Colony PCR

Transformation

PCR amplification

2D QSAR

Active site analysis

Inhibitors Dataset

Assay for Lipid peroxidase(LPO)

Cytomorphological analysis

Growth kinetic studies

Reducing power assay

TEM-EDAX analysis

Qualitative analysis of compounds

The table 6.1 gives the mixing probabilities and the associated parametricvalues fork(number of components) = 2,3, and 4. It may be noted thatthe Log likelihood value is smaller fork= 4 (the results fork= 5 , 6 etc.are not better than that fork= 4 and hence are not given here). The fourcomponents Poisson Mixture model is given in table 6.2. It may be notedthat 58% of wards may have higher incidence/relative risk and the remainingwards have lesser/lower incidence for the Cancer disease. We computed theposterior probability for each component for each ward (see table 6.3). Eachward is assigned to a particular component so that the posterior probability islarger. These results are also given in table 6.3 Finally we present Choroplethmaps based on those results

The Posterior Probability of Mixing Dis-tribution

Algorithm

EM Procedure

Poisson Mixture

Data Sources

Poisson Mixtures Distribution

Poisson Model

Haemolymph protein profile

Helicoverpa armiger

Spodoptera litura

Gut enzyme profile

Haemolymph protein profiling

Lactate dehydrogenase

VS preparation

Utilization of VS by the prey

Influence of prey on VS yield

Quantification of protein of VS yielded in the prey deprivation

Survival (SR) and venom milking rate (VMR

Venomous saliva optimization

Statistical analysi

Quantification of VS for protein

Venomous saliva utilization

Prey type

Starvation and collection method

Venomous saliva optimization

Insects Collection

VENOMOUS SALIVA: OPTIMIZATION AND UTILIZATION

Mandibular stylet

Head and stylet preparation

Micronuclei test

Malic dehydrogenase

Alkaline Phosphatase

Glycogen content

Analysis of Physico-chemical parameters of the water sampled

Increments in biomass:

Residual nutrients

Chlorophyll content in waters:

Growth maximum values

Rates of growth (OD678/day

Growth

Experimental Set up

BIOMASS PRODUCTION: ROLE OF N-P CONCENTRATION AND RATIOS

Soil pH and electrical conductivity (EC)

Experiment 2: Assessing the impacts of elevated temperature and N levels on yield and nutrient uptake in rice

Experiment 1: Assessing the impacts of elevated CO2and N levels on yield and nutrient uptake in rice

Experimental Design and Treatments

Temperature Gradient Tunnels (TGT)

Yield and its component traits:

Differences among cultivars for yield, biomass and HI

Kernel hardness

SDS-sedimentation test

Estimation of total N% of wheat grainsand straw

End use Quality parameters

Chlorophyllcontent

Root length (cm) and Root weight (mg)

Coleoptile length(cm)

Stomata / cm2

Leaf area index (LAI)

Physiological parameters

Laboratory observations

Days to physiological maturity

Static and shaken condition

Screening for proteolytic activity

Vaccination improved the Chemokine receptor CCR1, CCR3, CCR9 and Toll like receptor TLR2, TLR4 and TLR9 expression in HBsAgpositive newborns compared to healthy newborns.

IFN γ production by CD8 T cells upon stimulation with PMA and viral peptides

Pre-vaccination:Lower Chemokine and Toll like receptor expression in HBsAgpositive newborns:

T cell phenotypic distribution in HBsAgPositive, HBsAgNegative from HBsAg positive mothers and healthy newborns.

