Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function

we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants

Supplemental material

Supplemental material

We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.

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Kerry, the former US secretary of state, acknowledged that America had been absent from the international effort to contain dangerous global heating during Donald Trump’s presidency but added that “today no country and no continent is getting the job done”.

Oh, silly me, that won’t work. We can’t update index.html at all, so we certainly can’t remove the call to register!

the behavior you want happen is actually a chicken and egg situation, you need to initialise FancyList in order to get the value for let:prop from the FancyList, however you can't initialise FancyList without the value of id which comes from within FancyList.

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The backlash from gig economy companies was immediate, and Uber and similar app-based businesses have committed nearly $200 million to support a state ballot measure — making it the costliest in state history — that would exempt them from the law.

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if you lack the consent necessary to contact users, then you likely lack the consent needed to even email them to ask for consent.

Unfortunately, loading their code from their CDN is the only way those services permit their use.

Mozilla does not permit extensions distributed through https://addons.mozilla.org/ to load external scripts. Mozilla does allow extensions to be externally distributed, but https://addons.mozilla.org/ is how most people discover extensions. The are still concerns: Google and Microsoft do not grant permission for others to distribute their "widget" scripts. Google's and Microsoft's "widget" scripts are minified. This prevents Mozilla's reviewers from being able to easily evaluate the code that is being distributed. Mozilla can reject an extension for this. Even if an extension author self-distributes, Mozilla can request the source code for the extension and halt its distribution for the same reason.

To complicate things further, if you classify your social-sharing-plugins-usage as required functionality, and those need to set their own 3rd party cookies (as they themselves classify those as required), hello to 3rd party cookies being set by default and no way for users to opt-out (except by turning them off via browser, which means the whole thing is redundant, might as well just instruct users to disable third party cookies if they don't want to participate in social sharing crap?)

And this is where we can all see dark clouds forming: if Recaptcha is opt-in (as the GDPR requires) then all a spammer needs to do to bypass Recaptcha, is to not accept cookies, right?

Also note that the first two opt out tools are currently cookie-based and prevent Oracle from using, sharing, or selling your personal information for interest-based advertising on the browser on which they are installed. As a result, the opt out will only function if your browser is set to accept third-party cookies and may not function where cookies are sometimes automatically disabled or removed (e.g., certain mobile devices and operating systems). If you delete cookies, change your browser settings, switch browsers or computers, or use another operating system, you will need to opt out again. Oracle does not use persistent, unique identifiers to revive a previously opted-out profile or deleted cookie.

Note that the scope of personal data is truly broad, which makes processing complex and tricky. So, even though, for instance, you employ anonymization in Google Analytics to get rid of all information that falls under this category, you’re still in a catch-22 situation. This is because GA stores a visitor online identifier in a cookie, and under the GDPR that file constitutes a piece of personal data. That means you still need to obtain consent from visitors to process their data.

That's the problem with therapy: The people who really need help are often the onesnot trusting others (as a result of bad experiences)not recognizing they have a problem in the first placebeing too scared to open up (or leave the house)being too exhausted to goless likely to be able to afford it. (That's only an issue in countries without a proper health care system.)

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Immunoblotting

Cytotoxicity assay

StatisticalAnalysis

Somatotype Dispersion Distances (SDD)

5x105 L.major promastigotes were cultured in 5 mL modified DMEM supplemented with 10% FCS. At the end of 5 days of culture, the stationary phase promastigotes were harvested and resuspended in Hanks balanced salt solution at a cell density of 4x107/mL. The cell suspension was aspirated into a 1 mL syringe and 50 J.!L was injected into the footpad of mice. The mice were returned to the cage and fed ab-limitum. The onset and progression of cutaneous lesion was monitored at 2 weekly intervals by observing an increase in the thickness of the footpad

L.major infection in mouse footpad

The aggregation of the phospholipid vesicles, induced by ribotoxins, was assayed by titrating 40 nmoles of freshly prepared lipid vesicles with various molar concentrations of restrictocin and its mutants. The samples of I ml volume were prepared in 30 mM Tris-HCl buffer, pH 7.0, containing O.IM NaCl and incubated at 37 °C for I h. The change in absorbance due to an increase in turbidity of the lipid suspension was measured at 400 nm in Lambda Bio 20 spectrophotometer (Perkin Elmer) in the cells of I em optical path length. Appropriate control proteins, not interacting with the membranes and the vesicles without proteins were included in all the experiments. The change in absorbance of the lipid suspension was plotted against the toxin concentration.

Aggregation of Lipid Vesicles

~hCG and HBsAg sequences were accessed from GenBank or NBRF database on a Microvax II computer and sequence analysis performed using the HPLOT and AMPHI programmes. HPLOT is based on the algorithm of Kyte and Doolittle ( 1982 ) and plots the hydrophobic and hydrophilic segments of the protein by scanning the whole length of the sequence in blocks of a few residues. The window size used was 6 amino acids. AMPHI is based on the algorithm of Margalit et al., ( 1985 ) and predicts the amphipathic segments of the proetin which correspond to alpha helices and are therefore, likely candidates for being T cell epitopes.

