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  1. Jul 2019
    1. Transformation of ligation mixture in electro-competent E. coli host cells (DH5ααααstrain)
    2. Analysis of PCR amplicons using agarose gel electrophoresis
    3. Qualitative analysis of DNA by agarose gel electrophoresi
  2. Jun 2019
    1. The complementary segments of a globin needed for the semisynthesis of mutant chains were prepared by V8 protease digestion (Sivaram eta/, 2001). The a globin was dissolved in 0.01 M ammonium acetate buffer (pH 4) at a concentration of 1.0 mg/ml and digested at 37°C with V8 protease (1: 200, w/w) for 3 hours. The completion of digestion was ascertained by RPHPLC, after which the reaction was quenched by addition of neat TFA to a final concentration of 0.1 %. The complementary segments, al-30 and a31-141, from the digestion mixture were isolated in pure form by size-exclusion chromatography on a Sephadex G50 column (98cm x 2.8cm). The column was equilibrated and run in 0.1% TFA. The lyophilized sample of the digest was dissolved in the above solvent and loaded on to the column. The column was run at a flow rate of 30 mllhour and the elution profile monitored at 280 nm. The individual chromatographic profile of a globin digest showed only two peaks, a31-141 and a1-30 respectively, as expected from a single cleavage at the 30-31 peptide bond. The peak fractions were pooled separately and lyophilized
    2. Generation of complementary fragments, al-30 and a31-141, from heme-free a globin
  3. May 2019
    1. and fixed with 100μl of fixative solution per well, for 10 minutes at room temperature. The cells were then washed twice with PBS and 100μl of staining solution was added to each well. The plate was kept at 37° C, until the color development.
    2. 4x103-5x103 cells were plated in 96 well plate, well. Cells were transfected with reporter plasmid 18 -24 hrs after plating. After 48 hrs, cells were washed once with PBS
    3. Procedure:
    4. 1X PBS diluted in distilled water 1X fixative solution diluted in distilled water Staining Solution25 μl Solution A 25 μl Solution B 25 μl Solution C 125 μl 20 mg/ml X-gal in DMF
    5. Working Solutions:
    6. 20 mg/ml X-gal in dimethylformamide Solution A as 40 mM potassium ferricyanide. Solution B as 40 mM potassium ferrocyanide. Solution C as 200mM magnesium chloride. 10X fixative (20% formaldehyde; 2% glutaraldehyde in 10X PBS) 10X PBS as 0.017 M KH2PO4, 0.05 M Na2HPO4, 1.5 M NaCl, pH 7


    7. Stock Solutions:
    8. Transient transfection of plasmid DNA in culture cells was performed using Lipofectamine 2000 according to manufacturer’s protocol. Briefly, forty million cells were seeded in a 35mm tissue culture dish, one day before transfection. Transfection was performed 18-24 hrs after seeding the cells. 4μg DNA was mixed in 50μl of Opti-MEM in one eppendorf tube. In another tube, 5μl of Lipofectamine 2000 was diluted in 50μl Opti-MEM and incubated at room temperature for 5 minutes. After five minutes, DNA and Lipofectamine 2000 were mixed together and complexes, incubated for 30 minutes at room temperature. Meanwhile, the adherent cells were washed twice with PBS and 1ml of Opti-MEM was added. 100μl of complexes were then added to each dish containing cells and medium. After 6hrs, the medium containing complexes was removed and complete medium was added and transgene expression was accessed 24-48 hrs after transfection
    9. Transient transfections in adherent cells
    1. Trypsinization: The decolourized bands were dried in a vacuum dryer for 1 hr until the gel pieces were completely dry. 5 μl of 0.1 μg/μl trypsin and 25 μl of 25 mM NH4HCO3 (pH 8.0) were then added to the dried gel pieces. The tubes were sealed with parafilm and kept in a water bath at 37 ̊C, overnight. Care was taken that the gel pieces in the tubes did not dry up. If the gel pieces got dried, 25 μl of NH4HCO3 was added on top. Peptide extraction: A 1:1 mixture of ACN:5% TFA in water was added (30 μl) to overnight tryptic digests and kept for 30 min. The elutant was removed in a separate low binding tube. The extraction step was repeated once more. The elutant was then dried in a vacuum dryer (1-2 hr) and reconstituted in 5 μl of 25% ACN in 0.1% TFA
    2. Destaining of gel bands: The protein bands of differentially expressed proteins were cut out from the gel and put in low binding microfuge tubes. 150 μl of 50:50 Acetonitrile:Ammonium bi carbonate pH 8.0 (NH4HCO3) was then added and kept under shaking for 30 min. Coloured liquid was discarded and the washing step repeated until the bands decolourised
    3. Destaining of gel, trypsinization and peptide extraction
    4. 0.5-1 μg of DNA was used for each restriction enzyme digestion. 2-4 units of the restriction enzymes with the appropriate 10X buffers supplied by the manufacturers were used in a total reaction volume of 20 μl. The digestion was allowed to proceed for 6 h or 10min. (for FAST digest enzymes) at the temperature recommended by the manufacturer. The DNA fragments were visualized by ethidium bromide staining following electrophoresis on 1-1.5% agarose gels. Commercially available DNA size markers were run along with the digestion samples to compare with and to estimate the sizes of the restriction fragments
    5. Restriction enzyme digestion and analysis
    1. Band intensities in gel autoradiograms were determined by densitometry with the aid of the Fujifilm Multi Gauge V3.0 imaging system.Equal areas of radioactive bands (preferably the unbound probe) were boxed and the PSL (Photostimulated luminescence) valueswere further considered. For Kd(dissociation constant)calculations, the values thus obtained for each lane were expressed as a percentage with respect to the PSL for the lane without any protein taken as 100%
    2. Densitometry
    3. 5% glycerol)containing (i) 5′-end-labeled DNAfragmentof 1200 cpm radioactive count(ii) 1 μg each of bovine serum albumin andpoly(dIdC)(iii) the protein at the indicated monomer concentrations and (iv) when required,co-effectorsat specified concentrations. The reaction mixture was incubated at room temperature for 30-minsand the complexes were resolved by electrophoresis on a non-denaturing 5%polyacrylamide gel (39:1 acrylamide:bisacrylamide)in 0.5X TBE buffer pH8.3, at 12.5V/cm for 3 hrs at 18°C.The gels were then dried on a gel drier at 80°C for 45 minsand the radioactive bands were visualised with a Fujifilm FLA-9000 scanner.For DNA bending EMSA, co-effectors were not added in the binding reaction but at aconcentration of 0.1 mM in both the gel and running buffer
