Obtaining transposon insertions near gene of interest

run on an agarose gel and band size determined by comparison against DNA ladder with bands of known sizes. Clones that were positive for the presence of gene/ plasmid were further checked by restriction digestion. For restriction digestion, plasmid was first isolated by Miniprep as described below. The digestion reactions were set according to manufacturer's protocol appropriate for the enzymes used. The products were run on an agarose gel and band size determined by comparison against DNA ladder with bands of known sizes. Clones with the desired pattern of digestion were propagated further and used

Bacterial colonies obtained by transformation were checked for the presence of plasmid or gene inserted into the plasmid by colony PCR and restriction digestion. For PCR, a master mix for all the PCR reactions to be performed was made, aliquoted into PCR vials and stored on ice. Individual colonies were picked up, numbered and streaked onto an LB agar plate followed by deposition of a few cells into the PCR mix. PCR was carried out as described above for the respective primer pairs with the exception that the initial denaturation was carried out for 7 min at 94 °C. The products were

Screening of bacterial transformants

Quantitative measurement of periplasmic acid phosphatase activity in phosphate-starved C. glabratacells was performedas mentioned previously (Orkwis et al., 2010). A total of 0.5 OD600YNB-grown and phosphate-starved cells were collected, washed thrice with cold water and oncewith cold 0.1 M sodium acetatebuffer (pH 4.2). Washed cells were resuspendedin 500 μl sodium acetate (0.1 M)and incubated at 30 ̊C with constant stirring. After 10 min incubation, 500 μl freshly-prepared solution of 20 mM p-nitrophenyl phosphate in 0.1 M sodium acetate(pH 4.2) was added to the cell suspension. Enzymatic activity was stopped after incubation at 25 ̊C for 20 min by addition of 250 μl sodium carbonate (1 M)tothe reaction mix. Resultant colour change was measured by monitoring absorbance at 400 nm. Acid phosphatase activity was expressed as a ratio of OD400to OD600 to normalize against cell density

Determination of acid phosphatase activity

C. glabratacells grown either in RPMI medium or harvested from THP-1 macrophages were collected, washed with DEPC treated water and were disrupted with glass beads in trizol. Total RNA was isolated using acid phenol extraction method and frozen at -80C. Quality of RNA was examined by determiningtheRNAintegrity number (RIN) before microarrayanalysis.Microarray experiments wereperformed atOcimum Biosolutions Ltd., Hyderabad (http://www.ocimumbio.com). Briefly, a4x44K GE Agilent array comprised of 10,408 probes representing 5,205 ORFs of C. glabratawas used wherein average number of replicates for each probe was four to five. Feature Extraction software version 10.7.3.1. (Agilent) and Quantile normalization was used for data analysis. Hierarchical clustering was performed using Complete Linkage methodwith Euclidean Distance as distance measure. Data arethe average of two hybridizations from biological replicates ofeach sample and raw data sets for this study areavailable at the Gene Expression Omnibus database(Accession number -GSE38953)

Microarray Analysis

E. coliDH5α strain was transformed with plasmids carrying appropriate inserts to clone and generatedeletion strains of C. glabrataORFs(Sambrook, 2001). Ultracompetentcells stored at -70⁰C were thawed on icefor 5-10 min. 5 μlligated plasmid was added to100 μlultracompetent cells andcells were incubatedon ice. After 30 min, competent cells were subjected to heat shock at 42⁰C for 90 seconds. Cells were immediately transferredtoicefor 2-3min. Next, 800 μlSOC (or LB) medium was added and cells were allowed to recover for 45 minon a shaker incubator set at 37⁰C.After the recovery, cells were centrifuged at 2,500g for 4 min. Medium supernatant was discarded and cells were resuspended in 200 μlfresh sterile LBmedium. Cells were plated on LB agar medium containing appropriate antibiotics. Plates wereincubatedat37⁰C for 12-16 h

Bacterial transformation

Ammonium persulfate(APS)

