Sample records for batch rebinding assays
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Sorption of ochratoxin A from aqueous solutions using β-cyclodextrin-polyurethane polymer.
Appell, Michael; Jackson, Michael A
2012-02-01
The ability of a cyclodextrin-polyurethane polymer to remove ochratoxin A from aqueous solutions was examined by batch rebinding assays. The results from the aqueous binding studies were fit to two parameter models to gain insight into the interaction of ochratoxin A with the nanosponge material. The ochratoxin A sorption data fit well to the heterogeneous Freundlich isotherm model. The polymer was less effective at binding ochratoxin A in high pH buffer (9.5) under conditions where ochratoxin A exists predominantly in the dianionic state. Batch rebinding assays in red wine indicate the polymer is able to remove significant levels of ochratoxin A from spiked solutions between 1-10 μg·L(-1). These results suggest cyclodextrin nanosponge materials are suitable to reduce levels of ochratoxin A from spiked aqueous solutions and red wine samples.
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Sorption of Ochratoxin A from Aqueous Solutions Using β-Cyclodextrin-Polyurethane Polymer
Appell, Michael; Jackson, Michael A.
2012-01-01
The ability of a cyclodextrin-polyurethane polymer to remove ochratoxin A from aqueous solutions was examined by batch rebinding assays. The results from the aqueous binding studies were fit to two parameter models to gain insight into the interaction of ochratoxin A with the nanosponge material. The ochratoxin A sorption data fit well to the heterogeneous Freundlich isotherm model. The polymer was less effective at binding ochratoxin A in high pH buffer (9.5) under conditions where ochratoxin A exists predominantly in the dianionic state. Batch rebinding assays in red wine indicate the polymer is able to remove significant levels of ochratoxin A from spiked solutions between 1–10 μg·L−1. These results suggest cyclodextrin nanosponge materials are suitable to reduce levels of ochratoxin A from spiked aqueous solutions and red wine samples. PMID:22474569
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Sorption of Ochratoxin A from aqueous solutions using beta-cyclodextrin-polyurethane polymer
USDA-ARS?s Scientific Manuscript database
The ability of a cyclodextrin-polyurethane polymer to remove ochratoxin A from aqueous solutions, including wine, was examined by batch rebinding assays and equilibrium sorption isotherms. The results were fit to two parameter models. Freundlich analysis of the sorption isotherm indicates the polyme...
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Wu, Zhihui; Hou, Jiapeng; Wang, Yuyan; Chai, Miaolin; Xiong, Yan; Lu, Weiyue; Pan, Jun
2015-12-30
This paper reports studies on preparation and evaluation of amoxicillin loaded dual molecularly imprinted nanoparticles (Amo/Dual-MIPs) designed for anti-H. pylori therapy. Both MNQA and AmoNa were chosen as templates to prepare Dual-MIPs using inverse microemulsion polymerization method. NQA was modified with myristic acid (MNQA) to become amphiphilic and assist in leaving NQA cavities on the surface of Dual-MIPs for H. pylori adhesion. AmoNa was applied to produce imprinting sites in Dual-MIPs for rebinding AmoNa to exert its anti-H. pylori effect. Batch rebinding test demonstrated a preferential rebinding effect of NQA toward the Dual-MIPs. In vivofluorescence imaging showed the prolonged residence time of Dual-MIPs in H. pylori infected mice stomachs after intragastric administration of nanoparticles.In vivo H. pylori clearance tests indicated Amo/Dual-MIPs had a better aniti-H. pylori effect than amoxicillin powder did. In conclusion, Amo/Dual-MIPs may provide an alternative drug delivery strategy for anti-H. pylori therapy. Copyright © 2015 Elsevier B.V. All rights reserved.
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Effects of target binding kinetics on in vivo drug efficacy: koff , kon and rebinding.
Vauquelin, Georges
2016-08-01
Optimal drug therapy often requires continuing high levels of target occupancy. Besides the traditional pharmacokinetic contribution, target binding kinetics is increasingly considered to play an important role as well. While most attention has been focused on the dissociation rate of the complex, recent reports expressed doubt about the unreserved translatability of this pharmacodynamic property into clinical efficacy. 'Micro'-pharmacokinetic mechanisms like drug rebinding and partitioning into the cell membrane may constitute a potential fix. Simulations were based on solving differential equations. Based on a selected range of association and dissociation rate constants, kon and koff , and rebinding potencies of the drugs as variables, their effects on the temporal in vivo occupancy profile of their targets, after one or multiple repetitive dosings, have here been simulated. Most strikingly, the simulations show that, when rebinding is also taken into account, increasing kon may produce closely the same outcome as decreasing koff when dosing is performed in accordance with the therapeutically most relevant constant [Lmax ]/KD ratio paradigm. Also, under certain conditions, rebinding may produce closely the same outcome as invoking slow diffusion of the drug between the plasma compartment and a target-containing 'effect' compartment. Although the present simulations should only be regarded as a 'proof of principle', these findings may help pharmacologists and medicinal chemists to devise ex vivo and in vitro binding kinetic assays that are more relevant and translatable to in vivo settings. © 2016 The British Pharmacological Society.
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Synthesis of molecular imprinting polymers for extraction of gallic acid from urine.
Bhawani, Showkat Ahmad; Sen, Tham Soon; Ibrahim, Mohammad Nasir Mohammad
2018-02-21
The molecularly imprinted polymers for gallic acid were synthesized by precipitation polymerization. During the process of synthesis a non-covalent approach was used for the interaction of template and monomer. In the polymerization process, gallic acid was used as a template, acrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linker and 2,2'-azobisisobutyronitrile as an initiator and acetonitrile as a solvent. The synthesized imprinted and non-imprinted polymer particles were characterized by using Fourier-transform infrared spectroscopy and scanning electron microscopy. The rebinding efficiency of synthesized polymer particles was evaluated by batch binding assay. The highly selective imprinted polymer for gallic acid was MIPI1 with a composition (molar ratio) of 1:4:20, template: monomer: cross-linker, respectively. The MIPI1 showed highest binding efficiency (79.50%) as compared to other imprinted and non-imprinted polymers. The highly selective imprinted polymers have successfully extracted about 80% of gallic acid from spiked urine sample.
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Effects of target binding kinetics on in vivo drug efficacy: koff, kon and rebinding
2016-01-01
Abstract Background and Purpose Optimal drug therapy often requires continuing high levels of target occupancy. Besides the traditional pharmacokinetic contribution, target binding kinetics is increasingly considered to play an important role as well. While most attention has been focused on the dissociation rate of the complex, recent reports expressed doubt about the unreserved translatability of this pharmacodynamic property into clinical efficacy. ‘Micro’‐pharmacokinetic mechanisms like drug rebinding and partitioning into the cell membrane may constitute a potential fix. Experimental Approach Simulations were based on solving differential equations. Key Results Based on a selected range of association and dissociation rate constants, kon and koff, and rebinding potencies of the drugs as variables, their effects on the temporal in vivo occupancy profile of their targets, after one or multiple repetitive dosings, have here been simulated. Conclusions and Implications Most strikingly, the simulations show that, when rebinding is also taken into account, increasing kon may produce closely the same outcome as decreasing koff when dosing is performed in accordance with the therapeutically most relevant constant [Lmax]/K D ratio paradigm. Also, under certain conditions, rebinding may produce closely the same outcome as invoking slow diffusion of the drug between the plasma compartment and a target‐containing ‘effect’ compartment. Although the present simulations should only be regarded as a ‘proof of principle’, these findings may help pharmacologists and medicinal chemists to devise ex vivo and in vitro binding kinetic assays that are more relevant and translatable to in vivo settings. PMID:27129075
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Towards water compatible MIPs for sensing in aqueous media.
Horemans, F; Weustenraed, A; Spivak, D; Cleij, T J
2012-06-01
When synthesizing molecularly imprinted polymers (MIPs), a few fundamental principles should be kept in mind. There is a strong correlation between porogen polarity, MIP microenvironment polarity and the imprinting effect itself. The combination of these parameters eventually determines the overall binding behavior of a MIP in a given solvent. In addition, it is shown that MIP binding is strongly influenced by the polarity of the rebinding solvent. Because the use of MIPs in biomedical environments is of considerable interest, it is important that these MIPs perform well in aqueous media. In this article, various approaches are explored towards a water compatible MIP for the target molecule l-nicotine. To this end, the imprinting effect together with the MIP matrix polarity is fine-tuned during MIP synthesis. The binding behavior of the resulting MIPs is evaluated by performing batch rebinding experiments that makes it possible to select the most suitable MIP/non-imprinted polymer couple for future application in aqueous environments. One method to achieve improved compatibility with water is referred to as porogen tuning, in which porogens of varying polarities are used. It is demonstrated that, especially when multiple porogens are mixed, this approach can lead to superior performance in aqueous environments. Another method involves the incorporation of polar or non-polar comonomers in the MIP matrix. It is shown that by carefully selecting these monomers, it is also possible to obtain MIPs, which can selectively bind their target in water. Copyright © 2012 John Wiley & Sons, Ltd.
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Novel approach for extraction of quercetin using molecular imprinted membranes
NASA Astrophysics Data System (ADS)
Kamarudin, Siti Fatimah; Ahmad, Mohd Noor; Dzahir, Irfan Hatim Mohamed; Nasir, Azalina Mohamed; Ishak, Noorhidayah; Halim, Nurul Farhanah
2017-12-01
Quercetin imprinted membrane (QIM) was synthesized and applied for the extraction of quercetin. The quercetin imprinted membranes (QIM) were fabricated through a non-covalent approach via surface thermal polymerization. Polyvinylidene fluoride (PVDF) microfiltration membrane was used as a support to improve mechanical stability of the membrane. The thin imprinted layer was formed by copolymerization of acrylamide (AA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as crosslinker in the presence of quercetin as template in tetrahydrofuran (THF) solution. The Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscope (FESEM) were used to visualize the surface of membrane. Batch rebinding and binding kinetic experiments proved that the binding properties of the QIM are higher than non-imprinted membranes (NIM). QIM also have higher selectivity towards quercetin compared to sinensetin and rosmarinic acid.
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Feng, Zufei; Lu, Yan; Zhao, Yingjuan; Ye, Helin
2017-11-02
On the basis of magnetic molecularly imprinted polymer (MMIP) solid-phase extraction coupled with high performance liquid chromatography, we established a new method for the determination of the 4-methylimidazole (4-MEI) in soy sauce. Scanning electron microscopy (SEM), Fourier transform infrared (FT-IR), X-ray diffraction (XRD) and vibrating sample magnetometer (VSM) were used to characterize the synthesized MMIPs. To evaluate the polymers, batch rebinding experiments were carried out. The binding strength and capacity were determined from the derived Freundlich isotherm (FI) equation. The selective recognition capability of MMIPs was investigated with a reference compound and a structurally similar compound. As a selective pre-concentration sorbents for 4-methylimidazole in soy sauce, the MMIPs showed a satisfied recoveries rate of spiked samples, ranged from 97% to 105%. As a result, the prepared MMIPs could be applied to selectively pre-concentrate and determine 4-methylimidazole in soy sauce samples.
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Terao, E; Daas, A
2016-01-01
The European Pharmacopoeia (Ph. Eur.) prescribes the control of the activity of low molecular mass heparins by assays for anti-Xa and anti-IIa activities (monograph 0828), using a reference standard calibrated in International Units (IU). An international collaborative study coded BSP133 was launched in the framework of the Biological Standardisation Programme (BSP) run under the aegis of the Council of Europe and the European Commission to calibrate replacement batches for the dwindling stocks of the Heparin low-molecular-mass for assay Biological Reference Preparation (BRP) batch 8. Thirteen official medicines control and manufacturers laboratories from European and non-European countries took part in this study to calibrate two freeze-dried candidate batches against the 3rd International Standard (IS) for heparin, low molecular weight (11/176; 3rd IS). The Heparin low-molecular-mass for assay BRP (batch 8) was also included in the test panel to check the continuity between subsequent BRP batches. Taking into account the stability data, the results of this collaborative study and on the basis of the central statistical analysis performed at the European Directorate for the Quality of Medicines & HealthCare (EDQM), the 2 candidate batches were officially adopted by the Commission of the European Pharmacopoeia as Heparin low-molecular-mass for assay BRP batches 9 and 10 with assigned anti-Xa activities of 102 and 100 IU/vial and anti-IIa activities of 34 and 33 IU/vial respectively.
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Morgeaux, S; Manniam, I; Variot, P; Daas, A; Costanzo, A
2015-01-01
The current batch of the European Pharmacopoeia (Ph. Eur.) Biological Reference Reagents (BRRs) used for the in vitro potency assay of hepatitis A vaccines (HAV) by ELISA (enzymelinked immunosorbent assay) was established in 2012 for use in conjunction with Ph. Eur. general chapter 2.7.14 Assay of hepatitis A vaccine. It is composed of a coating reagent and a set of detection antibodies. As stocks of the latter are running low, the European Directorate for the Quality of Medicines & HealthCare (EDQM) organised a collaborative study to qualify replacement batches. The candidate BRR antibodies (primary monoclonal antibody and labelled secondary antibody) were prepared under appropriate conditions from starting materials similar to those used for the current batches. The new batches of antibodies were tested alongside previous batches of BRRs to ensure continuity, and the results confirmed that they were suitable for use in the potency assay of hepatitis A vaccines by ELISA using the standard method referenced in Ph. Eur. general chapter 2.7.14 at the same final concentrations as the previous batches, i.e. 1:500 for the primary monoclonal antibody and 1:400 for the secondary conjugated antibody. The outcome of the study allowed their establishment by the Ph. Eur. Commission in March 2015 as anti-hepatitis A virus primary detection antibody BRR batch 3 and conjugated secondary detection antibody BRR batch 3 respectively. They are available from the EDQM as hepatitis A vaccine ELISA detection antibodies set BRR batch 3.
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Cay, A B; Van der Stede, Y
2010-12-01
Although licensed batches of an enzyme-linked immunosorbent assay (ELISA) for Aujeszky's disease virus (ADV) were used, and the assays were performed within an ISO/IEC 17025 accredited quality control system, certain routine runs of the ADV ELISA were not validated using the quality system criteria, even when all technical parameters were controlled. Incubation at different temperatures and batch composition were identified as parameters that could result in non-validated assays/runs. Therefore, the effect of incubation temperature and batch composition on the analytical sensitivity of the ELISA was investigated. The World Organisation for Animal Health (OIE) standard reference serum ADV1 was diluted 1:8 and tested in 94 different glycoprotein E ELISA runs performed with different batches and different incubation temperatures. The incubation temperature and batch components had a significant influence on the qualitative result for the OIE standard reference serum. An incubation temperature of at least 22 degrees C was recommended, based on the results of this analysis. Which of the batch components caused these differences in sensitivity was not investigated further.
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Terao, E; Daas, A; Rautmann, G; Buchheit, K-H
2010-10-01
A collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM) in the context of the Biological Standardisation Programme (BSP), under the aegis of the Council of Europe and the European Commission, to establish replacement batches for the dwindling stocks of the Heparin low-molecular-mass for assay European Pharmacopoeia Biological Reference Preparation (BRP). The replacement batches of BRP are intended to be used in the assays for anti-Xa and anti-IIa activities, as described in the European Pharmacopoeia (Ph. Eur.) monograph Heparins, low-molecular-mass (0828). Three freeze-dried candidate batches were calibrated against the current International Standard (IS) for Heparin, lowmolecular- weight (2nd IS, 01/608). For the purpose of the continuity check between subsequent BRP batches, the current Heparin low-molecular-mass for assay BRP (batch 5) was also included in the test panel. Thirteen official medicines control and manufacturers laboratories from European and non-European countries contributed data. A central statistical analysis of the datasets was performed at the EDQM. On the basis of the results, the 3 candidate materials were assigned a potency of 104 IU/vial for the anti-Xa activity and 31 IU/vial for the anti-IIa activity. Taken into account the preliminary stability data and the results of this collaborative study, the 3 batches of candidate BRP were adopted in June 2010 by the Commission of the Ph. Eur. as Heparin low-molecular-mass for assay BRP batches 6, 7 and 8.
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Turner, Nicholas W.; Wright, Bryon E.; Hlady, Vladimir; Britt, David W.
2008-01-01
Protein imprinting leading to enhanced rebinding of ferritin to ternary lipid monolayers is demonstrated using a quartz crystal microbalance. Monolayers consisting of cationic dioctadecyldimethylammonium bromide, non-ionic methyl stearate, and poly(ethylene glycol) bearing phospholipids were imprinted with ferritin at the air/water interface of a Langmuir-Blodgett trough and transferred hydrated to hydrophobic substrates for study. This immobilization was shown by fluorescence correlation spectroscopy to significantly hinder any further diffusion of lipids, while rebinding studies demonstrated up to a six-fold increase in ferritin adsorption to imprinted versus control monolayers. A diminished rebinding of ferritin to its imprint was observed through pH reduction to below the protein isoelectric point, demonstrating the electrostatic nature of the interaction. Rebinding to films where imprint pockets remained occupied by the template protein was also minimal. Studies with a smaller acidic protein revealed the importance of the steric influence of poly(ethylene glycol) in forming the protein binding pockets, as albumin-imprinted monolayers showed low binding of ferritin, while ferritin-imprinted monolayers readily accommodated albumin. The controllable structure-function relationship and limitations of this system are discussed with respect to the application of protein imprinting in sensor development as well as fundamental studies of proteins at dynamic interfaces. PMID:17204279
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Molecularly imprinted polymers for separation of various sugars from human urine.
Okutucu, Burcu; Onal, Seçil
2011-12-15
Molecularly imprinted polymers were the new, simple and unexpensive materials that can be used in several clinical applications. Phenylboronic acid has been frequently used as functional monomer for the covalent imprinting of diols. In this study, the phenylboronic acid esters of fructose, galactose, glucose and raffinose were synthesized and then used as template analytes. The adsorption capacities of fructose, galactose and glucose-phenylboronic acid imprinted polymers were 75, 10 and 30%, respectively. The batch rebinding studies and Scatchard analysis were done for all sugar imprinted polymer. Glucose is one of the mostly found sugar in the urine. The glucose:phenylboronic acid imprinted polymer was used for the analysis of glucose, fructose, galactose, sucrose, maltose, lactose and raffinose in spiked urine. The selectivity of glucose:phenylboronic acid imprinted polymer to urine monosaccharides was found as nearly 45-55% and to di- and polysaccharides was found as 30-35%, respectively. Copyright © 2011 Elsevier B.V. All rights reserved.
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NASA Astrophysics Data System (ADS)
Zhang, Zhenyu
This thesis is written to summarize investigations of the mechanisms that underlie the kinetics of diatomic ligand rebinding to the iron atom of the heme group, which is chelated inside heme proteins. The family of heme proteins is a major object of studies for several branches of scientific research activity. Understanding the ligand binding mechanisms and pathways is one of the major goals for biophysics. My interests mainly focus on the physics of this ligand binding process. Therefore, to investigate the problem, isolated from the influence of the protein matrix, Fe-protophorphyrin IX is chosen as the prototype system in my studies. Myoglobin, the most extensively and intensively studied protein, is another ideal system that allows coupling the protein polypeptide matrix into the investigation. A technique to synchro-lock two laser pulse trains electronically is applied to our pump-probe spectroscopic studies. Based on this technique, a two color, fs/ps pump-probe system is developed which extends the temporal window for our investigation to 13ns and fills a gap existing in previous pump-probe investigations. In order to apply this newly-developed pump-probe laser system to implement systematic studies on the kinetics of diatomic ligand (NO, CO, O2) rebinding to heme and heme proteins, several experimental setups are utilized. In Chapter 1, the essential background knowledge, which helps to understand the iron-ligand interaction, is briefly described. In Chapter 2, in addition to a description of the preparation protocols of protein samples and details of the method for data analysis, three home-made setups are described, which include: a picosecond laser regenerative amplifier, a pump-probe application along the bore (2-inch in diameter) of a superconducting magnet and a temperature-controllable cryostat for spinning sample cell. Chapter 3 presents high magnetic field studies of several heme-ligand or protein-ligand systems. Pump-probe spectroscopy is used to study the ligand recombination after photolysis. No magnetic field induced rate changes are observed in any of these ligand recombination processes within the experimental detection limit. A magnetic field dependent CO rebinding behavior is observed for the FePPIX-CO sample in 80%glycerol/20%water environment. Careful data analysis indicates that this magnetic field induced change is due to the amplitude difference of a "fast" (<10ps) response with and without the magnetic field application (the amplitude changes from ˜55% at 0 Tesla to ˜45% at 10 Tesla). Kinetics of CO rebinding to FePPIX in 80%glycerol at the extremes of the magnetic field intensities (0Tesla vs. 10 Tesla) can be decomposed into a ligand rebinding process plus two 5ps decays heme cooling with different amplitudes. It leads to suggest a magnetic field induced change of a short-lived heme cooling response after photolysis. Also, CO rebinding kinetics to different heme compounds demonstrates a wide range for the Arrhenius pre-factors. This work reveals that the "spin-selection rule" does not play a key role in the recombination process of CO to heme iron. In Appendix 1, the recombination of oxymyoglobin and its mutants is investigated in the temperature range from 275K to 318K, using a home-made cryostat. Quite surprisingly, the O2 molecule rebinds to heme iron inside myoglobin with dramatically different behavior as the temperature is varied, depending on the protein environment. It shows little dependence (Mb), no dependence (V68W Mb mutant) and large dependence (L29W Mb mutant) in this 40K temperature window. To expand this temperature window, since the motor inside the cryostat is capable to work as low as 230K, glycerol is introduced into the protein preparation. It is observed that protein samples in a glycerol/water mixture, even with only 20% glycerol (in weight), the temperature dependences of the O2 rebinding to heme iron are dramatically altered. The O 2 rebinding behavior also shows a high dependence on the glycerol concentration in the solution. In Appendix 2, the absorption spectra of Fe-protoporphyrin IX in different monomeric complexes are investigated and compared. This work may suggest that, inside the CTAB micelles, ferrous Fe-PPIX exists in an equilibrium state of different species, CO probably can bind to FeII-PPIX with different trans ligand (H2O or OH-), and the trans effect, exhibiting while NO binds to H93G Mb mutants might also happen when NO binds to FeII-PPIX inside CTAB micelles. In Appendix 3, several ultrafast kinetics studies of CO rebinding to FePPIX are listed. They might be helpful for future studies on such systems. In Appendix 4, a series of systematic studies of the NO recombination to FeIIPPIX is performed with temperature and environment variation. A four-state model is proposed to explain the kinetics of NO-FeIIPPIX after photolysis. (Abstract shortened by UMI.)
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Corre, Guillaume; Dessainte, Michel; Marteau, Jean-Brice; Dalle, Bruno; Fenard, David; Galy, Anne
2016-02-01
Nonreplicative recombinant HIV-1-derived lentiviral vectors (LV) are increasingly used in gene therapy of various genetic diseases, infectious diseases, and cancer. Before they are used in humans, preparations of LV must undergo extensive quality control testing. In particular, testing of LV must demonstrate the absence of replication-competent lentiviruses (RCL) with suitable methods, on representative fractions of vector batches. Current methods based on cell culture are challenging because high titers of vector batches translate into high volumes of cell culture to be tested in RCL assays. As vector batch size and titers are continuously increasing because of the improvement of production and purification methods, it became necessary for us to modify the current RCL assay based on the detection of p24 in cultures of indicator cells. Here, we propose a practical optimization of this method using a pairwise pooling strategy enabling easier testing of higher vector inoculum volumes. These modifications significantly decrease material handling and operator time, leading to a cost-effective method, while maintaining optimal sensibility of the RCL testing. This optimized "RCL-pooling assay" ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.
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EDQM biological reference preparation for rabies vaccine (inactivated) for veterinary use.
Daas, A; Bruckner, L; Milne, C
2015-01-01
Rabies is a deadly zoonotic disease. Control of rabies in animals by vaccination is an important strategy to protect humans from infection and control the spread of the disease. Requirements for the quality control of rabies vaccines (inactivated) for veterinary use include an in vivo quantitative potency determination as outlined in the Ph. Eur. monograph 0451. Performance of this assay requires a reference preparation calibrated in International Units (IU). A European Pharmacopeia (Ph. Eur.) Biological Reference Preparation (BRP) for rabies vaccines (inactivated) for veterinary use, calibrated in IU, has been established for this purpose. Due to the dwindling stocks of the current batch (batch 4) of Ph. Eur. BRP for rabies vaccines (inactivated) for veterinary use, a collaborative study was run as part of the EDQM Biological Standardisation Programme to establish BRP batch 5. Ten laboratories, including Official Medicines Control Laboratories and manufacturers, participated. The candidate BRP5 was assayed against the 6(th) International Standard for rabies vaccine using the in vivo vaccination-challenge assay (monograph 0451) to assign a potency value. The candidate was also compared to BRP batch 4 to establish continuity. Taking into account the results from the comparisons a potency of 10 IU/vial was assigned and in March 2015 the Ph. Eur. Commission adopted the material as Ph. Eur. BRP for rabies vaccines (inactivated) for veterinary use batch 5. In addition to the in vivo assay 3 laboratories tested the candidate material using their in-house in vitro assays for information.
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Gao, Ruixia; Su, Xiaoqian; He, Xiwen; Chen, Langxing; Zhang, Yukui
2011-01-15
This paper reports the preparation of carbon nanotubes (CNTs) functionalized with molecularly imprinted polymers (MIPs) for advanced removal of estrone. CNTs@Est-MIPs nanocomposites with a well-defined core-shell structure were obtained using a semi-covalent imprinting strategy, which employed a thermally reversible covalent bond at the surface of silica-coated CNTs for a large-scale production. The morphology and structure of the products were characterised by transmission electron microscopy and Fourier transform infrared spectroscopy. The adsorption properties were demonstrated by equilibrium rebinding experiments and Scatchard analysis. The results demonstrate that the imprinted nanocomposites possess favourable selectivity, high capacity and fast kinetics for template molecule uptake, yielding an adsorption capacity of 113.5 μmol/g. The synthetic process is quite simple, and the different batches of synthesized CNTs@Est-MIPs nanocomposites showed good reproducibility in template binding. The feasibility of removing estrogenic compounds from environmental water using the CNTs@Est-MIPs nanocomposites was demonstrated using water samples spiked with estrone. Copyright © 2010 Elsevier B.V. All rights reserved.
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Dantsker, David; Roche, Camille; Samuni, Uri; Blouin, George; Olson, John S; Friedman, Joel M
2005-11-18
After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D --> A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B --> A and C --> A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.
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Effect of DNA Binding on Geminate CO Recombination Kinetics in CO-sensing Transcription Factor CooA*
Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan; Poulos, Thomas L.; Champion, Paul M.
2012-01-01
Carbon monoxide oxidation activator (CooA) proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding in two CooA homologues, Rhodospirillum rubrum (RrCooA) and Carboxydothermus hydrogenoformans (ChCooA). The effects of DNA binding and the truncation of the DNA-binding domain on the CO geminate recombination kinetics were specifically investigated. The CO rebinding kinetics in these CooA complexes take place on ultrafast time scales but remain non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the time scale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in the CooA proteins relaxes to form a very efficient hydrophobic trap for CO. DNA binding further tightens the narrow distal pocket and slightly weakens the iron-proximal histidine bond. Comparison of the CO rebinding kinetics of RrCooA, truncated RrCooA, and DNA-bound RrCooA proteins reveals that the uncomplexed and inherently flexible DNA-binding domain adds additional structural heterogeneity to the heme doming coordinate. When CooA forms a complex with DNA, the flexibility of the DNA-binding domain decreases, and the distribution of the conformations available in the heme domain becomes restricted. The kinetic studies also offer insights into how the architecture of the heme environment can tune entropic barriers in order to control the geminate recombination of CO in heme proteins, whereas spin selection rules play a minor or non-existent role. PMID:22544803
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Effect of DNA binding on geminate CO recombination kinetics in CO-sensing transcription factor CooA.
Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan; Poulos, Thomas L; Champion, Paul M
2012-06-22
Carbon monoxide oxidation activator (CooA) proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding in two CooA homologues, Rhodospirillum rubrum (RrCooA) and Carboxydothermus hydrogenoformans (ChCooA). The effects of DNA binding and the truncation of the DNA-binding domain on the CO geminate recombination kinetics were specifically investigated. The CO rebinding kinetics in these CooA complexes take place on ultrafast time scales but remain non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the time scale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in the CooA proteins relaxes to form a very efficient hydrophobic trap for CO. DNA binding further tightens the narrow distal pocket and slightly weakens the iron-proximal histidine bond. Comparison of the CO rebinding kinetics of RrCooA, truncated RrCooA, and DNA-bound RrCooA proteins reveals that the uncomplexed and inherently flexible DNA-binding domain adds additional structural heterogeneity to the heme doming coordinate. When CooA forms a complex with DNA, the flexibility of the DNA-binding domain decreases, and the distribution of the conformations available in the heme domain becomes restricted. The kinetic studies also offer insights into how the architecture of the heme environment can tune entropic barriers in order to control the geminate recombination of CO in heme proteins, whereas spin selection rules play a minor or non-existent role.
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[Evaluation of pipetting systems. III. Micropipette precision in a routine task].
