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  1. Last 7 days
  2. Jul 2021
  3. Jun 2021
    1. Angular is nothing but an open-source web application framework. It is being led by the Angular Team at Google and also by a community of individuals and corporations. It is based on TypeScript.Angular is a complete rewrite from the same team that had built AngularJS. It can be used as the frontend of the MEAN stack, which consists of the MongoDB database, Express.js web application server framework, Angular itself (or AngularJS), and the Node.js server runtime environment.

      Top Angular 8 Interview Questions to get you ready

  4. May 2021
  5. Mar 2021
    1. Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

      AssayResult: 7.3

      AssayResultAssertion: Normal

      StandardErrorMean: 0.08

    2. A total of 84 PALB2 patient-derived missense variants reported in ClinVar, COSMIC, and the PALB2 LOVD database were selected

      HGVS: NM_024675.3:c.1145G>T p.(Ser382Ile)

    1. SUPPLEMENTARY DATA

      AssayResult: 58

      AssayResultAssertion: Indeterminate

      PValue: < 0.0001

      Approximation: Exact assay result value not reported; value estimated from Figure 6C.

    2. SUPPLEMENTARY DATA

      AssayResult: -32

      AssayResultAssertion: Abnormal

      PValue: < 0.0001

    3. SUPPLEMENTARY DATA

      AssayResult: 76.45

      AssayResultAssertion: Indeterminate

      PValue: 0.0001

      Comment: Exact values reported in Table S3.

    4. To this end, 44 missense variants found in breast cancer patients were identified in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) and/or selected by literature curation based on their frequency of description or amino acid substitution position in the protein (Supplemental Table S1).

      HGVS: NM_024675.3:c.23C>T p.(Pro8Leu)

    1. Source Data

      AssayResult: 83.96

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardErrorMean: 9.89

      Comment: Exact values reported in “Source Data” file.

    2. Source Data

      AssayResult: 66.19

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardDeviation: 21.26

      StandardErrorMean: 15.03

      Comment: Exact values reported in “Source Data” file.

    3. We, therefore, analyzed the effect of 48 PALB2 VUS (Fig. 2a, blue) and one synthetic missense variant (p.A1025R) (Fig. 2a, purple)29 on PALB2 function in HR.

      HGVS: NM_024675.3:c.1250C>A p.(S417Y)

    1. Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function

      AssayResult: 1.2

      AssayResultAssertion: Abnormal

      ReplicateCount: 11

      StandardErrorMean: 0.7

      Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

    2. we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants

      HGVS: NM_198056.2:c.1156G>A p.(Gly386Arg)

    1. RRID:ZFIN_ZDB-ALT-120221-8

      DOI: 10.7554/eLife.64267

      Resource: (ZFIN Cat# ZDB-ALT-120221-8,RRID:ZFIN_ZDB-ALT-120221-8)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-120221-8


      What is this?

    2. RRID:ZFIN_ZDB-ALT-041129-8

      DOI: 10.7554/eLife.64267

      Resource: (ZFIN Cat# ZDB-ALT-041129-8,RRID:ZFIN_ZDB-ALT-041129-8)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-041129-8


      What is this?

  6. Feb 2021
    1. Supplemental material

      AssayResult: 86

      AssayResultAssertion: Normal

      Comment: See Table S3 for details

    2. Supplemental material

      AssayResult: 6.1

      AssayResultAssertion: Abnormal

      Comment: See Table S3 for details

    3. We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.

      HGVS: NM_000546.5:c.215C>A p.(Pro72His)

    1. cloud computing shall not be referred to as the future, but rather a trend deepening its root in the present. So when many businesses are migrating to cloud or hybrid servers, how can healthcare players sit back.

      As technology is evolving and growing, cloud computing is entering a diverse set of fields. Currently, it’s enjoying a high rise in the healthcare industry.

  7. Jan 2021
  8. Nov 2020
  9. Sep 2020
    1. of which the handle [was x minas] of gold, he provided for his friend.

      It shows the his friend Enkidu.

    2. o Enkidu, may the paths [of] the Forest of Cedar mourn you [without pause,] by day and by night!

      Message of Gilgamesh to Enkidu when he died.