Computer analysis.

f. 5 μl of water was then spotted on each spot for 30 sec and removed using Whatman filter paper strips. This step was repeated once. g. 1-2 μl of SAP matrix was then applied to each spot and allowed to dry. h. The chip was then placed in the SELDI machine

a. 5 μl of 10 mM HCl was added to each spot on the chip and removed after 5 min. using Whatman filter paper strips. b. Washing was given by spotting 3 μl of water for 30 sec on each spot followed by removal using Whatman filter paper strips. This step was repeated two times. c. 10 μl of low stringency/ high stringency buffer was then added to the spot and kept in humid chamber for 5 min. followed by removal using Whatman filter paper strips. d. 3 μl of sample prepared in low stringency/ high stringency buffer was then added to the spot and incubated in humid chamber for 30 min. e. Washed the spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH 7.0 for 30 sec and removed using Whatman filter paper strips. This step was repeated five times.

Activation of CM10 (weak cation exchange ) array

d. 3 μl of sample prepared in low stringency buffer was added to the spot activated with low stringency buffer and incubated in humid chamber for 30 min. and removed using whatman strips. (same protocol was repeated for the samples prepared in high stringency buffer on spots activated with high stringency buffer). e. Stringent washings were given to each spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH7.0 for 30 sec and removed using Whatman filter paper strips. f. 1-2 μl of SAP matrix was added to each spot and allowed to dry. g. The chip was then placed in the SELDI machine

One set of cell extracts was prepared in low stringency buffer by mixing cell extracts and low stringency buffet in 1:1 ratio and another in high stringency buffer. b. 10 μl of low stringency/high stringency buffer was added to the spots on the chip and incubated in a humid chamber for 5 min. c. Buffer was removed using Whatman strips without touching the spot surface. This step was repeated once

Activation of LSAX (strong anion exchange ) array

Activation of H50 protein chip array

b. 5 μl of ACN + TFA (25% ACN in PBS + 0.1% TFA) was added to the spot surface and removed after 30 sec. c. 5 μl of cell lysate sample was then spotted on the chip and kept in a humid chamber for 30 min. d. Stringent washes were given by spotting 5 μl water on the spot surface for 30 sec and removing using Whatman filter paper strips. This was followed with a 25% ACN wash or three washes with 25% ACN or 50% CAN or 75% ACN. e. Washing was performed by spotting 5 μl of water for 30 sec followed by removal using Whatman filter paper strips. f. Dried chip at room temperature. g. 1-2 μl of SAP matrix (5 mg of matrix + 200 μl ACN + 200 μl of 1% TFA) was then spotted on the chip surface and allowed to dry. h. The chip was then placed in the SELDI machine

5 μl of water was added to each spot on the chip and removed after 30 sec using Whatman filter paper strips. Care was taken not to touch the spot surface. This step was repeated once

5 μl of 0.1% TFA was applied to the spots on the SEND array and removed after 30 sec using Whatman paper (care was taken not to touch the spot surface). b. 5 μl of cell lysate sample was spotted on the SEND array and incubated in a humid chamber for 10 min. Removed after 30 min. c. 5 μl of 0.1% TFA was then added and removed after 30 sec. d. 2 μl of 25% ACN in 0.1% TFA was added to the spots and allowed to dry. e. The chip was then placed in the SELDI machine

Activation of SEND arrays for peptide analysis

Trypsinization: The decolourized bands were dried in a vacuum dryer for 1 hr until the gel pieces were completely dry. 5 μl of 0.1 μg/μl trypsin and 25 μl of 25 mM NH4HCO3 (pH 8.0) were then added to the dried gel pieces. The tubes were sealed with parafilm and kept in a water bath at 37 ̊C, overnight. Care was taken that the gel pieces in the tubes did not dry up. If the gel pieces got dried, 25 μl of NH4HCO3 was added on top. Peptide extraction: A 1:1 mixture of ACN:5% TFA in water was added (30 μl) to overnight tryptic digests and kept for 30 min. The elutant was removed in a separate low binding tube. The extraction step was repeated once more. The elutant was then dried in a vacuum dryer (1-2 hr) and reconstituted in 5 μl of 25% ACN in 0.1% TFA

Destaining of gel bands: The protein bands of differentially expressed proteins were cut out from the gel and put in low binding microfuge tubes. 150 μl of 50:50 Acetonitrile:Ammonium bi carbonate pH 8.0 (NH4HCO3) was then added and kept under shaking for 30 min. Coloured liquid was discarded and the washing step repeated until the bands decolourised

Destaining of gel, trypsinization and peptide extraction

12% resolving gel (for 25 ml)Water = 8.2 ml 30% Acrylamide = 10.0 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.01 ml 5% stacking gel (for 10 ml)Water = 6.8 ml 30% Acrylamide = 1.7 ml 1.5 mM Tris (pH 6.8) = 1.25 ml 10% SDS = 0.1 ml 10% APS = 0.1 ml TEMED = 0.01 ml

A double cylinder gradient former was used with 12% poly acrylamide gel mix in the inner cylinder and a 3% polyacrylamide gel mix in the outer cylinder that was stirred using a magnetic bead on a magnetic stirrer. A pump was connected to the flow tube and the flow rate adjusted at 5-8 to cast a 12-3% gradient gel. A 5% stacking gel was used. After the protein samples were run on the gradient gel, it was stained in instant blue over night under shaking. 3% resolving gel (for 25 ml)Water = 15.68 ml 30% Acrylamide = 2.5 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.02 ml

