Boyd I.L.,University of St. Andrews |
Frisk G.,Florida Atlantic University |
Urban E.,University of Delaware |
Tyack P.,Woods Hole Oceanographic Institution |
And 22 more authors.
Oceanography | Year: 2011
The effect of noise on marine life is one of the big unknowns of current marine science. Considerable evidence exists that the human contribution to ocean noise has increased during the past few decades: human noise has become the dominant component of marine noise in some regions, and noise is directly correlated with the increasing industrialization of the ocean. Sound is an important factor in the lives of many marine organisms, and theory and increasing observations suggest that human noise could be approaching levels at which negative effects on marine life may be occurring. Certain species already show symptoms of the effects of sound. Although some of these effects are acute and rare, chronic sublethal effects may be more prevalent, but are difficult to measure. We need to identify the thresholds of such effects for different species and be in a position to predict how increasing anthropogenic sound will add to the effects. To achieve such predictive capabilities, the Scientific Committee on Oceanic Research (SCOR) and the Partnership for Observation of the Global Oceans (POGO) are developing an International Quiet Ocean Experiment (IQOE), with the objective of coordinating the international research community to both quantify the ocean sound scape and examine the functional relationship between sound and the viability of key marine organisms. SCOR and POGO will convene an open science meeting to gather community input on the important research, observations, and modeling activities that should be included in IQOE. © 2011 by The Oceanography Society. All rights reserved.
News Article | March 1, 2017
No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment. Genomic DNA of Methanosarcina barkeri strain Fusaro DSM804 was provided by R. Thauer. A list of the plasmids used in this work is given in Extended Data Table 1. Genes were PCR amplified using a forward primer containing NdeI or AseI and a reverse primer with both SpeI and BamHI restriction sites (see Extended Data Table 1). The SpeI site was added on the reverse primer for subsequent link and lock cloning37. PCR fragments were digested with the relevant restriction enzymes and ligated into the pET14b plasmid. Genes were sequenced by GATC Biotech or Source BioScience LifeSciences. For the subcloning of Mbar_A0344 (cfbA), the gene was PCR amplified from pET14b-cfbA using primers cbiX_AscI_fo and cbiX_SalI_re (Extended Data Table 1). The resulting PCR fragment was digested with AscI and SalI and ligated into the correspondingly digested vector pETDuet-1 (Novagen/Merck Millipore). The cfbA gene Mbar_A0344 was then cut from this construct using the restriction enzymes NdeI and SalI, and the purified fragment was ligated into the correspondingly digested plasmid pET22b (Novagen), yielding expression plasmid pET22b-cfbA (Extended Data Table 1). For cloning of multi-gene constructs, sequenced genes were transferred into pET3a (to remove the His -tag), then constructed piecewise by the link-and-lock cloning method37 in the pETcoco-2KAN plasmid. E. coli Rosetta pLysS was transformed with plasmids containing putative coenzyme F biosynthesis genes cloned into pET14b and selected on LB agar with 34 μg ml−1 chloramphenicol and 100 μg ml−1 ampicillin. For protein production, an overnight pre-culture was grown in LB medium for 16 h at 37 °C, 150 r.p.m. The next day, 10 ml of pre-culture was transferred into 1–4 l of LB medium with 34 μg ml−1 chloramphenicol and 100 μg ml−1 ampicillin. The cells were grown at 37 °C, 150 r.p.m. until an OD of 1.0 was reached. Protein production was induced with 0.4 mM IPTG and cells were left overnight at 19 °C with 150 r.p.m. shaking. For increased production of iron-sulfur enzymes, 1 mM ammonium ferric citrate was added to the cultures at the induction stage. Proteins containing Fe-S clusters were purified in an anaerobic glovebox (Belle Technologies or Coy Laboratory Products), with O levels at less than 2 p.p.m. All buffers and solutions were purged with argon before use in the glovebox. E. coli cultures were centrifuged at 5,180g at 4 °C for 20 min. Cells were then resuspended in 15 ml of binding buffer (20 mM Tris-HCl, pH 8, 500 mM NaCl and 5 mM imidazole), followed by sonication under anaerobic conditions at 4 °C for 5 min with 10 and 30 s pulse and rest cycles, respectively. Cell lysates were centrifuged at 37,044g at 4 °C for 20 min. The supernatant was then purified using 5 ml of pre-charged nickel chelated Sepharose. This was then washed with 50 ml of binding buffer, followed by washing steps (25 ml) containing increasing concentrations of imidazole from 30 to 70 mM. Elution was performed with buffer containing 400 mM imidazole. Purified protein was desalted on a pre-packed PD-10 column equilibrated in buffer without imidazole. E. coli Rosetta pLysS containing plasmid pET22b-cfbA was cultivated as described above with the exception that the induction of protein production with IPTG was initiated when the cells had reached an OD of about 0.4. After overnight cultivation the cells were collected by centrifugation and the cell pellet from 1 l of culture was resuspended in 20 ml of buffer A (50 mM Tris-HCl, pH 8) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were disrupted by sonication and the resulting cell lysate was centrifuged in an ultracentrifuge at 175,000g at 4 °C for 60 min. The soluble protein fraction was loaded onto a 1 ml HiTrap Q XL column (GE Healthcare) at a flow rate of 1 ml min−1. The column was washed with 10 ml of buffer A and the bound proteins were then eluted using a linear NaCl gradient (0–400 mM NaCl in buffer A) developed over 20 ml. The CfbA-containing elution fractions were pooled, concentrated to 5 ml and then loaded onto a HiLoad 16/600 Superdex 75 prep grade column (GE Healthcare) equilibrated with 50 mM Tris-HCl, pH 8, 150 mM NaCl at a flow rate of 1 ml min−1. The elution fractions containing CfbA were pooled and the buffer of the purified protein was exchanged inside the anaerobic chamber using a PD-10 column equilibrated