Salas, R; Loría, A; Rocha, C
1995-01-01
To establish a norm of the precision achievable with a micropipette in an IRMA assay under routine conditions. A micropipette (Gilson) adjusted to dispense 100 microL was used by a single analyst with experience in its use. In each assay, ten aliquots of radioactive antiprolactin were pipetted in clean tubes (PRE-batch tubes), followed by pipetting of the tubes being processed in the assay, and at the end, a second pipetting of 10 aliquots in clean tubes (POST-batch tubes). The study includes the data of 15 consecutive batches during a seven month period with an overall mean of 283 tubes per batch. The PRE- and POST-tubes were read in a gamma counter (Crystal plus). The mean, SD and CV for PRE, POST and global (PRE+POST) tubes were calculated for each batch. The global CV of the 15 batches ranged from 1.6 to 6.9%, mean of 3.1%. We found no evidence of increased imprecision due to fatigue of the analyst, but surprisingly, we observed that in nine of the 15 batches there was a significant difference in the means of the PRE-tubes vs the POST-tubes (t test) without differences in precision. Thus, part of the global variability is due to what we have called pseudoimprecision (i.e. an increase in CV due to differences in means). In addition, the POST-tubes had higher values in the first 7 batches but the opposite occurred in the last 8 batches (table 2). This shift in the sign of the PRE-POST differences suggests the presence of opposite factors operating in time, i.e. one or more factors increased the volume of pipetting after using the pipette more than 150 times (batches 1-7) whereas other/others decreased it (batches 8-15). 1. Our first approximation to a norm of micropipetting precision in batches of 200-300 tubes was a CV of 3.1%. 2. This norm was influenced by a problem of pseudoimprecision detected ex-post-facto. 3. Our findings justify continuation studies to detect the pseudoimprecision and evaluate its causes prospectively.
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Jones, Sara; Wiesneth, Russ; Barry, Cathy; Webb, Erika; Belova, Larissa; Dolan, Peggy; Ho, Shiaolan; Abravaya, Klara; Cloherty, Gavin
2013-01-01
Diagnostic laboratories are under increasing pressure to improve and expand their services. Greater flexibility in sample processing is a critical factor that can improve the time to results while reducing reagent waste, making laboratories more efficient and cost-effective. The introduction of the Abbott mPLUS feature, with the capacity for extended use of amplification reagents, significantly increases the flexibility of the m2000 platform and enables laboratories to customize their workflows based on sample arrival patterns. The flexibility in sample batch size offered by mPLUS enables significant reductions in processing times. For hepatitis B virus tests, a reduction in sample turnaround times of up to 30% (105 min) was observed for batches of 12 samples compared with those for batches of 24 samples; for Chlamydia trachomatis/Neisseria gonorrhoeae tests, the ability to run batches of 24 samples reduced the turnaround time by 83% (54 min) compared with that for batches of 48 samples. Excellent correlations between mPLUS and m2000 standard condition results were observed for all RealTime viral load assays evaluated in this study, with correlation r values of 0.998 for all assays tested. For the qualitative RealTime C. trachomatis/N. gonorrhoeae assay, the overall agreements between the two conditions tested were >98% for C. trachomatis and 100% for N. gonorrhoeae. Comparable precision results were observed for the two conditions tested for all RealTime assays. The enhanced mPLUS capability provides clinical laboratories with increased efficiencies to meet increasingly stringent turnaround time requirements without increased costs associated with discarding partially used amplification reagents. PMID:24088850
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Lucic, Danijela; Jones, Sara; Wiesneth, Russ; Barry, Cathy; Webb, Erika; Belova, Larissa; Dolan, Peggy; Ho, Shiaolan; Abravaya, Klara; Cloherty, Gavin
2013-12-01
Diagnostic laboratories are under increasing pressure to improve and expand their services. Greater flexibility in sample processing is a critical factor that can improve the time to results while reducing reagent waste, making laboratories more efficient and cost-effective. The introduction of the Abbott mPLUS feature, with the capacity for extended use of amplification reagents, significantly increases the flexibility of the m2000 platform and enables laboratories to customize their workflows based on sample arrival patterns. The flexibility in sample batch size offered by mPLUS enables significant reductions in processing times. For hepatitis B virus tests, a reduction in sample turnaround times of up to 30% (105 min) was observed for batches of 12 samples compared with those for batches of 24 samples; for Chlamydia trachomatis/Neisseria gonorrhoeae tests, the ability to run batches of 24 samples reduced the turnaround time by 83% (54 min) compared with that for batches of 48 samples. Excellent correlations between mPLUS and m2000 standard condition results were observed for all RealTime viral load assays evaluated in this study, with correlation r values of 0.998 for all assays tested. For the qualitative RealTime C. trachomatis/N. gonorrhoeae assay, the overall agreements between the two conditions tested were >98% for C. trachomatis and 100% for N. gonorrhoeae. Comparable precision results were observed for the two conditions tested for all RealTime assays. The enhanced mPLUS capability provides clinical laboratories with increased efficiencies to meet increasingly stringent turnaround time requirements without increased costs associated with discarding partially used amplification reagents.
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Kinetics of CO Recombination to the Heme in Geobacillus Stearothermophilus Nitric Oxide Synthase†
Whited, Charlotte A.; Warren, Jeffrey J.; Lavoie, Katherine D.; Winkler, Jay R.; Gray, Harry B.
2012-01-01
We report the kinetics of CO rebinding to the heme in His134Ser, Ile223Val and His134Ser/Ile223Ser mutants of Geobacillus stearothermophilus nitric oxide synthase (gsNOS). The amplitudes of the two observed kinetics phases, which are insensitive to CO concentration, depend on enzyme concentration. We suggest that two forms of gsNOS are in equilibrium under the conditions employed (6.1–27 µM gsNOS with 20 or 100% CO atmosphere). The kinetics of CO rebinding to the heme do not depend on the identity of the NO-gate residues at positions 134 and 223. PMID:23976816
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A rapid and efficient assay for extracting DNA from fungi
Griffin, Dale W.; Kellogg, C.A.; Peak, K.K.; Shinn, E.A.
2002-01-01
Aims: A method for the rapid extraction of fungal DNA from small quantities of tissue in a batch-processing format was investigated. Methods and Results: Tissue (< 3.0 mg) was scraped from freshly-grown fungal isolates. The tissue was suspended in buffer AP1 and subjected to seven rounds of freeze/thaw using a crushed dry ice/ethanol bath and a boiling water bath. After a 30 min boiling step, the tissue was quickly ground against the wall of the microfuge tube using a sterile pipette tip. The Qiagen DNeasy Plant Tissue Kit protocol was then used to purify the DNA for PCR/ sequencing applications. Conclusions: The method allowed batch DNA extraction from multiple fungal isolates using a simple yet rapid and reliable assay. Significance and Impact of the Study: Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays that previously required specialized instrumentation, was time-consuming or was not conducive to batch processing.
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Shaikh, Huma; Memon, Najma; Khan, Hamayun; Bhanger, M I; Nizamani, S M
2012-07-20
The molecularly imprinted polymer (MIP) selective for di(2-ethylhexyl) phthalate (DEHP) an environmental endocrine disruptor was prepared by suspension polymerization using methacrylamide as functional monomer and N,N'-methylene-bis-acrylamide as cross-linker. The imprinted polymer was employed for solid-phase extraction of DEHP from water samples of environmental importance and characterized by FT-IR and SEM. The adsorption properties of the imprinted polymer were demonstrated by equilibrium rebinding experiments, Pseudo-second-order kinetic model, Sips isotherm and Scatchard analysis. The reusability of MIP was checked for at least six repeated batch adsorption cycles and the results showed almost no deterioration in the adsorption capacity. The competitive recognition studies were performed with DEHP and structurally similar compounds; dimethyl phthalate (DMP), diethyl phthalate (DEP), and dibutyl phthalate (DBP). The imprinting factor (IF) of DEHP was found to be 12.86 which was much higher than the imprinting factors (IF) of other phthalates. A method constituted by molecularly imprinted solid-phase extraction (MISPE) with GC-FID was developed for DEHP analysis in water samples under very simple conditions. Sample loading and desorption conditions were also optimized. The MISPE method's linearity ranged from 0.035 to 3.0 μg ml⁻¹ with r² = 0.9998. Intra-assay, interassay precision and accuracy ranged from 0.0168% to 1.017%, 1.130% to 4.799% and 94.98% to 99.35%, respectively. The LOD and LOQ were found to be 0.011 and 0.035 μg ml⁻¹, respectively. Synthesized MIP was employed in MISPE for cleaning up the spiked river water samples prior to gas chromatographic analysis. The river samples were found to contain DEHP in the range of 1.4 × 10⁻³ to 0.349 μg ml⁻¹. Copyright © 2012 Elsevier B.V. All rights reserved.
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Lühn, Susanne; Grimm, Juliane C; Alban, Susanne
2014-04-10
Sulfated polysaccharides (SP) from algae are of great interest due to their manifold biological activities. Obstacles to commercial (especially medical) application include considerable variability and complex chemical composition making the analysis and the quality control challenging. The aim of this study was to evaluate a simple microplate assay for screening the quality of SP. It is based on the fluorescence intensity (FI) increase of the sensor molecule Polymer-H by SP and was originally developed for direct quantification of SP. Exemplarily, 65 SP batches isolated from the red alga Delesseria sanguinea (D.s.-SP) and several other algae polysaccharides were investigated. Their FI increase in the Polymer-H assay was compared with other analytical parameters. By testing just one concentration of a D.s.-SP sample, quality deviations from the reference D.s.-SP and thus both batch-to-batch variability and stability can be detected. Further, structurally distinct SP showed to differ in their concentration-dependent FI profiles. By using corresponding reference compounds, the Polymer-H assay is therefore applicable as identification assay with high negative predictability. In conclusion, the Polymer-H assay showed to represent not only a simple method for quantification, but also for characterization identification and differentiation of SP of marine origin.
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Wang, Yongmei; Yang, Chongchong; Sun, Yan; Qiu, Fengtao; Xiang, Yang; Fu, Guoqi
2018-02-01
Surface molecular imprinting over functionalized nanoparticles has proved to be an effective approach for construction of artificial nanomaterials for protein recognition. Herein, we report a strategy for synthesis of core-shell protein-imprinted nanoparticles by the functionalization of nano-cores with ionic liquids followed by aqueous precipitation polymerization to build thermo-responsive imprinted polymer nano-shells. The immobilized ionic liquids can form multiple interactions with the protein template. The polymerization process can produce thermo-reversible physical crosslinks, which are advantageous to enhancing imprinting and facilitating template removal. With bovine hemoglobin as a model template, the imprinted nanoparticles showed temperature-sensitivity in both dispersion behaviors and rebinding capacities. Compared with the ionic-liquid-modified core nanoparticles, the imprinted particles exhibited greatly increased selectivity and two orders of magnitude higher binding affinity for the template protein. The imprinted nanoparticles achieved relatively high imprinting factor up to 5.0 and specific rebinding capacity of 67.7 mg/g, respectively. These nanoparticles also demonstrated rapid rebinding kinetics and good reproducibility after five cycles of adsorption-regeneration. Therefore, the presented approach may be viable for the fabrication of high-performance protein-imprinted nanoparticles with temperature sensitivity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Kinetic modelling of anaerobic hydrolysis of solid wastes, including disintegration processes
DOE Office of Scientific and Technical Information (OSTI.GOV)
García-Gen, Santiago; Sousbie, Philippe; Rangaraj, Ganesh
2015-01-15
Highlights: • Fractionation of solid wastes into readily and slowly biodegradable fractions. • Kinetic coefficients estimation from mono-digestion batch assays. • Validation of kinetic coefficients with a co-digestion continuous experiment. • Simulation of batch and continuous experiments with an ADM1-based model. - Abstract: A methodology to estimate disintegration and hydrolysis kinetic parameters of solid wastes and validate an ADM1-based anaerobic co-digestion model is presented. Kinetic parameters of the model were calibrated from batch reactor experiments treating individually fruit and vegetable wastes (among other residues) following a new protocol for batch tests. In addition, decoupled disintegration kinetics for readily and slowlymore » biodegradable fractions of solid wastes was considered. Calibrated parameters from batch assays of individual substrates were used to validate the model for a semi-continuous co-digestion operation treating simultaneously 5 fruit and vegetable wastes. The semi-continuous experiment was carried out in a lab-scale CSTR reactor for 15 weeks at organic loading rate ranging between 2.0 and 4.7 g VS/L d. The model (built in Matlab/Simulink) fit to a large extent the experimental results in both batch and semi-continuous mode and served as a powerful tool to simulate the digestion or co-digestion of solid wastes.« less
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Measurements of heme relaxation and ligand recombination in strong magnetic fields.
Zhang, Zhenyu; Benabbas, Abdelkrim; Ye, Xiong; Yu, Anchi; Champion, Paul M
2009-08-06
Heme cooling signals and diatomic ligand recombination kinetics are measured in strong magnetic fields (up to 10 T). We examined diatomic ligand recombination to heme model compounds (NO and CO), myoglobin (NO and O(2)), and horseradish peroxidase (NO). No magnetic field induced rate changes in any of the samples were observed within the experimental detection limit. However, in the case of CO binding to heme in glycerol and O(2) binding to myoglobin, we observe a small magnetic field dependent change in the early time amplitude of the optical response that is assigned to heme cooling. One possibility, consistent with this observation, is that there is a weak magnetic field dependence of the nonradiative branching ratio into the vibrationally hot electronic ground state during CO photolysis. Ancillary studies of the "spin-forbidden" CO binding reaction in a variety of heme compounds in the absence of magnetic field demonstrate a surprisingly wide range for the Arrhenius prefactor. We conclude that CO binding to heme is not always retarded by unfavorable spin selection rules involving a double spin-flip superexchange mechanism. In fact, it appears that the small prefactor ( approximately 10(9) s(-1)) found for CO rebinding to Mb may be anomalous, rather than the general rule for heme-CO rebinding. These results point to unresolved fundamental issues that underlie the theory of heme-ligand photolysis and rebinding.
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Dantsker, David; Samuni, Uri; Friedman, Joel M; Agmon, Noam
2005-06-01
Geminate CO rebinding in myoglobin is studied for two viscous solvents, trehalose and sol-gel (bathed in 100% glycerol) at several temperatures. Mutations in key distal hemepocket residues are used to eliminate or enhance specific relaxation modes. The time-resolved data are analyzed with a modified Agmon-Hopfield model which is capable of providing excellent fits in cases where a single relaxation mode is dominant. Using this approach, we determine the relaxation rate constants of specific functionally important modes, obtaining also their Arrhenius activation energies. We find a hierarchy of distal pocket modes controlling the rebinding kinetics. The "heme access mode" (HAM) is responsible for the major slow-down in rebinding. It is a solvent-coupled cooperative mode which restricts ligand return from the xenon cavities. Bulky side-chains, like those His64 and Trp29 (in the L29W mutant), operate like overdamped pendulums which move over and block the binding site. They may be either unslaved (His64) or moderately slaved (Trp29) to the solvent. Small side-chain relaxations, most notably of leucines, are revealed in some mutants (V68L, V68A). They are conjectured to facilitate inter-cavity ligand motion. When all relaxations are arrested (H64L in trehalose), we observe pure inhomogeneous kinetics with no temperature dependence, suggesting that proximal relaxation is not a factor on the investigated timescale.
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Isolation of a matrix that binds medial Golgi enzymes
1994-01-01
Rat liver Golgi stacks were extracted with Triton X-100 at neutral pH. After centrifugation the low speed pellet contained two medial-Golgi enzymes, N-acetylglucosaminyltransferase I and mannosidase II, but no enzymes or markers from other parts of the Golgi apparatus. Both were present in the same structures which appeared, by electron microscopy, to be small remnants of cisternal membranes. The enzymes could be removed by treatment with low salt, leaving behind a salt pellet, which we term the matrix. Removal of salt caused specific re-binding of both enzymes to the matrix, with an apparent dissociation constant of 3 nM for mannosidase II. Re-binding was abolished by pretreatment of intact Golgi stacks with proteinase K, suggesting that the matrix was present between the cisternae. PMID:8106542
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Pardo, Antonelle; Mespouille, Laetitia; Blankert, Bertrand; Trouillas, Patrick; Surin, Mathieu; Dubois, Philippe; Duez, Pierre
2014-10-17
Molecularly imprinted polymers (MIPs) based on quercetin and synthesized by either bulk, precipitation or suspension polymerization were characterized in terms of size and shape by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). After a study of rebinding protocols, the optimal materials were evaluated as sorbents for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) to confirm the presence of imprinted cavities and to assess their selectivity. Besides quercetin, other structurally related natural compounds, naringenin, daidzein and curcumin, were employed for selectivity tests of MIPs. Although rebinding protocols previously described for such MIPs are typically based on binding, washing and eluting methanol-based solutions, we show that this highly polar solvent leads to weak specific interactions (imprinting factor<1) and poor sorbent properties, most probably because of hydrogen-bonding interferences between the MIP and MeOH. Similar experiments performed in tetrahydrofuran yield to much more improved properties (imprinting factor>2.4). This calls for reviewing most of previously published data on quercetin-MIPs; in proper binding conditions, published MIPs may prove more performing than initially assessed. As expected, tested MIPs exhibited the highest selective rebinding towards quercetin template (imprinting effect, quercetin, 3.41; naringenin, 1.54; daidzein, 1.38; curcumin, 1.67); the differences in selectivity between quercetin analogues were explained by the ligand geometries and H-bonding patterns obtained from quantum-chemical calculations. The evaluation of MIPs under identical analytical conditions allowed investigating the effect of the production method on chromatographic performances. The MIPs in bead materials (for quercetin, peak width, 0.69; number of theoretical plates, 143; symmetry factor, 2.22) provided a significant improvement in chromatographic efficiency over the bulk materials (for quercetin, peak width, 1.25; number of theoretical plates, 115; symmetry factor, 2.92). Using the quercetin-beaded MIP as SPE sorbent, quercetin was selectively extracted from Allium cepa L. extract. The MIP developed in this work therefore appears highly promising for the enrichment and determination of quercetin in natural products. Copyright © 2014 Elsevier B.V. All rights reserved.
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Internal quality assurance in a clinical virology laboratory. II. Internal quality control.
Gray, J J; Wreghitt, T G; McKee, T A; McIntyre, P; Roth, C E; Smith, D J; Sutehall, G; Higgins, G; Geraghty, R; Whetstone, R
1995-01-01
AIMS--In April 1991 additional quality control procedures were introduced into the virology section of the Clinical Microbiology and Public Health Laboratory, Cambridge. Internal quality control (IQC) samples were gradually included in the serological assays performed in the laboratory and supplemented kit controls and standard sera. METHODS--From April 1991 to December 1993, 2421 IQC procedures were carried out with reference sera. RESULTS--The IQC samples were evaluated according to the Westgard rules. Violations were recorded in 60 of 1808 (3.3%) controls and were highest in the IQC samples of complement fixation tests (25/312 (8%) of controls submitted for complement fixation tests). CONCLUSIONS--The inclusion of IQC samples in the serological assays performed in the laboratory has highlighted batch to batch variation in commercial assays. The setting of acceptable limits for the IQC samples has increased confidence in the validity of assay results. PMID:7730475
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A general mechanism for competitor-induced dissociation of molecular complexes
Paramanathan, Thayaparan; Reeves, Daniel; Friedman, Larry J.; Kondev, Jane; Gelles, Jeff
2014-01-01
The kinetic stability of non-covalent macromolecular complexes controls many biological phenomena. Here we find that physical models of complex dissociation predict that competitor molecules will in general accelerate the breakdown of isolated bimolecular complexes by occluding rapid rebinding of the two binding partners. This prediction is largely independent of molecular details. We confirm the prediction with single-molecule fluorescence experiments on a well-characterized DNA strand dissociation reaction. Contrary to common assumptions, competitor–induced acceleration of dissociation can occur in biologically relevant competitor concentration ranges and does not necessarily implyternary association of competitor with the bimolecular complex. Thus, occlusion of complex rebinding may play a significant role in a variety of biomolecular processes. The results also show that single-molecule colocalization experiments can accurately measure dissociation rates despite their limited spatio temporal resolution. PMID:25342513
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Jasmine, Farzana; Shinkle, Justin; Sabarinathan, Mekala; Ahsan, Habibul; Pierce, Brandon L; Kibriya, Muhammad G
2018-03-12
Relative telomere length (RTL) is a potential biomarker of aging and risk for chronic disease. Previously, we developed a probe-based RTL assay on Luminex platform, where probes for Telomere (T) and reference gene (R) for a given DNA sample were tested in a single well. Here, we describe a method of pooling multiple samples in one well to increase the throughput and cost-effectiveness. We used four different microbeads for the same T-probe and four different microbeads for the same R-probe. Each pair of probe sets were hybridized to DNA in separate plates and then pooled in a single plate for all the subsequent steps. We used DNA samples from 60 independent individuals and repeated in multiple batches to test the precision. The precision was good to excellent with Intraclass correlation coefficient (ICC) of 0.908 (95% CI 0.856-0.942). More than 67% of the variation in the RTL could be explained by sample-to-sample variation; less than 0.1% variation was due to batch-to-batch variation and 0.3% variation was explained by bead-to-bead variation. We increased the throughput of RTL Luminex assay from 60 to 240 samples per run. The new assay was validated against the original Luminex assay without pooling (r = 0.79, P = 1.44 × 10 -15 ). In an independent set of samples (n = 550), the new assay showed a negative correlation of RTL with age (r = -0.41), a result providing external validation for the method. We describe a novel high throughput pooled-sample multiplex Luminex assay for RTL with good to excellent precision suitable for large-scale studies. © 2018 Wiley Periodicals, Inc.
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Tackling the widespread and critical impact of batch effects in high-throughput data.
Leek, Jeffrey T; Scharpf, Robert B; Bravo, Héctor Corrada; Simcha, David; Langmead, Benjamin; Johnson, W Evan; Geman, Donald; Baggerly, Keith; Irizarry, Rafael A
2010-10-01
High-throughput technologies are widely used, for example to assay genetic variants, gene and protein expression, and epigenetic modifications. One often overlooked complication with such studies is batch effects, which occur because measurements are affected by laboratory conditions, reagent lots and personnel differences. This becomes a major problem when batch effects are correlated with an outcome of interest and lead to incorrect conclusions. Using both published studies and our own analyses, we argue that batch effects (as well as other technical and biological artefacts) are widespread and critical to address. We review experimental and computational approaches for doing so.
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Rogers, Thomas A.
2011-01-01
The thermal deactivation of TEM-1 β-lactamase was examined using two experimental techniques: a series of isothermal batch assays and a single, continuous, non-isothermal assay in an enzyme membrane reactor (EMR). The isothermal batch-mode technique was coupled with the three-state “Equilibrium Model” of enzyme deactivation, while the results of the EMR experiment were fitted to a four-state “molten globule model”. The two methods both led to the conclusions that the thermal deactivation of TEM-1 β-lactamase does not follow the Lumry-Eyring model and that the Teq of the enzyme (the point at which active and inactive states are present in equal amounts due to thermodynamic equilibrium) is at least 10 °C from the Tm (melting temperature), contrary to the idea that the true temperature optimum of a biocatalyst is necessarily close to the melting temperature. PMID:22039393
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Martin, J; Daas, A; Milne, C
2016-01-01
Inactivated poliomyelitis vaccines are an important part of the World Health Organization (WHO) control strategy to eradicate poliomyelitis. Requirements for the quality control of poliomyelitis vaccines (inactivated) include the use of an in vitro D antigen quantification assay for potency determination on the final lot as outlined in the European Pharmacopoeia (Ph. Eur.) monograph 0214. Performance of this assay requires a reference preparation calibrated in International Units (IU). A Ph. Eur. biological reference preparation (BRP) for poliomyelitis vaccine (inactivated) calibrated in IU has been established for this purpose. Due to the dwindling stocks of batch 2 of the BRP a collaborative study was run as part of the European Directorate for the Quality of Medicines & HealthCare (EDQM) Biological Standardisation Programme to establish BRP batch 3 (BRP3). Twelve laboratories including Official Medicines Control Laboratories (OMCLs) and manufacturers participated. The candidate BRP3 (cBRP3) was from the same source and had the same characteristics as BRP batch 2 (BRP2). During the study the candidate was calibrated against the 3 rd International Standard for inactivated poliomyelitis vaccine using in-house D antigen ELISA assays in line with the Ph. Eur. monograph 0214. The candidate was also compared to BRP2 to evaluate the continuity. Based on the results of the study, values of 320 DU/mL, 78 DU/mL and 288 DU/mL (D antigen units/mL) (IU) for poliovirus type 1, 2 and 3 respectively were assigned to the candidate. In June 2016, the Ph. Eur. Commission adopted the material as Ph. Eur. BRP for poliomyelitis vaccine (inactivated) batch 3.
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Zhou, Xi; Wang, Anqi; Yu, Chenfei; Wu, Shishan; Shen, Jian
2015-06-10
A facilely prepared fluorescence sensor was developed for dopamine (DA) determination based on polyindole/graphene quantum dots molecularly imprinted polymers (PIn/GQDs@MIPs). The proposed sensor exhibits a high sensitivity with a linear range of 5 × 10(-10) to 1.2 × 10(-6) M and the limit of detection as low as 1 × 10(-10) M in the determination of DA, which is probably due to the tailor-made imprinted cavities for binding DA thought hydrogen bonds between amine groups of DA and oxygen-containing groups of the novel composite. Furthermore, the prepared sensor can rebind DA in dual-type: a low affinity type (noncovalent interaction is off) and a high affinity type (noncovalent interaction is on), and the rebinding interaction can be adjusted by tuning the pH, which shows a unique potential for adjusting the binding interaction while keeping the specificity, allowing for wider applications.
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Establishment of hepatitis A vaccine (inactivated, non-adsorbed) BRP batches 2 and 3.
Morgeaux, S; Manniam, I; Variot, P; Buchheit, K H; Daas, A; Wierer, M; Costanzo, A
2015-01-01
The current hepatitis A vaccine (HAV), inactivated, non-adsorbed, European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) is used for the in vitro potency assay of HAV as prescribed by the Ph. Eur. general chapter 2.7.14 Assay of hepatitis A vaccine. This reference preparation was calibrated in 2008 through an international collaborative study and was assigned a potency of 12 IU/mL. During use of this BRP it appeared to be inapplicable in certain cases due to a low nominal antigen content. Consequently, the European Directorate for the Quality of Medicines and HealthCare (EDQM) established replacement batches for this BRP, calibrated against the 1(st) WHO International Standard (IS) for HAV (inactivated), using the standard in vitro ELISA (enzyme-linked immunosorbent assay) method validated previously. The results of the study showed that the candidate BRPs were suitable for the intended purpose, and following completion of the study, they were adopted in November 2014 by the Ph. Eur. Commission as HAV (inactivated, non-adsorbed) BRP batches 2 and 3, with an assigned potency of 1350 IU/mL, for in vitro antigen content determination by ELISA. As the amount of material in each vial largely exceeds the amount required for the performance of a single assay, the BRPs are to be aliquoted by users as single-use aliquots and refrozen below -50 °C prior to their use as reference preparations.
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Dressler, Dirk; Mander, Gerd; Fink, Klaus
2012-01-01
The biological potency of botulinum toxin (BT) drugs is determined by a standardised LD50 assay. However, the potency labelling varies vary amongst different BT drugs. One reason for this may be differences in the LD50 assays applied. When five unexpired batches of onabotulinumtoxinA (Botox(®)) and incobotulinumtoxinA (Xeomin(®)) are compared in the Xeomin(®) batch release assay, the potency variability of both BT drugs fell within the range allowed by the European Pharmacopoiea. Statistical analyses failed to detect differences in the potency labelling of both products. Although the existence of a conversion ratio has been questioned recently, our experimental data are in line with previous clinical experience showing that Botox(®) and Xeomin(®) can be compared using a 1:1 conversion ratio. Identical potency labelling allows easy exchange of both BT drugs in a therapeutic setting, and direct comparison of efficacy, adverse effects and costs.
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2009-09-01
active scripting, file downloads, installation of desktop items, signed and unsigned ActiveX controls, Java permissions, launching applications and...files in an IFRAME, running ActiveX controls and plug-ins, and scripting of Java applets [49]. This security measure is very effective against DNS
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[Potency testing of anti-lymphocyte Globulins: In vitro alternatives for the monkey skin-graft assay
Conrad, Christoph; Kabelitz, Dieter; Schäffner, Gabriele
1998-01-01
Antilymphocyte globulins (ALG) are immunosuppressive agents of animal origin currently used in clinical transplantation medicine and for the treatment of severe aplastic anemia. The potency of each batch is tested in vivo using primates as hosts for allogeneic skin transplantation. The test is done with a maximum of three animals, one as a control and two after the treatment with ALG. The two in vitro methods in use are a cytotoxic assay and the rosette inhibition assay. These methods are evaluated with the microscope. Besides wellfare aspects these methods require a lot of experience, are subjective, difficult to validate and the information about the biological potency of the sera is questionable. The aim of our study is a better biological characterisation as a prerequisite to subsequently define an in vitro alternative for the potency test in monkeys. Using a competition assay with monoclonal antibodies we can identify several specificities directed against functional molecules on T cells (e.g., CD2, CD3, CD5, CD28), B Cells (CD19), macrophages and natural killer cells (CD16) and nonlineage specificities such as CD18, CD25, CD29, CD95. This method could describe a part of the biological potency and control homogeneity of batches. The cytotoxic capacity of ALG either with or without complement as well as DNA-fragmentation characteristic for apoptosis can be analysed by flowcytometry using propidiumiodide- (PI) incorporation. Immunoprecipitation of cell-lysate with ALG
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Potency control of modified live viral vaccines for veterinary use.
