Developmental delay

RRID:ZFIN_ZDB-GENO-190322-4

RRID:IMSR_OBS:4

RRID:ZFIN_ZDB-ALT-150424-4

RRID:ZFIN_ZDB-ALT-060821-4

4

seizures

intellectual disability

developmental delay

failure to thrive

a promising technology for decades that's never truly caught on. That's constantly changing with the current wave of VR products,

LC-MS Analysis

Lumbar gibbus

disproportionate stature

platyspondyly

Eventually, the term Communist became identified with the brand of Socialism that came to be called “Marxism.”

to my Celestial Court and to be in control of your country’s trade with China, this request is contrary to all usage of my dynasty and cannot possibly be entertained. It is true that Europeans, in the service of the dynasty, have been permitted to live at Peking, but they are compelled to adopt Chinese dress, they are strictly confined to their own precincts and are never permitted to return home.

You, O King, live beyond the confines of many seas, nevertheless, impelled by your humble desire to partake of the benefits of our civilisation, you have dispatched a mission respectfully bearing your memorial. Your Envoy has crossed the seas and paid his respects at my Court on the anniversary of my birthday. To show your devotion, you have also sent offerings of your country’s produce.

All those people in China who sell opium or smoke opium should receive the death penalty.

Preparation of distilled extracts

Conclusion

Preparation of distilled extracts

Tests for Saponins

Preparation of distilled extracts

Qualitative and quantitative estimation of DNA

Sporulation medium used for Trichodermasp

Collection of the diseased materia

Relative prevalence of extracellular virulence factors among Vibriofrom Cochin estuary, shrimp farm and seafood

Detection of type III secretion system genes

Production of gelatinase

Plasmid profiles among Vibriofrom Cochin estuary, shrimp farms and seafood

Relative antibiotic resistance amongVibrioisolated from Cochin estuary, shrimp farm and seafood

Plasmid profiling of the drug resistant strains

Detection ofblaNDM-1gene

Antibiotic resistance among Vibriofrom seafood

Seasonal variation in the diversity and distribution of Vibrioin Cochin estuary

Gel documentation and image analysis

Indole production

Isolation of Vibriospecies from water and sediment of Cochin estuary

Indole production

Isolation of Vibriospecies from water and sediment of Cochin estuary

Active site identification, metal detection and interaction of Dof domain structure

Superposition of the Dof domain with predicted 3D structure

Validation of the predicted 3D structure

Tertiary structural prediction

Secondary structural prediction

Secondary and tertiary structure prediction of SbDof proteins o

Gene structure prediction

Phylogenetic and motif analysis of sequenced Dof domains

In silico characterization of sequenced Dof domains of cereal

Sequencing of Dof domain and gene

Cloning of Dof genes of sorghum using pBSK vector

Cloning of Dof domain and Dof genes using pGEM-T Easy

Gel elution of PCR products

PCR based cloning, sequencing and in silico characterization of Dof domain and Dofgenes of cereals and millet

Comparative analysis of cereals and millets based on banding patterns generated by Dof domain and Dof genes-specific primers

Transformation of ligation mixture in electro-competent E. coli host cells (DH5ααααstrain)

Analysis of PCR amplicons using agarose gel electrophoresis

Qualitative analysis of DNA by agarose gel electrophoresi

Plasmids and bacterial strain

Estimation of antibody response in rLdADHT+BCG vaccinated hamsters

Statistical analysis

Post-challenge survival

Determination of antileishmanial antibody responses in hamster

RT - PCR

cDNA synthesis and amplification

Formaldehyde gel electrophoresis

RNA isolation

Quantification of mRNA cytokines and inducible NO synthase (iNOS) in hamsters by Real time-PCR

NO production

LTT assay

DTH

Immunological assays

Patients and isolation of peripheral blood mononuclear cells (PB

Purification of recombinant ADHT (rLdADHT)