Casting a gradient SDS-polyacrylamide gel

1μl of the cell lysate was mixed with 200 μl of 5X Bradford reagent and 800 μl of water. O.D was measured at 595 nm. Standard curve of BSA was plotted using various dilutions of BSA protein by Bradford method. Protein estimation of the cell lysate samples was performed using the standard curve equation y=0.0695x + 0.0329 μg/μl

Protein estimation by Bradford method

microfuge tubes and snap frozen in liquid nitrogen and were stored at ─80 ̊C. Protein estimation was performed simultaneously with one of these aliquots

The strains were grown to stationary phase in 500 ml LB supplemented with ampicillin (100 μg/ml) overnight. Cells were pelleted at 2100g for 30 min at 4 ̊C and dissolved in 5 ml of 1X PBS with 2X protease inhibitor and 3 mM DTT. Cells were lysed using French Press at 1500 psi for three cycles. The lysed cells were pelleted at 20,000g for 45 min at 4 ̊C. Clear supernatant was collected in sterile 2 ml

Preparation of cell extracts

These experiments were undertaken in the laboratories of Dr. Sylvie Rimsky and Dr. Malcolm Buckle at the Ecole Normale Superioure, Cachan, Paris (France)

Methods for SELDI (Surface Enhanced Laser Desorption/Ionization)

the membrane and sandwiched between 1 piece of buffer-soaked Whatman paper from Biorad on each side. The sandwich was placed between graphite electrodes with the membrane towards the anode. The transfer was done for 12-16 hr using a voltage of 40 V in the cold room. Protein transfer was viwed using Ponceau S staining. Blot was dipped in the Ponceau S stain under shaking and washed using PBST. After transfer, the membrane was blocked with 5% non-fat milk in PBST (1X PBS with 0.1% Tween-20) for 2 hr at room temperature. The membrane was then washed thrice with PBST under shaking and incubated with the primary antibody (1:1000 dilution in PBST) for 12-16 hr. The membrane was again washed thrice under shaking with PBST and incubated with 1:20000 dilution alkaline phosphatase conjugated anti-goat IgG secondary antibody (in PBST) for 2 hr. The membrane was once again washed thrice as described above and the signal developed using ECL kit from Amersham on X-ray films in a dark room. The reactive protein bands appeared as black bands upon gentle shaking at room temperature in 1X developer solution. The reaction was stopped by dipping and shaking the film in 1X Fixer solution followed by washing in water

The protein samples separated on SDS-PAGE were transferred to PVDF (polyvinyledene difluoride) membrane (Amersham, Buckinghamshire, UK) electrophoretically by a semi-dry method using BioRad apparatus. The gel and the membrane (pre-wetted with methanol) were wetted with transfer and the gel was placed in contact with

Western blotting with anti-Rho/anti-NusG antibody

(ii) Stacking gel buffer: 1.0 M Tris-Cl pH 6.8 (iii) Resolving gel buffer: 1.5 M Tris-Cl pH 8.8 (iv) SDS stock: 10% (w/v) solution (v) Ammonium persulphate (APS) stock: 10% (w/v) solution made fresh (vi) Gel running buffer (1X) (vii) Loading dye (6X): (viii) Lysis buffer (RIPA) Gels of 1.5 mm thickness were cast in the Biorad small gel apparatus. Resolving gel of 10% (10 ml) was made by mixing 4.2 ml 10% acrylamide, 3.1 ml water, 2.5 ml of 1.5 M Tris-Cl pH 8.8 and 0.1 ml of 10% SDS. Stacking gel (2 ml) was made by mixing 0.33 ml of 30% acrylamide, 1.4 ml of water, 0.25 ml of 1 M Tris-Cl pH 6.8 and 0.02 ml of 10% SDS. Gels were polymerized by the addition of TEMED (N,N,N′, N′-tetramethyl ethylene diamine) and APS (1/100th volume of gel mix). Sample preparation for gel loading was done as follows. Mid log and late log phase 10 ml cultures were centrifuged at 26000g and the cell pellet was resuspended in 0.5 ml RIPA buffer. Cells were sonicated on ice for 1 min at output power of 5 to get a cleared lysate. The culture lysate was centrifuged at 26000g to recover the clear supernatant. Total cell protein was quantified in the lysates using BCA kit reagents (BioRad) using the manufacturers protocol. Appropriate volume of cell lysate was mixed with the loading dye in a final concentration of 1X and loaded onto the gel. The gel was run at constant voltage of 60 V for stacking and 80 V for resolving gel

The method followed was as described in Sambrook and Russell (2001). The following solutions were used to cast and run SDS-PAGE gels. (i) Acrylamide stock: 29% (w/v) acrylamide and 1% N,N′-methylene bisacrylamide

Sodium dodecyl sulphate-polyacrlyamide gel electrophoresis (SDS-PAGE)

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 by either CDFD or an outsourced sequencing facility

