volume of 50 mM acetate buffer (pH-5.4), and dialyzed overnight with 10 mM Tris buffer, pH 7.5. Pellet was used for dilution plating for calculating CFUs. For whole cell protein isolation, bacterial pellet was dissolved in 50 mM sodium acetate buffer (pH-5.4) and sonicated for 30 min (1 min on and off, Amplitude 32) by adding phenylmethylsulfonyl fluoride (PMSF) at a final concentration of 1 mM in ice-cold solution. Both extracellular proteins and whole cell lysate fractions were aliquoted in 1.5 ml microcentrifuge tube, and protein quantification was performed using a Pierce BCA protein assay kit (Thermo Scientific) as per manufacturer’s instructions using bovine serum albumin as standard and stored at -80°C for further use. Cell normalized extracellular and whole cell lysate proteins fractions from different strains were resolved on 12% SDS-PAGE gel at 90 V till the dye front reached the bottom. One gel was processed for silver staining (Sambrook et al., 1989), and other for western-blot analysis by using anti-GFP antibody. For western blot analysis, resolved proteins were transferred to Hybond-ECL membrane (Amersham biosciences) at 35 V for overnight in the cold room. Transfer of the proteins were visually confirmed by examining marker’s lane and membranes were incubated in small box for 2-3 h in 5% fat free milk prepared in 1X PBST for blocking. Blocking solutions were discarded, and primary antibody, appropriately diluted in 5% fat free milk prepared in 1X PBST, was added to the box containing membrane. After 2-3 h incubation in primary antibody, membranes were washed thrice with 1X PBST for 10 min. Membranes were incubated for 2 h in appropriate secondary antibody (anti-Rabbit antibody)diluted in 5% fat free milk prepared in 1X PBST. Blots were either developed by chemiluminescence based ECL-plus western detection system or alkaline phosphatase method. For HRP based chemiluminescence method, detection was performed using the ECL plus kit (Amersham biosciences) and incubated for 3 min. Blot was exposed to the film and developed i

For protein extraction, Xanthomonas oryzaepv. oryzaestrains with eGFP plasmid were grown for 24-30 h in PS medium to an OD of 0.8 as described above and centrifuged at 12,000 g for 10 min. The supernatant was taken as extracellular fraction and protein was extracted as described previously (Ray et al., 2000). Extracellular proteins were precipitated from this fraction by constantly adding 50% (wt/vol) ammonium sulphate at 4°C. After precipitation, the solution was kept on ice for 15-20 min and centrifuged at 12,000 g for 30 min at 4°C. The pellet was dissolved in s

Protein extraction and immunoblotting

development. Absorbance was measured at 490 nm, and concentration of glucose production was calculated against glucose standard. Cellulase activity is expressed as micromoles of reducing sugar (glucose) released per minute per 109cells. For plate assay, cell-free culture supernatant of X. oryzaepv. oryzaestrains were inoculated in wells of 0.2% CMC agarose plates. In addition, cellulase assay was also performed by spotting the colony on 0.2% CMC PSA plates. Plates were incubated for 8 to 24 h and stained with congo red to observe the halo formation as described previously (Wood and Bhat, 1988). Extracellular xylanase activity in different X. oryzaepv. oryzae strains was measured using 0.2% 4-O-methyl-D-glucurono-D-xylanremazol Brilliant Blue R (RBB-Xylan) (Sigma-Aldrich) as substrate (Biely et al., 1988)on 1% agarose plates. Xylanase activity is indicated by production of halo around the bacterial colony (Ray et al., 2000). Similarly, for lipase activity p-nitrophenyl butyrate was used as substrate. Lipase activity was calculated by measuring the level of p-nitrophenol released upon hydrolysis of p-nitrophenyl butyrate at 410 nm (Acharya and Rao, 2002). Lipase activity was expressed as micromoles of p-nitrophenol released permin per109cells. For plate assay, colonies were spotted on 1% PSA plates containing 0.5% Tributyrin in 100 mM Tris (pH 8) and 25 mM CaCl2 and halo formation was observed for lipase activity

For extracellular enzyme assays, X. oryzaepv. oryzae strains were grown in PS, MM9 and XOM2 media to an OD of 0.6, and centrifuged at 12,000 g for 10 min to collect the supernatant. The supernatant was taken as an extracellular fraction and cell pellet was plated by dilutionplating to get the CFUs per milliliter of culture. Extracellular cellulase activity was measured using phenol-sulphuric acid (H2SO4) method, which measures pentoses and hexoses (concentration of glucose released) upon cellulase activity (DuBois et al., 1956). Briefly, a specific amount of supernatant was taken and volume was adjusted to 300 μl by adding 50 mM acetate buffer (pH-5.4). To this, 1% carboxy methyl cellulose (CMC) substrate solution was added and mixed well. This mixture was incubated at 28°C for 30 min, and the reaction was stopped by adding 1 ml ice-cold ethanol. Solution was mixed well, kept on ice for 5 min and centrifuged at 12,000 g for 5 min. Supernatant was recovered and 5% phenol was added to it, mixed well followed by adding 1 ml H2SO4. The tube was incubated at RT for 20 min for co

Extracellular enzyme assays

work were autoclaved twice and dried at 80°C for overnight before use. RNA was isolated from Xanthomonasculture using Trizol method. Xanthomonascells were harvested at 12,000 g for 5 min at 4°C, resuspended in approximately 1 ml Trizol (Invitrogen),mixed properly and incubated at room temperature (RT) for 5 min. 200 μl chloroform was added to the tube, shaken for 15 seconds and incubated at RT for 2-15 seconds. Next, tubes were centrifuged at 13,000 g for 15 min at 4°C. Aqueous phase was transferred to new 1.5 ml microcentrifuge tube and RNA was precipitated by adding 500 μl isopropanol and incubated for 5-10 min at RT. Precipitated RNA was collected by centrifugation at 10,000 gfor 10 min at 4°C. RNA pellet was washed with 70% ethanol and resuspended in 20 μl nuclease-free water. RNA concentration was determined by measuring absorbance at 260 nm. Quality of RNA was examined by gel electrophoresis on 0.8% agarose gel with TAE buffer prepared in DEPC treated water

For RNA experiments, all solutions were prepared in RNase free diethylpyrocarbonate (DEPC) treated water. Microcentrifuge and tips u

RNA extraction

Xanthomonas strains were grown in PS medium for 14-16 h at 28°C with continuous shaking at 200 rpm. 1 ml of bacterial cultures were ten-fold serially diluted in water and 100 μl volume of each dilution was plated on PS agar plates to get the colony forming units (CFUs). Similarly, 5 μl volume of each dilution was spotted on PS agar plates containing different concentration of streptonigrin and different detergents for intracellular iron and membrane sensitivity assay, respectively. Plates were incubated at 28°C and images were captured after 2-8 days of incubation depending upon m

Serial dilution plating and spotting assay

10% APS -30 μlTEMED -3 μlSDS loading buffer (2X)100 mM Tris-HCl (pH-6.8)20% (v/v) Glycerol4% (W/V) SDS0.02% Bromophenol Blue10% β-MercaptoethanolSDS-loading buffer was prepared as 2X stock solution in H2O and used at 1X concentration.SDS-PAGE running buffer14.4 g Glycine3.03 g Tris methylamine1 g SDSDissolved in H2O and volume was adjusted to 1L with H2O.Buffers for western blot analysisTransfer buffer (1 litre)14.4 g Glycine3.03 g Tris methylamine800 ml H2O 200 ml methanolBlocking and wash buffers (PBS-T)5% Fat-free milk0.05% Tween-20Volume was adjusted to 100 ml with1XPBS

