Benzamidine

Immunofluorescence assay was carried out as described by Bhattacharyyaet al., 2010.Adherent cells weregrown either on cover slips. After treatment, cells were fixed with 3.7% paraformaldehyde solution in PBS for 15 min and permeabilised with 0.5% Triton X-100 at room temperature for 10 min, followed by blockingin PBS containing 2% BSA for 1h. Post blocking, cells were incubated with a primary antibody in PBS (1:200 to 1:500) for 2 h. After washing, cells were incubated with fluorescent-conjugated secondary antibodyin PBS(Alexa Fluor 488 or 594 goat anti-rabbit or antii-mouse, 1:1000) for 30 min. After final wash with PBS, nuclei were counterstained with DAPI containing mounting medium (Vectashield, USA). All the steps were performed at room temperature, unless otherwise stated. Images were obtained using either the laser scanning confocal LSM510 (Carl Zeiss, Oberkochen, Germany) or fluorescence inverted (Olympus 1X51, Tokyo, Japan) microscopes

Immunofluorescence

After the RNA samples were resolved, the gel was placed in a tray and incubated for 5-10 min in DEPC treated water followed by 10X SSC buffer. A nylon membrane (N+Hybond, GE Life Sciences) was cut to the size of the gel and it was rinsed in 10X SSC buffer for 5 min. In the same tray, 10X SSC buffer was filled and a gel running boat (15 cm length) was placed in an inverted position. Whatmannumber 3 filter paper (wick) was cut to an appropriate size and placed on the inverted boat in such a way that the longer edges of the paper should touch the buffer. The gel was placed upside down on the wick and care was taken not to allow any air bubbles between the gel and wick. On top of the gel, three Whatman number 3 papers that were cut to the size of gel were placed by avoiding the air bubbles. A bundle of blotting sheets was placed on top of this, on which appropriate weightwas placed and was allowed the transfer to take place for 15 –16 h

Capillary transfer of RNA on to the membrane

A bacterial clone expressing the recombinant protein was grown overnight in 5ml of LB medium with 50ug/ml ampicillinand the next day culture was transferred into 500ml of LB medium with 50 ug/ml ampicillin. Bacterial culture was allowed to grow at 37°C until reached an OD600 of 0.6-0.8 and then induced with IPTG (1 mM) for 4 h at 37°Cor overnight at 16°C to induce fusion protein expression. Subsequently, bacterial culture was centrifuged at 4000 RPM for 10 minutes (At this point bacterial cell pellets can be stored at -20° C for later use). The bacterial cell pellet was washed once with PBS and resuspended in 10ml of lysis buffer (1X NETN and protease inhibitors) followed by sonication (45 sec. pulse was given three times). The cell lysate was centrifuged for 10 minutes at 14000 RPM. The supernatant containing the GST fusion proteins was added to the 200μl of 50% GST/MBP beads. After 2 hours of incubation at 4°C, beads were washed three times with 1 ml lysis buffer. Purified proteins were eluted by using GST/MBP elution buffer. The recombinant proteins were analyzed by SDS-PAGE followed by either Coomassie staining orwestern blotting

GST/MBP recombinant protein purification from bacteria

Intracellular iron content in different Xanthomonas oryzaepv. oryzicolastrains was measured by using atomic absorption spectroscopy as described previously with few modifications (Velayudhan et al., 2000). For estimation of intracellular iron, different strains of Xanthomonas oryzaepv. oryzicolawere grown overnight in 3 ml PS media with appropriate antibiotics for differentially marked strains. 0.2% of the overnight grown culture was inoculated in 250 ml PS medium alone or PS plus 2, 2’-dipyridyl for iron stravation, and grown to an OD600 of 1.2. Cells were then pelleted down by centrifuging at 7000 g for 10 min, and washed twice with phosphate buffer saline (PBS). After washing, cells were lyophilized, and their dry weights were determined. Lyophilized cells were then dissolved in 30% nitric acid at 80ºC for 12 h and diluted 10-fold with miliQ water. Iron content was determined by atomicabsorption spectroscopy using ICP-OES (JY 2000 sequential Inductively Coupled Plasma Optical Emisson spectrometer,Jobin Yvon, Horiba, France). Iron level was quantified against aqueous standard of iron traceable to NIST (National institute of standards and technology, India)