Subcloning of ADHT in pET-28a expression vector

Sequencing of ADHT gene

Cloning of gene in pTZ57R/T (T/A cloning) vecto

Results of Python Molecular Viewer

Molecular Docking

Catalase assay

Detection of intracellular ROS

Nitric oxide radical quenching activity

DLS particle size and zeta potential analysis

Synthesis of AgNPs

The Posterior Probability of Mixing Dis-tribution

Spreading inhibitory behavior

Inhibition of haemocytes spreading behavior

Oral toxicity bioassay

Influence of prey on VS yield

Quantification of VS for protein

Salivary gland-Morphology and Anatomy

Gross morphology and histology of salivary gland

Fish sampled from selected water bodies for further analysis

Increments in biomass:

Organic carbon

Soil pH and electrical conductivity (EC)

Mechanical composition

Methods of soil analyses

Trait plasticity and response to competition

Root length (cm) and Root weight (mg)

Number of productive tillers per meter

Concentration of ideal carbon source

Carbon sources

NaCl concentration

Temperature

pH

Static and shaken condition

Inoculum concentration

Optimization

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

Decreased CD3 ζ chain expression on CD8 T cells in HBsAgpositive newborns

Phenotypic and Functional Characterization of CD8 T cells in cord blood

CD107a expression (marker of cytotoxicity)

Serological and Virological Assessment

Lysis buffer for Ni-NTA and glutathione purification

Luciferase reporter assay

General reagents and plastic wares

GFP-β-catenin- C (634 -781 a.a)

Analysis of ROS

Plasmid

Tissue Culture Medium

Standard

Calculation

Procedure

Reagent

Proline

Fresh and Dry weight

Root and Shoot length

Morphological Studies

ELISA binding Buffer

Preparation of the vaccines

Large scale preparation of plasmid DNA

Ultra-competent cells

RS-1 Assay

RS-1 Assay

Chorioallantoic membrane (CAM) assay

Procedur

Reagents

Hydroxyl radical scavenging assay

Electrochemical Measurement

Single Cell Tes

Electrochemical Measurement

Physical Characterizatio

Catalyst Preparation

Material

STRUCTURAL STABILITY AND ELECTROCHEMICAL PERFORMANCE OF W-DOPED PLATINUM ALLOY NANO PARTICLES FOR SEAWATERELECTROLYSIS ON ALCOHOL OXIDATION INNON-MEMBRANE FUEL CELL SYSTEMS

Electrochemical Measuremen

Electrochemical Measuremen

Total alkalinity

Characterization of embelin isolated from E.ribes

High Performance Liquid Chromatography (HPLC) analysis

Foaming Index

Estimation of free aminoacids

Per cent Overlap:

Somatotype Categories:

Mean Somatotypes:

Urban-Rural Somatotype Comparisons

somatotype Categories:

Somatotype Categories:

Mesomorph-endomorph:

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¶PQRWVD\LQJRQHJUDSKLVEHWWHUWKDQWKHRWKHU,QIDFWWKH\FDQFRPSOHPHQWHDFKRWKHU+RZ\RXVSOLW\RXUGDWDGHSHQGVRQKRZPXFKGHWDLO\RXQHHGRUGRQ¶WQHHG

7KHPDLQSRLQWLVWKDWGDWDDQGYLVXDOL]DWLRQGRQ¶WDOZD\VKDYHWREHMXVWDERXWWKHFROGKDUGIDFWV6RPHWLPHV\RX¶UHQRWORRNLQJIRUDQDO\WLFDOLQVLJKW5DWKHUVRPHWLPHV\RXFDQWHOOWKHVWRU\IURPDQHPRWLRQDOSRLQWRIYLHZWKDWHQFRXUDJHVYLHZHUVWRUHIOHFWRQWKHGDWD