    4. The DNA templates were obtained by PCR from E. coligenomic DNA. After 5-end labeling, the PCR fragments were purified by electroelution following electrophoresis on 6% native polyacrylamide gels (Sambrook and Russell,2001). EMSA reactions were performed in 20 μl reaction volume inEMSA binding buffer(10 mM Tris-Cl at pH 7.5, 1 mM EDTA, 50 mM NaCl, 5 mM dithiothreitol, and
    5. Electrophoretic mobility shift assay (EMSA)
    6. Primer extension analysis to map thetranscription start site was carried out as describedby Conway et al. (1987) and Rajkumari et al. (1997). 20 pmolof primer was labelled at its 5′-end with 32P-γ-ATP as described above. 106cpm equivalent of labelled primer was mixed with 10μg of total cellular RNA. Sodium acetate pH-5.5 was added to a final concentration of0.3 M and the nucleic acids were precipitated with ethanol, washed with 70% alcohol,air-dried and dissolved in hybridization buffer (9 mM Tris-Cl, pH-8 and 0.35 mMEDTA) and incubated overnight at 43ºC for annealing. Reverse transcriptase reactionwas performed by the addition of 5 mM MgCl2, 1 mMdNTP’s, 1 X RT buffer, highconcentration (10 units) of Superscript III Reverse Transcriptase (Invitrogen) to the mixture of annealedlabelled primer and RNA. The reaction was incubated at 43ºC for 1-hr following whichthe nucleic acids were precipitated with absolute alcohol and 0.3 M CH3COONa, pH-5.5. The precipitate was air dried and dissolved in water and gel-loading dye (95%formamide, 20 mM EDTA, 0.05% each of xylene cyanol and bromophenol blue) wasadded. The samples were heated at 90ºC for 2-min before loading on a 6% denaturingpolyacrylamide gel for electrophoreticresolution alongside a sequencingladder
    7. Primer extension analysis
    8. Oligonucleotides and PCR products were end labeled using phage T4-polynucleotidekinase (PNK, New England Biolabs) with 32P-γ-ATP. The radiolabelling reactionmixture (50 μl) contained 1 X of buffer provided by the company, 10 units of T4-PNKand 50 μCi of32P-γ-ATP. The reaction mix was incubated for 1-hr at 37ºC and thereaction was stopped by adding 10 μl of 0.5 M EDTA. The labeled oligonucleotides andDNA fragments were purifiedeither by the Qiagen PCR purification or nucleotide removal kit.Labelling efficiency was checked by scintillation counting
    9. Radiolabelling of oligonucleotides and PCR products
    10. Site directed mutagenesis of plasmid DNA was carried out by using QuikChange kit(Stratagene) with a pair of complementary oligonucleotide primers carrying thenecessary sequence modifications. In this process, the plasmid (around 20-100 ng)containing the fragment of DNA where nucleotidehas to be altered, was used astemplate and “linear PCR” of 20 cycles was set up using Pfu Turbo DNA polymerase toamplify the whole plasmid with extension time calculated according to a rate of 500-bp/min. The reaction mix was digested with DpnIfor 1-hr(to destroy the original inputplasmid DNA) following which it was transformed directly to a highly competent DH5cells. The mutated plasmid was confirmed by sequencing
    11. Site directed mutagenesis
    12. Automated DNA sequencing on plasmid templates or on PCR products was carried outwith dye terminator cycle sequencing kits from Perkin-Elmer on an automatedsequencer (model 377, Applied Biosystems), following the manufacturer’s instructions.Manual sequencing was achieved using the SequenaseVersion2.0 DNASequencing Kit from USB Corp. as described in manufacturer’s instructions and thesequencing reaction products were resolved by electrophoresis on a 6% sequencing gel
    13. DNA sequencing
    14. and a colourless upper aqueous phase. The upper aqueous phase in which RNA existsexclusively, was transferred to a fresh microfuge tube and RNA was precipitated byadding 0.5 ml of isopropyl alcohol for each ml of Trizol used. Samples were incubatedat 15 to 30ºC for 10-min and centrifuged at 12000 rpm for 10-min at 4ºC. RNA formeda gel like precipitate at the bottom of the tube. Supernatant was removed and RNA waswashed with 75% ethanol (by adding 1 ml of ethanol per ml of Trizolemployed). RNAcould be stored after this step in –20 or –70ºC for more than a year. RNA pellet was airdried for 15-to 30-min following which it was dissolved in nuclease free water. Theconcentrations and purity of RNA samples were determined spectroscopically as wellas by visual inspection on formaldehyde-agarose gel in MOPS buffer (Goodet al., 1996). Before loading onto the gel, RNA was mixed with loading buffer and heated at90ºC for 3-min
    15. For isolation of RNA, cells were grown in minimal A medium supplemented with 0.2%glucose upto A600of 0.6. Cells were harvested by centrifugation and total RNA wasisolated by using Trizol (Invitrogen) according to manufacturer’s instructions. 1 ml ofTrizol was used to lyse cells equivalent of approximately 4 ml of overnight culture.Homogeneous lysis was achieved by gentle pipetting repeatedly. The homogenized samples were incubated at room temperature for 5-min to permit complete dissociationof nucleoprotein particles. Following homogenization, 0.2 ml of chloroform for each 1ml Trizol reagent was added and vigorously shaken with hand for 15-sec and incubatedfurther for 3-min at RT. It was then centrifuged at 12000 rpm for 10-min at 4ºC, whichseparates out the homogenate into lower phenol chloroform phase (red), an interphase
    16. Isolation of total cellular RNA
