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  1. May 2019
    1. Reverse transcriptase “Superscript III” (Invitrogen, 18080-051) was used to perform cDNA synthesis. Briefly, 500 ng of DNase I-digested RNA was incubated with 1 μl of 10 mM dNTP and 50 μM oligo(dT) at 65°C for 5 min in a 10 μl reaction mixture followed by cooling on ice for 5 min. Post incubation, 10 μl of cDNA synthesis mixture was added which contained 2 μl of 10XRT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, 1 μl of RNase out (40 units) and 1μl of Superscript III (200 units). Tubes were incubated at 50°C for 1 h and thereaction was terminated at 85°C for 5 min. The quality of synthesized cDNA was checked by using it as a template in a PCR reaction to amplify the housekeeping gene CgACT1. Amplification of CgACT1was indicative of proper cDNA synthesis
    2. Deoxyribonuclease I (DNase I) enzyme (Invitrogen) was used to remove the DNA contamination from RNA samples,if any. Briefly,1 μg of RNA was subjected to DNase I digestion by using 1 U of DNase I in a 10 μl reaction mixture which contained 1X DNase I buffer and appropriate volume of water. The reaction mixture was incubated at room temperature for 15 min. Post incubation,to inhibit DNase I enzyme activity,1 μl of 25 mM EDTA was added to the reaction mixture and tubes were heated at 65°C for 10 min. DNase I-digested RNA samples were used as template to perform PCR for the amplification of CgACT1geneandabsence of amplification product was used as criterion toconfirmproper DNase I digestion and lackof DNAcontaminationin the RNA sample
    3. microcentrifuge tube. For precipitation of RNA, 1/10thvolume of 3 M sodium acetate (pH 5.3) and 2.5 volume of 100% ice-coldethanol was added. In order to facilitate precipitation, tubes werekept at -20°C for 20 min. Tubes were centrifuged at 13,000 rpm for 10 min in a refrigerated centrifuge. The RNA pellet was washed with 70% ethanol,resuspendedin 100-200 μl of nuclease-free water and stored at -20°C untiluse.Care was taken to keep allreagents and tubes on ice to maintain the cold temperature throughout theRNA extractionprocess
    4. All reagents required for RNA extraction were preparedin DEPC-treated water. RNasecontamination from non-autoclavable items wasremoved by wiping them with RNaseZap® (Ambion). Total RNA from yeast cells was extractedusing acid phenolextractionmethod. Briefly, yeast cells were grown underappropriate conditions and at suitabletime points,cells were harvested by centrifugation at 4,000 rpm for 5 min. The cell pellet was washed twice with ice-cold DEPC-treated water, resuspended in 350 μl of AE buffer and transferred toa1.5 ml microcentrifuge tube. To this,40 μl of 10% SDS and 400 μl of acid phenol (pH 4.3) was added. The cell suspension was mixed well by vortexing thrice, short pulsesof10 seconds each,and incubated at 65°C for 15 min with continuous agitation at 800 rpm. Post incubation, cells were kepton ice for 5 min and centrifuged at 13,000 rpm in a refrigerated centrifuge set at 4°C for 10 min. After centrifugation, aqueous layer was transferred to a new1.5 ml microcentrifuge tube and 400 μl of chloroform was added. Tubes were mixed well by gentlyinverting them 4-5 times and centrifuged at 13,000 rpm for 10 min. The aqueous layer was separated and transferred to a new1.5 ml
    5. This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use
    6. Yeast genomic DNA was isolated by mechanically lysing the yeast cells. Briefly, 10 ml of overnight grown yeast culture was transferred toa 15 ml centrifuge tube andcells were spun down at 4,000 rpm for 5 min. Media was decanted and cells were washed with 10 ml sterile water. Washedcells were resuspended in 500 μl of Buffer A and transferred to a 1.5 ml microcentrifuge tube. Tubes were incubated at 65°C for 15 min. Post incubation,500 μl of PCI (25:24:1) solution was added. To this, 0.5 g of 0.5 mm glass beads were added and cells were lysed mechanically in a bead-beatinghomogenizer (MP Biomedicals,FastPrep®-24) thrice, 45 seceach, with intermittent cooling on ice. Tubes were spun at 12,000 rpm for 5 min and the aqueous layer was transferred to a new 1.5 ml microcentrifuge tube. To this, 500 μl of PCI solution was addedand mixed gently by inverting the tubes.Tubes were centrifuged again at 12,000 rpm for 5 min and aqueous layer was transferred to another 1.5 ml microcentrifuge tube. Next, 2.5 volume of absolute ethanol was added to the aqueous layer, mixed well and centrifuged at 13,000 rpm for 10 min. Supernatant was decanted and the DNA pellet was washed once with 70% ethanol and centrifuged at 13,000 rpm for 10min. Washed DNA pellet was air-dried and dissolved in 100-200 μl of 1X TE buffer by gently tapping the tubes

