Min Incubation Time Research Articles

Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus

Bovine ephemeral fever virus (BEFV), identified as the causative pathogen of bovine ephemeral fever (BEF), is responsible for increasing numbers of epidemics/outbreaks and has a significant harmful effect on the livestock industry. Therefore, a rapid detection assay is imperative for BEFV diagnosis. In this study, we described the development of lateral-flow dipstick isothermal recombinase polymerase amplification (LFD-RPA) assays for detection of BEFV. RPA primers and LF probes were designed by targeting the specific G gene, and the amplification product can be visualized on a simple lateral flow dipstick with the naked eyes. The amplification reaction was performed at 38 °C for 20 min and LFD incubation time within 5 min. The detection limit of this assay was 8 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses such as bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine vesicular stomatitis virus. In addition, the assay was performed with total 128 clinical specimens and the diagnostic results were compared with conventional RT-PCR, real-time quantative(q) PCR. The result showed that the coincidence rate of BEFV LFD-RPA and real-time qPCR was 96.09% (123/128), which was higher than conventional RT-PCR. The RPA combined with LFD assay probably provides a rapid and sensitive alternative for diagnosis of BEFV infections outbreak.

Single step green synthesis of stable nickel and nickel oxide nanoparticles from Calotropis gigantea: Catalytic and antimicrobial potentials

Nickel (Ni) and nickel oxide (NiO) nanoparticles (NPs) were successfully synthesized by using a simple, novel and green synthetic route using metal salt and leaves extract of a wild plant “Calotropis gigantea” which act as reducing as well as stabilizing agent due to extra availability of phenolic contents and antioxidants in it. Optimization was carried out by varying different parameters controlling the reaction and the best yield was obtained at 80°C temperature, 90min incubation time, pH 12, 1mM precursor concentration and 20% extract concentration. Characterization was made by using various characterizing techniques. UV/VIS absorption peak was at 400nm for synthesized Ni NPs and at 415 for NiO NPs. The average size of the particles was 20–40nm, in the case of NiO NPs and <60nm for Ni NPs confirmed by X-ray diffraction analysis and Scanning electron microscope (SEM). Functional groups indication was made by using Fourier transform Infrared (FT-IR) spectroscopic analysis of materials. Catalytic efficiency was investigated against methylene blue dye, the maximum degradation efficiency was 98.8%, followed by first order reaction kinetics. Synthesized NPs were highly stable i.e. till 3 months. Biological test results exposed that synthesized Ni and NiO nanoparticles have broad spectrum antibiotic abilities against E. coli and Bacillus subtilus.

Multiplex and Label-Free Relative Quantification Approach for Studying Protein Abundance of Drug Metabolizing Enzymes in Human Liver Microsomes Using SWATH-MS.

We describe a sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) based method for label-free, simultaneous, relative quantification of drug metabolism enzymes in human liver microsomes (HLM; n = 78). In-solution tryptic digestion was aided by a pressure cycling method, which allowed a 90 min incubation time, a significant reduction over classical protocols (12-18 h). Digested peptides were separated on an Acquity UHPLC Peptide BEH C18 column using a 60 min gradient method at a flow rate of 0.100 mL/min. The quadrupole-time-of-flight mass spectrometer (ESI-QTOFMS) was operated in positive electrospray ionization mode, and data were acquired by data-dependent acquisition (DDA) and SWATH-MSALL mode. A pooled HLM sample was used as a quality control to evaluate variability in digestion and quantification among different batches, and inter-batch %CV for various proteins was between 3.1 and 7.8%. Spectral library generated from the DDA data identified 1855 distinct proteins and 25 681 distinct peptides at a 1% global false discovery rate (FDR). SWATH data were queried and analyzed for 10 major cytochrome P450 (CYP) enzymes using Skyline, a targeted data extraction software. Further, correlation analysis was performed between functional activity, protein, and mRNA expression for ten CYP enzymes. Pearson correlation coefficient (r) between protein and activity for CYPs ranged from 0.314 (CYP2C19) to 0.767 (CYP2A6). A strong correlation was found between CYP3A4 and CYP3A5 abundance and activity determined using midazolam and testosterone (r > 0.600, p < 0.001). Protein-to-activity correlation was moderate (r > 0.400-0.600, p < 0.001) for CYP1A2, CYP2A6, CYP2B6, CYP2C9, and CYP2E1 and significant but poor (r < 0.400, p < 0.05) for CYP2C8, CYP2C19, and CYP2D6. The findings suggest the suitability of SWATH-MS based method as a valuable and relatively fast analytical technique for relative quantification of proteins in complex biological samples. We also show that protein abundance is a better surrogate than mRNA to predict the activity of CYP activity.

