text:
Machine Gun Kelly's Ex-Girlfriend Crashes Party Where He Performed
Machine Gun Kelly's ex-girlfriend Sommer Ray reportedly crashed a party this week that he was playing at and sat across the dance floor from his fiancée, Megan Fox.
According to Page Six, the couple seemingly didn't interact with Ray, who spent the evening dancing with friends.
The two dated in 2020 before his relationship with Fox was public. In March of last year, the social media star claimed that her relationship with the Texas native overlapped with his romance with Megan Fox.
Machine Gun Kelly's ex-girlfriend Sommer Ray reportedly crashed a party this week that he was playing at.
She sat across the dance floor from his fiancée, Megan Fox
Taylor Swift Invites Fans To 'Meet Me At Midnight' For New Music Video
Niall Horan Announces New Single
Quickies: Lewis Capaldi, Coldplay, Doja Cat, Sam Smith


If you are looking for a HVAC Company, Air Conditioning Repair Service or Heating Repair Service in Hamilton Branch, you are in the right place. The FindLocal-HVAC Directory displays Hamilton Branch, CA HVAC, Air Conditioning & Heating Repair Services. We also display HVAC, Air Conditioning Repair Services & Heating Repair Services throughout the United States.
Our goal is to provide a great experience for consumers looking for HVAC, Air Conditioning Repair Services or Heating Repair Services in Hamilton Branch, CA. We keep our directory free of clutter and unnecessary links and ads. Consumers can quickly locate Hamilton Branch HVAC, Air Conditioning Repair Services or Heating Repair Services using the FindLocal-HVAC Directory.
Please use our zip code search function at the top of the page to search for HVAC Companies, Air Conditioning Repair Services or Heating Repair Services in your area if Hamilton Branch is not the city where you are trying to find a Air Conditioning or Heating Repair Company.


Well that is a hole. Serger me, surgery….have to try again tomorrow. Whoops. #firstattempt #handmade #yardsoffabric #epicfail #dustyourselfoff #tryagain @darinrios you got those tutorials? Need help with the neckline.


Want to drive more business and expand value to your customers, particularly as they explore options for migrating data workloads to the cloud? We invite you—as a valued SentryOne partner—to join the SentryOne team for an in-depth workshop on the SentryOne data performance management platform.
Data drives every business, and your company's ability to proactively manage data performance is critical to business success. In this session, SentryOne CEO Bob Potter provides a brief overview of SentryOne strategic initiatives that will dramatically boost your company's ability to achieve peak database performance on the Microsoft Data Platform.
SentryOne CTO Greg Gonzalez provides a look at forthcoming technology that will help data professionals anticipate resource needs and proactively manage data workloads to ensure continuous delivery of business-critical information. He'll review the SentryOne roadmap, giving a glimpse of products on the horizon, and he'll show how organizations are getting optimized database performance with extreme scalability now. He'll also present the SentryOne strategy for offering end-to-end database lifecycle solutions with the recent Pragmatic Works Software acquisition.
SentryOne offers an array of SQL Server database performance monitoring tools that empower the Microsoft data professional to achieve breakthrough performance. But how can you unlock the SentryOne magic that helps you become more proactive about managing performance in your data platform?
Jason Hall provides a pragmatic tour of the tools available from SentryOne, and how you can use them to go from reactive to proactive as fast as possible. Whether you manage 5 SQL Server instances, or 500, you will learn how to quickly get a handle on—and ahead of—what's happening day to day. You'll learn best practices for proactive issue detection, preventative configuration, automated remediation, task automation, alerting and response, automated maintenance, query tuning, and indexing strategy using SentryOne tools and features.
Fast databases are built with speed in mind from the start, with components that allow developers and DevOps teams to deliver high-performing database applications. In this session, John Welch demonstrates how developers can turbo-charge productivity with optimized SSIS components and tasks that eliminate the need for programming, as well as automated testing tools that streamline testing of data-centric applications. He'll also cover documentation and metadata management tools that ensure organizations can confidently address compliance to GDPR and other regulations.
Andy and Kevin will lead a panel discussion with SentryOne customers who are planning cloud migrations and evaluating different approaches. We'll cover challenges inherent in any cloud migration, and new platform choices such as Amazon RDS and Azure SQL DB Managed Instance, which enables companies to retain many of the on-premises features of SQL Server along with Azure cloud implementation benefits. Come learn from your peers as they map their unique paths to the cloud.
If you're a current or prospective SentryOne partner, don't miss this session, where you'll learn from established SentryOne resellers and managed service providers (MSPs) how to expand the value proposition of your businesses by offering services grounded in SentryOne solutions. The panel discussion will also cover how to use SentryOne co-marketing opportunities to grow your business and keep current customers informed and engaged.
Troubleshooting performance problems with SQL Server Analysis Services (SSAS) can be a frustrating exercise. In this session, Steve Wright will discuss effective troubleshooting methodology and offer time-saving tips using BI Sentry to identify and resolve SSAS bottlenecks for both MDX and Tabular modes.
Where is everything and what do we need to move?
What will be the impact of making changes?
How do I know my system is processing data correctly?
How do I know that it is all working as planned?
These and many other questions normally mean a long and drawn-out process of discovery, planning, testing, and post-migration hand-holding. But what if there was a way to help automate and speed up these processes? SentryOne has many solutions that can help provide fast and comprehensive answers to these questions and more. Together we will walk through how we can de-risk and ensure successful cloud migrations with SentryOne solutions, providing insight at every stage of the process for both new and migrated systems.
Watch the SentryOne query masters as they untangle your worst-performing queries. You'll come away with guidelines you can use immediately for troubleshooting common query-writing mistakes and a list of red-flag query operators to watch for that can bog down databases.
Informal networking with the SentryOne team and fellow attendees.


text:
Blessed Charles of Austria
Charles of Habsburg
Carlo d'Austria
Karl I von Österreich
Karl IV von Österreich
1 April on some calendars
Son of Archduke Otto and Princess Maria Josephine of Saxony; great-nephew of Emperor Francis Joseph I. A stigmatic nun prophesied that he would be the victim of attacks and great suffering. A group of people were specifically assigned to pray for him at all times; after his death this group formed the League of Prayer of the Emperor Charles for the Peace of the Peoples (Gebetsliga Kaiser Karl für den Völkerfrieden), which became an ecclesiastically recognized prayer group in 1963. He received a strong Catholic education, and developed a strong devotion to the Holy Eucharist and the Sacred Heart of Jesus. Married Princess Zita of Bourbon and Parma on 21 October 1911. They had eight children over the next ten years.
With the assassination of Archduke Francis Ferdinand on 28 June 1914, the trigger for World War I, Charles became heir presumptive to the throne of the Austro-Hungarian Empire. On the death of Emperor Francis Joseph on 21 November 1916, Charles became Emperor of Austria; crowned apostolic king of Hungary on 30 December 1916. He saw his crown as a way to implement Christian charity and social reform. He worked for peace, for an end to the war, and was the only leader to support Pope Benedict XV's peace effort. After the war, Charles was exiled to Switzerland in March 1919. Trying to prevent the rise of Communism in Central Europe, he tried twice in 1921 to return to power, but since he refused to be the cause of civil war, he finally gave up. Since he considered his office a mandate from God, he never abdicated his throne or title, but he was exiled to the island of Madeira, Portugal and spent his remaining days in prayerful poverty. His widow, princess Zita, dressed in black and lived in mourning her remaining 67 years.
17 August 1887 in Persenbeug Castle, Melk, Lower Austria
1 April 1922 at Funchal, Madeira, Portugal of pneumonia
Venerated
12 April 2003 by Pope John Paul II (decree of heroic virtues)
Beatified
3 October 2004 by Pope John Paul II
his beatification miracle involved the cure of metastatic breast cancer in a Baptist women from Kissimmee, Florida
Catholic Saints Guy
Emperor Karl League of Prayer
Encyclopedia Britannica – 1922 edition
First World War: The Abdication
Hagiography Circle
Maccabee Society
Olivia Rao: Emperor Honored in Nation's Capital: Coordinator Meets Pope
Regina Magazine
Wikimedia Commmons
Austria Forum: After the Wedding
webseiten auf deutsch
Austria Forum
Directorio Franciscano
I strive always in all things to understand as clearly as possible and follow the will of God, and this in the most perfect way. – Blessed Charles' life motto
"Blessed Charles of Austria". CatholicSaints.Info. 14 April 2019. Web. 18 July 2019. <>

redpajama_set_name: RedPajamaCommonCrawl


BonTon Style | virtual runway: Oh Baby!
Every woman wants to look her best throughout her pregnancy. From those queasy first months, to the fabulous second trimester and on through the can-I-get-any-bigger last few weeks. We're here to help, with an amazing selection of flattering styles from Three Seasons Maternity. Many tops feature an adjustable tie at the back to take you through your pregnancy comfortably. And maternity pants are becoming more and more hip and comfortable, too, all the time. When I was expecting, I tried to shop sensibly by choosing a few pairs of versatile, comfortable pants, and then multiplying my options with different tops and accessories. You see, simply buying extra-large sizes of your old favorites won't cut it. Invest in some key quality maternity pieces so that you look, and FEEL, fabulous from now 'til your baby arrives.
Deal of the Day: Steak Knives!
Ryan Kwanten Appearance at Brookfield Square!
Keurig is coming town… Chi-Town!