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    7. E. coliDH5α ultra-competent cells were used for all bacterial transformations. Briefly, frozen DH5α ultra-competent cells were taken out from -80°C freezerandthawed on ice for 15 min. DNA to be transformed was added to the bacterial cell suspension and incubated on ice for 30 min. For transforming ligation mixtures and plasmids,5-10 μl and 100-500 ng of DNA was used, respectively. Followed by 30 min incubation on ice, heat shock was given for 60-90 sec at 42°C in a water bath and cells were immediately kept back onice for 2 min. To this,1 ml of sterile LB medium was added and tubes were incubated inashaker incubator set at 37°C, 200 rpm for 45 min.Next, cells were spun down and resuspended in 500 μl of LB medium. About 100-200 μl of resuspended cells were plated on LB-agar medium containing appropriate antibiotics and incubated for 12-16 h at 37°C. Transformants were purified on LB-agar plates containing appropriate antibiotics andpositive transformantscarrying desired DNAwere verified by PCR, restriction digestion and sequencing analyses
    8. ml YPD broth at an initial OD600of 0.1. Cultures were allowed to grow for 4-5 hin a shaker incubator setat 30°C, 200 rpm until the OD600of the cultures reached 0.4-0.6. Next,cells were harvested ina15 ml centrifuge tube by centrifugation, washed twice with 10 ml of sterile water, resuspended in 1 ml of sterile water and were transferred to a 1.5 ml microfuge tube. Cells were harvested by centrifugation at 4,000 rpm for 5 min,resuspended in 50 μl of100 mM lithium acetate solution and transformation mixture was added. Transformation mixture consisted of 240 μl polyethylene glycol (50%), 36 μl of lithium acetate (1 M), 5 μl of heat denatured single stranded carrier DNA (10 mg/ml), 500 ng to 1 μg of transforming DNA and final volume was made to 360 μl with sterile water. The tubes were incubated at 30°C for 45 min. To this, 43 μl of sterile DMSO was added and heat shock was given at 42°C for 15 min. Next, tubeswere transferred to ice for 10-15 sec, centrifuged at 4,000 rpm and transformation mixture reagents wereremoved completely by pipetting. Cells were resuspended in 200 μl of sterile water and spread-plated on appropriate selection medium. Plates were incubated at 30°C for 24-48 h
    9. Yeast transformation was performed as described previously (Gietz et al., 1992) with fewmodifications. Briefly, overnight grown C. glabratacultures were freshly inoculated in 10
    10. To phenotypically characterize C. glabratamutants,serial dilution spot growth assays were performed. Briefly, the optical density of overnight-grown C. glabratacultures wasnormalized to OD600of 1.0andnormalized cultures were further diluted 10-fold in 1X sterile PBS five times. 3 μl of serially diluted culture were spotted on test plates. Plates were incubated at 30°C (unless mentioned otherwise) for 24-48hand growth was recorded by capturing plate images. For experiments involvingchecking theability of mutants to utilize non-fermentable carbon sources,growth was scoredafter 6-7 days of incubation
    11. For time course growth analysis,C. glabratacells were inoculated in appropriate medium and grown for 14-16 h. Followed by overnight growth,yeast cells were sub-cultured in test medium at an initial OD600of 0.1 and growth was monitored by recording the absorbance of the culture at 600 nm at regular time-intervals till 72h. Absorbance values were plotted with respect to time and generation time of yeast strains were calculated between 4-8 h of growthwhen cells were in logarithmic phase of growth using following equation.Generationtime(G)=T2−T1Xlog2logNf/NiG = Generation time (h)T1 = Initial time point taken for analysisT2 = Final time point taken for analysisNf= Number of cells at time T2 (1 OD600of C. glabratacorresponds to 2 X 107cells)Ni = Number of cells at time T1 (calculated from OD600 value as mentioned above)For CFU-based viability assays, appropriate dilutions of yeast cultures were prepared in 1X sterile PBS and suitable volume of diluted cultures wasplated on YPD-agar medium. Plates were incubated at 30°C for 24-48 h and viable colonies that appeared on YPD-agar plate were counted.To obtain logarithmic phase cells, overnight-grown C. glabratacell suspension was inoculated in appropriate medium at an OD600of 0.1 and grown ina shakerincubator set at 30°C, 200 rpm for approximately 4 htill the culture density reached OD600of 0.4-0.6
    12. C. glabratastrains were routinely grown in rich YPD medium or synthetically defined YNB medium, or YNB medium supplemented with CAA, unlessstatedotherwise.To obtain overnight grown liquid cultures, C. glabratacells were inoculated in appropriate medium and incubated at 30°C under constant agitation (200 rpm) to maintain proper aeration.To revive the frozenstocks,about one tipfull of frozen culture was streaked either on YPD-agar or on CAA-agar medium. In general, frozen stocks of C. glabratastrains were revived on YPD-agar medium.However,C. glabratastrains harbouring plasmidscontainingURA3as a selectable marker were revived on CAA-agar medium. After streaking, plates were allowed to grow for 24-48 h at 30°C and were stored at 4°C for a maximum period of two weeks. For long term storage, freezer stocks of C. glabratastrainswere prepared in 15% glycerol and stored at -80° C.Escherichia colistrain DH5αwas revived on LB-agar medium from frozenstock and incubated at 37°C for 14-16 h. DH5α strainwas used for transformation purpose and maintaining plasmids. Bacterial strains harbouring plasmids containing selection markerswere revived on LB-agar medium supplemented with appropriate antibiotics.Bacterial liquid cultures were either grown in LB broth or LB broth containing suitable antibioticsand incubatedin a shakerincubator set at 37°C, 200 rpm for 14-16 h. For preparation of bacterial frozenstocks, 1 ml overnight grown bacterial culture was added to500 μl of 50% glycerolto obtain final concentration of ~16 % glyceroland stored at -80°Cuntil use
    13. PMA-treated THP-1 cells were seeded toa24-well tissue culture plate to a cell density of 1 million cells per well and allowed to grow for 12 h. After12 hincubation,spent medium was replaced with fresh pre-warmed RPMI-1640 medium andcells were allowed to recover for 12 h before use.C. glabratacells were grown in YNB medium for 14-16 h at 30°C and 200 rpm. 1 ml of theseC. glabratacells were harvested in 1.5 ml centrifuge tubes, washed twice with 1X sterile PBS and the cell density was adjusted to 2x107cells/ml. 50 μl of this cell suspension was used for infection to a MOI of 1:1. Two hours post infection, wells were washed thricewith 1X sterile PBS to remove the non-phagocytosed yeast cells and 1 ml of fresh pre-warmed RPMI-1640 medium was added. Plates were incubated under tissue culture conditions at 37°C and 5% CO2for 24 h. Supernatants were collected in 1.5 ml microfuge tubes, centrifuged at 3,000 rpmto remove the particulate matter, if any, and stored at -20°C until use. Estimation of different cytokines were performed using BD OptEIA ELISA kits as per the supplier’s instructions
    14. To perform survival analysis of C. glabratacells in macrophages, PMA-treated THP-1 cells were seeded to 24-well tissue culture platesto afinal celldensity of 1 millionper well. C. glabratacells were grown in YNB medium for 14-16 h at 30°C and 200 rpm. 1 mloftheseC. glabratacells were harvested in 1.5 ml centrifuge tubes, washed twice with 1X sterile PBS and the cell density was adjusted to 2x106cells/ml. 50 μl of this cell suspension was used to infect PMA-activated macrophages to a MOI (multiplicity of infection) of 0.1. Two hours post infection, THP-1 cells were washed thrice with 1X sterile PBS to remove the non-phagocytosed yeast cells and 1 ml of fresh pre-warmed complete RPMI-1640 medium was added.At different time points, infected THP-1 macrophages were osmolysed with1 ml sterile water. Post lysis,lysates were collected by scraping the wells using 1 ml microtip. Lysates were diluted in 1X sterile PBSand appropriate dilutions were plated onYPD-agar plates. Plates were incubated at 30°C for 24-48 h and colony forming units (CFUs) were counted. Final CFUs/ml were determined by multiplying CFUs withappropriate dilution factor and percentage phagocytosis was calculated by dividing CFUs obtained at 2 h post infection by total numberofyeast cells used for infection. Fold replication was calculated by dividing the CFUs obtained at 24 h post infection by CFUs obtained at 2 h post infection
    15. phorbol myristateacetate (PMA) (Tsuchiya et al., 1982). For PMA treatment, THP-1 cells were allowed to grow till 70-80% confluence and were collectedin a centrifuge tube by centrifugationat 1,000 rpm for 3 min. THP-1 cell pelletswere resuspended in 4-5 ml of pre-warmed complete RPMI-1640 medium, 100 μl of this cell suspension was appropriately diluted in PBS (1X) and viability was determined by counting trypan blue stained cellsusing hemocytometer. THP-1 cell suspension was diluted appropriately to obtainafinal cell density of 106cells/ml with pre-warmed complete RPMI-1640 medium. PMA was added totheTHP-1 cell suspension at a final concentration of 16 nM and mixed well by gently inverting the tubes. PMA-treated cells were seeded either in 24-well cell culture plates or in cell culture dishes and allowed to grow for 12 h under tissue culture conditions i.e. at 37°C and 5% CO2.After 12 h incubation, spent medium was replaced withfresh pre-warmed complete RPMI-1640 medium and cells were allowed to recover for another 12 h
    16. THP-1 monocytes getdifferentiated intophagocytic macrophages upon treatment with
    17. For cryopreservation of THP-1 and Lec-2 cells, 5-6 million cells wereresuspendedin 0.5 ml of eithercommercially procuredcell preservation medium from GIBCO(12648010)or complete medium supplemented with 10 % fetal bovine serum and 10 % DMSO.Cells were initially kept inanisopropanol bath and werelatertransferred to -70°C freezer. After 2-3 days, frozencells were transferred to liquid nitrogen container till further use. To revive the cells, frozenstockswere taken out of the liquid nitrogen container and immediately transferred to water bath set at 37°Cfor thawing. When freezing medium has thawed completely, cells were transferred to a 100 mm cell culture dishcontaining 12 ml completemedium and incubated under tissue culture conditions at 37°C and 5% CO2for 12 h. Afterincubation, medium was replaced by 12 ml fresh pre-warmed medium and incubated under tissue culture conditions till they reached 70-80% of confluencebefore splitting
    18. Human monocytic cell line THP-1 (ATCC TIB-202TM), derived form 1 year old acute monocytic leukemia patient,wasused to perform single cell infection assays. Differentiation of these THP-1 monocytic cellsto phagocytic cells was induced by using phorbol myristateacetate (PMA)(16 nM). Lec2 (ATCC CRL-1736TM),an ovary epithelial cell line,which has beenderived fromtheChinese hamster Cricetulus griseus, wasused to determine the adherence of C. glabratacells. THP-1 and Lec-2 cells were routinely cultured and maintained in RPMI-1640 and α-MEM media, respectively, supplemented with10 % heat inactivated fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml) andstreptomycin (100 μg/ml). Cultures were maintained in cell culture incubator (Thermo scientific)at 37°C and 5% CO2. Cultures were split after every 2-3 daysandspent medium was replaced with fresh pre-warmed medium. For splitting, cells were harvested at 1,000 rpm for 2-3 min, spent medium wasdiscarded and cell pellet was resuspended in 4-6 ml of pre-warmed medium. Cell density was determined by using hemocytometer. A total of3-4 million cells were seeded in a 100 mm cell culture dishcontaining 12 mlfresh medium