    17. require high fidelity,Taq DNA Polymerase from MBI Fermentas was used. However,for precise amplifications either Herculase Fusion or PfuDNA polymerasefrom Stratagene was used. Approximately, 10-20ng of plasmid or 100 to 200 ng ofchromosomal DNA was used as a template in a 50 μl reaction volume containing 200μM of each dNTP, 20 picomoleeach of forward and reverse primer and 1.5 units of DNA polymerase.In the case of colony PCR performed to examine multiple colonies for presence of the plasmid clones, E. coli cells from afreshly grown plate wereresuspended in 50 μl of sterile Milli-Q water to get a cell suspension (~109cells/ml)and 4 μl from this was usedas the source of DNA template. To verify various pMU575 clonesdescribed in this study, by colony PCR,the vector specific primer pairs JGJpMUF and JGJgalK were used. The expected amplicon for pMU575 alone is ~300-bp, while that carrying the cloned fragment would be >300-bp.For each PCR reaction, the samples were subjected to 30-cycles of amplification and the typical conditions were as follows (although there were slight alterations from one set of template/primerto another):The initial denaturation was carried out at 95°C for 4-min and the cycle conditionswere as given below:Annealing 45ºC to 50°C 1-minExtension 68°C (1-min/kb of DNA template to be amplified)Denaturation 95°C 1-minAfter 30 cycles of PCR, the final extension step was carried out again for 10-min at68°C
    18. For amplification of short length (100-200-bp)DNA fragmentsor that do not
    19. Polymerase chain reaction (PCR)
    20. Molecular techniques
    21. Gel-filtration chromatography was performed at room temperature on a BioLogic LP protein purification system (Biorad) with an in-house packed Sephadex G-100 column of size 1.5 X 43 cm; each protein sample was loaded in 0.8-ml volume, and the buffer used for chromatography was 20 mM Tris-Cl (pH 8) with 200 mM NaCl at a flow rate of 0.1 ml per min with 1.5-ml fractions being collected for analysis. Protein elution was detected by measurement of A295.The void volume, V0was determined using blue dextran (2X 106Daltons) and theelution parameter Kavfor each proteinwas calculated from elution volume Veand total bed volumeVtusing the equation:Kav= (Ve–V0)/(Vt–V0)Initially, acalibration curve was derived froma semilogarithmic plotof Kav of protein standardsalbumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa) and ribonuclease A (13 kDa) on the Y-axis against log10of their molecular masses on theX-axis. The Kavof the ArgPdproteins were calculated based on their elution volume and then the molecular masses were derived from the corresponding point on the calibration curve
    22. Around 0.5 to 1 μg of DNA was regularly used for each restriction digestion. 2to 5units of restriction enzyme were used in the total reaction volume of 20 μl containing 2μl of the corresponding buffer supplied at 10 X concentration by the manufacturer. Thereaction was incubated for 2 hrs at the temperature recommended by the manufacturer.The DNA fragments were visualised by ethidium bromide staining after electrophoresison a 0.8 to 1% agarose gels. Commercially available DNA size markers were run alongwith the digestion samples to compare with and to estimate the sizes of the restrictionfragments
    23. Restriction enzyme digestion and analysis
    24. Thialysine or thiosine (S-Aminoethyl-L-cysteine)is a toxic analog of Lys. Strains were testedfor sensitivity/resistance to thialysine by streaking them on minimal A-glucose platessupplemented without and with100-200 μg/ml thialysine(Steffes et al., 1992)
    25. Test for thialysine resistance
    26. For testing ArgR+/–phenotype, the colonies werestreaked on minimal A-glucose plates containing uracil (40 μg/ml) and CAN(65 μg/ml). Uracil wasadded to the medium to sensitize an argR+strain to CAN. An argR+strain is inhibited at65 μg/ml CANon a uracil-containing plate, whereas on a plate without uracil, argR+would grow even at 700-800 μg/ml CAN. Uracil represses the carAB transcription, whichencodes the carbamoyl phosphate synthase enzyme (CarAB). This results in reducedamounts of carbamoyl phosphate, which is the common intermediate between pyrimidineand Arg biosynthetic pathways. Reduced carbamoyl phosphate levels would result indecreased flux through the Arg biosynthetic pathways. This in turn would result indecrease in Arg pools inside the cell. An argR mutant would be derepressed for the Argbiosynthetic pathway and is resistant even to 300 μg/ml CANin a uracil-containing plate
    27. Test for ArgR+/–phenotype
    28. Test for canavanine (CAN) sensitivity
    29. CAN is a toxic analog of Arg and is an inhibitor of bacterial growth. Strains were tested for sensitivity/resistance to CAN by streaking them on minimal A-glucose platessupplemented withoutand with40 μg/ml CAN(or other concentrations as indicated) and 40 μg/ml uracil
    30. The colonies to be tested were streaked on the surface of minimal A-glucose plates containing either 0.4-0.7 M NaCl with 1 mM glycine betaine, and incubated at 37oC. NaCl-tolerant strains grew toform single colonies in 36-60 hrs whereas NaCl-sensitive ones did not. As controls, MC4100 (WT) and other previously identified NaCl sensitive mutants were streakedfor comparison
    31. NaCl-sensitivity testing
    32. agar platesLac+colonies will appear dark pink colonies whereas Lac–will remain colourless
    33. A. lacphenotype
    34. Scoring for phenotypes
    1. Log-phase yeastcells were collected, washed and suspendedin 10 mM Tris-HCl (pH 7.5) containing 50 mg/ml zymolyase-20T. Cell suspension was incubated at room temperature and absorbance was monitored at 600 nm every10mininterval. Initial absorbance of the cultures at 0 minwas normalized to 100%and the graph was plottedas%decrease in the absorbance with respect to time
    2. Zymolyasedigestion assay
    3. OD600of 0.5and transferred to a 1.5 ml microcentrifuge tube. Probe loading was carried out by adding freshly-prepared CFDA-SE solution (0.01 M stock in DMSO) tocell suspension to a final concentration of 160 μM. Cell suspension was mixed on vortex mixerfor 10 secand incubated at 37 ̊C for 1 hwith shaking at 300 rpmon thermo mixer.Cells were harvested, washed twice with 1 ml 50 mM CP buffer to remove unloaded probe,resuspendedin 250 μl CP buffer andwereincubated at 30 ̊C for 30 minwith shaking to recover from the stress induced during probe loading. Afterincubation,fluorescent intensitywasdetermined with spectrofluorophotometer (Varioskan flash-3001, Thermo Scientific) by excitation at 430 nm (pH-independent) and 490 nm (pH-dependent) with emission at 525 nm. Background fluorescence of the probe was removed by subtracting the fluorescence intensity of the probe in CP buffer from the fluorescence intensity of the probe-loaded cells