Whole cell lysis buffer50 mM Sodium acetate 410 mg Sodium acetate anhydrous was dissolved in 80 ml H2O. pH was adjusted to 5.4 with glacial acetic acid and finally volume was adjusted to 100 ml with H2O.1 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol.Dialysis buffer50 mM Trizma basepH was adjusted to 7.5 by using concentrated HCl.Silver stainingFixing solution50% ethanol10% glacial acetic acid0.05% formaldehydeFinal volume was adjusted with sterile H2O.0.2% Silver nitrate solution (AgNO3)0.2 g AgNO3

0.075% formaldehyde (37% stock) Dissolved in 100 ml of H2O. Stored at 4°C for 1 hour in brown colored bottle.Developing solution 6% Sodium carbonate (Na2CO3)0.05% Formaldehyde (37% stock)0.02% Sodium thiosulphateStorage buffer50% EthanolSDS-PAGE30% Acrylamide solution29 g Acrylamide1 g Bis-acrylamideAcrylamide solution was prepared in H2O.Resolving gel mix (12%) (10 ml)H2O -3.3 ml30% Acrylamide:Bisacrylamide mix (29:1) -4 ml1.5 M Tris-HCl (pH-8.8) -2.5 ml10% SDS -100 μl10% Ammonium persulphate (APS) -100 μlN, N, N’,N’,-Tetramethylethylenediamine (TEMED) -4 μlStacking gel mix (5%, 3 ml)H2O -2.1 ml30% acrylamide:bisacrylamide mix (29:1) -500 μl1.5 M Tris-HCl (pH-6.8) -380 μl 10% SDS -30 μl

Buffers and solutions for protein extraction, analysis by SDS-PAGE (sodium dodecyl sulphate-polyacrylamaide gel electrophoresis) and silver staining

Rifr, Apr, Kmr, Gmr, Tetrand Spcrindicate resistant to rifampicin, ampicillin, kanamycin, gentamicin and spectinomycin, respectively.Table 2.2: List of oligonucleotides used in the study

hydrochloric acid, sulphuric acid, methanol, acetic acid, acetone and nitric acid were purchased from Fischer Scientific. Protease inhibitor tablets were procured from Roche. Hybond-P membranes for protein transfer were purchased from Amersham Biosciences. Taq DNA polymerase and Hi-fidelity Taq DNA polymerase were purchased from Thermoscientific and Larova, respectively. SYBR-green kit for real-time PCRwas procured from Qiagen and Thermoscientific. Superscript SS-III RT kit was obtained from Invitrogen. Random hexamers were obtained from Qiagen. Different restriction enzymes used for cloning and mutation generation were purchased from New EnglandBiolabs (NEB). Plasmid DNA purification, PCR purification, gel extraction and reaction clean up kits were procured from Qiagen. Medium components for bacterial culture viz., sucrose, agar, Luria Bertani (LB), Nutrient Agar (NA), peptone, yeast extract, beef extract, magnesium chloride hexahydrate (MgCl2.6H2O) and potassium sulphate (K2SO4) were purchased from Himedia. Table 2.1: List of strains and plasmids used in the study

Agarose, phenol, dimethyl sulphoxide (DMSO), sodium acetate, sodium carbonate, sodium bicarbonate, mangenese sulphate, tris methylamine, trizma base, sodium dodecyl sulphate (SDS), formamide, ethylenediaminetetraacetic acid (EDTA), glycerol, polyethylene glycol, tributyrin, ammonium persulphate, TEMED, acrylamide, bis-acrylamide, coomassie brilliant blue (CBB), β-mercaptoethanol, chloroform, formaldehyde, nuclease free water, diethylpyrocarbonate (DEPC), isopropanol, ferrozine, glycine, sodium lauryl sarcosine, carbonylcyanidep-trifluoromethoxyphenylhydrazone (FCCP), benzyl amino purine (BAP), ferrozine, tween-20, triton-X-100, aniline blue, trisodium citrate dehydrate, remazol brilliant blue-xylan (RBB-xylan), lactic acid, nicotinic acid, hexadecyltrimethyl ammonium bromide (HDTMA), p-nitrophenol, carboxymethyl cellulose (CMC cellulose), sodium phosphate dibasic, sodium phosphate monobasic, rubidium chloride, ferrous sulphate, ferric chloride, ammonium sulphate, 2,5-diphenyloxazol (PPO), 1,4-bis (5 phenyl 1,2-oxazole) Benzene (POPOP) and 2, 2-dipyridyl were purchased from Sigma Chemicals. Sodium hypochloride, disodium hydrogen orthophosphate dehydrate, sodium chloride, sodium hydroxid

Chemicals, kits and culture medium components

A total of 150 fungal and 150 bacterial isolates were screened qualitatively and quantitatively for their ability to produce the enzyme, tannase.

Screening and selection of potential tannase producers

Buffer pH range Stock Solutions Volume of Stock A + Stock BCitrate Phosphate 3–5 A: 0.1M solution of citric acid B: 0.2M solution of Na2HPO4pH 3: 39.8 ml A + 10.2 ml B made up to 100 ml pH 4: 30.7 ml A + 19.3 ml B made up to 100 ml pH 5: 24.3 ml A + 25.7 ml B made up to 100 ml Phosphate 6–8 A: 0.2M solution of NaH2P04 B: 0.2M solution of Na2HPO4 pH 6: 87.7 ml A + 12.3 ml B made up to 200 ml pH 7: 39 ml A + 61 ml B made up to 200 ml pH 8: 5.3 ml A + 94.7 ml B made up to 200 ml Tris - HCI 9 A: 0.1M solution of (HOCH2)3CNH2B: 0.1M HCI solution (16.16 ml of 11.35N HCI /L) pH 9: 70 ml A + 30 ml B made up to 200 ml Glycine - NaOH 10 A: 0.1M glycine B: 0.1M NaOH solution pH 10: 50 ml A + 32 ml B made up to 200 ml Phosphate hydroxide 11 A: 0.05M Na2HP04B: 0.05M sodium hydroxide pH 11: 91 ml A + 9 ml B Hydroxide Chloride 12 A: 0.05M KCI solution B: 0.05M KOH solution pH 12: 82 ml A + 18 ml B

Composition of buffers

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

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

Adherence assay

using the GENESPRING GX (Version 12.0) software,normalized to 75 percentile shift and represent the average of two hybridizations from biological replicates for each sample. Functional annotation of differentially regulated gene set(≥1.5 Fold change with p≤0.05)was performed using the GENESPRING GX (Version 12.0) softwareand GO terms with p<0.05 were considered as statistically significant. Using the REVIGO tool(http://revigo.irb.hr), redundant and significantly overlapping GO terms were removed and summarized. In REVIGO analysis, S. cerevisiaedatabase was chosen for GOterm sizes andtheallowed similarity value was set to 0.5(small).Additionally, to identify the overlap among differentially expressed genes, functional category analysis was performed usingthefungal specific annotation tool FUNGIFUN (https://sbi.hki-jena.de/FungiFun/FungiFun.cgi). Significantly enriched FunCat (Functional Catalogue) associated pathways were extracted usingthewhole C. glabratagenome as background and compared across differentially regulated gene sets. The parameters used for FUNGIFUN analysis were cut-off p=0.05; Fisher’s exact test; FunCatlevel 3. Raw data sets for this study are available attheGene Expression Omnibus database (http://www.ncbi.nlm. nih.gov/geo; accession no. GSE60741