Estimation of intracellular iron content

confirmed through PCR (by using primers SC11 and SC10) and squencing. Double mutant was complemented for DSF production by cloning whole rpfFgene of Xoo, cloned in HindIII and EcoRI sites of pHM1 (a broad host range vector for Xanthomonas) to get pSC6 plasmid. The resultant pSC6 plasmid was introduced into double mutant by electroporation

To obtain the insertional nonpolar mutant in the motA (encodes flagellar motor stator protein)andfliC (flagellin)genes, a 321 bp internal fragment of the motAgene and a 450 bp internal fragment of fliC containing the EcoRI and XbaI site were amplified using respective primer listed in Table 2.2. These fragments were cloned in pk18mob suicide vector, in which the lacZpromoter drives the expression of downstream gene (Schäfer et al., 1994; Windgassen et al., 2000), to obtain pRR1 and pRR2,respectively (Table 2.2). The resulting plasmid (pRR1& pRR2) was introduced into XooBXO43 strain by electroporation. Single Kmrrecombinants were selected on PSA plate containing kanamycin. Insertion of the pK18mob vector in motA andfliCgene was confirmed with PCR and sequencing. To further confirm the mutation in the flagellar genes, we did swimming motility assay on 0.1% peptone-sucrose agar (PSA). Swimming plate assay indicated that both motA and fliCmutant of Xoowas deficient in motility. Further, to obtain motAand fliC insertional knock out mutants in rpfFbackground, we cloned spectinomycin cassette obtained from pUC1318Ω plasmid, into the HindIII site of pRR1 and pRR2 plasmid to obtain pRR3 and pRR4. The resulting plasmid (pRR3& pRR4) were transformed in rpfF. Single specrrecombinants were selected on PSA plate containing kanamycin and spectinomycin. Insertion of the vector was further confirmed by PCR and sequencing. T2SSrpfFdouble mutant was constructed by transforming the plasmid with rpfF::Tn7Kanamycin cassette in the T2SS (xpsF) mutant background, and Kmrrecombinants were selected on PSA plates containing kan

Construction of mutants in X. oryzaepv. oryzae

3.0–5.0, phosphate buffer for pH 6.0–8.0 and Tris-HCl buffer for pH 9.0) were used. •pH stability: The pH stability of the selected tannases was examined in the range of 3.0–9.0 by incubating the enzyme samples for 6 h in different buffers. Tannase activity was estimated under standard assay conditions. •Temperature tolerance: Temperature tolerance of the tannases was examined by assaying their activity at different temperatures in the range of 20 to 80ºC. •Temperature stability: Temperature stability of the tannases was determined by incubating them in the temperature range of 20 to 70 ºC for 6 h. After the incubation tannase activity (%) was determined under standard assay conditions. •Organic solvent stability: In order to determine the suitability of the selected tannases for organic synthesis, their stability was determined in different organic solvents. Experimentally, 10 mg of each of the crude lyophilized tannase from the selected cultures were mixed with 1.0 ml of the following organic solvent: a) Hexane b) Methanol c) Propanol d) Isoamyl alcohol e) Petroleum ether f ) Chloroform The mixture was incubated for 6 h at optimal temperature and the organic solvents were then decanted and the residues were dried in a vacuum desiccator. These dried samples were dissolved in 1.0 ml of citrate phosphate buffer (50 mM, pH 5.0) and the tannase activity was determined under standard assay conditions. The tannase activity thus obtained from each culture were compared with initial tannase activity. Finally, on the basis of tannase titres produced per ml and desirable biochemical properties, the best tannase producer was selected for further investigations