    19. Stripping buffer100 mM β-mercaptoethanol2 % SDS62.5 mM Tris-HCl (pH 6.7)Final volume was madeto 250 ml with water
    20. Transfer buffer (10 X stock solution)0.25 M Tris-HCl (pH8.0)1.92 M Glycine1% SDSThe stock solution was prepared asa10 X concentrate and was diluted to 1 X concentration prior to use.1X Transfer buffer (1 litre)200 ml Methanol100 ml 10X Transfer buffer700 ml WaterTris-Buffered Saline (TBS)50 mM Tris150 mM NaClFinal pHof the bufferwas adjusted to 7.4 with HCl.Blocking buffer5% Fat-free milk0.1% Tween-20Final volume was made to 100 ml with 1 X TBS.Wash buffer (TBS-T)TBS (1 X final concentration)0.1% Tween-20Final volume was prepared with water
    21. 2% DTTThe stock solution of SDS loading buffer was made asa4 X concentrateand was added to the protein sample to the final concentration of 1 X.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSThe stock solution was prepared as a 10 X concentrate and was diluted to 1 X concentration prior to use.Resolving gel mix (12%, 10 ml)3.3 ml H2O4 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix2.5 ml 1.5 M Tris-HCl (pH 8.8)100 μl 10% SDS100 μl 10% Ammonium persulfate (APS)4 μl N,N,N′,N′-Tetramethylethylenediamine (TEMED)Stacking gel mix (5%, 3 ml)2.1 ml H2O0.5 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8)30 μl 10% SDS30 μl 10% APS3 μl TEMED
    22. 2% DTTThe stock solution of SDS loading buffer was made asa4 X concentrateand was added to the protein sample to the final concentration of 1 X.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSThe stock solution was prepared as a 10 X concentrate and was diluted to 1 X concentration prior to use.Resolving gel mix (12%, 10 ml)3.3 ml H2O4 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix2.5 ml 1.5 M Tris-HCl (pH 8.8)100 μl 10% SDS100 μl 10% Ammonium persulfate (APS)4 μl N,N,N′,N′-Tetramethylethylenediamine (TEMED)Stacking gel mix (5%, 3 ml)2.1 ml H2O0.5 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8)30 μl 10% SDS30 μl 10% APS3 μl TEMED
    23. Total cell lysis buffer (Homogenization buffer)50 mM Tris-HCl (pH 7.5)2 mM EDTA10 mM Sodium fluoride*1 mM Sodium orthovanadate*1 X protease inhibitor cocktail (Sigma, P 8215)** Were added fresh before use.SDS-PAGE30% acrylamide solution29 g Acrylamide1 g N,N’-MethylenebisacrylamideDissolved in 100 ml H2O10% Sodium Dodecyl Sulfate (SDS)10 g SDS in 100 ml H2OSDS loading buffer130 mM Tris-HCl (pH 8.0)20% (v/v) Glycerol4.6% (w/v) SDS0.02% Bromophenol blue
    24. Phenol solution saturated with 0.1 M citrate buffer (pH 4.3 ± 0.2)was procured from Sigma (P4682)
    25. Buffer C100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSPhenol:Chloroform:Isoamyl alcohol (25:24:1) solution25 ml Tris-equilibrated phenol (pH 8.0)24 ml Chloroform1 ml Isoamyl alcoholDNA sample loading buffer0.25% Bromophenol blue0.25% Xylene cyanol15% FicollStock solution of the loading buffer was prepared in water as a 6 X concentrate and was added to the sample DNA to the final concentration of 1 X.RNA isolation bufferAE buffer3 M sodium acetate0.5 M EDTA (pH 8.0)Reagents used for RNA isolation were prepared in DEPC-treatedwater and stored at 4°C. For preparationof DEPC-treated water,0.1 ml DEPC was added to 100 ml waterand kept overnight onamagnetic stirrer. Followingincubation,the solution was autoclaved to remove any traces of DEPC.Acid phenol solution
    26. Genomic DNA isolation buffersBuffer A50 mM Tris-HCl10 mM EDTA150 mM NaCl1% Triton-X1% SDSBuffer B50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 M Sorbitol50 mM β-mercaptoethanol (Added freshbefore use)
    27. 10 mg/ml carrier DNA 5 μlAbove-mentioned reagents were added to prepare thetransformation mixture, and the volumes indicated wereused per transformation.500-1,000 ng of desired transforming DNA was added to this transformation mixture and final volume was adjusted to 360 μl with sterile water.Carrier DNA (Sonicated salmon sperm DNA, Stratagene, 201190) washeat denatured at 95⁰C for 10 min and transferred on ice before additionto the transformation mixture.43 μl DMSO was added to each transformation mixture before heat shock.Zymolyase cocktail buffer for yeast colony PCR2.5 mg/ml zymolyase (MP Biomedicals, 0832092)1.2 M SorbitolThe cocktail was prepared in sterile water
    28. Tris-acetic acid EDTA (TAE) buffer40 mM Tris Base0.5 M EDTAFinal pHof the bufferwas adjusted to 8.5 with glacial acetic acid.TAE buffer was prepared as a 50 Xconcentrate and diluted to 0.5X concentration prior to use as agarose gel electrophoresis running buffer and to cast agarose gels.HEPES [4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid] buffer1 M HEPESFinal pHof the bufferwas adjusted to 7.5 with NaOH.HEPES was used as a buffering agent forpreparationof different pHmedium. Buffer was filter-sterilizedby usinga0.22 μm membrane filterand stored at 4°C.INOUE transformation buffer10 mM PIPES15 mM CaCl2.2H2O250 mM KCl55 mM MnCl2.4H2OFor preparation ofINOUE transformationbuffer,above-mentioned solutes were dissolved in appropriate amount in 800 ml of water and then 20 ml of 0.5 M PIPES(piperazine-1,2-bis[2-ethanesulphonic acid])(pH 6.7) was added. Final volume was adjusted to 1 litre with water, buffer was filter sterilized by usinga0.22 μm membrane filter and stored at -20°C. Stock solution of PIPES was preparedseparatelyby dissolving 15.1 gm of PIPES in 80 ml of water, pH was adjusted to 6.7 using 5 M KOH and volume was adjusted to 100 ml.Yeast transformation reagents1 M lithium acetate 36 μl50 % polyethylene glycol 240 μl
    29. Phosphate-Buffered Saline (PBS)137 mM NaCl2.7 mM KCl10 mM Na2HPO42 mM KH2PO4Final pH of the buffer was adjusted to 7.3with 11.6 N HCland volume was adjusted to 1 Lbefore autoclaving.PBS was prepared as a 10X stock solution and diluted to 1 X concentration before autoclaving.Tris-HCl buffer0.5 M Trizma BaseFinal pHof the bufferwas adjusted to 7.6 using 11.6 NHCl.Tris-EDTA (TE) buffer10 mM Tris-HCl (pH 8.0)1 mM EDTATE buffer was prepared as a 10 X concentrate and diluted to 1 X concentration before use
    30. Casamino acid (CAA)0.67% Yeast Nitrogen Base2% Dextrose0.6% Casamino acid
    31. Yeast Extract-Peptone-Dextrose (YPD)1% Yeast Extract2% Peptone2% DextroseYeast Nitrogen Base (YNB)0.67% Yeast Nitrogen Base2% DextroseFor alternate carbon source utilization experiments, dextrose was replacedwith other carbon sources viz.,ethanol, glycerol, oleic acid and sodium acetate.Ethanol, oleic acid and sodium acetate were used at afinal concentration of 2%and glycerol was used at a final concentration of 3%
    32. 2.5 mM KCl10 mM MgCl210 mM MgSO4SOCSOB mediumwas modified to prepare the SOC medium.20 ml of sterile 1 M glucose solution was added to the autoclaved SOB medium to obtainafinal concentration of 20 mM glucosein 1 litre of medium.AntibioticsAmpicillin 60 μg/mlKanamycin 30 μg/mlStock solution of antibiotics (50 mg/ml) were prepared in sterile water. Prior to storage at -20°C,antibioticswere filter sterilizedthrougha0.22 μm membrane filter. Before pouring the plates,antibiotics were added to moderatelywarm LB-agar medium
    33. Luria Bertani (LB)0.5% Yeast Extract1% Tryptone1% NaClSuper Optimal Broth (SOB)0.5% Yeast Extract2% Peptone10 mM NaCl
    34. Allmedia and solutions were sterilizedby autoclaving at 121°C and15 psi for 20 min.For preparation of plates,2% agar was added to the medium before autoclaving.Heat labile components and reagents were filter sterilizedby passing them through a 0.22 μm membrane filter
    35. dipotassium hydrogen phosphate, disodium hydrogen orthrophosphate, acetone and citric acid were obtained from Qualigen Chemicals. Kitused for quantitationof histone deacetylase activity waspurchased from Cayman Chemical Company (#10011563). Hybond-P membranefor protein transferand ECL kit for immunoblottingwere purchased from Amersham Biosciences. Kits used for estimation of cytokines were procured fromBD Biosciences.Medium components used to culture C. glabrataand bacterial strains viz.,yeast extract, peptone, dextrose, casamino acid hydrolysate, yeast nitrogen base with aminoacids and ammonium sulphate, yeast nitrogen base without amino acids, yeast nitrogen base without aminoacids and ammonium sulphate and Luria-Bertani (LB) medium were purchased from BD (Becton, Dickinson and Company, USA). Animal cell culture media RPMI-1640, DMEM and α-MEM were purchased from Hyclone and Gibco-Invitrogen. Fetal bovine serum, glutamine and antibiotics for cell culture medium were procured from Gibco-Invitrogen
    36. Allchemicals used in thisstudy were obtained from commercial sourcesand were of molecular biology grade. The enzymes used for PCR amplification and molecular cloning viz.,restriction endonucleases, T4DNA ligase, Taq DNA polymerase were obtained from New England Biolabs, Fermentas, Sigma and Finnzymes. Kits used for first strand c-DNA synthesis, quantitative real time-PCR were purchased from Invitrogen andEurogentech, respectively.Kits used forplasmid isolation,PCR product purification, reaction clean up, and gel extraction of DNA fragmentswere procured fromQIAGEN.Agarose, phenol, dimethyl sulphoxide (DMSO), sodium acetate, sodium chloride, sodium hydroxide, sodium carbonate, sodium dodecyl sulphate (SDS), trizma base, bathophenanthrolinedisulfonic acid disodium salt (BPS), ferric chloride, formamide, ethylenediaminetetraacetic acid (EDTA), glycerol, polyethylene glycol, hydroxyurea, methylmethane sulphonate (MMS), ammonium persulphate, acrylamide, bis-acrylamide,N,N,N′,N′-Tetramethylethylenediamine(TEMED), diethylpyrocarbonate (DEPC), lithium acetate, polyethylene glycol (PEG), menadione, phorbol myristate acetate (PMA), isopropanol,tween-20, trypan blue, hydrogenperoxide, uracil and orthrophenylenediamine (OPD) were procured form Sigma Chemicals. INVIVO PROTWIN label mix, S35(Met:Cys-65:25) were obtained from from BRIT-Jonaki, CCMB, Hyderabad. Fluconazole was purchased from Ranbaxy.Ferrozine was purchased from HIMEDIA.Hydrochloric acid, sulphuric acid, acetic acid, methanol, potassium dihydrogen orthrophosphate,
    37. Table 2.4: List of antibodies used in thisstudy
    38. All antibodies used in thisstudy, their clonality and dilutions used,Manufacturers’ details,and catalogue numbersare listed in Table 2.4
    39. Oligonucleotides used for generation of C. glabratadeletion strains, for cloning and for quantitative Real time Polymerase Chain Reaction (qPCR)were commercially synthesized either at MWG Biotech Pvt. Limited, Bangalore, India or at Xcelris genomics Pvt. Limited, Ahemdabad, India. All the oligonucleotides used were designed by using freely available online tool Primer 3 plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) and are listed in Table 2.3
    40. All C. glabrataand bacterial strains,and plasmids used in thisstudy are listed in Table 2.1 and Table 2.2, respectively.Table 2.1: List of C. glabrataand bacterial strains used in the study
    1. Statistical analysis was performed using GraphPsssad Prism 5. Data are presented as mean ± SEM. The difference between two groups was analyzed using either a two-tailed Student’s t-test or a nonparametric two-tailed Mann-Whitney test, as appropriate. The differences between multiple groups were analyzed by one-way ANOVA, using Tukey’s multiple comparison test for parametric data and Kruskal-Wallis test for non-parametric data. P<0.05 was considered as statistically significant. The cell numbers used to obtain quantitative data (n) and the number of independent experiments performed is indicated in the respective figure legends