Sample Collection

1X TBS

Immunofluorescence

Secondary antibodies

Animals

GFP-β-catenin T41A mutant

Transfection of short interfering RNA (siRNA) and plasmid

Mammalian cell lines

Assay of IFN-yand IL-10 using sandwich ELISA

Anti-Stm and anti-E coli /gG subclass assay

Mice

Cellline

Extraction of Tannin

Extraction

Procedure

Standard curve of sugar

Extraction and determination of protein

Standard curve of protein

Reagents

Estimation of protein

Standard curve of ascorbic acid

Fresh and Dry weight

Dilution Formula

Cd (NO3)2

ZnSO4

Preparation of ppm solution

Youman's modified medium

Histopathological studies

Bacterial Cultures

Preparation of ultra competent E. coli

Antibiotics

Monoclonal Antibodies against the Human Glycophorin-

Monoclonal Antibodies against the Human Glycophorin

Isolation of endothelial cells from rat aorta

Procedur

Procedur

Procedure

Procedure

Reagent

Total antioxidant activit

Procedur

Catalyst Preparation

Catalyst Preparation

Catalyst Preparation

Single Cell Test

Electrochemical Measuremen

Physical Characterization

Catalyst Preparation

Material

ELECTROGENERATION OF SEAWATER ON ALCOHOL OXIDATION IN AN NON-MEMBRANE POWER SYSTEM

Total dissolved solids

High Performance Liquid Chromatography (HPLC) analysis

High Performance Thin Layer Chromatography (HPTLC) analysis

. Thin Layer Chromatography (TLC) analysis

Preliminary phytochemical screening (Ali, 1998; Evans, 2002)

Microbial Contamina

Foaming Index

pH values

Ash values

Extractive value

. Physico-chemical standardization

Standardization of ethanolic extract of E.ribes (WHO, 1998; IndianPharmacopoeia, 1996)

Estimation of Stevioside

Estimation of Steviol

Estimation of total phenols

Somatotype Categories:

somatotype Categories:

somatotype Categories:

Range of Component Ratings:

Somatocharts:

Mean Somatotypes:

SOMATOTYPE DISTRIBUTION OF RURAL SAMPLE

Somatocharts:

Balanced mesomorph:

Overnight grown primary culture of E. coli cells (1 % v/v final concentration) was inoculated into 1 litre of LB media containing antibiotics. Culture was incubated at 37 oc at 200 rpm. Growth was monitored by measuring absorbance of E. coli broth at 600 nm. Culture was induced by adding 1 mM IPTG at an OD of 0.6 and was harvested after 4 hrs of induction. Samples were taken on an hourly basis after induction to check the kinetics of protein expression. Un-induced and induced E. coli cells were analyzed by SDS-PAGE to check the expression of recombinant protein.

Growth and expression of recombinant proteins in E. coli cells

The [3-PMB and a-PMB chains were eluted with a linear gradient of 500 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) at a flow rate of 50 ml/hour. The chains were separately concentrated using Centriprep concentrators (Amicon) and stored in liquid nitrogen till further use

The heme bound a and ~ subunits were obtained as described by Bucci (1981 ). Briefly, hemoglobin was reacted with PMB in an eight fold molar excess (8 moles of PMB per mole of hemoglobin). The reaction mixture was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and then loaded onto a CM52 column (30cm x !Scm) that was pre-equilibrated with the same buffer.

Separation of the a and f3 subunits of hemoglobin

Paduans

Riding over the hills, and eating their fill,7 lying a little too long; these things are, perhaps, enough to explain what happened. How­ever, that may be: they woke suddenly from a sleep they had never meant to take. The standing stone was cold, and it cast a long pale shadow. The sun was gleaming through the mist; north, south, and east, the fog was thick, cold and white. The air was silent, heavy and chill.The hobbits8 sprang to their feet in alarm, and ran to the western rim. They found that they were upon an island in the fog. Even as they looked out in dismay towards the setting sun, it sank before their eyes into a white sea, and a cold grey shadow sprang up in the East behind. The fog rolled up to the walls and rose above them, and as it mounted it bent over their heads until it became a roof. They felt as if a trap was closing about them. They packed up as quickly as their chilled fingers would work.Soon they were leading their ponies in single file9 over the rim and down the long northward slope of the hill, down into a foggy sea. As they went down the mist became colder and damper, and their hair hung lank and dripping on their foreheads. When they reached the bottom it was so cold that they halted and got out cloaks and hoods, which soon became bedewed with grey drops. Then, mounting their ponies, they went slowly on again. To prevent their getting separated and wandering in different directions they went in file, with Frodo leading. Suddenly Frodo saw a hopeful sign. On either side ahead a darkness began to loom through the mist; and he guessed that they were at last approaching the gap in the hills. 'Come on! Follow me!' he called back over his shoulder, and he hurried forward. His pony reared, and he fell off. When he looked back he found that he was alone: the others had not fol­lowed him.