Paper-based amperometric sensor for determination of acetylcholinesterase using screen-printed graphene electrode

A simple paper-based sensor with electrochemical detection was first developed for rapid, selective, and sensitive determination of acetylcholinesterase (AChE). The screen-printed graphene electrode was used as working electrode providing sensitivity for the sensor. The amperometric detection of AChE is based on the determination of thiocholine (TCh) produced from hydrolysis of acetylthiocholine chloride (ATCh) by AChE. To detect TCh, the ATCh immobilized sheet was stacked onto the detection sheet using double adhesive tape, then samples of AChE were dropped on the back side of an ATCh immobilized sheet with only 1min of incubation time. To avoid interference, glutathione (GSH), the potential of 0.5V vs. Ag/AgCl was applied onto a graphene electrode and the current, which depends on AChE concentration, was measured. Under optimized conditions, the limit of detection (LOD) from the experiment of AChE determination was 0.1U/mL with AChE concentration in range of 0.1–15U/mL. The data correlated well with the data obtained using spectrophotometric method. The developed sensor had been successfully applied to detect AChE in blood samples.

Enzymes loaded chitosan/coconut fibre/zinc oxide nanoparticles strip for polyamine determination

Most often, the immobilized enzyme based quantification is an attractive alternative to other chromatographic, electrochemical and mass spectrometry based methods due to its specificity and simplicity. In the present study, polyamine oxidase specific for spermine and spermidine and diamine oxidase specific for putrescine, were co-immobilized onto a novel chitosan/coconut fibre/zinc oxide nanoparticles (CS/CF/nZnO) hybrid support to yield a polyamine sensing strip. The strip worked optimally at pH 7.0, temperature 25°C and 6min of incubation time. Pretty good values for kinetic constants Kmapp (6.60mM), Vmax (17.69μmol/min mg protein) and kcatapp (1987.64s−1) as well as for thermal (<50 % activity retained at 40°C), storage (half life-40days) and operational stabilities (<90 % activity retained after 20 reuses) were obtained. The strip was employed for polyamine determination in some of the locally grown fruit and vegetables and the results were found to be comparable, reliable and reproducible.

Preclinical evaluation of melanocortin-1 receptor (MC1-R) specific 68Ga- and 44Sc-labeled DOTA-NAPamide in melanoma imaging

PurposeAlpha melanocyte stimulating hormone (α-MSH) enhances melanogenesis in melanoma malignum by binding to melanocortin-1 receptors (MC1-R). Earlier studies demonstrated that alpha-MSH analog NAPamide molecule specifically binds to MC1-R receptor. Radiolabeled NAPamide is a promising radiotracer for the non-invasive detection of melanin producing melanoma tumors by Positron Emission Tomography (PET). In this present study the MC1-R selectivity of the newly developed Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using melanoma tumors. MethodsDOTA-NAPamide was labeled with Ga-68 and Sc-44 radionuclides. The MC1-R specificity of Ga-68- and Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using MC1-R positive (B16-F10) and negative (A375) melanoma cell lines. For in vivo imaging studies B16-F10 and A375 tumor-bearing mice were injected with 44Sc/68Ga-DOTA-NAPamide (in blocking studies with α-MSH) and whole body PET/MRI scans were acquired. Radiotracer uptake was expressed in terms of standardized uptake values (SUVs). Results44Sc/68Ga-labeled DOTA-NAPamide were produced with high specific activity (approx. 19 GBq/μmol) and with excellent radiochemical purity (99%<). MC1-R positive B16-F10 cells showed significantly (p≤0.01) higher in vitro radiotracer accumulation than that of receptor negative A375 melanoma cells. In animal experiments, also significantly (p≤0.01) higher Ga-68-DOTA-NAPamide (SUVmean: 0.38±0.02), and Sc-44-DOTA-NAPamide (SUVmean: 0.52±0.13) uptake was observed in subcutaneously growing B16-F10 tumors, than in receptor negative A375 tumors, where the SUVmean values of Ga-68-DOTA-NAPamide and Sc-44-DOTA-NAPamide were 0.04±0.01 and 0.07±0.01, respectively. Tumor-to-muscle (T/M SUVmean) ratios were approximately 15-fold higher in B16-F10 tumor-bearing mice, than that of A375 tumors, and this difference was also significant (p≤0.01) using both radiotracers after 60 min incubation time. ConclusionOur newly synthesized 44Sc-labeled DOTA-NAPamide probe showed excellent binding properties to melanocortin-1 receptor (MC1-R) positive melanoma cell and tumors. Due to its high specificity and sensitivity 44Sc-DOTA-NAPamide is a promising radiotracer in molecular imaging of malignant melanoma.