DNA sequencing

Inverse PCR is a technique to amplify unknown regions flanking the site of transposon insertion using the primers designed from the known sequence from one end of the transposon element. Genomic DNA was digested with a 4-base recognition restriction enzyme, Sau3A1 followed by intramolecular ligation set up at high dilutions. These ligated molecules were then used as templates for the PCR performed with a pair of divergently-oriented primers designed from one end of the transposon named AH1-AH2. The PCR product thus obtained was sequenced with the same set of primers to identify the junction sequence at the site of transposon insertion and hence the identity of the gene disrupted in each case. Typical PCR conditions used were as follows:- Annealing 55°C 2 min Elongation 72°C (1 min/kb of DNA template to be amplified) Denaturation 95°C 2 min After 30 cycles of PCR, the final elongation step was carried out again for 10 min at 72°C

Inverse PCR

Denaturation 95°C 1 min After 30 cycles of PCR, the final elongation step was carried out again for 10 min at 72°C

The PCRs were normally performed using a PCR amplification kit from Fermentas/Sigma (USA), following the company's protocols. Approximately, 10 ng of chromosomal or 1-2 ng of plasmid DNA was used as template in a 50 μl reaction volume containing 0.2 mMM of each dNTP, 20 picomoles each of forward and reverse primer and 0.5 units of Taq DNA polymerase. In some cases, freshly streaked E.coli cells from a plate were resuspended in 50 μl of sterile Milli Q water to get a cell suspension (~ 109 cells/ml) and 10 μl from this was used as the source of DNA template. The samples were subjected to 30 cycles of amplification and the typical conditions of PCR were as follows (although there were slight modifications from one set of template/primers to another): The initial denaturation was done at 95°C for 3 min and the cycle conditions were as given below. Annealing 55°C 1 min Elongation 72°C (1 min/kb of DNA template to be amplified)

Polymerase chain reaction (PCR)

Fresh overnight culture of the E. coli strain (DH5α) was subcultured 1:100 in 250 ml LB/SOB media at 18 ̊C and 2500g and allowed to grow to an A600=0.55. Culture was chilled on ice and centrifuged at 2500g at 4 ̊C for 10 min. The cell pellet was redissolved in 80 ml ice-cold Inoue transformation buffer (55 mM MnCl2, 15 mM CaCl2, 250 mM KCl, 10 mM PIPES pH6.7). This cell suspension was centrifuged at 2500g at 4 ̊C for 10 min. and the cell pellet was resuspend in 20ml ice-cold Inoue buffer with 1.5 ml DMSO. This mixture was then placed on ice for 10 min. Aliquots of this suspension were dispensed into chilled, sterile microfuge tubes that were snap-frozen in a bath of liquid nitrogen. Tubes were stored at ─70 ̊C until required. For transformation the cells were thawed on ice and plasmid DNA was added followed by the standard transformation protocol

Preparation of ultracompetent cells

Overnight cell culture raised in LB medium was subcultured 1:100 in LB with 20 mM MgCl2. When the A600 reached 0.4-0.6, the culture was centrifuged at 2800g for 5 min at 4 ̊C. To the cell pellet 0.4 volumes of ice-cold TBF-I buffer was added and incubated on ice for 15 min. The cell suspension was centrifuged at 2800g for 5 min at 4 ̊C and the cells recovered were dissolved in 0.04 volume of ice-cold TBF-II buffer and kept on ice for 45 min. 100 μl aliquots of these competent cells were used for transformation using the normal transformation protocol

TBF method for preparation of high competency cells

For routine plasmid transformations, where high efficiency is not required, the following method which is a modification of that described by Sambrook and Russell (2001) was used. An overnight culture of the recipient strain was subcultured in fresh LB and grown till mid-exponential phase. The culture was chilled on ice for 15 min, and the steps hereafter were done on ice or at 4°C. The culture was centrifuged, and the pellet was resuspended in one third volume of cold 0.1 M CaCl2. After 15 min incubation on ice, the cells were again recovered by centrifugation, and resuspended in one tenth volume of cold 0.1 M CaCl2. The suspension (0.1 ml) was incubated on ice for 1 h after which DNA was added (~10-100 ng of DNA in less than 10 μl volume). The mixture was again incubated on ice for 30 min, and then heat shocked for 90 seconds at 42°C. Immediately 0.9 ml of LB broth was added to the tube and incubated at 37°C for 45 min for phenotypic expression of the antibiotic marker before being plated on selective medium at various dilutions. A negative control tube (with no plasmid DNA addition) was also routinely included in each of the experiments

Calcium chloride method

Transformation protocols

Typically, 100-200 ng of DNA was used in each ligation reaction. The ratio of vector to insert was maintained between 1:3 and 1:5. The reactions were usually done in a 10 μl volume containing ligation buffer (provided by the manufacturer) and 0.05 Weiss units of T4 DNA ligase, at 16°C for 12-16 hr

Ligation of DNA

DNA fragments to be used for ligation were eluted from the agarose gel, after the electrophoresis. The gel piece containing the desired band of DNA was sliced out and the DNA was purified from the gel using the purification kits available for this purpose. The efficiency of elution was determined by checking a small aliquot of DNA sample on the gel