    2. Ip6k1-/-(4 male, 5 female)] were exposed to 4NQO (100 μg/mL) in their drinking water. Mice were allowed free access to drinking water containing the carcinogen, and the water was changed every week.Water bottles containing carcinogen was covered with foil to avoid exposure to light. All the apparatus used for this study and remaining water were decontaminated using 1% sodiumhypochlorite for 30 min. Every week water consumption and weight of the each animal was monitored. Mice displaying any characteristics of weight loss or dehydration before 24 weeks were euthanized and examined for lesions. After 24 weeks, mice were euthanized by CO2inhalation and a complete necropsy was performed. Tissues from the aerodigestive tract (tongue, esophagus, stomach,and duodenum) werefixed in formalin and paraffin-embedded sections were stained with hematoxylin and eosin (H&E) to examine thelesions by light microscopy.Images were acquired using a bright field light microscope (Nikon ECLIPSE Ni, NIS Elements acquisition software, 20x 0.5 N.A. objective). During the pathologicalexamination, the lesions observed in the various tissues were categorized into hyperplasia, dysplasia,and invasive carcinoma. Hyperplasia was defined as an increase in the layers or thickening of the epithelium with hyperkeratinization. Dysplasia was defined as loss of epithelial cell polarity, nuclear pleomorphism and abnormal mitoses confined to the epithelium. Invasive carcinoma was defined as invasion of dysplastic cells or lesions into subepithelial tissues such as submucosa and muscle
    3. Carcinogenesis studies were conducted as described previously for Ip6k2-/-mice (Morrisonet al., 2009). Briefly, the carcinogen, 4NQO stock solution (5 mg/mL) was prepared fresh in propylene glycol every week. Four-week old mice [11 Ip6k1+/+(5 male, 6 female) and 9
    4. HCT116 cells stably expressing NT or shRNA against IP6K1were grown to subconfluence. Logarithmic phase monolayer cultures were harvested by trypsinization, cell number was determined using a hemocytometer,and resuspended in complete DMEM. Before injection, cells were washedand resuspended in PBS at 2×107cells/mL. Approximately 2×106 cellswere injected subcutaneously into either flank of 6 week old female homozygous Foxn1nuathymic nude mice(n= 8 mice) and tumor size was monitored every 3 days for a period of 4 weeks. Mice were euthanized 4 weeks after injection and tumors were surgically excised and weighed
    5. hemocytometer. 2 mL of cell suspension containing 2×104cells/mL were aliquoted in 15 mL conical tubes. To prepare top agarose (0.3%), 3 mL of prewarmed 0.5% agarose mixture was added to the 2 mL cell suspension, mixed gently by pipetting up and down to avoid air bubbles. Quickly 2 mL of this mixture (cells+0.5% agarose mixture) was transferred to the plate containing 2 mL top agarose. All the samples plates were kept at room temperature (TC laminar flow hood) for at least 60 min to solidify the top agarose. After agarose solidification, plates were incubated at 37°C and 5% CO2for 28 days. Every 2 days, spent media was replaced with 0.5 mL fresh media to prevent it from drying. After 28 days, colonies were stained with 0.005% crystal violet (in 2% ethanol) for 1 h at 37°C. Excess stain was washed with 2% ethanol until clear visible colonies were seen. Plates were imaged using AlphaImager (Alpha Innotech), and the number of colonies was determined by ImageJ software. Multiple images of stained colonies were acquired on a phase contrast inverted microscope(Zeiss)at 5x objective with 1.2 N.A using ProgRes CapturePro v2.8 acquisition software. Area of the individual colonies was determined using ImageJ software
    6. Anchorage-independent cell growth was conducted in 6 well plates at least in triplicates. 1% of low temperature melting agarose (Difco) was prepared in sterile MQ water and mixed with 2X DMEM containing 20% FBS kept warm at 37ºC. Both the solutions were mixed in equal proportion to get 0.5% agarose in 1X DMEM and 10% FBS. This solution was kept warm throughout the experiment at 37ºCin a water bath or incubator. To prepare base agar (0.5% agarose), 2 mL of this solution (0.5% agarose+1x DMEM+10% FBS) was dispensed in 6 well plate to spread evenly without any bubbles and allowed to solidify at room temperature at least for 30 min. Cells were harvested and counted using a
    7. Scratch wound healing assay was performed as described previously (Raoet al., 2015). Cells were seeded in 6 well plates in triplicates to attain confluence. On the next day, a scratch was made on the confluent monolayer culture using a 200 μL pipette tip. Cells were washed gently twice with PBS to remove floating cells, replaced with complete DMEM and incubated at 37ºC with 5% CO2.Images were acquired using a Zeiss phase contrast inverted microscope (ProgRes CapturePro v2.8 acquisition software, 5x 0.12 N.A. objective). Images were acquired immediately after scratch (0 h) and after a period of 4 h (MEFs) or 24 h (HeLa and HCT116) to monitor wound closure. The area of wound closure was analyzed using ImageJ software, and the data was plotted as total area covered in square μm(μm2)
    8. shRNA were processed for invasion studies. A suspension of 1×105cells in 200μLof 1%FBS containing DMEM was prepared and plated as described in Section 2.2.16. Cells were incubated at 37ºC with 5% CO2for 24 h to allow invasion. Downstream processing of the samples i.e. removing non-invading cells, staining and imaging were followed as described in Section 2.2.16. The number of cells invadedto the lower surface was quantified by counting the total number of DAPI positive nuclei in at least 10 random fields. The total number of cells invaded was normalized to non-targeting control cells and expressed as percentage invasion
    9. Invasion assays were conducted as described previously (Raoet al., 2015).Invasion chambers with pre-coated matrigel (BioCoat Matrigel invasion chamber, 24 well, 8 μm pore size, Corning) were used to conduct the invasion assays. Packages containing invasion chambers were kept outside and allowed to come to room temperature. Inserts were equilibrated by adding with 500 μLwarm (37ºC) 1% FBS containing DMEM to the upper chamber of the insert and bottom well of the companion plate, and rehydrated for 2 h in a humidified tissue culture incubator at 37ºCwith 5% CO2. The rehydration media was removed carefully without disturbing the pre-coated matrigel matrix on the membrane. Meanwhile, HCT116 cells expressing either non-targeting or IP6K1 targeting
    10. and pelleted down at 1200 rpm for 5 min. The supernatant containing 10% FBS and trypsin were discarded and the cell pellet was resuspended in media containing 1% FBS. 5×104cells suspended in 200 μLDMEM (1% FBS) was added to the upper chamber and carefully transferred to the well of the companion plate containing 700 μLof complete DMEM (10% FBS) to serve as chemotactic agent. Inserts were lifted with sterile forceps and placed in the companion plate by avoiding any bubbles at the bottom surface of the insert. Cells were incubated at 37ºC with 5% CO2for 18 h (MEFs) and 24 h (HeLa and HCT116 cells) to allow migration. At the indicated time, cells on the upper surface of the filter were removed carefully by scrubbing with wet cotton swabs and inserts were dunked twice in excess PBS. Cells that migrated to the lower surface of the filter were rinsed twice with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature. Post fixation inserts were rinsed in PBS twice and air dried at room temperature. Filters were cut carefully through the edges using a scalpel blade and placed inverted on a clean slide so that the migrated cells faced the upward direction. Filters were mounted with vectashield DAPI and covered with a clean coverslip. Migrated cells stained with DAPI were counted by imaging multiple fields using anepifluorescence inverted microscope (Olympus lX51,Image-Pro AMS 6.0 acquisition software, 20x 0.45 N.A. objective).The number of cells migrated to the lower surface was quantified by counting the total number of DAPI positive nuclei in at least 10 random fieldsper sample
    11. Transwell migration assays were conducted as described previously (Raoet al., 2015). Transwell inserts (24 well, 8 μm pore size, Costar, Corning)were used to conduct migration assays. Inserts were equilibrated by adding 1% FBS containing media to the upper chamber, as well as lower chamber of the insert (companion plate) and placed in a 37ºC incubator with 5% CO2till use. Cells were harvested, counted using hemocytometer
    12. Cells were seeded in six well plates. After attaining optimal growth, cell lysates were prepared by scraping cells in 1x Laemmli buffer and samples were processed by standard western blot techniques. To detect FAKactivation samples were processed as described in Section 2.2.13. Briefly, 2 x106cells (6 x105cells per time point) were held in suspensionin complete medium containing 1% methylcellulosefor 90 min (Susp), andreplatedon fibronectin (2 μg/mL) coated surfacesfor 20 min (+FN)or for 4 h (SA -stably adherent). Cells at each time point were lysed in 1x Laemmli buffer and subjected to immunoblotting. Membranes were probed with specific antibodies(Table 2.3)and detected using the ECL detection system (GE Healthcare) as described in Section 2.2.10
    13. the selection marker.Knockdown was confirmed by immunoblot analysis with an IP6K1 specific antibody (Table 2.3) as described in Section 2.2.10
    14. lines were used for stable knockdown of IP6K1 expression. Viral particles harboring either non-targeting control or IP6K1directed shRNA were used to infect HeLa or HCT116 cell lines at 0.5 MOI, following treatment with polybrene (8 μg/mL)for 2 h.After 48 h, transduced cells were selected with 2 μg/mL puromycin. Medium was changed twice a week and observed for colony formation. After reaching the optimum growth, selected cells were maintained in DMEM supplemented with 10% FBS and 1 μg/mL puromycin as
    15. HEK293T packaging cellswere seeded at 30-40% confluency in 60 mm dishes. After 24 h, cells were co-transfected with three plasmids required for viral production i.e. VSV-G, psPAX2 (Addgene plasmid # 12260) and pLKO.1-puro-non-targeting and shIP6K1 clones using polyethyleniminereagent(PEI) and incubated at 37°C and 5% CO2 for virion formation. After 48 h, viral particles were harvested by collecting supernatant and filtered througha 0.45 μm syringe filter unit. Viral stock was aliquoted and stored at -80°C for further use. Viral titer was approximated on the number of cells plated for the production of lentivirus. Calculations were done as per Cell Bio Labs instruction. 2 x 106cells will yield 107infectious units/mL. All necessary precautions were taken while generating lentiviral particles such as wearing mask, double gloves, and sterile filter tips. All the consumables used were bleached (1% sodium hypochlorite solution) at least 1 h before being discarded
    16. fluorophore-conjugated phalloidin (Alexa Fluor 488 or rhodamine) for 45 min, followed by DAPI staining for 2 min. Cover slips were mounted onto glass slides using Fluoromount G (Southern Biotech) or Vectashield (VectorLabs),and imaged using an LSM 710 laser confocal-anisotropymicroscope (Zeiss, Zen acquisition software, 40x 1.3 N.A. objective) for 15 min spreading,or LSM 510laser confocal microscope (Zeiss, LSM acquisition software, 63x 1.4 N.A. objective) for 24 hspreading.To measure the cell spread area for serum depleted cells (0.2% serum), images captured at identical zoom settings were analyzed using the Image J software (NIH)as follows:thresholdvalues were set to define the cell edge and amaskwas then createdfor each cellto get the totalcellarea(with arbitrary units)within the mask.For 15 min spreading (10% serum) and 24 h spreading, the exact spread area was calculated based on pixel dimensions during image acquisition