A unique opportunity to join a successful advice charity at an exciting time. North Somerset Citizens Advice Bureau, part of the national Citizens Advice network is looking for an inspirational manager to lead them through the next stage of development. The successful candidate will be responsible for all aspects of strategic leadership, development, external relations and internal management and be accountable to the Trustee Board.
The ideal candidate will be a passionate and committed leader with excellent interpersonal skills to lead 23 paid staff and 67 volunteers as well as representing the Bureau authoritatively at all levels. The candidate will ideally have knowledge of advice issues, charity law and governance. The successful candidate will have the ability to inspire the confidence of funders and civic leaders as well as experience of financial management and generating income in the not-for-profit sector.


what are the disadvantages of impact crusher - Crusher . advantages of mobile crushers In a horizontal shaft impact crusher, material pours into a hopper with a rotating shaft.
advantage disadvantage cone crusher impact crusher 55 Views. The Zenith is the professional mining equipments manufacturer in the world, … jaw crusher disadvantages.
2012-03-18· Spring cone crusher (and jaw crusher compare the main advantages 🙂 1, crushing cavity depth big, work successive, high productive capacity, the unit consumption is low. It with the same to ore mouth width than jaw crusher, production capacity to than the latter twice as tall as above, and the consumption of every tons of ore than jaw type is 1.2 times; low 0.5.
Advantage And Disadvantage Of Jaw And Impact Crushers. the jaw crusher is used for primary crushers and impact crusher this series of cone crusher is used for ... advantages of impact crushers - advantages of impact crushers.
Mobile Cone Crusher . advantages of mining iron ore in the ph disadvantages of iron ore mining, Clinker Grinding Mill. disadvantages of . is specialized in the design, manufacture and supply of crushing equipment used in mining industry.
Hydraulic cone crusher technical advantages,cone crusher … The crusher avoids the disadvantages of impact crusher like low average crushing ratio, large energy consumption, fast abrasion of hammer, impact plate and lining board.


