diphenyl-1- picrylhydrazyl (34.14 ± 2.19 μg/mL), and hydrogen peroxide (44.96 ± 1.77 μg/mL), as well as ferric reducing properties (58.24 ± 6.98 μg/mL). The methanolic extract also showed strong lipoxygenase (71.42 ± 6.36 μg/mL) and nitric oxide inhibitory activities (94.23 ± 8.11 μg/mL). Cytotoxic activity against MCF7 cells was found to be higher (IC50= 37.62 ± 2.94 μg/mL), than that of MDAMB231 cells (IC50= 69.11 ± 5.02 μg/mL). The qPCR-based analysis indicated dose-dependent increase in the expression of the pro-apoptotic genes such as executioner caspases and apoptotic protease activating factor-1. Overall, the results indicated the possible use of methanol extract of A. tetracantha leaves as a chain-breaking antioxidant molecule and are capable of inhibiting inflammatory enzymes and the proliferative potential of breast cancer cells.     

The analysis of arildone in plasma,urine and feces by gas—liquid chromatography with electron-capture detection

The analysis of arildone in plasma, urine and feces by gas—liquid chromatography with electron-capture detection is described. O-(2,3,4,5,6-Pentafluorohenzyl)hydroxylamine is the derivatizing agent for the plasma and urine analysis; 3-nitrophenylhydrazine is utilized for fecal analysis. The mean (± S.E.) minimum quantifiable level of arildone was 1.4 (± 0.2) ng/ml in urine, 6.4 (± 0.1) ng/ml in plasma, and 12.6 (± 1.0) ng/g in feces. The chromatographic response was linear in the range of 0 and 10–120 ng/ml for plasma, 0 and 2.5–20 ng/ml for urine and 0 and 25–250 ng/g for feces. The estimated overall precision of the assay was 5.5%, 6.4% and 8.9% in urine, plasma and feces, respectively.     

Comparative effects of ischemic pre and postconditioning on ischemia-reperfusion injury in spontaneously hypertensive rats (SHR)

Brief episodes of myocardial ischemia-reperfusion applied early in reperfusion may attenuate the reperfusion injury, strategy called ischemic postconditioning (IPO). Our objective was to examine the effects of IPO compared with ischemic preconditioning (IP) on postischemic myocardial dysfunction in spontaneously hypertensive rats (SHR). Isolated hearts from SHR and normotensive WKY rats were subjected to the following protocols: (1) Ischemic control (IC): global ischemia 20 min (GI20) and reperfusion 30 min (R). (2) IPO: three cycles of R30sec–IG30sec at the onset of R; (3) IP: a cycle of IG5–R10 previous to GI20, (4) IPO in the presence of chelerythrine, an inhibitor of protein kinase C (PKC). Systolic and diastolic function were assessed through developed pressure (LVDP) and end diastolic pressure (LVEDP), respectively. Lipid peroxidation was estimated by thiobarbituric reactive substance (TBARS) concentration. IPO significantly improved postischemic dysfunction. At the end of R, LVDP recovered to 87 ± 7% in WKY and 94 ± 7% in SHR vs. 55 ± 11% and 58 ± 12% in IC hearts. LVEDP reached values of 24 ± 6 mmHg for WKY and 24 ± 3 mmHg for SHR vs. 40 ± 8 and 42 ± 5 mmHg in IC hearts. Similar protection was achieved by IP. TBARS contents of SHR hearts were significantly diminished by IP and IPO. PKC inhibition aborted the protection of myocardial function and attenuated the diminution of lipid peroxidation conferred by IPO. These data show that IPO was as effective as IP in improving the postischemic dysfunction of hearts from SHR hearts, and that this cardioprotection appears to be associated with a diminution of ROS-induced damage involving the PKC activation.     

Tissue distribution of puerarin and its conjugated metabolites in rats assessed by liquid chromatography–tandem mass spectrometry

Puerarin (an isoflavone C-glucoside from kudzu root) has been the focus of several studies investigating its potential effects on health benefits. In this study, we determined single dose tissue distribution of puerarin and its metabolites in order to examine whether they undergo selective uptake by specific organs. Puerarin was administered orally (50 mg/kg) to rats and the concentration of puerarin in tissue compartments was determined using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Puerarin was widely distributed in rat tissues with highest concentrations in lungs (799±411.6 ng/g wet tissues). In addition, we examined the excretion of puerarin into the bile. LC–MS/MS analysis of bile samples collected after infusing puerarin directly into the portal vein indicated that puerarin was excreted into the bile predominantly in the form of unconjugated puerarin. This report identifying puerarin in several organs including kidney and pancreas may explain its beneficial effects in diabetes.     

Determination of counter‐ions in synthetic peptides by ion chromatography,capillary isotachophoresis and capillary electrophoresis

The utility of three various analytical techniques [ion chromatography (IC), capillary electrophoresis (CE) and isotachophoresis (ITP)] was tested in the determination of counter‐ions in synthetic peptides. The analyzed ions were acetates, trifluoroacetates and chlorides. IC provided the best results; CE, except limit of detection and limit of quantification, exhibited the comparable results. ITP was classified as the less useful because of the problem with the determination of the chloride ions. Nevertheless, all the three techniques were able to analyze trifluoroacetates and acetates ions with satisfactory results. Except analytical methods, three procedures using hydrochloric acid (HCl) (at two different concentrations) and acetic acid as sample solvents processed by lyophilization were tested. It has been found that the lyophilization not only by HCl but also by acetic acid is a simple and cheap procedure for removal of toxic trifluoroacetic counter‐ions from peptides on satisfactory levels. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of ageing on human plasma glutathione concentrations as determined by high-performance liquid chromatography with fluorimetric detection