with anaerobic test buffer (25 mM Tris-HCl, pH 8, 150 mM NaCl, 10 mM MgCl , 10% (v/v) glycerol). The purified CfbA was stored at −80 °C until further use. The reconstitution of iron-sulfur clusters within CfbC and CfbD was performed as described previously38. After reconstitution, the excess of iron and sulfide was removed by centrifugation and subsequent passage of the protein solution through a NAP-25 column (GE Healthcare), which was used according to the manufacturer’s instructions. The iron and sulfide concentrations for Mbar_A0346 (CfbC) and Mbar_A0347 (CfbD) were determined as previously described39. Protein concentration was estimated separately using Bradford reagent (Bio-Rad Laboratories) with bovine serum albumin as a calibration standard. Samples were prepared and then flash frozen in liquid nitrogen. EPR experiments were performed on a Bruker ELEXSYS E500 spectrometer operating at X-band, using a Super High Q cylindrical cavity (Q factor ≈ 16,000) equipped with an Oxford Instruments ESR900 liquid helium cryostat linked to an ITC503 temperature controller. Experimental parameters: microwave power 0.5 mW, field modulation amplitude 7 G, field modulation frequency 100 KHz, temperature 15 K. Sirohydrochlorin was synthesized using the one-pot incubation method described previously40. For the CfbA activity assay, 5 μM sirohydrochlorin and 50 μM of NiSO were incubated at 37 °C with varying amounts of purified CfbA (0, 1, 1.5 and 2.5 μM) in anaerobic test buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 10 mM MgCl and 10% (v/v) glycerol) inside the anaerobic chamber. For each enzyme concentration the assay was performed at least three times. The deduced specific activity represents the mean value of all measurements. The chelation of nickel into sirohydrochlorin was monitored by recording UV/Vis absorption spectra at different time points using a V-650 spectrophotometer (Jasco). E. coli KRX auto-induction strain was transformed with the pETcoco-2KAN-cobA-sirC-cbiXS-nixA and pET14b-Mbar_A0348 plasmids using 0.2% (w/v) glucose to maintain the single copy state of the pETcoco-2KAN derived plasmid and 25 μg ml−1 kanamycin and 100 μg ml−1 ampicillin for antibiotic selection. An overnight pre-culture was grown for 16 h at 28 °C, 150 r.p.m. The next day 10 ml of pre-culture was transferred into 1 l of 2YT medium with 50 μg ml−1 kanamycin, 100 μg ml−1 ampicillin, 0.05% glucose (w/v), 0.1% rhamnose (w/v), 0.01% (w/v) arabinose and between 25 μM and 100 μM NiCl .6H O. The cells were grown at 28 °C and 150 r.p.m. for 24 h. This yields approximately 1–2 mg l−1 of nickel-sirohydrochlorin a,c-diamide in complex with the His -tagged amidotransferase Mbar_A0348 (CfbE) enzyme, which can be purified using IMAC purification under low-salt (100 mM) buffer conditions. The protocol for the antimony-phosphomolybdate colorimetric based stopped-assay41 was used for determining the ATPase activity of the M. barkeri CfbE amidotransferase in the presence of its substrate nickel-sirohydrochlorin. 0.2% (w/v) citric acid was added after a time delay of 2 min to prevent background increases in absorbance from acid hydrolysis of ATP. Assays were performed in buffer B (20 mM Tris-HCl, pH 8 and 100 mM NaCl buffer) at 20 °C. (15NH ) SO (Cambridge Isotope Laboratories) was used for labelling of the amide side chains. Single-turnover reactions were prepared in 10 ml of buffer B with 25 μM of pure M. barkeri CfbE, 25 μM nickel-sirohydrochlorin, 1 mM MgCl , 25 mM (15NH ) SO . Turnover was controlled by an ATP titration series of 0, 25, 50 and 100 μM. Reactions were left for 30 min at 37 °C. The reaction product was purified, dehydrated and dissolved in d -DMSO in order to reduce proton solvent exchange to allow observation of the NH amide signals, which are barely detectable in D O or acidic (pH 5) 1:10 H O/D O mixtures. Two-dimensional datasets were collected including 1H–15N HSQC, 1H–1H NOESY and 1H–15N HSQC-TOCSY spectra. The 1H–15N correlation spectra were collected by the SOFAST-HSQC method, which increases sensitivity using fast repetition rates42. This method resolved four clear amide peaks with no background signals (Extended Data Fig. 2). These were correlated to show clear NOE through space interactions with the ring A and C propionate side chains as indicated in the ROESY and NOESY spectra (Extended Data Fig. 3; Supplementary Table 1). This provides strong evidence for the positioning of the amide groups at the a and c positions, thus confirming the product of the CfbE amidation reaction as Ni2+-sirohydrochlorin a,c-diamide. Samples (10–100 μl) were injected onto an Ace 5 AQ column (2.1 × 150 mm, 5 μm, Advanced Chromatography Technologies) that was attached to an Agilent 1100 series HPLC coupled to a micrOTOF-Q (Bruker) mass spectrometer and equipped with online diode array and fluorescence detectors and run at a flow rate of 0.2 ml min−1. Tetrapyrroles were routinely separated with a linear gradient of acetonitrile in 0.1% TFA. Mass spectra were obtained using an Agilent 1100 liquid chromatography system connected to a Bruker micrOTOF II MS, using electro-spray ionisation in positive mode. UV/Vis absorption spectra were monitored by DAD-UV detection (Agilent Technologies). The assay for testing the reductase activity of CfbC/CfbD was performed under anaerobic conditions at 37 °C in anaerobic test buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 10 mM MgCl and 10% (v/v) glycerol). The assay contained 10 μM nickel-sirohydrochlorin a,c-diamide (formed in situ by the action of CfbE), 10 μM CfbC, 10 μM CfbD, 3.2 mM ATP, 3.2 mM sodium dithionite and residual amounts of the enzymes HemB, HemC, HemD, CobA, SirC, CfbA and CfbE, which were used for the formation of nickel-sirohydrochlorin a,c-diamide. The reaction was followed by UV/Vis absorption spectroscopy and by analysing the tetrapyrrole content of the assay mixtures after 0, 1.5, 14 and 22 h of incubation by HPLC. For HPLC analysis, the tetrapyrroles were extracted by denaturation of the proteins using guanidinium chloride. For this, 160 mg of guanidinium chloride were dissolved in 300 μl of the sample, and the mixture was incubated for 2 min at room temperature. Subsequently, the free tetrapyrroles were separated