Exosomes as diagnostic tools.

Exosomes have evolved into novel candidates as diagnostic tools due to their composition of proteins and nucleic acids and ability to cross hypoxic regions, the systemic circulation and blood vessel barriers. Exosomes are nano-sized extracellular vesicles that contain information from their source cells and are found in almost all body fluids. In this chapter, we have focused on basic biogenesis, contents, and functions of these unique particles, and provide a comprehensive discussion on their usefulness as novel diagnostic tools in various diseases. In addition, these unique features make them potential candidates for development of advanced therapeutics and monitoring thereof.

Exosomes: A new horizon in modern medicine.

Exosomes are a type of extracellular vesicles belonging to endocytic origin. These vesicles carry different biological cargo that play numerous physiological roles and is also indicative of different diseased state. Exosomes are considered as promising tools for therapeutic drug delivery, owing to their intrinsic features like stability, biocompatibility and a capacity of stealth. A clearer understanding of the composition, biogenesis and biology of exosomes can provide us with better insights into the pathophysiological, diagnostic, and therapeutic roles of these extracellular vesicles. In this review we have summarize existing literature regarding the production, efficacy, action mechanism, and potential therapeutic roles of exosomes in the contexts of various diseases such as cancer, renal disease, neurological disorders, cardio-vascular diseases, inflammatory diseases and some of the auto-immune diseases.

Ultrasound-assisted production of biodiesel using engineered methanol tolerant Proteus vulgaris lipase immobilized on functionalized polysulfone beads.

In the present study, Proteus vulgaris lipase (PVL) was engineered using directed evolution to increase methanol tolerance so that it would be more tolerant and efficient for harsh conditions employed in biodiesel synthesis, which is limiting their industrial use. The influence of ultrasound under different experimental conditions on the biodiesel conversion yield using methanolysis of non-edible neem oil was also emphasized. A special attention was also paid to the immobilization of lipase on Polysulfone (PS) beads and comparative studies with industrially used Burkholderia cepacia lipase. The Engineered Proteus vulgaris lipase showed >80% activity after 3 h when incubated in 50% methanol with simultaneous sonication. The lipase retained improved longevity (~70% residual activity) over wild-type PVL over repeated use.

Amelogenin-Ameloblastin Spatial Interaction around Maturing Enamel Rods.

Amelogenin and ameloblastin are 2 extracellular matrix proteins that are essential for the proper development of enamel. We recently reported that amelogenin and ameloblastin colocalized during the secretory stage of enamel formation when nucleation of enamel crystallites occurs. Direct interactions between the 2 proteins have been also demonstrated in our in vitro studies. Here, we explore interactions between their fragments during enamel maturation. We applied in vivo immunofluorescence imaging, quantitative co-localization analysis, and a new FRET (fluorescence resonance energy transfer) technique to demonstrate ameloblastin and amelogenin interaction in the maturing mouse enamel. Using immunochemical analysis of protein samples extracted from 8-d-old (P8) first molars from mice as a model for maturation-stage enamel, we identified the ~17-kDa ameloblastin (Ambn-N) and the TRAP (tyrosine-rich amelogenin peptide) fragments. We used Ambn-N18 and Ambn-M300 antibodies raised against the N-terminal and C-terminal segments of ameloblastin, as well as Amel-FL and Amel-C19 antibodies against full-length recombinant mouse amelogenin (rM179) and C-terminal amelogenin, respectively. In transverse sections, co-localization images of N-terminal fragments of amelogenin and ameloblastin around the prism boundary revealed the "fish net" pattern of the enamel matrix. Using in vivo FRET microscopy, we further demonstrated spatial interactions between amelogenin and ameloblastin N-terminal fragments. In the maturing mouse enamel, the association of these residual protein fragments created a discontinuity between enamel rods, which we suggest is important for support and maintenance of enamel rods and eventual contribution to unique enamel mechanical properties. We present data that support cooperative functions of enamel matrix proteins in mediating the structural hierarchy of enamel and that contribute to our efforts to design and develop enamel biomimetic material.

Antioxidant and antimutagenic activity of Curcuma caesia Roxb. rhizome extracts.