    4. Intracellular pH(pHi)in yeast cells was determinedusing fluorescent 5,(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Molecular Probes) asdescribed previously (Bracey et al.1998). For pHiprobe estimation,YNB medium-grown log-phase cells were inoculatedin YNB, YNB-pH 2.0 or YNB medium supplemented with acetic acid and incubated at 30 ̊C for different time points.Log-phase C. glabratacells were harvested and washed twice with 50 mM citric-phosphate (CP) buffer (pH 4.0). Washed cells were resuspendedin 1ml 50 mM CP buffer to an
    5. Measurementof intracellular pH (pHi)
    6. stranded DNA. Final reaction volume was adjusted to 20 μl with DEPC-treated waterandamplificationreaction was carried out usingthese parameters: initial denaturation at 95 ̊C for 5 min followed by 40 cycles of denaturationat 95 ̊C for 30 sec, annealing at 55 ̊C-57 ̊C for 30 sec, elongation at 72 ̊C for 40 sec and final extension at 72 ̊C for 10 min. Transcript levelswerequantified with an end-point value known as Ct (cycle threshold). The Ctdefines the number of PCR cycles required forthe fluorescent signal of SYBR green dye to cross more than the background level. The Ctvalue isinversely proportional to the amount of nucleic acid product. Ctvalues were obtained during exponential phase of amplification and used forcalculation of relative-fold change in gene expression after normalization to Ctvalues ofeither housekeeping gene ACT1 (gene encoding actin)orTDH3 (gene encoding Gapdh)with the help of the following formula. Fold change in expression = 2-∆∆Ct∆∆Ct= ∆Cttreated -∆Ctuntreated∆Cttreated = Ctvalue forgene of interest under test/treatedcondition -Ctvalue forinternal controlgene(ACT1/TDH3) under test/treatedcondition∆Ctuntreated = Ctvalue forgene of interest under untreatedcondition -Ctvalue forinternal control (ACT1/TDH3)gene under untreatedcondition
    7. Todeterminethe expression level of a specific gene, quantitative real-time polymerase chain reaction (qRT-PCR/qPCR)was performed oncDNA usinggene specific primers. Primers for qPCR weredesigned in such a way so as to get amplification products in a size range of 150 to 300 bp. Optimalprimer and cDNA concentrationswere standardized and qPCR was performed in ABI Prism 7000/7500 Real time PCR Machine (Applied Biosystems). Briefly, 0.4 μl cDNA was mixed with 0.1 to 0.2 picomolesof gene specific forward and reverse primers and 10 μl 2X MESA GREEN qPCR™Mastermix Plus containing SYBR green dye (Eurogentec) in awell of a96-well PCR plate (Axygen). SYBR green is a dye that specifically binds to double
    8. Quantitative real-time polymerase chain reaction (qRT-PCR)
    9. C. glabratastrains were grown overnighteither in YPDor YNBliquid mediumat 30 ̊C with shaking at 200 rpm. Cells were harvested and suspended in 1X PBS to a final OD600of 1.0.Five 10-fold serial dilutions of cell suspension wereprepared in PBS and3-4μlwasspotted on YPD/YNBplates containing various test compoundsusing a multi-channel pipette.Plates were incubated at 30 ̊C and growth profileswererecorded after2-4days
    10. Serial dilution spot assay
  4. sg.inflibnet.ac.in sg.inflibnet.ac.in
    1. activated TLC silicagel-60plate and transferred to theTLC chamber. After the solvent had migratedupwards (1.5 cm fromthetop), TLC plate was removed, air dried behind perspex shield, wrapped with cling plastic wrap and was exposed to phophorimager screenfor 2 h. Phosphorimager screen was scanned usingaFugi-FLA 9000 scanner
    2. To resolvephospholipids,a TLC chamber was prepared by pouring50 ml developing solution and sealing the chamber with aluminium foil so that developing solution can generate vapor. TLC silicagel-60plate(Merck)was incubated at 80ºCfor 4 h for activation. After 30 min of TLC chamber preparation, phospholipidsextracted from C. glabratacells werespotted at thebottom (1.5 cm fromthelowerend) of the
    3. Seperation of phospholipids by thin layer chromatography(TLC)
    4. PI-3kinase reaction was set up ina total volume of50μlin a 1.5 ml microcentrifuge tube as described below.PI-3 kinase reaction buffer = 25 μlSpheroplast lysate = 20 μl (equivalent to 10 μg protein)Sonicated phosphatidyl inositol = 5 μlReaction mix was incubated at 25ºC for 20 min and enzyme reaction was stopped by adding 80μlHCL (1N) solution. To extract phospholipids, 160 μl chloroform:methanol (1:1) was added to the reaction mix withcontinuous mixing. Organic phase containing phopholipidswas separated fromaqueous phase by centrifugation at 7,500g for 4 min at 4ºC and transferred to a new vial. Using vacuum evaporator apparatus, solvent was evaporated and phospholipidsweredissolved in 10 μl chloroform
    5. PI-3 kinase reaction set up and phopsholipid extraction
    6. 10 mg phosphatidylinositol-sodium salt(from Glycine max)was dissolved in 2 ml chloroform to prepare a 5 mg/ml stock solution. This solution was prepared in a small glass vial aschloroformis known to reactwith polypropylene. Small aliquots of stock solution were madeand stored at -20ºC till further use. To avoid spillage due to vapor pressure, vials containing phosphatidylinositol-sodium salt solutionwereopened very carefully.To prepare sonicated phosphatidylinositolfor one PI-3 kinase reaction, 2 μlof the stock phosphatidylinositolsolution (10 μg) wastransferredtoanew1.5 ml microcentrifuge tube. Using vacuum evaporator apparatus, chloroformwas evaporated from the solution and phosphatidylinositol-sodium saltwas resuspended in 5 μl sonication buffer.For sonication, a total of 20 pulses, each of 30 sec with30 sec resting time weregiven on ice
    7. Preparation and sonication of phosphatidylinositol-sodium salt solution
    8. A single colonyof desired C. glabratastrainwas inoculated in YPD-liquid mediumand grown for 14-16 h. 50 μl overnight culture was inoculated inYPD-liquid mediumfor 4 h. Log-phase-grownyeast cells were harvested,washedwith PBSandwereinoculated atinitial OD600of 2 and 4,into YNB-dextrose and YNB-sodium acetate liquid medium,respectively.After 4 hincubation,yeast cells were harvested by centrifugation at 2,500g for 5 minand treated with 1.2 M zymolyasefor 1 hto obtain spheroplasts.Post zymolyase treatment, spheroplasts were resuspended in 100 μl resuspension bufferandanequal amount of 0.25 mm glass beadswasadded to lyse the spheroplasts. Using bead beater apparatus, spheroplasts were lysed and protein concentration in spheroplast lysateswas determined usingbicinchoninic acid assay (BCA) method and samples were stored at -20ºC till further use