from the denatured proteins by ultrafiltration using an Amicon Ultra 10 k filter unit (Merck Millipore). The tetrapyrrole-containing filtrate (40 μl injection volume) was analysed by HPLC using a ReproSil-Pur C18 AQ column (Dr. Maisch HPLC GmbH) and a JASCO HPLC 2000 series system (Jasco). The separation was carried out at a flow rate of 0.2 ml min−1. Solvent A was 0.01% formic acid in H O and solvent B was acetonitrile. Tetrapyrroles eluted with a linear gradient system within 25 min: start conditions 95% A/5% B and end conditions 65% A/35% B. The tetrapyrroles were detected by photometric diode array analysis in the range of 220–670 nm. The masses of the eluting tetrapyrroles were confirmed by ESI-MS analysis on an Esquire 3000+ ESI ion trap mass spectrometer coupled to an Agilent 1100er series HPLC system using the same column, eluent, and gradient. Scan was carried out in alternating mode between m/z 500–2000, the target mass set to m/z 1,000, nebulizer pressure to 70 p.s.i., dry gas flow to 11 l min−1 and dry gas temperature to 360 °C. The CfbB assay was conducted under anaerobic conditions at 37 °C in anaerobic test buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 10 mM MgCl , 10% (v/v) glycerol). The assay contained 7.5 μM of either Ni2+-hexahydrosirohydrochlorin a,c-diamide or seco-F (formed as described for the CfbC/CfbD assay), 0.75 μM or 7.5 μM CfbB and 3.2 mM ATP. After 1 or 2 h of incubation, the tetrapyrroles were extracted and analysed by HPLC and HPLC–MS as described for the CfbC/CfbD assay. For structural determination an isotopically enriched sample (4 mM) of the seco-F intermediate was prepared using 15N-glutamine as the amide donor and the incorporation of two 15N atoms in the product was confirmed by HPLC–MS. Analysis of the data following assignment established the presence of the lactam attached to ring B. This was determined from the combination of the following pieces of information. Protons attached to C3-C4 -C5 are present in a single scalar coupled network and C5 (36.37 p.p.m.) appears sp3 hybridized with two germinal protons (1.56 and 1.84 p.p.m.). The chemical shift of C6, assigned from the 1H–13C HMBC spectrum, is 96.39 p.p.m.. Lastly, the 15N HSQC clearly shows 3 signals from which the germinal pair of protons was assigned to the NH of the a-sidechain (N23) and the single N–H resonance observed at lower field to the lactam formed from the c-sidechain of ring B (N73) (Extended Data Fig. 6). All data generated or analysed during this study are available within the paper (and its Supplementary Information files).
News Article | November 16, 2016
Molecular spectroscopy is the qualitative and quantitative study of molecules by observing their interaction with variousfrequencies and energy. In other way, it is the study of absorption of light by molecules. It is analyzed by ultraviolet (UV) light, visible light and infrared radiations using an instrument called a spectrometer. Thermo Fisher Scientific,Agilent Technologies, Danaher Corporation, PerkinElmer, Inc., Shimadzu Corporation, JASCO International Co., Ltd. and Jeol Ltd. are some major players engaged in the development, manufacturing and marketing of instruments used in molecular spectroscopy study. Antaris II FT-NIR Analyzer, picoSpin 80 spectrometer (Thermo Fisher Scientific) and Cary 630 FTIR Spectrometer, 4300 Handheld FTIR (Agilent Technologies) are some prominent brands available in the market. The global market for molecular spectroscopy has been segmented as follows by technology type: These technologies may be further segmented as well. For example, infrared spectroscopy may be segmented by the type of devices available on the market such as bench top IR spectroscopy, portable spectroscopy or terahertz IR spectroscopy. Similarly, nuclear magnetic resonance spectroscopy may be categorized as continuous wave nuclear magnetic resonance spectroscopy, fourier transform NMR and Solid-State NMR. Ultraviolet visible spectroscopy may be segmented as single-beam UV spectrometer, double-beam UV spectrometer and array based UV spectrometer. On the other hand, on the basis of application, the market has been segmented into: Governmental legislation that are compelling pharmaceutical companies to maintain very high quality of drugs and excipients during manufacturing and increasing concerns for food and beverages safety are some major factors driving the growth of molecular spectroscopy market.Governmental support by funding research related with molecular spectroscopy is also oneof the major factors driving the market growth worldwide.Technological developments in the area of molecular spectroscopy are also encouraging manufacturers to opt for this technology as it will give them competitive in terms of quality. On the other hand, high cost associated with acquiring molecular spectroscopy technology may hamper the market growth to some extent. Geographically, the market for molecular spectroscopy has been segmented into four major regions, namely, North America, Europe, Asia-Pacific and Rest of the World (RoW). North America holds the largest regional market, followed by Europe. Federal government’s investments to support research in the area of medical sciences are one of the reasons driving the market growth in the region. Presence of a large number of pharmaceutical manufacturing companies in the region also plays a key role in propelling the market growth. Europe represents the second largest market after North America. The market in Western European region which includes countries like Germany, France, Spain and United Kingdom is relatively mature than the markets of Eastern European region. Emerging economies of countries of Eastern region will help in driving the market growth in the region due to the significantly large contract manufacturing market.India and China in particular are expected to increase the number of companies offering contract manufacturing services to the western region. It is likely to result in growing demand for various molecular spectroscopy devices, and thus will help in driving the market growth in the region. In RoW region, Brazil, Mexico, Israel and Middle East countries are the potential markets for molecular spectroscopy.