    17. Cell spreading assays were done as described previously (Balasubramanianet al., 2010).Briefly, cells were cultured in complete medium (10% FBS) or subjected to serum starvation (0.2% FBS for 12-14 h), trypsinized, counted using a hemocytometer, and held in suspension in the same medium containing 1% methylcellulose at 37°Cand 5% CO2in a 50 mL tube in a slanted position. After 90 min, cells werepelleted down at 1350 rpm for 8 min at 4°Cand the supernatant was discarded leaving 5 mL at the bottom. Cells were dislodged or displaced gently without touching the walls of the tubes, which might lead to activation of adhesion dependent signalling. Now, cells were washed once with excess of cold media (4°C)and one more time with warm media (37°C). A day before the experiments coverslips were coated with fibronectin (2 μg/mL)overnight at 4°Cand washed once with PBS before plating the cells. Cell suspension equivalent to 105cells was plated onto each fibronectin (2 μg/mL) coated coverslip and allowed to spread for 15 min or24 hin serum depleted (0.2% FBS) or complete medium. At each time point, unadhered cells were washed off with PBS and the coverslips were fixedwith 3.5-4% paraformaldehyde for 20 minat room temperature. After fixation coverslips are washed thrice with PBS and cells were permeabilized with 0.1% Triton X-100 for 5 min,followed by blocking with 5% BSAfor 30 min at room temperature. Fixed cells were stained with
    18. Cell adhesion assayswereperformed as described previously (Hockinget al., 1998)withslight modifications. Fibronectin coating was done overnight at 4°C. 5×104 cells were seeded per well onto fibronectin (2 μg/mL) coated 24 well platesin triplicates. Cells were allowed to adhere for different time periods. At each time point, unadhered cells were washed away with PBS; adhered cells were trypsinized and counted with a hemocytometer. Percentage of adhesion was calculated by normalizing total number of adhered cells at each time point to number of cells adhered after 5 h
    19. supernatant was collected and filtered through a 0.45 μm syringe filter unit. Lentiviral particles carrying either non-targeting shRNA or both IP6K2 directed shRNA constructs were used to infect MEFs for 48 h. At the 36thhour, cells were treated with HU for 12 h, followed by recovery for 6 h, and immunofluorescence was carried out as described in 2.2.6.Immunoblotting with an IP6K2 antibody(Table 2.3) was performed to analyse knockdown levels as described in Section 2.2.10
    20. (Sigma-Aldrich SHC016) or shRNA directed against mouse Ip6k2(Sigma-Aldrich, TRCN0000202175 or TRCN0000202065), were co-transfected with VSV-G and VSV-GP encoding plasmids into thePhoenix amphotropic packaging cell line, using Polyfect reagent (Qiagen), and incubated at 37°C and 5% CO2for virion formation. After 48 h, the
    21. Dishes containing adherent cultures were placed on ice and washed once with cold PBS. Cells were scraped in 1x sample buffer on ice. Samples were sonicated in a probe sonicator (Misonix ultra sonic liquid processor, S-4000) 3 times for 15 sec each to complete cell lysis and shear DNA to reduce viscosity. Equal volumes of lysates were loaded and resolved using 12% SDS-PAGE. Samples were stored at -80ºC if necessary. Protein transfer onto PVDF membrane (GE Healthcare) was carried out at 200mA constant current for 2 h by placing the transfer tank in ice. Membranes were blocked with appropriate blocking buffer according to manufacturer’s instructions provided for each antibody. Preferentially TBST with 5% non-fat dry milk was used for blocking and for antibody dilutions. Briefly, non-specific interactions were blocked with blocking buffer (TBST+5%non-fat dry milk) for 1 h at room temperature. Membranes were probed with appropriate primary (overnight at 4ºC) and HRP-conjugated secondary antibodies (2 h at room temperature). Proteins were detected using ECL detection system. Chemiluminiscence was detected using the LAS4000 (GE Healthcare) or FlourChem E (Protein Simple) documentation system. Densitometry analysis of bands was done using ImageJ documentation software (Fiji) or the multiplex band analysis tool in AlphaView software (Protein Simple)
    22. dropped on clean prechilled slides held at a 45°angle from a height of 18 inches, dried at 55°Con a hot plate and stained with 3% geimsa in phosphate buffer (pH 6.8) for 10 min. Slides were washed three times and dried, individual metaphase spreads were analyzed using bright field microscopy (Zeiss Axio Scope), and chromosomal aberrations were scored manually from each spread
    23. MEFsplated in 6 cmdishes were treated with MMC (300 nM) for 12 h. Post damage cells were treated with colcemid (0.1 μg/mL) for 4 hto arrest cells in metaphase. After 4 h cells were washed twice with PBS to remove traces of colcemid and harvested by trypsinisation, pelleted down at 100 x g for 5 min in 15 mL conical tubes. The supernatant was removed by leaving 0.5 mL and resuspended thoroughlyby pippeting. Cells were subjected to hypotonic swelling in pre-warmed 0.075 M KCl for 15 min at 37°C[Note: key to this step is to add hypotonic solution drop by drop slowly otherwise cell clumps will form which are impossible to disperse. Add a few drops first then pipette the cells up and down to mix them thoroughly and add few more drops. Invert tubes 2-3 times during the incubation]. After 15 min cells were fixed by adding few drops of chilled fresh fixative (methanol: glacial acetic acid; 3:1) kept in the deep freezer. Cells were pelleted down at 100 x g for 8 min, the supernatant was removed, 0.5 mL of fixative was left behind and the pellet was resuspended very gently. Slowly fresh fixative was added in the same manner as the KCl solution and cells were pelleted down at 100 x g for 8 min; this step was repeated twice. The supernatant was removed by leaving 0.5 mL fixative and the cells were resuspended and mixed well before preparing metaphase spreads. Cell suspensions were
    24. were mixed or shaken every 15 min during the incubation as to keep them in suspension. At the end of the incubation, cells were washedagain with 800 μLof rinse bufferby centrifugation at 500 x gfor 6 min and the supernatant was aspirated. BrdU incorporation was detected by adding 100 μLof antibody staining solution containing 5 μLAlexa488 dye labeled anti-Brdu antibody with 95 μLof rinse buffer(1:20 dilution), and the cell suspension was incubated for 30 min at room temperature in the dark.At the end 350 μLof PI staining buffer was added to each sample and incubated for an additional 30 min at room temperature in the dark. Cells were analyzed forthe presence of DSBs by flow cytometry on a FACS ARIA instrument (BD). Viable cells were analyzed for the presence of DNA DSBs by excluding the hypodiploid (<G0/G1)population, using FACS DIVA software (BD). Medianfluorescence values of the treated cells were plotted as a fold difference over untreated controls, using GraphPad Prism 5
    25. Cells were grown in 35mm dishes at 30% initial confluence. At 60% confluence, cells were treated with 0.2 mMhydroxyurea for 12 h. After treatment,media containing drug was removed and fresh media was added for recovery of the cells from genotoxic stress.DNA DSBs were monitored during the treatment period and recovery period of 3, 6, 9 and 12 h. At each time point cells were harvested and fixed using 70% icecold ethanol by gentle vortexing at very low speed and kept overnight at -20ºC. A TUNEL assay to detect DSBs was conducted using Apo-DirectTunel assay kit (A35127, Invitrogen). After overnight fixation, cells were washed with 800 μLof wash buffer, and 50 μLof DNA labeling solution [10 μL reaction buffer, 0.75 μL of TdT enzyme, 8 μL of BrdUTP and 31.25 μLof dH2O (Sigma)]was added to each sample, incubated at 37ºC for 4 h. Samples
    26. The IP6K inhibitor TNP was dissolved in DMSO at a concentration of 22.6 mM and stored at -20°C. MEFs were seeded on coverslips in 12 well plates at 15% confluence. Once, cells adhered and attained their morphology, they were pre-treated with 5 μM TNP and DMSO 3 h prior to HU treatment. After 3 h, cells were treated with 0.5 mM HU for 12 h in presence or absence of TNP. Similarly, post drug removal,cells were incubated with or without TNP to monitorrecovery for 6 h. At each time point cells were processed for presence of nuclear BLM as described in Section 2.2.6. BLM was used as readout for initiation and completion of repair. Imageswere acquired as described in Section 2.2.6
    27. Inhibition of IP6Ksin MEFs
    28. ncubated with appropriate Alexa-488 conjugated secondary antibodies diluted in PBST with 2% BSA at room temperature for 1 h (for dilutions refer to Table-2.3). The cover slips were washed with PBST, mounted on glass slides using Vectashield mounting medium with DAPI. Images were acquired on a LSM 510 META confocal microscope (Zeiss, LSM acquisition software, 63x 1.4 N.A. objective) at a 0.7 scan zoom to collect maximum number of cells per field. The number of foci in each nucleus was manually counted by changing contrast identically across all the samples. A minimum of 10 random fields were imaged per sample. Data are represented as average number of foci per nuclei.
    29. Localization of the DNA damage response proteins γH2AX, Rad51 and BLM and mitotic marker H3S10 (Histone H3 phosphorylated on Ser10) upon genotoxic stress and recovery was analyzed by immunofluorescence, following hypotonic lysis. Cells were seeded on cover slips in 12 well plates at 10-15% confluenceand incubated overnight. Cells were treated with 0.5 mM hydroxyurea and 0.25 μg/mL neocarzinostatin for 12 h. Coverslips were washed twice with PBS and cells were incubated in hypotonic lysis buffer containing 10mMTrisHCl (pH 7.4), 2.5mM MgCl2, 1mM PMSF and 0.5% NP-40, on ice for exactly 8 min on a shaker at 50-60 rpm. Cells were washed twice on ice, with ice cold PBS for2 min, with continuous shaking. Fixation was carried out with ice-cold 100% ethanol (which hadbeenkept overnight in -20°C) for 4 min, shaking on ice. 500 μLof ethanol was added to each cover slip during fixation. Cells were washedthrice with 1 mL PBS containing 0.2% Tween-20 (PBST), for 8 min each,at room temperature with shaking.Nonspecific interactions were blocked by incubating the cells for 30 minwith 2% BSA diluted in 1X PBS. Cover slips were again washed twice with 1 mLPBST, incubated with appropriate primary antibody (Table-2.3) diluted in PBS + 0.2% BSA for 2 h at room temperature. 200 μLof antibodywasplaced directlyonthecover slips. Post incubation, the cover slips were washed thrice with 1 mL PBSTfor 3 min each and the cells were
    30. Immunofluorescence
    31. the same solution and stored overnight at 4ºC. Fixed cells were permeabilized with 100 μL saponin based permeabilisationbuffer and wash buffer for 2 minin the dark. Cells were pelleted down and washed twice with saponin based permeabilisation buffer, centrifuged to pellet the cells and the supernatant was aspirated leaving 30-40 μLbuffer to dislodge the pellet. EdU was detected by adding 350 μLof Click-iT reaction cocktail and incubated for 30 min at room temperature in the dark. Cells were washed once with permeabilisation buffer and DNA content was measured by adding 5 μL Ribonuclease A and 2 μLof cell cycle dye 633 red incubated for 15 min at room temperature in the dark. Cells were analyzed by flow cytometry (FACS ARIA, Becton Dickenson). Data analysis to determine the stages of thecell cycle was performed using FACSDiva (BectonDickenson), and results were plotted using GraphPad Prism 5