7KHIRUPHUUHDGVDVDQXPEHUZLWKRXWPXFKFRQWH[WZKHUHDVWKHODWWHULVPRUHUHODWDEOH

)LQDOO\DOZD\VFRQVLGHU\RXUDXGLHQFHDQGWKHSXUSRVHRI\RXUJUDSKLFV

%DVLFDOO\ZKDW\RX¶UHORRNLQJIRULVVWXIIWKDWPDNHVQRVHQVH0D\EHWKHUHZDVDQHUURUDWGDWDHQWU\DQGVRPHRQHDGGHGDQH[WUD]HURRUPLVVHGRQH0D\EHWKHUHZHUHFRQQHFWLYLW\LVVXHVGXULQJDGDWDVFUDSHDQGVRPHELWVJRWPXFNHGXSLQUDQGRPVSRWV:KDWHYHULWLV\RXQHHGWRYHULI\ZLWKWKHVRXUFHLIDQ\WKLQJORRNVIXQN\

%XEEOHVUHSUHVHQWFRXQWULHVDQGPRYHEDVHGRQWKHFRUUHVSRQGLQJFRXQWU\¶VSRYHUW\GXULQJDJLYHQ\HDU

7KHLQWHUDFWLYHSLHFHVFUDSHVVHQWHQFHVDQGSKUDVHVIURPSHUVRQDOSXEOLFEORJVDQGWKHQYLVXDOL]HVWKHPDVDER[RIIORDWLQJEXEEOHV

The complementary segments of a globin needed for the semisynthesis of mutant chains were prepared by V8 protease digestion (Sivaram eta/, 2001). The a globin was dissolved in 0.01 M ammonium acetate buffer (pH 4) at a concentration of 1.0 mg/ml and digested at 37°C with V8 protease (1: 200, w/w) for 3 hours. The completion of digestion was ascertained by RPHPLC, after which the reaction was quenched by addition of neat TFA to a final concentration of 0.1 %. The complementary segments, al-30 and a31-141, from the digestion mixture were isolated in pure form by size-exclusion chromatography on a Sephadex G50 column (98cm x 2.8cm). The column was equilibrated and run in 0.1% TFA. The lyophilized sample of the digest was dissolved in the above solvent and loaded on to the column. The column was run at a flow rate of 30 mllhour and the elution profile monitored at 280 nm. The individual chromatographic profile of a globin digest showed only two peaks, a31-141 and a1-30 respectively, as expected from a single cleavage at the 30-31 peptide bond. The peak fractions were pooled separately and lyophilized

Generation of complementary fragments, al-30 and a31-141, from heme-free a globin

Xylanolytic activity was determined according to Archana and Satyanarayana (1997). The reaction mixture containing 0.5 mL of 1% birchwood xylan in glycine NaOH buffer (0.1 M, pH 9.0) and 0.5 mL of cell free sonicated supernatant was incubated at 80 °C in a water bath for 10 min. After incubation, 1 mL DNSA reagent (Miller, 1959) was added to the reaction mixture and the tubes were incubated in a boiling water bath for 10 min, followed by the addition of 400 μL of 33% w/v sodium potassium tartrate. The absorbance values were recorded at 540 nm in a spectrophotometer (Shimadzu, Japan). The liberated reducing sugars were determined by comparing the absorbance values of these with a standard curve drawn with different concentrations of xylose. One unit (IU) of xylanase is defined as the amount of enzyme required for liberating one μmol of reducing sugar as xylose mL-1 min-1under the assay conditions. Composition of Dinitrosalicylic acid (DNSA) reagent NaOH - 10.0 g Phenol - 2.0 g DNSA - 2.0 g Distilled Water - 1000 mL DNSA reagent was stored in an amber bottle at 4 °C till further use. Sodium sulphite (0.05 % v/v) was added just before the use of the reagent.