text:
Research ArticleArticle
Absolute Quantification and Differential Expression of Drug Transporters, Cytochrome P450 Enzymes, and UDP-Glucuronosyltransferases in Cultured Primary Human Hepatocytes
Olaf Schaefer, Sumio Ohtsuki, Hirotaka Kawakami, Tae Inoue, Stephanie Liehner, Asami Saito, Atsushi Sakamoto, Naoki Ishiguro, Takehisa Matsumaru, Tetsuya Terasaki and Thomas Ebner
Olaf Schaefer
Sumio Ohtsuki
Hirotaka Kawakami
Tae Inoue
Stephanie Liehner
Asami Saito
Atsushi Sakamoto
Naoki Ishiguro
Takehisa Matsumaru
Tetsuya Terasaki
The levels of metabolizing enzymes and transporters expressed in hepatocytes are decisive factors for hepatobiliary disposition of most drugs. Induction via nuclear receptor activation can significantly alter those levels, with the coregulation of multiple enzymes and transporters occurring to different extents. Here, we report the use of a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for concurrent quantification of multiple cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and transporter proteins in cultured primary human hepatocytes. The effects of culture format (i.e., sandwich culture versus conventional culture) and of dexamethasone (DEX) media concentrations on mRNA, protein, and activity levels were determined for three donors, and protein expression was compared with that in liver. In general, P450 and UGT expression was lower in hepatocyte cultures than that in liver, and CYP2C9 was found to be the most abundant P450 isoform expressed in cultured hepatocytes. The sandwich culture format and 0.1 μM DEX in media retained the protein expression in the hepatocytes closest to the levels found in liver. However, higher in vitro expression was observed for drug transporters, especially for multidrug resistance protein 1 and breast cancer resistance protein. Direct protein quantification was applied successfully to study in vitro induction in sandwich cultured primary hepatocytes in a 24-well format using the prototypical inducers rifampicin, omeprazole, and phenobarbital. We conclude that targeted absolute LC-MS/MS quantification of drug-metabolizing enzymes and transporters can broaden the scope and significantly increase the impact of in vitro drug metabolism studies, such as induction, as an important supplement or future alternative to mRNA and activity data.
The assessment of potential drug-drug interactions by in vitro methods has become an indispensable part of the preclinical development process of new drug candidates. In addition to investigations on reversible or irreversible inhibition of drug-metabolizing enzymes, studies on the effects of enzyme induction and on the interaction with drug transporters have to be performed and are considered as mandatory by current regulatory guidelines and draft guidance documents [U.S. Food and Drug Administration, Draft Guidance for Industry: Drug Interaction Studies-Study Design, Data Analysis, and Implications for Dosing and Labeling (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm072101.pdf); European Medicine Agency, Draft Guideline on the Investigation of Drug Interactions (http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2010/05/WC500090112.pdf]. Primary human hepatocytes are the recommended and broadly used experimental system for these types of studies (Hewitt et al., 2007; Sahi et al., 2010; Swift et al., 2010). The use of hepatocytes provides, among others, the advantage of including active drug transport mechanisms as well as effects of drug metabolites that are formed during the experimental incubation period by phase I and II metabolism.
Cultured hepatocytes offer the possibility to assess long-term effects, such as enzyme induction (i.e., changes of the expression levels of molecular entities of the hepatocytes). Common culture formats used for in vitro enzyme induction studies comprise conventional and sandwich cultures, the former referring to plated hepatocytes maintained on rigid substratum (e.g., collagen), whereas in the latter cells are "sandwiched" by an additional layer of either collagen or a composite of extracellular matrix proteins, such as Matrigel (LeCluyse, 2001). Experimental protocols for in vitro enzyme induction studies vary substantially between laboratories across the industry, and no standard criteria for induction assays are applied currently (Sinz et al., 2008). Assay conditions, such as culture format and duration, media supplements etc., were reported previously to alter the phenotype and induction response of cultured hepatocytes (LeCluyse, 2001). Therefore, selection of the appropriate culture format and conditions for a specific in vitro study is crucial with regard to the in vivo predictability of the data generated. Although the quantification of enzymatic activities using suitable probe substrates has become a standard procedure to measure the changes in expression levels and activities of cytochrome P450 (P450) enzymes, other approaches are feasible but are not necessarily performed on a routine basis. The assessment of changes in expression at the mRNA or protein level is such an approach. Quantification of mRNA by reverse transcription polymerase chain reaction (RT-PCR) methodologies is relatively straightforward and often conducted to supplement data on enzyme activity. However, changes in mRNA levels may not necessarily reflect protein expression, especially for drug transporters (Ohtsuki et al., 2012).
Measuring changes of protein amounts is performed far less frequently (Sinz et al., 2008; Chu et al., 2009) and not required by current regulatory guidelines. This is because common experimental procedures are relatively tedious, require the availability of specific antibodies, and typically lack the necessary level of accuracy (e.g., compared with enzymatic activity assays). Yet, quantitative data on protein levels would be highly valuable for several reasons: first, to confirm the suitability of the experimental system for the respective study by comparing relevant in vitro protein expression (P450s, drug transporters, etc.) with in vivo data; second, because results for enzyme activity from in vitro induction studies can be confounded by concomitant enzymatic inhibition by the test compound, especially irreversible inhibition, therefore leading to false negative results. Such effects have been described for troleandomycin, mifepristone, verapamil, and several HIV protease inhibitors, specifically ritonavir (Hsu et al., 1998; Luo et al., 2002; McGinnity et al., 2006; Kenny et al., 2008). Even if enzyme inhibition by the test compound may be recognized beforehand during separate in vitro studies, effects of inhibitory metabolites formed by the hepatocytes can occur and may remain unrecognized (Ogilvie et al., 2006). However, using mRNA as the sole parameter to measure enzyme induction is prone to misinterpretation, most often by overestimating the induction potential. To avoid such problems, routine quantification of expression levels for a panel of important proteins during in vitro enzyme induction studies would be highly desirable. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS)-based techniques recently have been developed successfully for absolute quantification of membrane-bound enzymes and transporter proteins (Kamiie et al., 2008; Kawakami et al., 2011).
The purpose of the present study is to demonstrate the suitability of LC-MS/MS-based absolute protein quantification for sensitive and specific determination of molecular entities in primary human hepatocyte cultures that are involved in hepatic drug metabolism. This includes the effects from culture and induction not only for drug-metabolizing enzymes but also for drug transporters, which have been recognized as a decisive factor for the understanding of hepatobiliary drug disposition (Shitara et al., 2006; International Transporter Consortium et al., 2010). To our knowledge, this is the first report comparing enzyme activity and mRNA levels with absolute quantitative data on protein concentration in primary hepatocyte cultures measured by a targeted LC-MS/MS approach.
Amodiaquine, dexamethasone (DEX), diclofenac, dimethyl sulfoxide, extracellular matrix (ECM) gel from Engelbreth-Holm-Swarm murine sarcoma, omeprazole (OME), penicillin-streptomycin (100×), phenobarbital (PB), rifampicin (RIF), testosterone, trypan blue solution, and Williams' medium E (WME) were purchased from Sigma-Aldrich (St. Louis, MO). Bupropion was from Promochem (Wesel, Germany), S-mephenytoin was from Enzo Life Sciences GmbH (Lörrach, Germany), and phenacetin was from Acros Organics (Fairlawn, NJ). Rat tail collagen I was from Roche Diagnostics (Mannheim, Germany), fetal calf serum, phosphate-buffered saline, and stabilized l-glutamine (GlutaMAX, 100×) was from Invitrogen (Carlsbad, CA), and insulin-transferrin-selenous acid and bovine serum albumin premix (100×) was from BD Biosciences (San Jose, CA). Dulbecco's modified Eagle's medium (10×) was purchased from Biozol (Eching, Germany).
Hepatocyte Culture.
Fresh human hepatocytes from Caucasian donors were provided by the Human Tissue and Cell Research Foundation/Hepacult GmbH (Regensburg, Germany). Cells were prepared from surplus liver tissue obtained from tumor resections using the two-step collagenase perfusion technique as described previously (LeCluyse et al., 2005) and shipped as a suspension. Donor demographics, a list of latest medications, and informed consent were provided. WME supplemented with 6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 ng/ml selenous acid, 5.35 μg/ml linoleic acid, 1.25 mg/ml bovine serum albumin, 0.01 μM DEX, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine was used for human hepatocyte culture. Upon arrival, hepatocytes were sedimented for 5 min at 72g and 4°C. Cell viability was assessed by trypan blue exclusion. Hepatocyte suspensions were adjusted to 750,000 viable cells per milliliter in WME containing 6% fetal calf serum, and cells were plated on BD BioCoat collagen I-coated plates (BD Biosciences). Seeding density was 375,000 cells per well of a 24-well plate for enzyme activity and RNA expression analysis and 10 million (mio) cells per 100-mm dish for microsomal and plasma membrane preparations, respectively. After an incubation period of 4 h at 37°C, 5% CO2, and 95% humidity, nonattached cells were removed, and fresh WME without serum was supplied. One hundred-millimeter dishes were used to prepare microsomal and plasma membrane fractions for absolute protein quantification of P450s, UGT enzymes, and transporters by LC-MS/MS, whereas 24-well plates were used for P450 in situ enzyme activity assessment and subsequent mRNA expression analysis for P450s, UGTs, and drug transporters. Cultures were assessed daily by light microscopy to ensure morphological normalcy (i.e., polygonal shape and distinct bile canaliculi) as well as confluence adequate for experiments (Supplemental Table 1).
Sandwich Culture Using ECM Gel.
Twenty-four hours after plating, medium was removed, and hepatocytes were overlaid with 0.5 ml per 24-well or 9 ml per 100-mm dish, respectively, of ice-cold WME containing 0.25 mg/ml ECM gel and incubated for 24 h at 37°C, 5% CO2, and 95% humidity to obtain the sandwich cultures. For the high DEX cultures, medium containing 0.1 μM DEX was used starting from 24 h after plating.
Sandwich Culture Using Collagen I.
Lyophilized rat tail collagen I was dissolved overnight in 0.2% acetic acid to give a 0.83 mg/ml stock solution. A total of 0.55 ml of 10× Dulbecco's modified Eagle's medium was added to 4.95 ml of collagen I stock solution on ice, and the pH was adjusted to 7.2 to 7.4 with 1 N NaOH, resulting in a final collagen I solution of 0.75 mg/ml. Medium was removed 24 h after initial plating, and 75 μl of ice-cold collagen I solution was added per well in a 24-well plate or 1.5 ml per 100-mm dish, respectively. Culture plates were incubated for 45 min at 37°C, 5% CO2, and 95% humidity for collagen gelling before fresh WME was supplied.
Single Gel Monolayer Culture.
Hepatocytes were maintained in culture after initial seeding without any matrix overlay. All of the cultures were kept in total for 4 days in 24-well format before in situ enzyme activity assessment and RNA isolation and for 5 days in 100-mm format, respectively, before cell harvest for protein quantification. Fresh medium was supplied daily. Maintenance medium for all of the cultures was identical, containing 0.01 μM DEX, except for the high DEX condition where 0.1 μM DEX was used.
The research protocols for the present study were approved by the Ethics Committee of the Graduate School of Pharmaceutical Sciences of Tohoku University. The study has been carried out in accordance with the Declaration of Helsinki.
In Situ P450 Enzyme Activity Assessment.
P450 enzyme activities were determined in situ by monitoring metabolite formation in culture medium over time (0–45 or 0–90 min) with LC-MS/MS after incubation with specific probe substrates. Validated enzyme activity assays comprised separate probe reactions performed in 24-well format. Probe substrates and assay details are provided as supplemental information (Supplemental Table 3). The following equipment was used for LC-MS/MS analysis of incubation samples: HTS PAL autosampler (CTC Analytics, Zwingen, Switzerland), 1200 series binary pump and degasser (Agilent Technologies, Santa Clara, CA), column switch thermostat Spark 880 (May Lab, Vienna, Austria), and API 4000 QTRAP (Applied Biosystems/MDS Sciex, Foster City, CA). Chromatographic separation of incubation samples was performed on YMC-Pack Pro C18 (50 × 2.1 mm, 5 μm; YMC Europe, Dinslaken, Germany) and XTerra MS C18 (50 × 2.1 mm, 5 μm; Waters, Milford, MA) analytical columns. Twenty-five microliters of the incubation sample were diluted with 480 μl of water containing 5 to 10% acetonitrile, 0.1% formic acid, and the respective stable isotope-labeled metabolite as an internal standard. Five microliters was injected into the LC-MS/MS system operated with an atmospheric pressure electrospray ionization source. Details on analytical assays are provided in Supplemental Table 3.
Microsomal and Plasma Membrane Preparation.
Hepatocytes were scraped from culture dishes using a rubber policeman, washed with phosphate-buffered saline, sedimented at 800g for 10 min at 4°C, and snap-frozen in liquid nitrogen. After being thawed, cells were lysed by two-step nitrogen cavitation at 450 and 800 psi each for 15 min at 4°C using a pressure vessel (Parr, Moline, IL). The homogenates were centrifuged at 10,800g for 20 min at 4°C, and the supernatants were collected and ultracentrifuged at 100,000g for 60 min at 4°C. The microsomal pellet was suspended in buffer A [0.1 M KCl-phosphate buffer (pH 7.4) containing protease inhibitor cocktail] and ultracentrifuged at 100,000g for 60 min at 4°C. The resulting pellet was suspended buffer B (20 mM Tris-HCl buffer containing 0.25 M sucrose and 5.4 mM EDTA), and an aliquot of the suspension was stored as a microsomal fraction at −80°C. An enriched plasma membrane fraction was prepared from the microsomal fraction for the quantification of lower abundance transporter proteins. Therefore, the remaining volume of the microsomal fraction was layered on top of a 38% (w/v) sucrose solution and centrifuged at 100,000g for 30 min at 4°C. The turbid layer at the interface was recovered, suspended in buffer B, and centrifuged at 100,000g for 30 min at 4°C. The plasma membrane fraction, containing both canalicular and sinusoidal membranes, was obtained from the resulting pellet by suspension in buffer B. Levels of Na/K-ATPase and γ-glutamyl transpeptidase (γ-GTP), plasma membrane marker proteins used for data normalization, typically were increased in plasma membrane factions compared with those in microsomal fractions (Table 1). Protein concentrations were measured by the Lowry method using the detergent compatible protein assay reagent (Bio-Rad Laboratories, Hercules, CA).
Absolute protein levels and enzyme activities in human hepatocytes from three donors kept in sandwich culture (ECM overlay, 0.01 μM DEX)
P450 and UGT proteins were determined from microsomal fractions, and transporter proteins were determined from plasma membrane preparations. Concentrations are calculated as the total amount of microsomal and plasma membrane protein, respectively. P450 enzyme activities were determined in situ by measuring phenacetin O-deethylation (CYP1A2), bupropion 2-hydroxylation (CYP2B6), amodiaquine N-deethylation (CYP2C8), diclofenac 4′-hydroxylation (CYP2C9), S-mephenytoin 4′-hydroxylation (CYP2C19), and testosterone-6β hydroxylation (CYP3A4).
TaqMan RT-PCR Gene Expression Analysis.
Total RNA from hepatocytes cultured in 24-well plates was isolated using the RNeasy protocol (QIAGEN GmbH, Hilden, Germany). The yield of extracted RNA was analyzed spectrophotometrically at 260 nm. Purity and integrity of RNA were assessed using RNA 6000 LabChips and 2100 Bioanalyzer (Agilent Technologies). For first-strand cDNA synthesis, 200 ng of total RNA was reverse-transcribed in a final volume of 20 μl following the protocol for the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). cDNA samples were diluted to 60 μl with water and stored at −20°C. One microliter of diluted cDNA was used for quantitative real-time PCR. P450 and UGT analyses were performed on a 7500 Fast Real-Time PCR System (Applied Biosystems), and transporter analyses were performed on an iQ5 Real-Time PCR System (Bio-Rad Laboratories). Assays were performed as technical triplicates. Information on inventoried assays and primer and probe sequences of custom-made assays are provided in Supplemental Tables 4 and 5.
Data Normalization and Analysis.
Gene expression data were normalized to β-actin as an endogenous control quantified in the same sample (duplexed RT-PCR) or as a separate sample on the same assay plate for inventoried assays. Relative expression differences were calculated using the comparative ΔΔCt method, accounting for the actual PCR efficiency for each target and run.
P450, UGT, and Transporter Protein Quantification.
Absolute quantification of P450, UGT, and transporter proteins was conducted by LC-MS/MS measurement of tryptic protein fragments similar to the procedures described previously (Kamiie et al., 2008; Kawakami et al., 2011). In brief, protein samples were diluted in suspension buffer containing 7 M guanidine-HCl, 500 mM Tris-HCl (pH 8.0), and 10 mM EDTA. Samples were reduced by the addition of 1 mM dithiothreitol and alkylated under a nitrogen atmosphere with 10 mM iodoacetamide. The S-carbamoylmethylated proteins were precipitated by the addition of a mixture of methanol and chloroform. Precipitates were dissolved in 6 M urea, diluted with 100 mM Tris-HCl (pH 8.0), and digested with tosyl phenylalanyl chloromethyl ketone-treated trypsin at an enzyme/substrate ratio of 1:100 at 37°C for 16 h. The tryptic digests were spiked with stable isotope-labeled peptides of identical amino acid sequences as the corresponding analyte peptides as internal standards and acidified with formic acid for analysis with the nano-LC system or high-performance liquid chromatography system, which was connected to an electrospray ionization triple-quadrupole mass spectrometer [API 5000 or 4000 QTRAP (for CYP2B6 and 2C19); Applied Biosystems/MDS Sciex]. LC was performed with C18 capillary columns. Linear gradients of 1 to 45% acetonitrile in 0.1% formic acid were applied to elute the peptides at a flow rate of 100 nl/min for nano-LC and 50 μl/min for high-performance liquid chromatography. The mass spectrometer was prepared to run multiple reaction monitoring (MRM) experiments for peptides. Peak integration of ion count chromatograms was performed using Analyst software (Applied Biosystems/MDS Sciex). A specific tryptic fragment peptide was selected for the quantification of each target protein and monitored in four different MRM transitions. The amount of each peptide was determined as an average of three to four MRM transitions from one sample. In the cases where signal peaks were detected for less than three transitions, the amount of peptide was defined as below the limit of quantification. Quantification of CYP3A4 was performed indirectly by the subtraction of the values determined for the peptide specific for CYP3A43 and the one detecting both CYP3A4 and CYP3A43: CYP3A4 = CYP3A4/CYP3A43 − CYP3A43.
Absolute Protein Expression of P450, UGT, and Drug Transporter Proteins in Sandwich Cultured Human Hepatocytes.
The absolute amounts of P450, UGT, and transporter proteins were determined in primary human hepatocytes from three donors cultured for 5 days on 100-mm BD BioCoat collagen I-coated culture dishes, overlaid with a film of ECM protein gel and with 0.01 μM DEX present in culture medium (Fig. 1; Table 1). The expression levels of P450 and UGT enzymes were quantified in the microsomal fractions, and drug transporters were quantified in the plasma membrane fractions containing both sinusoidal and canalicular membranes, respectively. CYP2C9 and UGT2B7 showed the highest protein expression levels of the measured P450 and UGT enzymes (Fig. 1), whereas the levels of CYP2B6 and CYP3A5 were the lowest and even below the limit of quantification (LOQ) for two donors (Table 1). CYP2A6 concentrations varied the most among hepatocyte cultures from three donors (approximately 7.8-fold). Multidrug resistance protein 1 (MDR1) and multidrug resistance associated protein 2 (MRP2) showed the highest expression levels of canalicular transporters, and organic cation transporter 1 (OCT1) showed the highest expression level among the sinusoidal transporters (Fig. 1). The highest variability in expression levels was observed for ABCA8 (7.7-fold), ABCG8 (7.5-fold), and MRP1 (10.7-fold). This was predominantly due to higher concentrations of these transporters found in the plasma membrane fractions from donor HuHep10261, exhibiting up to 10.6-fold higher expression compared with the average of donors HuHep9966 and HuHep10901, whereas a 1.74-fold difference in expression was observed at most between these two donors (data not shown). MRP3 and MRP4 were below the LOQ for both of the latter two donors.
Absolute expression levels of P450, UGT (A), and transporter proteins (B) in sandwich cultured human hepatocytes. Expression levels of P450 and UGT enzymes were determined in microsomal fractions of human hepatocytes from three donors cultured for 5 days under standard conditions (i.e., ECM gel and 0.01 μM DEX). Expression levels of transporter proteins were determined in purified plasma membrane fractions. Data represent mean ± S.D. (n = 2 or 3, excluding data below LOQ). Open squares represent data determined from only one donor. *, amounts of CYP3A4 were determined indirectly as described under Materials and Methods.
Effects of Culture Conditions on P450 Protein Expression, mRNA Levels, and Enzymatic Activity in Human Hepatocytes.
Hepatocytes from the identical three donor preparations described above were cultured in parallel to control for three additional conditions (see Materials and Methods). The effects were assessed on enzyme activity and mRNA levels for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 after 4 days of culture in 24-well plates and on protein expression after 5 days in 100-mm dishes, respectively (Table 2). Enzyme activities were determined in situ from 24-well plates by measuring the metabolite formation in culture supernatants after the addition of specific probe substrates, whereas P450 protein was quantified from the microsomal preparations obtained from 100-mm dishes. Hepatocytes plated on BD BioCoat collagen I-coated culture dishes with ECM overlay and 0.01 μM DEX in culture medium served as a control. Compared with these culture conditions, hepatocytes also were cultured either using the same sandwich format with a 10-fold higher media concentration of DEX (0.1 μM DEX) or with an overlay of gelled collagen I (Collagen I) instead of ECM gel or using a conventional culture on BD BioCoat collagen I-coated culture dishes without any additional overlay (single gel). Mean relative changes (n = 3) in protein levels, enzyme activity, and mRNA expression of the selected P450 isoforms CYP1A2, CYP2B6, and CYP3A4 for the different culture conditions are depicted in Fig. 2. Table 2 summarizes the mean relative changes of protein levels, mRNA, and enzyme activity for the different culture conditions compared with the control condition (0.01 μM DEX). No consistently significant effect on P450 expression or activity was seen (Fig. 2), because donor variability superseded the effects caused by different culture conditions. However, the high DEX condition generally increased P450 expression, particularly CYP2A6, CYP2E1, and CYP3A7, mRNA levels of CYP1A2, CYP2A6, and CYP3A4, and enzymatic activity of CYP3A4. In contrast, the conventional single gel condition exhibited the lowest levels in P450 protein, mRNA, and enzyme activity. Collagen I cultures showed the lowest enzyme activity for CYP2C9 (0.49-fold) but the highest protein levels (1.53-fold, compared with control) for all of the tested culture conditions. Because this appears to be an obvious contradiction, it has to be taken into account that, unlike P450 protein that was measured in microsomal preparations, P450 activities were determined in situ; therefore, additional factors such as active transport and/or properties of matrix overlay may affect metabolite kinetics in the supernatant.
Mean relative change in mRNA expression, protein levels, and in situ enzyme activity in hepatocytes cultured at different conditions compared with control (ECM overlay, 0.01 μM DEX)
The same donor preparations (n = 3) were used for all of the tested conditions. An analysis of mRNA expression and enzyme activity was performed after 4 days in culture and protein after 5 days, respectively. P450 and UGT proteins were determined from microsomal fractions, and transporter proteins were determined from plasma membrane preparations of hepatocyte cultures.
