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  2. Sep 2021
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  7. Mar 2021
    1. Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

      AssayResult: 5.1

      AssayResultAssertion: Normal

      StandardErrorMean: 0.7

    2. A total of 84 PALB2 patient-derived missense variants reported in ClinVar, COSMIC, and the PALB2 LOVD database were selected

      HGVS: NM_024675.3:c.1421G>A p.(Ser474Asn)

    1. SUPPLEMENTARY DATA

      AssayResult: 53

      AssayResultAssertion: Indeterminate

      PValue: < 0.0001

      Approximation: Exact assay result value not reported; value estimated from Figure 6C.

    2. SUPPLEMENTARY DATA

      AssayResult: -25

      AssayResultAssertion: Abnormal

      PValue: < 0.01

    3. SUPPLEMENTARY DATA

      AssayResult: 77.32

      AssayResultAssertion: Indeterminate

      PValue: 0.0002

      Comment: Exact values reported in Table S3.

    4. To this end, 44 missense variants found in breast cancer patients were identified in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) and/or selected by literature curation based on their frequency of description or amino acid substitution position in the protein (Supplemental Table S1).

      HGVS: NM_024675.3:c.2841G>C p.(Leu947Phe)

    1. Source Data

      AssayResult: 11.06

      AssayResultAssertion: Abnormal

      ReplicateCount: 2

      StandardErrorMean: 2.4

      Comment: Exact values reported in “Source Data” file.

    2. Source Data

      AssayResult: 9.92

      AssayResultAssertion: Abnormal

      ReplicateCount: 2

      StandardDeviation: 1.93

      StandardErrorMean: 1.37

      Comment: Exact values reported in “Source Data” file.

    3. We, therefore, analyzed the effect of 48 PALB2 VUS (Fig. 2a, blue) and one synthetic missense variant (p.A1025R) (Fig. 2a, purple)29 on PALB2 function in HR.

      HGVS: NM_024675.3:c.172_175delTTGT p.(Q60Rfs)

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

      AssayResult: 41.7

      AssayResultAssertion: Abnormal

      ReplicateCount: 15

      StandardErrorMean: 10.8

      Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

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

      HGVS: NM_198056.2:c.2236G>A p.(Glu746Lys)

  8. Feb 2021
    1. Supplemental material

      AssayResult: 83

      AssayResultAssertion: Normal

      Comment: See Table S3 for details

    2. Supplemental material

      AssayResult: 4.1

      AssayResultAssertion: Abnormal

      Comment: See Table S3 for details

    3. 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.

      HGVS: NM_000546.5:c.329G>A p.(Arg110His)

  9. Oct 2020
    1. he extinction coefficient could be calculated using therelation:k=αλ/4π. Figure 15 shows the variation of extinc-tion coefficient as a function of wavelength; it shows a

      El coeficiente de extinción podría ser calculado usando la relación: (ecuación). La figura 15 muestra la variación del coeficiente extinción como una función de la longitud de onda; esto muestra un agudo/fuerte incremento en la región ultravioleta debido a la alta absorbancia fotones incidentes cerca del band gap.