    9. Preparation of cell lysate
    10. In vitroPI-3 kinase reactions wereset up to measure PI-3P synthesized as described earlier(Whitman et al., 1988)
    11. Phosphatidyl inositol-3 kinase (PI-3 kinase assay)
    12. Colony blot assay was performed to analyse secretion of carboxypeptidase Y(CPY)as described previously (Roberts et al., 1991). Single colony of a C. glabratastrain was inoculated in YPD medium andculture was grown till stationary phase. 0.1 OD600equivalent cellsfrom this culture were spotted on CAA medium,overlaidwith a nitrocellulose membrane and plate was incubated at30 ̊C for 18-20 h.Afterincubation, nitrocellulose membranewas washed with water to remove cells and membrane-bound CPYwas detected by immunoblotting with polyclonal anti-CPY antibody at a dilution of 1:10,000
    13. Colonyblot assay
    14. was washed three times with PBS to remove non-adherantC. glabratacellsand Lec-2 cells were lysed in 5% SDS. Lysates were transferred totubes containing scintillation fluidand radioactive counts obtained were considered as ‘output values’. Percentage adherence wasdetermined using following formula
    15. Adherence of C. glabratacells toLec-2 epithelial cells wasmeasured as described previously(Cormack et al., 1999).Lec2cells were seeded ina 24-well tissue culture plate at a seeding density of 5X105cells per well and allowed to adhere for 12 h. After 12 h,medium supernatant was discarded by inverting the plate in a reservoir and cells were washed thrice with PBS. Lec2 cells were fixed in 3.7% para-formaldehyde for 15 minfollowed by 2 PBS washes. PBS containing antibiotics, penicillin and streptomycin,was added toeach well of the 24-well plate and Lec-2 cellswere stored at 4°C.For adherence measurement,strains were taken out either on YPD or CAA mediumandgrown at 30°C for 2 days. Single colony of a C. glabratastrain wasinoculated in 10 ml CAA medium ina 100 ml culture flaskand allowed to grow at 30°C for 16-20 h. 100 μlyeast culture wasreinoculated in fresh 5 ml CAA liquid medium in a 15 ml polypropylene tube. 200 μCi of S35(Met:Cys-65:25) INVIVO PROTWIN labelmix(JONAKI, India) was added to thetube and cultures were grown at 30°C for 16-20 h for radiolabeling of C. glabratacells. C. glabratacells from 1 ml culture were harvested and washed threetimes with PBS to remove residual S35(Met:Cys-65:25) labeling mix from medium supernatant. Next,cells were resuspended in 1 ml PBS. OD600was measured and cell suspensions of 0.5 OD600were prepared. PBS was aspirated out of the wells of 24-well plate containing fixed Lec-2 cells. 200 μl of S35(Met:Cys-65:25)-labeled C. glabratacell suspensions were added to each well. To determine the total amount of radioactivity present in labeled C. glabratacell suspension, 200 μl of S35(Met:Cys-65:25)-labeledC. glabratacell suspensions were transferred to a scintillation vial containing scintillation fluid. Radioactive counts present in this fraction were considered as ‘input values’. For measurement of yeast adherence to Lec-2 cells, plates were centrifuged at 1,000g for 5 min and incubated for 30 min at room temperature. Following incubation, each wel
    16. Adherence assay
    17. 20 mg protein samples, isolated from RPMI-grown and macrophage-internalized yeast, were usedto measure KDAC activityusing HDAC Fluorimetric Assay/Drug Discovery Kit (EnzoLifeScience) as per manufacturer’s instructions
    18. Lysine deacetylase (KDAC) activity measurement
    19. For protein extraction, yeast cells were suspended in 50-100μl protein extraction buffer containing 320 mM (NH4)2SO4, 200 mM Tris-Cl (pH 8), 20 mM EDTA (pH 8), 10 mM EGTA (pH 8), 5 mM MgCl2, 1 mMDTT, 10% glycerol and protease inhibitorsand disrupted using glass beads.Cell lysate was centrifuged at 7,500g and4oC for 15 min. 30 μg of total protein was resolved on a 15% SDS-PAGE gelat 32 mA till the dye front reachedthe bottom. Resolved proteins were transferred to Hybond-P membrane at 350 mA for 1.5 h in the cold room.Transfer of the proteins was visually confirmed by examining marker’s lane and membranes wereincubated in a small box for 2 h in 5% fat free milkprepared in 1X TBST for blocking. Blocking solutions were discarded and primary antibody, appropriately diluted in 5% fat free milkprepared in 1X TBST,was added to the box containing membrane. After overnight incubation in primary antibody, membranes were washed thrice with 1X TBST for 10 min. Membranes wereincubated for 2 h inappropriate secondary antibodydiluted in 5% fat free milkprepared in 1X TBST. Blots were either developedby chemiluminescence based ECL-Plus western detection system orChemidocTMgel imagingsystem. CgGapdhwas used as a loading control. To exclude the possibility of any contribution of THP-1 proteins tocell extracts prepared frommacrophage-internalized yeast, two control experiments wereperformed. First, we probedthe blots with antibodies specific for mammalian tubulin and actin.As expected, we neitherdetectedanysignal for mammalian actin nor formammalian tubulin. In the second control experiment, we treated macrophage lysates with proteinase-K prior to the yeast pellet disruptionand probed yeast lysates for different histone modifications.This proteinase-K treatmentdid not alter the epigenetic signature of C. glabratacells.Together, these data indicate that yeast protein samples were devoid ofany mammalian protein contamination
    20. Protein extraction and immunoblotting
    21. homogenizedin 1 ml PBS and fungal burden was assessed by plating appropriate dilutions of tissue homogenate on YPD plates containing penicillin and streptomycinantibiotics (100units/mlpenicillin and 100μg/mlstreptomycin). All mice experiments were repeated twice with a set of 7-8 mice per strain in each experiment
    22. Experiments involving mice were conducted at VIMTA Labs, Hyderabad.100 l YPD-grown C.glabratacellsuspension(4 X 107cells)was injected into female BALB/c mice (6-8 weeks old) through tail vein. Seven dayspost infection, mice weresacrificedand kidneys, liver,spleenand brainwere harvested. Organs were
    23. Mouse infection assay