Terpstra, C; Kroese, A H
1996-04-01
This paper reviews various aspects of efficacy, and methods for assaying the potency of modified live viral vaccines. The pros and cons of parametric versus non-parametric methods for analysis of potency assays are discussed and critical levels of protection, as determined by the target(s) of vaccination, are exemplified. Recommendations are presented for designing potency assays on master virus seeds and vaccine batches.
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Potency control of modified live viral vaccines for veterinary use.
Terpstra, C; Kroese, A H
1996-01-01
This paper reviews various aspects of efficacy, and methods for assaying the potency of modified live viral vaccines. The pros and cons of parametric versus non-parametric methods for analysis of potency assays are discussed and critical levels of protection, as determined by the target(s) of vaccination, are exemplified. Recommendations are presented for designing potency assays on master virus seeds and vaccine batches.
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NASA Astrophysics Data System (ADS)
Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.
2011-09-01
Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.
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Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.
2011-01-01
Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. PMID:21974603
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Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R
2011-09-01
Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. © 2011 American Institute of Physics
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NASA Astrophysics Data System (ADS)
Coppock, Matthew B.; Farrow, Blake; Warner, Candice; Finch, Amethist S.; Lai, Bert; Sarkes, Deborah A.; Heath, James R.; Stratis-Cullum, Dimitra
2014-05-01
Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design.
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Molecularly imprinted polymers for RGD selective recognition and separation.
Papaioannou, Emmanuel; Koutsas, Christos; Liakopoulou-Kyriakides, Maria
2009-03-01
Molecularly imprinted polymers that could recognize the tripeptide Arg-Gly-Asp have been produced with the use of two functional monomers and three different cross-linkers, respectively. Methacrylic acid and acrylamide were used as functional monomers and the role of the ethylene glycol dimethacrylate, trimethylpropane trimethacrylate and N,N'-methylene-bisacrylamide as crosslinking monomers, was investigated on their recognition capability. The % net rebinding and the imprinting factor values were obtained, giving for the methacrylic acid-trimethylpropane trimethacrylate polymer the highest values 12.3% and 2.44, respectively. In addition, this polymer presented lower dissociation constant (K(D)) value and the higher B (max)% of theoretical total binding sites than all the other polymers. Rebinding experiments with Lys-Gly-Asp, an analogue of Arg-Gly-Asp, and other different peptides, such as cholecystokinin C-terminal tri- and pentapeptide and gramicidin, further indicated the selectivity of methacrylic acid-trimethylpropane trimethacrylate copolymer for Arg-Gly-Asp giving specific selectivity factor values 1.27, 1.98, 1.31 and 1.67, respectively.
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A viscoelastic-stochastic model of the effects of cytoskeleton remodelling on cell adhesion.
Li, Long; Zhang, Wenyan; Wang, Jizeng
2016-10-01
Cells can adapt their mechanical properties through cytoskeleton remodelling in response to external stimuli when the cells adhere to the extracellular matrix (ECM). Many studies have investigated the effects of cell and ECM elasticity on cell adhesion. However, experiments determined that cells are viscoelastic and exhibiting stress relaxation, and the mechanism behind the effect of cellular viscoelasticity on the cell adhesion behaviour remains unclear. Therefore, we propose a theoretical model of a cluster of ligand-receptor bonds between two dissimilar viscoelastic media subjected to an applied tensile load. In this model, the distribution of interfacial traction is assumed to follow classical continuum viscoelastic equations, whereas the rupture and rebinding of individual molecular bonds are governed by stochastic equations. On the basis of this model, we determined that viscosity can significantly increase the lifetime, stability and dynamic strength of the adhesion cluster of molecular bonds, because deformation relaxation attributed to the viscoelastic property can increase the rebinding probability of each open bond and reduce the stress concentration in the adhesion area.
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The Third International Standard for Corticotrophin
Bangham, D. R.; Mussett, M. V.; Stack-Dunne, M. P.
1962-01-01
At its meeting in September 1957, the WHO Expert Committee on Biological Standardization agreed with the recommendation of the International Conference on Corticotrophin, held in July 1957, that a new international standard for corticotrophin should be set up, since the Second International Standard was made from crude material and was unsuitable for the assay of the purer preparations of corticotrophin now in general clinical use. In this paper, the authors describe the steps taken to establish the Third International Standard for Corticotrophin, from the preparation and international collaborative assay of the new material to the choice of the ”subcutaneous assay” for deriving the potency. The clinical and pharmacological implications of this choice are discussed. Since the preparation, characterization and exact quantitative assay of standards for corticotrophin are so difficult, several batches of approximately 3500 ampoules were prepared in a similar way from the same material to serve as an international Working Standard. Samples from two batches were included in the collaborative assay and found to have the same potency as the Third Standard. Sufficient ampoules of the Working Standard are available for use as national and laboratory standards. PMID:13966359
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Naik, Umesh Chandra; Das, Mihir Tanay; Sauran, Swati; Thakur, Indu Shekhar
2014-03-01
The present study compares in vitro toxicity of electroplating effluent after the batch treatment process with that obtained after the sequential treatment process. Activated charcoal prepared from sugarcane bagasse through chemical carbonization, and tolerant indigenous bacteria, Bacillus sp. strain IST105, were used individually and sequentially for the treatment of electroplating effluent. The sequential treatment involving activated charcoal followed by bacterial treatment removed 99% of Cr(VI) compared with the batch processes, which removed 40% (charcoal) and 75% (bacteria), respectively. Post-treatment in vitro cyto/genotoxicity was evaluated by the MTT test and the comet assay in human HuH-7 hepatocarcinoma cells. The sequentially treated sample showed an increase in LC50 value with a 6-fold decrease in comet-assay DNA migration compared with that of untreated samples. A significant decrease in DNA migration and an increase in LC50 value of treated effluent proved the higher effectiveness of the sequential treatment process over the individual batch processes. Copyright © 2014 Elsevier B.V. All rights reserved.
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Friedl, Gregor F; Mockaitis, Gustavo; Rodrigues, José A D; Ratusznei, Suzana M; Zaiat, Marcelo; Foresti, Eugênio
2009-10-01
A mechanically stirred anaerobic sequencing batch reactor containing anaerobic biomass immobilized on polyurethane foam cubes, treating low-strength synthetic wastewater (500 mg COD L(-1)), was operated under different operational conditions to assess the removal of organic matter and sulfate. These conditions were related to fill time, defined by the following feed strategies: batch mode of 10 min, fed-batch mode of 3 h and fed-batch mode of 6 h, and COD/[SO(4)(2-)] ratios of 1.34, 0.67, and 0.34 defined by organic matter concentration of 500 mg COD L(-1) and sulfate concentrations of 373, 746, and 1,493 mg SO(4)(2-) L(-1) in the influent. Thus, nine assays were performed to investigate the influence of each of these parameters, as well as the interaction effect, on the performance of the system. The reactor operated with agitation of 400 rpm, total volume of 4.0 L, and treated 2.0 L synthetic wastewater in 8-h cycles at 30 +/- 1 degrees C. During all assays, the reactor showed operational stability in relation to the monitored variables such as COD, sulfate, sulfide, sulfite, volatile acids, bicarbonate alkalinity, and solids, thus demonstrating the potential to apply this technology to the combined removal of organic matter and sulfate. In general, the results showed that the 3-h fed-batch operation with a COD/[SO(4)(2-)] ratio of 0.34 presented the best conditions for organic matter removal (89%). The best efficiency for sulfate removal (71%) was accomplished during the assay with a COD/[SO(4)(2-)] ratio of 1.34 and a fill time of 6 h. It was also observed that as fill time and sulfate concentration in the influent increased, the ratio between removed sulfate load and removed organic load also increased. However, it should be pointed out that the aim of this study was not to optimize the removal of organic matter and sulfate, but rather to analyze the behavior of the reactor during the different feed strategies and applied COD/[SO(4)(2-)] ratios, and mainly to analyze the interaction effect, an aspect that has not yet been explored in the literature for batch reactors.
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Molecularly imprinted titania nanoparticles for selective recognition and assay of uric acid
NASA Astrophysics Data System (ADS)
Mujahid, Adnan; Khan, Aimen Idrees; Afzal, Adeel; Hussain, Tajamal; Raza, Muhammad Hamid; Shah, Asma Tufail; uz Zaman, Waheed
2015-06-01
Molecularly imprinted titania nanoparticles are su ccessfully synthesized by sol-gel method for the selective recognition of uric acid. Atomic force microscopy is used to study the morphology of uric acid imprinted titania nanoparticles with diameter in the range of 100-150 nm. Scanning electron microscopy images of thick titania layer indicate the formation of fine network of titania nanoparticles with uniform distribution. Molecular imprinting of uric acid as well as its subsequent washing is confirmed by Fourier transformation infrared spectroscopy measurements. Uric acid rebinding studies reveal the recognition capability of imprinted particles in the range of 0.01-0.095 mmol, which is applicable in monitoring normal to elevated levels of uric acid in human blood. The optical shift (signal) of imprinted particles is six times higher in comparison with non-imprinted particles for the same concentration of uric acid. Imprinted titania particles have shown substantially reduced binding affinity toward interfering and structurally related substances, e.g. ascorbic acid and guanine. These results suggest the possible application of titania nanoparticles in uric acid recognition and quantification in blood serum.
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Chevaliez, Stéphane; Dubernet, Fabienne; Dauvillier, Claude; Hézode, Christophe; Pawlotsky, Jean-Michel
2017-06-01
Sensitive and accurate hepatitis C virus (HCV) RNA detection and quantification is essential for the management of chronic hepatitis C therapy. Currently available platforms and assays are usually batched and require at least 5hours of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed Aptima HCV Quant Dx assay that eliminates the need for batch processing and automates all aspects of nucleic acid testing in a single step, to accurately detect and quantify HCV RNA in a large series of patients infected with different HCV genotypes. The limit of detection was estimated to be 2.3 IU/mL. The specificity of the assay was 98.6% (95% confidence interval: 96.1%-99.5%). Intra-assay and inter-assay coefficients of variation ranged from 0.09% to 5.61%, and 1.05% to 3.65%, respectively. The study of serum specimens from patients infected with HCV genotypes 1 to 6 showed a satisfactory relationship between HCV RNA levels measured by the Aptima HCV Quant Dx assay, and both real-time PCR comparators (Abbott RealTime HCV and Cobas AmpliPrep/Cobas TaqMan HCV Test, version 2.0, assays). the new Aptima HCV Quant Dx assay is rapid, sensitive, reasonably specific and reproducible and accurately quantifies HCV RNA in serum samples from patients with chronic HCV infection, including patients on antiviral treatment. The Aptima HCV Quant Dx assay can thus be confidently used to detect and quantify HCV RNA in both clinical trials with new anti-HCV drugs and clinical practice in Europe and the US. Copyright © 2017 Elsevier B.V. All rights reserved.
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Findlay, L; Desai, T; Heath, A; Poole, S; Crivellone, M; Hauck, W; Ambrose, M; Morris, T; Daas, A; Rautmann, G; Buchheit, K H; Spieser, J M; Terao, E
2015-01-01
An international collaborative study was organised jointly by the World Health Organization (WHO)/National Institute for Biological Standards and Control (NIBSC), the United States Pharmacopeia (USP) and the European Directorate for the Quality of Medicines & HealthCare (EDQM/Council of Europe) for the establishment of harmonised replacement endotoxin standards for these 3 organisations. Thirty-five laboratories worldwide, including Official Medicines Control Laboratories (OMCLs) and manufacturers enrolled in the study. Three candidate preparations (10/178, 10/190 and 10/196) were produced with the same material and same formulation as the current reference standards with the objective of generating a new (3(rd)) International Standard (IS) with the same potency (10 000 IU/vial) as the current (2(nd)) IS, as well as new European Pharmacopoeia (Ph. Eur.). and USP standards. The suitability of the candidate preparations to act as the reference standard in assays for endotoxin performed according to compendial methods was evaluated. Their potency was calibrated against the WHO 2(nd) IS for Endotoxin (94/580). Gelation and photometric methods produced similar results for each of the candidate preparations. The overall potency estimates for the 3 batches were comparable. Given the intrinsic assay precision, the observed differences between the batches may be considered unimportant for the intended use of these materials. Overall, these results were in line with those generated for the establishment of the current preparations of reference standards. Accelerated degradation testing of vials stored at elevated temperatures supported the long-term stability of the 3 candidate preparations. It was agreed between the 3 organisations that batch 10/178 be shared between WHO and EDQM and that batches 10/190 and 10/196 be allocated to USP, with a common assigned value of 10 000 IU/vial. This value maintains the continuity of the global harmonisation of reference materials and unitage for the testing of endotoxins in parenteral pharmaceutical products. Based on the results of the collaborative study, batch 10/178 was established by the European Pharmacopoeia Commission as the Ph. Eur. Endotoxin Biological Reference Preparation (BRP) batch 5. The same batch was also established by the Expert Committee on Biological Standardisation (ECBS) of WHO as the WHO 3(rd) IS for Endotoxin. Batch 10/190 was adopted as the USP Endotoxin Reference Standard, lot H0K354 and vials from this same batch (10/190) will serve as the United States Food and Drug Administration (USFDA) Endotoxin Standard, EC-7.
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Lackner, F; Daas, A; Terao, E
2015-01-01
An international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) to calibrate replacement batches for the current European Pharmacopoeia (Ph. Eur.) prekallikrein activator (PKA) in albumin biological reference preparation (BRP), whose stocks were dwindling. The study was run in the framework of the Biological Standardisation Programme (BSP) of the Council of Europe and the European Union (EU) Commission. Twenty three laboratories from official medicines control authorities and manufacturers in Europe and outside Europe took part in the study. Three candidate replacement batches were produced from the same material as the one used for the World Health Organization (WHO) 2(nd) International Standard (IS) for PKA in albumin (02/168) and the Ph. Eur. PKA in albumin BRP batches 1, 2 and 3. Participants were requested to evaluate the candidate batches against the current WHO IS using their routine assay method. The Ph. Eur. PKA in albumin BRP batch 3 (BRP3) was also included in the test panel to ensure the continuity of the consecutive BRP batches. The study confirmed the stability of the PKA content of the current BRP3. The candidate batches were found to be comparable. Previous data on the starting material support its high stability. Thermal stress study on the candidate batches confirmed the stability of their PKA activity. The Commission of the Ph. Eur. officially adopted in November 2013 the 3 candidate batches as Ph. Eur. PKA in albumin BRP batches 4, 5 and 6 with an assigned content of 38 IU/vial. The activity of the 3 new batches of Ph. Eur. PKA in albumin BRP will be regularly monitored.
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Vanantwerpen, Gerty; Berkvens, Dirk; De Zutter, Lieven; Houf, Kurt
2015-07-09
Pigs are the main reservoir of human pathogenic Y. enterocolitica, and the microbiological and serological prevalence of this pathogen differs between pig farms. The infection status of pig batches at moment of slaughter is unknown while it is a possibility to classify batches. A relation between the presence of human pathogenic Yersinia spp. and the presence of antibodies could help to predict the infection of the pigs prior to slaughter. Pigs from 100 different batches were sampled. Tonsils and pieces of diaphragm were collected from 7047 pigs (on average 70 pigs per batch). The tonsils were analyzed using a direct plating method and the meat juice collected from the pieces of diaphragm was analyzed by Enzyme Linked ImmunoSorbent Assay. The microbiological and serological results were compared using a mixed-effects logistic regression at pig and batch level. Yersinia spp. were found in 2031 (28.8%) pigs, antibodies were present in 4692 (66.6%) pigs. According to the logistic regression, there was no relation at pig level between the presence of Yersinia spp. in tonsils and the presence of antibodies. Contrarily, at batch level, a mean activity value of 37 Optical Density (OD)% indicated a Yersinia spp. positive farm and the microbiological prevalence in pig batches could be estimated before shipment to the slaughterhouse. This offers the opportunity to classify batches based on their potential risk to contaminate carcasses with human pathogenic Yersinia spp. Copyright © 2015 Elsevier B.V. All rights reserved.
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Blass, Johanna; Albrecht, Marcel; Bozna, Bianca L; Wenz, Gerhard; Bennewitz, Roland
2015-05-07
We introduce a molecular toolkit for studying the dynamics in friction and adhesion from the single molecule level to effects of multivalency. As experimental model system we use supramolecular bonds established by the inclusion of ditopic adamantane connector molecules into two surface-bound cyclodextrin molecules, attached to a tip of an atomic force microscope (AFM) and to a flat silicon surface. The rupture force of a single bond does not depend on the pulling rate, indicating that the fast complexation kinetics of adamantane and cyclodextrin are probed in thermal equilibrium. In contrast, the pull-off force for a group of supramolecular bonds depends on the unloading rate revealing a non-equilibrium situation, an effect discussed as the combined action of multivalency and cantilever inertia effects. Friction forces exhibit a stick-slip characteristic which is explained by the cooperative rupture of groups of host-guest bonds and their rebinding. No dependence of friction on the sliding velocity has been observed in the accessible range of velocities due to fast rebinding and the negligible delay of cantilever response in AFM lateral force measurements.
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Study on the molecularly imprinted polymers with methyl-testosterone as the template.
Yang, Minli; Gu, Wancheng; Sun, Li; Zhang, Feng; Ling, Yun; Chu, Xiaogang; Wang, Daning
2010-04-15
Molecularly imprinted polymers (MIPs) using methyl-testosterone as the template, methacrylic acid (MAA) as the monomer and ethylene glycol dimethacrylate (EDMA) as the crosslinker were prepared by precipitation polymerization. The morphology of the obtained particles was characterized by scanning electron microscopy (SEM) and the pore size was measured by BET. Then, the specificity and selectivity of the MIPs were evaluated using the equilibrium rebinding experiments. Besides, the MIPs were also used as the stationary phase of HPLC column and the retention behaviour to the template and analogues was confirmed using HPLC-MS-MS. Finally, the real application of the methyl-testosterone imprinted polymers was evaluated using SPE procedure with the spiked tap water and lake water. The results indicated that the prepared methyl-testosterone imprinted polymer showed specific rebinding ability to its template and could retain the template strongly compared with other structural analogues. At the same time, the MIPs could be used as SPE column to enrich methyl-testosterone in the lake water and show broad prospects in real samples. (c) 2009 Elsevier B.V. All rights reserved.
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A viscoelastic–stochastic model of the effects of cytoskeleton remodelling on cell adhesion
Li, Long; Zhang, Wenyan
2016-01-01
Cells can adapt their mechanical properties through cytoskeleton remodelling in response to external stimuli when the cells adhere to the extracellular matrix (ECM). Many studies have investigated the effects of cell and ECM elasticity on cell adhesion. However, experiments determined that cells are viscoelastic and exhibiting stress relaxation, and the mechanism behind the effect of cellular viscoelasticity on the cell adhesion behaviour remains unclear. Therefore, we propose a theoretical model of a cluster of ligand–receptor bonds between two dissimilar viscoelastic media subjected to an applied tensile load. In this model, the distribution of interfacial traction is assumed to follow classical continuum viscoelastic equations, whereas the rupture and rebinding of individual molecular bonds are governed by stochastic equations. On the basis of this model, we determined that viscosity can significantly increase the lifetime, stability and dynamic strength of the adhesion cluster of molecular bonds, because deformation relaxation attributed to the viscoelastic property can increase the rebinding probability of each open bond and reduce the stress concentration in the adhesion area. PMID:27853571
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Anti-A and anti-B hemagglutinin depletion during Cohn purification process of 5% immunoglobulin.
Salvatore, Alfonso; Esin, Semih; Batoni, Giovanna; Ascione, Ester; Farina, Claudio; Nardini, Claudia
2015-07-01
Polyvalent immunoglobulin G (IgG) products obtained by fractionation of human plasma are widely used to treat a broad range of conditions, including immunodeficiency syndromes and autoimmune, inflammatory, and infectious diseases. For high-quality products and to minimize adverse events related to the use of intravenous IgG (IVIG) it is very important to perform detailed analyses of their components. One of these components, that in rare cases can cause severe hemolytic conditions, is the amount of hemagglutinins, natural antibodies that bind A and/or B (anti-A or -B) antigens present in red blood cells (RBCs). To characterize different IgG batches and to monitor the efficacy of the production procedure in the hemagglutinin reduction, a direct agglutination test (DAT) and a new flow cytometry (FC)-based assay were used for measuring the activity and the content of hemagglutinins in IgG samples obtained at different stages of the purification process. A total of 113 batches of 5% IVIG, produced in 2013 by Kedrion Biopharma, were analyzed for the ability to agglutinate RBCs by DAT. All batches tested were within the limits set by the European Pharmacopoeia. Three batches of 5% IVIG were analyzed for their hemagglutinin levels. The finished products and the production intermediates were evaluated by the DAT and the FC assay. A significant decrease of anti-A and anti-B titer after the Fraction (F)III precipitation was observed in all batches tested and an evaluation of the results obtained by the two methods was performed. This study shows that the hemagglutinin titer, accurately measured in a high number of 5% IVIG batches, is within the allowed limits for the DAT method. The specific production process employed, in particular the FIII precipitation step, successfully removes IgM and significantly reduces IgG class hemagglutinins. © 2015 AABB.
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[Study on HPLC fingerprint of Oldenlandia diffusa].
Chen, Yan; Yao, Zhi-Hong; Dai, Yi; Cheng, Hong; Wen, Li-Rong; Zhou, Guang-Xiong; Yao, Xin-Sheng
2012-06-01
To establish the HPLC fingerprint chromatogram of Oldenlandia diffusa coupled with chemometrics means for the quality control of multi-batches of medicinal material. The separation was developed on C18 column(4.6 mm x 250 mm, 5 microm) by gradient elution with acetonitrile-water(both containing 0.1 per thousand (V/V) ocetic acid) as mobile phase at a flow rate of 0.8 mL/min, the detection wavelength at 238 nm and column temperature at 30 degrees C. The HPLC fingerprint chromatogram of Oldenlandia diffusa was set up and the main characteristic peaks were identified by comparing with chemical reference substance. The quality of 22 batches of medicinal material was evaluated by similarity assay as well as principal component analysis (PCA) and cluster analysis. The established HPLC fingerprint chromatogram of Oldenlandia diffusa was specific, precise, reproducible and stable. 11 peaks were chemically identified. The similarity of 17 batches of Oldenlandia diffusa was obviously higher than 5 batches of adulterants. PCA showed that 17 batches of Oldenlandia diffusa were in a domain and 5 batches of adulterants were far apart from the domain. The cluster analysis of the 22 batches of medicinal material showed that 17 batches of Oldenlandia diffusa were in a cluster while 5 batches of adulterants were excluded. Further cluster analysis was carried out for the quality consistency of 17 batches of Oldenlandia diffusa and accordingly they were devided into 4 clusters. With the combination of chemometrics means, the HPLC fingerprint chromatogram provides a method for evaluation of authenticity and quality control of Oldenlandia diffusa, which is favorable to improve overall quality control of Oldenlandia diffusa.
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Prevention of the Posttraumatic Fibrotic Response in Joints
2016-12-01
fibrotic deposits. Evaluation of the efficacy of the proposed approach was achieved by biochemical assays of collagen content and composition, then by...the amount of cross-links in collagen deposits, by histological assays of involved tissues, and by biomechanical evaluation of the flexion contracture...batches collected from each bioreactor run were evaluated by analyzing the binding affinity of the purified antibody to procollagen I standard that
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A batch assay to measure microbial hydrogen sulfide production from sulfur-containing solid wastes.
Sun, Mei; Sun, Wenjie; Barlaz, Morton A
2016-05-01
Large volumes of sulfur-containing wastes enter municipal solid waste landfills each year. Under the anaerobic conditions that prevail in landfills, oxidized forms of sulfur, primarily sulfate, are converted to sulfide. Hydrogen sulfide (H2S) is corrosive to landfill gas collection and treatment systems, and its presence in landfill gas often necessitates the installation of expensive removal systems. For landfill operators to understand the cost of managing sulfur-containing wastes, an estimate of the H2S production potential is needed. The objective of this study was to develop and demonstrate a biochemical sulfide potential (BSP) test to measure the amount of H2S produced by different types of sulfur-containing wastes in a relatively fast (30days) and inexpensive (125mL serum bottles) batch assay. This study confirmed the toxic effect of H2S on both sulfate reduction and methane production in batch systems, and demonstrated that removing accumulated H2S by base adsorption was effective for mitigating inhibition. H2S production potentials of coal combustion fly ash, flue gas desulfurization residual, municipal solid waste combustion ash, and construction and demolition waste were determined in BSP assays. After 30days of incubation, most of the sulfate in the wastes was converted to gaseous or aqueous phase sulfide, with BSPs ranging from 0.8 to 58.8mLH2S/g waste, depending on the chemical composition of the samples. Selected samples contained solid phase sulfide which contributed to the measured H2S yield. A 60day incubation in selected samples resulted in 39-86% additional sulfide production. H2S production measured in BSP assays was compared with that measured in simulated landfill reactors and that calculated from chemical analyses. H2S production in BSP assays and in reactors was lower than the stoichiometric values calculated from chemical composition for all wastes tested, demonstrating the importance of assays to estimate the microbial sulfide production potential of sulfur-containing wastes. Copyright © 2016 Elsevier B.V. All rights reserved.
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Progress in applying the Three Rs to the potency testing of Botulinum toxin type A.
Straughan, Donald
2006-06-01
Botulinum toxin type A (BTA) is being increasingly used for a range of therapeutic purposes and also for cosmetic reasons. For many years, the potency of BTA has been measured by using an LD50 assay in mice. This assay is a cause for concern due to its unpleasant nature and extreme severity, and the requirement for high numbers of mice to be used. Alternatives to this potency assay are presently reviewed with particular reference to the work at the National Institute for Biological Standards and Control (NIBSC), and to recent work by the UK manufacturer of the substance. An in vivo local paralysis assay with considerably less severity has been developed and is in use at the NIBSC. Alternative, ex vivo functional assays in use include the measurement of BTA-induced paralysis of neurally-stimulated rodent diaphragm or rat intercostal muscle. The latter method has the advantage of allowing more preparations to be derived from one animal. However, these ex vivo methods have not yet been fully validated and accepted by regulatory agencies as potency assays. Endopeptidase assays, although not measuring muscle paralysis directly, may provide a very useful consistency test for batch release and may replace the routine use of the LD50 test for that purpose. These assays measure the cleavage of the SNAP-25 protein (the final stage of BTA action), and have been validated for batch release by the National Control Laboratory (NIBSC), and are in regular use there. ELISA assays, used alongside the endopeptidase assay, also provide useful confirmatory information on the amounts of functional (and non-functional) BTA present. The UK manufacturer is further validating its endopeptidase assay, an ex vivo muscle assay and an ELISA. It is anticipated that their work will lead to a change in the product license, hopefully within the next two years, and will form a critical milestone towards the end of the LD50 potency test.
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Halaidych, Oleh V; Freund, Christian; van den Hil, Francijna; Salvatori, Daniela C F; Riminucci, Mara; Mummery, Christine L; Orlova, Valeria V
2018-05-08
Several studies have reported endothelial cell (EC) derivation from human induced pluripotent stem cells (hiPSCs). However, few have explored their functional properties in depth with respect to line-to-line and batch-to-batch variability and how they relate to primary ECs. We therefore carried out accurate characterization of hiPSC-derived ECs (hiPSC-ECs) from multiple (non-integrating) hiPSC lines and compared them with primary ECs in various functional assays, which included barrier function using real-time impedance spectroscopy with an integrated assay of electric wound healing, endothelia-leukocyte interaction under physiological flow to mimic inflammation and angiogenic responses in in vitro and in vivo assays. Overall, we found many similarities but also some important differences between hiPSC-derived and primary ECs. Assessment of vasculogenic responses in vivo showed little difference between primary ECs and hiPSC-ECs with regard to functional blood vessel formation, which may be important in future regenerative medicine applications requiring vascularization. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
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Testing of veterinary clostridial vaccines: from mouse to microtitre plate.
Redhead, K; Wood, K; Jackson, K
2012-01-01
Vaccines to protect against clostridial diseases are among the most common veterinary biologicals. Each batch of these materials is subjected to a variety of toxicity and antigenicity tests. The potency of the final vaccine is then assessed by Toxin Neutralisation Test (TNT). All of these tests use mice and have lethal endpoints. Development of alternatives for potency testing was based on ELISAs able to measure antibody levels to the specific toxins relative to a standard serum with a defined unitage. These alternative assays were shown to correlate with the relevant TNTs and have been accepted by European Regulatory Authorities as batch release potency tests. Recently we have developed in vitro cell line alternatives for the toxicity and antigenicity tests for Cl. septicum using the VERO cell line. With this cell line it has been possible to develop in vitro assays which, when compared with the in vivo tests, gave correlations of 87% to 100%. Having shown proof of principle, similar cell line assays have been developed for Cl. novyi and Cl. perfringens types C and D.
