centrifugation at 5,000 rpm for 4 minat room temperature. Harvested cells werewashed with PBS and treated with different compoundse.g.H2O2. After treatment,cells were harvested and further processed according to the type of experiments performed

For several experiments, log-phase C. glabratacells were harvested and treated with different compounds. For this, single colony of aC. glabratastrain was inoculated in YPD-liquid medium and grown for 14-16 h at 30ºC withcontinuous shaking at 200 rpm. Overnight cultures were reinoculated in YPD medium to an initial OD600of 0.1 andgrown for another 4 h. These log-phase cells were harvested by

Harvesting of and treatment to logarithmic phase C. glabratacells

To collectmacrophage-internalized yeast cellsfor RNA and protein extraction, 107THP-1 monocytes were seeded in 100 mm cell culture dishes and treated with PMA. PMA-differentiated THP-1 macrophages were infected with appropriateC. glabratastrainsto a MOIof 1:1. Equal numberof C. glabratacells wasinoculated inRPMI medium as control. Two hourspost infection,non-phagocytosed yeast cells were removed by washing THP-1 macrophages thrice with PBS. At different time points, culture dishes were washed twice with chilled PBS and 2 mlchilled sterile water was added toeach dish to lyse the macrophages. Corresponding cultures grown in RPMI medium were transferred to50 ml polypropylene tubesand transferred on ice. Lysates were collected by scrapping the macrophage monolayer and transferred to50 ml polypropylene tubes.RPMI-grown and macrophage-internalized C. glabratacells were harvested by centrifugation at 2,500g for 8 min. Macrophage cell debris were removed frommacrophage-internalized cells by repeated washing with chilled sterile water. Harvested C. glabratacells were stored at -20ºC till further use

Harvesting of macrophage-internalized C. glabratacellsfor RNA and protein extraction

nvolved use of GFP based vector system, the expression of the transgene was visualized under fluorescent microscope with excitation filter of 485+20 nm

Transient transfection of plasmid DNA in cellswas performed usingLipofectamine 2000transfection reagentaccording to manufacturer’s protocol. Briefly, 0.5 to 1million cellswere seeded in a 35mm tissue culture dish one day prior totransfection. For each 35mm dish, 4μg DNA was mixed in 250μl of Opti-MEMin one polypropylene tube. In another tube 10μl of Lipofectamine 2000 was diluted in250μl Opti-MEM and incubated at room temperature for 5 minutes. DNA and Lipofectamine 2000 were mixed together and allowed to form complexes for 30minutes at room temperature. Meanwhile, the adherent cells were washed twicewithPBS and 1ml of Opti-MEM was added. 500μl of complexes were then added to each dishcontaining cells and medium. After 6-8 hrs, the medium containing complexes wasremoved and complete medium was added and transgene expression was evaluated 24-48 hrs after transfection. Since most of the experiments

Transient transfection in adherent cells

Inoue buffer

For preparation of Ultra competent cells

Aprotinin

Co-Immunoprecipitation assays were performed essentially as described by Lee (Lee, 2007). For a typical immunoprecipitation assay, cells were washed with ice-cold PBS and scapped in ice-cold microfuge tube. Then, cells were lysed with NETN buffer (containing 1 μg/ml each of leupeptin, aprotinin, 10mMeach ofNaF and phenylmethylsulfonyl fluoride (PMSF))on shaking rotator in cold room for 30 min. After centrifugation, the whole cell lysate (500 μg-1 mg) obtained was incubated with 1 μg of antibody of interest(orwithisotype control)on shaking rotator in cold room for 3 h, followed by addition of 10-20 μl of ProteinSepharose A/G beads (Santa Cruz) for 1 h. The immuno-complexes bound to beads were then pelleted atlow speed centrifugation (2500 rpm for 3 min) and washed three times with NETN buffer. The proteins bound to beadswere resolved by SDS-PAGE and immunoblotting was performed accordingto standard protocol described earlie

Co-Immunoprecipitation (Co-IP)