    3. Gilgamesh [began mourning] his friend:

      It shows their friendship.

    1. À cette fin, comme le prévoit l'article D. 111-8 du Code de l'éducation, les directeurs d'école et les chefs d'établissement doivent communiquer aux associations de parents d'élèves qui en font la demande la liste des parents d'élèves de l'école ou de l'établissement scolaire mentionnant leurs noms, adresses postale et électronique, à la condition que ceux-ci aient donné leur accord exprès à cette communication.
  10. Jan 2020
    1. RRID:ZFIN_ZDB-ALT-011017-8

      DOI: 10.7554/eLife.42881

      Resource: (ZFIN Cat# ZDB-ALT-011017-8,RRID:ZFIN_ZDB-ALT-011017-8)

      Curator: @evieth

      SciCrunch record: RRID:ZFIN_ZDB-ALT-011017-8


      What is this?

    1. 8
    2. 8
    3. 8
    4. 8
    5. muscular hypotonia
    6. motor development
    7. speech and language development
  11. Dec 2019
  • Jul 2019
    1. Nobel Peace prize

      What did he do to win the noble prize?

    2. We deplore all forms of violence

      They are non-violent

    1. Distribution of antibiotic resistance genes in Vibriofrom Cochin estuary, shrimp farm and seafood
    1. Estimation of bioaccumulation of trace metals inmuscle and gill tissue of fish, Labeo rohita
  • Jun 2019
    1. Post vaccination enhanced expression of activation marker CD69 and Chemokine receptor CCR5 on memory B cells in HBV positive newborns
    1. concentrators (Amicon), and subjected to reduction with 0.0.5 M sodium dithionite. For this an appropriate amount of anaerobically prepared dithionite solution was added to the reconstituted Hb and the reaction mixture was quickly passed through a Sephadex G25 gel filtration column (30 em x 1.5 em) equilibrated with 0.05 M Tris HCI (pH 7.4), in order to minimize the duration of contact of dithionite with the protein. The reduced Hb was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and loaded onto a CM52 column (1 Ocm x 1.5cm) equilibrated with the same buffer. A linear gradient of 150 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) was employed to elute the protein from the column
    2. Construction of mutant a globins
    3. Reconstitution of a globin and rf chain into HbS tetramers
  • May 2019
    1. The reaction mixture contained 0.2 mL of enzyme sample, 0.3 mL of buffer and 0.5 mL of p-nitrophenyl-β-D-glucopyranoside (1.0 mM) prepared in 100 mM buffer as the substrate. The reaction was terminated after 30 min of incubation at 70 °C by adding 2 mL of sodium carbonate-bicarbonate buffer (0.1 M, pH 10.0). The liberation of p-nitrophenol was measured at 400 nm and its yield was determined using a standard curve of p-nitrophenol (1-10 μg mL-1) prepared in sodium carbonate-bicarbonate buffer
    2. β-Glucosidase
    3. The activities ofβ-xylosidase, xylan acetylesterase and arbinofuranosidase were measured using 1 mM p-nitrophenylxylopyranoside, p-nitrophenylacetate and p-nitrophenylarabinofuranoside, respectively prepared in sodium citrate buffer (0.1 M, pH 7.0). One mL of reaction mixture containing 0.2 mL of crude enzyme solution, 0.3 mL of sodium citrate buffer (0.1 M, pH 7.0) and 0.5 mL of substrate was incubated at 80 °C for 30 min. The reaction was terminated by adding 2 mL sodium carbonate-bicarbonate buffer (1.0 M, pH 10.0). The activities were determined using p-nitrophenol standard curve (1-10 μg mL-1) drawn using absorbance values measured in spectrophotometer at 400 nm. One unit of the enzyme is defined as the amount of enzyme that liberates 1μmole of p-nitrophenol mL-1min-1 under assay conditions.
    4. Assays for β-Xylosidase, acetylesterase and arbinofuranosidase
    5. 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.
    6. Enzyme Assays