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Wanandy, T; Dwyer, H E; McLean, L; Davies, N W; Nichols, D; Gueven, N; Brown, S G A; Wiese, M D
2017-11-01
Allergen immunotherapy uses pharmaceutical preparations derived from naturally occurring source materials, which contain water-soluble allergenic components responsible for allergic reactions. The success of in vivo and in vitro diagnoses in allergen sensitization and allergen immunotherapy largely depends on the quality, composition and uniformity of allergenic materials used to produce the active ingredients, and the formulation employed to prepare finished products. We aimed to examine the factors influencing batch-to-batch consistency of Jack Jumper (Myrmecia pilosula) ant venom (JJAV) in the form of active pharmaceutical ingredient (AI) and informed whether factors such as temperature, artificial light and container materials influence the quality of JJAV AIs. We also aimed to establish handling and storage requirements of JJAV AIs to ensure preservation of allergenic activities during usage in the diagnosis of allergen sensitization and in allergen immunotherapy. The quality and consistency of JJAV AIs were analysed using a combination of bicinchoninic acid assay for total protein quantification, HPLC-UV for JJAV allergen peptides quantification, ELISA inhibition for total allergenic potency, SDS-PAGE, AU-PAGE and immunoblot for qualitative assessment of JJAV components, and Limulus Amebocyte Lysate assay for the quantification of endotoxin concentration. API-ZYM and Zymogram assays were used to probe the presence of enzymatic activities in JJAV. Pharmaceutical-grade JJAV for allergen immunotherapy has good batch-to-batch consistency. Temporary storage at 4°C and light exposure do not affect the quality of JJAV. Exposure to temperature above 40°C degrades high MW allergens in JJAV. Vials containing JJAV must be stored frozen and in upright position during long-term storage. We have identified factors, which can influence the quality and consistency of JJAV AIs, and provided a framework for appropriate handling, transporting and storage of JJAV to be used for the diagnosis of allergen sensitization and in AIT. © 2017 John Wiley & Sons Ltd.
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Rebinding the Ties that Bind: Government Efforts to Preserve and Promote Marriage
ERIC Educational Resources Information Center
Brotherson, Sean E.; Duncan, William C.
2004-01-01
Governmental efforts to strengthen marriage through a variety of approaches have become increasingly common in the last decade. Societal trends related to family formation, marriage, and divorce have shaped interest in marriage and its stability as a social institution. The public sector has targeted efforts at key stages in the life history of…
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Mixture model normalization for non-targeted gas chromatography/mass spectrometry metabolomics data.
Reisetter, Anna C; Muehlbauer, Michael J; Bain, James R; Nodzenski, Michael; Stevens, Robert D; Ilkayeva, Olga; Metzger, Boyd E; Newgard, Christopher B; Lowe, William L; Scholtens, Denise M
2017-02-02
Metabolomics offers a unique integrative perspective for health research, reflecting genetic and environmental contributions to disease-related phenotypes. Identifying robust associations in population-based or large-scale clinical studies demands large numbers of subjects and therefore sample batching for gas-chromatography/mass spectrometry (GC/MS) non-targeted assays. When run over weeks or months, technical noise due to batch and run-order threatens data interpretability. Application of existing normalization methods to metabolomics is challenged by unsatisfied modeling assumptions and, notably, failure to address batch-specific truncation of low abundance compounds. To curtail technical noise and make GC/MS metabolomics data amenable to analyses describing biologically relevant variability, we propose mixture model normalization (mixnorm) that accommodates truncated data and estimates per-metabolite batch and run-order effects using quality control samples. Mixnorm outperforms other approaches across many metrics, including improved correlation of non-targeted and targeted measurements and superior performance when metabolite detectability varies according to batch. For some metrics, particularly when truncation is less frequent for a metabolite, mean centering and median scaling demonstrate comparable performance to mixnorm. When quality control samples are systematically included in batches, mixnorm is uniquely suited to normalizing non-targeted GC/MS metabolomics data due to explicit accommodation of batch effects, run order and varying thresholds of detectability. Especially in large-scale studies, normalization is crucial for drawing accurate conclusions from non-targeted GC/MS metabolomics data.
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Chen, Jing; Wang, Shu-Mei; Meng, Jiang; Sun, Fei; Liang, Sheng-Wang
2013-05-01
To establish a new method for quality evaluation and validate its feasibilities by simultaneous quantitative assay of five alkaloids in Sophora flavescens. The new quality evaluation method, quantitative analysis of multi-components by single marker (QAMS), was established and validated with S. flavescens. Five main alkaloids, oxymatrine, sophocarpine, matrine, oxysophocarpine and sophoridine, were selected as analytes to evaluate the quality of rhizome of S. flavescens, and the relative correction factor has good repeatibility. Their contents in 21 batches of samples, collected from different areas, were determined by both external standard method and QAMS. The method was evaluated by comparison of the quantitative results between external standard method and QAMS. No significant differences were found in the quantitative results of five alkaloids in 21 batches of S. flavescens determined by external standard method and QAMS. It is feasible and suitable to evaluate the quality of rhizome of S. flavescens by QAMS.
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Méndez-Hermida, F.; Castro-Hermida, J. A.; Ares-Mazás, E.; Kehoe, S. C.; McGuigan, K. G.
2005-01-01
The results of batch-process solar disinfection (SODIS) of Cryptosporidium parvum oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m−2) at 40°C. Viability assays (4′,6′-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively. PMID:15746372
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Kinetic modelling of anaerobic hydrolysis of solid wastes, including disintegration processes.
García-Gen, Santiago; Sousbie, Philippe; Rangaraj, Ganesh; Lema, Juan M; Rodríguez, Jorge; Steyer, Jean-Philippe; Torrijos, Michel
2015-01-01
A methodology to estimate disintegration and hydrolysis kinetic parameters of solid wastes and validate an ADM1-based anaerobic co-digestion model is presented. Kinetic parameters of the model were calibrated from batch reactor experiments treating individually fruit and vegetable wastes (among other residues) following a new protocol for batch tests. In addition, decoupled disintegration kinetics for readily and slowly biodegradable fractions of solid wastes was considered. Calibrated parameters from batch assays of individual substrates were used to validate the model for a semi-continuous co-digestion operation treating simultaneously 5 fruit and vegetable wastes. The semi-continuous experiment was carried out in a lab-scale CSTR reactor for 15 weeks at organic loading rate ranging between 2.0 and 4.7 gVS/Ld. The model (built in Matlab/Simulink) fit to a large extent the experimental results in both batch and semi-continuous mode and served as a powerful tool to simulate the digestion or co-digestion of solid wastes. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Nanosecond Absorption Spectroscopy of Hemoglobin: Elementary Processes in Kinetic Cooperativity
NASA Astrophysics Data System (ADS)
Hofrichter, James; Sommer, Joseph H.; Henry, Eric R.; Eaton, William A.
1983-04-01
A nanosecond absorption spectrometer has been used to measure the optical spectra of hemoglobin between 3 ns and 100 ms after photolysis of the CO complex. The data from a single experiment comprise a surface, defined by the time-ordered set of 50-100 spectra. Singular value decomposition is used to represent the observed spectra in terms of a minimal set of basis spectra and the time course of their amplitudes. Both CO rebinding and conformational changes are found to be multiphasic. Prior to the quaternary structural change, two relaxations are observed that are assigned to geminate recombination followed by a tertiary structural change. These relaxations are interpreted in terms of a kinetic model that points out their potential role in kinetic cooperativity. The rapid escape of CO from the heme pocket compared with the rate of rebinding observed for both R and T quaternary states shows that the quaternary structure controls the overall dissociation rate by changing the rate at which the Fe--CO bond is broken. A comparable description of the control of the overall association rates must await a more complete experimental description of the kinetics of the quaternary T state.
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NASA Astrophysics Data System (ADS)
Lu, Jian; Qin, Yingying; Zhang, Qi; Wu, Yilin; Cui, Jiuyun; Li, Chunxiang; Wang, Liang; Yan, Yongsheng
2018-01-01
High-selective multilayered Li+-imprinted membranes (Li-IIMs) with enhanced hydrophilicity and stability were developed based on polyether sulfone substrate membranes. The multilayered structure was prepared with polydopamine (pDA) as the interfacial adhesion layer, SiO2 nanoparticles as the hydrophilic layer and Li+-imprinted polymers as the imprinted layer. The selective ;Li+-recognition sites; were formed using 12-crown-4 (12C4) as the adsorbing units. The optimal relative selectivity coefficients (α) of Li+/Na+ and Li+/K+ reached up to 1.85 and 2.07 with the imprinting factor (β) of 2.51, and the high permselectivity factors (γ) of Na+/Li+ (7.39) and K+/Li+ (9.86) were achieved on Li-IIMs. The Langmuir isotherm model and the pseudo-second-order kinetics model best fitted the rebinding data of Li-IIMs, as well as the rebinding capacities reached up to 90.3% of initial binding after 5 cycles of adsorption/desorption and just declined to 88.1% after another 5 cycles a month later. Therefore, the as-prepared Li-IIMs would have potential applications for the separation of lithium ions from salt lake brines.
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Smart coumarin-tagged imprinted polymers for the rapid detection of tamoxifen.
Ray, Judith V; Mirata, Fosca; Pérollier, Celine; Arotcarena, Michel; Bayoudh, Sami; Resmini, Marina
2016-03-01
A signalling molecularly imprinted polymer was synthesised for easy detection of tamoxifen and its metabolites. 6-Vinylcoumarin-4-carboxylic acid (VCC) was synthesised from 4-bromophenol to give a fluorescent monomer, designed to switch off upon binding of tamoxifen. Clomiphene, a chlorinated analogue, was used as the template for the imprinting, and its ability to quench the coumarin fluorescence when used in a 1:1 ratio was demonstrated. Tamoxifen and 4-hydroxytamoxifen were also shown to quench coumarin fluorescence. Imprinted and non-imprinted polymers were synthesised using VCC, methacrylic acid as a backbone monomer and ethylene glycol dimethacrylate as cross-linker, and were ground and sieved to particle sizes ranging between 45 and 25 μm. Rebinding experiments demonstrate that the imprinted polymer shows very strong affinity for both clomiphene and tamoxifen, while the non-imprinted polymer shows negligible rebinding. The fluorescence of the imprinted polymer is quenched by clomiphene, tamoxifen and 4-hydroxytamoxifen. The switch off in fluorescence of the imprinted polymer under these conditions could also be detected under a UV lamp with the naked eye, making this matrix suitable for applications when coupled with a sample preparation system.
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Molecularly Imprinted Polymers with DNA Aptamer Fragments as Macromonomers.
Zhang, Zijie; Liu, Juewen
2016-03-01
Molecularly imprinted polymers (MIPs) are produced in the presence of a template molecule. After removing the template, the cavity can selectively rebind the template. MIPs are attractive functional materials with a low cost and high stability, but traditional MIPs often suffer from low binding affinity. This study employs DNA aptamer fragments as macromonomers to improve MIPs. The DNA aptamer for adenosine was first split into two halves, fluorescently labeled, and copolymerized into MIPs. With a fluorescence quenching assay, the importance of imprinting was confirmed. Further studies were carried out using isothermal titration calorimetry (ITC). Compared to the mixture of the free aptamer fragments, their MIPs doubled the binding affinity. Each free aptamer fragment alone cannot bind adenosine, whereas MIPs containing each fragment are effective binders. We further shortened one of the aptamer fragments, and the DNA length was pushed to as short as six nucleotides, yielding MIPs with a dissociation constant of 27 μM adenosine. This study provides a new method for preparing functional MIP materials by combining high-affinity biopolymer fragments with low-cost synthetic monomers, allowing higher binding affinity and providing a method for signaling binding based on DNA chemistry.
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Whole-Genome DNA Methylation Status Associated with Clinical PTSD Measures of OIF/OEF Veterans
Emerging knowledge suggests that post -traumatic stress disorder (PTSD) pathophysiology is linked to the patients epigenetic changes, but...promoter-bound CpGIs to identify networks related to PTSD. The identified networks were further validated by an independent test set comprising 31 PTSD /29...set. To improve the statistical power and mitigate the assay bias and batch effects, a union set combining both training and test set was assayed
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NASA Astrophysics Data System (ADS)
Semprini, L.; Bartow, S.; Radniecki, T.
2012-04-01
Understanding the toxicity of nanoparticles on ecologically significant wastewater microbiota, specifically ammonia oxidizing bacteria (AOB), is critical due to the exponential increase in commercialization of nanoparticles as well as the sensitivity of AOB to inhibitors. A high-throughput activity assay was developed to rapidly screen for nanoparticle toxicity on AOB, using a multi-well plate method and AOB Nitrosomonas Europaea. This method demonstrated good agreement with previously established batch bottle assays utilizing both silver ions (Ag+) and nanoparticles (Ag-NPs) as nitrification inhibitors. The method was used to study the inhibition of Ag+ and Ag-NPs (20 nm) on the nitrification by N. Europaea cells grown in fill-and-draw reactors compared exponentially grown batch cells. Results indicate longer hydraulic residence times increased some protection against inhibition as measured by the production of nitrite over a three hour assay. The cells were more sensitive to Ag+ than Ag-NP, which is consistent with our past observations. Studies are currently being conducted to determine the effects that the presence of humic acid and cations on the inhibition and toxicity. Our initial results show that the presence of Mg++ provides protect from Ag-NP inhibition, which partly results from the aggregation of the Ag-NP and a decrease in the rate of oxidation of the Ag-NP to Ag+. The presence of humic acid also provides for some protection from Ag-NP inhibition.
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Force probe simulations of a reversibly rebinding system: Impact of pulling device stiffness
NASA Astrophysics Data System (ADS)
Jaschonek, Stefan; Diezemann, Gregor
2017-03-01
We present a detailed study of the parameter dependence of force probe molecular dynamics (FPMD) simulations. Using a well studied calix[4]arene catenane dimer as a model system, we systematically vary the pulling velocity and the stiffness of the applied external potential. This allows us to investigate how the results of pulling simulations operating in the constant velocity mode (force-ramp mode) depend on the details of the simulation setup. The system studied has the further advantage of showing reversible rebinding meaning that we can monitor the opening and the rebinding transition. Many models designed to extract kinetic information from rupture force distributions work in the limit of soft springs and all quantities are found to depend solely on the so-called loading rate, the product of spring stiffness and pulling velocity. This approximation is known to break down when stiff springs are used, a situation often encountered in molecular simulations. We find that while some quantities only depend on the loading rate, others show an explicit dependence on the spring constant used in the FPMD simulation. In particular, the force versus extension curves show an almost stiffness independent rupture force but the force jump after the rupture transition does depend roughly linearly on the value of the stiffness. The kinetic rates determined from the rupture force distributions show a dependence on the stiffness that can be understood in terms of the corresponding dependence of the characteristic forces alone. These dependencies can be understood qualitatively in terms of a harmonic model for the molecular free energy landscape. It appears that the pulling velocities employed are so large that the crossover from activated dynamics to diffusive dynamics takes place on the time scale of our simulations. We determine the effective distance of the free energy minima of the closed and the open configurations of the system from the barrier via an analysis of the hydrogen-bond network with results in accord with earlier simulations. We find that the system is quite brittle in the force regime monitored in the sense that the barrier is located near to the closed state.
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Stalder, J; Costanzo, A; Daas, A; Rautmann, G; Buchheit, K-H
2010-04-01
A reference standard calibrated in International Units (IU) is needed for the in vitro potency assay of hepatitis A vaccines prepared by formalin-inactivation of purified hepatitis A virus grown in cell cultures. Thus, a project was launched by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to establish one or more non-adsorbed inactivated hepatitis A vaccine reference preparation(s) as working standard(s), calibrated against the 1st International Standard (IS), for the in vitro potency assay (ELISA) of all vaccines present on the European market. Four non-adsorbed liquid preparations of formalin-inactivated hepatitis A antigen with a known antigen content were obtained from 3 manufacturers as candidate Biological Reference Preparations (BRPs). Thirteen laboratories participated in the collaborative study. They were asked to use an in vitro ELISA method adapted from a commercially available kit for the detection of antibodies to hepatitis A virus. In-house validated assays were to be run in parallel, where available. Some participants also included commercially available hepatitis A vaccines in the assays, after appropriate desorption. During the collaborative study, several participants using the standard method were faced with problems with some of the most recent lots of the test kits. Due to these problems, the standard method did not perform satisfactorily and a high number of assays were invalid, whereas the in-house methods appeared to perform better. Despite this, the overall mean results of the valid assays using both methods were in agreement. Nonetheless, it was decided to base the assignment of the potency values on the in-house methods only. The results showed that all candidate BRPs were suitable for the intended purpose. However, based on availability of the material and on the results of end-product testing, 2 candidate reference preparations, Samples C and D, were selected. Both were from the same batch but filled on different days; no statistically significant difference in potency was observed. They were thus combined in 1 single batch. The candidate preparation (Sample C/D) was adopted at the June 2009 session of the European Pharmacopoeia (Ph. Eur.) Commission as the Ph. Eur. BRP batch 1 for hepatitis A vaccine (inactivated, non-adsorbed), with an assigned potency of 12 IU/ml for in vitro antigen content assays. Accelerated degradation studies have been initiated. The preliminary data show that the BRP is stable at the recommended storage temperature (< -50 degrees C). The BRP will be monitored at regular intervals throughout its lifetime.
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Witt, Lukas; Suzuki, Yosuke; Hohmann, Nicolas; Mikus, Gerd; Haefeli, Walter E; Burhenne, Jürgen
2016-08-01
Chlorzoxazone is a probe drug to assess cytochrome P450 (CYP) 2E1 activity (phenotyping). If the pharmacokinetics of the probe drug is linear, pharmacologically ineffective doses are sufficient for the purpose of phenotyping and adverse effects can thus be avoided. For this reason, we developed and validated an assay for the ultrasensitive quantification of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma. Plasma (0.5mL) and liquid/liquid partitioning were used for sample preparation. Extraction recoveries ranged between 76 and 93% for both analytes. Extracts were separated within 3min on a Waters BEH C18 Shield 1.7μm UPLC column with a fast gradient consisting of aqueous formic acid and acetonitrile. Quantification was achieved using internal standards labeled with deuterium or (13)C and tandem mass spectrometry in the multiple reaction monitoring mode using negative electrospray ionization, which yielded lower limits of quantification of 2.5pgmL(-1), while maintaining a precision always below 15%. The calibrated concentration ranges were linear for both analytes (2.5-1000pgmL(-1)) with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <15% and <11% and plasma matrix effects always were below 50%. The assay was successfully applied to assess the pharmacokinetics of chlorzoxazone in two human volunteers after administration of single oral doses (2.5-5000μg). This ultrasensitive assay allowed the determination of chlorzoxazone pharmacokinetics for 8h after microdosing of 25μg chlorzoxazone. Copyright © 2016 Elsevier B.V. All rights reserved.
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Rudi, Knut; Flateland, Signe L; Hanssen, Jon Fredrik; Bengtsson, Gunnar; Nissen, Hilde
2002-03-01
There is a clear need for new approaches in the field of microbial community analyses, since the methods used can be severely biased. We have developed a DNA array-based method that targets 16S ribosomal DNA (rDNA), enabling the direct detection and quantification of microorganisms from complex communities without cultivation. The approach is based on the construction of specific probes from the 16S rDNA sequence data retrieved directly from the communities. The specificity of the assay is obtained through a combination of DNA array hybridization and enzymatic labeling of the constructed probes. Cultivation-dependent assays (enrichment and plating) and cultivation-independent assays (direct fluorescence microscopy and scanning electron microscopy) were used as reference methods in the development and evaluation of the method. The description of microbial communities in ready-to-eat vegetable salads in a modified atmosphere was used as the experimental model. Comparisons were made with respect to the effect of storage at different temperatures for up to 12 days and with respect to the geographic origin of the crisphead lettuce (Spanish or Norwegian), the main salad component. The conclusion drawn from the method comparison was that the DNA array-based method gave an accurate description of the microbial communities. Pseudomonas spp. dominated both of the salad batches, containing either Norwegian or Spanish lettuce, before storage and after storage at 4 degrees C. The Pseudomonas population also dominated the batch containing Norwegian lettuce after storage at 10 degrees C. On the contrary, Enterobacteriaceae and lactic acid bacteria dominated the microbial community of the batch containing Spanish lettuce after storage at 10 degrees C. In that batch, the Enterobacteriaceae also were abundant after storage at 4 degrees C as well as before storage. The practical implications of these results are that microbial communities in ready-to-eat vegetable salads can be diverse and that microbial composition is dependent both on the origin of the raw material and on the storage conditions.
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Rudi, Knut; Flateland, Signe L.; Hanssen, Jon Fredrik; Bengtsson, Gunnar; Nissen, Hilde
2002-01-01
There is a clear need for new approaches in the field of microbial community analyses, since the methods used can be severely biased. We have developed a DNA array-based method that targets16S ribosomal DNA (rDNA), enabling the direct detection and quantification of microorganisms from complex communities without cultivation. The approach is based on the construction of specific probes from the 16S rDNA sequence data retrieved directly from the communities. The specificity of the assay is obtained through a combination of DNA array hybridization and enzymatic labeling of the constructed probes. Cultivation-dependent assays (enrichment and plating) and cultivation-independent assays (direct fluorescence microscopy and scanning electron microscopy) were used as reference methods in the development and evaluation of the method. The description of microbial communities in ready-to-eat vegetable salads in a modified atmosphere was used as the experimental model. Comparisons were made with respect to the effect of storage at different temperatures for up to 12 days and with respect to the geographic origin of the crisphead lettuce (Spanish or Norwegian), the main salad component. The conclusion drawn from the method comparison was that the DNA array-based method gave an accurate description of the microbial communities. Pseudomonas spp. dominated both of the salad batches, containing either Norwegian or Spanish lettuce, before storage and after storage at 4°C. The Pseudomonas population also dominated the batch containing Norwegian lettuce after storage at 10°C. On the contrary, Enterobacteriaceae and lactic acid bacteria dominated the microbial community of the batch containing Spanish lettuce after storage at 10°C. In that batch, the Enterobacteriaceae also were abundant after storage at 4°C as well as before storage. The practical implications of these results are that microbial communities in ready-to-eat vegetable salads can be diverse and that microbial composition is dependent both on the origin of the raw material and on the storage conditions. PMID:11872462
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Lyng, Fiona M; Desplanques, Maxime; Jella, Kishore Kumar; Garcia, Amaya; McClean, Brendan
2012-10-01
The aim of this study was to investigate the importance of serum serotonin levels in the measurement of bystander cell death. The study was undertaken as part of an intercomparison exercise involving seven European laboratories funded under the European Union Sixth Framework Programme (FP6) Non-Targeted Effects (NOTE) integrated project. Three batches of foetal bovine serum were tested; serum with high and low serotonin content from the intercomparison exercise as well as serum from the home laboratory. Three sets of human keratinocytes (HaCaT cell line) were cultured in DMEM:F12 medium supplemented with serum with high or low serotonin content or serum from the home laboratory and both donor and recipient HaCaT cells were plated. The donor HaCaT cells were irradiated (0.5 Gy) using a cobalt 60 teletherapy unit, the medium was harvested 1 hour post irradiation and transferred to the recipient HaCaT cells. Bystander induced cell death was measured by the clonogenic survival assay and the Alamar blue viability assay. A significant reduction in cell survival, as measured by the clonogenic assay, and in cell viability, as measured by the Alamar blue assay, was observed in the recipient HaCaT cells treated with medium from irradiated cells compared to the cells treated with medium from unirradiated cells. No significant difference was found between the three batches of serum. The data suggest that in our cell system and with our endpoints (clonogenic assay and Alamar blue assay), serum serotonin levels do not play a role in bystander-induced cell death.
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Costa, J C; Gonçalves, P R; Nobre, A; Alves, M M
2012-06-01
Biochemical methane potential of four species of Ulva and Gracilaria genus was assessed in batch assays at mesophilic temperature. The results indicate a higher specific methane production (per volatile solids) for one of the Ulva sp. compared with other macroalgae and for tests running with 2.5% of total solids (196±9 L CH(4) kg(-1)VS). Considering that macroalgae can potentially be a post treatment of municipal wastewater for nutrients removal, co-digestion of macroalgae with waste activated sludge (WAS) was assessed. The co-digestion of macroalgae (15%) with WAS (85%) is feasible at a rate of methane production 26% higher than WAS alone without decreasing the overall biodegradability of the substrate (42-45% methane yield). The use of anoxic marine sediment as inoculum had no positive effect on the methane production in batch assays. The limiting step of the overall anaerobic digestion process was the hydrolysis. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Water decontamination containing nitrate using biosorption with Moringa oleifera in dynamic mode.
Paixão, Rebecca Manesco; Reck, Isabela Maria; Gomes, Raquel Guttierres; Bergamasco, Rosângela; Vieira, Marcelo Fernandes; Vieira, Angélica Marquetotti Salcedo
2018-05-20
This study was conducted to assess the feasibility of using Moringa oleifera Lam. (MO) seeds in the biosorption of nitrate present in aqueous solutions by means of batch and fixed-bed column biosorption processes. The batch assays showed that nitrate biosorption is enhanced under experimental conditions of pH 3 and a biosorbent mass of 0.05 g. For the experiments in dynamic mode, the results obtained from the statistical parameters showed that lesser pH, lesser feed flow rate, and higher initial concentration will result in an increase of the maximum capacity of the bed. These conditions were confirmed by experimental analysis. The best experimental conditions, according to the values for percentage removal (91.09%) and maximum capacity (7.69 mg g -1 ) of the bed, were those used in assay 1, which utilized pH 3, feed flow rate of 1 mL min -1 , and initial nitrate concentration of 100 mg L -1 .
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Macdonald, Isabel K.; Allen, Jared; Murray, Andrea; Parsy-Kowalska, Celine B.; Healey, Graham F.; Chapman, Caroline J.; Sewell, Herbert F.; Robertson, John F. R.
2012-01-01
An assay employing a panel of tumor-associated antigens has been validated and is available commercially (EarlyCDT®-Lung) to aid the early detection of lung cancer by measurement of serum autoantibodies. The high throughput (HTP) strategy described herein was pursued to identify new antigens to add to the EarlyCDT-Lung panel and to assist in the development of new panels for other cancers. Two ligation-independent cloning vectors were designed and synthesized, producing fusion proteins suitable for the autoantibody ELISA. We developed an abridged HTP version of the validated autoantibody ELISA, determining that results reflected the performance of the EarlyCDT assay, by comparing results on both formats. Once validated this HTP ELISA was utilized to screen multiple fusion proteins prepared on small-scale, by a HTP expression screen. We determined whether the assay performance for these HTP protein batches was an accurate reflection of the performance of R&D or commercial batches. A HTP discovery platform for the identification and optimal production of tumor- associated antigens which detects autoantibodies has been developed and validated. The most favorable conditions for the exposure of immunogenic epitopes were assessed to produce discriminatory proteins for use in a commercial ELISA. This process is rapid and cost-effective compared to standard cloning and screening technologies and enables rapid advancement in the field of autoantibody assay discovery. This approach will significantly reduce timescale and costs for developing similar panels of autoantibody assays for the detection of other cancer types with the ultimate aim of improved overall survival due to early diagnosis and treatment. PMID:22815807
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Basco, Leonardo K
2003-08-01
In vitro drug sensitivity assay is an important tool for various on-going studies aiming to establish the correlation between candidate molecular markers for drug resistance and drug response in laboratory-adapted strains and field isolates of Plasmodium falciparum. A widespread use of this technique in the field would require a suitable substitute that can replace human serum. In this study, several alternative sources of serum substitutes and supplements were evaluated for their capacity to sustain parasite growth for a single life cycle and their compatibility with in vitro assays for clinical isolates that have not been adapted to in vitro culture. Albumax, a commercial preparation of lipid-enriched bovine albumin, did not support parasite growth as much as human serum and fetal calf serum in several isolates. Other serum supplements (AmnioMax and Ultroser) supported parasite growth relatively well. The 50% inhibitory concentrations (IC50s) of chloroquine and antifolates determined with 0.05% Albumax were generally two or three times higher than with human serum. With 10% fetal calf serum, IC50s for chloroquine and antifolates were approximately two times higher and three times lower than with human serum, respectively. The use of AmnioMax and OptiMAb resulted in a greater than two-fold increase in IC50s and several uninterpretable assays. Despite possible batch-to-batch differences, fetal calf serum may be a suitable substitute for in vitro drug assays while awaiting the results of further studies on other serum substitutes and supplements.
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2012-07-01
prostate lobes were dissected free of fat and connective tissue and weighed separately. 2.3. Hormone assays All assays were performed in a single batch...Ferrell, R.E., Roth, S.M., 2005. Androgen receptor CAG repeat polymorphism is associated with fat -free mass in men. J. Appl. Physiol. 98, 132–137. Wu, C.T...S., Kennemer, M.I., Mohan, S., Nazarenko, I., Watanabe, C., Sparks, A.B., Shames , D.S., Gentleman, R., de Sauvage, F.J., Stern, H., Pandita, A
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Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR.
Liu, Ya-Mei; Qiu, Liang; Sheng, An-Zhi; Wan, Xiao-Yuan; Cheng, Dong-Yuan; Huang, Jie
2018-01-01
A TaqMan probe and a pair of specific primers were selected from the small subunit ribosomal DNA (SSU rDNA) sequence of Enterocytozoon hepatopenaei (EHP); this real-time PCR assay was developed and optimized. It showed a good linearity in detecting standards of EHP SSU rDNA fragments from 4 × 10 2 to 4 × 10 8 copies/reaction using the established method. The detection limit of the qPCR method was as low as 4 × 10 1 copies per reaction, which was higher than the conventional PCR and SYBR Green I-based EHP qPCR reported. Using the qPCR assay, EHP was detected in four batches of slow-growing Penaeus vannamei specimens collected from Tianjin and Zhejiang Province in China was detected using qPCR. The results showed that all the hepatopancreas from the slow-growing P. vannamei specimens were detected as EHP-positive. EHP copies of hepatopancreas in some batches had a negative correlation with the body mass index (BMI) of shrimps; however, not all batches of specimens had this negative correlation between EHP copies of hepatopancreas and BMI. This qPCR technique is sensitive, specific and easy to perform (96 tests in <3 h), which provides technical support for the detection and prevention of EHP. Copyright © 2017 Elsevier Inc. All rights reserved.