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    1. Une authentique dérogation est à noter: la loi du 15 mars 2004 a introduit l’interdiction du « port de signes ou de tenues » manifestant « ostensiblement une appartenance religieuse » pour les élèves des écoles, collèges et lycées publics, qui sont des usagers du service public de l’éducation. Ainsi, les signes et tenues interdits sont ceux dont le port conduit à se faire immédiatement reconnaître par son appartenance religieuse, tels que le foulard, la kippa ou une croix de dimension manifestement excessive. La loi ne remet pas en cause le droit des élèves de porter des signes religieux discrets.Il est à noter que cette interdiction, strictement limitée aux écoles collèges et lycées publics, ne s’applique pas dans l’enseignement supérieur,et notamment au sein de l’université, que les élèves, usagers, sont libres de fréquenter en revendiquant leur religion, sous réserve, classiquement, de ne pas troubler l’ordre public.
  10. Aug 2020
  11. Jan 2020
    1. 15 Best Marketing Tools to Increase your Sales Whether it’s a small business or an enterprise, every business needs to have its marketing game on point. Big businesses invest heavily in marketing and promoting their products on various platforms but, a small business cannot afford big marketing budgets. This article lists best marketing tools By Jenny Targa13th Jan 20200:00/15:0323 claps+20 Share on23 claps+20 Share onShare onWhether it’s a small business or an enterprise, every business needs to have its marketing game on point.Big businesses invest heavily in marketing and promoting their products on various platforms like Google, social media, banner ads, etc. but, a small business that cannot afford big marketing budget hence, lacks behind and fails.Did you know around 90% of the startup fails within the first year of establishment? Do you know why?Well, there are lots of variable factors that result in business failures but one of the major ones is marketing. Marketing is all about running the right campaign based on the products, audience, and channel.To make your campaign optimized and organized you need to have the right tools. In this article, we will provide you with an 18+ marketing tool that will definitely increase your sales.Content Generation Tool“Content is King”You've got to have the best content to see business growth. Whether it’s Google or your customers, everyone expects content that helps them in solving their problems. If you can solve someone's problems, then you are most likely to make him your customer for life.To generate content that helps your customers in solving problems, you need to find the problem first. So, the following tool helps you in creating the best content-Google Google is the most used search engine in the world with 80-85% of market share. Ranking your website for keywords helps you in increasing sales.What do you do when you want to buy or search for something?Google it!But, how Google will help you in creating content that ranks and viewers find interesting?Google can help you in finding the topics or problems people are facing related to your industry. You can then create content like articles, videos, etc. solving that issue. Simple, isn’t it?Just put in the keyword related to your industry and you can get various topics to write about from the suggestion section as well as on auto-fill keywords. People are searching for all these keywords and questions.For example- Search for the keyword “How to start a business” on google.comYou’ll notice Google is providing you with auto refill options. All of these can be your potential content topics.When you scroll down you’ll notice there are few suggestions provided by Google, they are your content topic.And hey, it’s FreeGoogle TrendsGoogle Trends is yet another amazing product that is free and provides you with some amazing results. They are best if you want to find out what is trending and where is it trending.If your customers are from specific demographics like the US, Google Trends can give you location-specific results related to your industry. You can create content specific to that region.You can use related queries or interests by region to create a specific topic for your content. The content created not only ranks for the keyword for a specific region but also brings relevant traffic to your site.EveryDesigns When you are creating content or marketing your business you need to have graphic design services. Around 80-90% of all the people are visual meaning creating a moving design can influence the viewer in buying your products.EveryDesigns provides the best designs by organizing a design contest. You just have to provide the brief and you will receive different concepts crafted by multiple designers.You can then select your favorite design and pay for only that design. It’s the world's easiest way of getting the design, isn’t it?BuzzSumoBuzzSumo helps you in generating ideas, high-quality content, track performance, and identify influencers to promote your content.The above dashboard shows you the engagement with a variety of filters to narrow down your analytics. You can use this data to create content that gets engagement as well as social signals.You can also use this tool to find the best social media channels to get your content shared and increase engagement.Content Optimization ToolNow, you have created high-quality content that not only ranks but also engages your visitors. But, it is important for optimizing content for grammatical errors, keyword density, and other important optimization. The following tools can help you in optimizing your content.GrammarlyGrammarly can help you in removing any grammatical errors and provide you with replacement words. Grammarly also provides you with a content score that can make your job easy. Higher the score better than the content.  You also get to find whether it is engaging or boring based on the writing tone. If you want to make your writing your English professor then you got to have Grammarly.It is free as well as pro versions, you can for free if you are on a budget but, if you have bucks and find a grammar correction tool then this is it.Yoast SEO PluginYoast is an SEO plugin that helps you optimize your on-page SEO. If you use WordPress as your website CMS then this must be your go-to plugin. Yoast not only helps you in writing title, description but also helps in optimizing content by scoring content based on a variety of SEO factors.If everything is green like the title, description, Yoast Analysis then you are good to go, but if you find something in red or orange then improve that factor by highlighting it, until it turns green.Website ManagementHaving an online presence is important nowadays, and what is the best way to be online then having a website?A website helps your business in getting more customers and creating a global presence. If you have a website then you must require a website management tool. The following are the tools that can help in managing your website-WordPressWebsite management is the basic requirement that every business having a website needs. Using a CMS like WordPress can help you in making your job easy.You do not need to learn the programming language to work on WordPress. There are different plugins that help your website from the functioning part and you can use page builders to drag and drop.You can even use WordPress to build your own e-commerce website. About half the websites use WordPress to operate their website.Email MarketingIf you ask me, email marketing is the best way of getting quality sales. You only get those mail ids who are interested in your product. Everyone is not your customer and email marketing funnels downs only your customers.Using this method of marketing, people will visit your website regularly, which sends a good signal to Google and affects your ranking positively. The following are the best tools that you can use for marketing your brand via email-MailChimpMailChimp is one of the well-known email marketing tool that powers hundreds of webmasters in powering their marketing campaigns. The best part is it's free and comes with enough features to make small business.When you start to get more customer base then you can use their paid plan which is as low as $9, Cheap marketing tool isn’t it?HubspotHubspot is not just an email marketing tool but a group of inbound marketing tools that consist of conversational bots, email marketing, and other tools.These tools are designed specifically to create a marketing funnel that drives your customers through various stages of sales. These stages as defined by Hubspot are Attract, Engage, and Delight.Hubspot has free as well as paid software. You can start with the free version and eventually when your business starts getting bigger, you can purchase the paid version to increase the functionality of the software.Social Media MarketingSocial media is one of the biggest channels that has the potential of making or breaking your brand image. The daily visitor count on social media websites like Facebook, Instagram and Twitter is in millions, which makes it a potential source for brands to increase their customer engagement strategies.Tracking social media signals from users will ensure that your products or services are indeed perceived as good. If you find people posting a negative review for your service or product quality, you can quickly get into damage control and prevent spoiling the brand image.The tool that can help you in tracking, optimizing and using social media to its full potential is as follows-MeetEdgarMeetEdgar is a social media marketing tool that takes your social media campaign to auto-pilot. The biggest challenge that comes when handling a social media account is finding the relevant content, optimizing it and posting the content. You have to repeat the same process on different social media accounts. This is a lot of work.Tools like MeetEdgar automate these processes and speed up your campaign. There is an A/B testing option, tracking and unlimited library of content, so you don’t have to worry about producing the content yourself.The first month is free so you can try their service and if you find yourself liking the product then it is the best option for you to handle your social media channel.Crowdfire AppCrowdfire App is a software that helps you compile content to post on your social media account. The algorithm helps the user in finding the relevant content based on your niche.It assists you in optimizing your social media, it is a very powerful tool for handling your social media account. There are free as well as paid versions available in the market. The real potential of your social media can be utilized when you get the paid version of the app.Statistics Tracking ToolsStatistics or Data is the most important part of any marketing campaign. Data-driven marketing helps in keeping track of what you did right and what you did wrong?Planning different strategies and organizing different campaigns for your business can be overwhelming for you to track and certainly impossible.Data collection tools help you in tracking your progress and ranking your various campaigns. The tools that are best at tracking your data are as follows-Google Analytics & Search ConsoleGoogle Analytics & Search Console are free tools that help you in tracking your website visitors. These tools have become a marketing standard for every website.These tools are designed to provide data on user activity and engagements within your website as well as Google. The basic difference between search console and analytics is, the search console provides the data on the website performance on Google whereas analytics provides website performance on the visitor side.CrazzyEggCrazzyEgg is another marketing tool that provides valuable stats that the Google analytics and search console lacks. It adds another layer of information that can help you in improving your website performance.Features like heatmaps, scrolling, A/B testing, user site recording, etc. can provide an additional layer of data that can help you in optimizing your website.It is a paid product but the first 30 days are free.SEO ToolsSEO stands for Search Engine Optimization and it is one of the most important factors when it comes to online marketing. The main objective of SEO is to rank the website higher in the SERPs for specific keywords.To do so, you would need a variety of tools, the following are the list of tools that you can use to improve your site’s SEO-UberSuggestUberSuggest is another best marketing tool that helps you in making your business increase sales. It is a free tool and provides you with data like search volume, Keyword ideas, content ideas, web traffic, SEO difficulty and much more. If you are into digital marketing then this tool will help you in optimizing not only your content but also helps you in auditing your website and getting backlink data of your competitor.It is one of the best marketing tools that can make your website rank higher in the SERPs, free of cost.AhrefAhref provides you with a list of tools to use for optimizing your website as well as tracking your competitors. One of the most used tools that makes it best in the market is the content explorer. You can use this tool for content creation.The number of tools that you get from Aherf is enough to make your website rank higher. You can run a site audit, analyze competitors for their backlinks, track your backlinks, and much more.You can get a free trial for $7 and the full basic pack for $99.ConclusionMarketing is very important and when you are a small business it becomes a basic necessity. It is a method of helping your customers find your product.How many of the tools have you used before?What are your favorite tools?Or did I miss your favorite tool?