    7. A stock solution of xylose (1 mg mL-1) was prepared in distilled water. A dilution series ranging from 100-1000 μg mL-1 was prepared from the stock solution. To 1 mL of solution, 1mL of DNSA was added and kept in a boiling water bath for 10 min and then 400 μL of sodium potassium tartrate solution was added and kept it for cooling. The absorbance was recorded in a spectrophotometer (Shimadzu, UV-VIS) at 540 nm
    8. The clear cell-free supernatants were used as the source of crude recombinant xylanase.
    9. Preparation of standard curve of xylose
    1. Tris, pH 7.4, 1 mM dithiothreitol, and 10% glycerol. Protein concentration w~s detennined by densitometry analysis of Commassie stained gels. Protein samples were stored at -70°C until further use
    2. To facilitate the expression of recombinant GST-CDPK4 or its mutants, the desired regions of enzyme were PCR amplified using pGEMT-PfCDPK4 as template and PCR primers which possessed overhangs for XhoI and SmaI restriction enzymes (see List II). Often, the PCR products were cloned in TA cloning vector pGE¥T-I easy. Clones in pGEMT-easy vectors were digested with appropriate restriction enzymes to release the inserts. The released inserts were cloned in expression vector pGEX4T-l to facilitate the expression of recombinant proteins. In some cases, the PCR products were digested directly with restriction enzymes and ligated into expression vectors. The plasmid DNA for expression was used to transform E. coli BL21-RIL (Stratagene) strain for the expression of GST-PfCDPK4 and its mutants. Protein expression was induced by overnight incubation of cells with O.lmM IPTG at 18-20°e. Subsequently, cell pellets were suspended in ice cold lysis buffer, contaiJ;1ing 50 mM Tris, pH 7.4, 2 mM EDTA, 1 mM dithiothreitol, 1% TritonX-100, and protease inhibitors (lmM phenylmethylsulfonyl fluoride, 10~g/ml leupeptin, 1 O~g/ml pepstatin) and sonication was performed for 6 cycles of one minute each. The resulting cell debris was removed by centrifugation at 20,000g for 40 min at 4°C. Fusion proteins from the cell lysates were affinity-purified using glutathione-sepharose resin (Arnersham). Briefly, after the protein binding, the resin was washed with lysis buffer, and bound proteins were eluted with 50 mM Tris, pH 8.0 with 10 mM glutathione. Finally, purified proteins were dialyzed against 50 mM
    3. xpression and Purification of Recombinant GST (Glutath ion e-S-Transferase) fusion PfCDPK4 and its mutant
    4. For synchronization, mostly ring stage parasites (10 to 12 h post-invasion) wen~ used. The parasite culture was centrifuged at 200g for 5min and the supernatant was discarded. To the pellet, 4 ml of 5% sorbitol was added, mixed gently and incubated for 15min at 37°C. The mix was shaken 2 or 3 times and centrifuged at 200g followed by washing 3 times in complete medium (list I). The culture was then maintained at 5% hematocrit in a 37°C incubator
    5. orbitol-synchronization 0/ P./alciparum
    1. THP-1 macrophages and human peripheral blood monocyte derived macrophages were transfected with SMARTpool Bcl-2 siRNA (15 pmol), or ER-a siRNA (100 pmol), or ER-~ siRNA (100 pmol), or with negative control siRNA (15 pmol or 100 pmol) using TranspassR2 transfection reagent. Prior to transfection, the cells were depleted of serum by washing 2x with serum-free media. The transfection complex was prepared by diluting 0.5 J!L of transfection reagent A and 1.0 J!L of transfection reagent B to 400 J!L of serum-free media and siRNA's were added to the mix at an appropriate concentration and incubated for 20 min at room temperature. The formed transfection complexes were transferred gently using a large bore pipette tip to 105 cells/well grown in 24 well plates and incubated for 6 h, following which fresh complete medium was added. Transfection efficiency was estimated by observing Cy3-fluorescence of the negative control siRNA with a Nikon TE2000E fluorescence microscope using a tetramethyl rhodamine filter (530-580 nm). For all transfections, target protein knockdown was assessed 24 h after transfection by probing extracts oftransfected cells on Western blots using appropriate antibodies