    32. Cell cycle analysis by PI staining is based on the DNA content of cells and cannot distinguish G0from G1, and G2 from M phase. One more limitation is that it provides overlapping cell populations in different phases. Alternatively, multi parameter based cell cycle analysis can be performed using EdU (5-ethynyl-2’-deoxyuridine, a BrdU alternative)labeling which exclusively distinguishes cells in S phase from other phases of cell cycle. EdU labeling and cell cycle analysis was conducted using the Click-iT cell proliferation assay kit (C35002, Invitrogen), as per the manufacturer’s instructions. Briefly, cells were grown in 35mm dishes at 30% initial confluence. At 50-60% confluence, cells were treated with 0.2 mM HU for 12 h. After treatment, media containing drug was removed, gently washed twice with PBS and replaced with fresh media. Cells were allowedto recover for different time periods such as 3, 6, 9 and 12 h to observe arrest and release into S-phase. At each time point cells were labeled with 25μM EdUfor 30 min before harvestingby trypsinisation. Harvested cells were washed with 1% BSA in PBS and fixed with 100 μLof ClickiT-fixative containing 3% paraformaldehyde for 15 min at room temperature. After fixation, cells were again washed with 1% BSA in PBS, resuspended in
    33. Cell viability can be monitored by changes in the morphology of the cell or by membrane permeability to certain dyes such as PI. When exposed to PI, viable cells do not take up PI due to the presence of an intact membrane whereas dead cells take-up PI due to loss of membrane integrity. Briefly, cells were treated with HU for 12 h, and allowed to recover for different lengths of time as described in Section 2.2.2. At each time point, cells were harvested by trypsinisation and washed with PBS. Approximately 106cells were resuspended in 1 ml PBS and stained with 2μg/mL (final concentration) PI for 2 min at room temperature. The extent of PI staining was used to determine the viable and dead population by flow cytometry analysis (Accuri C6, Becton Dickenson). Nonviable cells were approximately 100 fold brighter than the unstained (viable cells) cells
    34. Cells were grown in 35 mm dishes at 20% initial confluence. At 40-50% confluence, cells were treated with different genotoxic agents as described in Section 2.2.2. Post treatment, cells were washed twice with PBS and replaced with fresh media to allow recovery for different lengths of time. At each time point, cells were harvested and fixed with 70% ethanol at -20°C overnight. Fixation was carried out by adding 70% ethanol drop by drop, while the cells were being vortexed at a low speed.The fixed samples were brought to room temperature, pelleted down at 2000 rpm for 3 min and washed twice with PBS. Cells were stained with PI solution containing 0.1% Triton X-100, 0.2 mg RNase and 20μg propidium iodide and incubated at 37ºC for 30 min in the dark. Samples were analyzed by flow cytometry (FACS ARIA, BD). Data was analyzed usingFACS DIVA (BD) and FlowJo (FLOWJO, LLC) softwares to identify different stages of the cell cycle
    35. Cells were plated at a density of 30,000 cells per well in triplicates in a 24-well plate. After 24 h, cells at a confluency of 30-40%, were treated with different genotoxic agents such as hydroxyurea (0.5 mM), neocarzinostatin (0.25 μg/mL) and mitomycin-C (1 μg/mL) for 12 h. Spent media containing drug was removed and cells were washed gently twice with PBS. Cells were then incubated for different lengths of time ranging from 24 h to 120 h, in fresh complete DMEMto allow them to recover from genotoxic stress. At each time point cell survival was analyzed by MTT assay as described in 2.2.1. Cell survival was expressed as a fold increase in cell population relative to cells treated with drug for 12 h
    36. cells convert MTT to insoluble purple formazan dye crystals which can besolubilized using detergent or DMSO and measured by colorimetry. At each time point mediacontaining MTT dye (250 μg/mL) was added to each well and incubated at 37ºC. After 2 h cells were lysed with 200 μL DMSO for 15 min on a rocker at room temperature to solubilize the formazan and absorbance was monitored at 570 nm using the EnSpire multimode plate reader (PerkinElmer). Doubling time was determined by plotting log [A570] vstime and conducting a linear regression analysis using GraphPad Prism 5
    37. Cells were seeded in 24 well plates at 10% confluence in triplicates and allowed to grow for different lengths of time. Spent medium was replaced with fresh medium every 48 h. Cells were incubated for different lengths of time ranging from 24 h to 120 h, to allow them to grow and cell survival was monitored by the MTT assay. Metabolically active
    38. 3.4 mL H200.63 mL 30% acrylamide solution (acrylamide:bis-acrylamide; 29:1)0.83 mL 1 M Tris (pH 6.8)0.05 mL 10% SDS0.05 mL 10% ammonium persulfate (APS)0.005 mL N,N,N',N'-Tetramethylethylenediamine (TEMED)Resolving gel solution (12%)10 mL3.3 mL H204 mL 30% acrylamide solution (acrylamide:bis-acrylamide; 29:1)2.5 mL 1.5 M Tris (pH 8.8)0.1 mL 10% SDS0.1 mL 10% ammonium persulfate0.004 mL N,N,N',N'-Tetramethylethylenediamine (TEMED)SDS-Running buffer(Tris/Glycine/SDS)25 mM Tris-Cl192 mM glycine0.1% SDSTransfer Buffer25mM Tris-Cl190 mM glycine20% MethanolMTT dyeMTT dye was dissolved in PBS at 5 mg/mL concentration. Filtered through 0.45 μm syringe filters, and stored in dark at 4ºC
    39. Hypotonic lysis buffer10mMTris Cl (pH 7.4)2.5mM MgCl2, 1mM PMSF 0.5% NP-40Hypotonic bufferPrewarmed 0.075 M KCl Fixatives used in this study100% ethanol kept overnight at -20ºCfor immunofluorescence by prelysis protocol70% ethanol kept overnight at -20ºCused for PI based cell cycle analysisMethanol: glacialactetic acid (3:1) for cytogenetic analysis4% Paraformaldehyde-4 g of paraformaldehyde dissolved in 100 mL waterPI staining solutionPBS containing, 0.1% Triton X-1000.2 mg RNase 20μg propidium iodideReagents required for Tris-Glycine SDS-PAGESDS-PAGE 30% Acrylamide solution29 g of acrylamide and 1 g bi-acrylamide (29:1 ratio) dissolved in 100 mL water10% SDS10 g SDS dissolved and 100 mL waterLaemmli buffer40% Glycerol 240 mM Tris/HCl pH 6.8 8% SDS 0.04% bromophenol blue 5% beta-mercaptoethanolStacking gel solution (5%) 5mL
    40. Table 2.3: Antibodies used in this study
    41. Click-iT cell proliferation kit (C35002, Invitrogen), Apo-BrdU TUNEL assay kit (A35127, Invitrogen)
    42. Cell culture reagents: fetal bovine serum (FBS, 26140-079), L-glutamine (25030-081), penicillin-streptomycin (15140-122) and trypsin (25200-056) were from Life Technologies. DNA damaging agents used for this study were obtained from following sources: hydroxyurea (HU; H8627, Sigma-Aldrich), neocarzinostatin (NCS, N9162, Sigma-Aldrich), mitomycin C (M0503, Sigma-Aldrich). Reagents used for cell spreading assays: methyl cellulose (Sigma-Aldrich), fibronectin (F2006, Sigma-Aldrich)andfluorophore conjugated phalloidin (Molecular Probes Inc).Antibiotic selection markers: puromycin (Sigma-Aldrich), G418 (Sigma-Aldrich), transwell inserts (24 well, 8 μm pore size, Costar, Corning), Invasion chambers (BioCoat Matrigel invasion chamber, 24 well, 8 μm pore size, Corning). Other chemicals: Propylene glycol (151957, MP Biomedicals), 4-Nitroquinoline-1-Oxide(4NQO; N8141, Sigma-Aldrich), MTT [(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide], TNP [N2-(m-(trifluoromethy)lbenzyl)N6-(p-nitrobenzyl)purine],DMSO (dimethyl sulfoxide), ethanol, paraformaldehude, vectashield DAPI (Vector labs),Tris, PMSF, NP-40, PBS, Tween-20, BSA, MgCl2, colcemid, KCl, methanol, glacial acetic acid, giemsa, SDS, sodium bicarbonate (S5761, Sigma-Aldrich),polyfect transfection reagent, crystal violet, propidium iodide, Triton X-100 and PEI were obtained from Sigma-Aldrich. Low melting agarose (Difco), ECL detection system (GE Healthcare)
    43. All animal experiments were conducted as per guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment, Forest, and Climate Change, Government of India,and these experiments were approved by the Institutional Animal Ethics Committee (Protocol numbers PCD/CDFD/02-version 2 and PCD/CDFD/08). Mice used for this study were housed in the Centre for DNA Fingerprinting and Diagnostics animal facility located within the premises of Vimta Labs, Hyderbad.Ip6k1+/-heterozygous mice were bred to generate age and sex matched Ip6k1+/+and Ip6k1-/-littermates for experiments. Foxn1numice were generated by breeding homozygous males with heterozygous females.These mice were used for in vivotumourigenic assays
    44. Other plasmids used for lentivirus generation: VSV-G, VSV-GP (gifts from Dr. Renu Wadhwa, AIST, Japan) and psPAX2 (a gift from Dr. Didier Trono, Addgene plasmid # 12260)
    45. Table 2.2:Plasmids expressing shRNAagainst mouse Ip6k2used for generating stable cells in MEFs are listed below
    46. Lentiviral vectors (pLKO.1)encoding various shRNA sequences against human IP6K1and mouse Ip6k2were obtained from Sigma-Aldrichto generate transient and stable knockdowns. shRNA clone IDs and their representation in the thesis are given below.Table 2.1:Plasmids expressing shRNA against human IP6K1used for generating stable cells in HeLa and HCT116
    47. The cell lines used in the study are mouse embryonic fibroblasts (MEFs) derived from wild type (WT) and Ip6k1knockout mouse embryos. The MEFs were immortalized with SV40 large T antigen (Bhandariet al., 2008)and single cell derived lines were generated in the lab. Ip6k1knockout MEFs display 70% lower levels of IP7compared with wild type MEFs (Bhandariet al., 2008). Ip6k1-/-MEFs expressing kinase active or inactive forms of IP6K1 were generated in the lab (Rescue MEFs). MEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 1 mM L-Glutamine (Life Technologies), 100U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies).Rescue MEFs were cultured in complete DMEM supplemented with G418 (200 μg/mL) as selection marker. HCT116 (colon cancer cells, a gift from Dr. Sagar Sengupta, NII, New Delhi) or HeLa (cervical cancer cells) expressing non-targeting control and shRNA against human IP6K1were cultured in complete DMEM containing puromycin (2μg/mL). The amphotropic Phoenix cells (a gift from Dr. Shweta Tyagi, CDFD, Hyderabad) and HEK293T packaging cells were usedfor generating lentiviral particles containing shRNA against human IP6K1or mouse Ip6k2and were maintained in complete DMEM
  2. Apr 2019
    1. Klotho-deficient mice have accelerated aging phenotypes, whereas overexpression of Klotho in mice extends lifespan. Klotho is an anti-aging single-pass membrane protein predominantly produced in the kidney, with shedding of the amino-terminal extracellular domain into the systemic circulation. Circulating levels of soluble Klotho decrease with age, and the klotho gene is associated with increased risk of age-related diseases. The three forms of Klotho protein have distinct functions. Membrane Klotho forms a complex with fibroblast growth factor (FGF) receptors, functions as an obligatory co-receptor for FGF23, which is involved in aging and the development of chronic diseases via regulation of Pi and vitamin D metabolism. Secreted Klotho functions as a humoral factor with pleiotropic activities including regulation of oxidative stress, growth factor signaling, and ion homeostasis. Secreted Klotho is also involved in organ protection. The intracellular form of Klotho suppresses inflammation-mediated cellular senescence and mineral metabolism. Herein we provide a brief overview of the structure and function and recent research about Klotho.
  3. Sep 2018
    1. rigid schedules

