Chae-Ryon Kong

Accurate Spectroscopic Calibration for Noninvasive Glucose Monitoring by Modeling the Physiological Glucose Dynamics

The physiological lag between blood and interstitial fluid (ISF) glucose is a major challenge for noninvasive glucose concentration measurements. This is a particular problem for spectroscopic techniques, which predominantly probe ISF glucose, creating inconsistencies in calibration, where blood glucose measurements are used as a reference. To overcome this problem, we present a dynamic concentration correction (DCC) scheme, based on the mass transfer of glucose between blood and ISF, to ensure consistency with the spectral measurements. The proposed formalism allows the transformation of glucose in the concentration domain, ensuring consistency with the acquired spectra in the calibration model. Taking Raman spectroscopy as a specific example, we demonstrate that the predicted glucose concentrations using the DCC-based calibration model closely match the measured glucose concentrations, while those generated with the conventional calibration methods show significantly larger deviations from the measured values. In addition, we provide an analytical formula for a previously unidentified source of limiting uncertainty arising in spectroscopic glucose monitoring from a lack of knowledge of glucose kinetics in prediction samples. A study with human volunteers undergoing glucose tolerance tests indicates that this lag uncertainty, which is comparable in magnitude to the uncertainty arising from noise and nonorthogonality in the spectral data set, can be reduced substantially by employing the DCC scheme in spectroscopic calibration.

Development of robust calibration models using support vector machines for spectroscopic monitoring of blood glucose.

Sample-to-sample variability has proven to be a major challenge in achieving calibration transfer in quantitative biological Raman spectroscopy. Multiple morphological and optical parameters, such as tissue absorption and scattering, physiological glucose dynamics and skin heterogeneity, vary significantly in a human population introducing nonanalyte specific features into the calibration model. In this paper, we show that fluctuations of such parameters in human subjects introduce curved (nonlinear) effects in the relationship between the concentrations of the analyte of interest and the mixture Raman spectra. To account for these curved effects, we propose the use of support vector machines (SVM) as a nonlinear regression method over conventional linear regression techniques such as partial least-squares (PLS). Using transcutaneous blood glucose detection as an example, we demonstrate that application of SVM enables a significant improvement (at least 30%) in cross-validation accuracy over PLS when measurements from multiple human volunteers are employed in the calibration set. Furthermore, using physical tissue models with randomized analyte concentrations and varying turbidities, we show that the fluctuations in turbidity alone causes curved effects which can only be adequately modeled using nonlinear regression techniques. The enhanced levels of accuracy obtained with the SVM based calibration models opens up avenues for prospective prediction in humans and thus for clinical translation of the technology.

Combined confocal Raman and quantitative phase microscopy system for biomedical diagnosis

We have developed a novel multimodal microscopy system that incorporates confocal Raman, confocal reflectance, and quantitative phase microscopy (QPM) into a single imaging entity. Confocal Raman microscopy provides detailed chemical information from the sample, while confocal reflectance and quantitative phase microscopy show detailed morphology. Combining these intrinsic contrast imaging modalities makes it possible to obtain quantitative morphological and chemical information without exogenous staining. For validation and characterization, we have used this multi-modal system to investigate healthy and diseased blood samples. We first show that the thickness of a healthy red blood cell (RBC) shows good correlation with its hemoglobin distribution. Further, in malaria infected RBCs, we successfully image the distribution of hemozoin (malaria pigment) inside the cell. Our observations lead us to propose morphological screening by QPM and subsequent chemical imaging by Raman for investigating blood disorders. This new approach allows monitoring cell development and cell-drug interactions with minimal perturbation of the biological system of interest.

Wavelength selection-based nonlinear calibration for transcutaneous blood glucose sensing using Raman spectroscopy

While Raman spectroscopy provides a powerful tool for noninvasive and real time diagnostics of biological samples, its translation to the clinical setting has been impeded by the lack of robustness of spectroscopic calibration models and the size and cumbersome nature of conventional laboratory Raman systems. Linear multivariate calibration models employing full spectrum analysis are often misled by spurious correlations, such as system drift and covariations among constituents. In addition, such calibration schemes are prone to overfitting, especially in the presence of external interferences that may create nonlinearities in the spectra-concentration relationship. To address both of these issues we incorporate residue error plot-based wavelength selection and nonlinear support vector regression (SVR). Wavelength selection is used to eliminate uninformative regions of the spectrum, while SVR is used to model the curved effects such as those created by tissue turbidity and temperature fluctuations. Using glucose detection in tissue phantoms as a representative example, we show that even a substantial reduction in the number of wavelengths analyzed using SVR lead to calibration models of equivalent prediction accuracy as linear full spectrum analysis. Further, with clinical datasets obtained from human subject studies, we also demonstrate the prospective applicability of the selected wavelength subsets without sacrificing prediction accuracy, which has extensive implications for calibration maintenance and transfer. Additionally, such wavelength selection could substantially reduce the collection time of serial Raman acquisition systems. Given the reduced footprint of serial Raman systems in relation to conventional dispersive Raman spectrometers, we anticipate that the incorporation of wavelength selection in such hardware designs will enhance the possibility of miniaturized clinical systems for disease diagnosis in the near future.

Multimodal spectroscopy detects features of vulnerable atherosclerotic plaque

Early detection and treatment of rupture-prone vulnerable atherosclerotic plaques is critical to reducing patient mortality associated with cardiovascular disease. The combination of reflectance, fluorescence, and Raman spectroscopy-termed multimodal spectroscopy (MMS)-provides detailed biochemical information about tissue and can detect vulnerable plaque features: thin fibrous cap (TFC), necrotic core (NC), superficial foam cells (SFC), and thrombus. Ex vivo MMS spectra are collected from 12 patients that underwent carotid endarterectomy or femoral bypass surgery. Data are collected by means of a unitary MMS optical fiber probe and a portable clinical instrument. Blinded histopathological analysis is used to assess the vulnerability of each spectrally evaluated artery lesion. Modeling of the ex vivo MMS spectra produce objective parameters that correlate with the presence of vulnerable plaque features: TFC with fluorescence parameters indicative of collagen presence; NC∕SFC with a combination of diffuse reflectance β-carotene∕ceroid absorption and the Raman spectral signature of lipids; and thrombus with its Raman signature. Using these parameters, suspected vulnerable plaques can be detected with a sensitivity of 96% and specificity of 72%. These encouraging results warrant the continued development of MMS as a catheter-based clinical diagnostic technique for early detection of vulnerable plaques.

Effect of photobleaching on calibration model development in biological Raman spectroscopy

A major challenge in performing quantitative biological studies using Raman spectroscopy lies in overcoming the influence of the dominant sample fluorescence background. Moreover, the prediction accuracy of a calibration model can be severely compromised by the quenching of the endogenous fluorophores due to the introduction of spurious correlations between analyte concentrations and fluorescence levels. Apparently, functional models can be obtained from such correlated samples, which cannot be used successfully for prospective prediction. This work investigates the deleterious effects of photobleaching on prediction accuracy of implicit calibration algorithms, particularly for transcutaneous glucose detection using Raman spectroscopy. Using numerical simulations and experiments on physical tissue models, we show that the prospective prediction error can be substantially larger when the calibration model is developed on a photobleaching correlated dataset compared to an uncorrelated one. Furthermore, we demonstrate that the application of shifted subtracted Raman spectroscopy (SSRS) reduces the prediction errors obtained with photobleaching correlated calibration datasets compared to those obtained with uncorrelated ones.