The rhizomes of Curcuma caesia Roxb. (zingiberacea) are traditionally used in treatment of various ailments and metabolic disorders like leukoderma, asthma, tumours, piles, bronchitis, etc. in Indian system of medicine. Considering the importance of natural products in modern phytomedicine, the antioxidant and antimutagenic activities of C. caesia Roxb. rhizome extract and its fractions were evaluated. The ethanolic fraction showed highest antioxidant activity by DPPH assay (86.91%) comparable to ascorbic acid (94.77%) with IC50 value of 418 µg/ml for EECC followed by MECC (441.90 µg/ml) > EAECC(561 µg/ml) > AECC(591 µg/ml). Based on the antioxidant activity, three of the rhizome extracts were evaluated for their antimutagenic properties against indirect acting mutagen cyclophosphamide (CP) using Salmonella typhimurium strains TA98 and TA100. The antimutagenic activity of the extracts against indirect acting mutagen cyclophosphamide in the presence of mammalian metabolic activation system was found to be significant (p < 0.01, p < 0.05). All the extracts showed similar antimutagenicity in dose dependent manner. The total phenolic content as well as reducing ability of the extracts was also determined.

Evaluation of drug interaction of glimepiride with phosphodiesterase inhibitors type V in diabetic nephropathy.

The present study investigate the drug interaction of Glimepiride (GLIM) with Sildenafil Citrate (SIL) in Diabetic Nephropathy (DN) rats. Diabetes was induced in rats by administering Streptozotacin i. e. STZ (60 mg/kg). In present investigation GLIM (0.5 mg/kg, P.O.) and SIL (2.5 mg/kg, P.O.) were given for 6 weeks after the confirmation of DN. Pharmacodynamic and kinetic parameters were estimated after 1(st) dose and at the end of 6(th) week of drug administration. There was significant (p<0.001) increase in the bioavailability of GLIM in the presence of SIL in DN after the 1(st) dose of administration of the drug as compared to GLIM treated rat, whereas at the end of 6(th) week of drug administration, there were significant (p<0.0001) decrease in the bioavailability of GLIM+SIL treated DN rats compared to GLIM treated rats. There was significant (P<0.01) reduction of blood glucose level in GLIM+SIL treated group as compare to only GLIM treated group on 1(st) dose of drug administration but after continuous treatment for next 6 weeks, GLIM treated group showed significant hypoglycemia which was found to be reduced in GLIM+SIL treated group significantly. The present study concluded that GLIM+SIL treatment reduces the hypoglycemic condition which was there for GLIM treated DN rat.

Development of health supplement probiotic in relation to physical strength and biological activity in experimental animal.

A probiotic nutritive health supplement having a healthy nutritive value was formulated to enhance body function. A 90 days study proved the formulated health supplement to improve the body weight with a maintained normal level of cholesterol and triglycerides in blood which is needful for the proper cardiac function. The supplement also maintained the blood parameters to avoid any illness in the body. In the present study, the health supplement was able to maintain the Haemoglobin level, RBC Count, WBC Count, Platelet Count in Sprague Dawley rats. It is also found that the supplement maintained the cardiac rhythm in the stressful condition and increased in the time of swimming in the force swimming test which indicates an increase in the physical strength.

Development and validation of RP-HPLC method for sildenafil citrate in rat plasma-application to pharmacokinetic studies.

Sildenafil citrate (SIL) is used in the treatment of erectile dysfunction and other chronic disorders. For the pharmacokinetic investigation of SIL we developed a simple and sensitive method for the estimation of SIL in rat plasma by reverse phase high-performance liquid chromatography (RP-HPLC). The drug samples were extracted by liquid-liquid extraction with 300 µl of acetonitrile and 5 ml of diethyl ether. Chromatographic separation was achieved on C18 column using methanol:water (85:15 v/v) as mobile phase at a flow rate of 1 ml/min and UV detection at 230 nm. The retention time of SIL was found to be 4.0 min having a separation time less than 5 min. The developed method was validated for accuracy, precision, linearity and recovery. Linearity studies were found to be acceptable over the range of 0.1-6 µg/ml. The method was successfully applied for the analysis of rat plasma sample for the application in pharmacokinetic study, drug interaction, bioavailability and bioequivalence.

Development and validation of RP-HPLC method for simultaneous estimation of glimepiride and sildenafil citrate in rat plasma-application to pharmacokinetic studies.