Log-phase C. glabratacells were grown either in YNB or YNB medium supplemented witheither50 μMBPS(iron limiting) or 500 μMferric chloride (iron excess) for 2 h. Cells were spun down at 4,000 rpm for 5 min and washed twice with ice-cold DEPC-treated water. Total RNA was extracted usingtheacid phenolisolationmethod, resuspended in nuclease-free water and stored at -80°C. The frozen RNA samples were sent to Genotypic Technology Ltd., Bangalore (http://www.genotypic.co.in) wherein quality of RNA samples wasdetermined by examining the RNA integrity number (RIN) before performing microarray analysis. Next, the 8x15 GE Agilent array,comprised of 60mer oligonucleotides representing a total of 5,503 C. glabrataORFs (three replicates of each probe on average),was used for single colour microarray experiments.Datawereextracted

Microarray analysis

To perform immunoblotting or western blotting, appropriate amounts of total protein(ranging from 20-40 μg) were separated ona SDS-PAGE gel of 12%acrylamide concentration in Tris-Glycine-SDS gel running buffer. Protein separation was done at 70-100 V for 2-3 h using a MINI PROTEAN®3 electrophoresis unit (Bio-Rad). Followingseparation, proteins weretransferredto polyvinylidene difluoride (PVDF) membrane, using a Bio-Rad Mini Trans-Blot electrophoretic transfer unit in Tris-Glycine transfer buffer at 4⁰C. Before setting transfer assembly, PVDF membrane was first activated in 100% methanolfollowedby washesin the transfer buffer. The transfer assembly was set inaBio-Rad Mini gel holder cassette (170-3931)according to manufacturer’s instructions. The transfer time and current settings varied depending on the size oftheprotein of interest. Post transfer, membranes wereseparated from the assembly and kept for blocking intheblocking buffer (0.1 % Tween-20, 5% w/v fat-free skimmed milk in 1X TBS) for 1 h at room temperature with shaking. Next,membranes wereincubated with appropriate dilutions of primary antibodiesin the blocking buffereitherfor 3-4 h at room temperature or overnight at 4°C with gentle shaking. Post incubation,membranes were washed thrice with 1X TBS-T, 10 min each,with constant agitation. After washes, membraneswere incubated with appropriate dilutions of secondary antibodiesconjugated with horseradish peroxidase (HRP) for 1 h at room temperature with gentle shaking. Next, membranes were washed thrice with 1X TBS-T, 10 min each,with constant agitation. To visualize proteins, membranes were removed from TBS-T, and theHRP substrate ECL plus (Amersham Biosciences, RPN2232) was uniformly added on top of the membrane. Chemiluminescent signalswere captured in the western blot imaging system (FluorChemTME system)

For protein extraction, cells were spun down at 4,000 rpm for 5 min and washed with ice-cold water. The cell pellet was resuspended in 250-500 μl of homogenisation buffer which contained50 mM Tris (pH 7.5), 2 mM EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF) (serine protease inhibitor), 10 mM sodium fluoride (serine/threonine and acid phosphatases inhibitor), 1 mM sodium orthovanadate (Tyrosine and alakaline phosphatases inhibitor) and 1X protease inhibitor cocktail (Sigma, P 8215). The cell suspensison was transferred to a 1.5 ml centrifuge tube and equal amounts of glass beads (0.5 mm size) were added. Cells were lysed mechanically by bead-beatinghomogenizer (MP Biomedicals, FastPrep®-24) atthemaximum speed for 60 seconds, five times each,with intermittent cooling on ice.After lysis, tubes were punctured at the bottom with the helpofasurgical needle, and the lysed cell suspension was collected in a fresh microcentrifuge tubes by putting the punctured tubes on top of the fresh tubes and centrifuging them at 3,000 rpm for 10 min. The supernatant was transferred toafresh microcentrifugetubeandprotein concentrationwasestimated usingtheBCA protein assay kit (Thermo scientific). Protein preparations werestored at -20°C until use

Total protein extraction and immunoblotting

For complementation studies, C. glabrataORFs, CgFTR1(1.22 kb), CgFET3(1.91 kb), CgYFH1(0.53 kb), CgCCW14(0.64 kb), CgMAM3(1.91 kb)andCgHOG1(1.34 kb) were PCR amplified from wild-type genomic DNA using Phusion high-fidelity DNA polymerase and cloned down-stream of the PGK1promoter intothe XmaIand XhoI, XbaIand XmaI, XmaIand XhoI, BamHIand SalI, XmaIand XhoI, and BamHIand XmaIsites, respectively, in the CEN-ARS containing plasmid pGRB2.2 (pRK74). For over-expression studies, C. glabrataORFs, CgCCC1(0.95 kb), CgYAP5(1.05 kb)andCgMRS4(0.92 kb) were PCR amplified from wild-type genomic DNA using Phusion high-fidelity DNA polymerase and cloned down-stream of the constitutive promoter PDC1into the BamHI and SalI, XbaIand XmaI, and XmaI and XhoIsites, respectively, in the CEN-ARS containing plasmid obtained from Addgene (Addgene-ID 45323). All clones were verified by PCR and sequencing analysis

Cloning of C. glabrataORFs

PCR-positive transformants were inoculated in 10 ml YPD medium, allowedto grow for 12 handgenomic DNAwas isolated.Another round of PCR was performed using genomic DNA asatemplate toconfirm the gene deletion

A homologous recombination-based strategy was used to disrupt C. glabraraORFs witha cassette containing the nat1gene, which codes for nourseothricin acetyltransferase and imparts resistance to nourseothricin. Briefly, 5’-and 3’-UTR region (nearly 500-700 bp) of the gene to be deleted were amplified by PCR using wild-type genomic DNA as template. Both 5’-and 3’-UTR amplified products were fused to one half each ofthenat1gene amplified from theplasmid(pRK625). The two nat1-amplified fragments share about 300-350 bp complimentary region. To obtain fusion products, primers were designed insucha way thatthereverse primer for 5’-UTR and the forward primer for 3’-UTRof the gene of interestshare 20 bp complimentary region with the forwardprimer for 5’nat1fragment andthereverse primer for 3’nat1fragment amplification, respectively. The fused PCR products were co-transformed in to the wild-type strain and transformants were plated on YPD-agar plates. Plates were incubated at 30°C for 16 h to allow the homologous recombination between nat1fragments,and 5’-and 3’-UTR atthegenomic loci. Post incubation,cells were replica plated ontoYPD-agar plate supplemented with 200 μg/ml nourseothricin and incubated for another 24 h. Nourseothricin-resistant colonies were purified and verified for gene disruption viahomologous recombination by PCR using appropriate set of primers

Generation of C. glabratadeletion strains

For Yeast colony PCR, yeast cells were subjected to zymolyase (MP Biomedicals, 0832092) digestion to obtain thespheroplast. To perform zymolyase digestion, a digestion cocktail was prepared in 1XPBS consisting of zymolyase (2.5mg/ml) and sorbitol (1.2 M). The cocktail was dispensed in 0.2ml PCR tubes in 10 μl aliquots anda tip-full of yeast cells wasadded to these tubes. Tubes were incubated at 37°C for 2-3 h and 1 μl of digested mixture was used as a template in a PCR reaction