    2. siRNA transfection
    1. resuspended in 0.2 ml TE, and extracted successively with phenol, phenol-chloroform, and chloroform. In the aqueous phase, 0.25 volume of J 0 M ammonium acetate and two volumes of chilled ethanol were added and the mixture was incubated at room temperature for 5 min. to precipitate the plasmid DNA. The pure plasmid DNA was recovered by centrifugation at J 2,000 g for J 0 min. at 4 °C, washed with 70 %ethanol, dried and resuspended in TE buffer (pH 8.0). The amount and the purity of the DNA was done spectrophotometrically by recording the absorbance at 260 nm.
    2. Plasmid DNA was prepared by alkaline lysis method of Ish-Horowicz ( 1981 ). 5 ml cultures were grown as described for small scale plasmid preparation. 0.5 ml from the growing culture was inoculated into 250 ml of LB containing ampicillin. The culture was grown for 12 h at 37 °C with vigorous shaking, centrifuged at 3000 g, at 4 °C, for 15 min. and the bacterial pellet was resuspended gently in 1 0 ml TEG buffer. The mixture was incubated at room temperature for 10 min., followed by addition of 20 ml of freshly prepared alkaline-SDS solution. The contents were mixed by inversion and the mixture was kept on ice for 10 min., followed by the addition of 15 ml of chilled potassium acetate solution. The contents were mixed by inverting the tube, and incubated on ice for 10 min. The lysed cell suspension was centrifuged at 5000 g, at 4 °C, for 20 min. The supernatant was taken, and nucleic acids we~,-.. precipitated by adding 0.6 volume of chilled isopropanol. The mixture was incubated on ice for 10 min. followed by centrifugation at 5000 gat 4 °C, for 10 min .. The pellet was washed with 70% ethanol, dried and resuspended in TE buffer. The plasmid DNA was purified further to remove the contaminating proteins and RNA following the PEG purification protocol as described by Sambrook et al ( 1989). Equal volume of chilled 5 M lithium chloride solution was added to DNA . suspension, mixed well and incubated on ice for 10 min. The precipitate was removed by centrifugation at 10,000 g at 4 °C, for 10 min. DNA was precipitated from the supernatant by adding equal volume of isopropanoL The mixture was centrifuged at 10,000 g for 10 min. at 4 °C and the pellet was washed with 70% ethanol. The DNA thus obtained was incubated in TE buffer containing 20 J.tg/ml of DNase free RNase A for 30 min. at room temperature. Afterwards, equal volume of 1.6 M NaCl containing 13% (w/v) PEG 8000 was added to DNA solution. The contents were. thoroughly mixed and centrifuged at 10,000 g, at 4 °C, for 10 min .. The pellet was
    3. Large Scale Plasmid DNA Preparation
    1. administered as and when required. Animals were put on continuous mating with males of proven fertility after administration of the three primary injections and monitored for menstrual cyclicity and conception. Ab titres were determined as described above except that anti-monkey HRPO conjugate was used as the revealing Ab.
    2. Female bonnet monkeys (Macaca radiata) reared at the Primate Facility (Nil, New Delhi) were selected and serum progesterone levels were estimated for atleast three months in samples which were collected biweekly. Animals showing atleast two consecutive normal ovulatory peaks (serum progesterone levels >2 ng/ml) (Bamezai, 1986) were selected for fertility trials. Five animals (MRA 375, 515, 640, 672, 770) immunized with 250 Jlg equivalent of r-bZP3, expressed in SG I3009[pREP4] cells, conjugated to DT, was emulsified with Squalene and Arlacel A, adjuvants permitted for human use, in a ratio of 4: I and administered intramuscularly at two sites. In addition, the primary dose also contained I mg/animal of SPLPS as an additional adjuvant. Animals were boosted at intervals of 4-6 weeks depending on