Relative changes in mRNA, enzymatic activity, and protein concentration of CYP1A2 (A), CYP2B6 (B), and CYP3A4 (C) in human hepatocyte cultures in response to culture conditions. The effects of 0.1 μM DEX, collagen I, and single gel conditions were compared relative to that of control (ECM gel sandwich and 0.01 μM DEX). Expression of mRNA was determined via RT-PCR, and in situ enzyme activity was determined by probe substrate reactions after 4 days of culture in 24-well format. P450 protein was quantified in microsomal preparations from the cultures of the same donors on 100-mm plates after 5 days. Data represent mean ± S.D. of three donors. Significance was determined applying a two-tailed, two-sample t test. *, p < 0.05; **, p < 0.01.
Effects of Culture Conditions on UGT Protein Expression and mRNA Levels.
Microsomal preparations from hepatocytes were analyzed for protein expression of UGT1A1 and UGT2B7 by LC-MS/MS (Table 2). Semiquantitative RT-PCR was applied to determine the mRNA expression of UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 relative to that of endogenous control β-actin. Results for relative mean changes under the different culture conditions are summarized in Table 2. Both mRNA and protein levels were within a 2-fold range for all of the conditions tested, showing less sensitivity for UGT enzyme expression toward external conditions compared with P450s. Single gel conditions reduced mRNA expression of UGT1A1 approximately 0.64-fold compared with that of control, and UGT1A1 protein likewise was reduced in the single gel cultures. However, the decrease was not statistically significant. UGT2B7 mRNA expression was the highest under control conditions, whereas protein levels were maximal in single gel culture (1.46-fold, compared with control).
Effects of Culture Conditions on Drug Transporter Protein Expression and mRNA Levels.
Mean expression levels of transporter proteins in plasma membrane fractions of hepatocytes from three donors cultured under the described conditions were within a 0.3- to 3.6-fold range compared with those of the control conditions (Table 2), and the control conditions showed overall the highest protein levels for both ATP-binding cassette (ABC) and solute carrier transporters. Except for MRP1, notable differences in the expression of the transporters among culture conditions were not observed. Protein expression of MRP1 under the collagen I condition was found to be on average 2.29-fold higher compared with that of the control. Mean MRP5 expression was increased by 3.6-fold in single gel culture; however, there was no statistically significant difference in the expression under control conditions. For mRNA, bile salt export pump (BSEP) and OCT1 showed the highest interculture differences in expression with approximately 3-fold higher levels in control condition compared with those in the single gel. MRP2 and OCT1 transcripts were the most abundant among the tested drug transporters; MRP1, breast cancer resistance protein (BCRP), and OATP1B3 mRNA was found to be the least abundant (data not shown).
Comparison of Human P450, UGT, and Transporter Protein Expression between Cultured Hepatocytes and Liver.
The expression of P450, UGT, and transporter proteins in hepatocytes under the tested culture conditions was compared with corresponding protein levels determined in liver tissue (Ohtsuki et al., 2012). Figure 3 shows the correlation between protein expression of P450 and UGT enzymes in human liver (n = 17) and that in hepatocytes from three donors kept over 5 days under four different culture conditions. Corresponding data for transporter protein expression are available in Supplemental Fig. 6.
Comparison of protein expression for P450 and UGT enzymes between human liver tissue and hepatocytes cultured under different conditions. Protein concentrations were determined from microsomal preparations. Data represent mean ± S.D. for human liver tissue (n = 17) and mean ± S.D. for the same human hepatocyte preparations (n = 3) cultured under different conditions. Values below LOQ were set to 0 fmol/μg protein for mean calculation. Dashed lines indicate 5-fold variation. Selected data are shown for clarity reasons.
As stated previously, relatively high interdonor variability in expression levels was observed among the three hepatocyte preparations, most markedly for CYP2C8, CYP2C19, and CYP2E1 (>10-fold) and irrespective of the culture condition applied. Variability of the lower abundance CYP2B6 and CYP3A5 protein in hepatocyte cultures was partly due to analytical sensitivity. Liver tissue samples obtained from 17 donors (donor demographics are given in Supplemental Table 2) showed the largest variability in CYP3A4 expression (>1000-fold), followed by CYP2C19 (approximately 100-fold) and CYP3A5 (>10-fold). Among the drug-metabolizing enzymes quantified, CYP2C9 and UGT2B7 were found to be the most abundant in both cultured hepatocytes and liver tissue. Mean expression of CYP2B6, CYP3A5, and CYP2E1 in cultures was the lowest relative to liver. For the other P450 and UGT proteins analyzed, expression was found to be within a 5-fold range compared with liver expression. Overall, relative effects of culture conditions on P450 expression were similar among the three donors, with 0.1 μM DEX retaining levels closest to liver. However, these effects were superseded altogether by donor variability in P450 expression.
In contrast to the P450 and UGT enzymes, transporter protein expression in cultured hepatocytes was generally higher than that in human liver (Supplemental Fig. 6), and different culture formats and DEX concentrations showed no relevant effects on protein levels. It is noteworthy that MRP3 was quantified in all of the liver samples but could not be detected in cultured hepatocytes, whereas the opposite was true for ABCG8. Mean expression levels of the canalicular transporter BCRP in cultured hepatocytes were >10-fold higher than those in the samples from liver biopsies, likely reflecting extensive bile canalicular network formation under in vitro conditions.
Induction of P450 and Drug Transporters by Prototypical Inducers in Sandwich Cultured Human Hepatocytes.
Fresh human hepatocytes from a separate donor preparation were cultured in 24-well format, applying our laboratory standard condition for in vitro induction studies as described above. Cells were exposed for 48 h to prototypical inducers RIF (at 25 μM), OME (at 50 μM), PB (at 1 mM), and solvent control. In situ enzyme activities and mRNA expression were determined. Cells from a parallel incubation were scraped from the 24 wells to prepare microsomal and plasma membrane fractions for protein quantification. Table 3 summarizes the results for absolute concentrations of P450, UGT, and transporter proteins and in situ P450 activities. Relative induction of CYP1A2, CYP2B6, and CYP3A4 mRNA, enzyme activity, and protein by RIF, OME, and PB is shown in Fig. 4. Marked response to the selective inducers was observed for P450 protein, yet exceeding the response of mRNA and activity for CYP1A2 and CYP3A4.
Absolute protein levels and enzyme activities in sandwich cultures from donor preparation HuHep10536 after 48 h of treatment with solvent control and prototypical inducers OME, PB, and RIF, respectively
Protein concentrations (except for Na/K-ATPase) were normalized to γ-GTP for each incubation condition. P450 enzyme activities were determined in situ by measuring phenacetin O-deethylation (CYP1A2), bupropion 2-hydroxylation (CYP2B6), amodiaquine N-deethylation (CYP2C8), and testosterone-6β hydroxylation (CYP3A4).
Induction of mRNA, enzyme activity, and protein of CYP1A2 (A), CYP2B6 (B), and CYP3A4 (C) in sandwich cultured human hepatocytes after 48 h of treatment with prototypical inducers. Hepatocytes from donor HuHep10536 were cultured in 24-well format under standard conditions and treated for 48 h with solvent control, 25 μM RIF, 50 μM OME, and 1 mM PB, respectively. In situ enzyme activity and mRNA were determined from the same culture plate. P450 protein was quantified in microsomal preparations from a separate 24-well plate per condition and concentration normalized to γ-GTP expression. Data represent mean ± S.D.
Figure 5 shows the effects of prototypical inducers on the relative expression levels of the canalicular transporters MDR1 and BCRP, sinusoidal transporters OATP1B1 and OCT1, and phase II enzyme UGT1A1. Clear induction of MDR1 protein by all three inducers RIF (1.78-fold), OME (2.46-fold), and PB (2.82-fold) was observed. A significant increase in BCRP protein expression was seen after treatment with OME (1.43-fold) and PB (1.37-fold). It is noteworthy that RIF induced the concentration of the sinusoidal transporter OATP1B1 (1.50-fold), whereas OCT1 expression was suppressed significantly (0.74-fold).
Induction of transporter and UGT proteins in sandwich cultured human hepatocytes after 48 h of treatment with prototypical inducers. Hepatocytes of donor HuHep10536 were cultured in 24-well format under standard conditions and treated for 48 h with solvent control, 25 μM RIF, 50 μM OME, and 1 mM PB, respectively. Protein concentrations of UGT1A1 were determined from microsomal preparations, and transporter proteins were quantified from purified plasma membrane preparations. Data were normalized to γ-GTP expression and represent mean ± S.D. Significance was determined by applying a two-tailed, two-sample t test. *, p < 0.05; **, p < 0.01.
LC-MS/MS assays were used in the present study to quantify P450, UGT, and transporter proteins in cultured primary human hepatocytes, and the data were compared with mRNA expression and enzyme activities. In addition, protein concentrations in cultured hepatocytes were compared with those from human liver tissues. We further demonstrated that targeted absolute protein quantification can be applied to standard in vitro induction studies.
MRM analysis of diagnostic peptides with stable isotope-labeled internal standards and reference peptides allows for sensitive and selective absolute quantification of protein concentrations with a linear dynamic range similar to that of small molecules (Elliott et al., 2009; Ohtsuki et al., 2011). Integral membrane proteins yet pose an analytical challenge due to their hydrophobic nature and low abundance, as seen for many drug transporters (Barnidge et al., 2003; Kamiie et al., 2008). Being a relatively new technique, LC-MS/MS-based absolute protein quantification has not yet been introduced widely as a standard method for the assessment of drug-metabolizing enzymes and drug transporter proteins; therefore, data on assay development and validation still are sparse. The validity of the quantification assays used in this study was demonstrated recently (Sakamoto et al., 2011) with regard to the acceptance criteria for robustness and reproducibility of bioanalytical assays that are used commonly in drug development. This underlines the potential and applicability for nonclinical studies on drug metabolism and pharmacokinetics, where validated analytical assays are highly desired—if not mandatory—that meet quality criteria required by regulatory guidance. Besides adding a valuable end point to standard in vitro studies, such as enzyme induction, these methods can be used to quantify transporter protein expression in cellular systems used for studies on drug transport, which may help the interpretation of results obtained from, for example, Caco-2 cultures and recombinant cells.
Primary hepatocytes are the preferred system to study in vitro hepatic metabolism and induction because they express the full complement of regulating nuclear cofactors, metabolic enzymes, and transporters. In vitro culture conditions, such as culture format and duration, media supplements, etc., can affect the phenotype of cultured hepatocytes (LeCluyse 2001; Pascussi et al., 2001). We found that, among the tested culture conditions, expression and activity of P450 proteins were the highest under high DEX and sandwich culture conditions, which is in line with previous reports (Pascussi et al., 2001). The synthetic glucocorticoid DEX is used commonly as a media supplement to compensate, at least to some extent, for reduced expression of P450 and activity that occur over time in primary culture, especially for CYP3A (Madan et al., 2003). We observed that sensitivity toward induction for CYP3A4 by prototypical inducer RIF in human hepatocyte sandwich cultures decreased markedly at near micromolar DEX (0.1–1 μM) media concentrations (unpublished data), reflecting substantially increased basal CYP3A4 activity caused by pregnane X receptor activation of DEX at these concentrations (McCune et al., 2000). Therefore, DEX at 0.01 μM medium concentration is used in our laboratory for induction studies to provide for adequate sensitivity of induction. Basal P450 activities are monitored routinely to ensure suitability of the culture. In contrast to P450, the highest levels of transporter expression were obtained under the lower DEX control condition. The single gel cultures showed lowest levels in mRNA, protein, and activity among the tested conditions, thus confirming reports that hepatocyte dedifferentiation occurs more rapidly in cultures lacking surrounding matrix protein (LeCluyse, 2001).
P450 and UGT protein levels in cultured hepatocytes from the three donors were generally lower compared with the expression in human liver tissues determined for 17 biopsy samples (Ohtsuki et al., 2012). Expression profiles under 0.1 μM DEX and collagen I showed the smallest differences compared with that of liver tissue. However, the impact of culture conditions on P450 expression was superseded by donor variability. Transporter protein expression was similar to that of human liver, yet showing higher expression of canalicular transporters such as BCRP and MDR1. This could possibly be explained by the extensive formation of bile canalicular networks in sandwich culture (LeCluyse et al., 1994), resulting in the larger formation of canalicular membranes, and consequently higher amounts of these canalicular transporters are found in the plasma membrane fractions prepared from hepatocytes than those in liver tissue.
In the course of in vitro studies on enzyme induction, coregulation of multiple enzymes and transporters by a single inducer can occur simultaneously but to a different extent. Inhibition, direct inhibition, or indirect inhibition by a formed metabolite can be a confounding factor when determining induction by enzymatic activity assessment (McGinnity et al., 2006). Because mRNA expression is a direct and very sensitive parameter to measure nuclear receptor-mediated transactivation, it is discussed and even recommended as the preferred end point for a quantitative assessment of the induction potential of drug candidates, because it allows detection of the intrinsic induction potential of a drug candidate independent of concurrent inhibition (Fahmi et al., 2010). Then again, an increase in mRNA and nuclear activation in response to the induction can be significantly greater than increases in protein and activity (LeCluyse, 2001; Almond et al., 2009), indicating a degree of redundancy in the amount of mRNA synthesized, and therefore such effects on mRNA may lack relevance to the changes of drug metabolism and disposition with respect to pharmacokinetics, drug safety, and efficacy. In addition, there is an even more remote correlation between mRNA expression and protein levels for drug transporters (Ohtsuki et al., 2012). LC-MS/MS-based protein quantification would certainly allow for more accurate evaluation of the intrinsic induction potential of drugs, including those that simultaneously inactivate drug-metabolizing enzymes.
The judgment whether a drug candidate may potentially cause enzyme induction in vivo depends on the application of thresholds that are proposed by regulatory guidance [U.S. Food and Drug Administration, Draft Guidance for Industry: Drug Interaction Studies-Study Design, Data Analysis, and Implications for Dosing and Labeling (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm072101.pdf); European Medicine Agency, Draft Guideline on the Investigation of Drug Interactions (http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2010/05/WC500090112.pdf]. Exceeding a predefined cut-off value at pharmacological concentrations compared with a positive control in vitro qualifies a compound as an inducer and usually triggers a clinical drug interaction study. Recently, more quantitative approaches to the assessment of in vitro induction data are discussed, applying mechanistic in silico models instead of empirical threshold values (Almond et al., 2009) to better predict the in vivo induction potential of a drug candidate. These in silico approaches include physiologically based pharmacokinetics and a combination of in vitro and in vivo pharmacokinetic data. For such approaches, however, absolute data on in vitro protein expression of a set of the most relevant drug-metabolizing enzymes and transporters in response to treatment with a drug candidate would certainly be of great benefit. This would be even more the case if such protein concentration data of drug-metabolizing enzymes and drug transporters also were available for the various organs involved in drug metabolism (e.g., kidney and intestine). Therefore, "pharmacoproteomics" (Ohtsuki et al., 2011) is a promising new approach adding to the established methodologies in drug research and development. Here, we demonstrated that LC-MS/MS quantification of major proteins involved in hepatic metabolism disposition can be performed in the course of common induction studies. Considering the data of this study together with the results of the accompanying study on protein expression in human liver tissue (Ohtsuki et al., 2012), valuable information is provided for a future better understanding and quantitative prediction of in vivo induction effects by mechanistic and physiologically based pharmacokinetics modeling.
In summary, we used novel LC-MS/MS assays, in conjunction with common enzyme activity and mRNA techniques, to quantify major drug-metabolizing enzymes and transporters in cultured primary human hepatocytes to elucidate differences in expression profiles in response to various culture conditions and in comparison to human liver. The methods described herein are suitable for absolute quantification of the expression of these molecular entities during in vitro induction studies.
Participated in research design: Schaefer, Ohtsuki, Ishiguro, Matsumaru, Terasaki, and Ebner.
Conducted experiments: Schaefer, Kawakami, Inoue, Liehner, Saito, and Sakamoto.
Performed data analysis: Schaefer, Ohtsuki, Kawakami, Inoue, Liehner, Saito, Sakamoto, and Ishiguro.
Wrote or contributed to the writing of the manuscript: Schaefer, Ohtsuki, Terasaki, and Ebner.
We thank Michelle Werner, Britta Koch, and Ikumi Washio for assistance with hepatocyte culture, Ikue Kitahara for assistance with mRNA analysis, and Takashi Igarashi for collaborative support.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
http://dx.doi.org/10.1124/dmd.111.042275.
ATP-binding cassette protein
breast cancer resistance protein
BSEP
bile salt export pump
concentrative nucleoside transporter
extracellular matrix protein
equilibrative nucleoside transporter
γ-GTP
γ-glutamyl transpeptidase
HuHep
human hepatocyte preparation
LC-MS/MS
liquid chromatography coupled to tandem mass spectrometry
limit of quantification
multidrug and toxin extrusion protein
multidrug resistance protein
multiple reaction monitoring
NTCP
sodium/taurocholate cotransporting polypeptide
organic cation transporter
reverse transcription-polymerase chain reaction
UDP-glucuronosyltransferase
Williams' medium E
cycle threshold.
Received August 8, 2011.
Accepted October 5, 2011.
Almond LM,
Yang J,
Jamei M,
Tucker GT,
Rostami-Hodjegan A
(2009) Towards a quantitative framework for the prediction of DDIs arising from cytochrome P450 induction. Curr Drug Metab 10:420–432.
Barnidge DR,
Dratz EA,
Martin T,
Bonilla LE,
Moran LB,
Lindall A
(2003) Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards. Anal Chem 75:445–451.
Chu V,
Einolf HJ,
Evers R,
Kumar G,
Moore D,
Ripp S,
Silva J,
Sinha V,
Sinz M,
Skerjanec A
(2009) In vitro and in vivo induction of cytochrome P450: a survey of the current practices and recommendations: a pharmaceutical research and manufacturers of america perspective. Drug Metab Dispos 37:1339–1354.
Elliott MH,
Smith DS,
Parker CE,
Borchers C
(2009) Current trends in quantitative proteomics. J Mass Spectrom 44:1637–1660.
Fahmi OA,
Kish M,
Boldt S,
(2010) Cytochrome P450 3A4 mRNA is a more reliable marker than CYP3A4 activity for detecting pregnane X receptor-activated induction of drug-metabolizing enzymes. Drug Metab Dispos 38:1605–1611.
Hewitt NJ,
Lecluyse EL,
Ferguson SS
(2007) Induction of hepatic cytochrome P450 enzymes: methods, mechanisms, recommendations, and in vitro-in vivo correlations. Xenobiotica 37:1196–1224.
Hsu A,
Granneman GR,
Bertz RJ
(1998) Ritonavir. Clinical pharmacokinetics and interactions with other anti-HIV agents. Clin Pharmacokinet 35:275–291.
Huang SM,
Tweedie DJ,
Benet LZ,
Brouwer KL,
Chu X,
Dahlin A,
Fischer V,
International Transporter Consortium, Giacomini KM, Huang SM, Tweedie DJ, Benet LZ, Brouwer KL, Chu X, Dahlin A, Evers R, Fischer V, et al. (2010) Membrane transporters in drug development. Nat Rev Drug Discov 9:215–236.
Kamiie J,
Ohtsuki S,
Iwase R,
Ohmine K,
Katsukura Y,
Yanai K,
Sekine Y,
Uchida Y,
Ito S,
Terasaki T
(2008) Quantitative atlas of membrane transporter proteins: development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria. Pharm Res 25:1469–1483.
Kawakami H,
Suzuki T,
(2011) Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with in silico target peptide selection. J Pharm Sci 100:341–352.
Kenny JR,
Chen L,
McGinnity DF,
Shakesheff KM,
Thomson B,
Riley R
(2008) Efficient assessment of the utility of immortalized Fa2N-4 cells for cytochrome P450 (CYP) induction studies using multiplex quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and substrate cassette methodologies. Xenobiotica 38:1500–1517.
LeCluyse EL
(2001) Human hepatocyte culture systems for the in vitro evaluation of cytochrome P450 expression and regulation. Eur J Pharm Sci 13:343–368.
Alexandre E,
Hamilton GA,
Viollon-Abadie C,
Coon DJ,
Jolley S,
Richert L
(2005) Isolation and culture of primary human hepatocytes. Methods Mol Biol 290:207–229.
Audus KL,
Hochman JH
(1994) Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration. Am J Physiol 266:C1764–C1774.
Luo G,
Cunningham M,
Kim S,
Burn T,
Lin J,
Hamilton G,
Rizzo C,
Gilbert D,
. (2002) CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes. Drug Metab Dispos 30:795–804.
Madan A,
Graham RA,
Carroll KM,
Mudra DR,
Burton LA,
Krueger LA,
Downey AD,
Czerwinski M,
Forster J,
Ribadeneira MD,
. (2003) Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes. Drug Metab Dispos 31:421–431.
McCune JS,
Hawke RL,
Gillenwater HH,
Ritchie J,
Lindley C
(2000) In vivo and in vitro induction of human cytochrome P4503A4 by dexamethasone. Clin Pharmacol Ther 68:356–366.
Berry AJ,
(2006) Evaluation of time-dependent cytochrome P450 inhibition using cultured human hepatocytes. Drug Metab Dispos 34:1291–1300.
Ogilvie BW,
Zhang D,
Li W,
Rodrigues AD,
Gipson AE,
Holsapple J,
Toren P,
Parkinson A
(2006) Glucuronidation converts gemfibrozil to a potent, metabolism-dependent inhibitor of CYP2C8: implications for drug-drug interactions. Drug Metab Dispos 34:191–197.
Kubo Y,
(2011) Quantitative targeted absolute proteomics-based ADME research as a new path to drug discovery and development: methodology, advantages, strategy, and prospects. J Pharm Sci 100:3547–3559.
Schaefer O,
Inoue T,
Liehner S,
Saito A,
Ishiguro N,
Kishimoto W,
Ludwig-Schwellinger E,
Ebner T,
. (2012) Simultaneous absolute protein quantification of transporters, cytochrome P450s and UDP-glucuronosyltransferases as a novel approach for the characterization of individual human liver: comparison with mRNA levels and activities. Drug Metab Dispos 40:000–000.
Pascussi JM,
Drocourt L,
Gerbal-Chaloin S,
Fabre JM,
Maurel P,
Vilarem MJ
(2001) Dual effect of dexamethasone on CYP3A4 gene expression in human hepatocytes. Sequential role of glucocorticoid receptor and pregnane X receptor. Eur J Biochem 268:6346–6358.
Sahi J,
Grepper S,
Smith C
(2010) Hepatocytes as a tool in drug metabolism, transport and safety evaluations in drug discovery. Curr Drug Discov Technol 7:188–198.
Sakamoto A,
Matsumaru T,
(2011) Reliability and robustness of simultaneous absolute quantification of drug transporters, cytochrome P450 enzymes, and Udp-glucuronosyltransferases in human liver tissue by multiplexed MRM/selected reaction monitoring mode tandem mass spectrometry with nano-liquid chromatography. J Pharm Sci 100:4037–4043.
Wallace G,
Sahi J
(2008) Current industrial practices in assessing CYP450 enzyme induction: preclinical and clinical. AAPS J 10:391–400.
(2006) Transporters as a determinant of drug clearance and tissue distribution. Eur J Pharm Sci 27:425–446.
Swift B,
Pfeifer ND,
(2010) Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity. Drug Metab Rev 42:446–471.
You are going to email the following Absolute Quantification and Differential Expression of Drug Transporters, Cytochrome P450 Enzymes, and UDP-Glucuronosyltransferases in Cultured Primary Human Hepatocytes
ABSOLUTE PROTEIN EXPRESSION IN CULTURED HUMAN HEPATOCYTES
Quantitative Proteomics of Clinically Relevant Drug-Metabolizing Enzymes and Drug Transporters and Their Inter-correlations in the Human Small Intestine
Quantitative Atlas of BAB Transporters in Pig
Carbonyl reductase 1 plays a significant role in converting doxorubicin to cardiotoxic doxorubicinol in mouse liver, but the majority of the doxorubicinol-forming activity remains unidentified