A convenient method for the determination of reduced glutathione (GSH) and oxidized glutathione (GSSG) in human plasma by high-performance liquid chromatography with fluorescence detection is reported. This assay involves direct addition of human plasma to methanolic monobrombimane. for simultaneous protein precipitation and thiol derivatization. The assay was validated by addition of authentic GSH and GSSG to plasma samples. Plasma glutathione levels in Chinese male and female volunteers were found to decrease with increasing age (age groups, 20–30, 30–40, 40–50, 50–60, and >60; mean ± S.E.M. 0.97 ± 0.03, 0.77 ± 0.02, 0.67 ± 0.03, 0.51 ± 0.02, 0.48 ± 0.02 μM for male volunteers and 1.11 ± 0.06, 0.76 ± 0.03, 0.61 ± 0.03, 0.53 ± 0.04 and 0.43 ± 0.04 μM for female volunteers). GSSG levels, in both males and females, did not show a correlation with age. There were no significant differences in GSH or GSSG levels among male and female volunteers of the same age group. These results suggest that elderly persons might be more susceptible to oxidative injury due to decreased plasma glutathione levels.     

Intracisternal TRH and RX 77368 Potently Activate Gastric Vagal Efferent Discharge in Rats

O-Lee, T. J., J. Y. Wei and Y. TachÉ. Intracisternal Trh and Rx 77368 potently activate gastric vagal efferent discharge in rats. Peptides 18(2) 213–219, 1997.—The influence of intracisternal (ic) TRH and the stable TRH analog, RX 77368, on gastric vagal efferent discharge (GVED) was investigated in urethane-anesthetized rats. Consecutive IC injections of TRH (3, 30, and 300 ng) at 60 min intervals stimulated dose dependently multi-unit GVED with a peak increase of 90 ± 21%, 127 ± 18% and 145 ± 16% respectively. In two separate studies, IC injection of RX 77368 at 1.5 or 15 ng stimulated multi-unit GVED by 142 ± 24% and 244 ± 95% respectively. Saline injection IC had no effect on GVED. RX 77368 (1.5 ng, ic) action was long lasting (84 ± 13 min) compared with TRH (3 ng: 44 ± 7 min). Single-unit analysis also showed that 13 of 13 units responded to ic RX 77368 (1.5 ng) by an increase in activity. These data indicate that low doses of TRH injected ic stimulate vagal efferent outflow to the rat stomach and that RX 77368 action is more potent than TRH.     

Neuroprotective effects of the mitochondria-targeted antioxidant MitoQ in a model of inherited amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motor neuron degeneration that ultimately results in progressive paralysis and death. Growing evidence indicates that mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in ALS. To further explore the hypothesis that mitochondrial dysfunction and nitroxidative stress contribute to disease pathogenesis at the in vivo level, we assessed whether the mitochondria-targeted antioxidant [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl]triphenylphosphonium methane sulfonate (MitoQ) can modify disease progression in the SOD1^G93A mouse model of ALS. To do this, we administered MitoQ (500 µM) in the drinking water of SOD1^G93A mice from a time when early symptoms of neurodegeneration become evident at 90 days of age until death. This regime is a clinically plausible scenario and could be more easily translated to patients as this corresponds to initiating treatment of patients after they are first diagnosed with ALS. MitoQ was detected in all tested tissues by liquid chromatography/mass spectrometry after 20 days of administration. MitoQ treatment slowed the decline of mitochondrial function, in both the spinal cord and the quadriceps muscle, as measured by high-resolution respirometry. Importantly, nitroxidative markers and pathological signs in the spinal cord of MitoQ-treated animals were markedly reduced and neuromuscular junctions were recovered associated with a significant increase in hindlimb strength. Finally, MitoQ treatment significantly prolonged the life span of SOD1^G93A mice. Our results support a role for mitochondrial nitroxidative damage and dysfunction in the pathogenesis of ALS and suggest that mitochondria-targeted antioxidants may be of pharmacological use for ALS treatment.     

Assessment of plant secondary metabolites in oil palm seedlings after being treated with calcium,copper ions and salicylic acid

The effect of calcium, copper ions and salicylic acid (SA) amendment on the incidence of basal stem rot and activity of secondary metabolites in oil palm seedlings were investigated in glasshouse study. Disease incidence (DI) in positive control (T8) was 75% at nine months after inoculation (9 MAI). However, weekly pre-immunisation with Ca^2+?+^?Cu^2+?+^?SA prior to inoculation significantly suppressed DI and delayed disease onset as noted in T7. In the present study, the lowest %DI was observed in T7 (15%) followed by T1, T5, T6, T3, T4 and T2. The Ca²⁺, Cu²⁺ and SA amendments were resulted in earlier and higher accumulation of plant secondary metabolites as noted in leaves, stems and root tissues in response to invasion by Ganoderma boninense. High total phenolic content concentration was detected in T7 (leaf: 233.38 ± 0.12 mg/g; stem: 132.78 ± 0.04 mg/g and root: 86.98 ± 0.28 mg/g). Similar trend was obtained in peroxidase activity, total lignin content and hydrogen peroxide scavenging activity. These results suggested that it could be due to the accumulation of phenolics, peroxidase activities, lignin content and hydrogen peroxide scavenging activities in oil palm seedling tissues which might have collectively contributed to induce resistance against G. boninense.