Yeast colony PCR

To perform restriction digestion of plasmid DNA and PCR-amplified DNA products, restriction enzymes were procured from NEW ENGLAND Biolabs(NEB). Restriction digestion was set in 50 μl reaction volume with appropriate buffer and 1X BSA. For ligation of DNA fragments obtained after restriction digestion, T4 DNA Ligase enzyme (NEB, M0202M) was used. All ligation reactions were set in 20 μl reaction volume containing 1X ligase buffer, 3-10 units of DNA ligase enzyme and vector to insert molar ratio of 1:3. The ligation mixture waseitherincubated at 16°C for 16h or at room temperature for 2-3 h. Post incubation, ligation reaction was inhibited by heatingtubes at 65°C for 15-20 min.2-5 μl of ligation mixture was used to transform ultra-competent E. coliDH5αcells

Restriction digestion and ligation

To extract DNA from agarose gels,the QIAquick® gel extraction kit (QIAGEN, 28706) was used. For purification of PCR amplified DNA products,the QIAquick® PCR purification kit (QIAGEN, 28106) was used. Clean-up of enzymatic reactions was performedusing the MinElute® Reaction Cleanup kit (QIAGEN, 28204). Allprotocolswere followed as per manufacturer’s instruction

Gel extraction, PCR purification and Reaction clean-up

Bacterial plasmid DNA was isolatedusing the QIAprep® spin Miniprep kit (QIAGEN, 27106). 10 ml of LB medium supplemented with appropriate antibioticswas inoculated withasingle bacterial colony and incubated at 37°C for 12-16 h. Cultures were spun down at 8,000 rpm for 5 min, supernatant was discarded and the cell pellet was processed to isolate plasmid DNA as per instructions given in the kit. DNA was eluted either in nuclease-free water or in elution buffer provided in the kit and stored at -20°C until use

Plasmid isolation

were chosen for qPCR. For all qPCR reactions,0.4 μl of cDNA template was used in a 20 μl reaction volume. Reactionswere performed anddata wereanalysed in ABI7500 real-time qPCR machine. Amplified products were run on 2% agarose gel to confirm amplification ofthecorrect size product. CTvalues of respective products were normalized with corresponding CTvalue of the housekeeping gene CgACT1. Relative change in expression was determined by comparative CTmethod,also referred as 2-∆∆CTmethod, utilizing following equation.Fold change upon treatment=2-∆∆CT∆∆CT=∆CT Treated-∆CT Untreated∆CTTreated= CTvalue for gene of interest upon treatment-CTvalue of internal control (CgACT1)upon treatment∆CTUntreated= CTvalue for gene of interest without treatment-CTvalue of internal control (CgACT1)without treatmentThe reaction cycling conditions were as follows1)95°C for 10 min (initial activation)2)95°C for 15 sec (denaturation)3)55°C for 30 sec (annealing)4)72°C for40 sec (extension)5)Go to step 2 (40 cycles)6)72°C for 10 min (final extension)

MESA GREEN qPCR mastermix (RT-SY2X-03+WOULR) supplied by Eurogentech was used in all qPCR experiments. Primers for real-time qPCR experiments were designed by using the Primer3 plus software to obtain 120-200 bp amplification products. Standardization of optimaltemplate and primer concentrationconditionswas done in a PCR reaction and concentrations resulting in good amplification withoutprimer dimers

Quantitative Real-time PCR(qPCR)

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

Complementary DNA (cDNA) synthesis

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

DNase I digestion

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

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

Total RNA isolation

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

Spheroplast lysis method

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

Glass bead lysis method

Total protein extraction and immunoblotting

For complementation studies, C. glabrataORFs, CgFTR1(1.22 kb), CgFET3(1.91 kb), CgYFH1(0.53 kb), CgCCW14(0.64 kb), CgMAM3(1.91 kb)andCgHOG1(1.34 kb) were PCR amplified from wild-type genomic DNA using Phusion high-fidelity DNA polymerase and cloned down-stream of the PGK1promoter intothe XmaIand XhoI, XbaIand XmaI, XmaIand XhoI, BamHIand SalI, XmaIand XhoI, and BamHIand XmaIsites, respectively, in the CEN-ARS containing plasmid pGRB2.2 (pRK74). For over-expression studies, C. glabrataORFs, CgCCC1(0.95 kb), CgYAP5(1.05 kb)andCgMRS4(0.92 kb) were PCR amplified from wild-type genomic DNA using Phusion high-fidelity DNA polymerase and cloned down-stream of the constitutive promoter PDC1into the BamHI and SalI, XbaIand XmaI, and XmaI and XhoIsites, respectively, in the CEN-ARS containing plasmid obtained from Addgene (Addgene-ID 45323). All clones were verified by PCR and sequencing analysis

Cloning of C. glabrataORFs

PCR-positive transformants were inoculated in 10 ml YPD medium, allowedto grow for 12 handgenomic DNAwas isolated.Another round of PCR was performed using genomic DNA asatemplate toconfirm the gene deletion

A homologous recombination-based strategy was used to disrupt C. glabraraORFs witha cassette containing the nat1gene, which codes for nourseothricin acetyltransferase and imparts resistance to nourseothricin. Briefly, 5’-and 3’-UTR region (nearly 500-700 bp) of the gene to be deleted were amplified by PCR using wild-type genomic DNA as template. Both 5’-and 3’-UTR amplified products were fused to one half each ofthenat1gene amplified from theplasmid(pRK625). The two nat1-amplified fragments share about 300-350 bp complimentary region. To obtain fusion products, primers were designed insucha way thatthereverse primer for 5’-UTR and the forward primer for 3’-UTRof the gene of interestshare 20 bp complimentary region with the forwardprimer for 5’nat1fragment andthereverse primer for 3’nat1fragment amplification, respectively. The fused PCR products were co-transformed in to the wild-type strain and transformants were plated on YPD-agar plates. Plates were incubated at 30°C for 16 h to allow the homologous recombination between nat1fragments,and 5’-and 3’-UTR atthegenomic loci. Post incubation,cells were replica plated ontoYPD-agar plate supplemented with 200 μg/ml nourseothricin and incubated for another 24 h. Nourseothricin-resistant colonies were purified and verified for gene disruption viahomologous recombination by PCR using appropriate set of primers

Generation of C. glabratadeletion strains

For Yeast colony PCR, yeast cells were subjected to zymolyase (MP Biomedicals, 0832092) digestion to obtain thespheroplast. To perform zymolyase digestion, a digestion cocktail was prepared in 1XPBS consisting of zymolyase (2.5mg/ml) and sorbitol (1.2 M). The cocktail was dispensed in 0.2ml PCR tubes in 10 μl aliquots anda tip-full of yeast cells wasadded to these tubes. Tubes were incubated at 37°C for 2-3 h and 1 μl of digested mixture was used as a template in a PCR reaction

Yeast colony PCR

To perform restriction digestion of plasmid DNA and PCR-amplified DNA products, restriction enzymes were procured from NEW ENGLAND Biolabs(NEB). Restriction digestion was set in 50 μl reaction volume with appropriate buffer and 1X BSA. For ligation of DNA fragments obtained after restriction digestion, T4 DNA Ligase enzyme (NEB, M0202M) was used. All ligation reactions were set in 20 μl reaction volume containing 1X ligase buffer, 3-10 units of DNA ligase enzyme and vector to insert molar ratio of 1:3. The ligation mixture waseitherincubated at 16°C for 16h or at room temperature for 2-3 h. Post incubation, ligation reaction was inhibited by heatingtubes at 65°C for 15-20 min.2-5 μl of ligation mixture was used to transform ultra-competent E. coliDH5αcells