News Article | November 3, 2016
— Segments The market for Liquid chromatography Instruments is segmented in mainly two parts i.e. by Type, by end users and its various sub-segments; by type include High pressure liquid chromatography (HPLC), Ultra high pressure liquid chromatography (UHPLC), Low pressure liquid chromatography (LPLC), Liquid-solid chromatography, Normal phase chromatography, Reverse phase chromatography, Flash chromatography, Partition chromatography, Ion chromatography, Size exclusion chromatography, Affinity chromatography and Chiral chromatography. by End Users include Biotechnology and pharmaceuticals industries, Hospitals, research laboratories, Agriculture industries and others. Market Synopsis of Liquid chromatography Instruments Market Scenario Globally the market for Liquid chromatography Instruments is increasing rapidly the main reason for this is the growth in pharmaceutical industry. The factors that influence the growth of Liquid chromatography Instruments market are the increasing development in pharmaceutical industry for understanding appropriate chemical for introducing new medicine and maintain pharmaceutical quality. The market is also growing due to usage of Liquid chromatography Instruments in industries such as Biotechnology and pharmaceuticals industries, Hospitals, research laboratories, Agriculture industries and others. Globally the market for Liquid chromatography Instruments is expected to grow at the rate of about XX% CAGR from 2016 to 2027. Ask for your specific company profile and country level customization on reports Key Players The key players that are involved in Global Liquid chromatography Instruments market are • AC Analytical Controls BV (Netherlands), • Thermo Fisher Scientific, Inc. (U.S.), • Phenomenex, Inc. (U.S.)., • Agilent Technologies (U.S.), • PerkinElmer, Inc. (U.S.), • Shimadzu Corporation (Japan), • Waters Corporation (U.S.), • JASCO, Inc. (U.S.), • Novasep Holding S.A.S. (France), • Pall Corporation (U.S.), • GL Sciences, Inc. (Japan). Study Objectives of Liquid chromatography Instruments • To provide detailed analysis of the market structure along with forecast for the next 10 years of the various segments and sub-segments of the global Liquid chromatography Instruments market • To provide insights about factors affecting the market growth • To Analyze the Liquid chromatography Instruments market based on various factors- price analysis, supply chain analysis, porters five force analysis etc. • To provide historical and forecast revenue of the market segments and sub-segments with respect to four main geographies and their countries- Americas, Europe, Asia, and Rest of the World (ROW) • To provide country level analysis of the market with respect to the current market size and future prospective • To provide country level analysis of the market for segment by Type, End Users and its sub-segments. • To provide strategic profiling of key players in the market, comprehensively analyzing their core competencies, and drawing a competitive landscape for the market Regional Analysis of Liquid chromatography Instruments North America dominated the Global Liquid chromatography Instruments market with the largest market share, accounting for $XX million and is expected to grow over $XX billion by 2027. The European market for Liquid chromatography Instruments is expected to grow at XX% GAGR (2016-2027). Asia-Pacific is expected to grow at CAGR of XX% from $ XX million in 2016 to $XX million by 2027. “Analysis also includes consumption. Import and export data for Regions North America, Europe, China, Japan, Southeast Asia, India.” The market is divided into the following segments based on geography: North America • US • Canada • Mexico Europe • Germany • France • Italy • U.K • Rest of Europe Asia– Pacific • China • India • Japan • Rest of Asia-Pacific RoW • Brazil • Argentina • Egypt • South Africa Others Reasons to Purchase this report: From an insight perspective, this research report has focused on various levels of analyses—industry analysis (industry trends), market share analysis of top players, supply chain analysis, and company profiles, which together comprise and discuss the basic views on the competitive landscape, emerging and high-growth segments of the Global Liquid chromatography Instruments Market. high-growth regions, and market drivers, restraints, and opportunities. Key questions answered in this report • What will the market size be in 2027 and what will the growth rate be? • What are the key market trends? • What is driving this market? • What are the challenges to market growth? • Who are the key vendors in this market space? • What are the market opportunities and threats faced by the key vendors? • What are the strengths and weaknesses of the key vendors? Related Report North America Drug Screening Market Research Report- Forecast To 2027 North America Drug Screening market Information, by products (rapid testing devices, breath analyzers, consumables) by samples, by end-users - Forecast to 2027 Know more about this report @ https://www.marketresearchfuture.com/reports/north-america-drug-screening-market-research-report-forecast-to-2027 About Market Research Future: At Market Research Future (MRFR), we enable our customers to unravel the complexity of various industries through our Cooked Research Report (CRR), Half-Cooked Research Reports (HCRR), Raw Research Reports (3R), Continuous-Feed Research (CFR), and Market Research & Consulting Services. MRFR team have supreme objective to provide the optimum quality market research and intelligence services to our clients. Our market research studies by products, services, technologies, applications, end users, and market players for global, regional, and country level market segments, enable our clients to see more, know more, and do more, which help to answer all their most important questions. For more information, please visit https://www.marketresearchfuture.com
News Article | February 24, 2017
According to a new market research report "High-performance Liquid Chromatography (HPLC) Market by Product (Instruments (Systems, Detectors), Consumables (Columns, Filters), and Accessories), Application (Clinical Research, Diagnostics, Forensics) - Analysis & Global Forecast to 2021", published by MarketsandMarkets, This report studies the global HPLC Market for the forecast period of 2016 to 2021. This market is expected to reach 4.13 Billion by 2021 from USD 3.23 Billion in 2016, growing at a CAGR of 5.1%. Browse 112 market data Tables and 42 Figures spread through 182 Pages and in-depth TOC on "High-performance Liquid Chromatography (HPLC) Market" Early buyers will receive 10% customization on this report. The global HPLC Market is segmented on the basis of product, application, and region. On the basis application, the HPLC is segmented into clinical research, diagnostics, forensics, and other applications (including food & environmental analysis and academic research). In 2016, the clinical research segment is expected to account for the largest share of the global HPLC Market. On the basis of product, the HPLC Market is categorized into instruments, consumables, and accessories. The instruments segment is estimated to account for the largest share of the global HPLC Market, by product. The consumables segment is projected to grow at the highest CAGR between 2016 