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Estimating Animal Abundance in Ground Beef Batches Assayed with Molecular Markers
Hu, Xin-Sheng; Simila, Janika; Platz, Sindey Schueler; Moore, Stephen S.; Plastow, Graham; Meghen, Ciaran N.
2012-01-01
Estimating animal abundance in industrial scale batches of ground meat is important for mapping meat products through the manufacturing process and for effectively tracing the finished product during a food safety recall. The processing of ground beef involves a potentially large number of animals from diverse sources in a single product batch, which produces a high heterogeneity in capture probability. In order to estimate animal abundance through DNA profiling of ground beef constituents, two parameter-based statistical models were developed for incidence data. Simulations were applied to evaluate the maximum likelihood estimate (MLE) of a joint likelihood function from multiple surveys, showing superiority in the presence of high capture heterogeneity with small sample sizes, or comparable estimation in the presence of low capture heterogeneity with a large sample size when compared to other existing models. Our model employs the full information on the pattern of the capture-recapture frequencies from multiple samples. We applied the proposed models to estimate animal abundance in six manufacturing beef batches, genotyped using 30 single nucleotide polymorphism (SNP) markers, from a large scale beef grinding facility. Results show that between 411∼1367 animals were present in six manufacturing beef batches. These estimates are informative as a reference for improving recall processes and tracing finished meat products back to source. PMID:22479559
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Zhang, Dezhi; Hegab, Hisham E.; Lvov, Yuri; ...
2016-01-20
Cellulase was immobilized onto silica gel surfaces pretreated with (3-aminopropyl) triethoxy-silane (3-APTES), and glutaraldehyde (GA) was used as a cross-linker. A carboxymethyl cellulose sodium salt (CMC) solution was used for activity experiments. Protein assay was performed to determine the mass immobilized and compare with free enzyme. Cellulase was successfully demonstrated to be immobilized on the modified silica gel surface, and no detectable amount of enzyme was stripped off during the hydrolysis of the CMC solution. The specific activity of the immobilized cellulase is 7 ± 2 % compared to the similar amount of free cellulase. Significant activity over multiple reusesmore » was observed. The seventh batch achieved 82 % activity of the initial batch, and the fifteenth batch retained 31 %. Lastly, it was observed that the immobilized cellulase retained 48 % of its initial activity after 4 days, and 22 % even after 14 days.« less
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Shumyantseva, Victoria V; Bulko, Tatiana V; Sigolaeva, Larisa V; Kuzikov, Alexey V; Archakov, Alexander I
2016-12-15
Electrosynthesis of molecularly imprinted polymer (MIP) templated with myoglobin (Mb) and the reference non-imprinted polymer (NIP) was examined with o-phenylenediamine (o-PD) as a monomer. Mass-sensitive quartz crystal microbalance with dissipation monitoring supplied by an electrochemical module (EQCM-D) was applied to characterize and optimize MIP/NIP electrosynthesis. Mb rebinding was detected by direct electrocatalytic reduction of Mb by square wave voltammetry (SWV) or differential pulse voltammetry (DPV). The results obtained showed high specificity of polymeric antibodies to template Mb, with an imprinting factor determined as a ratio Imax(MIP)/Imax(NIP) of 2-4. The prepared MIP sensor is characterized by an apparent dissociation constant of (3.3±0.5)×10(-9)M and has a broad range of working concentrations of 1nM-1μМ, with the detection limit of 0.5nM (9ng/ml). Mb rebinding was examined in Mb-free diluted human serum spiked with Mb as well as in plasma samples of patients with acute myocardial infarction (AMI) and in control plasma of healthy donors in order to demonstrate the potential medical application of developed MIP sensors. Copyright © 2016 Elsevier B.V. All rights reserved.
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Molecular oxygen migration through the xenon docking sites of human hemoglobin in the R-state.
Lepeshkevich, Sergei V; Gilevich, Syargey N; Parkhats, Marina V; Dzhagarov, Boris M
2016-09-01
A nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O2) rebinding to tetrameric human hemoglobin and its isolated α and β chains in buffer solutions equilibrated with 1atm of air and up to 25atm of xenon. Xenon binding to the isolated α chains and to the α subunits within tetrameric hemoglobin was found to cause a decrease in the efficiency of O2 escape by a factor of ~1.30 and 3.3, respectively. A kinetic model for O2 dissociation, rebinding, and migration through two alternative pathways in the hemoglobin subunits was introduced and discussed. It was shown that, in the isolated α chains and α subunits within tetrameric hemoglobin, nearly one- and two-third escaping molecules of O2 leave the protein via xenon docking sites, respectively. The present experimental data support the idea that O2 molecule escapes from the β subunits mainly through the His(E7) gate, and show unambiguously that, in the α subunits, in addition to the direct E7 channel, there is at least one alternative escape route leading to the exterior via the xenon docking sites. Copyright © 2016 Elsevier B.V. All rights reserved.
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ELISA reader does not interfere by mobile phone radiofrequency radiation.
Mortazavi, Seyyed Mohammad Javad; Baradaran-Ghahfarokhi, Hamid Reza; Abdi, Mohammad Reza; Baradaran-Ghahfarokhi, Milad; Mostafavi, Nayyer Sadat; Mahmoudi, Golshan; Berenjkoub, Nafiseh; Akmali, Zahra; Hossein-Beigi, Fahimeh; Arsang, Vajiheh
2016-01-01
The increasing number of mobile phones can physically cause electromagnetic interference (EMI) in medical environments; can also cause errors in immunoassays in laboratories. The ELISA readers are widely used as a useful diagnostic tool for Enzymun colorimetric assay in medicine. The aim of this study was to investigate whether the ELISA reader could be interfered by the exposure to the 900 MHz cell phones in the laboratory. Human serum samples were collected from 14 healthy donors (9 women and 5 men) and each sample was divided into four aliquots and was placed into four batches for the in-vitro quantitative determination of human chorionic gonadotropin (hCG). During colorimetric reading of the first, second, and third batches, the ELISA reader (Stat Fax 2100, Awareness Technology, Inc., USA) was exposed to 0.5, 1.0, and 2.0 W exposure of 900 MHz radiation, respectively. For the forth batch (control group), no radiation was applied. All experiments were performed comparing ELISA read out results of the I, II, and III batches with the control batch, using the Wilcoxon test with criterion level of P = 0.050. The final scores in the exposed batches I, II, and III were not statistically significant relative to the control batch (P > 0.05). The results showed that 900 MHz radiation exposure did not alter the ELISA measured levels of hCG hormone in I (P = 0.219), II (P = 0.909), and III (P = 0.056) batches compared to the control batch. This study showed that ELISA reader does not interfere by mobile phone RF radiation at a closed contact (less than 5 cm distance). However, we recommend that medical institutions discuss these issues in the context of their specific use of technologies and frame a policy that is clear and straightforward to guide staff, patients, and visitors.
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ELISA reader does not interfere by mobile phone radiofrequency radiation
Mortazavi, Seyyed Mohammad Javad; Baradaran-Ghahfarokhi, Hamid Reza; Abdi, Mohammad Reza; Baradaran-Ghahfarokhi, Milad; Mostafavi, Nayyer Sadat; Mahmoudi, Golshan; Berenjkoub, Nafiseh; Akmali, Zahra; Hossein-Beigi, Fahimeh; Arsang, Vajiheh
2016-01-01
Background: The increasing number of mobile phones can physically cause electromagnetic interference (EMI) in medical environments; can also cause errors in immunoassays in laboratories. The ELISA readers are widely used as a useful diagnostic tool for Enzymun colorimetric assay in medicine. The aim of this study was to investigate whether the ELISA reader could be interfered by the exposure to the 900 MHz cell phones in the laboratory. Materials and Methods: Human serum samples were collected from 14 healthy donors (9 women and 5 men) and each sample was divided into four aliquots and was placed into four batches for the in-vitro quantitative determination of human chorionic gonadotropin (hCG). During colorimetric reading of the first, second, and third batches, the ELISA reader (Stat Fax 2100, Awareness Technology, Inc., USA) was exposed to 0.5, 1.0, and 2.0 W exposure of 900 MHz radiation, respectively. For the forth batch (control group), no radiation was applied. All experiments were performed comparing ELISA read out results of the I, II, and III batches with the control batch, using the Wilcoxon test with criterion level of P = 0.050. Results: The final scores in the exposed batches I, II, and III were not statistically significant relative to the control batch (P > 0.05). The results showed that 900 MHz radiation exposure did not alter the ELISA measured levels of hCG hormone in I (P = 0.219), II (P = 0.909), and III (P = 0.056) batches compared to the control batch. Conclusion: This study showed that ELISA reader does not interfere by mobile phone RF radiation at a closed contact (less than 5 cm distance). However, we recommend that medical institutions discuss these issues in the context of their specific use of technologies and frame a policy that is clear and straightforward to guide staff, patients, and visitors. PMID:27376040
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González-García, I; García-Arieta, A; Merino-Sanjuan, M; Mangas-Sanjuan, V; Bermejo, M
2018-07-01
Regulatory guidelines recommend that, when a level A IVIVC is established, dissolution specification should be established using averaged data and the maximum difference between AUC and C max between the reference and test formulations cannot be greater than 20%. However, averaging data assumes a loss of information and may reflect a bias in the results. The objective of the current work is to present a new approach to establish dissolution specifications using a new methodology (individual approach) instead of average data (classical approach). Different scenarios were established based on the relationship between in vitro-in vivo dissolution rate coefficient using a level A IVIVC of a controlled release formulation. Then, in order to compare this new approach with the classical one, six additional batches were simulated. For each batch, 1000 simulations of a dissolution assay were run. C max ratios between the reference formulation and each batch were calculated showing that the individual approach was more sensitive and able to detect differences between the reference and the batch formulation compared to the classical approach. Additionally, the new methodology displays wider dissolution specification limits than the classical approach, ensuring that any tablet from the new batch would generate in vivo profiles which its AUC or C max ratio will be out of the 0.8-1.25 range, taking into account the in vitro and in vivo variability of the new batches developed. Copyright © 2018 Elsevier B.V. All rights reserved.
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Malherbe, Christiaan J.; de Beer, Dalene; Joubert, Elizabeth
2012-01-01
Biochemical detection (BCD) methods are commonly used to screen plant extracts for specific biological activities in batch assays. Traditionally, bioactives in the most active extracts were identified through time-consuming bio-assay guided fractionation until single active compounds could be isolated. Not only are isolation procedures often tedious, but they could also lead to artifact formation. On-line coupling of BCD assays to high performance liquid chromatography (HPLC) is gaining ground as a high resolution screening technique to overcome problems associated with pre-isolation by measuring the effects of compounds post-column directly after separation. To date, several on-line HPLC-BCD assays, applied to whole plant extracts and mixtures, have been published. In this review the focus will fall on enzyme-based, receptor-based and antioxidant assays. PMID:22489144
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Januskauskas, A; Johannisson, A; Rodriguez-Martinez, H
2003-09-01
This study investigated the use of annexin-V/PI assay to assess sub lethal changes in bull spermatozoa post-thawing, and to further relate these changes to results obtained by fluorometric assessment of sperm viability and sperm chromatin structure assay (SCSA), as well as field fertility (as 56-day non-return rates, 56-day NRR) after AI. Frozen-thawed semen samples were obtained from 18 Swedish Red and White bulls (one to three semen batches/bull) and fertility data was based on 6900 inseminations. The annexin-V/PI assay revealed that post-thaw semen samples contained on average 41.8+/-7.5% annexin-V-positive cells. Most of the annexin-V-positive cells were dying cells, i.e. also PI-positive. The incidence of annexin-V-positive cells was negatively related (r=-0.59, P<0.01) to the percentage of viable cells, as detected by fluorometry. The incidence of annexin-V-positive spermatozoa significantly correlated to the SCSA variable xalphat (r=0.53, P<0.05). The incidence of annexin-V-negative, dead cells was the only annexin-V/PI assay variable that correlated significantly with fertility both at batch (r=-0.40, P<0.05), and bull (r=-0.56, P<0.05) levels. Among sperm viability variables, subjectively assessed sperm motility (r=0.52-0.59, P<0.01), CASA-assessed sperm motility (r=0.43-0.61, P<0.05), and the incidence of live spermatozoa, expressed as total numbers (r=0.39-0.54, P<0.05), or percentage values (r=0.68-0.68, P<0.01), correlated significantly with field fertility both at batch, and bull levels. Among the SCSA variables, only the COMP alphat correlated significantly (r=0.33-0.51, P<0.05) with fertility results. The results indicate a certain proportion of bull spermatozoa express PS on their surface after thawing, e.g. they have altered membrane function, and that the incidence of such cells is inversely correlated to sperm viability, and positively correlated to abnormal sperm chromatin condensation since they eventually undergo necrosis.
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Buhule, Olive D; Minster, Ryan L; Hawley, Nicola L; Medvedovic, Mario; Sun, Guangyun; Viali, Satupaitea; Deka, Ranjan; McGarvey, Stephen T; Weeks, Daniel E
2014-01-01
Batch effects in DNA methylation microarray experiments can lead to spurious results if not properly handled during the plating of samples. Two pilot studies examining the association of DNA methylation patterns across the genome with obesity in Samoan men were investigated for chip- and row-specific batch effects. For each study, the DNA of 46 obese men and 46 lean men were assayed using Illumina's Infinium HumanMethylation450 BeadChip. In the first study (Sample One), samples from obese and lean subjects were examined on separate chips. In the second study (Sample Two), the samples were balanced on the chips by lean/obese status, age group, and census region. We used methylumi, watermelon, and limma R packages, as well as ComBat, to analyze the data. Principal component analysis and linear regression were, respectively, employed to identify the top principal components and to test for their association with the batches and lean/obese status. To identify differentially methylated positions (DMPs) between obese and lean males at each locus, we used a moderated t-test. Chip effects were effectively removed from Sample Two but not Sample One. In addition, dramatic differences were observed between the two sets of DMP results. After "removing" batch effects with ComBat, Sample One had 94,191 probes differentially methylated at a q-value threshold of 0.05 while Sample Two had zero differentially methylated probes. The disparate results from Sample One and Sample Two likely arise due to the confounding of lean/obese status with chip and row batch effects. Even the best possible statistical adjustments for batch effects may not completely remove them. Proper study design is vital for guarding against spurious findings due to such effects.
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Buhule, Olive D.; Minster, Ryan L.; Hawley, Nicola L.; Medvedovic, Mario; Sun, Guangyun; Viali, Satupaitea; Deka, Ranjan; McGarvey, Stephen T.; Weeks, Daniel E.
2014-01-01
Background: Batch effects in DNA methylation microarray experiments can lead to spurious results if not properly handled during the plating of samples. Methods: Two pilot studies examining the association of DNA methylation patterns across the genome with obesity in Samoan men were investigated for chip- and row-specific batch effects. For each study, the DNA of 46 obese men and 46 lean men were assayed using Illumina's Infinium HumanMethylation450 BeadChip. In the first study (Sample One), samples from obese and lean subjects were examined on separate chips. In the second study (Sample Two), the samples were balanced on the chips by lean/obese status, age group, and census region. We used methylumi, watermelon, and limma R packages, as well as ComBat, to analyze the data. Principal component analysis and linear regression were, respectively, employed to identify the top principal components and to test for their association with the batches and lean/obese status. To identify differentially methylated positions (DMPs) between obese and lean males at each locus, we used a moderated t-test. Results: Chip effects were effectively removed from Sample Two but not Sample One. In addition, dramatic differences were observed between the two sets of DMP results. After “removing” batch effects with ComBat, Sample One had 94,191 probes differentially methylated at a q-value threshold of 0.05 while Sample Two had zero differentially methylated probes. The disparate results from Sample One and Sample Two likely arise due to the confounding of lean/obese status with chip and row batch effects. Conclusion: Even the best possible statistical adjustments for batch effects may not completely remove them. Proper study design is vital for guarding against spurious findings due to such effects. PMID:25352862
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Valero-Navarro, Angel; Medina-Castillo, Antonio L; Fernandez-Sanchez, Jorge F; Fernández-Gutiérrez, Alberto
2011-07-15
The first polyurethane based magnetic-MIP for the selective detection of 1-naphthylamine (1-NA) in drinking water has been synthesised. The synthesis has been carried out in a two-step process: first,the incorporation of magnetite-coated-oleic acid nanoparticles (-Fe₃O₄-OA) into a lipophilic polymeric matrix (poly-MMA-co-EDMA) and second, the encapsulation of these magnetic seeds into the MIP structure by precipitation polymerisation. The mag-MIP was first RHTEM imaged showing a well-organised material with magnetite within the material and the imprinted polymer coating the magnetic core. Thereafter,it was evaluated by batch rebinding analysis and the derived Freundlich isotherm, calculating the number of binding sites (N(K(min)-K(max))=2.63 and 0.79 mmol g⁻¹, for mag-MIP and mag-NIP, respectively)and apparent average adsorption constant (K(K(min)-K(max))=3.31 and 3.06 mmol⁻¹, for mag-MIP and mag-NIP, respectively) showing a very effective imprinting process.We have also developed a magnetic optical sensor MIP by using an optical fiber coupled with a magnetic separator. An unexpected selectivity for 1-NA was revealed allowing the detection of this molecule in water, even in the presence of 4 structurally related compounds (2-naphthylamine, 1-naphthol, 2-naphthol and 1-naphthalenemethylamine), with a low limit of detection (LOD) = 18 ng mL⁻¹. Finally, we applied this new hybrid material to the analysis of 1-NA in tap and mineral waters, obtaining a 91.6%average recovery rate. Copyright © 2011 Elsevier B.V. All rights reserved.
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Commercial antibodies and their validation
Voskuil, JLA
2014-01-01
Despite an impressive growth in the business of research antibodies a general lack of trust in commercial antibodies remains in place. A variety of issues, each one potentially causing an antibody to fail, underpin the frustrations that scientists endure. Lots of money goes to waste in buying and trying one failing antibody after the other without realizing all the pitfalls that come with the product: Antibodies can get inactivated, both the biological material and the assay itself can potentially be flawed, a single antibody featuring in many different catalogues can be deemed as a set of different products, and a bad choice of antibody type, wrong dilutions, and lack of proper validation can all jeopardize the intended experiments. Antibodies endorsed by scientific research papers do not always meet the scientist’s requirements either due to flawed specifications, or due to batch-to-batch variations. Antibodies can be found with Quality Control data obtained from previous batches that no longer represent the batch on sale. In addition, one cannot assume that every antibody is fit for every application. The best chance of success is to try an antibody that already was confirmed to perform correctly in the required platform. PMID:25324967
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Effects of anti-foaming agents on biohydrogen production.
Sivagurunathan, Periyasamy; Anburajan, Parthiban; Kumar, Gopalakrishnan; Bakonyi, Péter; Nemestóthy, Nándor; Bélafi-Bakó, Katalin; Kim, Sang-Hyoun
2016-08-01
The effects of antifoaming agents on fermentative hydrogen production using galactose in batch and continuous operations were investigated. Batch hydrogen production assays with LS-303 (dimethylpolysiloxane), LG-109 (polyalkylene), LG-126 (polyoxyethylenealkylene), and LG-299 (polyether) showed that the doses and types of antifoaming agents played a significant role in hydrogen production. During batch tests, LS-303 at 100μL/L resulted in the maximum hydrogen production rate (HPR) and hydrogen yield (HY) of 2.5L/L-d and 1.08mol H2/mol galactoseadded, respectively. The following continuously stirred tank reactor operated at 12h HRT with LS-303 at 100μL/L showed a stable HPR and HY of 4.9L/L-d and 1.17mol H2/mol galactoseadded, respectively, which were higher than those found for the control reactor. Microbial community analysis supported the alterations in H2 generation under different operating conditions and the stimulatory impact of certain antifoaming chemicals on H2 production was demonstrated. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Tsapekos, P; Kougias, P G; Vasileiou, S A; Treu, L; Campanaro, S; Lyberatos, G; Angelidaki, I
2017-06-01
Bioaugmentation with hydrolytic microbes was applied to improve the methane yield of bioreactors fed with agricultural wastes. The efficiency of Clostridium thermocellum and Melioribacter roseus to degrade lignocellulosic matter was evaluated in batch and continuously stirred tank reactors (CSTRs). Results from batch assays showed that C. thermocellum enhanced the methane yield by 34%. A similar increase was recorded in CSTR during the bioaugmentation period; however, at steady-state the effect was noticeably lower (7.5%). In contrast, the bioaugmentation with M. roseus did not promote markedly the anaerobic biodegradability, as the methane yield was increased up to 10% in batch and no effect was shown in CSTR. High-throughput 16S rRNA amplicon sequencing was used to assess the effect of bioaugmentation strategies on bacterial and archaeal populations. The microbial analysis revealed that both strains were not markedly resided into biogas microbiome. Additionally, the applied strategies did not alter significantly the microbial communities. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Liu, Jiang; Zhang, Lu; Li Han Song, Le; Liu, Yuan; Tang, Hui; Li, Yingchun
2015-04-01
Metronidazole-imprinted polymers with superior recognition properties were prepared by a novel strategy called distillation-precipitation polymerization. The as-obtained polymers were characterized by Fourier-transform infrared spectroscopy, laser particle size determination and scanning electron microscopy, and their binding performances were evaluated in detail by static, kinetic and dynamic rebinding tests, and Scatchard analysis. The results showed that when the fraction of the monomers was 5 vol% in the whole reaction system, the prepared polymers afforded good morphology, monodispersity, and high adsorption capacity and excellent selectivity to the target molecule, metronidazole. The optimal binding performance is 12.41 mg/g for metronidazole just before leakage occurred and 38.51 mg/g at saturation in dynamic rebinding tests. Metronidazole-imprinted polymers were further applied as packing agents in solid-phase extraction and as chromatographic filler, both of which served for the detection of metronidazole in fish tissue. The results illustrated the recoveries of spiked samples ranged from 82.97 to 87.83% by using molecularly imprinted solid-phase extraction combined with a C18 commercial column and 93.7 to 101.2% by directly using the polymer-packed chromatographic column. The relative standard deviation of both methods was less than 6%. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Factorization of the association rate coefficient in ligand rebinding to heme proteins
NASA Astrophysics Data System (ADS)
Young, Robert D.
1984-01-01
A stochastic theory of ligand migration in biomolecules is used to analyze the recombination of small ligands to heme proteins after flash photolysis. The stochastic theory is based on a generalized sequential barrier model in which a ligand binds by overcoming a series of barriers formed by the solvent protein interface, the protein matrix, and the heme distal histidine system. The stochastic theory shows that the association rate coefficient λon factorizes into three terms λon =γ12
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Nickerson, Beverly; Harrington, Brent; Li, Fasheng; Guo, Michele Xuemei
2017-11-30
Drug product assay is one of several tests required for new drug products to ensure the quality of the product at release and throughout the life cycle of the product. Drug product assay testing is typically performed by preparing a composite sample of multiple dosage units to obtain an assay value representative of the batch. In some cases replicate composite samples may be prepared and the reportable assay value is the average value of all the replicates. In previously published work by Harrington et al. (2014) [5], a sample preparation composite and replicate strategy for assay was developed to provide a systematic approach which accounts for variability due to the analytical method and dosage form with a standard error of the potency assay criteria based on compendia and regulatory requirements. In this work, this sample preparation composite and replicate strategy for assay is applied to several case studies to demonstrate the utility of this approach and its application at various stages of pharmaceutical drug product development. Copyright © 2017 Elsevier B.V. All rights reserved.
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Parvovirus B19V DNA contamination in Chinese plasma and plasma derivatives
2012-01-01
Background To ensure the safety of plasma derivatives, screening for human parvovirus B19V genomic DNA in donated plasma using a pooling strategy is performed in some countries. We investigated the prevalence of B19V DNA and anti-B19V antibodies in Chinese plasma pools, plasma derivatives and plasma donations to evaluate the risk posed by B19V. Methods Using a Q-PCR assay developed in-house, we tested for B19V genomic DNA in 142 plasma pools collected between January 2009 and June 2011 from two Chinese blood products manufacturers. Plasma derivatives collected between 1993–1995 (10 batches of albumin, 155 batches of intravenous immunoglobulin, IVIG) and 2009–2011 (50 batches of albumin, 54 batches of IVIG, 35 batches of factor VIII, 7 batches of fibrinogen, and 17 batches of prothrombin complex concentrate, PCC) were also tested for B19V contamination. In addition, B19V genome prevalence in minipools(including 90 individual donations) of 49680 individual plasma samples collected between August 2011 and March 2012 by a single Chinese manufacturer was investigated. IgM/IgG was also investigated in plasma pools/derivatives and in minipools with B19V-DNA titers above 1x104 and 1x106 geq/mL using B19 ELISA IgM/IgG assay(Virion-Serion, Würzburg, Germany), respectively. Results B19V-DNA was detected in 54.2% of plasma pools from two Chinese blood product manufacturers; among recently produced blood products, B19V was detected in 21/54 IVIG samples, 19/35 factor VIII samples, 6/7 fibrinogen samples, and 12/17 PCC samples, but not in albumin samples. The levels of B19V-DNA in these samples varied from 102-107 geq/mL. In samples with >104 geq/mL genome DNA, B19V-specific IgG was also found in all corresponding plasma pools and IVIG, whereas none was detected in the majority of other plasma derivatives. Screening of plasma donations indicated that most minipools were contaminated with B19V-DNA (102-108 geq/mL) and one donation had 1.09 × 1010 geq/mL B19V genomic DNA along with a non-classical IgG/IgM profile. Conclusions Despite the implementation of some inactivation/removal methods designed to prevent viral contamination, B19V DNA was detectable in Chinese plasma pools and plasma derivatives. Thus, the introduction of B19V screening and discard donation with high viramic concentration for Chinese plasma donors would be desirable. PMID:22978673
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Low, Mitchell; Khoo, Cheang S; Münch, Gerald; Govindaraghavan, Suresh; Sucher, Nikolaus J
2015-02-07
The anti-inflammatory activity of Andrographis paniculata (Acanthaceae), a traditional medicine widely used in Asia, is commonly attributed to andrographolide, its main secondary metabolite. Commercial A. paniculata extracts are standardised to andrographolide content. We undertook the present study to investigate 1) how selective enrichment of andrographolide in commercial A. paniculata extracts affects the variability of non-standardised phytochemical components and 2) if variability in the non-standardised components of the extract affects the pharmacological activity of andrographolide itself. We characterized 12 commercial, standardised (≥30% andrographolide) batches of A. paniculata extracts from India by HPLC profiling. We determined the antioxidant capacity of the extracts using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, oxygen radical antioxidant capacity (ORAC) and a Folin-Ciocalteu (FC) antioxidant assays. Their anti-inflammatory activity was assessed by assaying their inhibitory effect on the release of tumor necrosis factor alpha (TNF-α) in the human monocytic cell line THP-1. The andrographolide content in the samples was close to the claimed value (32.2 ± 2.1%, range 27.5 to 35.9%). Twenty-one non-standardised constituents exhibited more than 2-fold variation in HPLC peak intensities in the tested batches. The chlorogenic acid content of the batches varied more than 30-fold. The DPPH free radical scavenging activity varied ~3-fold, the ORAC and FC antioxidant capacity varied ~1.5 fold among batches. In contrast, the TNF-α inhibitory activity of the extracts exhibited little variation and comparison with pure andrographolide indicated that it was mostly due to their andrographolide content. Standardised A. paniculata extracts contained the claimed amount of andrographolide but exhibited considerable phytochemical background variation. DPPH radical scavenging activity of the extracts was mostly due to the flavonoid/phenlycarboxylic acid compounds in the extracts. The inhibitory effect of andrographolide on the release of TNF-α was little affected by the quantitative variation of the non-standardised constituents.
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UV-absorbing and other sun-protecting substances: genotoxicity of 2-ethylhexyl P-methoxycinnamate.
Bonin, A M; Arlauskas, A P; Angus, D S; Baker, R S; Gallagher, C H; Greenoak, G; Brown, M M; Meher-Homji, K M; Reeve, V
1982-11-01
The mutagenicity of 2-ethylhexyl p-methoxycinnamate was demonstrated when 25 sunscreen ingredients were tested in the Salmonella/microsome assay. This substance also increased the frequency of sex-linked recessive lethals in Drosophila melanogaster. A trace contaminant may be implicated because many samples were obtained from several sources and the results were batch-related.
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NASA Astrophysics Data System (ADS)
Feldmann, Daniel P.; Xie, Yuran; Jones, Steven K.; Yu, Dongyue; Moszczynska, Anna; Merkel, Olivia M.