Purification of DNA fragments by elution from the gel

0.5-1 μg of DNA was used for each restriction enzyme digestion. 2-4 units of the restriction enzymes with the appropriate 10X buffers supplied by the manufacturers were used in a total reaction volume of 20 μl. The digestion was allowed to proceed for 6 h or 10min. (for FAST digest enzymes) at the temperature recommended by the manufacturer. The DNA fragments were visualized by ethidium bromide staining following electrophoresis on 1-1.5% agarose gels. Commercially available DNA size markers were run along with the digestion samples to compare with and to estimate the sizes of the restriction fragments

Restriction enzyme digestion and analysis

The DNA samples were mixed with the appropriate volumes of the 6X loading dye (0.25% bromophenol blue, 0.25% xylene cyanol and 30% glycerol in water) and subjected to electrophoresis through 1-1.5% agarose gel in either 1X TBE or 1X TAE buffer. The gel was stained in 1 μg/ml of ethidium bromide solution for 30 min at room temperature and the bands were visualized by fluorescence under UV-light

Agarose gel electrophoresis

Quiagen/HiPura following the manufacturer's protocols

The rapid alkaline lysis method of plasmid isolation, as described by Sambrook and Russel (2001), was followed with minor modifications. Bacterial pellet from 3 ml of stationary-phase culture was resuspended in 200 μl of ice-cold solution I (50 mM glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA pH 8.0 containing 1 mg/ml lysozyme) by vortexing. After 5 min incubation at room temperature, 400 μl of freshly prepared solution II (0.2 N NaOH, 1% SDS) was added and the contents were mixed, by gently inverting the tube several times. This was followed by the addition of 300 μl of ice-cold solution III (5 M potassium acetate, pH 4.8) and gentle mixing. The tube was incubated on ice for 5 min and centrifuged at 20,0000g for 15 min at 4°C. The clear supernatant was removed into a fresh tube and, if required, was extracted with an equal volume of phenol:chloroform mixture. The supernatant was precipitated with either two volumes of cold 95% ethanol or 0.6 volumes of isopropanol at room temperature for 30 min. The nucleic acids were pelleted by centrifugation, washed with 70% ethanol, vacuum dried, and dissolved in appropriate volume of TE buffer. If required, the sample was treated for 30 min with DNase free RNase at a final concentration of 20 μg/ml. The plasmid DNA was checked on a 0.8% agarose gel and stored at −20°. The plasmid DNA thus isolated was suitable for procedures such as restriction digestion, ligation, and preparation of radiolabeled probes. Plasmid isolation was also done with any of the commercially available kits from

Isolation of plasmid DNA

and the aqueous phase transferred to a fresh tube. The aqueous phase was further extracted successively, first with phenol:chloroform:isoamyl alcohol (25:24:1) and then with chloroform:isoamyl alcohol (24:1). DNA was precipitated from the clear supernatant by the addition of 0.6 volumes of isopropanol. The chromosomal DNA was either spooled out or pelleted at this stage, washed with 70% ethanol, air-dried, and dissolved in suitable volume of TE buffer

The method as described in the manual Current Protocols in Molecular Biology was followed for preparation of chromosomal DNA. Cells from 1.5 ml stationary phase culture were recovered by centrifugation and resuspended in 567 μl of TE buffer. To this, 30 μl of 10% SDS, and 3 μl of proteinase K (20 mg/ml) were added in that order and the cell suspension mixed and incubated at 37°C for 1 h. Next, when the suspension looked cleared, 100 μl of 5 M NaCl was added, thoroughly mixed, followed by the addition of 80 μl of CTAB/NaCl (10% cetyltrimethylammonium bromide in 7 M NaCl) and vigorous mixing (by inverting the microfuge tube). The suspension was incubated at 65°C for 10 min, brought to room temperature, extracted with an equal volume of chloroform-isoamyl alcohol (24:1 v/v)

Extraction of chromosomal DNA from bacterial cells

Molecular and genetic techniques

For TEM, C. glabrata cells were digested with zymolyase 20T for 3 h at 30◦C, centrifuged at 1,000 g and washed with YPD medium. Cell fixation was performed as described for SEM and dehydrated samples were embedded in araldite 6005 resin. After complete polymerization at 80 ̊C for 72 h, ultra-thin (50-70 nm) sections were preparedwith a glass knife on Leica Ultra cut (UCT-GA-D/E-1/00)microtomeand mounted on copper grids. Aqueous uranyl acetate-stained and Reynolds lead citrate-counterstained samples were viewed under Hitachi H-7500 transmission electron microscope

Transmission electron microscopy

For SEM, C. glabratacells were fixed for 24 h in 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.2) at 4 ̊C, post-fixed in 2% aqueous osmium tetroxide for 4 h and dehydrated. After drying to critical point, mounted samples were coated with a thin layer of gold for 3 min using an automated sputter coater and visualized by SEM (JEOL-JSM 5600)

Scanning electron microscopy

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed at the Electron Microscope Facility, RUSKA LABs, Acharya N. G. Ranga Agricultural University, Hyderabad