One-Step Facile Synthesis of Highly Magnetic and Surface Functionalized Iron Oxide Nanorods for Biomarker-Targeted Applications.

We report a one-step method for facile and sustainable synthesis of magnetic iron oxide nanorods (or IONRs) with mean lengths ranging from 25 to 50 nm and mean diameters ranging from 5 to 8 nm. The prepared IONRs are highly stable in aqueous media and can be surface functionalized for biomarker-targeted applications. This synthetic strategy involves the reaction of iron(III) acetylacetonate with polyethyleneimine in the presence of oleylamine and phenyl ether, followed by thermal decomposition. Importantly, the length and diameter as well as the aspect ratio of the prepared IONRs can be controlled by modulating the reaction parameters. We show that the resultant IONRs exhibit stronger magnetic properties compared to those of the widely used spherical iron oxide nanoparticles (IONPs) at the same iron content. The increased magnetic properties are dependent on the aspect ratio, with the magnetic saturation gradually increasing from 10 to 75 emu g-1 when increasing length of the IONRs, 5 nm in diameter, from 25 to 50 nm. The magnetic resonance imaging (MRI) contrast-enhancing effect, as measured in terms of the transverse relaxivity, r2, increased from 670.6 to 905.5 mM-1 s-1, when increasing the length from 25 to 50 nm. When applied to the immunomagnetic cell separation of the transferrin receptor (TfR)-overexpressed medulloblastoma cells using transferrin (Tf) as the targeting ligand, Tf-conjugated IONRs can capture 92 ± 3% of the targeted cells under a given condition (2.0 × 104 cells/mL, 0.2 mg Fe/mL concentration of magnetic materials, and 2.5 min of incubation time) compared to only 37 ± 2% when using the spherical IONPs, and 14 ± 2% when using commercially available magnetic beads, significantly improving the efficiency of separating the targeted cells.

Impact of shear stress on Src and focal adhesion kinase phosphorylation in fibrinogen-adherent platelets

: Shear stress alone can activate platelets resulting in a subsequent platelet aggregation, so-called 'shear-induced platelet aggregation'. In our work, we analyzed how differently elevated shear stress impacts the Src and focal adhesion kinase (FAK) activation in fibrinogen-adherent human platelets. We detected the extents of Src pY418 and FAK pY397 activations in platelets on immobilized fibrinogen and over BSA under shear conditions. Moreover, we analyzed the role of αIIbβ3 in the shear-induced platelet signaling by performing our experiments in the presence of the αIIbβ3-antagonist Abciximab. Abnormally high shear rates (5000 s) significantly increased the extent of phosphorylation of both tyrosine kinases after short (2 min) incubation time independently of the presence or absence of the integrin αIIbβ3 ligand, fibrinogen. We could see considerably greater Src activation on immobilized fibrinogen than on BSA, but the extent of FAK Y397 phosphorylation was independent on the matrix. Abciximab not only reduced the Src and FAK signaling in platelets exposed to 5000 s on immobilized fibrinogen, but in platelets exposed to 5000 s over BSA as well. Our data indicate that whereas Src activation under shear stress is dominantly ligand-dependent, FAK signaling seems to be mostly shear induced.