Enzyme Assays

Transformation of calcium-competent cells was carried out by the procedure detailed below: •The competent bacterial cells were thawed briefly and 200 μL of cells was mixed rapidly with plasmid DNA (10-50 ng) in fresh, sterile microcentrifuge tubes and maintained on ice for 30 min. A negative control with competent cells only (no added DNA) was also included. •Cell membranes were disrupted by subjecting cells to heat-pulse (42 °C) for 90 sec. •After heat shock, cells were incubated on ice for 5 min. •Cells were then mixed with 1 mL LB medium and incubated with shaking at 37 °C for 1 h. •For blue/white screening 40 μL of X-gal solution (20 mg mL-1 in dimethylformamide) and 4 μL of the IPTG (200 mg mL-1) was spread on LB-ampicillin (LB-amp) plates with a sterile glass rod. The plate was allowed to dry for 1h at 37 °C prior to spreading of bacterial cells. •Bacterial cells (100-200 μL) were spread and the plate was incubated at 37 °C for overnight. •White colonies were picked from the plates and suspended into LB-amp broth and cultivated to OD600=0.5

Transformation procedure

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

Preparation of electrocompetent cells (E. coli cells) A protocol was employed. The procedure was carried out in cold under sterile conditions as follows: •A single colony of E. coli DH10B/ DH5α/XL1blue was inoculated in 20 mL of LB medium and grown overnight at 30 °C. •500 mL LB medium was inoculated with 5mL of this overnight grown culture of the E. coli and incubated with vigorous shaking (250 rpm) at 30 °C until an A600of 0.5 - 0.8 was achieved. •The cells were chilled in ice for 10-15 min and transferred to prechilled Sorvall® centrifuge tubes and sedimented at 4,000 rpm for 20 min at 4 °C. •The supernatant was decanted and cells were resuspended in 500 mL of sterile ice-cold water, mixed well and centrifuged as described above. •The washing of the cells described above was repeated with 250 mL of sterile ice-cold water, following which cells were washed with 40 mL of ice-cold 10 % (v/v) glycerol and centrifuged at 4,000 rpm for 10 min. •The glycerol solution was decanted and the cell volume was recorded. The cells were resuspended in an equal volume of ice-cold 10 % glycerol. •Cells were then dispensed in 40 μL volumes and stored at -80 °C until required.

Electrotransformation

BACTERIAL TRANSFORMATION

Ligation of insert DNA with dephosphorylated vector

In order to minimize self ligation of vector during cloning experiments, the digested DNA was subsequently treated with calf intestinal phosphatase (CIP) [NEB, UK]. The reaction conditions and amount of CIP were optimized and varied from (0.06-1) unit/picomole DNA termini. The dephosphorylation reaction was carried out in 50 μL reaction as follows. Reaction mixture containing no restriction enzyme was treated as control. Reaction was incubated for 1 h at 37 °C and stopped by heat inactivation at 65 °C for 20 min. 2.5.5. Composition of restriction mixture (50 μL) Linearized Plasmid DNA X μL (1 μg) CIP 1 μL (0.06-1 U μL-1) Reaction buffer (10X) 5.0 μL Distilled water Y μL Total volume 50 μL Linearized and dephosphorylated plasmids from each reaction were purified from low melting agarose gel using gel extraction method according to the manufacturer’s protocol (Qiagen gel extraction kit, Germany). 100 ng DNA from each reaction was then ligated in15 μL reaction volume containing 1.5 μL of 10X ligation buffer (NEB, England) and 0.2 μL of T4 DNA ligase to check the efficiency of self ligation after dephosphoryaltion. The ligation mixture was incubated at 16 °C for overnight and transformed into E. coli DH5αcompetent cells.

Dephosphorylation of the restricted plasmid

The ligation reaction consisted of 10 ng of vector, appropriate amount of insert (insert:vector ratio :: 3: 1), 1 x ligation buffer and 1 U of T 4 DNA ligase (NEB, England). The total volume was made up to 10 III with autoclaved water. The ligation reaction mixture was incubated at 16°C for 12 hrs

Ligation

cryopreserved culture vial was obtained from the liquid nitrogen tank, and thawed quickly at 37°C in a water bath. To the vial, O.lv of 12% NaCl was addeq I slowly, dropwise, while shaking the tube gently. Subsequently, 10v of 1.6% NaCl I was added slowly, dropwise while swirling the tube, followed by centrifugation at 200 g at 20°C for 5 min. The supernatant was discarded and 10v of RPMI 1640 complete media was added, followed by centrifugation at 200 g at 20°C for 5 min.' After removal of the supernatant, pelleted parasites were resuspended in complete I media at 0.5% hematocrit. Cultures were gassed with 5% C02, 3% 02, and 92%' N2 and maintained at 37°C