      Improve practices for scheduling that take into account workers needs

    2. commute

      Assessment and implementation of protective measures for long commutes particularly those linked to long hours of work to avoid safety risks

    3. share agreement

      Adoption of mechanisms like the "Fair Share Agreement" in B.C. or similar to get funds for improving infrastructure in municipalities and regions.

    4. availability of housing

      Improve the need of affordable housing in host, hub and source communities

    5. travel

      Improve information for LDLC workers about travel; assess the existing gaps to protect workers during their work related travel and develop or improve the tools for their protection

    6. information LDLC workers

      Improve information for LDLC workers about their rights related to OH&S and WC

    7. Lack of metrics

      Develop or strengthen statistical information of new industry developments, ups and downs of existing industries.

    8. welfare support

      Implement a timely response of welfare support linked to slow-downs of industries that involve numerous workers in given communities

    9. mental health and addiction services

      Assess the prevalence and incidence of mental health issues and addictions and establish sufficient services for caring and prevention. This phenomena affect workers, their families and communities.

    10. medical care

      Improve medical care infrastructure and inter-provincial agreements to be able to cover LC-LD workers and their families in source, host and hub communities in a timely manner. the improvement of such services should be flexible enough to adapt to the ups and downs of the predominant industries.

    11. environmental impacts

      Improve environmental assessments in the planning stages of industrial and housing developments in source, host and hub communities

    12. community participation

      Establishment of bodies and paths for community participation in the development of new industrial activities and the assessments of their needs imposed by the labour market linked to the industry and its ups and downs.

    13. prior preparation

      Anticipation through planning of community needs due to new developments and ups and downs of predominant industries for example mining.