Gastrothylax crumenifer: ultrastructure and histopathology study of in vitro trematodicidal effect of Microlepia speluncae (L.) Moore

ABSTRACTAn in vitro incubation study was carried out to determine the efficacy of Microlepia speluncae (MS) fronds’ extract on the worm motility and viability, morphology and histopathological changes in/of Gastrothylax crumenifer. The methanolic extract of MS fronds was used for in vitro study at 1, 2, 3, 4 and 5 mg/ml concentration as test extracts. Oxyclozanide (OXY) at 1% (250 mg/25 ml) as standard control and Hedon–Fleig (H–F) salt solution as negative control. The solutions (25 ml), with 25 amphistomes each, distributed to 7 petriplates (90 mm diameter) and placed in an incubator with 5% CO2 at 37°C. The motility of control and treated flukes was observed at regular time intervals, 0, 10, 15, 30 and 60 min respectively with specific score 3, 2, 1, 0 for motility. Relative Motility assay and LC50 were determined by probit regression analysis. In vitro incubation study revealed death of all trematodes, lethal at 10 min incubation time at 5 mg/ml concentration, indicated RM value is 0, very much effective than Oxyclozanide, RM value was 0. 05. The LC50 was determined as 3.666. The worms subjected to in vitro trials were studied under Scanning Electron Microscope and Light Microscope for significant morphological changes.

Use of ionic liquids as headspace gas chromatography diluents for the analysis of residual solvents in pharmaceuticals

In this study, two ionic liquids (ILs), 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([BMIM][NTf2]) and trihexyltetradecylphosphonium bis[(trifluoromethyl)sulfonyl]imide ([P66614][NTf2]) were examined as contemporary diluents for residual solvent analysis using static headspace gas chromatography (SHS-GC) coupled with flame ionization detection (FID). ILs are a class of non-molecular solvents featuring negligible vapor pressure and high thermal stabilities. Owing to these favorable properties, ILs have potential to enable superior sensitivity and reduced interference, compared to conventional organic diluents, at high headspace incubation temperatures. By employing the [BMIM][NTf2] IL as a diluent, a 25-fold improvement in limit of detection (LOD) was observed with respect to traditional HS-GC diluents, such as N-methylpyrrolidone (NMP). The established IL-based method demonstrated LODs ranging from 5.8 parts-per-million (ppm) to 20ppm of residual solvents in drug substances. The optimization of headspace extraction conditions was performed prior to method validation. An incubation temperature of 140°C and a 15min incubation time provided the best sensitivity for the analysis. Under optimized experimental conditions, the mass of residual solvents partitioned in the headspace was higher when using [BMIM][NTf2] than NMP as a diluent. The analytical performance was demonstrated by determining the repeatability, accuracy, and linearity of the method. Linear ranges of up to two orders of magnitude were obtained for class 3 solvents. Excellent analyte recoveries were obtained in the presence of three different active pharmaceutical ingredients. Owing to its robustness, high throughput, and superior sensitivity, the HS-GC IL-based method can be used as an alternative to existing residual solvent methods.

Ultrasonic hyperactivation of cellulase immobilized on magnetic nanoparticles

In the present work, effect of low power, low frequency ultrasound on cellulase immobilized magnetic nanoparticles (cellulase@MNPs) was studied. To gain maximum activity recovery in cellulase@MNPs various parameters viz. ratio of MNPs:cellulase, concentration of glutaraldehyde and cross-linking time were optimized. The influence of ultrasonic power on cellulase@MNPs was studied. Under ultrasonic conditions at 24kHz, 6W power, and 6min of incubation time there was almost 3.6 fold increased in the catalytic activity of immobilized cellulase over the control. Results also indicated that there was improvement in pH and temperature stability of cellulase@MNPs. Furthermore, thermal deactivation energy required was more in cellulase@MNPs than that of the free cellulase. Secondary structural analysis revealed that there were conformational changes in free cellulase and cellulase@MNPs before and after sonication which might be responsible for enhanced activity after ultrasonication. Finally, the influence of ultrasound and cellulase@MNPs for biomass hydrolysis was studied.