Stripping buffer

After the HPLC run, vials numbered from 50 to 65 were surveyed using a Geiger counter, 4 vials with high counts were pooled into one vial and 5 μL of this 4 mL solution was measured in a liquid scintillation counter (Perkin Elmer). To the remaining solution, 800 μL of 50% ammonia solution was added to neutralise the pH and the tube was kept on ice. In a 50 mL conical tube, 45 mL of chilled water was taken, to which the neutralised IP7solution was added and kept on ice. A Sep-Pak cartridge [Waters, WAT020545] was equlibrated with 10 mL of ice cold deionized water using a 10 mL syringe. The Sep-Pak cartridge was attached to a 60 mL syringe and 50 mL of diluted IP7 solution was passed slowly through ot so that IP7would bind to the Sep-Pak column. The cartridge was washed with 8 mL of chilled water, followed by chilled 8 mL 0.2 M triethylammonium bicarbonate solution pH 8.5 (4 mL of 1.5 M triethylammonium bicarbonate, pH 8.5 + 26 mL chilled water). The bound IP7 was eluted in 4 mL of 1.5 M triethylammonium bicarbonate solution, pH 8.5, into three 1.5 mL microfuge tubes. The eluted IP7 was concentrated in a vacuum concentrator (Scanvac) at 2000 rpm, 25°C, to obtain 30-50 μL of a 1-2 μCi/μL solution of radiolabelled IP7

A mixture of 40 mL of DEPC treated water and 0.6 gof agarose was meltedby boiling. After cooling down the temperature to 55°C, 8.4 mL of formaldehyde (final concentration 2.2 M)and 5 mL of 10X MOPS were added, mixed well and poured into a boat to cast a gel

Preparation of formaldehyde agarose gel(50 mL) and RNA sample

protein agarose beads for 1 hour at 4 c and then washed three times with 1X NETN. The proteins bound to S-protein agarose beads were resolved by SDS-PAGE and visualized by Coomassie staining. Proteinspresent in the gels were analyzed by Mass Spectroscopy

HEK293T cells were transfected with S-protein/FLAG/SBP (streptavidin binding protein)-triple tagged WWP2/Dvl2 and then three weeks later puromycin-resistant colonies were selected and screened for WWP2/Dvl2 expression. The positive stable cells were then maintained in RPMI1640 supplemented with 10% FBS and 2 g/ml puromycin. The stable cells(harvested cells from ~30 tissue culture plates of 10cm size)were collected in 1X PBS by scraping them off the plates, and were lysedwith NETN buffer (20 mM Tris-HCl,pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 % Nonidet P-40) containing 50 mM -Glycerophosphate, 10 mm NaF, 0.5 mM PMSF, 1 g/ml of each Pepstatin and Aprotinin on ice for 20 minutes. After removal of cell debris by spinning, cell lysates were incubated with streptavidin sepharose beads for 1 hour at 4C. The bound proteins were washed three times with 1X NETN and then eluted twice with 2 mg/ml Biotin for 60 minutes at 4 C. The eluateswere incubated with S-

Tandem affinity purification

culture dishes and dishes were incubated at 28ºC. OD600 was measured after 16 and 42 h of incubation, and percentage inhibition of growth was determined with respect to the growth in the corresponding control cultures containing PS media without streptonigrin

For streptonigrin sensitivity assay, different strains of Xanthomonas oryzaepv. oryzicolawere grown overnight with appropriate antibiotics as described earlier. 0.2% of primary inoculum was added into fresh PS medium and grown for 24 h till the OD600reached 0.6. Serial dilution of bacterial cultures were performed as mentioned earlier, and 5μl diluted cultures were spotted on PSA plates containing different concentration of streptonigrin (0.05 μg/ml, 0.1 μg/ml and 0.15 μg/ml). Plates were incubated at 28ºC for 72 h and plate images were captured and analyzed for comparative growth inhibitionin different strains caused by streptonigrin. Further, streptonigrin sensitivity assay in liquid broth was performed by growing different strains as described previously (Wilson et al., 1998).Briefly, Xanthomonas oryzae pv. oryzicolastrains were grown to an OD of 1 in PS medium with appropriate antibiotics. Cells were pelleted down, and resuspended in fresh PS medium at an OD600of 0.6. Next, 100 μl culture was inoculated in 4 ml PS medium with or without streptonigrin. Streptonigrin was added to a final concentration of 0.1μg/ml into