2017-06-01
The triblock copolymer polyethylenimine-polycaprolactone-polyethylene glycol (PEI-PCL-PEG) has been shown to spontaneously assemble into nano-sized particulate carriers capable of complexing with nucleic acids for gene delivery. The objective of this study was to investigate micelleplex characteristics, their in vitro and in vivo fate following microfluidic preparation of siRNA nanoparticles compared to the routinely used batch reactor mixing technique. Herein, PEI-PCL-PEG nanoparticles were prepared with batch reactor or microfluidic mixing techniques and characterized by various biochemical assays and in cell culture. Microfluidic nanoparticles showed a reduction of overall particle size as well as a more uniform size distribution when compared to batch reactor pipette mixing. Confocal microscopy, flow cytometry and qRT-PCR displayed the subcellular delivery of the microfluidic formulation and confirmed the ability to achieve mRNA knockdown. Intratracheal instillation of microfluidic formulation resulted in a significantly more efficient (p < 0.05) knockdown of GAPDH compared to treatment with the batch reactor formulation. The use of microfluidic mixing techniques yields an overall smaller and more uniform PEG-PCL-PEI nanoparticle that is able to more efficiently deliver siRNA in vivo. This preparation method may prove to be useful when a scaled up production of well-defined polyplexes is required.
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Nalakath Abubackar, Haris; Veiga, María C.; Kennes, Christian
2015-01-01
The effect of different sources of nitrogen as well as their concentrations on the bioconversion of carbon monoxide to metabolic products such as acetic acid and ethanol by Clostridium autoethanogenum was studied. In a first set of assays, under batch conditions, either NH4Cl, trypticase soy broth or yeast extract (YE) were used as sources of nitrogen. The use of YE was found statistically significant (p < 0.05) on the product spectrum in such batch assays. In another set of experiments, three bioreactors were operated with continuous CO supply, in order to estimate the effect of running conditions on products and biomass formation. The bioreactors were operated under different conditions, i.e., EXP1 (pH = 5.75, YE 1g/L), EXP2 (pH = 4.75, YE 1 g/L) and EXP3 (pH = 5.75, YE 0.2 g/L). When compared to EXP2 and EXP3, it was found that EXP1 yielded the maximum biomass accumulation (302.4 mg/L) and products concentrations, i.e., acetic acid (2147.1 mg/L) and ethanol (352.6 mg/L). This can be attributed to the fact that the higher pH and higher YE concentration used in EXP1 stimulated cell growth and did, consequently, also enhance metabolite production. However, when ethanol is the desired end-product, as a biofuel, the lower pH used in EXP2 was more favourable for solventogenesis and yielded the highest ethanol/acetic acid ratio, reaching a value of 0.54. PMID:25608591
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Abubackar, Haris Nalakath; Veiga, María C; Kennes, Christian
2015-01-20
The effect of different sources of nitrogen as well as their concentrations on the bioconversion of carbon monoxide to metabolic products such as acetic acid and ethanol by Clostridium autoethanogenum was studied. In a first set of assays, under batch conditions, either NH4Cl, trypticase soy broth or yeast extract (YE) were used as sources of nitrogen. The use of YE was found statistically significant (p < 0.05) on the product spectrum in such batch assays. In another set of experiments, three bioreactors were operated with continuous CO supply, in order to estimate the effect of running conditions on products and biomass formation. The bioreactors were operated under different conditions, i.e., EXP1 (pH = 5.75, YE 1g/L), EXP2 (pH = 4.75, YE 1 g/L) and EXP3 (pH = 5.75, YE 0.2 g/L). When compared to EXP2 and EXP3, it was found that EXP1 yielded the maximum biomass accumulation (302.4 mg/L) and products concentrations, i.e., acetic acid (2147.1 mg/L) and ethanol (352.6 mg/L). This can be attributed to the fact that the higher pH and higher YE concentration used in EXP1 stimulated cell growth and did, consequently, also enhance metabolite production. However, when ethanol is the desired end-product, as a biofuel, the lower pH used in EXP2 was more favourable for solventogenesis and yielded the highest ethanol/acetic acid ratio, reaching a value of 0.54.
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Analytical validation of a new point-of-care assay for serum amyloid A in horses.
Schwartz, D; Pusterla, N; Jacobsen, S; Christopher, M M
2018-01-17
Serum amyloid A (SAA) is a major acute phase protein in horses. A new point-of-care (POC) test for SAA (Stablelab) is available, but studies evaluating its analytical accuracy are lacking. To evaluate the analytical performance of the SAA POC test by 1) determining linearity and precision, 2) comparing results in whole blood with those in serum or plasma, and 3) comparing POC results with those obtained using a previously validated turbidimetric immunoassay (TIA). Assay validation. Analytical validation of the POC test was done in accordance with American Society of Veterinary Clinical Pathology guidelines using residual equine serum/plasma and whole blood samples from the Clinical Pathology Laboratory at the University of California-Davis. A TIA was used as the reference method. We also evaluated the effect of haematocrit (HCT). The POC test was linear for SAA concentrations of up to at least 1000 μg/mL (r = 0.991). Intra-assay CVs were 13, 18 and 15% at high (782 μg/mL), intermediate (116 μg/mL) and low (64 μg/mL) concentrations. Inter-assay (inter-batch) CVs were 45, 14 and 15% at high (1372 μg/mL), intermediate (140 μg/mL) and low (56 μg/mL) concentrations. SAA results in whole blood were significantly lower than those in serum/plasma (P = 0.0002), but were positively correlated (r = 0.908) and not affected by HCT (P = 0.261); proportional negative bias was observed in samples with SAA>500 μg/mL. The difference between methods exceeded the 95% confidence interval of the combined imprecision of both methods (15%). Analytical validation could not be performed in whole blood, the sample most likely to be used stall side. The POC test has acceptable accuracy and precision in equine serum/plasma with SAA concentrations of up to at least 1000 μg/mL. Low inter-batch precision at high concentrations may affect serial measurements, and the use of the same test batch and sample type (serum/plasma or whole blood) is recommended. Comparison of results between the POC test and the TIA is not recommended. © 2018 EVJ Ltd.
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Multidimensional Normalization to Minimize Plate Effects of Suspension Bead Array Data.
Hong, Mun-Gwan; Lee, Woojoo; Nilsson, Peter; Pawitan, Yudi; Schwenk, Jochen M
2016-10-07
Enhanced by the growing number of biobanks, biomarker studies can now be performed with reasonable statistical power by using large sets of samples. Antibody-based proteomics by means of suspension bead arrays offers one attractive approach to analyze serum, plasma, or CSF samples for such studies in microtiter plates. To expand measurements beyond single batches, with either 96 or 384 samples per plate, suitable normalization methods are required to minimize the variation between plates. Here we propose two normalization approaches utilizing MA coordinates. The multidimensional MA (multi-MA) and MA-loess both consider all samples of a microtiter plate per suspension bead array assay and thus do not require any external reference samples. We demonstrate the performance of the two MA normalization methods with data obtained from the analysis of 384 samples including both serum and plasma. Samples were randomized across 96-well sample plates, processed, and analyzed in assay plates, respectively. Using principal component analysis (PCA), we could show that plate-wise clusters found in the first two components were eliminated by multi-MA normalization as compared with other normalization methods. Furthermore, we studied the correlation profiles between random pairs of antibodies and found that both MA normalization methods substantially reduced the inflated correlation introduced by plate effects. Normalization approaches using multi-MA and MA-loess minimized batch effects arising from the analysis of several assay plates with antibody suspension bead arrays. In a simulated biomarker study, multi-MA restored associations lost due to plate effects. Our normalization approaches, which are available as R package MDimNormn, could also be useful in studies using other types of high-throughput assay data.
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O'Connor, C; Kiernan, M G; Finnegan, C; O'Hara, M; Power, L; O'Connell, N H; Dunne, C P
2017-05-04
Rapid detection of patients with carbapenemase-producing Enterobacteriaceae (CPE) is essential for the prevention of nosocomial cross-transmission, allocation of isolation facilities and to protect patient safety. Here, we aimed to design a new laboratory work-flow, utilizing existing laboratory resources, in order to reduce time-to-diagnosis of CPE. A review of the current CPE testing processes and of the literature was performed to identify a real-time commercial polymerase chain reaction (PCR) assay that could facilitate batch testing of CPE clinical specimens, with adequate CPE gene coverage. Stool specimens (210) were collected; CPE-positive inpatients (n = 10) and anonymized community stool specimens (n = 200). Rectal swabs (eSwab™) were inoculated from collected stool specimens and a manual DNA extraction method (QIAamp® DNA Stool Mini Kit) was employed. Extracted DNA was then processed on the Check-Direct CPE® assay. The three step process of making the eSwab™, extracting DNA manually and running the Check-Direct CPE® assay, took <5 min, 1 h 30 min and 1 h 50 min, respectively. It was time efficient with a result available in under 4 h, comparing favourably with the existing method of CPE screening; average time-to-diagnosis of 48/72 h. Utilizing this CPE work-flow would allow a 'same-day' result. Antimicrobial susceptibility testing results, as is current practice, would remain a 'next-day' result. In conclusion, the Check-Direct CPE® assay was easily integrated into a local laboratory work-flow and could facilitate a large volume of CPE screening specimens in a single batch, making it cost-effective and convenient for daily CPE testing.
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Gebrekidan, Hagos; Gasser, Robin B; Stevenson, Mark A; McGrath, Sean; Jabbar, Abdul
2017-02-01
Oriental theileriosis caused by multiple genotypes of Theileria orientalis is an important tick-borne disease of bovines. Here, we assessed the performance of an established multiplexed tandem PCR (MT-PCR) for the diagnosis of the two recognized, pathogenic genotypes (chitose and ikeda) of T. orientalis in cattle using pooled blood samples. We used a total of 265 cattle blood samples, which were divided into two groups according to previous MT-PCR results for individual samples. Samples in group 1 (n = 155) were from a herd with a relatively high prevalence of T. orientalis infection; and those in group 2 (n = 110) were from four herds with a low prevalence. For group 1, 31 and 15 batches of five- and ten-pooled samples (selected at random), respectively, were formed. For group 2, 22 and 11 batches of five- and ten-pooled samples (selected at random), respectively, were formed. DNAs from individual pooled samples in each batch and group were then tested by MT-PCR. For group 1, the apparent prevalences estimated using the 31 batches of five-pooled samples (97%) and 15 batches of ten-pooled samples (100%) were significantly higher compared with individual samples (75%). For group 2, higher apparent prevalences (9% and 36%) were also recorded for the 22 and 11 batches of pooled samples, respectively, compared with individual samples (7%). Overall, the average infection intensity recorded for the genotypes of chitose and ikeda were considerably lower in pooled compared with individual samples. The diagnostic specificities of MT-PCR were estimated at 95% and 94%, respectively, when batches of five- and ten-pooled samples were tested, and 94% for individual samples. The diagnostic sensitivity of this assay was estimated at 98% same for all individual, five- and ten-pooled samples. This study shows that screening batches of five- and ten-pooled blood samples from cattle herds are similar to those obtained for individual samples, and, importantly, that the reduced cost for the testing of pooled samples represents a considerable saving to herd managers. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Novel method for the high-throughput processing of slides for the comet assay
Karbaschi, Mahsa; Cooke, Marcus S.
2014-01-01
Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. “Scoring”, or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure. PMID:25425241
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Novel method for the high-throughput processing of slides for the comet assay.
Karbaschi, Mahsa; Cooke, Marcus S
2014-11-26
Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. "Scoring", or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure.
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Gomes, Rui S; Moreira, Felismina T C; Fernandes, Ruben; Sales, M Goreti F
2018-01-01
This work presents an alternative device for cancer screening in liquid biopsies. It combines a biomimetic film (i) with electrochemical detection (ii). The biomimetic film (i) was obtained by electro-polymerizing amine-substituted benzene rings around a CA 15-3 target. This protein target was previously adsorbed on a gold (Au) support and incubated in charged monomers (4-Styrenesulfonate sodium and 3-Hydroxytyraminium chloride). The protein was further eliminated by enzymatic activity, leaving behind vacant sites for subsequent rebinding. Electrochemical detection (ii) was achieved on an Au working electrode, designed on commercial screen-printed electrodes. Raman spectroscopy, atomic force microscopy and ellipsometric readings were used to follow the chemical modification of the Au surface. The ability of the material to rebind CA15-3 was monitored by electrochemical techniques. The device displayed linear responses to CA15-3 ranging from 0.25 to 10.00 U/mL, with detection limits of 0.05 U/mL. Accurate results were obtained by applying the sensor to the analysis of CA15-3 in PBS buffer and in serum samples. This biosensing device displayed successful features for the detection of CA 15-3 and constitutes a promising tool for breast cancer screening procedures in point-of-care applications. Moreover, its scale-up seems feasible as it contains a plastic antibody assembled in situ, in less than 1 minute, and the analysis of serum takes less than 30 minutes.
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Golker, Kerstin; Karlsson, Björn C. G.; Rosengren, Annika M.; Nicholls, Ian A.
2014-01-01
In this report, principal component analysis (PCA) has been used to explore the influence of template complexation in the pre-polymerization phase on template molecularly imprinted polymer (MIP) recognition and polymer morphology. A series of 16 bupivacaine MIPs were studied. The ethylene glycol dimethacrylate (EGDMA)-crosslinked polymers had either methacrylic acid (MAA) or methyl methacrylate (MMA) as the functional monomer, and the stoichiometry between template, functional monomer and crosslinker was varied. The polymers were characterized using radioligand equilibrium binding experiments, gas sorption measurements, swelling studies and data extracted from molecular dynamics (MD) simulations of all-component pre-polymerization mixtures. The molar fraction of the functional monomer in the MAA-polymers contributed to describing both the binding, surface area and pore volume. Interestingly, weak positive correlations between the swelling behavior and the rebinding characteristics of the MAA-MIPs were exposed. Polymers prepared with MMA as a functional monomer and a polymer prepared with only EGDMA were found to share the same characteristics, such as poor rebinding capacities, as well as similar surface area and pore volume, independent of the molar fraction MMA used in synthesis. The use of PCA for interpreting relationships between MD-derived descriptions of events in the pre-polymerization mixture, recognition properties and morphologies of the corresponding polymers illustrates the potential of PCA as a tool for better understanding these complex materials and for their rational design. PMID:25391043
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Golker, Kerstin; Karlsson, Björn C G; Rosengren, Annika M; Nicholls, Ian A
2014-11-10
In this report, principal component analysis (PCA) has been used to explore the influence of template complexation in the pre-polymerization phase on template molecularly imprinted polymer (MIP) recognition and polymer morphology. A series of 16 bupivacaine MIPs were studied. The ethylene glycol dimethacrylate (EGDMA)-crosslinked polymers had either methacrylic acid (MAA) or methyl methacrylate (MMA) as the functional monomer, and the stoichiometry between template, functional monomer and crosslinker was varied. The polymers were characterized using radioligand equilibrium binding experiments, gas sorption measurements, swelling studies and data extracted from molecular dynamics (MD) simulations of all-component pre-polymerization mixtures. The molar fraction of the functional monomer in the MAA-polymers contributed to describing both the binding, surface area and pore volume. Interestingly, weak positive correlations between the swelling behavior and the rebinding characteristics of the MAA-MIPs were exposed. Polymers prepared with MMA as a functional monomer and a polymer prepared with only EGDMA were found to share the same characteristics, such as poor rebinding capacities, as well as similar surface area and pore volume, independent of the molar fraction MMA used in synthesis. The use of PCA for interpreting relationships between MD-derived descriptions of events in the pre-polymerization mixture, recognition properties and morphologies of the corresponding polymers illustrates the potential of PCA as a tool for better understanding these complex materials and for their rational design.
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Gomes, Rui S.; Moreira, Felismina T. C.; Fernandes, Ruben
2018-01-01
This work presents an alternative device for cancer screening in liquid biopsies. It combines a biomimetic film (i) with electrochemical detection (ii). The biomimetic film (i) was obtained by electro-polymerizing amine-substituted benzene rings around a CA 15–3 target. This protein target was previously adsorbed on a gold (Au) support and incubated in charged monomers (4-Styrenesulfonate sodium and 3-Hydroxytyraminium chloride). The protein was further eliminated by enzymatic activity, leaving behind vacant sites for subsequent rebinding. Electrochemical detection (ii) was achieved on an Au working electrode, designed on commercial screen-printed electrodes. Raman spectroscopy, atomic force microscopy and ellipsometric readings were used to follow the chemical modification of the Au surface. The ability of the material to rebind CA15-3 was monitored by electrochemical techniques. The device displayed linear responses to CA15-3 ranging from 0.25 to 10.00 U/mL, with detection limits of 0.05 U/mL. Accurate results were obtained by applying the sensor to the analysis of CA15-3 in PBS buffer and in serum samples. This biosensing device displayed successful features for the detection of CA 15–3 and constitutes a promising tool for breast cancer screening procedures in point-of-care applications. Moreover, its scale-up seems feasible as it contains a plastic antibody assembled in situ, in less than 1 minute, and the analysis of serum takes less than 30 minutes. PMID:29715330
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Aldridge, Andrew; Kouroupis, Dimitrios; Churchman, Sarah; English, Anne; Ingham, Eileen; Jones, Elena
2013-01-01
Background aims Mesenchymal stromal cells (MSCs) are regenerative and immuno-privileged cells that are used for both tissue regeneration and treatment of severe inflammation-related disease. For quality control of manufactured MSC batches in regard to mature fat cell contamination, a quantitative method for measuring adipogenesis is needed. Methods Four previously proposed methods were validated with the use of bone marrow (BM) MSCs during a 21-day in vitro assay. Oil red staining was scored semiquantitatively; peroxisome proliferator activated receptor-γ and fatty acid binding protein (FABP)4 transcripts were measured by quantitative real-time polymerase chain reaction; FABP4 protein accumulation was evaluated by flow cytometry; and Nile red/4′,6-diamidino-2-phenylindole (DAPI) ratios were measured in fluorescent microplate assay. Skin fibroblasts and MSCs from fat pad, cartilage and umbilical cord were used as controls. Results Oil red staining indicated considerable heterogeneity between BM donors and individual cells within the same culture. FABP4 transcript levels increased 100- to 5000-fold by day 21, with large donor variability observed. Flow cytometry revealed increasing intra-culture heterogeneity over time; more granular cells accumulated more FABP4 protein and Nile red fluorescence compared with less granular cells. Nile red increase in day-21 MSCs was ∼5- and 4-fold, measured by flow cytometry or microplate assay, respectively. MSC proliferation/apoptosis was accounted through the use of Nile red/DAPI ratios; adipogenesis levels in day-21 BM MSCs increased ∼13-fold, with significant correlations with oil red scoring observed for MSC from other sources. Conclusions Flow cytometry permits the study of MSC differentiation at the single-cell level and sorting more and less mature cells from mixed cell populations. The microplate assay with the use of the Nile red/DAPI ratio provides rapid quantitative measurements and could be used as a low-cost, high-throughput method to quality-control MSC batches from different tissue sources. PMID:23260089
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Yamada, Kentaro; Henares, Terence G; Suzuki, Koji; Citterio, Daniel
2015-11-11
"Distance-based" detection motifs on microfluidic paper-based analytical devices (μPADs) allow quantitative analysis without using signal readout instruments in a similar manner to classical analogue thermometers. To realize a cost-effective and calibration-free distance-based assay of lactoferrin in human tear fluid on a μPAD not relying on antibodies or enzymes, we investigated the fluidic mobilities of the target protein and Tb(3+) cations used as the fluorescent detection reagent on surface-modified cellulosic filter papers. Chromatographic elution experiments in a tear-like sample matrix containing electrolytes and proteins revealed a collapse of attractive electrostatic interactions between lactoferrin or Tb(3+) and the cellulosic substrate, which was overcome by the modification of the paper surface with the sulfated polysaccharide ι-carrageenan. The resulting μPAD based on the fluorescence emission distance successfully analyzed 0-4 mg mL(-1) of lactoferrin in complex human tear matrix with a lower limit of detection of 0.1 mg mL(-1) by simple visual inspection. Assay results of 18 human tear samples including ocular disease patients and healthy volunteers showed good correlation to the reference ELISA method with a slope of 0.997 and a regression coefficient of 0.948. The distance-based quantitative signal and the good batch-to-batch fabrication reproducibility relying on printing methods enable quantitative analysis by simply reading out "concentration scale marks" printed on the μPAD without performing any calibration and using any signal readout instrument.
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Post-use assay of vaginal rings (VRs) as a potential measure of clinical trial adherence.
Spence, Patrick; Nel, Annalene; van Niekerk, Neliëtte; Derrick, Tiffany; Wilder, Susan; Devlin, Bríd
2016-06-05
Adherence measurement for microbicide use within the clinical trial setting remains a challenge for the HIV prevention field. This paper describes an assay method used for determining residual dapivirine levels in post-use vaginal rings from clinical trials conducted with the Dapivirine Vaginal Matrix Ring-004 developed by the International Partnership for Microbicides to prevent male to female HIV transmission. Post-use assay results from three Ring-004 clinical trials showed that of the 25mg drug load, approximately 4mg of dapivirine is released from the matrix ring over a 28-day use period. Data obtained by both in vitro and in vivo studies indicate that dapivirine is released according to a diffusion mechanism, as determined by conformance of both data sets to the Higuchi equation. This, coupled with the low variability associated with batch production over two manufacturing sites and 20 batches of material, provides evidence that post-use ring analysis can contribute to the assessment of adherence to ring use. Limitations of this method include the potential of intra-participant and inter-participant variability and uncertainty associated with measuring the low amount of dapivirine actually released relative to the drug load. Therefore, residual drug levels should not serve as the only direct measurement for microbicide adherence in vaginal ring clinical trials but should preferably be used as part of a multi-pronged approach towards understanding and assessing adherence to vaginal ring use. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
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Quantitative determinations using portable Raman spectroscopy.
Navin, Chelliah V; Tondepu, Chaitanya; Toth, Roxana; Lawson, Latevi S; Rodriguez, Jason D
2017-03-20
A portable Raman spectrometer was used to develop chemometric models to determine percent (%) drug release and potency for 500mg ciprofloxacin HCl tablets. Parallel dissolution and chromatographic experiments were conducted alongside Raman experiments to assess and compare the performance and capabilities of portable Raman instruments in determining critical drug attributes. All batches tested passed the 30min dissolution specification and the Raman model for drug release was able to essentially reproduce the dissolution profiles obtained by ultraviolet spectroscopy at 276nm for all five batches of the 500mg ciprofloxacin tablets. The five batches of 500mg ciprofloxacin tablets also passed the potency (assay) specification and the % label claim for the entire set of tablets run were nearly identical, 99.4±5.1 for the portable Raman method and 99.2±1.2 for the chromatographic method. The results indicate that portable Raman spectrometers can be used to perform quantitative analysis of critical product attributes of finished drug products. The findings of this study indicate that portable Raman may have applications in the areas of process analytical technology and rapid pharmaceutical surveillance. Published by Elsevier B.V.
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Collaborative study for the establishment of erythropoietin BRP batch 4.
Burns, C; Bristow, A F; Daas, A; Costanzo, A
2015-01-01
The European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for erythropoietin (EPO) is used as a working standard for potency determination of EPO preparations by in vivo bioassay as prescribed in the Ph. Eur. monograph Erythropoietin concentrated solution (1316). The BRP batch 3 was calibrated in 2006 and its stocks are depleted. The European Directorate for the Quality of Medicines & HealthCare (EDQM) thus initiated a project to calibrate a replacement batch in International Units against the WHO 3(rd) International Standard (IS) for Erythropoietin, recombinant, for bioassay (11/170). A Ph. Eur. Chemical Reference Substance (CRS) was established recently for use as reference in some of the physicochemical tests prescribed in the monograph. Therefore, the EPO BRP batch 4 was only calibrated for the normocythaemic and polycythaemic mouse in vivo bioassays described in the Assay section of the Ph. Eur. monograph (1316). The collaborative study involved seven laboratories from Europe, the USA and South America. The results confirmed that the candidate BRP (cBRP) is suitable for use as a reference preparation in the potency determination of EPO medicinal products by bioassay (using the normocythaemic or polycythaemic mouse methods). The outcome of the study enabled the Ph. Eur. Commission to establish the proposed standard as erythropoietin BRP batch 4 in November 2014 for use as a reference preparation solely for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 13 000 IU/vial. Furthermore, the potency of BRP3 was confirmed during the study, thus warranting a good continuity of the IU.
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HIV RNA testing in the context of nonoccupational postexposure prophylaxis.
Roland, Michelle E; Elbeik, Tarek A; Kahn, James O; Bamberger, Joshua D; Coates, Thomas J; Krone, Melissa R; Katz, Mitchell H; Busch, Michael P; Martin, Jeffrey N
2004-08-01
The specificity and positive predictive value of human immunodeficiency virus (HIV) RNA assays have not been evaluated in the setting of postexposure prophylaxis (PEP). Plasma from subjects enrolled in a nonoccupational PEP study was tested with 2 branched-chain DNA (bDNA) assays, 2 polymerase chain reaction (PCR) assays, and a transcription-mediated amplification (TMA) assay. Assay specificity and positive predictive value were determined for subjects who remained negative for HIV antibody for >or=3 months. In 329 subjects examined, the lowest specificities (90.1%-93.7%) were seen for bDNA testing performed in real time. The highest specificities were seen with batched bDNA version 3.0 (99.1%), standard PCR (99.4%), ultrasensitive PCR (100%), and TMA (99.6%) testing. Only the 2 assays with the highest specificities had positive predictive values >40%. For the bDNA assays, increasing the cutoff point at which a test is called positive (e.g., from 50 copies/mL to 500 copies/mL for version 3.0) increased both specificity and positive predictive values to 100%. The positive predictive value of HIV RNA assays in individuals presenting for PEP is unacceptably low for bDNA-based testing and possibly acceptable for PCR- and TMA-based testing. Routine use of HIV RNA assays in such individuals is not recommended.
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Parreño, Viviana; Romera, S Alejandra; Makek, Lucia; Rodriguez, Daniela; Malacari, Darío; Maidana, Silvina; Compaired, Diego; Combessies, Gustavo; Vena, María Marta; Garaicoechea, Lorena; Wigdorovitz, Andrés; Marangunich, Laura; Fernandez, Fernando
2010-10-01
Two ELISAs to quantify antibodies to BoHV-1 in the sera of cattle and immunized guinea pigs were developed and validated using ISO/IEC 17025 standards. The cut-off value of the assay was established at 20% positivity of a high positive control for screening of cattle. Using this threshold, the assay properly classified the OIE bovine reference sera EU1, EU2 and EU3. For vaccine potency testing, a cut-off of 40% was selected for both species. The reliability of the assays, given by their diagnostic sensitivity and specificity, using the threshold of 40% was 89.7% and 100%, respectively, for bovines and 94.9% and 100% for guinea pigs, respectively. There was almost perfect agreement between the ELISA and virus neutralization results. In addition, after vaccination, there was a good correlation between the neutralizing and ELISA antibody titers of the serum from the same bovine or guinea pig, sampled at 60 and 30 days post-vaccination, respectively (R(bovine)=0.88, R(guinea pig)=0.92; p<0.0001). A similar correlation was observed when analyzing the mean antibody titers of groups of vaccinated animals (R(bovine)=0.95 and R(guinea pig)=0.97; p<0.0001), indicating the relevance of the ELISAs for batch to batch vaccine potency testing in the target species and in the laboratory animal model. The intermediate precision of the assays expressed as the relative coefficient of variation (CV) of the positive control assayed over a 3-year period in the same laboratory was 22.2% for bovines and 23.1% for guinea pigs. The reproducibility of both techniques obtained in inter-laboratory assays was CV=12.4% for bovines and CV approximately 0 for guinea pigs, which met the requirements of the OIE (CV<30%). The validated ELISAs represent important methods for vaccine potency testing and for controlling BoHV-1 infections. Copyright (c) 2010 Elsevier B.V. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Muryn, J; Wilkinson, D
Purpose: The purpose of this work is to evaluate a method for confirming source strength of I-125 seeds in a bulk assay while maintaining sterility and time efficiency. Methods: The I-125 seeds used in this study (STM 1251, Bard Brachytherapy, Inc.) were available as loose seeds or linked in 3, 4, or 5 seed configurations. A third party 10% assay (NIST traceable) is provided. Source strengths ranging from 0.395 to 0.504 U were available for this study. A stand was built out of aluminum to hold an exposure meter (Inovision (Fluke) 451P) at 25 cm above the I-125 sources tomore » measure the exposure rate. Three different seed configurations were measured: loose, linked, and loaded needles (Bard FastFil Seed Implant Needle). The measurements were made in an operating room, and a sterile sheet was used under the non-sterile aluminum stand. Seeds and needles were placed in a sterile tray. Results: One hundred forty-two loose seeds in 5 batches (0.395, 0.395, 0.409, 0.444, 0.444 U/seed) and 902 seeds in 7 batches containing various strands (0.444, 0.444,.0444, 0.466, 0.466, 0.504, 0.504 U/seed) were measured. The average exposure rate per unit activity was measured to be 0.593 mR per hr per U with a standard deviation of 0.016. The Result for loaded needles was 0.261 mR per hr per U with a standard deviation of 0.014. Once the apparatus is set up, measurements of 180 linked sources as supplied in the Bard package requires only a few minutes. Conclusion: The proposed method can confirm the activity of a batch of loose or stranded I-125 seeds within a range of 5%.« less
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2010-02-25
metabolic activation mixture was prepared by SITEK Research Laboratories and it consisted of phenobarbital -S,6-Benzoflavone (phenobarbitallB-naphthoflavone... Phenobarbital -S,6-Benzoflavone <-70°C May 21, 2011 Detailed infonnation about the S-9 batch used in the Assay is provided in Appendix N. 13 SITEK Study No
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Research and Development in Preventive Dentistry.