      Whether it’s a small business or an enterprise, every business needs to have its marketing game on point. Big businesses invest heavily in marketing and promoting their products on various platforms but, a small business cannot afford big marketing budgets. This article lists best marketing tools

  12. Dec 2019
  13. Jun 2019
    1. Electrostatic potentials were calculated by the Finite Difference Poisson-Boltzmann (FDPB) method using the program MEAD running within the PCE web server (http://bioserv.rpbs.jussieu.fr/PCE) (Miteva et al, 2005; Bashford et al, 1992). Additions of hydrogen atoms as well as assigning of atomic radii and charges were performed automatically within the server. MEAD numerically solves the Poisson-Boltzmann equation to yield the distribution of electrostatic potential on the protein surface. Calculations were performed on one of the native a-chains of the 2HbS crystal structure (Harrington et al, 1997) as well as its SCWRL (Dunbrack et al, 1993) generated mutants. All calculations were performed by setting the internal protein dielectric constant to 4 and the external solvent dielectric constant to 80. The ionic strength parameter was held at 0.1
    2. Electrostatic Potentia
  14. May 2019
    1. A 96-well microplate was coated overnight at 4°C with ovalbumin conjugated peptide in 100 mM carbonate buffer, pH 9.5 (2 /J-g/well). The plate was washed 3 times with PBST and blocked with PBS containing 2% BSA (200/J-l/well) at 37°C for 1 h. Serum samples (diluted in PBS) were added in duplicates (50 (/J-lIwell) at different dilutions (1: 1 00, 1: 1000, 1: 10,000) and the plate was incubated at 37°C for 1 h. The plate was washed and incubated with HRP-conjugated appropriate antibody (1: 1 0,000 dilution in PBS containing 2% BSA) at 37°C for 1 h. The plate was washed thoroughly with PBST and freshly prepared TMB substrate (100/J-lIwell) was added and the reaction was stopped with 2 N H2S04 (50 (/J-l/well) and the absorbance at 450 nm was recorded in an ELISA reader
    2. ELISA
    1. Nitric oxide (NO) generation within the macrophage was detected using the fluorescent NO-sensitive probe DAF-FM diacetate (7). THP-1 macrophages were harvested and resuspended in serum and phenol-red free RPMI-1640 medium and incubated at room temperature for 30 min in the presence of 1 llM DAF-FM diacetate dye. The cells were washed once with fresh medium to remove the excess probe and kinetic fluorescent measurements were performed on a spectrofluorimeter (BMG Fluostar Optima) at an excitation of 480 nm and emission of 520 nm. Time kinetic measurements were performed after appropriate treatment and the values were represented as arbitrary fluorescence units with the comparisons being made against the fluorescence of the control cells. SNAP (S-nitroso-N-acetylpenicillamine), a photoactivatable nitric oxide donor (8) was used as positive control in the assay.
    2. Measurement of intracellular nitric oxide (NO) generation
    1. The specific ribonucleolytic activity of restrictocin and its mutants was followed by detecting the release of the characteristic 400 nucleotide long a-fragment from 28S rRNA of eukaryotic ribosomes. All the reagents, water and glassware used during the experiment were treated with O.I% DEPC to get rid of contaminating ribonucJeases. Rabbit reticulocyte lysate (30 J.Ll) was incubated with different concentrations of the toxin in 40 mM Tris-HCl (pH 7.5) containing 10 mM EDTA at 37 °C for 30 min. in a 50 J.Ll reaction volume. The control reaction did not contain an)' toxin. The reaction was stopped by adding 2 J.Ll of I 0% SDS and incubated at ambient temperature for 5 min. Total RNA was extracted using Trizol reagent. 200 J.LI of the reagent was added to the reaction mixture, mixed well and incubated at room temperature for 5 min. Subsequently, 50 J.LI chloroform was added to each tube, mixed thoroughly and incubated at ambient temperature for 2 min. followed by centrifugation at I2,000 rpm, at 4 °C, for I5 min. in a microfuge (Plastocraft). The aqueous phase was mixed with 125 J.Ll isopropanol to precipitate the RNA, allowed to stand at ambient temperature for I 0 min. and centrifuged at 12,000 rpm at 4 °C for I5 min. The RNA pellet was washed with 75% ethanol, dried in air, dissolved in 10 J.Ll of 0.5% SDS solution and electrophoresed on a 2% agarose gel after heating at 65°C for 2 min. The RNA was visualized by ethidium bromide staining and photographed using Polaroid camera. The photographs were scanned, printed using a laser printer to present as figures in this thesis.
    2. Specific Ribonucleolytic Activity Assay
    1. Lipofectin was kindly provided by Syntex, Inc., USA as an aqueous solution containing 1 mg I ml of 1 ipid ( DOTMA DOPE; 50 50 ). The procedure used was as described by Feigner et al., 1987 with appropriate I modifications as suggested in the user s notes. Lipofection was done with 0.5 x 106 cells seeded on a 60 mm plate. For each plasmid, the lipofection was performed in duplicate. The amount and quality of the plasmid DNA used ranged from 400 ng of crude DNA prepared by the mini prep method, to 5 ug of highly purified, cesium banded DNA. The appropriate amount of DNA was suspended in 1.5 ml of serum free DMEM. In another tube, 30 ug of lipofectin was suspended in 1.5 ml of serum free DMEM. The two solutions were mixed. The cells were washed twice with HBSS to totally wash off all traces of serum. The DNA 1 lipofectin mix was then applied to the cells and the cells incubated for 4 hours at 37°C. Next, 3 ml of media containing 10 % FCS was added and the incubation continued at 3 7°C for 16 hours. The culture supernate was then aspirated off and fresh medium added to the cells. The selection for stable clones was started after 48 hours by the procedure described below.