Electron Microscopy

For preparing agarose gels,appropriate amount of agarose(0.8-2%) was dissolved by boilingin TAEbuffer, until clear slurry was formed.It wasthen poured in a casting tray containing a comb for desired number of wells. The gel was allowedto solidify andshifted to horizontal electrophoresis tank containing TAE bufferwith 1 μg/ml ethidium bromide. Appropriate volumesof 6X DNA loading dye were added in the samples and subjected to electrophoresis atconstant voltage (generallyat80 V), along with appropriate DNA ladder,untilband were resolved. The gel was visualised underUV-light in a Gel-Doc or UV-transilluminator

Agarose gel electrophoresis

recovery, cells were centrifuged at 2,500x gfor 4 min. The medium supernatant was discarded and cells were resuspended in 200 μLfresh sterile LB medium. Cells were plated on LB agar medium containing appropriate antibiotics. Plates were incubated at 37°C for 12-16 hto allow growh of individual colonies

E. coli DH5α strain was transformed with yeast plasmids carrying appropriate inserts that express S.cerevisiaeproteins (Sambrook and Russell, 2001). Ultracompetent cells stored at -80°C were thawed on ice for 5-10 min. 20μLligated plasmid was added to 100 μLultracompetent cells,and the cells were incubated on ice. After 30 min, competent cells were subjected to heat shock at 42°C for 90 seconds. Cells were immediately transferred to ice for 2-3 min. Next, 900 μLLB medium was added and cells were allowed to recover for 45 min on a shakerincubator set at 37°C. After the

Bacterial transformation

Statistical analysis was performed using GraphPsssad Prism 5. Data are presented as mean ± SEM. The difference between two groups was analyzed using either a two-tailed Student’s t-test or a nonparametric two-tailed Mann-Whitney test, as appropriate. The differences between multiple groups were analyzed by one-way ANOVA, using Tukey’s multiple comparison test for parametric data and Kruskal-Wallis test for non-parametric data. P<0.05 was considered as statistically significant. The cell numbers used to obtain quantitative data (n) and the number of independent experiments performed is indicated in the respective figure legends

Data analysis

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That the Federal, Government should be composed of a Governor General, or Viceroy, to be appointed by the Queen; of an Upper House, or Senate, elected upon a territorial basis of representation and of a House of Assembly, elected on the basis of population., The Executive to be composed of Ministers, responsible to the Legislature:

We ought to look at the question apart from party considerations, and on its own merits: that is to say, we ought to place in the Constitution a counterpoise to prevent any party legislation, and to moderate the precipitancy of any government which might be disposed to move too fast and go too far,—I mean a legislative body able to protect the people against itself and against the encroachments of power. (Hear, hear.) In England, the Crown has never attempted to degrade the House of Peers by submerging it, because it knows well that the nobility are a bulwark against the aggressions of the democratic element. The House of Lords, by their power, their territorial possessions, and their enormous wealth, are a great defence against democratic invasion, greater than anything we can oppose to it in America. In Canada, as in the rest of North America, we have not the castes—classes of society—which are found in Europe, and the Federal Legislative Council, although immutable in respect of number, inasmuch as all the members belonging to it will come from the ranks of the people, without leaving them, as do the members of the House of Commons, will not be selected from a privileged class which have no existence. Here all men are alike, and are all equal; if a difference is to be found, it arises exclusively from the industry, the intelligence, and the superior education of those who have labored the most strenuously, or whom Providence has gifted with the highest faculties. (Hear, hear.) Long ago the privileges of caste disappeared in this country. Most of our ancient nobility left the country at the conquest, and the greater number of those who remained have sunk out of sight by inaction. Accordingly, whom do we see in the highest offices of state? The sons of the poor who have felt the necessity of study, and who have risen by the aid of their intellect and hard work. (Hear, hear.) Everything is democratic with us, because everyone can attain to everything by the efforts of a noble ambition. The legislative councillors appointed by the Crown will not be, therefore, socially speaking, persons superior to the members of the House of Commons; they will owe their elevation only to their own merit.

He said that even if the Lower House were altogether liberal, the Upper House would remain composed of conservatives; this was his fear. He has been a long while trying to gain predominance for his democratic notions, but it is evident he will not succeed.

relative to the constitution of the Legislative Council, and said that he had not looked at the question, while speaking the other evening, in the same light as the honorable member for the county of Quebec. He spoke of the conservatives as a party, and his fear was, not that the Upper House would not be conservative enough, but that it would be too much so.

MR. GEOFFRION—YOU have equality between the two provinces. HON. ATTY. GEN. CARTIER—Yes, we have equality, but not as a race, nor in respect of religion. When the leader for Lower Canada shall have sixty-five members belonging to his section to support him, and command a majority of the French-Canadians and of the British from Lower Canada, will he not be able to upset the Government if his colleagues interfere with his recommendations to office? That is our security. At present, if I found unreasonable opposition to my views, my remedy would be to break up the Government by retiring, and the same thing will happen in the Federal Government.

HON. ATTY. GEN. CARTIER—Am I not in a minority at present in appointing judges? And yet when I propose the appointment of a judge for Lower Canada, is he not appointed?

the objection of the honorable member for the county of Quebec is well founded, because the Federal Government may appoint all English or all French-Canadians as legislative councillors for Lower Canada. If the honorable member had read the resolutions, he would have found that the appointments of legislative councillors are to be made so as to accord with the electoral divisions now existing in the province. Well, I ask whether it is probable that the Executive of the Federal Government, which will have a chief or leader as it is nowVI ask whether it is very probable that he will recommend the appointment of a French-Canadian to represent divisions like Bedford or Wellington for instance?