A novel non-imaging optics based Raman spectroscopy device for transdermal blood analyte measurement

Due to its high chemical specificity, Raman spectroscopy has been considered to be a promising technique for non-invasive disease diagnosis. However, during Raman excitation, less than one out of a million photons undergo spontaneous Raman scattering and such weakness in Raman scattered light often require highly efficient collection of Raman scattered light for the analysis of biological tissues. We present a novel non-imaging optics based portable Raman spectroscopy instrument designed for enhanced light collection. While the instrument was demonstrated on transdermal blood glucose measurement, it can also be used for detection of other clinically relevant blood analytes such as creatinine, urea and cholesterol, as well as other tissue diagnosis applications. For enhanced light collection, a non-imaging optical element called compound hyperbolic concentrator (CHC) converts the wide angular range of scattered photons (numerical aperture (NA) of 1.0) from the tissue into a limited range of angles accommodated by the acceptance angles of the collection system (e.g., an optical fiber with NA of 0.22). A CHC enables collimation of scattered light directions to within extremely narrow range of angles while also maintaining practical physical dimensions. Such a design allows for the development of a very efficient and compact spectroscopy system for analyzing highly scattering biological tissues. Using the CHC-based portable Raman instrument in a clinical research setting, we demonstrate successful transdermal blood glucose predictions in human subjects undergoing oral glucose tolerance tests.

Robust Spectroscopic Calibration for Transcutaneous Glucose Monitoring by Modeling of Diffusion Kinetics

Noninvasive blood glucose measurement techniques have been extensively investigated due to its important implications for diabetes management and therapeutics. NIR Raman spectroscopy has provided successful predictions of glucose at physiologically relevant concentrations in serum, whole blood and even in human volunteers [1, 2]. However, the development of a clinically accurate and robust algorithm capable of prospective prediction has proven to be challenging. In particular, correction for the presence of a physiological lag between blood and interstitial fluid (ISF) glucose has been difficult [3]. The lag time introduces systematic errors in calibration algorithms, due to the mismatch between the reference blood glucose concentrations and the acquired tissue spectra that predominantly measure ISF glucose. We propose a novel spectroscopic calibration scheme based on “dynamic concentration correction” (DCC), which is based on a two-compartment mass transfer model of blood and ISF glucose. The DCC transformations are performed iteratively in conjunction with an implicit calibration method, such as partial least squares (PLS) or principal component regression (PCR), to enable the development of an accurate and consistent regression model.