A short report on the effect of decreased incubation time on the architectural profile of autologous conditioned serum (ACS)

If present in high enough concentrations, IL-1-Ra has the potential to inhibit Interleukin-1, the chief offender that promotes the pro-inflammatory cascade causing pain, swelling and joint dysfunction associated with osteoarthritis (OA). IL-1-Ra and growth factor levels were quantified from whole blood in this retrospective chart review investigation (n=20) using Zero and 15min incubation times respectively. The hypothesis that this process can significantly (p<0.0001) increase levels of IL-1-Ra was confirmed. Mean Arthrokinex™ induced IL-1-Ra levels reached a concentration of 13,288pg/mL and 12,809pg/mL compared to 518pg/mL at baseline, representing a 26-fold increase. Post conditioning levels of pro-inflammatories IL-1β, IL-6 and TNF α were not changed to any significant degree. The Arthrokinex™ blood conditioning process induces adequate levels of IL-1-Ra to alter the IL-1-Ra: IL-1β ratio and mitigate the inflammatory cascade, while increasing growth factors PDGF and TGF respectively.

Partial purification and characterization of amylase enzyme under solid state fermentation from Monascus sanguineus.

Amylase is an important enzyme having a varied range of industrial applications from food to cosmetics, from pharmaceutical to detergent industry, etc. The present study was carried out considering these important applications of amylase enzyme. Monascus sanguineus also has not been explored for its efficiency to produce amylase enzymes under solid state fermentation.In the present study, various substrates were screened and among them beetroot as a solid substrate has given maximum yield (0.029 U/mL). Enzyme activity was further optimized by response surface methodology (RSM) and maximum experimental yield of 0.014 U/mL was obtained at optimized conditions of pH 5, incubation temperature of 50 °C and 10 min incubation time. A MATLAB software package was used for the graphical and regression analysis of the experimented data. Enzyme kinetics was calculated with different concentrations of starch and observed Km value was 0.055 mM from linear regression analysis. The enzyme was moderately inhibited (44.7%) by NaCl and KCl (0.105 U/mL) with minimum inhibition (14.8%) observed with SDS. Molecular weight calculation and amylase confirmation in protein sample was done by SDS–PAGE and Zymography. Calculated molecular weight was 56 kDa. Alkaline amylase produced by M. sanguineus has exhibited high efficiency towards removal of stains on cloths in combination with commercial detergent (Surf excel) at 20 °C.It can be concluded that the fungus M. sanguineus is a good source of amylase production under solid state fermentation. Application of amylase produced by M. sanguineus in detergent industry was also carried out and it was proven very effective in stain removal from the fabrics.

Comparative preclinical evaluation of 68Ga-NODAGA and 68Ga-HBED-CC conjugated procainamide in melanoma imaging

Malignant melanoma is the most aggressive form of skin cancer. The early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of this disease. Previous studies have shown that benzamide derivatives (e.g. procainamide) conjugated with PET radionuclides specifically bind to melanin pigment of melanoma tumors. 68Ga chelating agents can have high influence on physiological properties of 68Ga labeled bioactive molecules, as was experienced during the application of HBED-CC on PSMA ligand. The aim of this study was to assess this concept in the case of the melanin specific procaindamide (PCA) and to compare the melanin specificity of 68Ga-labeled PCA using HBED-CC and NODAGA chelators under in vitro and in vivo conditions.Procainamide (PCA) was conjugated with HBED-CC and NODAGA chelators and was labeled with Ga-68. The melanin specificity of 68Ga-HBED-CC-PCA and 68Ga-NODAGA-PCA was investigated in vitro and in vivo using amelanotic (MELUR and A375) and melanin containing (B16-F10) melanoma cell lines. Tumor-bearing mice were prepared by subcutaneous injection of B16-F10, MELUR and A375 melanoma cells into C57BL/6 and SCID mice. 21±2days after tumor cell inoculation and 90min after intravenous injection of the 68Ga-labelledlabeled radiopharmacons whole body PET/MRI scans were performed.68Ga-NODAGA-PCA and 68Ga-HBED-CC-PCA were produced with excellent radiochemical purity (98%). In vitro experiments demonstrated that after 30 and 90min incubation time 68Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p≤0.01) higher than the 68Ga-HBED-CC-conjugated PCA accumulation in the same cell line. Furthermore, significant difference (p≤0.01 and 0.05) was found between the uptake of melanin negative and positive cell lines using 68Ga-NODAGA-PCA and 68Ga-HBED-CC-PCA. In vivo PET/MRI studies using tumor models revealed significantly (p≤0.01) higher 68Ga-NODAGA-PCA uptake (SUVmean: 0.46±0.05, SUVmax: 1.96±0.25,T/M ratio: 40.7±4.23) in B16-F10 tumors in contrast to 68Ga-HBED-CC-PCA where the SUVmean, SUVmax and T/M ratio were 0.13±0.01, 0.56±0.11 and 11.43±1.24, respectively.Melanin specific PCA conjugated with NODAGA chelator showed higher specific binding properties than conjugated with HBED-CC. The chemical properties of the bifunctional chelators used for 68Ga-labeling of PCA determine the biological behaviour of the probes. Due to the high specificity and sensitivity 68Ga-labeled PCA molecules are promising radiotracers in melanoma imaging.