1979-12-01
Characterization 16 B. Core Material Preparation 18 C. Microencapsulation 20 D. Characterization of Microcapsules 22 1. Size Distribution 22 2. Assays 22 3... microencapsulated with a biodegradable polymer, poly-L(-)- lactide, using a fluidized bed coating technique. A series of microcapsule batches with different...lbs/hr. Material was less than 15 iim (99%), and most of the lidocaine was in the 1 micron range, * C. MICROENCAPSULATION Lidocaine microcapsules were
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Catelani, Tiago A; Santos, João Rodrigo; Páscoa, Ricardo N M J; Pezza, Leonardo; Pezza, Helena R; Lopes, João A
2018-03-01
This work proposes the use of near infrared (NIR) spectroscopy in diffuse reflectance mode and multivariate statistical process control (MSPC) based on principal component analysis (PCA) for real-time monitoring of the coffee roasting process. The main objective was the development of a MSPC methodology able to early detect disturbances to the roasting process resourcing to real-time acquisition of NIR spectra. A total of fifteen roasting batches were defined according to an experimental design to develop the MSPC models. This methodology was tested on a set of five batches where disturbances of different nature were imposed to simulate real faulty situations. Some of these batches were used to optimize the model while the remaining was used to test the methodology. A modelling strategy based on a time sliding window provided the best results in terms of distinguishing batches with and without disturbances, resourcing to typical MSPC charts: Hotelling's T 2 and squared predicted error statistics. A PCA model encompassing a time window of four minutes with three principal components was able to efficiently detect all disturbances assayed. NIR spectroscopy combined with the MSPC approach proved to be an adequate auxiliary tool for coffee roasters to detect faults in a conventional roasting process in real-time. Copyright © 2017 Elsevier B.V. All rights reserved.
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High quality epoxysilane substrate for clinical multiplex serodiagnostic proteomic microarrays
NASA Astrophysics Data System (ADS)
Ewart, Tom; Carmichael, Stuart; Lea, Peter
2005-09-01
Polylysine and aminopropylsilane treated glass comprised the majority of substrates employed in first generation genetic microarray substrates. Second generation single stranded long oligo libraries with amino termini provided for controlled terminal specific attachment, and rationally designed unique sequence libraries with normalized melting temperatures. These libraries benefit from active covalent coupling surfaces such as Epoxysilane. The latter's oxime ring shows versatile reactivity with amino-, thiol- and hydroxyl- groups thus encompassing small molecule, oligo and proteomic microarray applications. Batch-to-batch production uniformity supports entry of the Epoxysilane process into clinical diagnostics. We carried out multiple print runs of 21 clinically relevant bacterial and viral antigens at optimized concentrations, plus human IgG and IgM standards in triplicate on multiple batches of Epoxysilane substrates. A set of 45 patient sera were assayed in a 35 minute protocol using 10 microliters per array in a capillary-fill format (15 minute serum incubation, wash, 15 minute incubation with Cy3-labeled anti-hIgG plus Dy647-labeled anti-hIgM, final wash). The LOD (3 SD above background) was better than 1 microgram/ml for IgG, and standard curves were regular and monotonically increasing over the range 0 to 1000 micrograms/ml. Ninety-five percent of the CVs for the standards were under 10%, and 90% percent of CVs for antigen responses were under 10% across all batches of Epoxysilane and print runs. In addition, where SDs are larger than expected, microarray images may be readily reviewed for quality control purposes and pin misprints quickly identified. In order to determine the influence of stirring on sensitivity and speed of the microarray assay, we printed 10 common ToRCH antigens (H. pylori, T. gondii, Rubella, Rubeola, C. trachomatis, Herpes 1 and 2, CMV, C. jejuni, and EBV) in Epoxysilane-activated slide-wells. Anti-IgG-Cy3 direct binding to printed IgG calibration spots could be detected (3 x LOD) above background at 100 pg/ml (0.13 femtomoles sample content) in a 10 minute incubation. The LOD for detection of serum anti-H. pylori antibody level was 9 ng/ml in the same incubation time.
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Specific detection of tetanus toxoid using an aptamer-based matrix.
Modh, Harshvardhan B; Bhadra, Ankan K; Patel, Kinjal A; Chaudhary, Rajeev K; Jain, Nishant K; Roy, Ipsita
2016-11-20
Batch-to-batch variation of therapeutic proteins produced by biological means requires rigorous monitoring at all stages of the production process. A large number of animals are employed for risk assessment of biologicals, which has low ethical and economic acceptability. Research is now focussed on the validation of in vitro and ex vivo tests to replace live challenges. Among in vitro methods, enzyme-linked immunosorbent assay (ELISA) is considered to be the gold standard for estimation of integrity of tetanus toxoid. ELISA utilizes antibodies for detection, which, because of their biological origin and limited modifiability, may have low stability and result in irreproducibility. We have developed a method using highly specific and selective RNA aptamers for detection of tetanus toxoid. Using displacement assay, we first identified aptamers which bind to different aptatopes on the surface of the toxoid. Pairs of these aptamers were employed as capture-detection ligands in a sandwich-ALISA (aptamer-linked immobilized sorbent assay) format. The binding efficiency was confirmed by the fluorescence intensity in each microtire plate well. Using aptamers alone, detection of tetanus toxoid was possible with the same level of sensitivity as antibody. Aptamers were also used in the capture ALISA format. Adjuvanted tetanus toxoid was subjected to accelerated stress testing, including thermal, mechanical and freeze-thawing stress conditions. The loss in antigenicity of the preparation determined by ALISA in each case was found to be similar to that determined by conventional ELISA. Thus, it is possible to replace antibodies with aptamers to develop a more robust detection tool for tetanus toxoid. Copyright © 2016 Elsevier B.V. All rights reserved.
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Brulé, Mathieu; Bolduan, Rainer; Seidelt, Stephan; Schlagermann, Pascal; Bott, Armin
2013-01-01
Batch biochemical methane potential (BMP) assays to evaluate the methane yield of biogas substrates such as energy crops are usually carried out with undiluted inoculum. A BMP assay was performed on two energy crops (green cuttings and grass silage). Anaerobic digestion was performed both with and without supplementation of three commercial additives containing trace metals in liquid, solid or adsorbed form (on clay particles). In order to reveal positive effects of trace metal supplementation on the methane yield, besides undiluted inoculum, 3-fold and 10-fold dilutions of the inoculum were applied for substrate digestion. Diluted inoculum variants were supplemented with both mineral nutrients and pH-buffering substances to prevent a collapse of the digestion process. As expected, commercial additives had no effect on the digestion process performed with undiluted inoculum, while significant increases of methane production through trace element supplementation could be observed on the diluted variants. The effect of inoculum dilution may be twofold: (1) decrease in trace metal supplementation from the inoculum and (2) reduction in the initial number of bacterial cells. Bacteria require higher growth rates for substrate degradation and hence have higher trace element consumption. According to common knowledge of the biogas process, periods with volatile fatty acids accumulation and decreased pH may have occurred in the course ofanaerobic digestion. These effects may have led to inhibition, not only ofmethanogenes and acetogenes involved in the final phases of methane production, but also offibre-degrading bacterial strains involved in polymer hydrolysis. Further research is required to confirm this hypothesis.
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Genotoxicity of processed food items and ready-to-eat snacks in Finland.
Omoruyi, Iyekhoetin Matthew; Pohjanvirta, Raimo
2014-11-01
Processed foods are an insufficiently characterized source of chemical mutagens for consumers. Here, we evaluated the genotoxicity of selected food products in Finland. Mutagenicity was determined by the standard plate incorporation assay followed by methylcellulose overlay and treat-and-wash assays, using the Salmonella strains TA 100 and 98 with and without metabolic activation. Generally, the mutagenic activity of food samples was low, but exhibited lot-wise variation. Cold cuts of cold-smoked beef, grilled turkey, and smoked chicken (a single batch of each) were mutagenic in all three assays with the TA 100 strain with and without metabolic activation, indicating the mutagenic effect was not secondary to histidine release from the food products. However, none of the food extracts showing mutagenic potential induced DNA damage in vitro using the Comet Assay. Our findings imply that in Finland today, there are still products the production methods of which should be refined to reduce the potential risk of mutagenicity to consumers. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Saydam, Manolya; Rigsby, Peter; Mawas, Fatme
2014-01-01
Current Haemophilus influenzae b conjugate vaccines (Hib), which are made of purified capsular polysaccharide (poly-ribosyl-ribitol-phosphate; PRP) conjugated to a carrier protein, are almost completely evaluated by physico-chemical methods to ensure the integrity and stability of the vaccine and consistency of manufacture of batches. The absence of a potency assay makes the quantification of total PRP content (in SI units) and of % free polysaccharide in final fills or bulk components of Hib vaccines critical release tests for both manufacturers and national control authorities. Here we describe a simple and sensitive Enzyme-Linked Immuno-sorbent Assay (ELISA) which has been developed to quantify total and free PRP content in Hib-TT vaccine alone or when in combination with other vaccines. The assay is robust, specific and highly sensitive. Copyright © 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
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Mattsson, Leena; Xu, Jingjing; Preininger, Claudia; Tse Sum Bui, Bernadette; Haupt, Karsten
2018-05-01
We developed a competitive fluorescent molecularly imprinted polymer (MIP) assay to detect biogenic amines in fish samples. MIPs synthesized by precipitation polymerization using histamine as template were used in a batch binding assay analogous to competitive fluoroimmunoassays. Introducing a complex sample matrix, such as fish extract, into the assay changes the environment and the binding conditions, therefore the importance of the sample preparation is extensively discussed. Several extraction and purification methods for fish were comprehensively studied, and an optimal clean-up procedure for fish samples using liquid-liquid extraction was developed. The feasibility of the competitive MIP assay was shown in the purified fish extract over a broad histamine range (1 - 430µM). The MIP had the highest affinity towards histamine, but recognized also the structurally similar biogenic amines tyramine and tryptamine, as well as spermine and spermidine, providing simultaneous analysis and assessment of the total amount of biogenic amines. Copyright © 2018 Elsevier B.V. All rights reserved.
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Timoumi, Asma; Cléret, Mégane; Bideaux, Carine; Guillouet, Stéphane E; Allouche, Yohan; Molina-Jouve, Carole; Fillaudeau, Luc; Gorret, Nathalie
2017-01-01
Yarrowia lipolytica, a non-conventional yeast with a promising biotechnological potential, is able to undergo metabolic and morphological changes in response to environmental conditions. The effect of pH perturbations of different types (pulses, Heaviside) on the dynamic behavior of Y. lipolytica W29 strain was characterized under two modes of culture: batch and continuous. In batch cultures, different pH (4.5, 5.6 (optimal condition), and 7) were investigated in order to identify the pH inducing a stress response (metabolic and/or morphologic) in Y. lipolytica. Macroscopic behavior (kinetic parameters, yields, viability) of the yeast was slightly affected by pH. However, contrary to the culture at pH 5.6, a filamentous growth was induced in batch experiments at pH 4.5 and 7. Proportions of the filamentous subpopulation reached 84 and 93 % (v/v) under acidic and neutral conditions, respectively. Given the significant impact of neutral pH on morphology, pH perturbations from 5.6 to 7 were subsequently assayed in batch and continuous bioreactors. For both process modes, the growth dynamics remained fundamentally unaltered during exposure to stress. Nevertheless, morphological behavior of the yeast was dependent on the culture mode. Specifically, in batch bioreactors where cells proliferated at their maximum growth rate, mycelia were mainly formed. Whereas, in continuous cultures at controlled growth rates (from 0.03 to 0.20 h -1 ) even closed to the maximum growth rate of the stain (0.24 h -1 ), yeast-like forms predominated. This pointed out differences in the kinetic behavior of filamentous and yeast subpopulations, cell age distribution, and pH adaptive mechanisms between both modes of culture.
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Sarkar, Srijata; Zhang, Lin; Subramaniam, Prasad; Lee, Ki-Bum; Garfunkel, Eric; Strickland, Pamela A. Ohman.; Mainelis, Gediminas; Lioy, Paul J.; Tetley, Teresa D.; Chung, Kian Fan; Zhang, Junfeng; Ryan, Mary; Porter, Alex; Schwander, Stephan
2014-01-01
Acting as fuel combustion catalysts to increase fuel economy, cerium dioxide (ceria, CeO2) nanoparticles have been used in Europe as diesel fuel additives (Envirox™). We attempted to examine the effects of particles emitted from a diesel engine burning either diesel (diesel exhaust particles, DEP) or diesel doped with various concentrations of CeO2 (DEP-Env) on innate immune responses in THP-1 and primary human peripheral blood mononuclear cells (PBMC). Batches of DEP and DEP-Env were obtained on three separate occasions using identical collection and extraction protocols with the aim of determining the reproducibility of particles generated at different times. However, we observed significant differences in size and surface charge (zeta potential) of the DEP and DEP-Env across the three batches. We also observed that exposure of THP-1 cells and PBMC to identical concentrations of DEP and DEP-Env from the three batches resulted in statistically significant differences in bioreactivity as determined by IL-1β, TNF-α, IL-6, IFN-γ, and IL-12p40 mRNA (by qRT-PCR) and protein expression (by ELISPOT assays). Importantly, bioreactivity was noted in very tight ranges of DEP size (60 to 120 nm) and zeta potential (−37 to −41 mV). Thus, these physical properties of DEP and DEP-Env were found to be the primary determinants of the bioreactivity measured in this study. Our findings also point to the potential risk of over- or under- estimation of expected bioreactivity effects (and by inference of public health risks) from bulk DEP use without taking into account potential batch-to-batch variations in physical (and possibly chemical) properties. PMID:24825358
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Multivariate statistical process control in product quality review assessment - A case study.
Kharbach, M; Cherrah, Y; Vander Heyden, Y; Bouklouze, A
2017-11-01
According to the Food and Drug Administration and the European Good Manufacturing Practices (GMP) guidelines, Annual Product Review (APR) is a mandatory requirement in GMP. It consists of evaluating a large collection of qualitative or quantitative data in order to verify the consistency of an existing process. According to the Code of Federal Regulation Part 11 (21 CFR 211.180), all finished products should be reviewed annually for the quality standards to determine the need of any change in specification or manufacturing of drug products. Conventional Statistical Process Control (SPC) evaluates the pharmaceutical production process by examining only the effect of a single factor at the time using a Shewhart's chart. It neglects to take into account the interaction between the variables. In order to overcome this issue, Multivariate Statistical Process Control (MSPC) can be used. Our case study concerns an APR assessment, where 164 historical batches containing six active ingredients, manufactured in Morocco, were collected during one year. Each batch has been checked by assaying the six active ingredients by High Performance Liquid Chromatography according to European Pharmacopoeia monographs. The data matrix was evaluated both by SPC and MSPC. The SPC indicated that all batches are under control, while the MSPC, based on Principal Component Analysis (PCA), for the data being either autoscaled or robust scaled, showed four and seven batches, respectively, out of the Hotelling T 2 95% ellipse. Also, an improvement of the capability of the process is observed without the most extreme batches. The MSPC can be used for monitoring subtle changes in the manufacturing process during an APR assessment. Copyright © 2017 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.
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Electrosynthesized MIPs for transferrin: Plastibodies or nano-filters?
Zhang, Xiaorong; Yarman, Aysu; Erdossy, Júlia; Katz, Sagie; Zebger, Ingo; Jetzschmann, Katharina J; Altintas, Zeynep; Wollenberger, Ulla; Gyurcsányi, Róbert E; Scheller, Frieder W
2018-05-15
Molecularly imprinted polymer (MIP) nanofilms for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of ~5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered. Copyright © 2018 Elsevier B.V. All rights reserved.
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Deshpande, Kanchanmala; Mishra, Rupesh K.; Bhand, Sunil
2010-01-01
A highly sensitive and specific enzyme inhibition assay based on alcohol oxidase (AlOx) and horseradish peroxidase (HRP) for determination of mercury Hg(II) in water samples has been presented. This article describes the optimization and miniaturization of an enzymatic assay using a chemiluminescence reaction. The analytical performance and detection limit for determination of Hg(II) was optimized in 96 well plates and further extended to 384 well plates with a 10-fold reduction in assay volume. Inhibition of the enzyme activity by dissolved Hg(II) was found to be linear in the range 5–500 pg·mL−1 with 3% CV in inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II) was measurable as low as 1 pg·mL−1 within 15 min. About 10-fold more specificity of the developed assay for Hg(II) analysis was confirmed by challenging with interfering divalent metal ions such as cadmium Cd(II) and lead Pb(II). Using the proposed assay we could successfully demonstrate that in a composite mixture of Hg(II), Cd(II) and Pb(II), inhibition by each metal ion is significantly enhanced in the presence of the others. Applicability of the proposed assay for the determination of the Hg(II) in spiked drinking and sea water resulted in recoveries ranging from 100–110.52%. PMID:22163555
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ho, S.M.; Fehrer, S.; Yu, M.
1988-06-01
Specific (3H)estradiol-17 beta ((3H)E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds (3H)E2 with highmore » affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of (3H)E2 binding activity in both cytosolic and nuclear fractions. The exchange between (3H)E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species.« less
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2008-06-13
ACTIVATION SYSTEM The metabolic activation mixture was prepared by SITEK Research Laboratories and it consisted of phenobarbital -S,6-Benzoflavone...2147 31.0mg/mL Phenobarbital -S,6-Benzoflavone <-70°C April 19. 2009 Detailed information about the S-9 batch used ill the Assay is provided in
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Protein relaxation without a geminate phase in nanosecond photodissociated CO carp hemoglobin
NASA Astrophysics Data System (ADS)
Loupiac, Camille; Kruk, Nicolay; Valat, Pierre; Alpert, Bernard
1999-03-01
Transient heme-protein interactions upon passing from ligated to deligated carp hemoglobin were observed through time-resolved optical spectra following nanosecond CO photodissociation. The spectral evolution of the heme, in the nanosecond and microsecond time ranges, shows a protein conformational relaxation and the absence of a geminate CO recombination in carp hemoglobin. The comparison of the phenomena in carp and human hemoglobin implies that the physical basis of the geminate rebinding in human hemoglobin should involve an out-of-equilibrium protein conformation, close to a dissipative structure defined by the thermodynamics of Prigogine.
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NASA Astrophysics Data System (ADS)
Hung, Chih-Chang; Yabushita, Atsushi; Kobayashi, Takayoshi; Chen, Pei-Feng; Liang, Keng S.
2017-09-01
Ultrafast dynamics of endothelial nitric oxide synthase (eNOS) oxygenase domain was studied by transient absorption spectroscopy pumping at Soret band. The broadband visible probe spectrum has visualized the relaxation dynamics from the Soret band to Q-band and charge transfer (CT) band. Supported by two-dimensional correlation spectroscopy, global fitting analysis has successfully concluded the relaxation dynamics from the Soret band to be (1) electronic transition to Q-band (0.16 ps), (2) ligand dissociation and CT (0.94 ps), (3) relaxation of the CT state (4.0 ps), and (4) ligand rebinding (59 ps).
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Succinic acid production from cellobiose by Actinobacillus succinogenes.
Jiang, Min; Xu, Rong; Xi, Yong-Lan; Zhang, Jiu-Hua; Dai, Wen-Yu; Wan, Yue-Jia; Chen, Ke-Quan; Wei, Ping
2013-05-01
In this study, cellobiose, a reducing disaccharide was used to produce succinic acid by Actinobacillus succinogenes NJ113. A final succinic acid concentration of 30.3g/l with a yield of 67.8% was achieved from an initial cellobiose concentration of 50 g/l via batch fermentation in anaerobic bottles. The cellobiose uptake mechanism was investigated and the results of enzyme assays revealed that the phosphoenolpyruvate phosphotransferase system (PEP-PTS) played an important role in the cellobiose uptake process. In batch fermentation with 18 g/l of cellobiose and 17 g/l of other sugars from sugarcane bagasse cellulose hydrolysates, a succinic acid concentration of 20.0 g/l was obtained, with a corresponding yield of 64.7%. This study found that cellobiose from incomplete hydrolysis of cellulose could be a potential carbon source for economical and efficient succinic acid production by A. succinogenes. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Effect of arsenic on nitrification of simulated mining water.
Papirio, S; Zou, G; Ylinen, A; Di Capua, F; Pirozzi, F; Puhakka, J A
2014-07-01
Mining and mineral processing of gold-bearing ores often release arsenic to the environment. Ammonium is released when N-based explosives or cyanide are used. Nitrification of simulated As-rich mining waters was investigated in batch bioassays using nitrifying cultures enriched in a fluidized-bed reactor (FBR). Nitrification was maintained at 100mg AsTOT/L. In batch assays, ammonium was totally oxidized by the FBR enrichment in 48 h. As(III) oxidation to As(V) occurred during the first 3h attenuating arsenic toxicity to nitrification. At 150 and 200mg AsTOT/L, nitrification was inhibited by 25%. Candidatus Nitrospira defluvii and other nitrifying species mainly colonized the FBR. In conclusion, the FBR enriched cultures of municipal activated sludge origins tolerated high As concentrations making nitrification a potent process for mining water treatment. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Metabolic Engineering toward Sustainable Production of Nylon-6.
Turk, Stefan C H J; Kloosterman, Wigard P; Ninaber, Dennis K; Kolen, Karin P A M; Knutova, Julia; Suir, Erwin; Schürmann, Martin; Raemakers-Franken, Petronella C; Müller, Monika; de Wildeman, Stefaan M A; Raamsdonk, Leonie M; van der Pol, Ruud; Wu, Liang; Temudo, Margarida F; van der Hoeven, Rob A M; Akeroyd, Michiel; van der Stoel, Roland E; Noorman, Henk J; Bovenberg, Roel A L; Trefzer, Axel C
2016-01-15
Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.
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In vitro and in vivo characterization of a reversible synthetic heparin analog.
Whelihan, Matthew F; Cooley, Brian; Xu, Yongmei; Pawlinski, Rafal; Liu, Jian; Key, Nigel S
2016-02-01
The global supply of unfractionated heparin (UFH) and all commercially available low molecular weight heparins (LMWH) remain dependent on animal sources, such as porcine intestine or bovine lung. Recent experience has shown that contamination of the supply chain (with over-sulfated chondroitin sulfates) can result in lethal toxicity. Fondaparinux is currently the only commercially available synthetic analog of heparin. We recently described a new class of chemoenzymatically synthesized heparin analogs. One of these compounds (S12-mer) is a dodecasaccharide consisting of an antithrombin-binding moiety with repeating units of IdoA2S-GlcNS6S and two 3-O-sulfate groups that confer the ability to bind protamine. We sought to further characterize this new compound in vitro using biochemical and global coagulation assays and in vivo using thrombosis and hemostasis assays. The anticoagulant activities of the Super 12-mer (S12-mer) and Enoxaparin in anti-factor Xa and plasma-based thrombin generation assays were roughly equivalent with a 50% reduction in peak thrombin generation occurring at approximately 325nM. When protamine was titrated against a fixed concentration of S12-mer in plasma or blood, the S12-mer displayed a significant restitution of thrombin generation and clot formation. In vivo, S12-mer inhibited venous thrombosis to a similar extent as Enoxaparin, with similar bleeding profiles. These data show that the S12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the ability to ensure purity and homogeneity from batch to batch, the S12-mer is a promising new synthetic heparin analog with a potentially enhanced safety profile. Copyright © 2015 Elsevier Ltd. All rights reserved.
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False-positive pregnancy test after transfusion of solvent/detergent-treated plasma.
Jilma-Stohlawetz, Petra; Wreford-Bush, Tim; Mills, Francesca; Davidson, Fiona; Kursten, Friedrich W; Jilma, Bernd; Birchall, Janet
2017-12-01
The transmission of pathogens, antibodies, and proteins is a possible consequence of blood product transfusion. A female patient had an unexpected positive serum β-human chorionic gonadotropin result, indicative of pregnancy, after she had received a transfusion with 1 unit of platelet concentrate, 4 units of red blood cells, and 4 units of pooled solvent/detergent-treated plasma (Octaplas). To investigate the possibility of passive transfusion of β-human chorionic gonadotropin from the plasma transfusion, one additional unit from the same batch was thawed and analyzed. To validate the β-human chorionic gonadotropin assay for use in solvent/detergent-treated plasma and to investigate any interference in the assay, dilution experiments were performed using the implicated plasma batch diluted with male and non-pregnant female sera. Also, plasma from a known pregnant woman was diluted with Octaplas (tested negative for β-human chorionic gonadotropin) and with a male serum to validate the assay for use in solvent/detergent-treated plasma. The implicated solvent/detergent-treated plasma had a mean β-human chorionic gonadotropin level of 91.5 mIU/mL. Results from the dilution experiments revealed an excellent correlation (r > 0.99) between β-human chorionic gonadotropin measurement in solvent/detergent-treated plasma and male serum and no over or under recovery of the expected results. Further measurements of β-human chorionic gonadotropin levels in the female recipient revealed an estimated half-life of 6 hours. This case demonstrates the importance of considering the possibility of passive transmission of analytes to a patient from the transfusion of blood products. Furthermore, the measurement of β-human chorionic gonadotropin is valid in solvent/detergent-treated plasma using a Roche Cobas analyzer. © 2017 AABB.
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Tsai, Huey-Pin; Tsai, You-Yuan; Lin, I-Ting; Kuo, Pin-Hwa; Chen, Tsai-Yun; Chang, Kung-Chao; Wang, Jen-Ren
2016-01-01
Quantitation of cytomegalovirus (CMV) viral load in the transplant patients has become a standard practice for monitoring the response to antiviral therapy. The cut-off values of CMV viral load assays for preemptive therapy are different due to the various assay designs employed. To establish a sensitive and reliable diagnostic assay for preemptive therapy of CMV infection, two commercial automated platforms including m2000sp extraction system integrated the Abbott RealTime (m2000rt) and the Roche COBAS AmpliPrep for extraction integrated COBAS Taqman (CAP/CTM) were evaluated using WHO international CMV standards and 110 plasma specimens from transplant patients. The performance characteristics, correlation, and workflow of the two platforms were investigated. The Abbott RealTime assay correlated well with the Roche CAP/CTM assay (R2 = 0.9379, P<0.01). The Abbott RealTime assay exhibited higher sensitivity for the detection of CMV viral load, and viral load values measured with Abbott RealTime assay were on average 0.76 log10 IU/mL higher than those measured with the Roche CAP/CTM assay (P<0.0001). Workflow analysis on a small batch size at one time, using the Roche CAP/CTM platform had a shorter hands-on time than the Abbott RealTime platform. In conclusion, these two assays can provide reliable data for different purpose in a clinical virology laboratory setting. PMID:27494707
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Synthesis and Study of Guest-Rebinding of MIP Based on MAA Prepared using Theophylline Template
NASA Astrophysics Data System (ADS)
Nurhayati, T.; Yanti; Royani, I.; Widayani; Khairurrijal
2016-08-01
A molecularly imprinted polymer (MIP) based on methacrylic acid (MAA) monomer and theophylline template has been synthesized using a modified bulk polymerization method. Theophylline was employed as a template and it formed a complex with MAA through hydrogen bonding. Self-assembly of template-monomer was followed by cross-linking process using ethylene glycol dimethacrylate (EGDMA) cross-linker. The polymerization process was initiated by thermal decomposition of benzoyl peroxide (BPO) as the initiator at 60oC after cooling treatment at -5oC. After 7 hours, a rigid polymer was obtained and followed by grinding the polymer and removing the template. As a reference, a nonimprinted polymer (NIP) has also been synthesized using similar procedure by excluding the template. FTIR study was carried out to investigate the presence of theophylline in the as- prepared polymer, MIP, and NIP. The spectra indicated that theophylline was successfully incorporated in the as-prepared polymer. This result was also confirmed by EDS analysis showing that N atoms of the as-prepared polymer were derived from amino group of theophylline. Furthermore, the polymer particles of MIP were irregular in shape and size as shown by its SEM image. The capability of guest-rebinding of the MIP was analyzed through Batchwise guest-binding experiment. The results showed that for initial concentration of theophylline in methanol/chloroform (1/1, v/v) of 0.333 mM, the binding capacity of the MIP was 23.22 /mol/g. Compared to the MIP, the adsorption capacity of the NIP was only 3.73 /mol/g. This result shows that MIP has higher affinity than NIP.
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Lawrence, J Josh; Brenowitz, Stephan; Trussell, Laurence O
2003-08-01
The mechanism of action of aniracetam on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors was examined in outside-out patches and at glutamatergic synapses in neurons of the chick cochlear nucleus. A combination of rapid-flow analysis, using glutamate as an agonist, and kinetic modeling indicated that aniracetam slows both the rate of channel closing, and the microscopic rates of desensitization, even for partially liganded receptors. Little effect was observed on the rate of recovery from desensitization or on the response to the weakly desensitizing agonist kainate. Aniracetam's effects on receptor deactivation saturated at lower concentrations than its effects on desensitization, suggesting that cooperativity between homologous binding sites was required to regulate desensitization. Analysis of responses to paired pulses of agonist also indicated that AMPA receptors must desensitize partially even after agonist exposures too brief to permit rebinding. In the presence of aniracetam, evoked excitatory synaptic currents (EPSCs) and miniature EPSCs in low quantal-content conditions had decay times similar to the time course of receptor deactivation. Under these conditions, the time course of both transmitter release and clearance must be <1 to 2 ms. However, in high quantal-content conditions, the evoked EPSC in aniracetam decayed with a time course intermediate between deactivation and desensitization, suggesting that the time course of transmitter clearance is prolonged because of pooling of transmitter in the synaptic cleft. Moreover, by comparing the amounts of paired-pulse synaptic depression and patch desensitization prevented by aniracetam, we conclude that significant desensitization occurs in response to rebinding of transmitter to the AMPA receptors.