    2. Using lipofectin.
    3. PBS and then replenished with the complete medium. Two days following transfection, the cells were subcultured into the appropriate selective medium for selection of stable clones as described below.
    4. Calcium phosphate mediated stable transfections were performed by the method of Graham and Van der Eb ( 1973 with modifications as described by Gorman ( 1986 ). For each plasmid, two petri dishes each containing 0. 5 x 106 CHO-K1 cells were used, with 10 ug of cesium purified DNA for each transfection. A mock transfection which did not contain any DNA, was performed simultaneously as negative control. Precipitation of the DNA was done with great care to ensure the obtention of a fine, translucent precipitate rather than a dense and opaque precipitate. The calcium phosphate I DNA precipitate was added in 4 ml medium to the cells and the cells incubated for 3 hours at 37°C. At this stage, the cells were examined under the microscope and a fine precipitate appeared as small grains all over the cells. The cells were washed once with serum free medium and a glycerol shock given for 3 minutes at 37°C. The cells were washed twice again with
    5. Using calcium phosphate.
    6. Stable transfection was performed into CHO-K1 cells by the following procedures
    7. stable transfection.
    8. rinsed twice with serum free medium and replenished with 4 ml of DMEM containing 10 % FCS and 100 uM chloroquine. The incubation was continued for another 3 hours at the cells were washed and fed with the normal growth medium containing 10 % FCS. As in the case of FWIL cells, the supernate was collected after 72 hours of transfection and assayed for BhCG activity by RIA.
    9. ayed for BhCG activity by RIA. In case of the other five monolayer forming cell lines, a slightly different protocol was used. Only 1.8 ug plasmid DNA was used for each transfection using 0.5 x 106 cells, and 70 uM chloroquine was included in the DNA 1 DEAE-dextran mixture. Cells were fed 3 hours prior to transfection and washed twice with serum free medium just before exposure to DNA. Cells were exposed to DNA 1 DEAE-dextran mix for approximately 3 hours at 37°C. Following this, the cells were
    10. 1 - 5 ug of plasmid DNA using the DEAE -dextran procedure. DEAE dextran M.Wt. 500,000 was used to perform transient transfection by the method of Luthman and Magnusson 1983 ) , with modifications as described by Gorman ( 1986 ) . Six cell lines ( described above ) with two petri dishes ( 60 mm ) for each cell line were used. In case of FWIL, 5. 4 ug plasmid DNA was used to transfect approximately 5 x 106 cells. No exposure to chloroquine was given. The cells were treated with the DNA 1 DEAE -dextran mixture for 20 minutes at 37°C in a tightly capped tube, mixed gently and reincubated at 37°C for 10 minutes. The sample was then diluted with 3 ml of IMDM supplemented with 10 % FCS, centrifuged and the pellet washed once with normal growth medium. Finally, the pellet was resuspended in 4 ml of growth medium and transferred to a T-25 flask. After incubating for 24 hours at 37°C, 3 ml of fresh medium was added to the cells. The cells were harvested after 72 hours post transfection and the culture supernate was ass
    11. Transient expression of the cloned gene product was studied by transfection performed with
    12. Transient expression.
    13. Transfection.
    1. Turku, Finland). Data were expressed as mean counts per minute (cpm) ± SE of triplicate cultures.
    2. Single cell suspensions of splenocytes in RPMI-1640 medium were prepared from plasmid DNA immunized mice, on day 45, by mechanical disruption of the spleen. Red blood cells were lysed by exposing the cell pellet to 1 OX concentration of 50 mM PBS and immediately bringing the concentration to IX PBS by addition of water. Cells were diluted to a final concentration of 3 x 106 cells/ml in RPMI-1640 medium supplemented with l 0% FCS. A 100 J.ll aliquot of splenocytes was added to each well of a 96-well microtitration plate containing serial dilutions of refolded recombinant proteins (r-bmZP1, r-dZP3 orr-rG), diluted in the same medium, as a source of antigen. All assays were carried out in . triplicates. Three days after the addition of the cells, culture were pulsed with 1 J.lCi/well of eH] thymidine (NEN, Life Science Products, Boston, MA) for 16 h. Cells were lysed and harvested onto glass fibre filaments for liquid scintillation counting (Betaplate; Wallac,
    3. T CELL PROLIFERATION ASSAY
    1. Thegillandmuscleswereisolatedfromcontrolandeffluentexposed(7%)fishes.Physiologicalsalinesolution(0.75%NaCl)wasusedtorinseandcleanthetissues.Theywerethenimmediatelystudiedandphotographed.
    2. Aftertheperiodofexposure,thecaudalfinwasseveredtogetthebloodforsmearing.BufferedLeishman'sstainofpH6.8gaveexcellentresults.Theworkreportedhereisbasedontheanalysisofslidesoffishestreatedwith7%effluentconcentrationsastheobservedchangesaremaximuminthesefishes.
    3. Bloodandorganstudies