Raman Spectroscopy: An Emerging Tool for Clinical Diagnostics

U of different forms of nicotine (smoking and smokeless tobacco products) have been a major public health issue for many years. It is related with several cancers, cardiovascular disorder and many other diseases. Low dietary protein possesses a constraint on the metabolic activity and results in impaired detoxification machinery. The adverse effects of nicotine on different organs under protein restricted condition are still unclear. This study was performed to investigate the ameliorative effect of curcumin against nicotine induced changes in different tissues under protein restricted condition. Albino-rats were maintained under normal/ protein-restricted diets and subcutaneously injected with nicotine tartrate (2.5 mg/ kg body weight/day) and orally supplemented with curcumin (80 mg/kg body weight/ day) for 21 days. The effects of the drugs were monitored by comet assay, UVvisible and CD-spectroscopy, and molecular docking. Cytokine profile and gene expression studies were performed by ELISA and Real-time-PCR respectively. Nicotine binds to DNA and distorts its structure at low concentrations and induces strand breaks at high concentration (>1mM). Curcumin revives nicotine induced structural changes of DNA. Nicotine elevates IL-6, TNF-α and STAT3 expression more in protein restricted condition and enhances p65 and Bcl-2 expression more in normal condition. Over-expression of antiapoptotic protein and DNA structural alteration by nicotine in normal diet condition indicates higher chances of malignant transformation of cells, whereas extensive DNA damage and inflammatory responses in protein restricted diet indicates cell-necrosis. Curcumin effectively ameliorates nicotine induced changes in both dietary conditions. Amelioration of Curcumin of Nicotine Induced Necrosis of Different Tissues of Female Rats in Protein Restricted ConditionC officinalis, belonging to the family of Asteraceae, commonly known as English Marigold or Pot Marigold is an aromatic herb which is used in Traditional System of Medicine. It is mainly used because of its various biological activities to treat diseases like analgesic, antidiabetic and anti-inflammatory. It is also used for ingastro-intestinal, gynecological, eye disease, skin injuries and in some cases of burn. The plant is rich in many pharmaceutical active ingredients like Carotenoids, flavonoids, glycosides, steroids and sterols. Thus the present study was designed to evaluate the antidiabetic and antihyperlipidemic effect of hydroalcoholic extract of calendula officinalis (CRHAt) in alloxan induced diabetic rats. The extract was prepared by soxhlet extraction technique with a ratio of Water : Alcohol (70:30) for 36 hrs which ensured complete extraction of active constituents.Diabetes was induced by single intraperitoneal injection of alloxan (150 mg/kg) of body weight. Oral administration of alcoholic CRHAt to diabetic rats, at a dose of 100 mg/kg body weight, resulted in a significant reduction in blood glucose, urine sugar and serum lipids in alloxan diabetic rats. The extract also increases the total haemoglobin lever. The extract effect was similar to that of insulin. Thus, the investigation clearly shows that alcoholic CRHAt has both antidiabetic and antihyperlipidaemic effects. Antidiabetic and Antihyperlipidaemic Effect of Hydro-alcoholic Extract of Calencula OfficinalisN nanoparticulate delivery system encapsulating gallic acid into the nanogels has been developed through aqueous inverse miniemulsion ATRP and encapsulation of the gallic acid was carried out at three drug loading percentages i.e., 10, 20 and 30 wt% of the nanogels to study its in vitro kinetics in order to ensure its effective delivery in a controlled fashion. Nanogels synthesized through this technique have uniformly crosslinked network and degrade in reducing environment to individual polymer chain with a very narrow molecular weight distribution (Mw/Mn=1.6). The nanogels have been shown to degrade in the presence of glutathione, which is a natural reducing agent present in the body, to individual water soluble chains and are non-toxic to the cells and biocompatible. Size distribution of the nanogel was determined with and without drug loading by Dynamic Light Scattering. Release profile of the gallic acid was studied in PBS (pH7.4), and various mathematical models i.e., Zero order, First order, Higuchi, HixsonCrowell and Korsmeyer-Peppas equations were applied for initial 60% drug release. Further the antioxidant activity of the as released gallic acid i.e., at 6, 12, 24, 48 h, was determined using FRAP assay and compared with free Gallic acid. Gallic acid was successfully encapsulated into the nanogels and size of the nanogels was increased on drug loading. It can be concluded from the in vitro release study that the release of gallic acid is slow and in a controlled fashion from the nanogels, exhibiting release mechanisms explained by more than one mathematical model. Release of the gallic acid from nanogels in PBS (pH-7.4) was following anomalous behavior indicating more than one release mechanism involved. Biodegradable Nanogels as Potential Drug Delivery Carriers for Development of Target Specific Chemotherapeutic AgentsA new Reversed Phase-High Performance Liquid Chomatographic method was developed and validated for the quantitation of Amlodipine besylate and Telmisartan in pharmaceutical formulations. Determination was performed on a HPLC Binary gradient system (Agilent Technologies, 1120 Compact LC HPLC) using a Hypersil-BDS (C-18) column (5 μm, 250 mm 4.60 mm), a mobile phase containing 0.05 M potassium dihydrogen ortho phosphate: acetonitrile (60:40% v/v) in isocratic mode at a flow rate of 1.4 mL/min with UV detection at 237.0 nm. The retention time (tR) for Amlodipine besylate and Telmisartan were 5.47min and 8.53 min respectively. The response was linear in the concentration range of 1-50 μg mL-1 for both Amlo and Tel with regression coefficient (r2) 0.9987 and 0.999 respectively. The method was validated for precision, accuracy robustness and ruggedness as per ICH guidelines. The LOD and LOQ were found to be 0.03512 μg mL-1, 0.10664 μgmL-1 and 0.04663 μg mL-1, 0.14132 μg mL-1 for Amlodipine besylate and Telmisartan respectively. The mean recoveries of Amlodipine besylate and Telmisartan were found to be 99.78% and 100.02% respectively. The robustness of the developed RP-HPLC method evaluated by making deliberate variations in the method parameters such as change in flow rate [-0.1 level1.3 ml/min; 0.0 level1.4 ml/min; +0.1 level -1.50ml/min], ratio of aqueous: organic composition and pH of the mobile phase [+1:-1 level-(61:39/pH-6.41); 0:0 level -(60:40/pH-6.50);-1:+1 level (59:41/ pH-6.47)] on the retention time, tailing factor and % content indicated that the developed method was unaffected by small changes in method parameters and is robust. The ruggedness of the developed method which was also evaluated by studying the effect of parameters like different analysts and chemicals and solvents (QualigensThermo Fisher Scientific India Pvt. Ltd., Mumbai, India and Universal Labs. Mumbai, India) employed exhibited low % R.S.D values for retention time, tailing factor and % content indicating that the developed method is rugged. The specificity of the method evaluated by forced degradation studies suggest that the method could effectively separate the drugs from its degradation products and can be used for stability-indicating analysis. Due to its simplicity, high precision and accuracy, the proposed RP-HPLC method can be used for the estimation of Amlodipine besylate and Telmisartan in pharmaceutical preparations. A Validated Stability Indicating RP-HPLC Method for the Determination of Amlodipine Besylate and Telmisartan in Pharmaceutical FormulationF and environmental safety is a prime concerned of modern human society. The economy of India is mainly depending on agriculture. The green revolution achieved in India has been possible only because of the effective management of land and water resources along with inputs like fertilizers and pesticides. The increasing use of pesticides/herbicides/insecticides in recent years for achieving higher agricultural yields has posed considerable problems in general health programs. These organic toxins enter animals and human beings directly as well as indirectly through the food chain or drinking water. The high toxicity of organophosphorus neurotoxins and their large use in modern agriculture practices has generated public concerns. Short duration exposure of these pesticides can potentially create health hazards. Thus, there is a need for the detection of these pesticides at high sensitive level with fast, reliable and economically feasible analytical technique. Biosensor is an alternative tool to detect the pesticide at sensitive level with rapidly and cost effectiveness. There is also a need to develop a simple dipstick technique having field applicability and rapidity for the detection of food toxins like pesticides. Following useful system have been developed at CFTRI for the detection of pesticides and food borne pathogens/toxins. 1. Biosensor based sensitive detection techniques. 