Introducing Mauro as a wild card investor was done specifically to intrigue the reader. Investors can certainly guess or plan and succeed a few times, even in a row, but is it sustainable?
This is the foundation for how the future can be predicted with enough data and intelligent algorithm. However if you only think about how online shopping sites can track what you bought and predict your next desire opens us up to buying without thought. Do you do that? Let us know your thoughts after you read or listen to this story.
Wolfgang then explained, "There is a bright star named Mauro, who came from the small town of San Rafael Mendoza, and leads an oil futures trading desk. He has a small team of analysts in Buenos Aires, and one of them is his wife, Luisa. Together they have bet successfully on oil price movements for the last six months. Last month this team started doing the same thing with currency arbitrage bets, only the time horizons are greater than anything I've ever seen.
Otto nodded and agreed, "Yes, I remember the case. He was found shot in the back with a rifle. As I recall, the authorities ruled it suicide, though I never heard how that was justified with him shot in the back.


I was in Seattle when I was looking at my nanny camera and realized there was water leaking from the third floor of my home all the way down to the first floor. I was in a panic but SERVPRO of Lexington-Bedford responded to my call so fast.
There was extensive damage to my home but they took care of it as if it were their own.
We were told that due to the storm we had mold under our home that was affecting our crawlspace. SERVPRO remediated it with no problem and I definitely recommended them to other people I knew who were facing losses.
Mr. John and the crew was great! They serviced our crawlspace that was affected with the river water rising from Hurricane Matthew.
Due to Hurricane Matthew much of our home was flooded but SERVPRO had no problem servicing us by doing demolition and drying out our home. I recommend them to everyone who is facing a disaster!
After we had ripped out the flooring ourselves, SERVPRO came in and dried out the home for us just 3 days. I was happy they were able to service us.
Our commercial business was badly affected by mold due to Hurricane Matthew and we were lucky to have SERVPRO remediate our office as quickly as they could taking.
I was really happy to have SERVPRO of Lexington-Bedford help me out with my flooded home when the local SERVPRO's were overwhelmed.