Riding over the hills, and eating their fill,7 lying a little too long; these things are, perhaps, enough to explain what happened. How­ever, that may be: they woke suddenly from a sleep they had never meant to take. The standing stone was cold, and it cast a long pale shadow. The sun was gleaming through the mist; north, south, and east, the fog was thick, cold and white. The air was silent, heavy and chill.The hobbits8 sprang to their feet in alarm, and ran to the western rim. They found that they were upon an island in the fog. Even as they looked out in dismay towards the setting sun, it sank before their eyes into a white sea, and a cold grey shadow sprang up in the East behind. The fog rolled up to the walls and rose above them, and as it mounted it bent over their heads until it became a roof. They felt as if a trap was closing about them. They packed up as quickly as their chilled fingers would work.Soon they were leading their ponies in single file9 over the rim and down the long northward slope of the hill, down into a foggy sea. As they went down the mist became colder and damper, and their hair hung lank and dripping on their foreheads. When they reached the bottom it was so cold that they halted and got out cloaks and hoods, which soon became bedewed with grey drops. Then, mounting their ponies, they went slowly on again. To prevent their getting separated and wandering in different directions they went in file, with Frodo leading. Suddenly Frodo saw a hopeful sign. On either side ahead a darkness began to loom through the mist; and he guessed that they were at last approaching the gap in the hills. 'Come on! Follow me!' he called back over his shoulder, and he hurried forward. His pony reared, and he fell off. When he looked back he found that he was alone: the others had not fol­lowed him.

The clear cell-free supernatants were used as the source of crude recombinant xylanase.

Quantitative screening for determination of xylanase in shake flask

2 mL of an overnight culture of E. coli cells was inoculated into 100 mL LB medium and incubated with vigorous shaking at 30 °C until A600 of 0.8 was reached. •Cells were collected in 50 mL plastic (Falcon) tubes, cooled for 15 min on ice and centrifuged in a pre-cooled centrifuge (4,000 rpm for 10 min at 4 °C). •The pellet was suspended in 20 mL of ice-cold 50 mM CaCl2-15% glycerol solution, maintained on ice for 15 min and centrifuged again at 4,000 rpm for 10 min at 4 °C. •Pellet was resuspended in 2 mL of ice-cold 50 mM CaCl2-15 % glycerol solution, kept on ice for 30 min and aliquoted in 400 μL in microcentrifuge tubes. These were stored at -80 °C until required.

Preparation of calcium-competent cells

Two hundred μL of alkaline-SDS solution was added to the above suspension, mixed by inverting the tubes up and down 3 times and incubated for 5 min at room temperature. ƒTo the above mixture, 250 μL of 3 M Na-acetate (pH 4.8) was added, mixed by inverting the tubes up and down 3 times, and centrifuged at 12,000 x g for 10 min. ƒThe supernatant was collected in another micro centrifuge tube (MCT), 200 μL of phenol:chloroform solution was added, inverted two times and centrifuged at 12, 000 x g for 8 min at room temperature. ƒThe aqueous phase was transferred to new tubes and 500 μL of chilled (-20 °C) ethanol (96 %) was added. ƒThe tubes were centrifuged at 13,000 x g for 25 min at 4 °C, supernatant discarded and pellet dried for 15 min at room temperature. ƒThe pellet was washed with 500 μL of chilled 70 % (v/v) ethanol and centrifuged at 13, 000 rpm for 4 min at 4 °C. ƒThe pellet was dried at room temperature and dissolved in 50 μL of 1X TE buffer (pH 8.0) containing RNase and stored at -20 °C till further use.