    14. transportation in Greater Toronto

      Improve urban and peri-urban transportation for LC workers particularly those with low income

    15. improving infrastructure

      Improve infrastructure in the host and hub communities according to the needs imposing by industrial developments

    16. child care

      Improve child care services for LC workers particularly those with low income

    17. Child and elder care

      Improve in a timely manner services of child and elder care according to the needs imposed by the labour market (work shifts; LD-LC work).

    18. Transportation

      Improve in a timely manner transportation services according to the needs of LDLC workers taking into account those with low income.

    19. LDLC workers and their needs

      Develop or strengthen statistics regarding LDLC workers and their needs

    20. housing, infrastructure

      Anticipation to fulfill housing needs and assessment of the ups and downs of the industry that affect the housing needs. Decisions should be taken about short, medium and long term needs.

    21. infrastructure

      Timely response for the maintenance of roads and development of new routes when needed according to the traffic needs due to industrial developments and labour market that involve drive-in drive-out mobility and heavy transit during construction phase

  4. Aug 2018
    1. I really enjoyed this preprint. I have one comment: Throughout this manuscript, you reference univariate effect sizes. It seems relevant to reference a multivariate effect (i.e., Mahalanobis D) if only to make the point that even small mean differences in univariate preferences, personality traits, and cognitive abilities can combine to create perceivably large gender/sex differences. I know this topic is debated (and the authors are critical of D and what is means), but this thesis seems incomplete without reference to how preferences/traits/abilities could combine to create differences in what majors/jobs/careers men and women are good at/pursue (on average). I think the commentaries here (https://marcodgdotnet.files.wordpress.com/2014/04/delgiudice_etal_2012_comments_reply.pdf) and the examples described here (https://marcodgdotnet.files.wordpress.com/2014/04/delgiudice_2013_is-d_valid_ep.pdf) make the debate and the conceptual utility of D clear.

    1. 3 Steps for Licensing Your 3d Printed Stuff by Michael Weinberg. CC BY-SA 3.0 A set of instructions for how to license 3d printed materials https://www.publicknowledge.org/assets/uploads/documents/3_Steps_for_Licensing_Your_3D_Printed_Stuff.pdf

      Relevant content in the unit: Unit 3.2, Acquiring Essential Knowledge, What types of content can be CC-licensed, suggested additional content (related to both paragraphs in current content).

      While the primary purpose of this paper is about 3-D printing, this resource is a great overview of copyright law related to electronic files, whether they be photographs or the files for a 3-D printing project.

      This is an especially good resource for those interested in specific examples of the delineation of the functional, non-copyrightable aspect of a work and the artistic expression, copyrightable aspects of a work.

  5. Mar 2018
  6. Feb 2018
  7. Jan 2018
    1. maginativeintervention

      Connection to "3-D print your way to freedom and prosperity." Where as 3-D printing is an imaginative intervention.

    1. President Barack Obama, in his 2013 State of the Union address

      President Obama believed that tools like 3-D printing would be very useful for things we do in our everyday lives.

    2. 3-D print your way to freedom and prosperity

      3-D printing is the process of creating an object that is new, by breaking something down and then fabricating it into something of use. This process is very convenient and fast. Unlike the Haltman's essay this discusses the effects of 3-D printing on both social and economical scales.

  8. Dec 2017
    1. For example, if there are multiple "Submit" buttons on a screen, the Name for each must be unique

      This seems questionable. If there are "edit" buttons beside each element in a list of elements, must they all have different names, or can their function be inferred from context? I don't think WCAG requires this.

    2. A web page that has multiple form controls (e.g. buttons) with the same visual label must include unique identifiers that describe their unique purpose.

      This seems questionable. If there are "Add" buttons beside each element in a list of elements, must they all have different names, or can their function be inferred from context? I don't think WCAG requires this.

  9. Aug 2017
    1. Later, while shopping at a Cairo bazaar, Indy and Marion are attacked by sword-wielding Arabs working for Nazi agents. Indy fights them off but in the confusion Marion is trapped in a large basket and taken by two of the terrorists. The effort to track her down is held up by a man brandishing a sword in intimidating fashion. The swordsman is shot down in short order by a thoroughly unimpressed Indy.
  10. Jun 2017
  11. May 2017
    1. National Research Council
      The National Research Council (NRC) is an organization within the Government of Canada dedicated to research and development. Today, the NRC works with members of the Canadian industry to provide meaningful research and development for many different types of products. The areas of research and development that the NRC participates in include aerospace, aquatic and crop resource development, automotive and surface transportation, construction, energy, mining, and environment, human health therapeutics, information and communications technologies, measurement science standards, medical devices, astronomy and astrophysics, ocean, coastal, and river engineering, and security and disruptive technologies. The NRC employs scientists, engineers, and business experts. The mission of the NRC is as follows: “Working with clients and partners, we provide innovation support, strategic research, scientific and technical services to develop and deploy solutions to meet Canada's current and future industrial and societal needs.” The main values of the NRC include impact, accountability, leadership, integrity, and collaboration. The most recent success stories of the NRC include research regarding “green buildings,” math games, mechanical insulation, and many more (Government of Canada 2017). Here is a link to their achievement page where these stories and more are posted: http://www.nrc-cnrc.gc.ca/eng/achievements/index.html. Here is a link to the NRC webpage: http://www.nrc-cnrc.gc.ca/eng/index.html.  
      

      References

      Government of Canada. 2017. National Research Council Canada. May 5. Accessed May 8, 2017. http://www.nrc-cnrc.gc.ca/eng/index.html.

    1. Another study, published at the end of March, included 2,303 healthy postmenopausal women randomly assigned to take vitamin D and calcium supplements or a placebo. The supplements did not protect the women against cancer, the researchers concluded.

      This is interesting in that it is techinically accurate - the difference between the groups was statistically not significant at p=0.06. "A new diagnosis of cancer was confirmed in 109 participants, 45 (3.89%) in the vitamin D3 + calcium group and 64 (5.58%) in the placebo group (difference, 1.69% [95% CI, -0.06% to 3.46%]; P = .06)"

      The key points is that cancer is that only 5% of people in the study got a new diagnosis of cancer in 4 years, which is a small percentage, and yet the p value was very close to significance.

      Now, non-signifcant is non-significant, however it is unfortunately incredibly common for studies to report higher p valiues as a trend towards significant, a clinically significant change that almost reached statistical significance etc. That the authors adhered so strictly to the standards for significance in the case of vtiamin D, where so few do anymore in other studies, is curious.

    2. One study with 5,108 participants, published this month in JAMA Cardiology, found that vitamin D did not prevent heart attacks.

      This study is one of a number recently that for some incomprehensible reason has decided that monthly dosing of vitamin D is perfectly fine to test. "Interventions Oral vitamin D3 in an initial dose of 200 000 IU, followed a month later by monthly doses of 100 000 IU, or placebo for a median of 3.3 years (range, 2.5-4.2 years)."

      The conclusion from this study is valid only for monthly dosing, not for daily dosing.

      Another example of a study that used monthly dosing and saw a negative effect in falls in elderly - http://jamanetwork.com/journals/jamainternalmedicine/fullarticle/10.1001/jamainternmed.2015.7148

    3. randomized trials had found no particular benefit for healthy people to have blood levels above 20 nanograms per milliliter.

      This is just a straight out lie, as well as an example of misdirection. There are an incredible amount of trials showing associations between low vitamin D levels and various diseases such as cancer, heart disease, depression. Randomized trials are not the only evidence. However, there are randomized trials - as an example - "Cancer incidence was lower in women who received vitamin D/calcium than in those who received the placebo (HR = 0.68, 95% CI = 0.46-0.99; P < 0.05). When analysis was confined to cancers diagnosed after the first year, the HR for the group who received vitamin D/calcium was 0.65 (95% CI = 0.42 to 0.99; P <0.05). In proportional hazards modeling, both treatment group and serum 25(OH)D concentration after one year of intervention were significant predictors of cancer risk. Conclusions Supplementing with 2000 IU/day of vitamin D3 and 1500 mg/day of calcium substantially reduced risk of all cancers combined. This finding provides great impetus for improving vitamin D status through advances in vitamin D nutritional policy." https://apha.confex.com/apha/144am/meetingapp.cgi/Paper/368368

    4. ill-defined complaints as malaise or fatigue.

      Vitamin D deficiency has been shown to be prevalent in people with fatigue, and correction of the deficiency improves the fatigue- "Results:

      Prevalence of low vitamin D was 77.2% in patients who presented with fatigue. After normalization of vitamin D levels fatigue symptom scores improved significantly (P < 0.001) in all five subscale categories of fatigue assessment questionnaires.

      Conclusion:

      The prevalence of low vitamin D is high in patients who present with fatigue and stable chronic medical conditions, if any. Normalization of vitamin D levels with ergocalciferol therapy significantly improves the severity of their fatigue symptoms." https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158648/

      Of not also is that the researchers used ergocalciferol, the synthetic D2 form which is less effective than the cholecalciferol D3 form - https://www.ncbi.nlm.nih.gov/pubmed/15531486 https://www.ncbi.nlm.nih.gov/pubmed/21177785

  12. Mar 2017
    1. Central Committee man

      Dignity and Gender both. A man is put in charge to maintain order. Also, even through all that they've been through they still choose to elect a person of power to maintain their dignity throughout the situation.

  13. Feb 2017
  14. Nov 2016
  15. Oct 2016
    1. 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 D

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