Uniquitination assay was performed as described by Choo and Zhang, 2009. Ubiquitination is an enzymatic process of the covalent attachment of polypeptide ubiquitin on specific lysine residuesof protein, which is thendegraded by proteasome complex. MG132 (carbobenzoxy-Leu-Leu-Leucinal), a proteolytic activity inhibitor of proteasome complex, is widely used to assess the stability of protein in vivo. Briefly, parental and profilin-stable cells were treated with 10 μM MG132 for 6 h. The whole cell extracts prepared in NTEN lysis buffer were then subjected to immunoprecipitation with anti-ubiquitin antibody. The analysis of ubiquitination was performed by immunoblotting with anti-PTEN antibody

invivo Ubiquitination assay

Cellswere grown in YPD to an OD600of 0.5-0.7. Cells equivalent to 1OD600were washed with synthetic complete medium without uacil twice and suspended in SC-Ura containing 3 μCi/mLof [14C]uracil for 5 min. Cells were pelleted and washed with SC-Ura medium twice and suspended in 0.5 mLof AE solution(Section 2.1.6.2). 1OD600of this cell suspension was counted in a liquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were and converted into moles based on thespecific activity of [14C] uracil and plotted using GraphPad Prism5

Uracil uptake assay

Cells were grown overnight on coverslips and transfected with various combinations of plasmids. Post 24 hrs. of transfection, cells were washed with PBS and then fixed in 3% w/v paraformaldehyde in 1X PBS containing 50 mM sucrose for 15 minutes at room temperature. Cells were permeabilized with permeabilization buffer i.e. 0.5% Triton X-100 buffer containing20mM HEPES at pH 7.4, 50mM NaCl, 3mM MgCl2 and 300mM sucrose and were incubated for 5 min.at room temperature.Cells were washed twice with 1X PBS and blocked with 3% BSA/PBS for 30 minutes. Cells were incubated with specificprimary antibody diluted in the blocking buffer. After 2 hours of incubation, cells were washed thrice with 1X PBS (each washfor 5 minutes). The 1X PBS was removed,and the cellswere incubatedwith specific FITC or Rhodamine-conjugated secondary antibodyat 37°C for 30 min.To visualize nuclei, cells were co-stained with DAPI (10 μg/ml). Cellswere washed thrice with 1X PBS and after final wash, coverslips containing cell weremounted on the slidesusing glycerine containing paraphenylenediamine. The cells were analyzed using confocal microscopy facility at CDFD

Immunofluorescence

CFUs/ml) onto fully expanded leaf, and pricking with sterile needle to facilitate the entry of bacteria inside the leaves through wound. To detrmine the growth of bacteria inside leaves, 1 cm2 leaf area surrounding the inoculation site was cut at regular time intervals, surface sterilized by dipping in 2% (vol/vol) sodium hypochlorite for 2 min, and washed twice in sterile water. For getting the CFUs, leaves were crushed using mortar and pestle, serially diluted, and plated on PSA medium containing appropriate antibiotics

Exogenous iron supplementation was performed as described previously (Chatterjee and Sonti, 2002). Briefly, leaves of 40-day-old greenhouse-grown rice plants of the susceptible rice cultivar Taichung Native-1 (TN-1) were cut with scissors 2 cm above the junction of the leaf blade and leaf sheath. These cut leaves (25 leaves per flasks) were dipped in 250 ml conical flasks containing 200 ml 1μg/mlof Benzyl amino purine (BAP) in double distilled water. BAP (a cytokine hormone) maintain the detached rice leaves in fresh condition for longer period. For iron supplementation, FeCl3 was added to a final concentration of 50 μM (stock-10 mM). Prior to inoculation with different strains of Xanthomonas oryzaepv. oryzicola, the leaves were maintained overnight on a laboartory bench top. Strains were inoculated into the leaves by needle pricking method by dropping 20μl of bacterial suspension (approx. 1 × 108bacterial

Exogenous iron supplementation and bacterial growth assay in rice leaves

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

Cell adhesion assayswereperformed as described previously (Hockinget al., 1998)withslight modifications. Fibronectin coating was done overnight at 4°C. 5×104 cells were seeded per well onto fibronectin (2 μg/mL) coated 24 well platesin triplicates. Cells were allowed to adhere for different time periods. At each time point, unadhered cells were washed away with PBS; adhered cells were trypsinized and counted with a hemocytometer. Percentage of adhesion was calculated by normalizing total number of adhered cells at each time point to number of cells adhered after 5 h

Cell adhesion assay

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Now, these rights, at the very least, ought certainly to be confided to the highest legislative authority. I go further and maintain that guarantees for those rights ought to be placed in the written Constitution, that they ought to be beyond the power of interference by the legislative authority, and that they should be guarded by the judicial decisions of the highest courts in the country. In that case there would be a protection for property, but in this Constitution there is no such protection for property either in Upper or Lower Canada.