    24. Experiments involving mice were conducted at VIMTA Labs Limited, Hyderabad in strict accordance withguidelines of The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The protocol was approved by the Institutional Animal Ethics Committee (IAEC) of the Vimta Labs Ltd. (IAEC protocol approval number: PCD/OS/05). Procedures used in this protocol were designed to minimizeanimalsuffering
    25. Ethics statement
    26. Othermethods
    27. After14-16 hincubation, hybridization buffer was decanted to a radioactive liquid waste container.Membraneswere washedtwice with 2X SSC (saline-sodium citrate) containing 0.1% SDS for 15 min at 55°C followed by two washes with 1X SSC containing 0.1% SDS for 15 min at room temperature. Post washes,membranes were rinsed with 1XSSC buffer at room temperature and exposed to phosphorimager screen for 2-3 h
    28. Post-hybridization washes
    29. Identified mutants were phenotypically characterized in 96-well plate format. Mutant cultures were grown in YPD medium for overnight, diluted 150-fold in PBS and 5 μl of cell suspension was spotted on different plates with a 96-pin replicator. Growth was recorded after 1-2 daysof incubation at 30°C
    30. Phenotypic profiling
    31. THP-1 monocytes were treated with phorbol myrsitylacetate (PMA) to differentiate them to macrophages(Tsuchiya et al., 1982). For PMA treatment, THP-1 cells grown upto 70-80% confluencewere harvested from the culture dishes at 1,000 rpm for 3 min. Harvested THP-1 cells were resuspended in 5-10 ml fresh and prewarmed complete RPMI medium. 100μlof thiscell suspensionwasappropriatelydilutedinPBS and numberof viable cells was determined by trypan blue stainingusing hemocytometer. Cell suspension was diluted with prewarmed RPMI medium to a final density of 106cells/ml. PMA was added to this THP-1 cell suspension to a final concentration of 16 nM and mixedwell.PMA-treated THP-1 cellswere seeded either in 24-well cell culture plate or culture dishes and transferred to the incubator set at 37°C and 5%CO2.After 12 hincubation, medium was replaced with fresh prewarmed medium and cells wereallowed to recover for 12 h
    32. Treatmentof THP-1 monocytic cells with phorbol myrsityl acetate
    1. The immunoblots were quantified by densitometry software ImageJ 1.17 developed by Wayne Rasband, NIH Bethesda, MD (http://rsb.info.nih.gov/nih-image).All experiments were done at least in triplicates and results were expressed as mean ±s.e.m. A two tailedStudent’s t-test was done in Graph pad to arrive at p values and differences were considered statistically significant when p-value was less than 0.05 (*p≤ 0.05), highly significant(**p≤ 0.01)andextremely significant (***p≤ 0.001)
    2. Quantification of blots and statistical analysis
    3. A total of 100-200ng of DNA was used in each ligation reaction. Vector to insert ratio of 1:3 to 1:5was maintained. The reaction volume was generally maintained at 10μl containing 1μl of 10X ligation buffer (provided by the manufacturer) and 0.05 Weissunit of T4-DNA ligase. The reaction was carried outat 16ºC for 14-to 16-hrs or at room temperature for 4hours
    4. Ligation of DNA
    5. Agarose gels were preparedby boiling appropriate amount of agarose in TAEbuffer. After dissolution, it was cooled and then poured in a casting tray containing a comb for desired number of wells. The gel was allowed to solidify and then shifted to horizontal electrophoresis tank containing TAE buffer. The DNA samples were mixed with appropriate volumes of 6X DNA loading dye, loaded on the gel andelectrophoresedat appropriate voltage and current conditions (generally 80 V,400 mA). The gel was stained in ethidium bromide solution(1 μg/ml)for 15-min at room temperature and visualisedby fluorescence under UV-light in a UV-transilluminator
    6. Agarose Gel Electrophoresis
    7. For preparation ofcellular homogenate from adherent cell culture, the medium was first removed and cells were washed with ice cold 1X PBS. The cells were then scraped in 1X PBS and pellet down by gentle centrifugation (4000 rpm for 2 minutes) at 40C. Cell lysis buffer was then added to the cell pellets and lysis was allowed for 30 minutes on a rotor at 4⁰C. Post lysis, cellswere centrifuged at 13000 rpm for 10min at 4°C. The pellet was discarded and supernatantwascollectedas cell homogenate
    8. Extraction of total cellular protein
    1. Wound healing assay(Liang et al., 2007) was employed to study the difference in migration rates ofprofilin-stable compared to parentalcells. Briefly, cells were cultured upto 90-95% confluent monolayer and a scratch was created through the cell monolayer with sterile needle. Cell debris was then removed by washing with PBS before adding the media. Images of the open gap created by this “wound” were then captured at three random locations immediately (0 h) and then at the same locations after regular interval using phase contrast microscopy untilthey are closed by migrating cells. Captured images were then used to quantify wound closure by the percentage change in the wound area per unit time and averaged for three locations for each experimental condition. During the course of the experiment, cells were maintained in 0.1% FBS containing DMEM media to ensure that wound closurewas due to the migration of cells rather thandivisonof cells
    2. Wound healing assay
    1. Analysis of sensitivity to translation inhibitors was conducted in theDDY1810 S. cerevisiaestrain background, whichdoes not contain the kanrselection marker. Sensitivity to 6-azauracil (6AU) was monitored in the DDY1810, BY4741 or NOY222 strain backgrounds (Table 2.1). As uracil is a competitive inhibitor of 6AU, the plasmid p416GPD, carrying the URA3gene (Mumberget al., 1995)was introduced into BY4741-derived strainswhereas DDY1810 derived yeast strains were supported by the pYesGex plasmid carrying the URA3gene.Yeast strains were grown in YPD or SC-Ura medium,for 14-16 h at 30°C under continuous shaking at 200 rpm. Cultures were diluted to 0.25OD600, followed by 5 fold serial dilutions, and 3μL of each dilution was spotted on a YPD-agar plate containing the translation inhibitors G418 (8 μg/mL), paromomycin (100μg/mL or 200μg/mL), or hygromycin B (8 μg/mL), or an SC-Ura agar plate, containing 6AU 50 μg/mL or100 μg/mL and growth was monitored at 30°C or 37°C for 2-3 days. To perform an analysis of 6AU sensitivity with yeast carrying pYesGex6p2 plasmid, cells were grown overnight in SC-Uramedium and the serial dilutions were plated on SC-Ura medium containing 6AU, with galactose instead of glucose to express proteins under the GAL4promoter