The cells of E. coli DH10B having p18GFP vector were cultivated for overnight at 37 °C in LB medium containing ampicillin (100 μg mL-1). ƒThe E. coli culture having p18 GFP vector (~1.5 mL) was taken in Eppendorf tubes and centrifuged at 10, 000 x g for 5 min. ƒThe pellet was homogenized by vortex mixing in 100 μL of homogenizing solution

Plasmid isolation from miniprep method

An attempt was made to study the effect of storage of DNA extracts on DNA yield and purity. The DNA extracts were centrifuged and the supernatants were dispensed into 2.0 mL Eppendorf tubes and stored at -20 oC for a month. DNA precipitation and its quantification were carried out at a week intervals.

Effect of storage on soil/sediment DNA extracts

Attempts have been made to amplify the signature sequences of bacterial, archaeal and fungal specific regions by using respective sets of primers shown in Table2.2. The reactions were carried out in 50 μL reaction mixtures in a Thermal Cycler (Bio-Rad, USA) using respective primers (Table 2.2). The PCR conditions were optimized as follows: for Bacterial 16S rDNA, initial denaturation of 3 min at 94 oC followed by 30 cycles of 30 sec at 93 oC, 60 sec at 55 oC and 90 sec at 72 oC; Archaeal 16S rDNA, 5 min at 95 oC, 35 cycles of 50 sec at 94 oC, 60 sec at 62 oC and 60 sec at 72 oC; fungal specific ITS regions, 3 min at 95 °C, 30 cycles of 60 sec at 94 °C, 56 °C at 45 sec and 50 sec at 72 °C. Final extension time was 7 min at 72 °C in all PCR runs. Amplifications were visualized on 1.2 % w/v agarose gels

PCR amplification of microbial population

Purity of the DNA extracted from various environmental samples was confirmed by subjecting the extracted DNA to restriction digestion. DNA was digested with Sau3AI (New England Biolabs). One μg of metagenomic DNA in 20 μL reaction mixture was treated with 0.5 U of Sau3AI and incubated at 37 °Cfor 10 min. The reaction was terminated at 80 °C for 20 min and the digested DNA was fractionated on 1.2 % (w/v) agarose gel.

Restriction digestion

VALIDATION OF METAGENOME OBTAINED BY THE PROTOCOL DEVELOPED IN THIS INVESTIGATION

as well as commercial methods (MN kit, Germany; Mo-Bio kit, CA, USA; Zymo soil DNA kit, CA, USA) according to the manufacturer’s protocols and compared in terms of DNA yield and purity.

The soil DNA from Pantnagar and Lonar soil samples were also extracted by various manual (Desai and Madamwar, 2007; Agarwal et al., 2001; Yamamoto et al., 1998

Comparison of yield and purity of crude DNA

Various strains of Escherchia coli (DH5α, XL1Blue, DH10B) were used as hosts for the propagation of recombinant vectors. In addition, Bacillus subtilis was used as a host for the expression of xylanase gene from the recombinant vector pWHMxyl. Different vectors used in this investigation are listed in

BACTERIAL STRAINS

Parasites from synchronized cultures were harvested at different time points of growth to obtain ring, trophozoite and schizont stage parasites. RNA was isolated from these stages by using RNAeasy kit (Qiagen) following manufacturer's protocol. The concentration of total RNA was determined by measuring the absorbance at 260 nm. Purity of nucleic acid preparations were determined by calculating OD26onm / OD28onm ratio, a value of near ~ 1.6-1.8 was taken as a standard of purity. To get stage specific cDNA from RNA, reverse transcription was performed using RT-PCR kit (Invitrogen) that contained random hexamers. Subsequently, the gene of interest was amplified using gene specific primers

Isolation of the parasite RNA

Human 0+ or AB+ RBC was obtained from a donor and mixed with heparin (50 units/ml of blood) and centrifuged at 500 g for 10 min with minimu1p. de-acceleration. The supernatant was removed carefully and the pelleted RBCS were washed 3 times with RPMI 1640 to remove serum and buffy coat. Equal amount of RPMI 1640 media was added to packed RBC volume to achieve 50% hem~tocrit and stored at 4°C till: further use

Preparation of RBCsfor culture

proteins of interest were pooled and 1 mM TCEP was added. The protein of interest was collected and stored at -80°C for further use after adding 1 mM TCEP.