The tannases obtained (at high titres) from selected cultures were evaluated for the following important biochemical properties. 1. pH tolerance and stability 2. Temperature tolerance and stability 3. Organic solvent stability •pH tolerance: pH-tolerance of the selected tannases was examined in the range of 3.0–9.0. Buffers (0.05 M) of different pH (citrate phosphate for pH

Preliminary biochemical characterization of tannases from the potent tannase producers

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

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

Dishes containing adherent cultures were placed on ice and washed once with cold PBS. Cells were scraped in 1x sample buffer on ice. Samples were sonicated in a probe sonicator (Misonix ultra sonic liquid processor, S-4000) 3 times for 15 sec each to complete cell lysis and shear DNA to reduce viscosity. Equal volumes of lysates were loaded and resolved using 12% SDS-PAGE. Samples were stored at -80ºC if necessary. Protein transfer onto PVDF membrane (GE Healthcare) was carried out at 200mA constant current for 2 h by placing the transfer tank in ice. Membranes were blocked with appropriate blocking buffer according to manufacturer’s instructions provided for each antibody. Preferentially TBST with 5% non-fat dry milk was used for blocking and for antibody dilutions. Briefly, non-specific interactions were blocked with blocking buffer (TBST+5%non-fat dry milk) for 1 h at room temperature. Membranes were probed with appropriate primary (overnight at 4ºC) and HRP-conjugated secondary antibodies (2 h at room temperature). Proteins were detected using ECL detection system. Chemiluminiscence was detected using the LAS4000 (GE Healthcare) or FlourChem E (Protein Simple) documentation system. Densitometry analysis of bands was done using ImageJ documentation software (Fiji) or the multiplex band analysis tool in AlphaView software (Protein Simple)

Western blot analysis

ZFIN_ZDB-GENO-050809-10

ZFIN_ZDB-GENO-100513-10

ZFIN_ZDB-GENO-101227-10

ZFIN_ZDB-GENO-101227-10

Top 10 Augmented Reality Development Companies

Top 10 Web Developers

HON. MR. REESOR—There are several other provisions in the proposed Constitution which seem to be ambiguous in their meaning, and before discussion upon them it would be well to have them fully explained. In the eleventh clause of the twenty-ninth resolution, for instance, it is declared that the General Parliament shall have power to make laws respecting ” all such works as shall, although lying wholly within any province, be specially declared by the acts authorizing them to be for the general advantage.” It would appear from this, that works like the Welland canal, which yield a very large revenue, will be given over to the General Government; and this being the case, surely this is a sufficient setoff, five times over, for the railways given by New Brunswick, without the annual subsidy proposed to be given to that province of $63,000. HON. MR. MACPHERSON—The cost of these works forms part of the public debt of Canada, which is to be borne in part by the Lower Provinces under the Confederation. HON. MR. CAMPBELL—The honorable gentleman will see that there are some works which, although local in their geographical position, are general in their character and results. Such works become the property of the General Government. The Welland canal is one of them, because, although it is local in its position, it is a work in which the whole country is interested, as the chief means of water communication between the western lakes and the sea. Other works, in the Lower Provinces, may be of the same character, and it is not safe to say that because a certain work lies wholly in one province, it is not to belong to the General Government. HON. MR. REESOR—I do not object to the General Government having the control of these works. It is, I believe, a wise provision to place them under such control. But I do say that it is unfair that an express stipulation should be made to pay one province a large sum per annum for certain works, while, at the same time, we throw in our public works, such as the Welland and St. Lawrence canals, without any consideration whatever. This, I think, is paying quite too much for the whistle. Then the answer of the Commissioner of Crown Lands about the export duty on minerals in Nova Scotia is not at all satisfactory. Whatever dues may be levied on minerals in Canada—and Canada, although it may contain no coal, is rich in gold, silver, copper, iron, and other ores—in the shape of a royalty or otherwise, go to the General Government, while in Nova Scotia they accrue for the benefit of the Local Government. HON. MR. ROSS—NO, they will not go to the General Government. HON. MR. REESOR—Well, there is nothing to the contrary in the resolutions, and you may depend upon it that whatever revenues the General Government may claim, under the proposed Constitution, will be fully insisted upon.