and 2021, primarily due to the recurring requirement of consumables. The instruments segment is further categorized into systems, detectors, pumps, and fraction collectors. In 2016, the systems segment is expected to command the largest share and the highest growth of the instruments market. The consumables segment is further categorized into columns, filters, vials, and tubes. The columns segment is estimated to grow at the highest CAGR during the forecast period. This segment is further categorized into reverse-phase HPLC columns, normal-phase/hydrophobic interaction HPLC columns, ion exchange HPLC columns, and other columns. Based on region, the HPLC Market is divided into North America, Europe, Asia-Pacific, and the Rest of the World (RoW). The RoW region comprises Latin America, the Middle East, and Africa. In 2016, North America is projected to account for the largest share of the HPLC Market, followed by Europe and Asia-Pacific. Increasing funding for R&D, preclinical activities by CROs and pharmaceutical companies, and the growing food industry in Ontario are propelling the growth of the North American HPLC Market. The major players in the global HPLC Market are Waters Corporation (U.S.), Agilent Technologies (U.S.), and Shimadzu Corporation (Japan). These companies are dominant in the HPLC Market mainly due to their well-established presence in the field of chromatography, presence in over 50 countries, high R&D investments, and strong sales and distribution force. The other players in the market include Thermo Fisher Scientific Inc. (U.S.), GE Healthcare (U.S.), PerkinElmer, Inc. (U.S.), Bio-Rad Laboratories, Inc. (U.S.), Gilson, Inc. (U.S.), Phenomenex, Inc. (U.S.), and JASCO, Inc. (U.S.). PREPARATIVE AND PROCESS CHROMATOGRAPHY MARKET By Type (Preparative, Process), Products (Systems, Columns, Empty, Glass, Resins, Protein A, Affinity, Ion exchange, Mixed mode, Services), End User (Biotechnology, Pharmaceutical) - Global Forecasts to 2021. CHROMATOGRAPHY INSTRUMENTS MARKET By System (LC (HPLC, UHPLC, Flash), GC, Other Components (Autosamplers, Detectors, Fraction Collectors), Consumable (Reverse Phase Columns, Syringe Filters, Vials) - Analysis & Global Forecasts to 2020. 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Kamarei F.,University of Tennessee at Knoxville |
Gritti F.,University of Tennessee at Knoxville |
Guiochon G.,University of Tennessee at Knoxville |
Journal of Chromatography A | Year: 2014
The implementation of the traditional FA method is difficult with classical supercritical fluid chromatography (SFC) instruments. The instrument mixer and other sources of extra-column volumes are large and significantly broaden the fronts of injected plugs, which diminishes the precision and accuracy of the FA method. An SFC instrument was modified to permit more accurate determinations of adsorption isotherm data. The sample, the modifier, and CO2 are separately pumped via small volume connection tubes into a small volume mixer (250μL), where they are mixed into a homogeneous fluid fed to the column. The extra-column volumes and the column hold-up volume were accurately measured at each back pressure from the retention times of tracers. This modified instrument was used to measure the adsorption isotherm of S-naproxen by frontal analysis (FA) on a (R, R)-Whelk-O1 column, using a mixture of methanol (20%, v/v) and CO2 as the mobile phase. Its performance is studied at several different back pressures from 100 to 210bar. In all the experiments, the total flow rate was kept to a low value (1mL/min) in order to minimize the variation of the equilibrium constant along the column. Although a suitable breakthrough curve could not be obtained at low back pressures (<150bar) due to the closeness to the critical point pressure of the methanol/CO2 mixture, excellent results were obtained at higher back pressures (>150bar), conditions remote from the critical point and breakthrough curves with very sharp front shocks are obtained. The RSDs of the profiles recorded at each back pressures are excellent, better than 1%. © 2013 Elsevier B.V.
Ishikawa M.,Osaka University |
Katsura M.,Osaka University |
Nakashima S.,Osaka University |
Aizawa K.,JASCO |
And 3 more authors.
e-Journal of Surface Science and Nanotechnology | Year: 2011
The goal of the present study is to obtain broadband near-field infrared (IR) spectra by combining Fouriertransform infrared spectroscopy (FTIR) with scattering near-field optical microscopy (s-SNOM). A stage was added to the IR spectrometer with a ceramic light source in order to modulate the probe-sample distance, and the second harmonic component was extracted by a lock-in amplifier. The detected IR signal intensity decreased exponentially with the distance between the probe tip and an Au mirror, with a localization scale of approximately 100 nm. An area with Au islands formed by electron beam lithography was scanned with the modulation system with mapping steps of X = 80 nm and Y = 133 nm. The obtained IR intensity image matches the topographic image, indicating sub-micron spatial resolution. These results indicate that the addition of the modulation system to the broadband near-field IR spectrometer was successful in obtaining localized near-field signals and sub-micron spatial resolution, even using a ceramic IR light source. © 2011 The Surface Science Society of Japan.
News Article | December 23, 2015
The repeat module design process applied here consisted of an initial diversification round of large-scale sampling followed by filtering and clustering and then a second intensification round of sampling focused on successful topologies identified in the first round. Starting backbone models for sequence design were built using a fragment assembly protocol which is based on the standard Rosetta ab initio protocol31 with the following modifications: (1) fragment replacement moves were performed symmetrically across all repeats, guaranteeing that backbone torsion angles were identical at corresponding positions across repeats; (2) a pseudo-energy term (equal to the deviation between actual and desired curvature, in degrees, plus the deviation in rise multiplied by a factor of 5) was added to the potential to favour satisfaction of the geometric constraints; (3) the amino-acid sequence used for low-resolution scoring was assigned randomly at the start of each simulation from secondary-structure-specific distributions (helix: Ala+Ile+Leu+Asp+Ser; turn: Gly+Ser), which had the effect of increasing the diversity in helix packing distances and geometries compared with using a constant sequence such as poly-Val or poly-Leu. At the start of each independent design trajectory, the lengths of the secondary structure elements and turns were chosen randomly, defining the target secondary structure of the repeat module and its length. Together with the number of repeats, this defined the total length of the protein and the complete secondary structure, which was used to select 3- and 9-residue backbone fragments for use in the low-resolution fragment assembly phase. The design calculations reported here sampled helix lengths from 7 to 20 residues, turn lengths from 1 to 5 residues, and total repeat lengths ranging from 20 to 40 residues. The low-resolution fragment assembly simulation was followed by an all-atom sequence design stage consisting of two cycles alternating