Synergistic Effect of Multiple Enzymes on Apple Juice Clarification

Objectives: To clarify the apple juice enzymatically using different commercial enzymes like amylase, pectinase and cellulase at a standard concentration of 1U/ml both in isolation and in combination. To standardize the clarification conditions like temperature (40°C and 50°C) and different incubation time intervals (15 and 30 minutes). Methods/ Statistical Analysis: Apple juice was prepared from good cleaned fruits using distilled water by blending and extraction of juice. The extracted juice was filtered centrifuged and pasteurized before subjecting it to clarification. Different commercial enzymes like amylase, pectinase and cellulose were taken at a concentration of 1U/ml in sodium acetate buffer (pH 5) for clarification studies both in isolation and in combination. The clarification was measured in terms of viscosity reduction. The tested parameters for the present study were time of incubation (15-30 min), concentration of enzyme ( 1 U/ml) and temperature (40-50 °C). Findings: The apple juice was clarified using pectinase at 1U/ml in 30 min with reduction of viscosity to 1500 cP units at 40°C. When amylase and cellulase are used separately for apple juice clarification under similar enzymatic conditions there was viscosity reduction to 500 cP units at 40°C for 30 min incubation time and viscosity reduction to 600 cPunits 50° C for 30 min incubation time respectively. Pectinases in combination with amylases showed viscosity reduction up to 510 cP units and with cellulases 609 cP units at 50°C for 30 min of incubation time. Clarification of apple juice was found to be taking 30 min and there was reduction of viscosity to 400 (cP) when I U/ml concentration of amylase, pectinase and cellulase was used at 50°C in combination. Application/Improvements: The results indicated that there was a significant viscosity reduction of apple juice when enzymes were used in combination for fruit juice clarification. This can be applied for clarification of other fruit juices The study is significant as perishable and seasonal Indian apples grown in abundance can be used for commercial production of clarified juices.

In situ affinity purification of his-tagged protein A from Bacillus megaterium cultivation using recyclable superparamagnetic iron oxide nanoparticles

This paper discusses the use of recyclable functionalized nanoparticles for an improved downstream processing of recombinant products. The Gram-positive bacterium Bacillus megaterium was used to secrete recombinant protein A fused to a histidine tag into the culture supernatant in shaker flasks. Superparamagnetic iron oxide nanoparticles functionalized with 3-glycidoxypropyl-trimethoxysilane-coupled-nitrilotriacetic-acid groups (GNTA-SPION) were synthesized and added directly to the growing culture. After 10min incubation time, >85% of the product was adsorbed onto the particles. The particles were magnetically separated using handheld neodymium magnets and the product was eluted. The GNTA-SPION were successfully regenerated and reused in five consecutive cycles. In the one-step purification, the purity of the product reached >99.9% regarding protein A. A very low particle concentration of 0.5g/L was sufficient for effective product separation. Bacterial growth was not influenced negatively by this concentration. Particle analysis showed similar properties between freshly synthesized and regenerated GNTA-SPION. The overall process efficiency was however influenced by partial disintegration of particle agglomerates and thus loss of particles. The demonstration of very fast in situ product removal from growing bacterial culture combined with a very high product purity within one step shows possibilities for automated large scale purification combined with recycling of biomass.