Streptonigrin sensitivtity assay

insert of 1:3 for sticky end ligations. Ligation mix was incubated either at 22°C for 30 min or 16°C for 14-16 h. After incubation, T4DNA ligase was inactivated at 65°C for 20 min

After restriction enzyme digestion, digested products were resolved on agarose gels, and desired DNA fragments were extracted from the gel. Otherwise digested DNA fragments were precipitated by Phenol-choloroform-isoamyl alcohol method. Concentration of gel extracted or precipitated fragments were determined using spectrophotometer and ligation reactions were set up using a molar ratio

Ligation

The reaction mixture contained 10 μl of culture filtrate, 490 μl of double distilled water (DDW) and 300 μl of methanolic rhodanine solution. This mixture was incubated for 5 min at 30°C in a water bath. The reaction was stopped by adding 0.3 ml of methanolic rhodanine solution (0.667 %), which resulted in the formation of complex between gallate and rhodanine. This was followed by the addition of 0.2 ml of KOH solution (0.5N) and the tubes were further incubated at 30°C for 5 min. The total reaction mixture in each tube was diluted with 4.0 ml of distilled water. Tubes were further incubated at 30°C for 10 min. The absorbance was measured at 520 nm against a control having distilled water in place of culture filtrate. The absorbance thus obtained was used to calculate the amount of gallic acid present in the culture filtrate, from the standard gallic acid curve prepared in the range of 100-1000 μg/ml.

The procedure of Sharma et al. (2000) was used to estimate the gallic acid in the culture filtrate. Reagents: Methanolic rhodanine solution (0.667% w/v): Prepared by dissolving 0.667 g of rhodanine in 100 ml of methanol.Potassium hydroxide (0.5 N): 2.8 gpotassium hydroxide dissolved in100 ml of distilled water.

Gallic acid estimation (Sharma et al., 2000)

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

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

dropped on clean prechilled slides held at a 45°angle from a height of 18 inches, dried at 55°Con a hot plate and stained with 3% geimsa in phosphate buffer (pH 6.8) for 10 min. Slides were washed three times and dried, individual metaphase spreads were analyzed using bright field microscopy (Zeiss Axio Scope), and chromosomal aberrations were scored manually from each spread

MEFsplated in 6 cmdishes were treated with MMC (300 nM) for 12 h. Post damage cells were treated with colcemid (0.1 μg/mL) for 4 hto arrest cells in metaphase. After 4 h cells were washed twice with PBS to remove traces of colcemid and harvested by trypsinisation, pelleted down at 100 x g for 5 min in 15 mL conical tubes. The supernatant was removed by leaving 0.5 mL and resuspended thoroughlyby pippeting. Cells were subjected to hypotonic swelling in pre-warmed 0.075 M KCl for 15 min at 37°C[Note: key to this step is to add hypotonic solution drop by drop slowly otherwise cell clumps will form which are impossible to disperse. Add a few drops first then pipette the cells up and down to mix them thoroughly and add few more drops. Invert tubes 2-3 times during the incubation]. After 15 min cells were fixed by adding few drops of chilled fresh fixative (methanol: glacial acetic acid; 3:1) kept in the deep freezer. Cells were pelleted down at 100 x g for 8 min, the supernatant was removed, 0.5 mL of fixative was left behind and the pellet was resuspended very gently. Slowly fresh fixative was added in the same manner as the KCl solution and cells were pelleted down at 100 x g for 8 min; this step was repeated twice. The supernatant was removed by leaving 0.5 mL fixative and the cells were resuspended and mixed well before preparing metaphase spreads. Cell suspensions were

Cytogenetic analysis

the manuscripts that were discovered nine years ago, now in the University of Arkansas library with many of her other papers, are mostly complete and easily performed.

And he cast himself upon his bed, being overcome with the spirit and the things which he had seen. And being thus overcome with the spirit, he was carried away in a vision

Sectional interest and animosity will deepen the irritation, and all hope of remedy is rendered vain, by the fact that public opinion at the North has invested a great political error with the sanction of more erroneous religious belief.