    1. otal RNA was isolated from cell lines after 48 hrs of transfection using trizol (Invitrogen, U.S.A.). 32p labeled antisense HBx mRNA was in vitro transcribed using T7 RNA Polymerase and Riboprobe kit (Promega, U.S.A.), as described earlier. For generating antisense HBx probe, plasmid DNA was linearized with Bam HI and subjected to transcription. Total RNA was quantitated and equal concentration (15-20 pg) was loaded after adding loading dye (50%glycerol, 1 mM EDTA, 0.25% bromophenol blue, 0.25% xylene cyanol FF) on 1% formaldehyde-agarose gel and 1X MOPS was used as the running buffer. The gel was then run at 5 V /em length of the gel. The gel was then treated with 2.5% HCl for 15 min for depurination, 0.4N NaOH for another 15 min and then in 3 M sodium acetate for 15 min, before the transfer was set. Also the nylon membrane prior to transfer was first treated with distilled water for 5 min and then in 0.4 N NaOH for 20 min. The overnight transfer was set up using 20X sse buffer as the transfer buffer at room temperature. Thereafter, the membrane was cross-linked by uv and then dipped in 2X sse for 20 min. For pre-hybridization the membrane was soaked in Rapid hybridization buffer (Amersham Biosciences, U.K.) for 2 hr at 65oC in the hybridizing oven. The probe was then added and further incubation for 4 hrs was carried out. Post hybridization the membrane was washed thrice with 6X sse at 37oC on a shaker. The membrane was then dried on a filter paper and wrapped in a saran wrap. The membrane was then analyzed by autoradiography. For ensuring the equal loading, the formaldehyde-agarose gel was also stained with EtBr for 23s and 18s rRNA
    2. Northern Blot Analysis
    1. Cells growing in culture medium were harvested by trypsinization and washed twice with ice cold PBS. Cells were fixed by adding ice cold 70% ethanol and stored at 4°C. Before harvesting cells were washed twice with PBS and re-suspended in adequate amount of PBS containing Propidium Iodide (PI) to a final concentration of 50μg/ml and RNase to a final concentration of 10μg/ml. Thereby the cell suspension was incubated at 37°C for 30 minutes in dark. Analysis was done by running the samples in BD FACS Vantage System according to the standard procedures after calibration of instrument with Calibrite beads
    2. Flow Cytometry
    1. Chromatography is the technique of separation of compounds on the basis of their distribution/ partition between two phases. Thin Layer Chromatography (TLC) is a solid-liquid form of chromatography where the stationary phase is normally polar absorbent and the liquid phase is the mobile phase made up of a single or combination of solvents depending on the solutes to be separated. The sterol isolated by method described in 3.2.C.14, were also run on a Thin Layer Chromatogram. Standard ergosterol dilutions and samples from wild-type and half knock out parasites were spotted on a Silica Gel G plate. The sterols were resolved using a binary solvent [hexane/ ethyl acetate (75/25)]. The sterols were visualized using Mo' s stain (12.5g Ammonium molybdate (VI) tetrahydrate, 5.0g Ammonium cerium (IV) sulphate, 50mL concentrated sulphuric acid, water upto 500mL
    2. hin Layer Chromatography of ergosterol
    3. thoroughly by inverting the tube 4-6 times before keeping at RT for 5 min. 4mL of chilled Buffer P3 was added and mixed immediately and thoroughly by inverting the tube 4-6 times. A cartridge was capped and the entire contents were poured into it and allowed to settle for 10 min at RT. In the meantime, a Qiagen tip was equilibrated with 20mL of buffer QBT (750mM NaCl; SOmM MOPS, pH 7.0; 15%v /v isopropanol and 0.15% triton X-100). After the 10 min incubation, using a plunger, the contents of the cartridge were transferred into the equilibrated tip and allowed to drain by gravity. The tip was then washed with lOmL of Buffer QC (1M NaCl; SOmM MOPS, pH 7.0 and 15%v /v Isopropanol). The DNA was then eluted using SmL Buffer QF (125mM NaCl; SOmM Tris-Cl, pH 8.5 and 15%v /v Isopropanol) into a corex (glass) tube by gravity flow. 3.5mL of isopropanol was added to the eluted DNA and incubated at RT for 30 min. The DNA was then precipitated at 16000 x g at 4°C for 30 min. The supernatant was discarded and the pellet was washed in 2mL 70% ethanol at 16000 x g at 4°C for 10 min. The supernatant was gently decanted; the pellet was dried to remove any traces of alcohol. Then the DNA was resuspended in ~200]lL of Buffer EB (10mM Tris-Cl, pH 8.5) provided with the kit, or alternatively with nuclease-free water. The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A26o) and using the known formula: DNA concentration = A260 X SOX dilution factor. Purity of DNA was monitored by looking at the A26o/ A2so ratio (should be above 1.6)
    4. Plasmid DNA was isolated in large scale using QIAprep Midiprep kit according to manufacturer's protocol. Briefly, 100mL for a high copy number plasmid and 200mL for a low copy number plasmid was cultured overnight followed by centrifugation at 4629 x g for 15 min at 4°C. The pellet was washed once with PBS and then resuspended well in 4mL Buffer P1 by vortexing. To this, 4mL of Buffer P2 was added an
    5. MidiPrep for large scale isolation of plasmids
    1. colonies come up after 48 hr. A freshly grown overnight culture of the TetR strain was washed once with an equal volume of citrate buffer and resuspended at 10- or 100-fold dilution in the same buffer. 0.1 ml aliquots were then spread on Maloy plates. Colonies were obtained at a frequency of ~ 4 x 10−5/plated cell. The colonies from the selection plate were purified on medium of the same composition and then scored for the Tets phenotype
    2. The method described by Maloy and Nunn (Maloy and Nunn, 1981) was followed for obtaining spontaneous TetS mutants of a TetR strain. Freshly grown cells of the TetR strain of O.D 0.7-0.8 was washed once with an equal volume of citrate buffer and resuspended at 10- or 100-fold dilution in the same buffer. 0.1 ml aliquots were then spread on Maloy agar plates and TetS colonies, which came up after 48 hr of incubation at 37 ̊C with a frequency of 5-8 big colonies/106-107 cells plated were purified on the same medium. This is not a clean selection since in a background lawn of slow growing TetR colonies, few faster growing TetS
    3. Selection for Tets colonies on medium described by Maloy et al