The 29th section of the scheme submitted to us says : ” The Federal Parliament shall have the power of making laws for the peace, the well-being, and the good government of the Confederate provinces, and in particular in respect of the following matters.” The powers of the Federal Government will be in reality unlimited. The fact of the enumeration of these thirty-seven heads does not in the least restrain the power of the Federal Government from legislating on everything. The exceptions are few. I would ask the Honorable Premier, for instance, whether the Federal Government has not the power to enact that marriage is a civil contract ? He cannot deny it, and I do not believe that that clause will in any way suit Lower Canada. In a matter of divorce, I consider that the power of legislating upon it ought to be vested in the Federal Government ; but as to the passing of a marriage act, we have the authority of the past to convince us that Lower Canada will never be satisfied with what is proposed in the plan of Confederation. On a former occasion, when a member of the Parliament of Canada moved to enact that marriage should be made a civil contract, all the members for Lower Canada voted against the motion, and the whole country was opposed to it. I shall also inquire whether the Federal Government will not have the right to enact that religious corporations shall no longer exist in the country, or that they shall not be allowed to hold real property, except what is absolutely necessary for their lodging accommodation. According to the resolutions which have been submitted to us, the Federal Government would certainly have this right. It has been said that article 15 of the 43rd resolution replies to this objection, but I can see nothing in that article which restricts the right of the Federal Government to legislate on this matter. The 43rd resolution defines the powers of the local governments, and article 15 of that resolution declares that they may make laws respecting ” property and civil rights, excepting those portions thereof assigned to the General Parliament.” That article reserves to the local legislatures nothing relative to religious corporations, and the Federal Government would have full power to decree that those corporations shall not hold immovable property. The supreme power is that which has the right to legislate upon, and regulate the existence of, the corporations in question, and they can only possess civil rights so long as the Government permits them to exist. The same might be said of most of the institutions to which Lower Canada is attached. I am therefore right in saying that, so far as those things which Lower Canada most holds to are concerned, Confederation is in fact a Legislative union, because upon the Federal Government is conferred the right of legislating upon those subjects which Lower Canada holds most dear.

10

But this precedent could not be urged as an objection to Federation, inasmuch as it would be for the General Government to deal with our commercial matters. There could be no reason for well-grounded fear that the minority could be made to suffer by means of any laws affecting the rights of property.

The control of property and civil rights, the administration of justice, including the constitution, maintenance, and organization of the courts of civil jurisdiction, and the procedure in civil matters, were also left to the local legislatures. From the peculiar position of Lower Canada it was felt impossible to confide the matter of civil law to the General Legislature. The principles upon which the civil law of Lower Canada were founded differed entirely from those of the English law. Under it property was secured, and civil rights of every kind maintained, and the people had no particular wish to see it changed, especially at this moment, when the work of codifying and simplifying it was about completed, and when they knew that within the next three or four months they would have it put into their hands in one volume. He thought it was undesirable to do away with that law, which had been beneficial to the country and under which it had prospered. It was necessary to have it left to the local Legislature, because all in Lower Canada were unwilling to have substituted another law with which they were unacquainted.

But on April 13, 1970, an oxygen tank explosion aboard the Apollo 13 spacecraft set a harrowing mission into motion—and its success would turn a team of heartland boys into national heroes. A little more than two days into the mission’s voyage to the moon, the command module began to lose its supply of electricity and water. That’s when astronaut John Swigert uttered the phrase that would implant mission control in the public’s consciousness: “Houston, we’ve had a problem here.”

10 Oct 2016 22:13:49 UTC

by misrepresenting to them that concussions did not present serious, life-altering risks," the suit filed Wednesday charges.

The lawsuits claim the National Football League hid evidence linking concussions to permanent brain injuries and seek millions in compensation.

October 13-16, 2016