    2. Drug sensitivity analysis
    1. The LR reaction mixturewas incubated at room temperature for 1hr, followed by proteinase K treatment (2μL) at 37°C for 10 min. 5uL of the reaction mix was transformed into DH5α competent cells. Bacterial cells were spread on LB agar plates containing antibiotic ampicillin (50ug/ml). Plates were incubated at 37°C for overnight. The bacterial colonies were inoculated into 5mL LB broth containing 5uL ampicillin and incubated overnight in shaking incubator at 220 rpm. Next day, Plasmids were prepared using Plasmid miniprep kit (QIAprep miniprep). The destination plasmids obtained by LR reaction were given for plasmid-DNA sequencing to confirm thepositive clones. The positive clones were amplified through DH5α transformation and plasmid DNA maxiprep (Invitrogen). The expression plasmids generated through LR were used for studies in the mammalian and the bacterial cells
    2. BP reaction mix was incubated at room temperature for 1hr, followed by proteinase Ktreatment (2μL) at 37°C for 10 min. 5uL of the reaction mix was transformed into DH5α competent cells (Transformation into DH5α competent cells; plasmid constructs added into DH5α competent cells and incubated on ice for 30 min. followed by heat shock at 42°C for 1 min. 800μL LB broth was added and transformed DH5α cells were incubated at 37°C in shaking incubator at220 rpm for 1hr.;after 1 hr. cells were centrifuged at 6000 rpm for 1 min.;the supernatant was discarded,and the pellet was resuspended in 100μL of LB broth). Bacterial cells were spread on LB agar plates containing antibiotic kanamycin (30ug/ml). Plates were incubatedat 37°C for overnight. The bacterial colonieswereinoculated into 5mL LB broth containing5uL kanamycinand incubated overnight in shaking incubator at 220 rpm. Next day, Plasmids were prepared using Plasmid miniprep kit(QIAprep miniprep). The donor plasmids generatedby BP reaction were given for plasmid-DNA sequencing to confirm the positive clones.The donor plasmids obtained by BP reaction were cloned into the Gateway destination vectors by LR reaction (Table 7).Table 7: LR reaction mixture
    3. Gateway cloning is the highly efficient gene cloning technology. It comprises two primary steps of cloning; the BP reaction and the LR reaction. The PCR products of gene of interest were cloned into the Gateway donor vector by BP reaction(Table 6).Table 6: BP reaction mixture
    4. Gateway cloning
    1. biosensor strain 8523/KLN55was inoculated in fresh medium, and grown with the ethyl acetate extract isolated from the test strain as described earlier. After 30 h of growth, cells were pelleted by centrifugation, washed once with sterile water and resuspended in sterile miliQ waterfor measuring the GFP fluorescence intensity at excitation and emission wavelength of 472 and 512 nm, respectively. 1 DSF unit is equivalent to increase in fluorescence by 1 arbitary unit in DSF biosensor strain
    2. For DSF extraction, X. oryzaepv. oryzicolastrains were grown in PS media to an OD600 of 1.2 as described earlier. Supernatant was collected by pelleting down the cells at 7000 g for 10 min. Next, water-saturated ethyl acetate was added to the cell-free culture supernatant in a ratio of 2:1, and mixed properly for 5-10 min. The mixture was centrifuged at 5000 g to separate the DSF containing organic phase. The ethyl acetate layer (organic phase) was evaporated at 37°C, remaining residue was dissolved in methanol, and assayed for DSF by using Xccbiosensor strain 8523/KLN55 (Newman et al., 2004). Biosensor strain is a DSF minus strain comprised of DSF responsive endoglucanase promoter fused to promoterless gfpand expressed through plasmid (Peng::gfp). To check the DSF production by a particular strain, 0.2% inoc
    3. Isolation and detection of DSF
    4. Complementary-DNA synthesis was performed using reverse transcriptase enzyme (Invitrogen) and random hexamers (Qiagen). For this, 1 μg good quality RNA was treated with 1 μl (1 unit) DNase I (Invitrogen) for 20 min to remove DNA contamination. Next, Superscript III Reverse Transcriptase kit (Invitrogen) was used to synthesize cDNA according to the manufacturer’s instructions. cDNA synthesized was further confirmed by using it as a template for amplification in PCR. cDNA was stored at -20°C till further use
    5. Synthesis of complementary DNA (cDNA)
    6. A single colony of E.coliDH5α strain was inoculated in 5 ml LB medium and incubated at 37°C for overnight. 1% of overnight grown culture was inoculated in 500 mlfresh LB medium and incubated at 37°C for 2-3 h till the OD600 reached to 0.4-0.5. Culture was chilled on ice for 5 min followed by centrifugation at 3000 g for 15 min at 4°C. Harvested cells were washed gently with 200 ml ice-cold TFb-I buffer. Cells were collected by centrifugation at 3000 g for 5 min at 4°C and gently resuspended in 20 ml ice-cold TFb-II buffer. Bacterial cell suspension was kept on ice for 15 min and was aliquoted in 100 μl volumes in chilled sterile microcentrifuge tubes. Cells were immediately snap-frozen in liquid nitrogen and stored at -80°C
    7. Preparation of E.coliultracompetent cells
    1. Overnight-grown C. glabratacells were freshly inoculated either in YNBmedium or YNBmedium supplemented with BPS (50 μM) or FeCl3(500 μM) and allowed to grow for 4 h at 30°C, 200 rpm. After 4 h growth, cells were spun down at 4,000 rpm for 5 min in a refrigeratedcentrifuge set at 4°C and total protein was isolated. For estimation of histone deacetylase (HDAC) activity, 40 μg of protein samples were taken and HDAC Fluorometric Activity Assay Kit (#10011563; Cayman Chemical Company, Ann Arbor, MI, USA) was used as per manufacturer’s instructions. Fluorescence intensity values obtained inthepresence of the HDAC inhibitor, trichostatin A, were subtracted from those of the samples without inhibitorand plotted as relative arbitrary fluorescence units
    2. Estimation of histone deacetylase (HDAC) activity
    3. spectro-photometrically at 340 nm. For wild-type cells,mitochondrial aconitae activity was normalized to 100 % and for mutants the relative aconitase activity percentages were calculated