    1. Vacuolar morphology of C. glabratacells was examinedby staining vacuoleswith FM4-64 (Molecular Probes, Invitrogen). FM4-64 is a lipophilic dye that exhibits long wavelength red fluorescence when boundto lipids. FM4-64 binds to the plasma membrane and follows the endocytic pathway to reach the vacuole(Vida and Emr, 1995).Log-phase,YPDmedium-grown cells were harvested and washed with 1X PBS. 1 ODcells were resuspendedin 50 μl YPDmedium containing 30 μM FM4-64 andincubated at 30 ̊C for 30-45 min. After incubation, cells were washed thricewith YPD mediumand resuspendedin 100 μl of the samemedium. Cells were observed under confocal laser scanning microscope(Zeiss LSM 510 Meta)with 63X objective lens,2.5X final zoom, pinhole set at 108 μm and emission filterset to LP 565nmto capture fluorescence image.Along with the fluorescenceimage, aphase contrastimage was alsocaptured for each sample
    2. Staining of yeast vacuoleswith FM4-64
    1. QIAGEN QIAquick PCR purification kit containing buffers, spin columns and collection tubes wasused topurify DNA fragments from PCR andenzymatic digestion reactions as per the kit manufacturer’s instructions
    2. Purification of restriction enzyme-digestedand PCR amplifiedproducts
    3. E. coli BW23473 electro-competent cell aliquots were taken out from -70ºC freezer, thawed on ice and were mixed with 1-2 lplasmid DNA. Mixture was pulsed with the Gene Pulser® electroporation apparatus (Bio-Rad),set at 1800 Volts, 25 μF and 200 Ω,in a chilled 0.1 cm electroporation cuvette. After electric pulse, 1 ml LB medium was immediately added to the cuvette and suspension was transferred to a 1.5 ml sterile microcentrifuge tube. Cells were incubatedat 37°C and 200 rpm for 1 h, centrifuged and were plated on LB-agar plates containing kanamycin (30 μg/ml). Transformants were colony purifiedon LB-kanamycin plates. Positive clones were verified by colony PCR and inoculated in LB-liquid medium containing kanamycin (30 μg/ml) for plasmid isolation
    4. Transformation of E. coliBW23473 cells by electroporation
    1. The reaction was carried out by incubating at 37⁰C for 30 min. The reaction was stopped by adding 2μl of 0.5M EDTA, pH 8.0 and keeping on ice. A spin column was prepared using 1ml syringe and packed with sterile Sephadex G50 slurry and reaction mixture is applied on the top. The eluate is collected in different microcentrifuge tubes and radioactivity was counted using Geiger counter. The tube showing 7 to 9X106was used for experiment. The column containing the unincorporated [γ-32P] ATP was discarded in radioactive waste bin. The radiolabelled oligonucleotides were annealed with their corresponding complementary unlabelled oligonucleotides. A 50 fold molar excess of the latter was used for annealing for conversion of labelled single strand to double strand. Thetubes were kept in boiling waterbath for 3 min followed by room temperature for 30 min. The tubes were transferred to ice and the oligonucleotides were diluted to 4fmoles/μl using sterile H2O
    2. The oligonucleotides were labelled at their 5'end with 32P using T4 polynucleotide kinase (T4 PNK) enzyme in a reaction given belo
    3. end labelling of the oligonucleotides
    1. and stained with ‘Live & Dead’ cell assay reagent (5 μM ethidium homodimer, 5 μM calcein-AM) for 30 min at room temperature. Red (as dead) and green (as live) cells were analyzed under a fluorescence microscope (Labophot-2, Nikon, Tokyo, Japan).For flow cytometry, cells were transiently transfected with either empty vector or various constructs. After 12 h, cells were treated with a combination of CHX (cycloheximide, 25 μg/ml) and TNF (5 nM) for 24 h. Cells were washed, trypsinised and then subjectedto flow cytometry (FACS Aria, BD Biosciences) using Live-Dead Cytotoxicity assay kit (Invitrogen). Live versus dead cells were analysed using FlowJo software
    2. Cytotoxicity assay: The drug-induced cytotoxicity was measured by the 3-(4,5-Dimethylthiazolyl-2)-2,3-diphenyltetrazoliumbromide (MTT) assay as essentially described by Mosmann et al., 1983. Briefly, 5x104cells/well were seeded in 96-well plate. After 12 h, cells were treated in triplicates with different agents for different concentrations and time (in a final volume of 100μl). After completion of treatment, 25 μl of MTT solution (5 mg/ml in PBS) was then added and incubated for 2 h. The cytotoxicity was evaluated by uptake and cleavage of yellow MTT dye to purple formazan crystals by dehydrogenase activity in mitochondria of the living cells. Thereafter, 100 μl of extraction buffer (20% SDS in 50% dimethlylformamide) was added. After an overnight incubation at 37ºC, the absorbance at 570 nm was measured using 96-well multiscanner autoreader(Bio-Rad) with the extraction buffer as blank. Absorbance values were normalized to untreated cells and represented in percent cell viability for different concentrations or treatments.Determination of nuclear fragmentation: The morphology of live and dead cells was observed by staining the nucleus with DNA intercalating dye, propidium iodide (PI).Briefly, cells were treated with several apoptotic inducers for different concentration or time. Thereafter, cells werewashed with PBSand fixed in ice-cold 80% methanol for overnight at 4°C. Following day,cells were washed and suspended in 100 μl of PI solution (0.1% Triton, 0.2 mg/ml RNase A and 50 μg/ml PI in PBS) for 30 min in dark. Cells were then mounted on slides and viewed under fluorescence microscope (in 560 nm filter) to determine morphology of intact or fragmentednucleus.Live &dead assay: The cytotoxicity of various drugs was determined using the commercially available Live/Dead assay kit (Molecular Probes, Eugene, OR). Live cells have intact membraneand active cellular metabolism,which allow Calcien-AM to permeate inside and get cleaved into green fluorescent compound, Calcein (ex/em ~495 nm/~515 nm) due to intrinsic cellular esterase activity. On the other hand, Ethidium homodimer-1 (EthD-1) enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (ex/em ~495 nm/~635 nm). Hence, the cell viability can be assayed by either flow cytometry or fluorescence microscopy.For imaging, cells with different drugs treatments were washed with PBS