2. Application of nanoparticles for bioanalytical applications. Bioanalytical Techniques to Detect Traces of Pesticides and Toxins in the Foods and Environmental SamplesT present work describes a rapid and sensitive advance liquid chromatographic technique, ultra high-pressure liquid chromatography (UHPLC) method with UV detection to quantify antiviral drug ganciclovir (GCV) in rabbit aqueous humor. After deproteinisation with acetonitrile, gradient separation of GCV was achieved on a Waters Acquity BEH C18 (50 mm x 2.1 mm, 1.7 μm) column at 50°C. The mobile phase consisted of 0.1% trifluoroacetic acid in water (pH 3.5) and acetonitrile (95:5, v/v) at a flow rate of 0.45 mL/min. GCV analysis was performed at a wavelength of 254 nm with total run time of 3 min. Method was found to be selective, linear (r2 = 0.999), accurate (recovery, 97.0–100.2%) and precise (CV, ≤ 3.1%) in the selected concentration range of 0.1–1.0 μg/mL. Detection and quantitation limit of GCV in aqueous humor were 3.0 and 10.0 ng/mL, respectively. The method was applied to compare aqueous humor levels of GCV after single topical instillation of GCV solution, GCV nanoparticles, GCV nanocomplexes and GCV niosomal dispersions. Topical instillation of GCV-NCs (AUC0→t, 3440.7±26.2 ng.hr/mL) and GCV-NDs (AUC0→t, 3380.5±29.3 ng.hr/mL) provided approximately 5 fold increase in the relative ocular bioavailability compared with GCV solution (AUC0→t, 650.8±14.9 ng.h/ mL) and nearly 2.5 fold higher than the GCV-NPs (AUC0→t, 1350.2±18.5 ng.h/mL). The results indicate that the nanocomplexes and niosomal dispersions increases ocular bioavailability of GCV and prolong its residence time in the eye. Ocular Pharmacokinetics of Antiviral Nanoformulations by Ultra High-pressure Liquid Chromatographic MethodT control of drug abuse in athletes has become a highly specialized complex task which requires use of sophisticated testing procedures. Doping control analysis using instrumental analytical tools have been performed since the 1960s when various measures were initiated to control the misuse of performance-enhancing drugs in sports. Each major game viz. Olympics, Asian games and Commonwealth Games sees enormous advances in doping control. The analytical strategies employed for the XIX Commonwealth games testing in India is in compliance with the WADA ‘prohibited list’ which includes hundreds of substances, ranging from volatile stimulants to modified polysaccharides and glycoproteins. Apart from prohibited substances, there are few methods which are also prohibited viz. blood transfusion and other forms of blood doping. The determination of low molecular weight (700-800 Da) substances (stimulants, narcotics, anabolic agents, glucocorticosteroids, beta2-agonists, beta blockers, diuretics, antiestrogens, cannabinoids) was performed mainly by chromatographicmass spectrometric technique which is considered as the gold standard for antidoping analysis. The protein chemistry and molecular biology components (EPO, CERA, Blood transfusion, Human Growth Hormone) are analyzed by dedicated analytical techniques for the identification of high molecular masses viz. electrophoresis, luminometry etc. The presentation will focus on the various analytical strategies employed in the testing of the mega event so as to set a milestone of excellence in the field of doping control. Analytical Strategies Employed for Doping Analysis in XIX Commonwealth Games-2010, in IndiaD is the one of the most potent antitumor drug of Bone Cancer, Breast cancer. A simple and sensitive HPLC method was developed and validated for the assay of doxorubicin. The method used a column C18 (250mm*4.6mm) with a UV. Detector is 254nm wavelength. The mobile phase consists of buffer solution (Sodium Lauryl Sulphate & OrthoPhosphoric Acid): Acetonitrile. Flow rate was found 1.0ml/min. All the system suitability parameter was found according to the ICH standards. The method was extensively validated for specificity, linearity, accuracy and precision, recovery robustness. The present assay of drugs was found to be in range. Method Development and Validation of Anti-cancer Drug: DoxorubicinE to cadmium is among the main threats to human health from heavy metals. Among heavy metals cadmium and lead are the main contaminant of milk (Alonso et al., 2003, Ayar et al., 2008). The permissible limit of Cd in milk has not been defined though it is 3 ppb for mineral water as defined in CODEX STAN 193-1995 rev.2009. In the present study an electrochemical biosensor has been developed for the detection cadmium in milk. Bacillus badius whole cells, an isolate of biosensor technology lab, were used as biocomponent in the study, immobilized at the tip of carbon paste electrode. The principle of biosensor was based on the inhibition of urease activity, was measured in terms of NADPH through cyclic voltammetry at a scan rate of 100mV/s. Bacillus badius is a urease producing micro-organism; urease and Glutamate dehydrogenase of the cell were used as bienzymatic machinery in the assay. Ammonium ion produced by urease activity along with NADPH was utilized in the reaction catalyzed by Glutamate dehydrogenase. Excess of NADPH was measured through cyclic voltammetry. Inhibition of urease leads to lesser production of ammonium ion thereby increase in unutilized NADPH and therefore increases the current. The developed biosensor was applied to natural milk and spiked milk samples. A detection limit of as low as 0.5ppb has been achieved. Cadmium is showing specificity over lead there is no interference of lead. Application of whole cells in the study as bi-enzymatic machinery is making the practice cost effective. Electrochemical Biosensor for Detection of Cadmium in MilkA method was developed for the determination of the major storage lipids, wax ester and triglycerides, in the copepod Calanus finmarchicus. A variation of the Folch method was used to extract the lipid. The method was scaled down to enable the extract ion of either pooled (~l mg) or individual (~200 μg) copepods. The major lipid classes were identified using TLC and quantified using HPLC coupled with evaporative light scattering detection. Analysis of laboratory reference materials indicated that this method underestimated the minor triglyceride component, but gave a good estimate of the major wax ester component. The fatty acid and fatty alcohol composition of the C. finmarchicus were determined following transesterification of the lipid extract in methanol. Fatty acids and fatty alcohols were initially identified by comparison with authentic standard and by mass spectroscopy. Using GC with flame ionisation detection the normalised area percentage of the fatty alcohols and fatty acid m ethyl esters was deter mined simultaneously in one run for either pooled or individual copepod samples. These methods were applied to C. finmarchicus collected from the Irminger Sea, North Atlantic in 2001 and 2002. Development of an Analytical Method for the Determination of Storage Lipids in Calanus finmarchicusI of the glycosidases as well as alteration of lectin properties by chemically modified glycoconjugates can have profound effect in biology. Several C1-imino conjugates of D-galactose, D-lactose and D-ribose and C2-imino conjugates of D-glucose, where the nitrogen center was substituted by the salicylidene or naphthylidene, were synthesized and characterized. Those glyco-imino-aromatic conjugates, which are transition state analogues, exhibited 100% inhibition of glycosidases extracted from soybean and jack bean meal. Some of these conjugates exhibited IC50 values in the range of 20 to 50 μM and hence are potent inhibitors of glycosidases. The kinetic studies suggested non-competitive inhibition. Similar studies have been carried out by treating the lectins of both glucose/mannose specific (DLL-I, pea lectin, lentil lectin), galactose specific (DLL-II, PNA, SBA, moringa lectin) as well as lactose specific (unio lectin) with these glycoconjugates. Those conjugates which exhibit highest glycosidase inhibition also inhibit the agglutination of lectins and thereby modify the property of lectin accordingly. Both the experimental and computational docking studies revealed differences in the binding strengths of naphthylidene vs. salicylidene as well as galactosyl vs. lactosyl moieties present in these conjugates. The differential interactions of these glyco-conjugates have been addressed by computational docking studies to quantify the same exists between the enzyme (Figure) or lectin and the corresponding glycoconjugate. The present studies clearly supported the binding mainly through polar interactions in addition to exhibiting some nonpolar/hydrophobic ones. Interaction of Synthetic Glycoconjugates with Glycosidases and LectinsI is well known that the biological properties of bioactive compounds are closely related to their stereostructure as well as their physicochemical and chemical properties. Thus, the development of practical methods allowing for the unequivocal and reliable determination of the absolute configuration is actively researched. The high level of interest in this subject is, not withstanding the other reasons, related to the fact that the enantiomers of a particular bio-active compound may demonstrate differences of up to several orders of magnitude in their pharmaceutical effects and potency at the same receptor. The fact that one of the enantiomers is usually more active than its counterpart, which may even be toxic, necessitates the need to use enantiomerically pure products to avoid adverse effects. In this context, the chiroptical methods and the circular dichroism (CD) spectroscopy specifically, appear to be sensitive, fast, and convenient methods for the stereochemical assignment provided that the compounds studied are chiral and non-racemic. During the present lecture, the most recent results on the application of electronic circular dichroism spectroscopy in the structure elucidation of a broad variety of important bioactive compounds will be presented. The scope of the present lecture includes, among others, b-lactam antibiotics, amino acids, amino alcohols, vitamins, and carbohydrates. For the representative derivatives, the CD spectra computed applying the time-dependent density functional theory (TDDFT) will be compared to the experimental CD curves. The high sensitivity of the CD spectroscopy to the minor structural changes will be demonstrated. Structure Elucidation of Bioactive Compounds by Means of Circular Dichroism SpectroscopyT growing demand of crude drugs (raw materials and their products) by populations is forcing to develop standards for quality control and to evaluate the health climes. Much more studies in this aspect have been done on plant originated crude drugs/raw materials. Meager