the Ab titers with 250 Jlg of r-bZP3-DT using Squalene and Arlacel A as adjuvants. A second group of 3 monkeys (MRA 384, 502, 661) were immunized using a slightly different protocol. The primary immunization consisted of 125 J.lg of r-bZP3-DT and 125 J.lg of r-bZP3-TT (expressed in BL2I(DE3) cells) using the same adjuvants and immunization protocols mentioned above except that boosters were administered alternately with 250 Jlg of r-bZP3-DT or -TT conjugates using Squalene and Arlacel A as adjuvants. Following completion of the primary immunization and 2 boosters at monthly intervals, bleeds (1-2 ml) were collected biweekly from the antecubital vein for estimation of progesterone levels and Ab titres. Boosters were
    3. Immunization of Female Bonnet Monkeys
    4. Radiolabeling of Recombinant Proteins in Infected S/9 Cells
    5. Peptide antisera were generated in the laboratory against peptides PI, 23-45 aa residues with an extra lysine at the N-terminus (KQPFWLLQGGASRAETSVQPVL VE), P2, 300-322 aa residues (CSFSKSSNSWFPVEGPADICQCC) and P3, 324-347 aa residues (KGDCGTPSHSRRQPHVVSQWSRSA) corresponding to bZP3 precursor protein in rabbits and were used to determine their reactivity with the r-bZP3 protein expressed in E. coli in an enzyme linked immunosorbent assay (ELISA). Microtitration plates were coated with 200 ng of r-bZP3 or I J.tg/well of the peptide. HRPO conjugated goat anti-rabbit Ig at I :5000 dilution was used as revealing Ab.
    6. Reactivity with Anti-peptide Sera
    7. The bZP3 sequence was analyzed using PCgene and Lasergene DNA and protein analysis softwares. The alignment of the bZP3 aa sequence with the homologous sequences from other species was carried out using the Cluster V Multiple Alignment Programme (Higgins and Sharp, 1989).
    8. Analysis of Sequence
    1. Transformation was performed in chilled 1.5 ml eppendorf tubes, using 200 ul of competent cells and about 50 ng of ligated plasmid DNA. Frozen competent cells were thawed in ice and the DNA was added immediately after thawing. The DNA volume was always kept under 30 ul. The DNA was mixed well with the cells by gentle tapping, and the tube incubated in ice for 3 0 minutes with occasional gentle shaking. The tube was then immersed in a 42°C water bath for 2 minutes, to give a heat shock to the cells. The cells were then incubated in ice for 10 minutes. Next, 1 ml LB was to the cells, and the cells incubated in a 37°C water bath without shaking, for one hour. 50 ul aliquots were plated in triplicate from the transformed cell mixture on suitable antibiotic containing agar plates and incubated 0/N at 37°C to select the transformants. In case of JM105 cells, the transformed cells were plated on antibiotic containing agar plates on which 50 ul of 2 % X-gal ( made in dimethyl formamide ) , and 10 ul of 100 mM IPTG had been spread in advance, to select for the lac-phenotype. The lac-colonies appeared colourless while the lac+ colonies were blue. For each batch of transformations, a negative control was included in which no DNA was added to the cells while keeping the rest of the procedure the same as for the test transformations.
    2. Transformation procedure.
    3. were stored at -70°C for at least six months without any significant loss in the competence.
    4. A single ~.coli colony taken from an agar plate was used to inoculate 10 ml of LB and incubated 0/N at 37°C in an incubator-shaker. Next day, 0. 5 ml of this freshly grown culture was used to inoculate 100 ml of LB in a 500 ml flask. The culture was incubated at 37°C in an incubator -shaker and absorbance of the growing culture was monitored at 620 nm. When the A620 reached 0. 4 -0. 