    3. Assays for Apoptosis
    1. Yeast were grown in appropriate medium while the logarithmicphase and 1 OD600cells wereharvested and chilled on an ice(Elion and Warner, 1986). Allsubsequent steps,unless specified, werecarried out at4°C or on ice. Cells were collected by centrifugation at 2500 gfor 6min and washed with2 mLof TMN (Section 2.1.6.4). Cells were suspended in 1 mL of ice cold permeabilization buffer (Section 2.1.6.4) and incubated for 15 min.Cells were pelleted and incubated with 100μLof transcription assay buffer (Section 2.1.6.4) containingradiolabelled [α-32P]UTP. After incubation for 10 min at30°C and 300 rpm in a shaking drybath (Eppendorf),1mLof cold TMN containing1 mM nonradioactive UTP was added, the cells were collected by centrifugation, and RNA was prepared by the hot phenol methodas described in Section 2.2.8. Equal counts of labelled RNA were used for hybridization. The membrane was pre incubated with 50 mL of hybridization buffer for 20-30 min, and hybridization was performed with labelled RNA at 65°C for 15 h in 20 mL of hybridization buffer
    2. Transcription run on analysis
    1. immunoprecipitation by using substrate specific antibody or pull-down by affinity trapping the substrate tag. The IP/pull-down complexes wereanalyzed by detecting the ubiquitination of substrate protein by using either substrate specificantibody or ubiquitin antibody through western blotting
    2. HeLa cells were transfected with various combinations of plasmids. At 24 h post-transfection, cells were treated with MG132 (10μM) for 6 h and the whole-cell extracts were prepared by NETN lysis. The cell lysatewas subjected to
    3. In vivoubiquitylation assay
    1. 2 bed volumes of methanol, and equilibrated with 5 bed volumes of distilled water. In order to reduce the water solubility of siderophore in the supernatant, it was acidified to pH 2 using concentarted HCl. This acidified supernatant was passsed through the column, and finally eluted with 160 ml methanol by collecting approximately 60 fractions (2 ml each) of the flow through. Siderophore assay was done on CAS plate with each collected fraction. Fraction that gave orangish-yellow halo for the siderophore on CAS plate, was combined together, dried in rotary evaporator and finally reconstituted in 1 ml methanol for further quantification using HPLC as described previously (Amin et al., 2009).For HPLC analysis, siderophore samples were filtered through filter membrane (porosity, 0.45 μ). Next, 10 μl sample was injected into Agilent C18 (4.6mm×250mm×5μm) column (gradient:(A=H2O/0.1%TFA), (B= CH3CN/0.1%TFA) 0-30% B in 10 min, 30-45% B in 15 min,45-0%B in 20 min at a flow rate of 1 ml/min). Similarly, standard vibrioferrin (siderophore produced by Xanthomonas) was also estimated through HPLC for comparison. Fe(III) bound vibrioferrin complex was prepared by incubating FeCl3.6H2O and apo-vibrioferrin for overnight. This complex was detected at300 nm (RT 10.998 min), whereas apo-vibrioferrin was detected at 220 nm at RT 10.988 min. The siderophore concentration in the samples were determined by peak area and calculated against the standard curves obtained from standard vibrioferrin. The siderophore from the test samples were detected at 300 nm, which confirms that majority of the vibrioferrin isolated from the culture was present in bound form
    2. Different Xanthomonas oryzaepv. oryzicola strains were grown overnight in PS medium at 28 °C and 200 rpm. 0.2% of the overnight grown culture was inoculated in the the fresh PS medium supplemented with 50 μM 2, 2’-dipyridyl, and grown till OD600 reached to 1. Cultures were centrifuged at 12,000 g for 50 min to get the cell free culture supernantant, which was collected into acid treated bottles. Excess exoplysaccharide was removed by centrifugation for longer time. Siderophore was initially isolated by column chromatography as described previously (Wright, 2010). Briefly, 220 g of XAD-16 resin was soaked overnight and packed into the column (2.4×30 cm), column was wa
    3. HPLC based siderophore estimation from culture supernatant of different strains of X. oryzaepv. oryzicola
    1. Cells were seeded in six well plates. After attaining optimal growth, cell lysates were prepared by scraping cells in 1x Laemmli buffer and samples were processed by standard western blot techniques. To detect FAKactivation samples were processed as described in Section 2.2.13. Briefly, 2 x106cells (6 x105cells per time point) were held in suspensionin complete medium containing 1% methylcellulosefor 90 min (Susp), andreplatedon fibronectin (2 μg/mL) coated surfacesfor 20 min (+FN)or for 4 h (SA -stably adherent). Cells at each time point were lysed in 1x Laemmli buffer and subjected to immunoblotting. Membranes were probed with specific antibodies(Table 2.3)and detected using the ECL detection system (GE Healthcare) as described in Section 2.2.10
    2. Immunoblot analysis to detect pFAK
  15. Apr 2019
    1. April 15, the day when you pay your taxes, gives you a good index of how democracy is functioning. If democracy were functioning effectively, April 15 would be a day of celebration. That’s a day on which we get together to contribute to implementing the policies that we’ve decided on. That’s what April 15 ought to be. Here it’s a day of mourning. This alien force is coming to steal your hard-earned money from you. That indicates an extreme contempt for democracy. And it’s natural that a business-run society and doctrinal system should try to inculcate that belief.
  16. Dec 2018
    1. prayed unto the Lord, yea, even with all his heart