To extract DNA from agarose gels,the QIAquick® gel extraction kit (QIAGEN, 28706) was used. For purification of PCR amplified DNA products,the QIAquick® PCR purification kit (QIAGEN, 28106) was used. Clean-up of enzymatic reactions was performedusing the MinElute® Reaction Cleanup kit (QIAGEN, 28204). Allprotocolswere followed as per manufacturer’s instructions

Restriction digestion and ligation

Gel extraction, PCR purification and Reaction clean-up

Bacterial plasmid DNA was isolatedusing the QIAprep® spin Miniprep kit (QIAGEN, 27106). 10 ml of LB medium supplemented with appropriate antibioticswas inoculated withasingle bacterial colony and incubated at 37°C for 12-16 h. Cultures were spun down at 8,000 rpm for 5 min, supernatant was discarded and the cell pellet was processed to isolate plasmid DNA as per instructions given in the kit. DNA was eluted either in nuclease-free water or in elution buffer provided in the kit and stored at -20°C until use

Plasmid isolation

were chosen for qPCR. For all qPCR reactions,0.4 μl of cDNA template was used in a 20 μl reaction volume. Reactionswere performed anddata wereanalysed in ABI7500 real-time qPCR machine. Amplified products were run on 2% agarose gel to confirm amplification ofthecorrect size product. CTvalues of respective products were normalized with corresponding CTvalue of the housekeeping gene CgACT1. Relative change in expression was determined by comparative CTmethod,also referred as 2-∆∆CTmethod, utilizing following equation.Fold change upon treatment=2-∆∆CT∆∆CT=∆CT Treated-∆CT Untreated∆CTTreated= CTvalue for gene of interest upon treatment-CTvalue of internal control (CgACT1)upon treatment∆CTUntreated= CTvalue for gene of interest without treatment-CTvalue of internal control (CgACT1)without treatmentThe reaction cycling conditions were as follows1)95°C for 10 min (initial activation)2)95°C for 15 sec (denaturation)3)55°C for 30 sec (annealing)4)72°C for40 sec (extension)5)Go to step 2 (40 cycles)6)72°C for 10 min (final extension)

MESA GREEN qPCR mastermix (RT-SY2X-03+WOULR) supplied by Eurogentech was used in all qPCR experiments. Primers for real-time qPCR experiments were designed by using the Primer3 plus software to obtain 120-200 bp amplification products. Standardization of optimaltemplate and primer concentrationconditionswas done in a PCR reaction and concentrations resulting in good amplification withoutprimer dimers

Quantitative Real-time PCR(qPCR)

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

Complementary DNA (cDNA) synthesis

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

DNase I digestion

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

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

Total RNA isolation

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

Spheroplast lysis method

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

Glass bead lysis method

Yeast genomic DNA isolation

Molecular biology methods

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

Serial dilution spot growth assay

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

THP-1 monocytes getdifferentiated intophagocytic macrophages upon treatment with

PMA (Phorbol myristateacetate) treatment of THP-1 monocytic cells

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

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

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

Buffers for protein extraction and separation by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

Table 2.4: List of antibodies used in thisstudy

All antibodies used in thisstudy, their clonality and dilutions used,Manufacturers’ details,and catalogue numbersare listed in Table 2.4

Antibodies

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

Cell cycle analysis by PI staining

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

Mice

Viewing slides under microscope

A drop of immersion oil was put on top of the cover-slip before viewing it under microscope. The cells were viewed at 100X resolution of Nikon Eclipse 80i microscope.Thedifferential interference contrast images of the cells were captured using NIS-Elements D3.0 software also used to find out mean cell size using at least 100 randomly selected cells.Fluorescence images were captured on Zeiss LSM 710 Meta inverted confocal microscope

The RNA was cross-linked onto the membrane after transfer by exposing it to the UV light of 200KJ/cm2 energy in a UV-crosslinker

UV-crosslinking of the RNA

Band intensities in gel autoradiogramswere determined by densitometry with the aid of the Fujifilm Multi Gauge V3.0 imaging system. Equal areas of radioactive bands were boxed and the PSL (Photo stimulated luminescence) values were further considered. Background signal (obtained from equal area as that of the radioactive band but from other part of the gel/blot) is subtracted from the signal intensities obtained from radioactive bands to get the final values

Densitometry

Oligonucleotides and PCR products were end-labelled using phage T4-polynucleotidekinase (PNK, New England Biolabs or Fermentas or Sigma) with 32P-γ-ATP. The radiolabelling reaction mixture (20μl) contained 1X of buffer provided by the company, 10 units of T4-PNK and 40μCi of 32P-γ-ATP. The reaction mix was incubated for 1 hrat 37ºC and the reaction was heat-inactivated at 65oC for 20 minutes. The labelled oligonucleotides and DNA fragments were purifiedby the Qiagen nucleotide removal kit. Labelling efficiency was checkedeither by using Geiger-Muller (GM) counter orusing liquid scintillation counter.For scintillation counting, 1μl of radioactive sample wasadded to the 5ml scintillation cocktail, and radioactivity count was determined in the 32P channel of scintillation counter (Perkin Elmer, Liquid Scintillation analyzer, Tri-Carb 2910 TR, USA). Liquid scintillation cocktail consists of 5g PPO (2,5-diphenyloxazol) and 0.3g POPOP (1,4-bis (5 phenyl 1,2-oxazole) Benzene, adjusted to a volume of 1L in toluene

Radiolabelling of oligonucleotides

DNA sequencing

Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits on an automated sequencer following the manufacturer's instructions byan outsourced sequencing facility

The semi-quantitative reverse transcription-PCR (RT-PCR) involves the synthesis ofcomplementary DNA (cDNA)from RNA. For this, 1μgof RNAwas treated with 1μl (1 unit) DNase I enzyme (Sigma, amplification grade) for20 min to remove DNA contamination. DNase I was inactivated by heating at 70oC for 10 min. Next, 5pmol reverse primer wasadded along with dNTPs and volume made to 10μlwith DEPC-treated water; the mix washeated at 65oC for 5 min and incubated on ice forat least 1 min. The reverse transcription reaction was set up with this mix using the Superscript III RT kit (Invitrogen) as per manufacturer’s protocolto obtain cDNA. The cDNA servedas the template for setting up a PCR for requirednumber of cycles. The samples were finally run on agarose gels

Reverse transcription(RT)-PCR

Concentrations of DNApreparations were estimated by nanodrop or by gel electrophoresis followed by densitometric analysis.Concentration of RNA preparations were estimated by nanodrop

Estimation of DNA and RNA concentrations

Total RNA extraction from E. colicells was doneusing Qiagen RNeasy minikit. Cells were grown to an A600of 0.6 and harvested(amaximumof107cells)at 6000rpm for 5min at room temperature to prevent cells for encountering any stress in cold. Rest of the steps were followed exactly as mentioned in the manufacturer’s protocol. The quality of RNA preparations was assessed following electrophoresison 1.4% agarose-formaldehyde-MOPS gels.Ingeneral,forawild-typestrainRNAyieldwouldbe~0.5-1μg