The mutant proteins were expressed and purified analogous to wild type RaPt protein. Mutant clones pAC36, pAC50 and pAC38 were transformed in BL21 strain of E. coli. Analogous to the wild type RGPL protein the cells harbouring the mutant expression plasmids were cultured at 37°C to an O.D6oonm of 0.6 and uninduced at 30°C for 6-8 hrs. After harvesting, the cells were resuspended in lysis buffer (1 00 mM phosphate pH: 7 .0, I 0% glycerol) and disrupted using french press at 1100 psi pressure. Cell debris was removed by centrifugation at 50,000 g for 40 min at 4°C. 0. 75 ml L.1 of Ni2+ -NT A slurry was added to the supernatant and incubated at 4°C for 1 hr. This suspension was loaded onto a column working under gravity flow. The resin was washed with wash buffer (100 mM phosphate pH: 7.0, 10% glycerol and 5 mM imidazole) till all unbound proteins were removed. The protein was eluted using elution buffers containing increasing concentration of imidazole. Fractions containing the

Expression and purification of RcPL mutant proteins

Hair from the skin overlying the left and right dorsal flanks were removed using electrically operated razor. The skin overlying the abdomen was sterilized by wiping with 70% ethanol. Ketamine (1 00 mg/kg) and xylocaine (2%) (20 mg/kg) were mixed and administered intraperitoneally. The mice were returned to the cage and the onset of anesthetic effect was monitored. The mice were considered to be in surgical anesthesia when there was loss of palpebral reflex, righting reflex, and toe pinch reflex. Respiratory rate and heart rate were monitored continuously.

General anesthesia:

treatment were harvested by centrifugation at 250 x g for 5 min following which they were resuspended in 1x PBS (pH 7.5). PI was added at a final concentration of 1 J.tg/mL and incubated for 5 minutes following which the cells were pelleted by centrifugation and washed once with PBS. These cells were analyzed for uptake of PI by either flow cytometry in FL2 channel (570 nm) or by fluorescence microscopy using a G2A filter block.

Propidium iodide (PI) is a DNA intercalating fluorescent dye which is excluded by viable cells with intact membranes, however, dead and dying cells with damaged membranes take up the dye. To assess viability, cells after appropriate

population was determined by analyzing cells immunostained with an antibody against CD14 conjugated to FITC and the purity obtained was approximately 85% monocytes, the remaining being lymphocytes. The monocytes were further cultured in the presence of human AB serum for 7 days to allow differentiation to macrophages. At the end of 7 days post-isolation, greater than 95% of cells in culture are monocytes, with the majority of lymphocytes undergoing neglect induced death.

Peripheral blood (30 mL) was collected by venipuncture from healthy male volunteers after obtaining an informed consent and in accordance to the regulations of the Institutional Human Ethics Committee (National Institute of Immunology, New Delhi, India). The peripheral blood mononuclear cell (PBMC) population was isolated by density gradient centrifugation using Histopaque 1077, where, human whole blood was layered on Histopaque 1077 and centrifuged at 400 x g for 35 min at 25°C. The mononuclear cell population was isolated from the plasma-histopaque interface, and the monocytes were further purified by washing off the non-adherent cells after incubating the total PBMC for 1 h at 3 7°C. The homogeneity

Peripheral blood monocy.te isolation and macrophage differentiation

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include SORTING, that sorts, packs and assesses the quality of the experimentally measured diffraction data, and is run in the first step. The program TABLING calculates the continuous Fourier coefficients from the model placed in the artificial cell. The cross-rotation function is carried out by the program ROTING, which uses Crowther's algorithm (Crowther, 1972). TRAING is used to calculate the translation function. Finally FITING is used to refine the orientational and positional parameters of the molecule corresponding to the potential solutions, as a rigid body.