evaluation is available considering animal originated raw materials. Use of insect galls as medicine is not new but evaluation of such materials is unique. The ethnobotanical and Ayurvedic literature indicates that ‘Kakadshringi’ leaf galls are used for the treatment of diarrhoea. The leaf galls occurring on botanically three different plant species viz. Pistacia integrrima Stew. ex Brindis, Terminalia chebula Retz., and Garuga pinnata Roxb. are commerced in trade. These samples were standardized using pharmacognostic and phytochemical parameters to establish identification markers. To investigate the text clime, antidiarrheal activity was evaluated using animal modal. The result suggested that ethanol extract of P. integerrima and loperamide, a standard antidiarrheal drug showed significant reduction in fecal output in castor oil and magnesium sulphate induced diarrhoea and castor oil induced intraluminal fluid accumulation. Also it inhibited dose dependently (100-500 mg/kg) the intestinal propulsion of charcoal meal in normal and barium chloride induced changes in gastrointestinal tract. The ethanol extract of P. integerrima has antidiarrhoeal, antisecretory and antipropulsive activities and it may be due to their high phenolic and tannin content. Our studies indicated that P. integerrima can be equated to ‘Kakadshringi’ and its indication for the treatment of diarrhoea. Standardization and Preclinical Studies on ‘Kakadshringi’: Leaf Galls Used in Ayurvedic System of MedicineT use of stable carbon isotopes in metabolic research on humans has expanded significantly since the early 1980s. This is due to a combination of factors such as the availability of increasing variety of labeled compounds, absence of health risk from radiation and more significantly due to the development of sophisticated instrumentation and greater availability of analytical facilities. Usage of 13C labeled substrates in human studies has provided an array of information especially on the biochemistry, physiology and disease status. Some examples of this include the utilization of 13C-octanoate in the Gastric Mobility Breath Test, 13C-Methionine in the methionine Breath Test for measuring hepatic mitochondrial function, and 13C-Phenylalanine in 13C-Phenylalanine Breath Test. While simpler 13C substrates such as sugars, amino acids and fatty acids are readily commercially available, proteins and carbohydrates generally are not. One can obtain these by labeling photosynthetic organisms with 13CO2 during photosynthesis and then isolating the compound of interest. One can also take advantage of the naturally enriched substrates for metabolic studies by a prudent selection of these material from plants following different photosynthetic pathways. Most of the human diet is derived from food items from plants following the C3 photosynthetic pathway which display more negative carbon-13 signature than the plants that follow C4 type of photosynthetic metabolism. Thus by a simple and imaginative manipulation of the diet by mixing material obtained from both C3 and C4 plants, one could obtain a significant physiological information which might lead to the synthesis of newer pharmacological compounds. Carbon Isotope Ratio Mass Spectrometric Studies in MedicineM compounds are ever interesting to study their antitumor activity. This talk deals with the syntheses, characterization and structure determination of a Cu (II)-complex of anthtracenyl terpyridine and its plasmid cleavage, and cytotoxicity towards different cancer cell lines. The plasmid cleavage studies were carried out in presence of 1 in dark, under UV light and under visible light. While all the three forms of the plasmid DNA are seen when carried out under visible light, only closed and nicked circular forms were seen under UV light. In dark the plasmid cleavage was spontaneous. 1 showed remarkable antiproliferative activity for cancer cell lines, viz., cervical (HeLa, SiHa, CaSki), breast (MCF–7), liver (HepG2), and lung (H1299). The IC50 values are in the range of 0.8 to 6.3 μM, which is far superior as compared to the platin-drugs. Incubation of cells with 1 results in granular structures only with the HPV infected ones and not with others as studied by phase contrast and fluorescence microscopy (Figure). The role of HPV has been further confirmed by transfecting the MCF-7 cells with E6. To our knowledge this is the first copper complex that causes the cell death by interacting with HPV viral protein. We have also shown antiproliferative activity of Cu(II)-complexes of glycoconjugates. Therefore, our current focus is to modify the anthracenyl terpyridine ligand with some glycosylation and/or by attaching some cellular receptors, so that cell targeting can be achieved. Results of all these will be discussed. Antiproliferative and Anticancer Activity of Copper Complexes that Works without Chemical or hn as StimulantT 2 diabetes is reaching epidemic proportions worldwide, with an estimated 140 million people suffering from it and current projections suggest that this figure is set to increase to 300 million by 2025. Type 2 insulin resistant diabetes mellitus accounts for 90-95% of all diabetes. Diabetes is characterized by impaired insulin secretion from pancreatic β-cells, insulin resistance or both. Moreover, insulin resistance syndrome is responsible for the excess of cardiovascular disease. Majority of type-2 diabetic patients can be treated with agents that reduce hepatic glucose production (glucagon antagonist), reduce glucose absorption from gastro intestinal track GIT, stimulate β-cell functions (insulin secretagogues) or with agents that enhance the tissue sensitivity of the patients towards insulin (insulin sensitizes). Older antidiabetic agents such as sulfonylureas, and insulin are more effective than lifestyle modification in reducing microvascular complications of type-2 diabetes, but overall do not reduce cardiovascular risk. Metformin or thiazolidinedione used in type-2 diabetes also reduce cardiovascular risk. The drugs presently used to treat type-2 diabetes include α-glucosidase inhibitors, insulin sensitizers, insulin secretagogues and KATP channel blockers. However, almost half of type-2 diabetic subjects lose their response to these agents over a period of time and thereby require insulin therapy. Problem with current treatment necessitates new therapies to treat type-2 diabetes. In this regard glucagon like peptide 1 (GLP-1) agonist which promote glucose dependent insulin secretion in the pancreas and glucagon receptor antagonist, which inhibit hepatic glucose production by inhibiting glycogenolysis and gluconeogenesis, were found to be therapeutically potential. But native or synthetic GLP-1 peptidase are rapidly metabolized (they have very short half life) by proteolytic enzymers, such as dipeptidyl peptidase 4 (DPP-4) into inactive metabolite, thereby limiting the use of GLP-1 as a drug. Thus because of multiple benefits of GLP-1 augmentation, DPP-4 inhibition has been recognized as a mechanistic approach of potential value in the treatment of type-2 diabetes. Various aspects of DPP-4 inhibitors as antidiabetic agents will be discussed. DPP-4 Inhibitors as Antidiabetic AgentsN tropical diseases represent one of the most serious burdens to public health. Many of these diseases can be treated cost-effectively, but most of them could not attract the attention of policy makers associated with global health policy formulations. The 13 parasitic and bacterial infections known as the neglected tropical diseases include three soil-transmitted helminth infections (ascariasis, hookworm infection, and trichuriasis), lymphatic filariasis, onchocerciasis, dracunculiasis, schistosomiasis, Chagas’ disease, human African trypanosomiasis, leishmaniasis, Buruli ulcer, leprosy, and trachoma. An expanded list could include dengue fever, Japanese Encephalitis, Chikungunya, treponematoses, leptospirosis, strongyloidiasis, foodborne trematodiases, neurocysticercosis, and scabies, as well as other tropical infections. Polyparasitism has become very common rather than the exception in many under developed and developing countries. It has been reported that a large number of individuals harbor three or more parasites in remote areas of Sub-Saharan Africa, due to lack of adequate facilities of health and hygiene. Coinfection with malaria and HIV has recently been reported as a source of increased severity of both of these diseases in sub-Saharan Africa. Scientific literature focused on co-infection with diverse combinations of helminths, HIV, malaria, and tuberculosis is growing tremendously. Neglected tropical diseases have been ignored for a long time due to the negligence of health policymakers at national, regional, and global levels. During the past few years, several research and development agencies have shown their interest and started supporting the programs related to control of neglected tropical diseases. There is a need of concerted effort to face the challenges associated with diversity of disease control approaches and health policy structures-both nationally and internationally in controlling neglected tropical diseases. Neglected Tropical Disease Management: Problems and ProspectsT author focus on research topics related to synchrotron-based tomography, mainly on medical, biomedical, biological, and environmental science imaging with synchrotron X-rays. Also highlight on nanoscience by utilizing the nanosized high energy photon beam with light source / accelerator facilities to investigate the properties of novel biomaterials and nanomaterials and imaging tools for medical, biomedical, and environmental science imaging research. New and Improved Tools: Light source / accelerator facilities will provide unprecedented capabilities for coherence-sensitive approaches, including scanning microscopes and microprobes. This will reshape the technical choices one would make compared to other facilities, so that whole-cell tomography would be done with a tenfold