Isolation of total cellular RNA

For high fidelity PCR, Herculase II fusion DNA polymerase (AgilentTechnologies)was used. Approximately 0.5μg of chromosomal DNAwas used as a template in a 50μl reaction volume

The PCRs were normally performed using Taqpolymerasefrom Roche or Fermentas. Approximately 1-5ng of plasmid or 5-100ng of chromosomal DNA was used as a template in a50μlreaction volume containing 200μM of each dNTP, 20pM each of the forward and reverse primers and 1 unit of Taq DNA polymerase. For colony PCR E. coli cells from a freshly grown plate were resuspended in 10μl of sterile Milli-Q water to get a cell suspension and this was used as a template in a PCR reaction at a final volume of 50μl. The samples were typically subjected to 30 cycles of amplification with the following general conditions: Initial denaturation 95ºC5minutes Denaturation 95ºC 1 minute Annealing 55ºC 1 minute Extension 72ºC 1 minute/kb of DNA template to be amplified Final extension 72ºC 10 minutes

Polymerase Chain Reaction (PCR)

Molecular techniques

Restriction enzyme digestion and analysis

desired temperaturefor 45 minutes and plated on an appropriate selective medium at various dilutions. An aliquot of cell suspension to which plasmid DNA was not added served as a negative control. B. Inoue method i. Preparation of high efficiency competent cells Competent cells for high efficiency transformation were prepared by the method of (Inoueet al., 1990)with few modifications. An overnight culture of the strain (routinely DH5α) was subculturedinto fresh sterile LB broth in 1:100 dilutions and grown at 18ºC to anA600of 0.55. The cells were harvested by centrifugation at 2500rpm for 10 minutes at4ºC. Thesecells wereresuspended in0.4 volumes of INOUE buffer andincubated in ice for 10 minutes. The cells were recovered by centrifugation at 2500rpm at 4ºC for 10 minutes and finally resuspended in 0.01 volume of the same buffer. Sterile DMSO was added to a final concentration of 7%. After incubating for 10 minutes in ice, the cells were aliquoted in 100μl volumes, snap frozen in liquid nitrogen and stored at –80ºC. ii. Transformation protocolFor transformation, the required number of vials wasthawed on ice and the transformation protocol as described for CaCl2method was employed

A. Calcium chloride(CaCl2)method For routine plasmid transformation, following method which is a modification of that described by(Cohenet al.,1972)was used. An overnight culture of recipient strain was subcultured 1:100 in fresh LB medium and grown till mid-exponential phase. The culture was chilled on ice for 20 minutes, and the steps thereafter performed at 4ºC. 10 ml of culture was centrifuged and pelletwas resuspended in 5 ml of 0.1M CaCl2. After 5 minutes of incubation on ice, the cells were again centrifuged and resuspended in 1ml of 0.1M CaCl2. The suspension was incubated onice for 45 minutes. To the 100μl aliquot of the cellsuspension plasmid DNA (20-200ng in less than 10μl volumes) was added, incubated for 30-40 minutes on ice and given a heat shock for 90 seconds at 42ºC. The cultures were rapidly chilled for 1 minute, mixed with 0.9ml of LB broth and incubated at

Transformation

Table 2.3: Oligonucleotide primers

Primers

What other colours do you have this in?' asked Sayako of the assistants, who were packing her suits, loafers, bags and wig.'Just one other colour,' said an assistant (who thought, Jesus, we'll have a drink after work tonight).She hurried to the back of the shop and quickly returned with a toffee-brown version of the sumptuous coat.8'Yes,' said Sayako. 'I take both and, of course, boots to match, size four.' She pointed to the boots worn by the red-haired manne­quin.The pile on the counter grew. Her bodyguard standing inside the shop door shifted impatiently.When the Princess and her purchases had been driven away, the manageress and her assistants screamed and yelled and hugged each other for joy.

'What other colours do you have this in?' asked Sayako of the assistants, who were packing her suits, loafers, bags and wig.'Just one other colour,' said an assistant (who thought, Jesus, we'll have a drink after work tonight).She hurried to the back of the shop and quickly returned with a toffee-brown version of the sumptuous coat.8'Yes,' said Sayako. 'I take both and, of course, boots to match, size four.' She pointed to the boots worn by the red-haired manne­quin.The pile on the counter grew. Her bodyguard standing inside the shop door shifted impatiently.When the Princess and her purchases had been driven away, the manageress and her assistants screamed and yelled and hugged each other for joy.

ZFIN_ZDB-GENO-140423-3

ZFIN_ZDB-ALT-080321-3

ZFIN: ZDB-ALT-120409-3

ZFIN_ZDB-GENO-140423-3

ZFIN ID: ZDB-ALT-070612-3

involves a complex interaction of conscious and unconscious processes.

latent learning

Remember, the best way to teach a person or animal a behavior is to use positive reinforcement

Why is shaping needed?

In his operant conditioning experiments, Skinner often used an approach called shaping

In discussing operant conditioning, we use several everyday words—positive, negative, reinforcement, and punishment—in a specialized manner. In operant conditioning, positive and negative do not mean good and bad

There are specific steps in the process of modeling that must be followed if learning is to be successful.

It was then that Claire knew she wanted to discipline her children in a different manner.

PICKERING[zait/?enthusiasm]IcamefromIndiatomeetyou.HIGGINS.IwasgoingtoIndiatomeetyou.

Type hints cheat sheet (Python 3)¶ This document is a quick cheat sheet showing how the PEP 484 type annotation notation represents various common types in Python 3.

three types of gymnastics

DC3

Wherefore, after I have abridged the record of my father, then will I make an account of mine own life.

foolish imaginations of his heart

The evidence strongly suggests that the Canadiens adopted a casual attitude toward the clergy, which could (and did) sometimes express itself as contempt

accustom the people to direct taxation

however, have said that they were in favor of direct taxation for the support of the local governments, because it would lead those who have to pay the taxes to look more closely into what was going on, and the manner in which their money was expended. (Hear, hear.) There seems also to have been a feeling in the Lower Provinces in favor of a legislative union, and the Hon. Mr. GREY seems to be combatting that idea. He says that with a legislative union, municipal institutions, and direct taxation in every province, would be the only means of getting along. He expressed himself as opposed to that and in favor of a Federal union, which he thought would afford them all the advantage that could be attained, commercially, by union, and would allow each province to retain control over its own local affairs. The local legislatures, he said, were to be deprived of no power over their own affairs that they formerly possessed. But in Canada it was represented that the local legislatures were to be only the shadow of the General Legislature—that they were to have merely a shadow of power, as all their proceedings were to be controlled by the Federal Government. That is the position taken by the advocates of the measure on this floor. So it seems that those gentlemen who have represented to us that they acted in great harmony, and came to a common decision when they were in conference, take a widely different view of the questions supposed to have been agreed upon, and give very different accounts of what were the views of parties to the conference on the various subjects. (Hear, hear.) In the Lower Provinces they were strongly opposed to direct taxation, while here it was present end as one of the advantages to accrue from the Federation. (Cries of No, no.) Well, Mr. SPEAKER, I say yes. That view of the case has been taken. If the amount allowed for the expenses of local legislation—the 80 cents per head—was found insufficient, the local parliaments must resort to direct taxation to make up the deficiency, while in tile Lower Provinces, it seems, nothing of that kind was to follow.