5 ( in about 120 -150 minutes), the flask was rapidly chilled by shaking in ice. The cells were harvested in sterile, chilled centrifuge bottles at 4, ooog for 10 minutes at 4 °c. The pellet was gently resuspended in 50 ml sterile, ice cold 100 mM cacl2 and the cells incubated in ice for 30 minutes. The cells were again centrifuged as above and the pellet resuspended in 6.5 ml of sterile, chilled, 100 mM cac12 containing 15 % glycerol. The cells were resuspended very gently, and a 200 ul aliquot was transformed with a standard plasmid DNA to check the competence of the cells. Meanwhile, the rest of the competent cells were incubated in ice for 16 -18 hours, to increase the competence of the cells a further few fold. After ascertaining high transformation efficiency of the competent cells, the cells were dispensed as 200 ul aliquots into prechilled, sterile 1.5 ml eppendorf tubes. These cells
    5. Preparation of competent E.coli cells.
    6. All glassware I plasticware used for transformation procedure was sterile and prechilled.
    7. Transformation of E.coli.
    8. All antisera were obtained from the reagent bank at National Institute of Immunology, New Delhi.
    9. Antisera.
    1. collected by centrifugation at 12,000 X g for 15 min, and washed with 70% ethanol. The pellet was air-dried and resuspended in 20 Jll TE. The clones were checked for the pres~nce of the insert by restriction analysis. The digestion products were checked on 1% agarose gel for the release of the insert. One positive clone was selected from each set of transformations and the plasmid DNA was purified in large amount for the insert preparation.
    2. Transformants picked following blue-white selection were inoculated in 5 ml LB medium containing 100 j...tg/ml ampicillin (LBamp) and grown 0/N. Following day, 1.5 ml aliquots of 0/N culture were harvested by centrifugation at 10,000 X g in a microfuge. The supernatant was discarded and the pellet was resuspended in 100 j...tl of chilled TEG (25 mM Tris-Cl, pH 8.0, 10 mM EDTA and 50 mM glucose) and incubated for 10 min at RT. After incubation, 200 j...tl of freshly prepared alkaline-SDS (0.2 N NaOH, 1% SDS; sodium dodecyl sulfate) was added and the contents were mixed gently by inversion. This was followed by incubation on ice for 10 min. Post-incubation, 150 j...tl of ice-cold sodium acetate solution (3 M, pH 5.2) was added to the mixture and incubated on ice for 15 min. After incubation, the contents were centrifuged at 12,000 X g for 15 min at 4°C and the supernatant was carefully transferred to a fresh tube. DNA was precipitated by adding 0.6 volumes of isopropanol and incubating at RT for 10 min. The DNA pellet was obtained by centrifugation at 12,000 X g at RT for 15 min, air-dried and dissolved in 200 j...tl of TE. To remove RNA contamination, 50 j.lg of DNase free RNase was added and incubated for 1 h at 37°C. Plasmid DNA was then extracted once with an equal volume of phenol equilibrated with TE (I 0 mM Tris, pH 8.0 and 1 mM EDT A) followed by extraction with phenol : chloroform : isoamyl alcohol (25 : 24 : 1) and then with chloroform : isoamyl alcohol (24 : 1 ). DNA was precipitated by addition of 2 volumes of chilled 100% ethanol to the aqueous phase and incubating the contents at -70°C for 30 min. The DNA pellet was
    3. Small scale plasmid DNA isolation and restriction
    1. ITALYusinga96wellmicrolitreplatereadbyELISAmicroplatereader(modelEL3/Sx,BioTeKInstrumentsINC).Thefinalsolutionwasreadatawavelengthof450nm.Theplasmacortisolconcentrationwascalculatedbasedonaseriesofstandards.
    2. PlasmacortisolwasmeasuredbyadirectimmunoenzymaticdeterminationofcortisolkitmanufacturedbyEquiparSriviaG.Ferrari,21/N-21047,SARONNO
    3. Plasmacortisol
    4. phosphoricacidformedisreducedbytheadditionof1-amino2napthol~4-sulphonicacid(ANSA)reagenttoproducethebluecolor.Theactivityofthebluecolorwasreadat680nmagainstreagentblankusingaU.V.Spectrophotometer.Suitablestandardswererunthrougheachbatchofassays.Theenzymeactivitywasexpressedintermsofpgofinorganicphosphorusformedhr'1mg'1protein.