reduction in radiation dose, and chemical state mapping and trace element mapping would be done with improved sensitivity, spatial resolution, and speed. Extensive and innovative investigations related to biomaterials and nanomaterials with nanosized high energy photon beam are valuable. Multi-Technique Integration: Because scientific questions are often not completely answered by just one technique, an integrated suite of beamlines ranging from infrared, to soft and hard X-ray, should be developed with common sample preparation facilities and maximum compatibility of sample handling and mounting schemes. Cross-Disciplinary Approaches: A significant part of environmental science today addresses questions of the role of bacterial exudates and organic coatings on metal and radionuclide transport and reactivity in hydrated systems, as well as the health effects of contaminants. The overlap between this area of environmental science and bioimaging involves both scientific insight and technical approaches, so that this overlap should be embraced. Bioimaging Using Micro-Ct, Dei-Ct and X-Ray Microprobe with Synchrotron X-RaysB applications of lasers and laser spectroscopy are changing the face of medicine as it is currently practiced. Spectroscopy is a promising means of extracting biochemical and morphological information from tissue that is relevant to disease progression and diagnosis. In particular, Raman spectroscopy is a powerful tool for non-invasive and real time diagnosis due to its exquisite molecular specificity and lack of sample preparation requirements. Raman spectroscopy, which measures the molecular vibrations of a sample, is currently being used to study atherosclerosis, measure blood analytes, and detect dysplasia and cancer in various tissues including the breast, cervix, prostate, and skin. In this talk, we present our results on quantitative biological spectroscopy for non-invasive blood analyte detection. Our work in this area is primarily motivated by the necessity for accurate and frequent measurement of blood glucose levels, which is most commonly achieved by withdrawal of blood. Given the inconvenience and invasiveness of this procedure, a non-invasive method would greatly benefit the increasing number of diabetics. Our laboratory has successfully demonstrated the ability to measure glucose, urea and other blood analytes in serum, whole blood and individual human volunteers. In addition, we present our results for turbidity correction and suppression of tissue autofluorescence in biological Raman spectroscopy. We show that correction for these non-analyte specific variances provides a clinically accurate and robust calibration algorithm that can be used for prospective prediction in human population. Finally, we discuss our plans for miniaturization of the device for point of care and commercial applications. Raman Spectroscopy: An Emerging Tool for Clinical DiagnosticsT pharmaceutical sector represents a significant career opportunity for students in the life sciences. However, given the interdisciplinary nature of the field, it is imperative that the following aspects relevant to the sector be incorporated in curricula to the extent possible. (a) General concepts – the scientific method and hypothesis testing. (b) Specific scientific topics from physics, chemistry and mathematics. (c) Laboratory skills – intensive hands-on training, exposure to international regulatory requirements. (d) Public engagement and outreach – communication skills, regulations, ethics and scientific integrity. Using this framework, educators can identify how their own degree programs could potentially interface with the pharmaceutical sciences and orient the curriculum accordingly. Historical materials have much utility in illustrating general concepts and promoting discussions on topics related to development of a field of study. The reliance of modern biology on computers and instrumentation necessitates the incorporation of appropriate topics from physics, chemistry and mathematics. Laboratory work needs to be intensified, and formal exposure to international regimes such as GLP (good laboratory practices) would enhance the utility of a program, besides improving operations in academic labs. Finally, the inclusion of a formal component of public engagement and outreach will equip students to explore non-traditional careers away from bench science. These core principles will enable students from other life sciences streams, not just pharmacy proper, to fruitfully and creatively explore a career in the pharmaceutical industry. Enhancing Students’ Employability in the Pharmaceutical Industry Through Targeted Instructional InputsT enhance the skin permeability of a drug, the barrier function of the skin must be overcome at least temporarily. The barrier function of stratum corneum –the rate limiting membrane for skin permeation, depends upon the quality and quantity of its constituent lipids and a decrease in their concentration affects its barrier properties. In general, barrier disruption is followed by quick recovery responses. In the metabolic approach, permeation rate is enhanced by delaying this natural recovery processes by application of chemicals/drugs that interfere with the skin metabolism. However, to use this strategy, one has to have a clear understanding of the constituents of the skin as well as the mechanism of Skin homeostasis. The present article discusses some important aspects related to it. 1) the liquid crystalline nature of stratum corneum (the cholesterol and ceramides) 2) the biophysical aspects of the barrier lipids, 3) the sequence of events at stratum granulosum-stratum corneum interface, 4) role of different enzymes/ drugs (HMGCoA Reductase inhibitors) that interfere with barrier recovery and thereby enhance permeation rate of drugs. Important research on this aspect is also analyzed along with the advantages and limitations of the strategy. Enhancement of Skin Permeation: the Metabolic ApproachF design tool was applied for development of isocratic reversed-phase stability indicating HPLC method for the analysis of Mycophenolate mofetil (MMF), its degradation products Mycophenolic acid (MPA) and degradation products (DP3). Separation was achieved on a Symmetry C18 (250 mm × 4.6 mm, 5.0 μ) column using a Methanol: acetate buffer (75:25 v/v), pH 6.0 (adjusted with acetic acid), at 0.5 ml flow rate, column maintained at 55 0C and data was integrated at 251 nm. MMF was subjected to hydrolysis, oxidation, heat degradation, etc. under all these conditions degraded products were well separated. The method validation characteristics included accuracy, precision, linearity, range, specificity, LOD and LOQ. Robustness testing was conducted to evaluate the effect of minor changes to the chromatographic conditions and to establish appropriate system suitability parameters. The proposed method was used to investigate kinetics of acid, alkali hydrolysis and oxidation process. Major degradation product MPA and DP3 were isolated and quantitated. Characterization of MPA by NMR and LC-MS/MS and other degraded products by LC-MS/MS was attempted successfully. The method was used successfully for the quality assessment of three MMF drug products and its acid, alkali and oxidative degradation kinetics study. A simple and efficient stability indicating reverse-phase HPLC method was developed and was found to be accurate, precise and linear across the analytical range and is reported for the first time. The method is simple, fast, sensitive and specific for the determination and quantification of MMF, MPA and DP3. Development and Validation of Stability Indicating LcPda Method for Mycophenolate Mofetil (Mmf) in Presence of its Impurities and Major Degradation Product Mycophenolic Acid (Mpa) Using Factorial Design Tool and Use of Mass SpectroscopyB resistance towards to common antibiotics, results development of antibacterial metallopharmaceuticals (silver nanoparticles) (AgNP). AgNPs of different shapes were synthesized by solution phase routes, and their interactions with E.coli were studied. The antibacterial activity of silver compounds loaded on porous host matrices are evaluated under short contact time where in the bacterial load was exposed not directly to the metal-loaded material but to distilled water pretreated with them, was mainly attributed to generation of reactive oxygen species (ROS). In this work we first investigated the shape dependence of the antibacterial activity of silver nanoparticles. We also developed highly antibacterial porous carbon matrices supporting nano-silver by simple and cost effective way. EFTEM micrographs of the bacterial cells showed considerable changes in the cell membranes upon AgNP-treatment.T study describes a fast and efficient method for radiolabeling of etodolac with iodine-125, where both chloramine-T (CAT) and iodogen were used as oxidizing agents. The labeling reaction was carried out via electrophilic substitution of hydrogen atom with the iodonium atom I+. The labeling yield was found to be influenced by different factors such as drug concentration, pH of the reaction mixtures, different oxidizing agents, reaction time, temperature and different organic media. The radiochemical yield was determined by TLC system using methylene chloride: ethyl acetate (3:7 v/v) as a developing solvent and by electrophoresis using cellulose acetate moistened with 0.02M phosphate buffer pH 7. The maximum radiochemical yield of 125I-etodolac (87.7 %) was obtained. Labeled etodolac shows a good localization in inflamed muscle. It excretes mainly via kidney and to some via liver. Comparative Study between Chloramine-T and Iodogen to Prepare Radio Iodinated Etodolac for Imaging of InflammationT International Diabetes Federation estimates that 285 million people around the world have diabetes. This total is expected to rise to 438 million within 20 years. Each year a further 7 million people develop diabetes. Around 90 % of the diabetes patients are found to