Antioxidant response and biocompatibility of curcumin-loaded triblock copolymeric micelles

To evaluate the safety profile of cationic micelles, based on triblock copolymer poly(dimethylaminoethyl methacrylate)–poly(e-caprolactone)–poly(dimethylaminoethyl methacrylate) (PDMAEMA9– PCL70–PDMAEMA9), the effects of empty (PM) and curcumin loaded micelles (PM-Curc) on nonenzyme induced lipid peroxidation (LPO) in vitro, hemolytic activity and morphological changes in some organs after repeated intraperitoneal administration in vivo were studied. To induce LPO, rat liver microsomes were incubated with a solution of iron sulfate and ascorbinic acid (Fe2+/AA). The effect of empty PM (40 and 100 μg/ml), PM-Curc and free curcumin (both at 3.48 and 8.7 μg curcumin/ml) was assessed at 20 min incubation time. In the non-enzyme induced LPO model, the investigated substances at all concentrations significantly decreased the formation of malondialdehyde (MDA), compared to the Fe2+/AA induced LPO group. According to the results it can be concluded that curcumin alone and loaded in PM, exert significant antioxidant activity. In the biocompatibility safety studies, the mean hemolytic index for polymeric carrier was less than 2%, indicating it was non-hemolytic. The general appearance of the organ tissues from Wistar rats, treated in vivo with curcumin loaded PM was similar to that of controls, thus showing no apparent toxicity after repeated 14-days treatment.

In vitro study of trematodicidal action of Dicranopteris linearis (Burm.f.) Underw. extracts against Gastrothylax crumenifer.

The anthelmintic activity of column fractioned Dicranopteris linearis (Burm.f.) Underw. extracts was used for in vitro incubation study against adult Gastrothylax crumenifer. The qualitative analysis showed ethanol extract performed well to express 12 secondary metabolites studied except anthocyanin. Quantitative phytochemical analysis of terpenoids, phenols, flavonoids and tannins showed predominant amount of terpenoids (97.0±1.15)mg/g and tannins (30.8±0.44)mg tannic acid equivalents/gram. GC-MS analysis of fraction separated under column (Fraction V-79.5%) showed presence of QUERCETIN 7,3',4' TRIMETHOXY, a flavonoids (polyphenol derivative), PHYTOL, a diterpenoids, feren-8-ene, a triterpenoids, hexdecanoic acid, a fatty acid derivative shown to have potent anthelmintic property. 25ml of Hedon-Fleig (H-F) salt solution containing various concentrations (1, 2, 3, 4 and 5mg/ml) respectively as test extracts, Standard control Oxyclozanide @1(*)% (0.25g/25ml) and negative control H-F salt solution, distributed to 7-Petri plates in an incubator with 5% CO2 at 37°C. Twenty five amphistomes were incubated and the motility of control and drug treated flukes was observed under dissection microscope at a regular time interval of 0, 10, 15, 30min and 1hr respectively. The motility response of the parasites was categorized with specific score 3, 2, 1, 0 respectively. Relative Motility (RM) value based on mean mortality and drug concentration for analysis of Lethal Concentration (LC50) and were determined by Probit regression analysis. In vitro incubation study revealed death of all trematodes, lethal at 10min incubation time at 5mg/ml concentration, indicated RM value is 0, equivalent effective to Standard control Oxyclozanide, where RM value is 0 at 10min. Confirmative study on gross morphology, histopathology, and ultra-structural changes showed considerable effect on suckers, teguments and internal organs and were dose dependent. It is indicative that presence of terpenoids, flavonoids, tannins and phenolics which are contributing factors for anthelmintic activity, producing various degree of damage to suckers, teguments and internal organs. Hence D. linearis (Burm.f.) Underw. can be a lead for synthesis of a new trematodicidal drug as alternative for other drugs and overcome anthelmintic resistance and cost effective.