      All contents of his heart poured out to the Lord in prayer. Returning that which was inherited from Mythos in the First Place. On the first page of the BOM we have the illustration of a Full Imagining cycle and a witness to the fruit resulting from the recursion. (see 1 Nephi 1:15 or the paragraph at the bottom of this page)

  17. Nov 2018
    1. DISOLUCIÓN ANTICIPADADE LAS CÁMARAS

      Es un mecanismo jurídico que representa la contrapartida de la moción de censura: la posibilidad de disolver anticipadamente el parlamento y convocar nuevas elecciones. Para ello, el Presidente del Gobierno podrá proponer, bajo su exclusiva responsabilidad, la disolución del Congreso, del Senado o de las Cortes generales. Dicha disolución será decretada por el Rey y en ella se fijará la fecha de las elecciones. El protagonista de la disolución es el Presidente del Gobierno, no pudiéndose negar el Rey a firmar el decreto. El texto constitucional establece que no podrá presentarse propuesta de disolución anticipada del parlamento cuando esté en trámite una moción de censura. Disuelto el parlamento, desaparece el mandato parlamentario de sus miembros, salvo el de los que formen parte de las Diputaciones permanentes de las cámaras. La disolución anticipada de las cámaras aparece en la Constitución Española en el artículo 115, en el que muestra lo siguiente: El Presidente del Gobierno, previa deliberación del Consejo de Ministros, y bajo su exclusiva responsabilidad, podrá proponer la disolución del Congreso, del Senado o de las Cortes Generales, que será decretada por el Rey. El decreto de disolución fijará la fecha de las elecciones. La propuesta de disolución no podrá presentarse cuando esté en trámite una moción de censura. No procederá nueva disolución antes de que transcurra un año desde la anterior, salvo lo dispuesto en el artículo 99, apartado 5.

  18. Sep 2018
    1. We should, probably, in time aspire to have foreign relations of our own, to have our own army and navy, and to seek for that complete emancipation which with communities as with individuals, maturity prompts. But independence in a state must always be relative, and none of us can expect to live to see the day when the British dominions in this part of the world will be peopled to such an extent, and become so powerful, that they can afford to be independent of England. We must, from the necessities of our geographical position—so long as the United States continue to be as powerful as they are ; and even if they were divided into two or three portions—we must always find in them a source of danger which must force upon us a dependence on England.

      §§.15, 91(7), and 132 of the Constitution Act, 1867.

    1. Speaking of the Conference at Quebec, he stated that “the delegates unanimously resolved that the United Provinces of British North America shall be placed at the earliest moment in a thorough state of defence.” Hon. gentlemen, I was not aware that the Imperial Government had ever cast off the burden of the defence of this province.

      §.91(15) of the Constitution Act, 1867.

  19. Aug 2018
    1. when the time comes in the history of any colony that it has overcome the burdens and embarrassments of early settlement, and has entered on a career of permanent progress and prosperity, it is only fair and right that it should contribute its quota to the defence of the Empire.

      §.15 of the Constitution Act, 1867.

  20. Jul 2018
    1. 11

      Step 15:

      Secure piece assembled in the previous step using the 4 102509 with a Flat-Head screwdriver. Consult the graph for proper alignment.

      Step 16:

      Insert the 8 101345 studs into the pre-drilled holes.

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    Annotators

  21. Jun 2018
  22. Mar 2018
    1. The incorporation of private or local companies, except such as related to matters assigned to the General Parliament, would be reserved to the local Governments, being matters of a local character. Even the present law permitted the incorporation of companies under a very simple system, which would probably be continued.

      §.91(15) of the Constitution Act, 1867.

    2. It was desirable the General Government should have the control of the medium through which the trade and commerce of the country was carried on, and that in the establishment of banks, the issue of paper money and in offering to the public the paper representative of their labor, in whatever part of the country, there should be the same legislative security for the people

      §§.91(2)(14)(15)(16) of the Constitution Act, 1867.

    3. The control of the Militia was certainly a subject which they must all feel ought to be in the hands of one central power. If them was one thing more than another which required to be directed by one mind, governed by one influence and one policy, it was that which concerned the defence of the country.

      §§.15 and 91(7) of the Constitution Act, 1867.

  23. Jun 2017
  24. Mar 2017
  25. Feb 2017
  26. Dec 2016
  27. Sep 2016