text:
Welcome to Our New 6th Grade Teacher!
Back to School! - Principal's Message
Ware Middle School is Filling Buckets
Ware Middle School Open House 9/14/16
Ware Middle School Staff Dress Down for Charity.
New classroom laptop computers have arrived at Ware Middle School!
Ware Middle School Leadership Council Members Have Been Selected!
Grade 6 Starts Music Lessons thanks to Gerry's Music Shop in South Hadley
The Ware Middle School Leadership Council has begun the annual non-perishable food drive!
First Student Reviews Bus Safety with Students!
Drama Club is up and running at Ware Middle School!
WMS Chorus Sings for Students, Faculty, and Office Staff
Mrs. Shipman and Her Book Club Members just completed Terror at Bottle Creek by Watt Key.
Changes to Academic Excellence Award at WMS
Grade Six Students in Mr. Brach's and Mr. Coll 's Classes Make Pinatas!
WMS Competing in BANANAGRAMS Challenge!
Patriot's Pen Youth Essay Competition
Congratulations Tony He!! Ware Middle School's 'Best of the Bunch'!
New Books at WMS!
Ware Middle School Drama Club presents Charlotte's Web
WMS Book Club raises awareness for Autism
Dance Theater Ensemble visits WMS
Year Two of Band at WMS!
Can Food Drive
Christmas Cards for Jacob
WMS Stuffed the Bus!
CAN't Do It Without You!
WMS Participates in Anti-Bullying Essay Contest
LEAP Program participates in Special Olympics
Art Club Beautifies the Walls of WMS
Trapping Leprechaun's in LEAP
Ware Middle School MCAS Spring 2019 Schedule
Spring Showcase
Grades 4 & 6 Planting Marigolds and Receiving Earth Day Education
Play Summer Reading BINGO to Help Keep Your Kids Reading!
STEM Learning Labs in 2020
Title I: Literacy Around the World Night
WMS Documents
WMS PTO
Substance Use Prevention & Support
Nurse Newsletter
Help with Head Lice
Go Math Portal
Ware Middle School » News » What's New » Pie Face
Beginning with our study of integers and rational numbers, students in Mr. Brach and Ms. Rowden's homeroom were given the challenge of studying the digits of Pi, one of the irrational numbers we talked about in class. Over the course of 4 weeks, students were to study, independently, and memorize as many digits of Pi as they could. On Pi day (March 14), students were tested individually in class to see how far they had gotten. As a reward for their hard work, the top 4 winners between the two classes were given the chance to pie a teacher in the face. The students really stepped up to the challenge and pushed themselves to learn as many digits as they could. On the day of the challenge, each student was given the chance to recite the digits twice. The top student was able to recite 160 digits of Pi, quite impressive.
239 West Street, Ware, MA 01082
It is the policy of the Ware Public Schools not to discriminate on the basis of age, sex, race, color, religion, national origin, ethnicity, disability, sexual orientation, gender identity, homelessness, military service, union activity or genetics in its educational programs, activities or employment policies as required by Title IX of the 1972 Education Amendments, Chapter 622 of the Massachusetts General Laws, Section 504 of the Rehabilitation Act of 1973, Title VI of the Civil Rights Act of 1964, the Equal Educational Opportunities Act of 1974, Title II of the Americans with Disabilities Act of 1990, M.G.L. c. 71A and c. 76, s. 5.