Lower Canada is in a peculiar position. We have two races of people whose interests are distinct from each other in respect to origin, language and religion. In preparing the business of the Confederation at Quebec, we had to conciliate these two interests, and to give the country a Constitution which might reconcile the conservative with the democratic element; for the weak point in democratic institutions is the leaving of all power in the hands of the popular element. The history of the past proves that this is an evil. In order that institutions may be stable and work harmoniously, there must be a power of resistance to oppose the democratic element. In the United States the power of resistance does not reside in the Senate, nor even in the President.

It is true that the House of Lords, Conservative though it be, finds itself removed from all popular influence; but its numbers may be increased upon the recommendation of the responsible advisers of the Crown, if such a measure were to become necessary to obtain the concurrence of both Houses, or to prevent a collision between them. The position which its members occupy in it establishes a sort of compromise between the Crown and the popular element. But this new House, after Confederation, will be a perfectly independent body; its members will be nominated for life, and their number cannot be increased. How long will this system work without bringing about a collision between the two branches of the Legislature? Let us suppose the Lower House composed in a great part of Liberals, for how long a time would it submit to an Upper House named by Government? Be kind enough to observe, Mr. SPEAKER, that under the old system, the Legislative Council possessed the same elements of existence as the House of Lords, and that the Crown could increase its numbers at need; it augmented it in 1849, as it threatened to augment the House of Lords in 1832. Observe, again, that it is precisely this control exercised by the Crown over the Upper House that the hon. gentleman found so fatal to legislation previous to 1856. But there is a more rational manner of appreciating the part sustained by the House of Lords in the British Constitution. No one denies to the Sovereign the abstract right of increasing at will the House of Lords; but such right has never been exercised but for the purpose of rewarding men distinguished for great national services and when, in 1832, WILLIAM IV. granted Earl GREY the tremendous power to swamp the representative body of the great landed nobility, it was because the country was moving with rapid strides towards revolution, and because there remained to the Sovereign but two alternatives, either to lessen the moral weight of the House of Lords, or to see his own throne knocked to pieces from under his feet.

It must then have been a real revolution, this nomination of one hundred new peers, a revolution as real as that which menaced the Throne; and do we not feel persuaded that if one day our Federal Legislative Council were to place itself obstinately and systematically in opposition to popular will, matured and strengthened by ordeals, it would not be swept away by a revolutionary torrent such as threatened to sweep away the House of Lords in 1832? This Council, limited as to numbers , because the provinces insist on maintaining in it an equilibrium without which they would never have consented to a union, this Council, sprung from the people—having the same wants, hopes and even passions, would resist less the popular will in America, where it is so prompt and active, than could the House of Lords in England, where the masses are inert because they have not political rights; reason tells us thus because they would be a less powerful body socially or politically.

We have accorded the principle of representation based upon population in the House of Commons of the Federal Government, and that is without doubt a great sacrifice; but we ought only to make so important a concession on the condition that we shall have equality of representation in the Legislative Council, and the right reserved to ourselves to appoint our twenty-four legislative councillors, in order that they may be responsible to the public opinion of the province and independent of the Federal Government.—Without this essential guarantee I affirm that the rights of Lower Canada are in danger. For my part I am ready, on behalf of Lower Canada, to give up her right to elect directly her twenty-four legislative councillors, although the retention of the elective principle might perhaps be the surest means of preserving our institutions; but I am anxious that the new Constitution now proposed should give us adequate guarantees that the legislative councillors to be appointed for life should, at all events, be selected by the Local Government of Lower Canada, which would be responsible to the people. These not ill-grounded sources of anxiety I should like to see removed.

In order to protect local interests, and to prevent sectional jealousies, it was found requisite that the three great divisions into which British North America is separated, should be represented in the Upper House on the principle of equality. There are three great sections, having different interests, in this proposed Confederation.

To the Upper House is to be confided the protection of sectional interests ; therefore is it that the three great divisions are there equally represented, for the purpose of defending such interests against the combinations of majorities in the Assembly.

But the very essence of our compact is that the union shall be federal and not legislative. Our Lower Canada friends have agreed to give us representation by population in the Lower House, on the express condition that they shall have equality in the Upper House. On no other condition could we have advanced a step ; and, for my part, I am quite willing they should have it. In maintaining the existing sectional boundaries and handing over the control of local matters to local bodies, we recognize, to a certain extent, a diversity of interests ; and it was quite natural that the protection for those interests, by equality in the Upper Chamber, should be demanded by the less numerous provinces.