between fixed-backbone sequence design and fixed-sequence structure relaxation. Symmetry of backbone and side-chain torsion angles and sequence identities was maintained across all repeats. Since the starting backbones for design were built by relatively coarse sampling in a low-resolution potential, sequences designed with the standard all-atom potential were dominated by small amino acids and the resulting structures tended to be under-packed. To correct for this tendency, a softened Lennard–Jones potential32 was used for the sequence design steps, while the standard potential was used during the relaxation step. The Rosetta score12prime weights set was used as the standard potential for these design calculations. Final design models (typically 10,000–100,000 in this study) were first sorted by per-residue energy (total energy divided by the number of residues, to account for varying repeat length) and the top 20% filtered for packing quality (sasapack_score <0.5), satisfaction of buried polar groups (buried unsatisfied donors per repeat <1.5, buried unsatisfied acceptors per repeat <0.5), and sequence-structure compatibility via a fast, low-resolution symmetric refolding test (40 trajectories, requiring at least 1 under an r.m.s.d. threshold of 2 Å for 3-repeat designs and 4 Å for larger designs). Designs that passed these filters were clustered by C-α r.m.s.d. (allowing for register shifts when aligning helices with unequal lengths) to identify recurring architectures. The clusters were ranked by averaging residue energy, packing quality, and refolding success over all cluster members. During the intensification round of designs, representative topologies from successful design clusters were specifically resampled by enforcing their helix and turn lengths as well as their turn conformations (defined using a five-state, coarse-grained backbone torsion alphabet27; Extended Data Fig. 1e) during fragment selection. Selected low-energy designs from the second round that pass the filters described above were evaluated by a large-scale refolding test in which 2,000–10,000 ab initio models were built by standard (asymmetric) fragment assembly followed by all-atom relaxation. Success was measured by assessing the fraction of low-energy ab initio models with r.m.s.d. values to the design model under a length-dependent threshold. For designed toroids with an open, polar central pore, perfect symmetry may not allow optimal electrostatic interactions between nearby side chains corresponding to the same repeat position in successive repeats. We therefore explored symmetry-breaking mutations at a handful of inward-pointing positions via fixed-backbone sequence design simulations in which the length of the repeating sequence unit was doubled/tripled (for example, whereas perfect six-fold repeat symmetry would require K-K-K-K-K-K or E-E-E-E-E-E, doubling the repeat length allows charge complementarity with K-E-K-E-K-E). Solutions from these designs were accepted if they significantly lowered the total energy. The 12x31L design construct was generated by duplicating the final three repeats of the 9x31L design. To build a ‘design model’ for comparison with the experimentally determined structure, we followed the resampling protocol now forcing the 12x31L amino-acid sequence in addition to the number of repeats (12) and the helix and turn lengths (H14-L3-H11-L3) and turn conformations (GBB). Thus the sequence design steps were reduced to rotamer optimization (since the amino-acid identities were fixed). This symmetric structure prediction process was repeated 10,000 times and the lowest-energy final model was taken as the computational model. For a single representative of the 3x31L and 6x31R families, we performed lattice docking and design simulations to select mutations that might promote crystallization. Core positions were frozen at the design sequence. Candidate space groups were selected from those most commonly observed in the protein structural database. Theoretical models of crystal packing arrangements were built by randomly orienting the design model within the unit cell and reducing the lattice dimensions until clashes were encountered. Symmetric interface design was performed on these docked arrangements, and final designs were filtered by energy, packing, satisfaction of polar groups, and number of mutations from the original design model. To compute the handedness of helical bundles formed by tandem repeat proteins, we generated an approximate helical bundle axis curve by joining the location of repeat-unit centres of mass in a sliding fashion along the protein chain. The handedness was then estimated by computing the directionality of the winding of the polypeptide chain about this axis curve. To assess similarity between design models and proteins in the structural database, we performed searches using the structure–structure comparison program DALI33 as well as consulting the protein structure classification databases CATH34, SCOPe35, and ECOD36. Further details are given in Supplementary Discussion. Repeat protein design methods were implemented in the Rosetta software suite (www.rosettacommons.org) and will be made freely available to academic users; licenses for commercial use are available through the University of Washington Technology Transfer office. The plasmids encoding individual constructs were cloned into previously described bacterial pET15HE expression vectors37 containing a cleavable N-terminal His-tag and an ampicillin resistance cassette. Sequence-verified plasmids were transformed into BL21(DE3)RIL Escherichia coli cells (Agilent Technologies) and plated on lysogeny broth (LB) medium with ampicillin (100 μg ml−1). Colonies were individually picked and transferred to individual 10 ml aliquots of LB–ampicillin media and shaken overnight at 37 °C. Individual 10 ml aliquots of overnight cell cultures were added to individual 1 l volumes of LB–ampicillin, which were then shaken at 37 °C until the cells reached an absorbance at 600 nm of 0.6–0.8. The cells were chilled for 20 min at 4 °C, then isopropyl-β-D-thiogalactoside (IPTG) was then added to each flask to a final concentration of 0.5 mM to induce protein expression. The flasks were shaken overnight at 16 °C, and then pelleted by centrifugation and stored at − 20 °C until purification. Construct dTor_6x35L(SeMet), incorporating a single methionine residue at position 168 in the original design construct, was generated using a QuikChange site-directed mutagenesis kit (Agilent) and corresponding protocol from the vendor. The resulting plasmid construct was again transformed into BL21(DE3)RIL E. coli cells (Agilent Technologies) and plated on LB plates containing ampicillin (100 μg ml−1) and chloramphenicol (35 g ml−1). Subsequent cell culture and protein expression in minimal media, along with incorporation of selenomethionine, was incorporated during protein expression according to ref. 38. Cell pellets from 3 l of cell culture were resuspended in 60 ml of PBS solution (140 mM NaCl, 2.5 mM KCl, 10 mM NaHPO , 2 mM KH PO ) containing 10 mM imidazole (pH 8.0). Cells were lysed via sonication and centrifuged to remove cell debris. The supernatant was passed through a 0.2 μm filter, and then incubated on a rocker platform at 4 °C for 1 h after adding 3 ml of resuspended nickel-NTA metal affinity resin (Invitrogen). After loading