8

Local works naturally fell within the scope of local governments, and would undoubtedly be under the immediate influence of the municipal councils, but all the works of a really public character would be under the General Legislature; such, he meant, as were connected with the general policy of the whole country.

Lines of steam or other ships, railways, as well as canals and other works connecting any two or more of the Provinces together, or extending beyond the limits of any Province, would be under the control of the General Government.

a Governor General, who should be appointed by our Gracious Sovereign.

Go to Control Panel > Uninstall a Program > Turn Windows features on or off to turn off Hyper-V.

sity faculty member) describes her temporomandibular joint disorder in this way: "It's like a shadow that throws the other parts of my life into brighter contrast. You see my brights are brighter, 'cause I have this darkness hovering all around the edges" (1992:129). Weapon or demon metaphors are often used to describe pain (see also Good 1992). Scarry's respon- dents refer to pain as a "knife, or bones that cut through

God

and may even cause insanity.

Cindy A. Buckmaster: Animal research Is a labor of love for animals and people

You gotta go for what you knowMake everybody see, in order to fight the powers that beLemme hear you sayFight the Power

Some blocks in my neighborhood are getting downright spooky – front yards are filling with spider webs and tombstones, and ghosts peek through the bushes. Along with the piles of pumpkins and inevitable candy corn appearing in the supermarket, they are a reminder that Halloween is just around the corner. Americans celebrate Halloween on October 31 by trick-or-treating, displaying jack-o’-lanterns (carved pumpkins) on their porches or windowsills, holding costume parties, and sharing scary stories

10 Oct 2016 22:13:49 UTC

Reid, Clinton supporters hit Trump over Nevada pronunciation Published October 06, 2016 FoxNews.com Facebook0 Twitter0 livefyre9791 Email Print Now Playing What's Trump doing to prepare for 2nd presidential debate? Never autoplay videos Supporters for Democratic presidential nominee Hillary Clinton and Sen. Harry Reid attacked Donald Trump after the Republican presidential nominee told his supporters about the “correct” way to pronounce Nevada. Trump, during a rally Wednesday in Reno, insisted the correct way to pronounce the name of the Silver State was “Neh-VAH-da.” He declared that “nobody says it the other way.” Clinton supporters and Reid, a Democrat from Nevada, both used the moment to assail Trump. American Bridge immediately put up a video declaring that Trump was “looking like an idiot” for getting the name wrong. A statement from Reid declared that Trump’s stop in Reno was “disastrous.” "If Donald Trump wants to come down from the penthouse his daddy bought him to lecture us on how to say Nevada, he could at least pronounce it correctly,” Reid said in a statement. "Instead, Trump told us we pronounce the name of our state wrong minutes before he refused to take a position on Yucca Mountain. Predictions Map See the Fox News 2016 battleground prediction map and make your own election projections. See Predictions Map → “I have news for Donald: it's pronounced Nev-AD-a and Yucca Mountain is dead.” Trump made a stop at the International Church of Las Vegas and the International Christian Academy before his rally in Reno. He said the Pledge of Allegiance with schoolchildren at the school. He also visited with Hispanic business leaders at a Mexican restaurant before departing for northern Nevada. Fox News’ Chad Pergram and the Associated Press contributed to this report.

Without the tax-related reduction, Mylan’s profits on the EpiPen two-pack were about 60% higher than the figure given to Congress, or $166, it said in a new regulatory filing to the Securities and Exchange Commission Monday.

While we have yet to receive any definitive answer as to exactly what group hacked the DNC, the vast majority of experts believe that the attacks came from Russia or Russian-backed actors.

And research on both people and animals suggests the reason is that a brain injury can disrupt circuits that normally dampen the response to a frightening event.

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.

Matthew S. MacLennan