The memorial consists of two parts, of which the following is the first :— The Government are prepared to state that immediately after the prorogation, they will address themselves, in the most earnest manner, to the negotiation for a Confederation of all the British North American Provinces. That failing a successful issue to such negotiations, they are prepared to pledge themselves to legislation, during the next session of Parliament, for the purpose of remedying existing difficulties by introducing the Federal principle for Canada alone, coupled with such provisions as will permit the Maritime Provinces and the North-Western territory to he hereafter incorporated into the Canadian system. In other words, the Government promises, in the first part of the memorial in question, to direct its attention to a Confederation of all the British North American Provinces; and, in the event of its not succeeding in carrying out that object, to turn its attention to a Confederation of the two Canadas. And now here are the contents of the second part :— The Government are prepared to pledge themselves to bring in a measure, next session, for the purpose of removing existing difficulties, by introducing the Federal principle into Canada, coupled with such provision as will permit the Maritime Provinces and the North-West territory to be incorporated into the same system of government. And the Government will seek, by sending representatives to the Lower Provinces and to England, to secure the assent of those interests which are beyond the control of our own legislation, to such a measure as may enable all British North America to be united under a General Legislature based upon the Federal principle.

In the Legislative Council we shall have 24 members like Upper Canada and the Lower Provinces. I assert, then, that there is a vast difference between the argument s of the hon. member for Hochelaga and the measure of the Government ; the Legislative Council will protect our interests, and the measures of general interest will come under the jurisdiction of the Federal Parliament.

position—I am sure it was not pressed upon him by the delegates from the Maritime Provinces— comes forward and says, ” I will give you ten members as a set-off against the twenty-one members who are now members for life in the Canadian Legislative Council.” If I am not wrong in my arithmetic, ten are not a third of twenty-one. If the honorable gentleman had given seven members to the Lower Provinces as a set-off against the life memo. ers of this House he would have acted with strict justice, but he is generous enough to give them three more—ten, or nearly one half.

I have just said the agreement was that there should be equality in the representation in the Legislative Council. But the honorable gentleman has moved that the elective members as they now stand should form the Legislative Council in the Federal Government, and that also the life members should continue for the remainder of their days ; and, as a set-off against the life members, he proposes to allow the other provinces a certain number of new members who should have the right to sit in the Legislative Council of the Federal Government. But what does he do ? Does he preserve the proportion as laid down at the Convention ? Not a bit of it. The proportion agreed upon at the Convention was one-third to the Maritime Provinces ; the Lower Provinces grouped together had a right to send one-third of the representatives. The honorable gentleman, however, I suppose out of the fullness of his good dis-

When the gentlemen who composed the Conference met, they had to lay down a broad basis, as it were, for the foundation of their superstructure. Well, it so happened that the corner-stone was that which concerned the representation in both Houses. It was agreed on the one hand that in the House of Commons of the Confederate Government representation should be according to numbers, and that in the other branch of the Legislature it should be fixed that this representation should be equal for all the provinces—that is to say, Upper Canada, and Lower Canada, and the Maritime Provinces, grouped into one, should each be allowed to send the same number of representatives, so as to secure to each province its rights, its privileges, and its liberties. We acted upon this principle, because we felt that if the House of Commons’ representation was based upon population, equality should be secured in the other branch of the Legislature.

If we gained anything by introducing the elective principle, we propose to keep that advantage, by retaining it just in the same form, and bear: ing the same relation to the proposed Legislative Council as it is retained in and bears relation to this House. HON. MR. CAMPBELL—But, under the present union, there is no federative necessity tor relative equality of numbers in the Legislative Council, as there will be under the proposed union. HON. MR. SANBORN—I admit no necessities of the kind. These necessities are entirely artificial. In that respect, I think hon. gentlemen are entirely in error in the position they take. And, though I concede to my hon. friend from Erie Division (Hon. Mr. CHRISTIE) every credit for great candor and soundness of judgment, still L must say that, when he enters into the province of law, he is travelling a little, as we say in the profession, out of the record—and that any one who is familiar with the doctrine of trusts eould not fail to see the falseness of his reasoning in that particular. As regards a trust, of course, the person who has a mandate given to him, must aet according to his discretion under the circumstances. But then he must do so within the trust that is given him, and not beyond the trust. HON. MR. CHRISTIE—Of course. HON. MR. SANBORN—My hon. friend cites the act empowering the Legislature of Canada to change the constitution of the Legislative Council, and on this act he bases his whole argument. If I convince him that that act does not cover his argument, will he then concede the point ? That act, to which my hon. friend refers, was passed for a specific purpose, to enable Parliament to reconstruct this House. It had answered its purpose when the constitution of this House was changed, but it cmnot properly be invoked as giving authority with reference to bringing in other provinces to form a new Confederacy. HON. MR. CHRISTIE—But my hon. friend will observe, that we are not legislating now—that we are merely passing an Address.

alluded the other day to the conservative feature of the Senate in the United States, in allowing the same representation to small states as to the larger states. But this does not at all affect the general arrangement, because the large majority are large states. But while my honorable friend approves of this portion, he should have expressed an opinion on the whole system. In the United States, no change of Constitution can be effected without the consent of two-thirds of both branches of the Legislature, and that must afterwards be sanctioned by three fourths of the state governments. This is a conservative feature also.

As to local taxation, all the provinces will be put upon the same footing, and nothing can be fairer. If Upper Canada, which it is asserted is so much wealthier than the other portions of the Confederation, requires more than the eighty cents per head allowed to all the provinces, its greater wealth will cause it to feel the taxation so much the less.