suffer from type 2 diabetes mellitus. Many patients suffering from type 2 diabetes require treatment with more than one antihyperglycemic drug to achieve optimal glycemic control. The combination of metformin hydrochloride – Biguanides with any one of the Sulphonylureas glipizde, gliclazide, glibenclamide, glimperide or with Alpha glucosidase inhibitors voglibose can present daunting challenges to analytical scientist during HPLC analysis. This talk will drill into the details of method development and discuss the common problems encountered as in case of metformin & sulphonyl urea – vast differences in their polarity, pka, makes extraction from the biological fluids very difficult which may lead to low recovery and low sensitivity. Differences based on their protein binding characteristics sulphonyl urea strongly bound to proteins lead to low recovery. Strategies to optimize the mobile phase, extraction procedures in biological fluids, to increase the recovery by disrupting drugprotein binding will be highlighted. Challenges to improve the detection of low UV absorbing component as in the case of voglibose will be dealt. These methods are inevitable to monitor a population of diabetic patients who take several diabetic medications without changing HPLC colums. Strategies for Developing Bioanalytical Method for Certain Oral AntidiabeticsT aim of this study was to characterize two ceramide subclasses, CER[NP] and CER[EOS], of human stratum corneum (SC) and to identify the chemical structures of their subspecies. High performance thin layer chromatography (HPTLC) and normal phase high performance liquid chromatography (NP-HPLC) were used for the separation of CER fractions of interest, whereas nanoelectrospray tandem mass spectrometry was applied to identify the chemical structures in detail. Thus, CER[EOS] fragmentation revealed that in addition to linoleic acid other esterified fatty acids occur in the ω-hydroxylated-position (part E). Of particular interest is the identification of a 17:2 fatty acid located in this part of the molecule.A gaining relevant information on the harmful effects of a compound, the levels for its safe usage or the degree of its safeness is established. A wide range of toxicological studies are being undertaken as per national and International guidelines. Out of which the toxicological studies on Central Nervous System becoming the area of attraction. The present study highlights the possible CNS toxicities with a drug entity and their prevention and treatment. Fluoroquinolones can induce a wide range of serious adverse psychiatric effects showing depressant activity on the CNS. Its concomitant use of NSAIDs may increase seizure risk. Neurologic complications of cancer therapy are an increasingly important concern in patient management. Prompt recognition of these problems and their causes will have an impact on patient care in all areas of oncology. It is not surprising that clinical trails evaluating Biological Response Modifiers have also demonstrated that CNS toxicity is very common. The role of amifostine (WR-2721) in ameliorating radiationinduced central nervous system (CNS) toxicity is effective. Greater awareness of severe and complex CNS neurotoxicity even with low dose Cyclosporin A treatment in rheumatoid arthritis is of the utmost importance and so with Ciprofloxacin. Central nervous system (CNS) toxicity of tricyclic antidepressants (TCAs) is serious, costly, frequent, and difficult to diagnose early in its course. CNS lidocaine toxicity is biphasic and the most common cause for it is dosing error. This study concludes that the CNS toxicological studies of any new chemical entity will lead to its better use for the ailment of different diseases of mankind. Additional studies are warranted to investigate the protective effect with differing regimens of administration, more clinically relevant fractionation regimens and longer follow-up. A New Frontier on Toxicological Studies on Central Nervous SystemI will talk about two complementary spectroscopic techniques, namely Laser Induced Breakdown Spectroscopy (LIBS) and Raman Spectroscopy for the pharmaceutical applications. While Raman spectroscopy which is widely used in the industry to get the molecular information, LIBS is a relatively new technique that gives atomic information. It is an atomic emission based technique involving the interaction of a target with an intense laser pulse which generates plasma. The spectral analysis of the fluorescence emission from the cooling plasma plume contains specific signature of atoms of the material under investigation. The amount of sample and time needed to prepare the sample are far less compared to any other technique used for the elemental analysis such as Absorption, Fluorescence, FT-IR, Raman or Cavity Ring down Spectroscopy. LIBS is particularly attractive for the detection due to its stand-off detection capability, requirement of microscopic quantities of material, and rapid detection. The detection times are very fast which is very ideally suited for the industrial applications. LIBS and Raman studies on pharmaceutical samples have been reported in a recent study. A combined system capable of recording both the Raman and the LIBS spectra can give complementary information. Combined Laser Induced Breakdown Spectroscopy and Raman Spectroscopy for Pharmaceutical ApplicationsT methods of optics of scattering medium were developed and used for deciding some tasks linked with diagnostics of blood and erythrocytes. With reference to problems of an optical coherent tomography, the influence of glucose addition to specimens of diluted blood on the transmission spectrums have been investigated by experimentally and by simulation. Results of numerical modeling have shown, that the essential optical cleaning of blood can be achieved, but only at the concentrations of glucose, considerably exceeding physiological norm. Our experimental data for erythrocyte suspended in mixtures of blood plasma with glucose did not confirm blood optical clearing by using of glucose as the immersion liquid: transmission is decreasing for all wavelengths with the addition of glucose solution, as a result of erythrocyte interaction with an environment. At the glucose concentration grater than 20-25%, the spectral transmissions were increased as result of erythrocyte hemolysis. A model of changes in erythrocyte sizes at osmotic imbalance in a hypotonic medium is designed under the assumption of free diffusion of water throught erythrocyte membranes. The mechanisms of the formation of the scattering properties of erythrocyte suspensions depending on a degree of osmotic balance infringement are studied with accounting for erythrocyte swelling and its hemolysis. Satisfactory agreement between computational and experimental data certifies the adequacy of the proposed model. The method differs by simplicity and can be put into practice with the help of standard spectrophotometers. Suggested methods, based on measurement of characteristics of scattered light, can be used for research of influence of druges on the viscous-elastic properties of erythrocyte membranes and on processes of transmembrane water transferring and also erythrocyte osmotic resistance. Methods of Light Scattering for Investigation of the Structurallyfunctional Properties of ErythrocytesC is a group of diseases that arise from uncontrolled growth, spread of an abnormal cell and can result in death. The inefficiency (Tong et al, 1995) to treat several distinct classes of tumours led researchers to source for potential natural-based therapeutic compounds. Many Botanists (Etta, 2008) believed that the extract of P. niruri, (30 40 cm in height) originated from India by late of 1980s showed pharmacological, clinical efficacy against viral Hepatitis B (Padma et al., 1999; Paranjpe, 2001; Blumberg et al., 1990; Venkateswaran et al., 1987) antibacterial activity (Mazumder et al., 2006); Kloucek et al., 2005), anti-hepatotoxic or liver-protecting activity (Houghton et al., 1996; Rajeshkumar et al., 2000; Jeena et al., 1999), as well as anti-tumor and anti-carcinogenic properties (Rajeshkumar et al., 2001). In addition, it also exhibits hypoglycaemia properties (Mazunder et al., 2005; Raphael et al., 2002).H adult hemoglobin (HbA) has four subunits, namely, two-a subunits and two b-subunits. X-ray crystallographic analysis have indicated that there are two distinct quaternary structures, namely, the deoxy state, represented by the tense (T), low-affinity structure, and oxy state, represented by the relaxed (R), high-affinity structure. A characteristic spectral change of HbA in the near-UV CD occurs: from a small positive band in the oxy-R form to a negative CD band with a distinct peak at 287nm in the deoxy-T form. This negative CD band of deoxyHbA known as T-state marker has been supposed to derive from the changes of Tyr and Trp residues at the α1β2 subunit interface. To identify the aromatic residue responsible for the CD band, we have synthesized five recombinant Hbs in E. coli in which non aromatic residue is substituted for Tyr or Trp residue; rHb (α14Trp→Leu), rHb (β15Trp→Leu), rHb (β37Trp→His), rHb (α42Tyr→Ser), and rHb (β145Tyr→Thr). We examined the near-UV CD spectra of these rHbs and a natural mutant, Hb Rouen (α140Tyr→His). The CD spectra of individual aromatic residue were extracted from the difference between Hb A and each mutant. We concluded that changes in CD bands arising fromβ37Trp, α140Tyr, β145Tyr and α42Tyr residues contributed to the appearance of the negative CD bands at 287nm. To examine different signals of CD band among aromatic residues, the effects of environments on CD spectra were examined using model compounds of Tyr and Trp dissolved in various solvants. Quaternary Structure Analysis of Human Hemoglobin by a NearUV CD SpectroscopyA drug discovery cycle, to identify, to optimize and eventually take a compound to the market is generally along process (approx 12–15 years) and is very expensive (approx