onto a gravity-fed column, the resin was washed with 45 ml of the same lysis buffer described above, and the protein was eluted from the column with three consecutive aliquots of PBS containing 150 mM imidazole (pH 8.0). Purified protein was concentrated to approximately 5–25 mg ml−1 while buffer exchanging into 25 mM Tris (pH 7.5) and 200 mM NaCl and then further purified via size-exclusion chromatography using HiLoad 16/60 Superdex 200 column (GE). Protein samples were then split in half; one sample was used directly for crystallization while the other had the His tag removed by an overnight digest with biotinylated thrombin (Novagen), before additional crystallization trials. The digested sample was incubated for 30 min with streptavidin-conjugated agarose (Novagen) to remove the thrombin. All samples were tested for purity and removal of the His tag via SDS–polyacrylamide gel electrophoresis. The final protein samples, both with and without the N-terminal poly-histidine affinity tag, were concentrated to values of 5–25 mg ml−1 for crystallization trials. Proteins at a concentration of 4–10 mg ml−1 were run over a Superdex 75 10/300 GL column (GE Healthcare) in 25 mM Tris pH 8.0 plus 100 or 750 mM NaCl at a rate of 0.4 ml min−1 on an AKTAprime plus chromatography system (GE Healthcare). All fractions containing eluted toroid protein (visualized via electrophoretic gel analyses) were pooled, concentrated, and run over the column a second time to assess their solution oligomeric behaviour using protein with a minimal background of contaminants. Gel filtration standards (Bio-Rad) were run over the same column in matching buffer, and the ultraviolet trace of the proteins was overlaid onto the standards using UNICORN 5 software (GE Healthcare). For measurements of protein stability using circular dichroism spectroscopy, purified recombinant toroid constructs were diluted to between 10 and 20 μM concentration and dialysed overnight into 10 mM potassium phosphate buffer at pH 8.0. Circular dichroism thermal denaturation experiments were performed on a JASCO J-815 circular dichroism spectrometer with a Peltier thermostat. Wavelength scans (190–250 nm) were performed for each construct at 20 °C and 95 °C. Additional thermal denaturation experiments were conducted by monitoring circular dichroism signal strength at 206 nm over a temperature range of 4–95 °C (0.1 cm path-length cell), with measurements taken every 2°. Sample temperature was allowed to equilibrate for 30 s before each measurement. Purified proteins were initially tested for crystallization via sparse matrix screens in 96-well sitting drops using a mosquito (TTP LabTech). Crystallization conditions were then optimized with constructs that proved capable of crystallizing in larger 24-well hanging drops. Out of 11 constructs that were purified to homogeneity, 10 were crystallized, of which 5 yielded high quality X-ray diffraction that resulted in successful structure determination. dTor_6x35L was crystallized in 160 mM sodium chloride, 100 mM Bis-Tris pH 8.5 and 24% (w/v) polyethylene glycol 3350 at a concentration of 26 mg ml−1. The crystal was transferred to a solution containing 300 mM, then 500 mM sodium chloride and flash frozen in liquid nitrogen. Data were collected on a R-AXIS IV++ at wavelength 1.54 Å and processed on an HKL2000 (ref. 39). dTor_6x35L(SeMet) was crystallized in 140 mM sodium chloride, 100 mM Tris pH 8.5 and 22% (w/v) polyethylene glycol 3350 at a concentration of 26 mg ml−1. The crystal was transferred to a solution containing 300 mM, then 500 mM sodium chloride and flash frozen in liquid nitrogen. Data were collected at ALS Beamline 5.0.2 at wavelength 0.9794 Å and processed on an HKL2000 (ref. 39). dTor_3x33L_2-2 was crystallized in two different conditions, producing two different crystal lattices. The first condition had 30% polyethylene glycol 3350, 100 mM Tris pH 6.5, 200 mM NaCl with a protein concentration of 1.8 mM. The protein was soaked in a 15% ethylene glycol cryoprotectant for 1 min before being flash frozen in liquid nitrogen. Data were collected on a Saturn 944+ (Rigaku) at wavelength 1.54 Å for 180° at φ = 0 and another 180° at φ = 180. Data were then processed on an HKL2000 (ref. 39) out to 1.85 Å in space group P2 2 2 . The second condition had 45% polyethylene glycol 400 and 100 mM Tris pH 7.7 with a protein concentration of 1.8 mM. Protein crystal was flash frozen without being cryoprotected. Data were collected on a Saturn 944+ (Rigaku) at wavelength 1.54 Å for 180° at φ = 0 and another 180° at phi = 180. Data were then processed on an HKL2000 (ref. 39) out to 1.85Å in space group P4 2 2. dTor_9x31L_sub was crystallized in 100 mM Tris pH 8.5 and 15% (v/v) ethanol at a concentration of 11.5 mg ml−1. The crystal was transferred to a solution containing 75 mM Tris pH 8.5, 7.5% (v/v) ethanol and 25% (v/v) glycerol and flash frozen in liquid nitrogen. Data were collected at ALS Beamline 5.0.2 at wavelength 1.0 Å and processed on an HKL2000 (ref. 39) out to 2.9 Å in space group P4 2 2/P4 2 2. dTor_9x31L was crystallized in 0.1 M sodium citrate pH 5.4 and 1.0 M ammonium phosphate monobasic at a concentration of 8.8 mg ml−1 in 3 μl drops containing 1 μl protein and 2 μl well solution. The crystal was transferred to a solution containing the well plus 25% (v/v) glycerol and flash frozen in liquid nitrogen. Data were collected on a Saturn 944+ charge-coupled device at wavelength 1.54 Å and processed on an HKL2000 (ref. 39) out to 2.5 Å in space group P2 2 2 . dTor_12x31L was crystallized in 0.9 M sodium malonate pH 7.0, 0.1 M HEPES pH 7.0 and 0.5% Jeffamine ED-2001 pH 7.0 at a concentration of 8.8 mg ml−1 in 2 μl drops containing 1 μl protein and 1 μl well solution. The crystal was transferred to a solution containing 0.675 M sodium malonate pH 7.0, 0.075 M HEPES pH 7.0, 0.375% Jeffamine ED-2001 pH 7.0 and 25% glycerol, and flash frozen in liquid nitrogen. Data were collected on a Saturn 944+ charge-coupled device at wavelength 1.54 Å and processed on an HKL2000 (ref. 39) out to 2.3 Å in space group R3:H. The dTor_6x35L and both dTor_3x33L_2-2 structures were solved by Molecular Replacement with Phaser40 via CCP4i41 using the Rosetta-designed structure as a search model. The structures were then built and refined using Coot42 and Refmac543, respectively. The structure of dTor_6x35L(SeMet) was solved by Molecular Replacement with Phaser40 via PHENIX44 using the best refined model of dTor_6x35L as a phasing model. The structure was then built and refined using Coot42 and PHENIX45, respectively. The structures of dTor_9x31L_sub and dTor_9x31L were solved by Molecular Replacement with Phaser40 via PHENIX44 using the Rosetta-designed structure as a search model. The structure was then built and refined using Coot42 and PHENIX45, respectively. The structure of dTor_12x31L was solved by Molecular Replacement with Phaser40 via PHENIX44 using a 4-repeat subunit the Rosetta-designed structure as a search model. The structure was then built and refined using Coot42 and PHENIX45, respectively. Final Ramachandran statistics after refinement were as follows (given as % preferred, % allowed, % outliers, respectively): dTor_6x35L(SeMet): 98.06, 1.94, 0.0; dTor_3x33L_2-2a: 99.48, 0.0, 0.52; dTor_3x33L_2-2b: 98.96, 0.52, 0.52; dTor_9x31L_sub: 98.31, 1.69, 0.0; dTor_9x31L: 99.28, 0.36, 0.36; dTor_12x31L: 99.0, 1.0, 0.0.