Revival of cryo-preserved Plasmodiumfalciparum cultures

Enzymatic assays using acyl-peptidyl substrates were set up as follows: 100-120 llmoles of purified ~PL/RNRP protein, 200 l!M valeryl-FT AA-CoA/ valery 1-FT AAlaninal and 2 mM NADPH were incubated at 30°C for 2 hrs. The protein was precipitated with acetonitrile and the reaction was loaded on C 18 RP HPLC column (250 x 4.6 mm, 5l!, phenomenex). The products could be resolved using following gradient: 0 to 48% B in 25 min, 48% B in 40 min and 70% B in 50 min (A-water with 0.1% TF A and B-acetonitrile with 0.1% TF A) at a flow rate 0.6 ml/min. The elution profile was monitored at 220 nm. The identity of peaks obtained was confirmed by TOF-MS and tandem mass spectrometric analysis using ESI-MS (API QSTAR Pulsar i MS/MS, Applied Biosystems).

The enzymatic assays were performed as described for wild type ~Pl. in chapter 2. The standard reaction mixture contained 100 J.lM fatty acyl-CoA (30 J.lM [1-14C] fatty acyl-CoA (55 mCi/mmole,ARC) and 70 J.lM of unlabeled fatty acyl CoA), 2 mM NADPH and 10-20 nmoles protein for l-2 hrs. Lauroyl aldehyde [ l-14C] was obtained enzymatically from [ l-14C] lauric acid (55mCi/mmole,ARC) using the FadD9 protein, and extracted from TLC by using ethyl acetate. Assays were set up using a total of 100 J.lM ('4C labeled + unlabeled) of lauroyl aldehyde in the presence of 2 mM NADPH and 10-20 nmoles protein for 1-2 hrs. The products were extracted twice in 300 J.ll of hexanes and resolved on silica gel 60 F2s4 TLC plates (Merck) using hexanes:ethyl acetate (80:20, v/v) solvent system. The radiolabeled product was detected by using phosphorimager (Fuji BAS500)

nzymatic assays and product characterization

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

Assay for cell viability by propidium iodide dye exclusion method

Biochemical and cell biology techniques

Phaser is a program for phasing macromolecular crystal structures by both molecular replacement and experimental phasing methods (A. J. McCoy, 2007). The novel algorithms in Phaser are based on maximum likelihood probability theory and multivariate statistics rather than the traditional least-squares and Patterson methods. For molecular replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solutions from noise. One of the design concepts of Phaser was that it be capable of a high degree of automation. Phaser has novel maximum likelihood phasing algorithms for the rotation functions and translation functions in MR, but also implements other non-likelihood algorithms that are critical to success in certain cases.

Automated molecular replacement program (Phaser)

merging data, and symmetry equivalent positions, space group-specific systematic absences, total percentage of data collected and the linear Rmerge for data reduction. Finally, truncate program was used to obtain structure factor or amplitudes from averaged intensities (output from SCALA, or SCALEPACK) and write a file containing mean amplitudes and the original intensities. If anomalous data is present then F(+), F(-), with the anomalous difference, plus I(+) and 1(-) are also written out. The amplitudes are put on an approximate absolute scale using the scale factor taken from a Wilson plot. For all the Fab-peptide complexes and unliganded Fab of BBE6.12H3 antibody, the diffraction data were collected and processed using MOSFLM and subsequently merged using SCALA. For all the Fab-peptide complexes of 36-65 Fab, the diffraction data were collected and processed using DENZO and subsequently merged using SCALEPACK. The cell dimensions and space groups were unambiguously determined for each crystal. The solvent content and Matthews's constant were calculated (Matthews, 1968). The merged and scaled intensities were used for structure determination.