A multimodal spectroscopy system for real-time disease diagnosis

500million R&D expense). The enormous pressure that pharmaceutical and biotech companies are facing, has created the need to apply all available techniques to decrease attrition rates, costs and the time to market. Pharmacophore identification is one such technique. As case study example of drug design against Cycloxygenase (COX) enzyme is being discussed here using Catalyst Software. COX enzyme catalyze the biosynthesis of prostaglandins and thromboxane from arachidonic acid (AA). In the present paper we summarize, the development of hypotheses of a dataset comprising six chemically diverse series of known inhibitors for COX-2, by using the Catalyst/ Hypogen module. The most predictive pharmacophore model, consisting of four features, namely, one hydrogen bond donors, one hydrogen bond acceptor, one hydrophobic aliphatic and one ring aromatic feature, had a correlation (r) of 0.954 and a root mean square deviation of 0.894. The model was validated on a test set consisting of six different series of structurally diverse 27 compounds and performed well in correctly classifying active and inactive molecules correctly. The resultant best hypothesis was used to screen databases viz. NCI and maybridge to produce hit compounds. 264 hits were obtained which were arranged according to their fit value in 8 categories and subjected to secondary screening using Lipnski’s rule of five. The resultant compounds were then docked into the COX-2 binding site to study the ligand – protein interaction and binding energies were evaluated in terms of LUDI scores. Several new structural scaffolds have been obtained as a result of the virtual screening. The Compounds were screened in in-vitro assay and novel scaffolds were identified as lead compounds for development of COX-2 inhibitors. Drug Design And Development Using Pharmacophore Modeling And Virtual ScreeningI is becoming increasingly imperative to asses the teratogenecity of new chemical entities (NCEs) and drugs before they enter the market. Ethical limitation inhibit such studies in pregnant women. The current study attempts to create a 3-Dimentional in vitro platform that mimics human peri-implantation embryonic development using embryoid bodies (EBs) derived from human embryonic stem cells (hESCs). A layer of human endometrial cell line (CRL4003) was coated on layers of extracellular matrix proteins eg; fibronectin and collagen on low melting agarose (0.5%), to create a bio-mimetic platform similar to the implantation site. A time course study on EBs was done over a period of 20 days, to understand the induction of germ lineages. The expression profile of the ectoderm, endoderm, mesoderm and trophectoderm lineage markers, such as beta III-tubulin, GATA4, BMP2, Brachury hANP, cTnT, ABCG2, GATA2, BMP4, HAND1 and beta-hCG, were studied, by SQ and QRT-PCR and immunofluorescence. The lineage composition of smooth surfaced EBs (SSEBs) at day-6 closely resembled the human periimplantation blastocyst. Inhibition in the induction of any of the lineages caused by exposure to a NCE was confirmed by lack of biomarker expression and by loss of the ability of the SSEBs to functionally differentiate into particular lineages. Using proven embryotoxic componds we demonstrated that the model closely mimiced peri-impantation development and was sensitive even at very low doses. This model is adoptable to a 96 well plate format for high through put screening of NCEs. Human Embryonic Stem Cell Based High Throughput Screening Platform: A Biomarkers Based Approach for Predicting Human Developmental ToxicityT is a greater cause of death and disability than any other single disease. India is the 3rd largest tobacco producing country after USA and China. Use of different forms of tobacco is increasing day by day. Nicotine, the acute acting pharmacological agents of tobacco, plays the major role for drug of addiction. Elucidation of the complex effect of nicotine has worldwide implications. Nutritional status alters the actions, potencies and detoxification of toxicants. Any strategy through natural diet that prevents or slows the progression and severity of nicotine toxicity has a significant health impact. Nicotine induces oxidative stress. In search for natural antioxidants, present study was undertaken to evaluate the antioxidant efficacy of sesame lignans and conjugated linolenic acid, in nicotine treated rats. Experiments were conducted on male albino rats (120 – 130 g body weight) by injecting nicotine tartrate (3.5 mg/kg body wt. /day for 15 days) subcutaneously and thereby supplementing sesame lignans (0.2 g/100 g diet) and conjugated linolenic acid (0.2 g/100 g diet) orally to them simultaneously. Results showed that serum and liver lipid profile, activities of antioxidant enzymes, lipid peroxidation altered significantly due to oxidative stress generated by nicotine. Supplementation of sesame lignans and conjugated linolenic acid attenuates all the altered parameters by their antioxidant property. Results also showed that antioxidant activity of conjugated linolenic acid was more prominent than that of sesame lignans. Comparative Study of the Antioxidative Activity of Sesame Lignans and Conjugated Linolenic Acid Against Nicotine Toxicity