    5. Aftereffluentexposure,thecontrolandeffluentexposedfishtissueswereremovedandplacedinabeakercontainingice-coldSEIbuffer(300mMsucrose,200mMNa2EDTA,50mMimidazole,pH7.23)foranalysisofNa+-K+ATPaseactivity.ThetissueswereimmediatelyfrozeninliquidN2andstoredat-80°Cuntilanalyzed. Thespecificactivitiesofsodium,potassium,magnesiumandcalciumdependentATPaseswereassayedaccordingtothemethodsdescribedbyWatsonandBeamish(1980)and Boeseetal.(1982).AdenosinetriphosphatasecatalysestheconversionofATPandADP.Duringthisconversion,onemolecule ofphosphorusisliberated.ATPaseAdenosinetriphosphate^...^Adenosinediphosphate+PTheinorganicphosphorusliberatedwasassayedaccordingtothemethodofFiskeandSubbarow(1925).Inthismethodtheproteinisprecipitatedwithtrichloroaceticacid.Theproteinfreefiltrateistreatedwithaceticacidmolybdatesolutionandthe
    6. Na+K+ATPase,Mg2+ATPaseandCa2+ATPase
    7. ThecircadianrhythmofbimodalO2uptakeofcontrolandeffluenttreatedfisheswerestudiedseparatelyat28°±1°C.TheamountsofO2extractedfromwaterandairwereseparatelydeterminedforadayatregularintervalsof3hreach.TotalO2uptakeateachtimewasobtainedbysummingupthevaluesforaquaticandaerialrespirationobtainedatthecorrespondingtime.Throughoutthepresentstudy,theinitialO2contentofthewaterwaskeptconstant(6±0.5mgF1)
    8. CircadianrhythmofbimodalO2uptake
    9. Forstudyingtheaerialrespirationoffishesinair,respirometersweredesignedinvolvingtheprinciplesofmonometrictechniques.Thesetup(Figure10and11)consistsofarespiratorychamberconnectedtoagraduated‘U’tubecontainingBrodie’sfluid.KOHisusedasCO2absorbent.Thedifferenceinthelevelofthefluidinthemanometerforagiventimeisusedinthefollowingequationandthegasutilizediscalculated.VixhV=-...........-10,000Where,‘V’isthevolumeofthegasutilized‘Vi’isthevolume ofgasintherespiratorychamber‘h’isthedifferenceintheleveloftheBrodie’sfluidinthemanometerand10,000isthepressureofmanometricfluid(Brodie’sfluid)inmm
    10. AerialRespiration
    11. Theexperimentalsetup(Figure8and9)forthedeterminationofO2uptakesimultaneouslyfromairandwaterwassimilartothatusedearlierbyNatarajan(1972),Rani(1994)andVijayalakshmi(1996).Aclosedglassrespirometerof5litrecapacitywasfilledwith3.5litrefreshtapwater.Athermocolfloatwithasemicircularholeatitsperipherywasplacedoverthewater,whichseparatedtheair-waterinterphaseoftherespirometer.Theair-phaseoftherespirometerwasattachedtoafluidmanometer.Asthefishcomestothewatersurfaceandtakesair-gulp,thereisapressurechangeintheair-phasecausinganimbalanceinthemanometricfluid.AgraduatedsyringefilledwithpureO2(takenfrommedicalO2cylinder)isusedtorestoretheimbalanceofthemanometricfluid.TheamountthusneededshowstheaerialO2uptakeofthefish.TheexpiredCO2wasabsorbedbythepelletsofKOHinthepetridishoverthemanometricfluid.Theconcentrationofdissolvedoxygenoftheambientwaterwasestimatedbefore andaftertheexperimenttomeasuretheaquaticO2uptakebythefish.ThedifferenceintheDOandtheamountofwaterindicatestheactualaquaticO2uptake.Winkler’svolumetricmethod(Welch,1948)wasusedtoestimatetoDOofthewatersamples.Darkenedrespiratorychamberswereusedwithdimensionsthatwereclosetothoseofthefishinorderthatthefishshouldremaininmoreorlessthesamepositionbut havesufficientroomtomoveitsopercula.Theflowofwaterthroughtherespirometer wasregulatedandmeasuredbymeansofaflowmeter.APhilipsO2electrode(PI1056)waskeptinawaterjacketmaintainedatthesametemperatureastheclosedcirculation.SamplesoftheinflowandoverflowwatercouldalsobeledovertheO2electrode
    12. Bimodalrespiration