Another question on which the hon. member has also called us to account, relates to the export duties on timber and coals. In clause 29, which relates to the powers of the Federal Parliament, the third section reads as follows : This imposition or regulation of duties of customs on imports or exports, except on exports of timber, logs, masts, spars, deals, and sawn lumber from New Brunswick, and of coal and other minerals from Nova Scotia. The fact that this power has been conferred on the Government does not imply that it will be exercised. The power was granted simply because it might be necessary in certain cases mentioned. Now this is the reason for the second part of the clause which I have just read to the House, and which I cannot better explain than by citing some expressions of a speech by the Hon. the Minister of Finance on the subject. Nevertheless, as there are several honorable members in the House who do not understand English, I think it will perhaps be better to explain them in French. Here then was the thought of the Convention : as in New Brunswick the Government had found that it was a great disadvantage to collect the duties on timber according to the system formerly adopted, and they had substituted an export duty which superseded all other dues on that product, it was no more than right that this source of revenue should remain in New Brunswick, to which province it was an object of absolute necessity to defray its local expenses. In Canada we retain, under the new Constitution, our own method of collecting similar duties. As to New Brunswick, the duty on the article in question is their principal revenue, as coal is almost the sole revenue of Nova Scotia ; and if they had been deprived of them, they would have peremptorily refused to join the Confederation. (Hear, hear.) Their demand was perfectly just, and could not therefore be refused. Moreover, we have no right to complain, for they leave us all our mines and our lands, and we shall now, as heretofore, collect the proceeds for our own use and profit. The honorable member for Hochelaga says that it will be impossible to administer the affairs of the local legislatures without having recourse to direct taxation ; but a man of his experience ought not to have made that assertion. Instead of attempting to trade on popular prejudice, he ought to have admitted at once that the right granted by the new Constitution of levying direct taxes, is the same that already exists in the present Constitution ; it is the same right that all our municipalities possess.

reference to the meaning of the 5th sub-section of the 29th clause, which commits to the General Parliament ” the raising of money by all or any other modes or system of taxation.” Am I to understand that he General Government are to have the power of imposing local taxation upon the lands of the provinces ? HON. MR. CAMPBELL—The general national power of taxation is to be in the General Government.

Stadtinfo Köln (City Info Cologne) is a research project financed by the German Federal Ministry of Research that centres around the collection of various traffic data to be distributed to diverse platforms including the Internet, portable devices such as PDAs and mobile telephones, in-car navigation systems and variable message signs throughout the city. The project was implemented over a four-year period from 1998 to October 2002 by 15 partners in co-operation with the city of Cologne at a cost of €16.1 million.

The Cologne-based TÜV Rheinland headquarters is revitalizing its approximately 100,000 square meter business park with ten buildings in Poll. The management of TÜV Rheinland Immobiliengesellschaft mbH & Co. KG has developed an innovative concept with the engineering experts from Drees & Sommer as energy designer, building physicist and TGA planner: In future, there should only be one energy center. All buildings in the property are supplied with heat and cooling via the power grid of the new energy center. For heat supply, hybrid energy sources are used. These consist of the renewable raw material wood, a wood pellet boiler plant, as well as the fossil energy natural gas, gas condensing boilers and an integrated combined heat and power plant. The cold is generated by free-cooling, high-efficiency compression machines and absorption chillers. This can save 30 percent of primary energy compared to today. In addition, CO2 emissions will be reduced by more than 30 percent. The overall concept is modular in design and adaptable for the future.

KVB cycle hire Smart mobility   Smart mobility is climate-friendly, sustainable, space-saving and networked. It relies on diversity and multimodality. The resident of a smart city does not remain loyal to one mode of transport. The result is a mobility patchwork that is tailored to the individual circumstances and that can be configured quickly and easily at any time. Energy-efficient and space-saving mobility has priority here. "Sharing" is smart! The sharing of things and information already establishes itself under the term "sharing economy" and places the function before the property, in order to use existing resources more efficiently. Smart mobility in urban areas is therefore primarily a matter of sharing a networked mobility offer from buses, trains, bicycles and cars. Smart mobility is not just a technological task. Especially in the inner cities, walking and cycling will provide space for quality of life and urban development through active mobility. This is where the bicycle rental system of the Cologne Transport Company (KVB) comes in by closing a gap in the combination of environmentally conscious and mobility-active mobility. The bicycle rental system of KVB stands for an open architecture. It is therefore not a system with only fixed station terminals after the well-known role models from other major cities, because a template for all cases, the complex events of a city can consider insufficient. The system offers users fully flexible rental and return in the street, but also stationary station terminals depending on the available options and needs. The rental terminals cover the entire span between conventional stations and purely virtual stations.  

n times of energy transition and scarce resources, the architectural concept of Concrete Apartments Cologne is based on the requirements of the future - it is designed as an energy-saving passive house. This contains • a 26 cm thick external insulation made of rock wool, • triple glazed windows, • optimum recovery of radiated heat from residents and household appliances, • a ventilation system with a constant base temperature of 20 ° C - summer and winter - as well as • a digital control system that directs the use of luminaires and large consumers. Only those who like it even warmer must turn on the heating controller. All rooms are equipped with presence detectors, which automatically switch off lamps, for example, when not in use - this also saves energy. Of course, residents can also make the scheme manually. The energy and heat for the Boarding House creates its own, energy-efficient combined heat and power plant. State-of-the-art technology is also used here: surplus electricity is optionally fed into the public grid or used for the charging station for electric vehicles in the courtyard.

The diesel exhaust gases of the Rhine ships pollute the Cologne air with pollutants and fine dust and the climate with a significant amount of CO 2 . A part of it does not arise during the journey, but while the ships are at anchor. Because their generators must also run to generate the necessary electricity. Here, "Landstrom" provides a remedy: Since 2015, RheinEnergie has gradually been equipping a large part of the moorings along the Rhine with uniform power connections. Consequence: During the lay times the ship diesels can be turned off.

As well as energy-saving lighting, Smart Home is an important building block for an energy-efficient and comfortable future. With smart homes and smart meters in the network, homeowners and store owners can reduce their electricity and heating costs by an average of 7%! Add to that the great comfort of making the apartment burglar-proof and controlling almost every aspect of heating, electricity or security in the building. So you can control from your smartphone whether the stove is still on at home, a window has been left open, the heating is running at full speed or the light is on. In addition, before the house is on fire, modern, networked smoke detectors report any alarm directly to the owner's smartphone. It can automatically be initiated various steps, such. B. that the fire department is called. In order to test some scenarios and saving opportunities in everyday life and to make known the possibilities offered by these modern technologies, Smart Home applications were installed on the Klimastraße in nine private apartments of the Nippes Tower and in the bookstore Neusser Straße. This was financed by the project Klimastraße or the company RocketHome . In addition, it is planned to equip the entire climate road with smart meters from RheinEnergie.

Neusser Straße in the district of Nippes shows what a future SmartCity could look like, because a section of the street becomes Cologne's climatic road. There, the most important energy projects are implemented. All facets of climate protection are taken into account: from optimal building insulation and maximum heat efficiency to charging stations for electric vehicles and low-energy street lighting. Klimastraße offers innovative companies the opportunity to test their new products and services in everyday life. If possible, companies finance their projects themselves, promising projects are funded from the project budget of RheinEnergie AG. Companies also gain additional value by exchanging valuable information and innovative ideas with other companies, including at climate road events. For all the enthusiasm for innovation, of course, only technology is used that meets the very strict German safety requirements. In addition, RheinEnergie and the City of Cologne make sure that the high Cologne supply standards are adhered to. For all new projects, safety comes first - technically as well as logistically. That is why not everything changes in the climate route - but certainly much better. The following section deals in more detail with the individual projects.