A simple and sensitive method was developed for simultaneous estimation of Glimepiride (GLIM) and Sildenafil citrate (SIL) in rat Plasma by reverse phase high performance liquid chromatography (RP-HPLC). The drug samples were extracted by liquid-liquid extraction with 300 µl of acetonitrile and 5 ml of diethyl ether. Chromatographic separation was achieved on C18 column using methanol: water (85:15 v/v) as mobile phase at a flow rate of 1 ml/min and UV detection at 230 nm. The retention time of GLIM and SIL was found to be 2.5 and 4.0 min respectively with total run time of 7 min. The developed method was validated for accuracy, precision, linearity and recovery. The method was linear and found to be acceptable over the range of 100-12 000 ng/ml. The method was successfully applied for the analysis of rat plasma sample for application to pharmacokinetic.

Comparative Study of Hypolipidemic Profile of Resinoids of Commiphora mukul/Commiphora wightii from Different Geographical Locations.

The antihyperlipidemic activity of resinoids of guggul (Commiphora mukul/Commiphora wightii), belonging to family Burseraceae (Genus: Commiphora), collected from different parts of India (Gujarat, Madhya Pradesh and Rajasthan), was studied on cholesterol-rich high fat diet-induced model of hyperlipidemia in rats. The resinoids of these exudates were prepared in ethyl acetate and ethyl alcohol. The physicochemical characterization of these resinoids was carried out to determine their appearance, %yield, %moisture, %ash, acid value (mg/KOH/g), saponification value (mg/KOH/g), ester value and iodine value (g/g). Antihyperlipidemic study was carried out on all resinoids in high fat diet induced model of hyperlipidemia in Wistar albino rats. The result demonstrated that the resinoids of exudates of Commiphora wightii and Commiphora mukul, collected from Gujarat and extracted in ethyl acetate, and the resinoids of exudates of Commiphora mukul, collected from Madhya Pradesh and extracted in ethyl acetate, possessed significantly higher antihyperlipidemic activity compared with other resinoids, which may be due to regional/geographical variations.

Novel pattern forming process due to the coupling of convection and phase change.

We present a novel mechanism of pattern formation behind a flat interface during directional solidification of peritectic alloys. It is shown through computational modeling that irregular oscillatory thermosolutal convection can develop in the vertical Bridgman system, even with bottom seeding and bottom cooling. The coupling of the flow oscillation near the interface with solidification leads to ordered layered structures in the solidified crystal, which agree closely with earlier experimental results.

Mycotoxins - limits and regulations.

Since early years, a need has always been felt for some control on the quality of foodstuffs. With the discovery of aflatoxins in the early sixties, health authorities in man countries have become active in establishing regulations to protect their citizens and livestock fro t potential harm caused by mycotoxins. FDA mycotox-ins-in-foods sampling program is continuing with an objective to remove those foods from interstate commerce that contain Aflatoxins "at levels judged to be of regulator significance" Aflatoxins, Fumonisin B1 and B2, Deoxynivalenol (DON) Ochratoxin A and Patulin occur in a number of food products. FDA workers were instructed to sample and analyze all products for different types of mycotoxins. All baby foods should always be analyzed for all type of mycotoxins. The limits of Aflatoxins B1,B2,! < G2, and M1 in foods and feed stuffs varies from (0-40) ppb for foods & 0-1000ppb for food); for Ochratoxin A(0-50 ppb in food and 0-1000ppb in feed); for Don (500-2000ppb in food & 5-10,000 ppb in feed); for Zearalenone (0-1000 ppb in food); for Patulin (0-50 ppb in foods), for Diacetoxyscirpenol (0-100 ppd in feed); for chetomin (0ppb I feed); for stachybotryotoxin (0ppb in feeds and for Fumonisins (0-1000 ppb in food 5000-50,000 ppb in feedstuffs).

Protective efficacy of calcium channel blockers in sulphur mustard poisoning.

The present study was designed to ascertain the in vivo protective efficacy of Ca(2+)-channel blockers against dermally applied sulphur mustard (SM). Male albino mice were exposed to 1.5 LD50 of SM (232 mg/kg) percutaneously and the control group received an equal volume of vehicle (polyethylene glycol 300). Prior to SM application, the animals were administered nifedipine and dextrose saline containing antibiotic by intraperitoneal route. The protection assessed by the mean survival time (MST) was determined by Dunnett's method. The MST was significantly increased in nifedipine treated group. The characteristic biochemical indices of SM intoxication, i.e. lipid peroxidation and reduced glutathione (GSH) were determined in liver from animals sacrificed at 24, 48 and 72 h after exposure. SM application (1 LD50) caused a reduction in GSH level which was restored in nifedipine treated group. SM-induced lipid peroxidation was also prevented by nifedipine administration. The protective effect of nifedipine may be related to its capacity of attenuating SM-induced lipid peroxidation and glutathione depletion.