text:
<?xml version="1.0" encoding="UTF-8" standalone="no"?>
<PolicySet xmlns="urn:oasis:names:tc:xacml:3.0:core:schema:wd-17" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" PolicySetId="root"
   PolicyCombiningAlgId="urn:oasis:names:tc:xacml:3.0:policy-combining-algorithm:deny-overrides" Version="1.0">
   <Description>
      Purpose: Test detection of duplicate PolicyId across PolicySets
   </Description>
   <Target />
   <PolicySet PolicySetId="PS0" PolicyCombiningAlgId="urn:oasis:names:tc:xacml:3.0:policy-combining-algorithm:deny-overrides" Version="1.0">
      <Description>
         Purpose: Test detection of duplicate PolicyId within PolicySet
      </Description>
      <Target />
      <PolicySet PolicySetId="PS1" Version="1.0" PolicyCombiningAlgId="urn:oasis:names:tc:xacml:3.0:policy-combining-algorithm:deny-overrides">
         <Target />
      </PolicySet>
      <PolicySet PolicySetId="PS2" Version="1.0" PolicyCombiningAlgId="urn:oasis:names:tc:xacml:3.0:policy-combining-algorithm:deny-overrides">
         <Target />
      </PolicySet>
      <PolicySet PolicySetId="PS3" Version="1.0" PolicyCombiningAlgId="urn:oasis:names:tc:xacml:3.0:policy-combining-algorithm:deny-overrides">
         <Target />
      </PolicySet>
   </PolicySet>
   <PolicySet PolicySetId="PS4" PolicyCombiningAlgId="urn:oasis:names:tc:xacml:3.0:policy-combining-algorithm:deny-overrides" Version="1.0">
      <Description>
         Purpose: Test detection of duplicate PolicyId within PolicySet
      </Description>
      <Target />
      <PolicySet PolicySetId="PS2" Version="1.0" PolicyCombiningAlgId="urn:oasis:names:tc:xacml:3.0:policy-combining-algorithm:deny-overrides">
         <Target />
      </PolicySet>
      <PolicySet PolicySetId="PS5" Version="1.0" PolicyCombiningAlgId="urn:oasis:names:tc:xacml:3.0:policy-combining-algorithm:deny-overrides">
         <Target />
      </PolicySet>
   </PolicySet>
</PolicySet>

redpajama_set_name: RedPajamaGithub


text:
Joseph-Golven Tuault de La Bouvrie (, Ploërmel - , Ploërmel) es un político francés, diputado a los Estados Generales de 1789, diputado de Morbihan bajo el Primer Imperio y la Restauración .

Biografía
Hijo del maestro François-Marie Tuault de La Bouvrie, consejero del rey, senescal de la ciudad y policía de Ploërmel, estudió con los jesuitas de Vannes, fue admitido como abogado en el parlamento en 1766, y se convirtió en senescal de Ploërmel. Varias veces diputado de los Estados de Bretaña, se negó a permitir que se pidieran cartas de nobleza para él, preparó la redacción de la lista de agravios del tercero, y fue elegido, el 17 de abril de 1789, diputado del tercer estado del senescal de Ploërmel en los Estados Generales. Votó con la mayoría, desaprobó la destitución de Necker, leyó una protesta del municipio de Ploërmel contra la conducta del parlamento de Rennes, y propuso admitir el Día de la Federación del 14 de julio de 1790 las antiguas milicias de las provincias.

Afligido por la marcha de los acontecimientos, se alejó de las funciones públicas después de la sesión, fue arrestado como sospechoso bajo el Terror, escapó una noche de la prisión de las Ursulinas donde había sido encerrado. levantado, se escondió en el campo durante unos meses, luego, cansado de esta existencia, y procedente de la emigración, volvió a constituirse prisionero. "Llamamos a Tuault ante el comité, dice René Kerviler. - ¿Qué destino esperas de nosotros? ellos le dicen - ¡Libertad! respondió con orgullo. ¿Así que tienes un depósito? - Abre la ventana, respondió el preso, y pregunta al primer transeúnte si quiere ser fiador del ciudadano Tuault. Pasaba por la plaza un labrador de La Noe Verte, llamado Sebillot. Fue desafiado y aceptó esta peligrosa responsabilidad sin dudarlo. Tuault quedó libre y sus conciudadanos lo nombraron inmediatamente juez del tribunal de distrito y comandante de su caseta de vigilancia. »

Unos días más tarde, Prieur (de la Marne), que estaba depurando la administración de Morbihan, dio órdenes de arrestarlo; Tuault fue liberado solo después de más de cinco meses de detención. Fue nuevamente arrestado por cuarta vez ante el 9 Thermidor; estaba a punto de ser enviado ante el tribunal revolucionario de París, cuando Robespierre fue derrocado. Sus dos sobrinos, de Landivy-Tredion, habiendo sido tomados y fusilados en Quiberon, Tuault fue nuevamente comprometido, arrestado por quinta vez (Ventôse año IV), liberado después de un mes de prisión en Vannes, y puesto bajo vigilancia en esta ciudad.

El gobierno consular (Consulado (historia de Francia)) lo nombró consejero del distrito y, el 4 de Vendémiaire del año XIII, Tuault fue elegido, por el Senado conservador, diputado por Morbihan al Cuerpo legislativo (Consulado y Premier Empire (Cuerpo legislativo)]]; su mandato fue renovado el 3 de octubre de 1808; se convirtió, el mismo año, juez adjunto de paz de Ploërmel, luego caballero de la Légion d'honneur (6 de noviembre de 1810), y presidente del cantón.

Se sentó en el Cuerpo Legislativo hasta 1814 y fue reelegido bajo la Restauración (Restauración (historia francesa)) a la Cámara de Diputados (Cámara de Diputados (Restauración)). Fue nombrado caballero por Luis XVIII el 25 de noviembre de 1814, ascendido a Oficial de la Legión de Honor el 26 de enero de 1815 y nombrado al año siguiente , consejero general de Morbihan y presidente de la corte de Ploërmel a los 72 años. Miembro de varias sociedades literarias, publicó traducciones de obras y cuentos ingleses en verso.

Murió en el cargo. El epitafio que había compuesto para sí mismo estaba grabado en su lápida: "Al pasar, no lo pisotees, el que no ha pisoteado a nadie". »

Es el padrastro de François-Mathieu-Marie Dahirel.

Bibliografía
 Joseph-Golven Tuault de La Bouvrie, dans Adolphe Robert et Gaston Cougny, Dictionnaire des parlementaires français, Edgar Bourloton, 1889-1891.

Enlaces y referencias externas
 Ficha en el sitio web de la asamblea national francesa

Notas y Referencias

Véase también

 Revolución francesa

Personas de la Revolución francesa
Diputados de Francia del tercer estado en 1789-1791
Abogados de Francia

redpajama_set_name: RedPajamaWikipedia


The seven-time All-Star last played for the Mets in May 2016 due to a series of neck and back injuries.
The 35-year old began his latest comeback in August at Class A St. Lucie, where he hit.171 with a double and two RBIs in 41 at-bats during 10 games for St. Lucie and two with Triple-A Las Vegas, according to ESPN. He garnered MVP votes in six of those 13 seasons, finishing as high as fourth in the voting back in 2007.
To be clear, it does not sound as though Wright will formally announce his retirement. Joel Sherman of the New York Post tweets that, indeed, Wright will not formally retire but will not return as a player during his contract.
Wright has salaries of $20 million this year, $15 million next season and $12 million in 2020, the final year of his deal. He earned $20 million this season, a portion of which the Mets have recouped via insurance while he's been on the disabled list. As is typical for the Mets there has been confusion as to what the actual plan was, with Wright strongly suggesting he was ready to play while the Mets seemed inclined to hold him back.


Patapaa ft Sista Afia – There Is Nothing Music Mp3.
Patapaa released new song Ft Sista Afia titled 'There Is Nothing' Download Free MP3 By Patapaa Ft Sista Afia – There Is Nothing (Prod By Willis Beatz) Lyrics There is nothing oo i mean nothing i raised my head see what in the world i sight my eye see what in the sky there is nothing oo i see nothing oo i love you baby come back to me i need you baby you made me cry there is nothing i mean nothing ahhh.
Download "There Is Nothing " By Patapaa ft Sista Afia Below.


text:
https://version-11-0.string-db.org/cgi/network.pl?networkId=Q449OIA7fv4k
prmC
Release factor glutamine methyltransferase; Methylates the class 1 translation termination release factors RF1/PrfA and RF2/PrfB on the glutamine residue of the universally conserved GGQ motif (293 aa)
tsaD
tRNA N6-adenosine threonylcarbamoyltransferase; Required for the formation of a threonylcarbamoyl group on adenosine at position 37 (t(6)A37) in tRNAs that read codons beginning with adenine. Is involved in the transfer of the threonylcarbamoyl moiety of threonylcarbamoyl-AMP (TC-AMP) to the N6 group of A37, together with TsaE and TsaB. TsaD likely plays a direct catalytic role in this reaction; Belongs to the KAE1 / TsaD family (349 aa)
16S rRNA (cytosine(967)-C(5))-methyltransferase; Specifically methylates the cytosine at position 967 (m5C967) of 16S rRNA (451 aa)
NAD kinase; Involved in the regulation of the intracellular balance of NAD and NADP, and is a key enzyme in the biosynthesis of NADP. Catalyzes specifically the phosphorylation on 2'-hydroxyl of the adenosine moiety of NAD to yield NADP (306 aa)
Signal recognition particle protein; Involved in targeting and insertion of nascent membrane proteins into the cytoplasmic membrane. Binds to the hydrophobic signal sequence of the ribosome-nascent chain (RNC) as it emerges from the ribosomes. The SRP-RNC complex is then targeted to the cytoplasmic membrane where it interacts with the SRP receptor FtsY; Belongs to the GTP-binding SRP family. SRP54 subfamily (496 aa)
16S rRNA (cytosine(967)-C(5))-methyltransferase; Specifically methylates the cytosine at position 967 (m5C967) of 16S rRNA
ABB56992.1 ffh Synpcc7942_0962 Synpcc7942_1771
Signal recognition particle protein; Involved in targeting and insertion of nascent membrane proteins into the cytoplasmic membrane. Binds to the hydrophobic signal sequence of the ribosome-nascent chain (RNC) as it emerges from the ribosomes. The SRP-RNC complex is then targeted to the cytoplasmic membrane where it interacts with the SRP receptor FtsY; Belongs to the GTP-binding SRP family. SRP54 subfamily
ABB56992.1 prmC Synpcc7942_0962 Synpcc7942_1863
Release factor glutamine methyltransferase; Methylates the class 1 translation termination release factors RF1/PrfA and RF2/PrfB on the glutamine residue of the universally conserved GGQ motif
NAD kinase; Involved in the regulation of the intracellular balance of NAD and NADP, and is a key enzyme in the biosynthesis of NADP. Catalyzes specifically the phosphorylation on 2'-hydroxyl of the adenosine moiety of NAD to yield NADP