The energy transition presents network operators and energy providers with particular challenges. Both have to deal with an increasing share of electricity from renewable sources in the electricity grid. Wind turbines and photovoltaic systems produce electricity, however, depending on the weather and therefore fluctuating. For a secure supply, it is necessary that electricity production and consumption are always balanced as much as possible. In order for this to succeed, utilities and network operators must always know where and in what quantity energy is generated and consumed. Only then can production and consumption be optimally coordinated. However, continuous metering is not possible with today's metering technology.The solution to the problem is smart metering. In the future, so-called "smart metering systems" will transfer consumption data to grid operators and energy providers. This ensures that they can optimally control the network at any time. The technology is mainly used in households and businesses with high annual consumption.Consumers can access the data at any time. The additional transparency helps them to further increase their energy efficiency and thereby reduce costs. New services provided by energy suppliers are intended to reinforce the positive effects.

In the framework of the project "Celsius" we investigate which method leads to the best possible results in order to increase the chances of realization. For this purpose, demonstration plants were built at three different locations in the city. In Cologne-Wahn and Cologne-Mülheim, the heat is extracted directly from the sewer using so-called gutter heat exchangers. The heat exchangers with a length of 60 and 120 meters are installed at the bottom of the canal. The heat transfer medium transports the heat from there to the heat pumps with a capacity of 150 or 200 kW in the boiler rooms of the schools supplied. In Cologne-Nippes, a total of three schools and a sports hall are supplied by sewage heat. Here, the wastewater is pumped through a newly laid, 400-meter-long bypass to the boiler room of the Edith Stein-.Realschule. There, in the largest direct evaporator in Germany (400 kW), heat is transferred directly to the heating circuit of the schools. With the three demonstration plants, an environmental relief of a total of 500 t CO2 / year is achieved. The use of wastewater heat is technically mature and well developed. Nevertheless, this form of waste heat utilization has so far been a niche existence. This is partly because it is still little known, often the necessary information is not available locally, their implementation is relatively complex and requires high investment. Further reducing these barriers is the goal of the Cologne CELSIUS project.  

District heating is one of the key pillars of our sustainable energy action plan. This plan has been decided by the local parliament in 2008 and renewed in 2015. Our first priority is to cut in half the total energy demand of the city until 2050 and then cover the rest with renewable energy and/or waste heat. To use large amounts of waste heat (e.g. from a waste incineration plant, industry, datacentres …) you need a distribution system, because it is not useable only locally. This is why we want to increase the share of district heating in the city. For the future we see a district heating system which will be “open source technology” – everyone can use the heat and also be a prosumer, delivering surplus energy, e.g. from a solar – thermal plant, to the system. There will not be any longer central DH-Stations but smaller plants and the use of all waste heat sources we can get.

The Federal Government will have the right of imposing taxes on the provinces without the concurrence of the local governments. Under article five of the 29th resolution, the Federal Government may raise moneys by all modes or systems of taxation, and I look upon this power as most excessive. Thus, in case it should happen, as I said a moment ago, that the Lower Canada Government refused to undertake the payment of the debt contracted for the redemption of the Seigniorial Tenure, the Federal Government would have two methods of compelling it to do so. First, by retaining the amount out of the eighty cents per head indemnity to be accorded to the Local Government, and secondly, by imposing a local and direct tax. The Lieutenant Governor of the Local Government will be appointed by the Federal Government, and will be guided by its instructions. We are not told whether the Local Government will be responsible to the Local Legislature; whether there will be only one or two branches of the Legislature, nor how the Legislative Council will be composed, if there is to be one ; we are refused any information whatsoever on these points, which are nevertheless of some importance.

smart­PORT lo­gis­tics Thanks to in­tel­li­gen­t so­lu­tions for the flow of traf­fic and goods, the HPA is im­prov­ing the port's ef­fici­ency. smart­PORT lo­gis­tics com­bines eco­no­mic and eco­lo­gical as­pec­ts in three sub-sec­tors: traf­fic flows, in­fra­structure and the flow of goods. An in­ter­mo­da­l Port­Traf­fic cen­tre for sea, rail and road trans­port forms the ba­sis for net­work­ing the flow of traf­fic. In­tel­li­gen­t net­work­ing is a pre­req­ui­site for smooth, ef­fici­en­t trans­port in the port of Ham­bur­g and ul­ti­mately for the flow of goods: op­ti­mum da­ta cap­ture and rapid in­for­ma­ti­on shar­ing al­low lo­gis­tics man­agers, car­ri­ers and agen­ts to se­lect the most efficien­t means of trans­port for their goods.

The Green-Space Information System (GRIS) is an EDP procedure of the borough departments of green spaces and the Senate Department for Environment, Transport and Climate Protection, Commission III C, Open-Space Planning and Urban Green Spaces.

Further, in Lower Canada, each locality is told that it may rest satisfied it will not be overlooked, for each is to be represented in the Legislative Council by a gentleman residing or holding property in it

Crime increasing in the past 3 years47.75

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

Teach Source EvaluationSkillsIf you want to teach source evaluation skills, have small groups conduct research to answer a three-part problem such as this:1.How high is Mt. Fuji in feet?2.Find a different answer to this same question.3.Which answer do you trust and why do you trust it?

5

“I support a social transition for a kid who is in distress and needs to live in a different way. And I do so because I am very focused on what the child needs at that time,” said Johanna Olson-Kennedy, medical director of the Center for Transyouth Health and Development at Children’s Hospital Los Angeles, the largest transgender youth clinic in the United States with some 750 patients. A social transition to the other gender helps children learn, make friends, and participate in family activities. Some will decide later they are not transgender, but Olson-Kennedy says the potential harm in such cases may be overstated.

I finally want to express how much easier both my waking and my sleeping hours would be if there were one book in existence that would tell me something specific about my life. One book based in Black feminist and Black lesbian experience, fiction or non-fiction. Just one work to reflect the reality that I and the Black women whom I love are trying to create. When such a book exists then each of us will not only know better how to live, but how to dream.

they found in each other's eyes the intimacy they were looking for.

Nel imagines a "fiery prince" who never quite arrives while Sula gallops like a prince "on a gray

Thou wilt see (him) and [like a woman(?)] thou wilt rejoice.

the Central Government would have the power of raising money by all the other modes and systems of taxation—the power of taxation had been confided to the General Legislature—and there was only one method left to the Local Governments, if their own resources became exhausted, and this was direct taxation.

It was adopted by the Imperial Legislature with the view of remedying difficulties which then existed between the two Provinces. The inherent defect in the Imperial Act for the Union of the two Canadas was this : it attempted to combine the federal principle with unity of action. It endeavoured to give equal representation to the two sections of the Province, while it brought them together for the purpose of dealing as one with all subjects both (general and local,

Those claims were in themselves undoubtedly founded in justice— but at the same time there was great reason in the objections taken to them—they involved an interference with the Federal principle recognized in the Union Act, an interference which amounted to an entire change in the principles on which the Government of the country was to be administered, and could not be received otherwise than with dread by a large class, if not by the whole of the population of Lower Canada.

should have been a concession to Upper Canada, of additional members in proportion to its population, but that concession would, as be had already remarked, have been an invasion of the Federal principle, contained in the Union Act, and would unquestionably have been represented to the uttermost by a large proportion if not by the whole of Lower Canada.