MR. CORNELLIER—All the confederations which you have mentioned were or are republican, and had the common fate of republican institutions. You have not said a word about monarchical confederations. MR. JOLY—I have made no mention of monarchical confederations, because none have ever existed, and none can exist. The principle of a monarchy is that the power resides in one person; the principle of confederation is that it resides in all the members of the confederation. A confederation would, therefore, always be a republic, even if formed of several states subject to a monarchy; because the power would not be vested in one person, but in each of the several states, of which no one would acknowledge a head ; it would be a republic consisting of a very small number of members. Before I take leave of all the confederations, the names of which I have mentioned, I intend to say one word, at least, in their favor. We understand that states

The Prime Minister stated that the object of Confederation was to strengthen the monarchical principle in this country. I do not see that it is necessary to confer upon the Crown greater privileges than it already possesses in England itself. In England the members of the House of Lords are not appointed by the Crown ; succession in the peerage goes down hereditary from father to son ; but here it is proposed that the members of the Legislative Council, which body corresponds to the House of Lords, should be selected by the Crown. Why should this be ? Why go beyond what is done in England itself? Is it that the Crown complains that it has not sufficient power here ?

The app "Berliner Badestellen" uses the bathing place data of the LaGeSo to provide an overview of the water quality, temperature and other data of the Berlin bathing lakes. The app also provides a link to a corresponding request in the LOB connection planner, as well as to a display of the bathing place in various map services.

London Parking

A web portal and mobile application launched in January 2016, that is slated to be Singapore’s first one-stop online health information and services portal. Functions as the digital healthcare companion for every citizen by equipping citizens with the information, knowledge, tools and services to help them take greater ownership of their own health and wellness. A milestone project under Ministry of Health’s (MOH) Health IT Masterplan (HITMAP), healthcare institutions are now also connected with one another to provide continuity of care for patients.

WELCOME TO HEALTHHUB

ActiveSG is an all-encompassing and inclusive national movement for sport, brought to you by Sport Singapore. Poised to be a lifestyle destination for Singaporeans, ActiveSG will offer individuals, families and communities ample opportunities to experience and share the joy of living better through sport. Come join our national movement for sports and get active with a diverse and exciting line-up of sporting activities suited for all! Our sport facilities which are conveniently located all over Singapore are open to all! You can also sign up for ActiveSG membership registration to enjoy further privileges. Membership registration is free for all Singaporeans and Singapore Permanent Residents!

8

Some good men, and even of respectable information, consider the learned sciences as useless acquirements

to notice their conduct with diligence with candor & judgment.

that education like private & individual concerns, should be left to private & individual effort; not reflecting that an establishment,

the objects of education in the primary schools, whether private or public, in them should be taught reading, writing & numerical arithmetic, the elements of mensuration (useful in so many callings) and the outlines of geography and history,

Also the whole of his Slaves amounting to 57 in number.

[4 August 1818]

The human character is susceptible of other incitements to correct conduct, more worthy of employ, and of better effect. Pride of character, laudable ambition, & moral dispositions are innate correctives of the indiscretions of that lively age; and when strengthened by habitual appeal & exercise, have a happier effect on future character, than the degrading motive of fear; hardening them to disgrace, to corporal punishments, and servile humiliations, cannot be the best process for producing erect character. The affectionate deportment between father & son offers, in truth, the best example for that of tutor & pupil

Rockfish gap on the blue ridge

It was the degree of centrality to the white population of the state which alone then constituted the important point of comparison between these places

Education, in like manner engrafts a new man on the native stock, & improves what in his nature was vicious & perverse, into qualities of virtue and social worth

who, in the same act make other provision for the primary instruction of poor children

"American Idiot" - Green Day

Now everybody do the propaganda,And sing along to the age of paranoia.

We're not the ones who're meant to follow.

Television dreams of tomorrow.

“well then who the fuck did it?”

I confess to having reasonable doubt about whether Adnan killed Hae. I’m not talking about the courtroom kind, I’m talking about the normal person kind.

So, there he was. Mute through two trials, about five weeks total which isreally hard for anyone.

how could the cops have missed a detail like that

Because many of the theories deal with issues of power, students on the margin for particular reasons--ethnicity, class, ability--;ire often more receptive to the basic ideological premises of these theories than are their more privileged peers, who sometimes respond to theories such as gender and class as using the master's tools to dismantle the tnaster's house.