News Article | November 4, 2016
OKLAHOMA CITY--(BUSINESS WIRE)--Jasco Products, GE licensee and leader in Z-Wave wireless technologies, today announces the availability of three new products joining the largest line of Z-Wave connected lighting controls. Featuring the new Z-Wave Plus® Smart Door Sensor, Portable Smart Motion Sensor and 40 Amp Outdoor Direct Wire Smart Switch, the new GE branded Z-Wave products by Jasco are powered by the latest advancements in Z-Wave technology and now available in major retail stores and online. “These new innovations reinforce Jasco's position as a leader in the industry with over 2 million Z-Wave products installed in homes today and the largest ecosystem of Z-Wave connected lighting controls in the market,” said Cameron Trice, Jasco CEO. “A key for us moving forward is our continued commitment to developing connected home solutions with enhanced functionality and seamless connectivity with leading home automation systems.” The new patent-pending GE branded Z-Wave Plus Smart Door Sensor is the industry’s first battery-operated door sensor that discreetly fits onto the home’s door hinge to monitor the door opening and closing. The small, unobtrusive sensor is fixed in place by the door hinge pin and moves with the door without altering the room’s aesthetics. This innovative design means not having to worry about wires, door trim or crown molding interfering with the door sensor. By simply opening or closing the door, the sensor uses Z-Wave technology to wirelessly trigger scenes throughout your home and send and receive information to your Z-Wave hub, helping you stay safe and in control of your home at all times. Automate your lights to turn on and welcome you when you walk in, automatically adjust your smart thermostat, receive alerts or trigger alarms when doors unexpectedly open. The GE branded Z-Wave Plus Smart Door sensor works with leading Z-Wave hub providers such as SmartThings, Train, LG, Nexia and many others. The product MSRP is $39.99 and is available online at ezzwave.com and amazon.com. The GE branded Portable Smart Motion Sensor with Z-Wave Plus technology takes convenience to the next level. Placed in key locations like laundry rooms, hallways or the garage, the Portable Z-Wave Motion Sensor can signal lights to turn on and off based on occupancy, giving homeowners the convenience of hands-free operation and added energy savings. The discreet, versatile sensor can be mounted on the wall or ceiling or simply placed on a shelf, table, or desk–making them perfect for any room with a 180-degree detection range up to 45-feet away. The motion sensor can be powered by the built-in batteries or from USB power. The GE branded Portable Motion Sensor MSRP is $49.99 and is available at Wal-Mart and online at ezzwave.com and amazon.com. The GE branded Z-Wave Direct Wire Outdoor Smart Switch delivers simple automation for hard-wired appliances like water heaters, landscape lights, spas, pool pumps, baseboard heaters and more. Perfect for year-round use, the GE branded Direct Wire Smart Switch works with all Z-Wave compatible hubs for wireless scheduling and remote energy monitoring of appliances up to 40 Amps. Housed in a lockable, tamper-resistant metal case, the weather-resistant design is durable enough for extreme weather conditions and ensures wiring and settings are secure while keeping out dirt and debris. Save up to $2,200 each year on pool pumps or up to $150 on your water heater by scheduling runtimes and monitoring energy usage with state-of-the-art energy monitoring capabilities*. The GE branded 40 Amp Z-Wave Direct Wire Smart Switch has an MSRP of $179.99 and is available at Home Depot and online at ezzwave.com and amazon.com. For more information about GE branded Z-Wave products, visit ezzwave.com or follow the Jasco blog for the latest news and updates. *Cost savings based on 2HP pool pump operating 24-hours/day, 365-days/year at $0.12kwh vs. scheduling just 4-hours/day. Cost savings based on 50 gallon electric water heater operating 24-hours/day, 365-days/year at $0.12kwh vs. scheduling just 10-hours/day. Z-Wave technology and is an open internationally recognized ITU standard (G.9959). It is the leading wireless home control technology in the market today, with over 1400 certified interoperable products worldwide. Represented by the Z-Wave Alliance, and supported by more than 325 companies around the world, the standard is a key enabler of smart living solutions for home safety and security, energy, hospitality, office and light commercial applications. Z-Wave® is a registered trademark of Sigma Designs (NASDAQ: SIGM) and its subsidiaries in the United States and other countries. At Jasco, we design and develop products to simplify your life and connect your home. As a GE licensee, Jasco provides comprehensive product offerings in home automation, lighting, security, home entertainment, power and mobility products. While providing our retail partners full and far-reaching product assortments, we use our commitment to design, research and development to bring to market a steady flow of product innovations that energize and invigorate the home and mobile solution landscape. For more information, please visit www.byjasco.com. GE is a trademark of General Electric Company and under license to Jasco Products LLC, 10 E. Memorial Rd., Oklahoma City, OK 73114.
Mangabhai R.,Mangabhai Consulting |
Cave S.,JASCO |
Concrete (London) | Year: 2010
As part of CE marking under EN 1504 Parts 2 and 3(1), identification tests using BS EN 1767:1999(2) are required on the products at yearly intervals.