    13. Theexperimentalfishwasacclimatedtoglassrespirometersforabout24hrandtheywerenotgivenanyfoodduringthisperiod.TheeffluentexposedfishesalongwiththecontrolsweresubjectedtoO2consumptionseparately.Theexperimentswereperformedinaninsulatedroombetween8to10AMwithlightson.TherateoftotalO2uptakethroughgillsfromflowingwaters(DO=7.2mg021'1)wasmeasuredinfishesofdifferent body weights.Forthis,acylindricalglassrespirometerof2litrecapacitywasused.Thefishwasintroducedintherespirometerwhichwasconnectedtoalargeconstantlevelwatertanktomaintaintheflowofwaterunderconstanthydrostaticpressure.Thewaterenteredtherespirometeratonesideanditsflowperminutewasmeasuredasitlefttheotherside.Theflowwasadjustedaccordingtothesizeofthefish.Thefishwasacclimatizedtotherespirometeratleast12hrbeforereadingswere taken.ConcentrationofdissolvedoxygeninthesampleswasmeasuredbyWinkler’svolumetricmethod(Welch,1948).ThedifferenceinO?levelsbetweentheambientwaterandthatsuppliedtotherespirometeraswellaswiththerateofwaterflowandtheweightofthefishwasusedtocalculatetherateof O2uptakeintermsoftime(ml02hr'1)withthehelpoftheequation:V02=Vw(Ci02CE02)Where,VO2=02uptake(ml02hr'1)Vw=water(mlm'1)andCi02-CE02respectivelythe02concentrationofinletandoutletwaters.Arespirometercontainingnoanimalsservedasacontrolforadjustingcalculationsfor02uptakeinthewater.Uponremoval,fisheswereblottedwithpapertoweling, andweighed
    14. Aquaticrespiration
    15. Theeffectof2%,5%and7%effluentexposureontheoxygenuptakewasmeasuredatexperimentalconditions,viz.,(a)whenaccesstoairwasprevented(aquaticconsumption),(b)whenitwasallowed(bimodalrespiration)and(c)underaerialconditions(aerialrespiration)
    16. Effectofeffluentexposureontheoxygenconsumption
    17. Respirationstudies
    1. All the cell lines were grown and maintained in Dulbecco' s modified Eagle's medium (DMEM) with 10% Fetal bovine serum (FBS) and 1% antibiotic-antimycotic (penicillin, streptromycin and amphotericin B). The cells were maintained at 37<>C with 5% C02 in a humidified CD2 incubator (Nuaire-IR Autoflow CD2 Water-Jacketed incubator)
    2. ell culture media and cell lines
    1. appropriate secondary antibody (conjugated with horse-radish peroxidase) diluted in 5% fat free milk solution (in PBST) and incubated for 45 minutes at room temperature. After incubation the membrane was washed and processed for the detection of protein bands using ECL-plus detection reagent (Amersham Biosciences) followed by detection of signal on X-ray film (Hyperfilm-ECL, Amersham Biosciences)
    2. The proteins were resolved using denaturing SDS-PAGE gel and after completion of the run, the gel was over laid on a nitrocellulose paper cut to the size of gel and kept in the blotting cassette in the presence of blotting buffer. Finally the cassette was put in the mini transblot apparatus (Bio Rad) and blotting was done for 4 hours at a constant voltage of 60 V. Then the membrane was taken out and rinsed in PBS containing 0.1% Tween - 20 (PBST) for 5 minutes by gentle shaking. Later the membrane was immersed in 5% non-fat milk solution in PBST with gentle shaking for 1 hour at 37°C. The membrane was washed off from the traces of the fat free milk with PBST and the membrane was over laid with primary antibody diluted in PBST for 3 hours at 4°C with shaking. After incubation the membrane was washed with PBST and layered with
    3. Immunobloting
    1. f. 5 μl of water was then spotted on each spot for 30 sec and removed using Whatman filter paper strips. This step was repeated once. g. 1-2 μl of SAP matrix was then applied to each spot and allowed to dry. h. The chip was then placed in the SELDI machine
    2. a. 5 μl of 10 mM HCl was added to each spot on the chip and removed after 5 min. using Whatman filter paper strips. b. Washing was given by spotting 3 μl of water for 30 sec on each spot followed by removal using Whatman filter paper strips. This step was repeated two times. c. 10 μl of low stringency/ high stringency buffer was then added to the spot and kept in humid chamber for 5 min. followed by removal using Whatman filter paper strips. d. 3 μl of sample prepared in low stringency/ high stringency buffer was then added to the spot and incubated in humid chamber for 30 min. e. Washed the spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH 7.0 for 30 sec and removed using Whatman filter paper strips. This step was repeated five times.
    3. Activation of CM10 (weak cation exchange ) array