Calcium modulatory properties of O,O-diphenyl O-N-cyclooctyl phosphoramidate [corrected] isolated from Ptychodiscus brevis in rat atria and smooth muscle.

Calcium modulatory activity of a marine toxin has been studied employing in vitro preparations. The toxin induced contracture in rat diaphragm was not modified by denervation, d-tubocurarine and tetrodotoxin (TTX). In contrast, varying concentrations of calcium, EGTA and ryanodine inhibited the contracture significantly. The toxin produced a series of repeating contractions in vas deferens. Experiments with TTX, adrenoceptor blockers and other agents exclude a release of neuromediators or direct stimulation of post synaptic receptors to account for the rhythmic effect in vas deferens. The dependence of rhythmicity on external Ca2+ concentration and inhibiting effect of Mn2+, ryanodine and nifedipine indicate a direct activation of voltage-sensitive calcium channel. The toxin also evoked a similar pattern of response in paced atria mediated through Ca2+ influx.

Mechanism of cardiotoxicity induced by a marine toxin isolated from Ptychodiscus brevis.

An organophosphate toxin of marine origin isolated from red tide dinoflagellate P. brevis produced a dose-dependent dual effect on rat atria, i.e. positive inotropic effect at low concentrations (2.8 x 10(-8) to 8.4 x 10(-7) M) and negative inotropic and chronotropic responses at an elevated dose (4.8 x 10(-6) to 7.2 x 10(-4) M). The negative chronotropic and inotropic responses of the toxin were potentiated with physostigmine and ouabain whereas antagonized by atropine and hemicholinium-3 pretreatments and those effects remained unaltered by isoproterenol, phenylephrine and ouabain pretreatments. The results indicate that the toxin induced negative inotropic and chronotropic effects are mediated through release of acetylcholine from the nerve endings and consequent activation of muscarinic receptor. In atria exposed to guanethidine, bretylium, propranolol and tyramine tachyphylaxis, the positive inotropic response of the toxin was not modified. However, the response was antagonized by EGTA, nifedipine, ryanodine, calcium-free ringer and potentiated with caffeine and amiloride pretreatments. The results suggest that the positive inotropic effect of the toxin is mediated through Ca2+ influx and impairment of Na+/Ca2+ exchange process.

Cardiovascular effects of an organophosphate toxin isolated from Ptychodiscus brevis.

The dose-dependent hypotensive and bradycardic effects induced by an ichthyotoxic organophosphate compound isolated from the marine dinoflagellate Ptychodiscus brevis were studied. These effects were not antagonized by atropine, but potentiated by alpha-adrenoceptor blocker and hexamethonium. The toxin abolished the vasopressor effect elicited by phenylephrine, indicating an alpha-adrenergic blocking activity. The cardiovascular depressor responses were antagonized by tetraethylammonium while blockade of cholinergic and histaminergic receptors or inhibition of prostaglandin synthesis failed to modify these effects. The results indicate that the cardiovascular depressor effects of the toxin are probably mediated through alpha-adrenergic and ganglionic blockade accompanied by modulation of potassium channel activity.

Latex agglutination test for detection of antibodies to Toxoplasma gondii.

A resurgence of interest in Toxoplasma gondii has occurred because this coccidian parasite causes lethal infections in immunologically compromised hosts and is responsible for at least 3,000 congenitally infected infants in the United States annually. Thus, rapid, specific, and inexpensive serologic tests are required for routine screening of patients, especially pregnant women. We have developed a latex agglutination test for antibodies to T. gondii which utilizes covalently coupled T. gondii antigens. When compared with an indirect immunofluorescence assay, the latex test had a sensitivity of 94% and specificity of 100%. Compared with an enzyme-linked immunosorbent assay, the latex test had 86% sensitivity and 100% specificity. When testing samples which exhibited nonspecific polar staining by the immunofluorescence assay, the enzyme-linked immunosorbent assay had a 50% false-positive rate, whereas the latex agglutination test yielded no false-positive results. Thus, the latex agglutination test provided an efficacious method for routine serological screening for antibodies to T. gondii.