    4. To determine aconitase activity, mitochondria were isolated as described by Meisinger et al. Briefly, YPD-grown C. glabratacells (500 OD600) were subjected to spheroplasting followed by homogenization (15 strokes) with glass Teflon homogenizer. To collect mitochondria, homogenate was centrifuged at 13200 g for 20 min in a refrigerated centrifuge set at 4°C. The mitochondrial pellet was resuspended in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM Mops-KOH, pH 7.2) and stored at -80°C until further use. Mitochondrial aconitase activity was estimated by using method as described by Bulteau et al. Mitochondrial protein samples (5 μg) were prepared in KH2PO4buffer (25 mM, pH 7.2) containing 0.05 % Triton X-100. The samples were incubated with sodium citrate (1 mM), MnCl2(0.6 mM), NADP (0.2 mM) and isocitrate dehydrogenase (1 U/ml) for 20 min at room temperature. Isocitrate dehydrogenase catalysed reduction of NADP was recorded
    5. Measurement of aconitase activity
    6. For quantification of intracellular iron content, BPS-based colorimetric method as described by Tamarit et al.,was used. Briefly, overnight grown C. glabratacells were inoculated in fresh YPDmedium and allowed to grow at 30°Cand200 rpm for 6 h. Cultures were spun down and 50 OD600cells were subjected to nitric acid digestion (500 μl, 3%) for 16 h at 96°C.Next, lysates were spun down at 13,000 rpm to remove the cell debrisand 400 μl of the lysatewas incubated with 126 μl of ammonium acetate (1 M), 320 μl of BPS (1.7 mg/ml) and 160 μl of sodium ascorbate (38 mg/ml)for 5 min at room temperature. Absorbance of the samples was takenagainst the reagent blank at 535 nm and 680 nm which correspond to BPS-Fe-specific and BPS-Fe-non-specific absorbance, respectively. Non-specific absorbance was subtracted from specific absorbance and the iron content in each sample was calculated from the standard curve prepared using FeCl3and expressed as μM per OD600cells. In each experiment performed, total iron content of wild-typecellswas normalized to 100% and the iron content of mutants were calculated with respect tothe iron content of wild-type cells
    7. Estimation of total iron
    8. Radioactive counts measured in2x106labelled C. glabratacells and lysates were considered as ‘input’ and ‘output’ values, respectively. Percentage adherence was calculated by following equation.%Adherence=Output radioactive countsInput radioactive countsX 100
    9. Adherence of C. glabratacells to Lec2, Chinese hamster ovarian (CHO) cells, wasdetermined as described previously (Cormack et al., 1999). Briefly, Lec2 cells were seeded at a cell density of 5x105cells per wellin a 24-well tissue culture plate.Cells were incubated in a cell culture incubator (Thermo Scientific) set at 37°C and 5%CO2for 12 h. Post incubation, the medium was discarded in a reservoir and Lec2 monolayer was washed thrice with sterile 1X PBS without disturbing the monolayer. Lec2 cells were fixed with 3.7% para-formaldehyde for15 min followed by twoPBS washes. 1 mlof 1X PBS containing antibiotics, penicillin (100 units/ml) and streptomycin (100 μg/ml), was added to each well, plates were sealed with PARAFILM, Cole-Parmer(PM-996) and stored at 4°C until use.C. glabrata cells,to be tested for their adherence potential, were grown in CAAmedium for 24 h.100 μl of 24 h-grownculture was re-inoculated in fresh 5 ml CAAmedium containing 200 μCi of S35(Met:Cys-65:25) INVIVO PROTWIN label mix (JONAKI, India)in a 15 ml polypropylene tube.Cultures were allowed to grow for 16-20 h at 30°C with shakingat200 rpm to radiolabel the cells. Radiolabelled C. glabratacells were harvested by spinning down1 ml of labelled yeast cultures,andthe cell pellet was washed thrice with sterile 1X PBS to remove any residual S35(Met:Cys-65:25) labelling mix from the medium. Post washes, the pellet was resuspended in 1 ml PBS, OD600was measured andcell suspension of 0.4 OD600wasprepared.Next, 24well plates containing fixed Lec2 cells were taken out from 4°C and PBS from the wells wasdiscarded by inverting the plates. Wells were washed once with PBS and 2x106labelled yeast cells were added to eachwell, andincubatedfor 30 min at room temperature.Post incubation, plates were centrifuged at 1,000 rpm and the wells were washed thrice with 1X PBS to remove non-adherent C. glabratacells. Lec2 cells were lysed with 5% SDS in PBS by scraping the wells, lysates were collected and transferred to a vial containing scintillation fluid
    10. Adherence assay
    11. For mouse infection assay, 10 ml YPD medium was inoculated with different C. glabratastrains and allowed to grow at 30°C for 12-16 h. After growth,cultures were washed twice in sterile 1X PBS and the cell pellet was resuspended in appropriate volume of 1X PBSto obtain a cell density corresponding to20OD600. 100 μlcell suspension(4x107yeast cells)was injected into female BALB/c mice (6-8 weeks old) through tailvein. Seven days post
    12. infection, mice were sacrificed and kidneys, liver, brain and spleen were harvested. Organs were homogenised in 1 ml PBS and appropriate dilutions of tissue homogenate were plated onYPD-agar mediumsupplemented with penicillin and streptomycin antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). Plates were incubated at 30°C for 24-48 h and CFUs were counted. Fungal burden in different organs wasdetermined by multiplying the CFUsobtainedwithanappropriate dilution factor
    13. For mouse infection assay, 10 ml YPD medium was inoculated with different C. glabratastrains and allowed to grow at 30°C for 12-16 h. After growth,cultures were washed twice in sterile 1X PBS and the cell pellet was resuspended in appropriate volume of 1X PBSto obtain a cell density corresponding to20OD600. 100 μlcell suspension(4x107yeast cells)was injected into female BALB/c mice (6-8 weeks old) through tailvein. Seven days post
    14. Mouse infection assay
    15. Experiments involving mice were performed at the CDFD animal facility, VIMTA Labs Ltd., Hyderabad, India(www.vimta.com) in strict accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The protocol was approved by Institutional Animal Ethics Committee (IAEC) of the Vimta Labs Ltd. (IAEC protocol approval number: PCD/CDFD/05). Procedures used in this protocol were designed to minimize animal suffering
    16. Ethics statement
    17. Other methods