262 - Potential Involvement of Peroxymonocarbonate and SOD in the Oxidation of the Anticancer Drug Metabolite, 6-Mercaptopurine

Introduction 6-mercaptopurine (6MP), an immunosuppressive and cytotoxic metabolite of azathioprine, is used therapeutically in the treatment of many inflammatory and immunological diseases. 6MP is known to undergo metabolism through oxidation by xanthine oxidase, S-methylation via thiopurine methyltransferase, and can be metabolized to form 6-thioguanine, which is believed to be the active metabolite. At sites of inflammation, reactive oxygen species (ROS) are generated, including H2O2 that inactivates superoxide dismutase. In the presence of HCO3-, peroxymonocarbonate (HCO4–) can potentially be produced, which enhances the oxidation potential of H2O2. H2O2 can also produce CO3●– through SOD peroxidase activity. Thiols have been shown to be oxidized by HCO4– or CO3●– but no studies have investigated the effect on 6MP. Hypothesis HCO4– and CO3●– will oxidize 6-mercaptopurine at the sulfur atom, and SOD peroxidase activity will act as a catalyst. Methods and Results UV-Vis spectrometry was used at room temperature to monitor the spectral changes (200 – 500 nm) using 20 µM 6MP and other purine analogs including its parent drug, azathioprine, and related metabolites including 6-methyl-MP, 6-thioguanine, 6-thioxanthine, and 6-thiouric acid. Reactions were run in 0.1 M KPO4 buffer pH 7.4 containing 250 µM DTPA, and were initiated by the addition of 25 mM HCO3– and 1 mM H2O2. The most prominent changes were observed with 6MP and 6-thioguanine; less changes were observed with 6-thioxanthine and 6-thiouric acid. Oxidation of 6MP by H2O2 was observed in the absence of HCO3– but was significantly enhanced using 12.5 and 25 mM HCO3–. The reactions were analyzed by high resolution LC/MS and were compared with reactions that contained SOD to catalyze SOD peroxidase activity. 6MP was detected at its expected m/z (151.0086, C5H4N4S) and the major product peak found corresponded to an m/z of 182.9981 (C5H4N4O2S), representing a sulfoxide product. SOD inclusion in these reactions produced the same products but with only 30 min incubation time vs. 24 h without SOD. The reaction of CO3●– with 6-MP was confirmed by EPR and spectrometric measurements. SOD activity was significantly enhanced by 6-MP but not azathioprine and the CO3●– radicals were scavenged by only 6-MP. 6MP was oxidized by H2O2/HCO3– and treated with HEK293 and HepG2 cells. 6MP-sulfoxide was significantly less cytotoxicity to both cell lines compared to unadulterated 6MP. Conclusions Exposure of 6MP to H2O2/HCO3– with and without SOD significantly enhanced its oxidation, and led to the formation of a 6MP-sulfoxide, which loses its cytotoxic activity and consequently ameliorates SOD activity.

Enhanced pretreatment, characterization and utilization of Prosopis juliflora stem for bioethanol production

Purpose –Prosopis juliflora is a raw material for long-term sustainable production of bioethanol. The purpose of this paper is to identify the best combination of pre-treatment strategy implemented on the lignocellulosic biomass Prosopis juliflora for bioethanol production. Design/methodology/approach – Pre-treatment of lignocellulosic material was carried out using acid, alkali and sonication in order to characterize the biomass for bioethanol production. Prosopis juliflora stem was subjected to steam at reduce temperature (121°C) for one hour residence time initially. Further acid and alkali treatment was carried out individually followed by combinations of acid and sonication, alkali and sonication. Sodium hydroxide, potassium hydroxide, hydrochloric acid, sulphuric acid and nitric acid were used with 3 per cent (w/v) and 3 per cent (v/v) concentration under temperature range of 60-90°C for 60 min incubation time. Sonication under 60°C for 5 min and 40 KHz frequency was carried out. Pre-treated sample were further characterised using field emission scanning electron microscope and Fourier transform infrared spectroscopy to understand the changes in surface morphology and functional characteristics. Findings – In sono assisted acid treatment-based method, nitric acid yields better cellulose content at 70°C and removes lignin that even at increased temperatures no burning was observed. Originality/value – The paper adds to the scarce research available on the combination of auto hydrolysis coupled with sono assisted acid/alkali hydrolysis which is yet to be practiced.