While more divorces settle outside of court and are able to avoid litigation, the fact is that some must still go through the court system. Part of this process involves a deposition, which both parties are entitled to engage in. What is a deposition? It occurs when a party is under oath, as one would be during any other testimony given in a court room. He or she will be asked a range of questions related to the divorce case. The answer will be documented by a court reporter and the session might even be videotaped. Depositions are crucial to divorce cases because the answers provided can be used against either party later on during the case. If the same question is posed again and that party answers it differently, the deposition could be used to discredit him or her.
Depositions are used to gather background, evidence, and to lock in the stories of both spouses and any available witnesses. Obtaining these facts and admissions can help prepare a divorce attorney for trial.
When this critical tool is properly handled, a divorce deposition can be incredibly effective in assisting an attorney in negotiating a divorce agreement, including issues related to child custody or property division. However, even if an agreement cannot be reached, depositions are still incredibly powerful, allowing an attorney to obtain discovery into financial matters regarding the other spouse, for example, which can potentially change the outcome of a case.
Meet with your attorney before the deposition to help him or her prepare. This is something that your attorney will likely take the initiative to set up and it might not even be necessary for this to be an in-person meeting. Simply go over the deposition together and, if necessary, provide any additional information.
Be present at the deposition unless it will not be helpful or result in unnecessary distractions.
While present, make sure you focus on the process rather than on your spouse. Remember, this is not about revenge, but rather about gather information and collecting the facts that are needed to move the case forward.
Family law matters are rarely easy and divorce can be particularly emotional and overwhelming. At Goldblatt, Marquette & Rashba, P.C., our team skillfully handles all types of family law matters and applies more than 60 years of shared professional experience to every case. There is no need for you to endure this difficult time alone, so do not hesitate to reach out to us.
Contact our firm at (203) 687-4050 to speak with a Hamden divorce attorney today.


This "IS" the page you want to check first!
We try to help our customers however we can and developed our Special Consignments page because of it. It also save's a lot of unnecessary paperwork!
Stock # : N/A Price : $ 7,250 GVWR : 23,900 lb.


Originating in the 1800's, Diamine Ink has a long history and has been developed into one of the finest quality fountain pen inks on the market. It's reliability has lead it to become one of the most sought after inks by both professionals and beginners. This Diamine Majestic Blue Ink is suitable for any brand of fountain pens, making it a convenient purchase too. It is a water-based non-toxic ink, making it a safe option for vintage pens in particular. The majestic blue colour of this ink is not overly saturated and thus has a beautiful vibrancy to it. It is also versatile in the sense that it has the ability to be easily mixed with other colour inks and create new colours. This Diamine ink also has anti-clogging properties, ensuring even and smooth precision across paper.
The red sheen this ink produces is wonderful, and the colour is a super saturated blue. I really enjoy using this ink because of the incredible sheen, but unfortunately no matter which paper I use, if I run my hand over it – even a day later it smudges.
The "Majestic Blue" is a deep blue 'standard issue' ink. The shading depth is decent and has beautiful tints of red sheen when dry.
The ink has a medium-long dry time , however tends to dry out reasonably quickly on up-capped pens.
It is a quality, classic blue ink that is a 'must have' in any ink collection.


text:
Home Community Obituary: Joseph Sherman Moses
Obituary: Joseph Sherman Moses
Joseph Sherman Moses
Joseph Sherman Moses, 79, originally of Montclair, died on Jan. 24, 2019, after a courageously long battle with Alzheimer's disease.
Mr. Moses was an airman first class and a cryptologist serving in the United States Air Force between 1958 and 1965. He received the Air Force Outstanding Unit Award, and was a member of the Air Force Refueling Squadron GB-132 Department of the Air Force 1964. He received the Air Force Longevity Service Award with one bronze Oak Leaf Cluster. His last assignment was in the 1926th Communications Squadron, AFCS, Robins Air Force base. He also worked for many years for the United States Postal Service in Montclair.
He was an avid fisherman who loved the outdoors. He also enjoyed traveling, cooking, and spending time with family and friends.
Mr. Moses was predeceased by sisters Cynthia (Moses) Drakeford and Sharon (Moses) Webb, and parents Primes and Victoria (Fisher) Moses.
He is survived by his siblings Charles Moses, Clarence Moses, Wayne Moses, Ruth (Moses) Washington, Lillian (Moses) Waters, and Kathleen Coppedge as well as a host of nieces, nephews, cousins and friends.
A public memorial service will be held from 10 to noon on Saturday, Feb. 2. Arrangements are entrusted to Hugh M. Moriarty Funeral Home, 76 Park St., Montclair.
Previous articleMontclair High School, Catherine's Closet seek donated prom dresses, accessories
Next articleMontclairites offer freebies for furloughed workers
Deborah Ann Tripoldi
Obituary: Patricia Anne Balsamini
Obituary: Marjorie Brown


ACO's sales and distribution centre in Auckland services New Zealand with sales representation in both islands.
ACO Limited NZ is part of the ACO Group, a multinational company specializing in products for the construction and building industries.
ACO introduced the concept of modular Polymer Concrete surface drainage systems to New Zealand over 25 years ago. Today ACO Limited in New Zealand is a sales and marketing company with access to ACO's global strong manufacturing bases. Established in 2012, ACO uses a variety of materials for its extensive range of surface water drainage, utility enclosure, building drainage and residential products.
ACO NZ offers onsite support for all aspects of the business - from specification advice to installation expertise. Through dedicated training programs, ACO is recognized for providing architects and engineers surface drainage education.
As market leader, ACO is constantly innovating to bring new products to the NZ market. ACO NZ has access to the ACO group's fully established R&D department responsible for continuous development, quality and testing to ensure ACO products continue to lead the market.


text:
Sarah Singh
New Online Magazine The Wild Launches
As editorial jobs rapidly move online, those on the production and creative side of things--the photographers, stylists, shoot coordinators--have been slower to transition. Why? Well, words look the same on a computer and a page; images do not. But slickly-produced, web-only magazines like Contributing Editor are trying to prove that high-concept editorials can be just be just as compelling online as they are on paper. Contributing Editor--launched in September 2008 by former magazine editor Matthew Edelstein and creative director Ryan Schmidt--certainly paved the way for newer renditions of the new medium, like DIS, and even more recently The Wild, which launched on Friday. The Wild is the brainchild of Brooklyn-based photographer Giovanna Badilla and friends.
By Lauren Sherman


The Comprehensive Plan is the City's "roadmap to the future." It contains a future land use map, and goals and policies that provide guidance to the City as we grow and change to meet the demands of a growing region.
In addition to the City's growth policies, demographic and other background information pertaining to SeaTac is provided in the Background Reports.
The Comprehensive Plan Map sets land use policy by establishing areas of the City for residential use, commercial use, etc. Where these categories differ from the current zoning, the Comprehensive Plan establishes a potential for a property to be rezoned, if owners apply for a rezone, and required conditions can be met.
Current Comprehensive Plan (Adopted December 12, 2017).
The SeaTac Comprehensive Plan consists of ten chapters ("elements"). Each element is linked below. A series of related Background Reports contains demographic information (Housing and Human Services Background Report), information about population and employment forecasts (Land Use Background Report) and other factual information supporting the Plan's policies.
• The City Center Plan, adopted December, 1999. Note: Portions of the City Center Plan were amended through the 2010 Comprehensive Plan Amendment Process.
Copies of the Comprehensive Plan and Plan Map are available for viewing or purchase at the Department of Community and Economic Development, or contact Kate Kaehny at kkaehny@seatacwa.gov.


The BBC's nightmarish vision of Britain under attack by a rabies outbreak from 1983.
Starring Richard Heffer, Barbara Kellerman and Ed Bishop.
The Mad Death is a chilling three-part thriller that examined in terrifying detail what would happen if rabies hit the UK and infected the human population.
First shown in 1983, at the height of Britain's paranoia about the potential outbreak of the disease, The Mad Death horrified audiences and is still remembered for its grotesque depictions of victims foaming at the mouth, crazed foxes attacking the public and a haunting rendition of All Things Bright and Beautiful.
When a French woman cannot bear to leave her cat at home on a trip to Scotland, she smuggles it along. What she does not know is her pet is infected with deadly rabies, and passes the infection on in an encounter with a fox.
When a kind American (Ed Bishop) discovers the fox and takes it in, he soon becomes the unfortunate first human victim of The Mad Death. And once the horrified doctors have come to this realisation, it is too late. The virus has already infected most the animal population, and more of humankind.
It's up to Michael Hillard (Richard Heffer) and Ann Maitland (Barbara Kellerman) to join forces to fight the dreadful disease.


Sydney, Australia 29 Aug 2017 – Enov8, the recognised leaders in Enterprise IT & Test Environment Management solutions and Enterprise Release, today announced the release of a new set of enterprise behavioural dashboards for Ecosystem. The new capability will allow senior IT managers & stakeholders to look across the organization holistically and identify strengths, weaknesses and gaps of the different systems, products, teams and vendors and provide scorecards on core areas of interest.
Equipped with this intelligence executives will be able to see where to concentrate their effort across areas like Operational Baselining & Standardisation, Release Practices, Automation (including Health Checks & CICD i.e. Continuous Integration Continuous Deployment), Data Security Compliance and IT Cost Management.
"Ecosystem will now change this, starting from this new release any CIO, CTO or IT Executive will be able to see exactly what each team is responsible for, which systems are automated, which teams are compliant, which teams are providing timely service and which areas are causing footprint blowouts and cost overruns".
"Ecosystem will bring a change to enterprise behaviour, and using these new insights organizations should be able to cost optimize, save tens of millions of dollars a year and reduce traditional delivery cycles by up to 20% ".
Availability: Behavioral dashboards are now available with ecosystem 4.3.1.
Note: Key Performance Areas (KPAs) can be added to using the tools extensible engine.
About Enov8: Enov8 is an Australian Innovator, established in 2013, with a platform focused on supporting IT & Test Environment Management and key sub-disciplines including Release and Data Management. Our philosophy is to help organizations be "Agile with Discipline" (& implement DevOps at scale) through better understanding and control of the IT landscape.