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Lee, Jinho; Geiss, Gary K; Demirkan, Gokhan; Vellano, Christopher P; Filanoski, Brian; Lu, Yiling; Ju, Zhenlin; Yu, Shuangxing; Guo, Huifang; Bogatzki, Lisa Y; Carter, Warren; Meredith, Rhonda K; Krishnamurthy, Savitri; Ding, Zhiyong; Beechem, Joseph M; Mills, Gordon B
2018-06-01
Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed "batch effects" which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
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Surface plasmon resonance immunoassay analysis of pituitary hormones in urine and serum samples.
Treviño, Juan; Calle, Ana; Rodríguez-Frade, José Miguel; Mellado, Mario; Lechuga, Laura M
2009-05-01
Direct determination of four pituitary peptide hormones: human thyroid stimulating hormone (hTSH), growth hormone (hGH), follicle stimulating hormone (hFSH), and luteinizing hormone (hLH) has been carried out using a portable surface plasmon resonance (SPR) immunosensor. A commercial SPR biosensor was employed. The immobilization of the hormones was optimized and monoclonal antibodies were selected in order to obtain the best sensor performance. Assay parameters as running buffer and regeneration solution composition or antibody concentration were adjusted to achieve a sensitive analyte detection. The performance of the assays was assessed in buffer solution, serum and urine, showing sensitivity in the range from 1 to 6 ng/mL. The covalent attachment of the hormones ensured the stability of the SPR signal through repeated use in up to 100 consecutive assay cycles. Mean intra- and inter-day coefficients of variation were all <7%, while batch-assay variability using different sensor surfaces was <5%. Taking account both the excellent reutilization performance and the outstanding reproducibility, this SPR immunoassay method turns on a highly reliable tool for endocrine monitoring in laboratory and point-of-care (POC) settings.
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Oviedo, Adriana E.; Bernardi, María E.; Guglielmone, Hugo A.; Vitali, María S.
2015-01-01
Summary Background Immunoglobulin (IG) products, including intravenous (IVIG) or subcutaneous (SCIG) immunoglobulins are considered safe and effective for medical therapy; however, a sudden and unexpected increase in thromboembolic events (TE) after administration of certain batches of IVIG products has been attributed to the presence of activated coagulation factors, mainly factor XIa. Our aims were to examine the presence of enduring procoagulant activity during the manufacturing process of IGs, with special focus on monitoring factor XIa, and to evaluate the presence of in vitro procoagulant activity attributed to coagulation factors in different lots of IVIG and SCIG. Methods Samples of different steps of IG purification, 19 lots of IVIG and 9 of SCIG were analyzed and compared with 1 commercial preparation of IVIG and 2 of SCIG, respectively. Factors II, VII, IX, XI and XIa and non-activated partial thromboplastin time (NAPTT) were assayed. Results The levels of factors II, VII, IX, X and XI were non-quantifiable once fraction II had been re-dissolved and in all analyzed lots of IVIG and SCIG. The level of factor XIa at that point was under the detection limits of the assay, and NAPTT yielded values greater than the control during the purification process. In SCIG, we detected higher concentrations of factor XIa in the commercial products, which reached values up to 5 times higher than the average amounts found in the 9 batches produced by UNC-Hemoderivados. Factor XIa in commercial IVIG reached levels slightly higher than those of the 19 batches produced by UNC-Hemoderivados. Conclusion IVIG and SCIG manufactured by UNC-Hemoderivados showed a lack of thrombogenic potential, as demonstrated not only by the laboratory data obtained in this study but also by the absence of any reports of TE registered by the post marketing pharmacovigilance department. PMID:26733772
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Einstein, Mark H; Smith, Katherine M; Davis, Thomas E; Schmeler, Kathleen M; Ferris, Daron G; Savage, Ashlyn H; Gray, Jermaine E; Stoler, Mark H; Wright, Thomas C; Ferenczy, Alex; Castle, Philip E
2014-06-01
High-risk human papillomavirus (hrHPV) testing is now being introduced as a potential primary screening test for improved detection of cervical precancer and cancer. Current U.S. Food and Drug Administration-approved tests are batch tests that take several hours to complete. A rapid, non-batch test might permit point-of-care (POC) testing, which can facilitate same-day screen and management strategies. For a non-batch, random-access platform (GeneXpert; Cepheid, Sunnyvale, CA), a prototype hrHPV assay (Xpert) has been developed where testing for 14 hrHPV types can be completed in 1 h. In the first clinical evaluation, Xpert was compared to two validated hrHPV tests, the cobas HPV test (cobas, Roche Molecular Systems) and Hybrid Capture 2 (hc2, Qiagen), and to histologic outcomes using specimens from colposcopy referral populations at 7 clinical sites in the United States (n = 697). The sensitivity of Xpert for cervical intraepithelial neoplasia grade 2 or more severe diagnoses (CIN2+) (n = 141) was equal to that of cobas (90.8% versus 90.8%, P = 1) and greater than that of hc2 (90.8% versus 81.6%, P = 0.004). Xpert was more specific than cobas (42.6% versus 39.6%, P = 0.02) and less specific than hc2 (42.6% versus 47.7%, P < 0.001). Similar results were observed for cervical intraepithelial neoplasia grade 3 or higher (CIN3+) (n = 91). HPV16 detection by Xpert identified 41.8% of the CIN2+ specimens with a positive predictive value (PPV) of 54.6%. By comparison, HPV16 detection by cobas identified 42.6% of the CIN2+ specimens with a PPV of 55.0%. hrHPV detection by the Xpert demonstrated excellent clinical performance for identifying women with CIN2+ and CIN3+ that was comparable to that of currently available clinically validated tests. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Zoonoses action plan Salmonella monitoring programme: an investigation of the sampling protocol.
Snary, E L; Munday, D K; Arnold, M E; Cook, A J C
2010-03-01
The Zoonoses Action Plan (ZAP) Salmonella Programme was established by the British Pig Executive to monitor Salmonella prevalence in quality-assured British pigs at slaughter by testing a sample of pigs with a meat juice enzyme-linked immunosorbent assay for antibodies against group B and C(1) Salmonella. Farms were assigned a ZAP level (1 to 3) depending on the monitored prevalence, and ZAP 2 or 3 farms were required to act to reduce the prevalence. The ultimate goal was to reduce the risk of human salmonellosis attributable to British pork. A mathematical model has been developed to describe the ZAP sampling protocol. Results show that the probability of assigning a farm the correct ZAP level was high, except for farms that had a seroprevalence close to the cutoff points between different ZAP levels. Sensitivity analyses identified that the probability of assigning a farm to the correct ZAP level was dependent on the sensitivity and specificity of the test, the number of batches taken to slaughter each quarter, and the number of samples taken per batch. The variability of the predicted seroprevalence was reduced as the number of batches or samples increased and, away from the cutoff points, the probability of being assigned the correct ZAP level increased as the number of batches or samples increased. In summary, the model described here provided invaluable insight into the ZAP sampling protocol. Further work is required to understand the impact of the program for Salmonella infection in British pig farms and therefore on human health.
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Rafsanjany, Nasli; Sendker, Jandirk; Lechtenberg, Matthias; Petereit, Frank; Scharf, Birte; Hensel, Andreas
2015-09-01
The dried stigmata from Zea mays L. are used traditionally for the treatment of uncomplicated urinary tract infections. A recent screening has indicated that hydroalcoholic extract of the herbal material inhibits the adhesion of uropathogenic Escherichia coli (UPEC) to T24 bladder cells. For verification of these data EtOH-water (1:1) extracts from 4 different batches of Maydis stigmata were investigated. Within an in vitro adhesion assay (UPEC strain 2980 and human T24 bladder cells) a dose-dependent antiadhesive activity against UPEC was verified (IC50 1040μg/mL). Bioassay guided fractionation of M. stigmata, batch S1, by EtOH-water extraction, followed by chromatography on Sephadex LH20 revealed two active fractions (I and XI). Further purification of fraction I and structure elucidation of the isolated compound revealed the presence of significant amounts of the biocide benzethonium chloride as contaminant. Benzethonium chloride was also identified in subsequent investigations in 2 different batches of M. stigmata. The presence of such nondeclared and illegal contaminants in the herbal raw material market has to be discussed intensively. From benzethonium-free raw material (batch S2) as well as from batch S1 fraction XI was further fractionated by MPLC and preparative HPLC, leading to a still complex subfraction XIG, which was analyzed by UHPLC/+ESI-QTOF-MS analysis. Advanced data processing and species-metabolite relationship database revealed the tentatively existence of the unusual C-glycosidic flavones derhamnosylmaysin (6), 3'-deoxyrhamnosylmaysin (4), 3'-O-methylderhamnosylmaysin (3), apiferol (2) and alternanthin (8) which might be related to the antiadhesive activity of this subfraction against UPEC. Copyright © 2015 Elsevier B.V. All rights reserved.
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Bravery, Christopher A; Carmen, Jessica; Fong, Timothy; Oprea, Wanda; Hoogendoorn, Karin H; Woda, Juliana; Burger, Scott R; Rowley, Jon A; Bonyhadi, Mark L; Van't Hof, Wouter
2013-01-01
The evaluation of potency plays a key role in defining the quality of cellular therapy products (CTPs). Potency can be defined as a quantitative measure of relevant biologic function based on the attributes that are linked to relevant biologic properties. To achieve an adequate assessment of CTP potency, appropriate in vitro or in vivo laboratory assays and properly controlled clinical data need to be created. The primary objective of a potency assay is to provide a mechanism by which the manufacturing process and the final product for batch release are scrutinized for quality, consistency and stability. A potency assay also provides the basis for comparability assessment after process changes, such as scale-up, site transfer and new starting materials (e.g., a new donor). Potency assays should be in place for early clinical development, and validated assays are required for pivotal clinical trials. Potency is based on the individual characteristics of each individual CTP, and the adequacy of potency assays will be evaluated on a case-by-case basis by regulatory agencies. We provide an overview of the expectations and challenges in development of potency assays specific for CTPs; several real-life experiences from the cellular therapy industry are presented as illustrations. The key observation and message is that aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development. Copyright © 2013. Published by Elsevier Inc.
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2010-06-01
Materials and Methods Pv spz A chimpanzee at the Yerkes Primate Center, Emory University, Atlanta, GA was infected with Pv (India VII strain) in order to have...a source of Pv gametocytes. This strain of Pv was earlier adapted to infect and grow in various new world monkeys and chimpanzees [23]. Multiple...batches of Anopheles dirus mosquitoes were fed on blood from the infected chimpanzee , and it was documented at Center for Disease Control & Prevention
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Effect of leachate recirculation on mesophilic anaerobic digestion of food waste.
Shahriari, Haleh; Warith, Mostafa; Hamoda, Mohamed; Kennedy, Kevin J
2012-03-01
The effects of using untreated leachate for supplemental water addition and liquid recirculation on anaerobic digestion of food waste was evaluated by combining cyclic water recycle operations with batch mesophilic biochemical methane potential (BMP) assays. Cyclic BMP assays indicated that using an appropriate fraction of recycled leachate and fresh make up water can stimulate methanogenic activity and enhance biogas production. Conversely increasing the percentage of recycled leachate in the make up water eventually causes methanogenic inhibition and decrease in the rate of food waste stabilization. The decrease in activity is exacerbated as the number cycles increases. Inhibition is possibly attributed to accumulation and elevated concentrations of ammonia as well as other waste by products in the recycled leachate that inhibit methanogenesis. Copyright © 2011 Elsevier Ltd. All rights reserved.
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Zan, Ke; Cui, Gan; Guo, Li-Nong; Ma, Shuang-Cheng; Zheng, Jian
2018-05-01
High price and difficult to get of reference substance have become obstacles to HPLC assay of ethnic medicine. A new method based on quantitative reference herb (QRH) was proposed. Specific chromatograms in fruits of Capsicum frutescens were employed to determine peak positions, and HPLC quantitative reference herb was prepared from fruits of C. frutescens. The content of capsaicin and dihydrocapsaicin in the quantitative control herb was determined by HPLC. Eleven batches of fruits of C. frutescens were analyzed with quantitative reference herb and reference substance respectively. The results showed no difference. The present method is feasible for quality control of ethnic medicines and quantitative reference herb is suitable to replace reference substances in assay. Copyright© by the Chinese Pharmaceutical Association.
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Janthanomsuk, Panyawut; Verduyn, Cornelis; Chauvatcharin, Somchai
2015-11-01
Fed-batch, pH auxostat cultivation of the docosahexaenoic acid (DHA)-producing microorganism Aurantiochytrium B072 was performed to obtain high cell density and record high productivity of both total fatty acid (TFA) and DHA. Using glucose feeding by carbon excess (C-excess) and by C-limitation at various feeding rates (70%, 50% or 20% of C-excess), high biomass density was obtained and DHA/TFA content (w/w) was improved from 30% to 37% with a 50% glucose feed rate when compared with C-excess. To understand the biochemistry behind these improvements, lipogenic enzyme assays and in silico metabolic flux calculations were used and revealed that enzyme activity and C-fluxes to TFA were reduced with C-limited feeding but that the carbon flux to the polyketide synthase pathway increased relative to the fatty acid synthase pathway. As a result, a new strategy to improve the DHA to TFA content while maintaining relatively high DHA productivity is proposed. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Effect of temperature and benzalkonium chloride on nitrate reduction.
Hajaya, Malek G; Tezel, Ulas; Pavlostathis, Spyros G
2011-04-01
The effect of temperature and benzalkonium chloride (BAC) on nitrate reduction was investigated in batch assays using a mixed nitrate reducing culture. Nitrate was transformed completely, mainly through denitrification, to dinitrogen at 5, 10, 15 and 22 °C. In the absence of BAC, reduction of individual nitrogen oxides had different susceptibility to temperature and transient nitrite accumulation was observed at low temperatures. When the effect of BAC was tested up to 100 mg/L from 5 to 22 °C, denitrification was inhibited at and above 50mg BAC/L with transient nitrite accumulation at all temperatures. The effect of BAC was described by a competitive inhibition model. Nitrite reduction was the denitrification step most susceptible to BAC, especially at low temperatures. BAC was not degraded during the batch incubation and was mostly biomass-adsorbed. Overall, this study shows that low temperatures exacerbate the BAC inhibitory effect, which in turn is controlled by adsorption to biomass. Copyright © 2011 Elsevier Ltd. All rights reserved.
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Choi, Yoonha; Liu, Tiffany Ting; Pankratz, Daniel G; Colby, Thomas V; Barth, Neil M; Lynch, David A; Walsh, P Sean; Raghu, Ganesh; Kennedy, Giulia C; Huang, Jing
2018-05-09
We developed a classifier using RNA sequencing data that identifies the usual interstitial pneumonia (UIP) pattern for the diagnosis of idiopathic pulmonary fibrosis. We addressed significant challenges, including limited sample size, biological and technical sample heterogeneity, and reagent and assay batch effects. We identified inter- and intra-patient heterogeneity, particularly within the non-UIP group. The models classified UIP on transbronchial biopsy samples with a receiver-operating characteristic area under the curve of ~ 0.9 in cross-validation. Using in silico mixed samples in training, we prospectively defined a decision boundary to optimize specificity at ≥85%. The penalized logistic regression model showed greater reproducibility across technical replicates and was chosen as the final model. The final model showed sensitivity of 70% and specificity of 88% in the test set. We demonstrated that the suggested methodologies appropriately addressed challenges of the sample size, disease heterogeneity and technical batch effects and developed a highly accurate and robust classifier leveraging RNA sequencing for the classification of UIP.
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Tavares, Ana P M; Silva, Rui P; Amaral, António L; Ferreira, Eugénio C; Xavier, Ana M R B
2014-02-01
Image analysis technique was applied to identify morphological changes of pellets from white-rot fungus Trametes versicolor on agitated submerged cultures during the production of exopolysaccharide (EPS) or ligninolytic enzymes. Batch tests with four different experimental conditions were carried out. Two different culture media were used, namely yeast medium or Trametes defined medium and the addition of lignolytic inducers as xylidine or pulp and paper industrial effluent were evaluated. Laccase activity, EPS production, and final biomass contents were determined for batch assays and the pellets morphology was assessed by image analysis techniques. The obtained data allowed establishing the choice of the metabolic pathways according to the experimental conditions, either for laccase enzymatic production in the Trametes defined medium, or for EPS production in the rich Yeast Medium experiments. Furthermore, the image processing and analysis methodology allowed for a better comprehension of the physiological phenomena with respect to the corresponding pellets morphological stages.
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Kapoor, V; Elk, M; Li, X; Santo Domingo, J W
2016-08-01
The effect of cyanide (CN(-) ) on nitrification was examined with samples from nitrifying bacterial enrichments using two different approaches: by measuring substrate (ammonia) specific oxygen uptake rates (SOUR), and by using RT-qPCR to quantify the transcripts of functional genes involved in nitrification. The nitrifying bioreactor was operated as a continuous reactor with a 24 h hydraulic retention time. The samples were exposed in batch vessels to cyanide for a period of 12 h. The concentrations of CN(-) used in the batch assays were 0·03, 0·06, 0·1 and 1·0 mg l(-1) . There was considerable decrease in SOUR with increasing dosages of CN(-) . A decrease of more than 50% in nitrification activity was observed at 0·1 mg l(-1) CN(-) . Based on the RT-qPCR data, there was notable reduction in the transcript levels of amoA and hao for increasing CN(-) dosage, which corresponded well with the ammonia oxidation activity measured via SOUR. The inhibitory effect of cyanide may be attributed to the affinity of cyanide to bind ferric haeme proteins, which disrupt protein structure and function. The correspondence between the relative expression of functional genes and SOUR shown in this study demonstrates the efficacy of RNA-based function-specific assays for better understanding of the effect of toxic compounds on nitrification activity in wastewater. The effect of cyanide on nitrifying bacteria was characterized by measuring physiological and transcriptional response. Cyanide was inhibitory to nitrification at concentrations that may be found in industrial waste. The RNA-based function-specific assays represent a mechanistic approach for better understanding the effect of toxic compounds on nitrification activity in wastewater. Moreover, the relative abundance of RNA transcripts can be used to closely track in situ nitrifying bacterial activity which can be used to predict inhibition events, thereby providing a metric to potentially improve performance of wastewater nitrifying systems. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.
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Petrov, Kaloyan; Popova, Luiza; Petrova, Penka
2017-06-01
Lactobacillus paracasei DSM 23505 is able to produce high amounts of lactic acid (LA) by simultaneous saccharification and fermentation (SSF) of inulin. Aiming to obtain the highest possible amounts of LA and fructose, the present study is devoted to evaluate the impact of bivalent metal ions on the process of inulin conversion. It was shown that Mn 2+ strongly increases the activity of the purified key enzyme β-fructosidase. In vivo, batch fermentation kinetics revealed that the high Mn 2+ concentrations accelerated inulin hydrolysis by raise of the inulinase activity, and increased sugars conversion to LA through enhancement of the whole glycolytic flux. The highest LA concentration and yield were reached by addition of 15 mM Mn 2+ -151 g/L (corresponding to 40% increase) and 0.83 g/g, respectively. However, the relative quantification by real-time reverse transcription assay showed that the presence of Mn 2+ decreases the expression levels of fosE gene encoding β-fructosidase. Contrariwise, the full exclusion of metal ions resulted in fosE gene expression enhancement, blocked fructose transport, and hindered fructose conversion thus leading to huge fructose accumulation. During fed-batch with optimized medium and fermentation parameters, the fructose content reached 35.9% (w/v), achieving yield of 467 g fructose from 675 g inulin containing chicory flour powder (0.69 g/g). LA received in course of the batch fermentation and fructose gained by the fed-batch are the highest amounts ever obtained from inulin, thus disclosing the key role of Mn 2+ as a powerful tool to guide inulin conversion to targeted bio-chemicals.
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Heger, A; Janisch, S; Pock, K; Römisch, J
2016-10-01
The solvent/detergent treatment enables effective and robust inactivation of all lipid-enveloped viruses, but also inactivates partly sensitive plasma proteins such as protein S. The aim of this study was to investigate the thrombin generation capacity of octaplasLG ® , in particular focusing on the function of protein S in thrombin generation assay and the impact of assay settings. Sixteen octaplasLG ® batches and 32 units of single donor fresh frozen plasma (FFP) were investigated. For protein S, both functional activity and free antigen levels were measured. Thrombin generation assay was performed using two fluorogenic tests with different triggers. Finally, rotational thromboelastometry was performed. Mean protein S levels were lower in octaplasLG ® , but a wider range of values was found for FFP. Clotting parameters and thrombin generation capacities overlapped between the two plasma groups as demonstrated using both thrombin generation assays and different triggers. Spiking studies with protein S-depleted plasma, human purified protein S or antibodies against protein S confirmed a correlation between protein S and thrombin generation capacity under specific assay conditions, especially in an assay with low tissue factor concentration. Correlation between protein S and thrombin generation capacity was demonstrated in the TGA. Due to higher variability in protein S content in the FFP group, overlapping haemostatic potentials of the two plasma groups were found. © 2016 International Society of Blood Transfusion.
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Vanol, Pravin G; Singhal, Puran; Shah, Priyanka A; Shah, Jaivik V; Shrivastav, Pranav S; Sanyal, Mallika
2016-08-01
A rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method is described for determination of letrozole in human plasma. Following solid phase extraction (SPE) of letrozole and letrozole-d4 on Orochem DVB-LP cartridges, chromatography was performed on Acquity UPLC BEH C 18 (50 mm×2.1 mm, 1.7 µm) column using methanol-0.1% formic acid in water (85:15, v/v) as the mobile phase. Detection was carried out on a triple quadrupole mass spectrometer with an electrospray source, operated under positive ionization mode. Quantitation of letrozole and letrozole-d4 was done using multiple reaction monitoring (MRM) following the transitions at m/z 286.2→217.0 and m/z 290.2→221.0, respectively. The calibration plots were linear through the concentration range of 0.10-100 ng/mL ( r 2 ≥0.9990) using 100 µL human plasma. The extraction recovery of letrozole ranged from 94.3% to 96.2% and the intra-batch and inter-batch precision was ≤5.2%. The method was successfully applied to a bioequivalence study of letrozole after oral administration of 2.5 mg tablet formulation to 16 healthy postmenopausal Indian women. The assay reproducibility was also established through incurred sample reanalysis (ISR) of 74 subject samples.
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Inhibitory effect of cyanide on wastewater nitrification ...
The effect of CN- (CN-) on nitrification was examined with samples from nitrifying wastewater enrichments using two different approaches: by measuring substrate (ammonia) specific oxygen uptake rates (SOUR), and by using RT-qPCR to quantify the transcripts of functional genes involved in nitrification. The nitrifying bioreactor was operated as a continuous reactor with a 24 h hydraulic retention time. The samples were exposed in batch vessels to cyanide for a period of 12 h. The concentrations of CN- used in the batch assays were 0.03, 0.06, 0.1 and 1.0 mg/L. There was considerable decrease in SOUR with increasing dosages of CN-. A decrease of more than 50% in nitrification activity was observed at 0.1 mg/L CN-. Based on the RT-qPCR data, there was notable reduction in the transcript levels of amoA and hao for increasing CN- dosage, which corresponded well with the ammonia oxidation activity measured via SOUR. The inhibitory effect of cyanide may be attributed to the affinity of cyanide to bind ferric heme proteins, which disrupt protein structure and function. The correspondence between the relative expression of functional genes and SOUR shown in this study demonstrates the efficacy of RNA based function-specific assays for better understanding of the effect of toxic compounds on nitrification activity in wastewater. Nitrification is the first step of nitrogen removal is wastewater, and it is susceptible to inhibition by many industrial chemical. We looked at
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Baylis, S A; Terao, E; Blümel, J; Hanschmann, K-M O
2017-01-01
A new European Pharmacopoeia (Ph. Eur.) biological reference preparation (BRP) had to be established further to the decision to include nucleic acid testing (NAT) for the detection of hepatitis E virus (HEV) RNA in the monograph Human plasma (pooled and treated for virus inactivation) (1646). To this purpose, an international collaborative study was launched in the framework of the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM) and the Commission of the European Union (EU). The study was run in conjunction with the establishment of the 1 st World Health Organization (WHO) international reference panel (IRP) for hepatitis E virus RNA genotypes (8578/13). Twenty-three laboratories used in-house developed and commercially available assays to calibrate a lyophilised candidate BRP prepared from a HEV 3f strain positive human plasma against the 1 st WHO International Standard (IS) for HEV RNA (6329/10). Results from quantitative and qualitative assays were in good agreement and were combined to calculate an assigned potency. Real-time stability studies indicated that the candidate BRP is very stable at lower temperatures and is thus suitable for long-term use. Based on these results, in February 2016, the Ph. Eur. Commission adopted the candidate material as the hepatitis E virus RNA for NAT testing BRP batch 1, with an assigned unitage of 2.1 × 10 4 IU/vial (4.32 log 10 IU/vial).
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Effect of pasteurization on selected immune components of donated human breast milk.
Ewaschuk, J B; Unger, S; O'Connor, D L; Stone, D; Harvey, S; Clandinin, M T; Field, C J
2011-09-01
Pasteurized, donated milk is increasingly provided to preterm infants in the absence of mother's own milk. The aim of this study was to determine the effect of pasteurization on the concentration of selected components in donated human breast milk. Donated milk from 34 mothers was pooled into 17 distinct batches (4 mothers per batch). Aliquots of each batch were then Holder pasteurized (62.5 °C for 30 min). Interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70 and IL-13 were measured in a multiplex enzyme-linked immunosorbent assay (ELISA). Granulocyte colony-stimulating factor (G-CSF), heparin-binding epidermal-like growth factor (HB-EGF) and hepatocyte growth factor (HGF) were measured by ELISA. Lipids were assessed by gas chromatography and gangliosides by the resorcinol-HCl reaction. IFN-γ, TNF-α, IL-1β, IL-10 and HGF were significantly reduced by pasteurization (P<0.05). Gangliosides were not affected, but the proportion of medium-chain saturated fats was increased (P<0.05) with a trend towards a decreased proportion of oleic acid (P=0.057). Pasteurization significantly reduced the concentration of several immunoactive compounds present in breast milk, but did not have an impact on others.
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Establishment of the Ph. Eur. erythropoietin chemical reference substance batch 1.
Burns, C; Bristow, A F; Buchheit, K H; Daas, A; Wierer, M; Costanzo, A
2015-01-01
The Erythropoietin (EPO) European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch 3 was calibrated in 2006 by in vivo bioassay and was used as a reference preparation for these assays as well as for the physicochemical methods in the Ph. Eur. monograph Erythropoietin concentrated solution (1316). In order to avoid the frequent replacement of this standard and thus reduce the use of animals, a new EPO Chemical Reference Substance (CRS) was established to be used solely for the physicochemical methods. Here we report the outcome of a collaborative study aimed at demonstrating the suitability of the candidate CRS (cCRS) as a reference for the physicochemical methods in the Ph. Eur. monograph. Results from the study demonstrated that for the physicochemical methods currently required in the monograph (capillary zone electrophoresis (CZE), polyacrylamide gel electrophoresis (PAGE)/immunoblotting and peptide mapping), the cCRS is essentially identical to the existing BRP. However, data also indicated that, for the physicochemical methods under consideration for inclusion in a revised monograph (test for oxidised forms and glycan mapping), the suitability of the cCRS as a reference needs to be confirmed with additional work. Further to completion of the study, the Ph. Eur. Commission adopted the cCRS as "Erythropoietin for physicochemical tests CRS batch 1" to be used for CZE, PAGE/immunoblotting and peptide mapping.
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Absence of anti-hypocretin receptor 2 autoantibodies in post pandemrix narcolepsy cases.
Luo, Guo; Lin, Ling; Jacob, Louis; Bonvalet, Mélodie; Ambati, Aditya; Plazzi, Giuseppe; Pizza, Fabio; Leib, Ryan; Adams, Christopher M; Partinen, Markku; Mignot, Emmanuel Jean-Marie
2017-01-01
A recent publication suggested molecular mimicry of a nucleoprotein (NP) sequence from A/Puerto Rico/8/1934 (PR8) strain, the backbone used in the construction of the reassortant strain X-179A that was used in Pandemrix® vaccine, and reported on anti-hypocretin (HCRT) receptor 2 (anti-HCRTR2) autoantibodies in narcolepsy, mostly in post Pandemrix® narcolepsy cases (17 of 20 sera). In this study, we re-examined this hypothesis through mass spectrometry (MS) characterization of Pandemrix®, and two other pandemic H1N1 (pH1N1)-2009 vaccines, Arepanrix® and Focetria®, and analyzed anti-HCRTR2 autoantibodies in narcolepsy patients and controls using three independent strategies. MS characterization of Pandemrix® (2 batches), Arepanrix® (4 batches) and Focetria® (1 batch) was conducted with mapping of NP 116I or 116M spectrogram. Two sets of narcolepsy cases and controls were used: 40 post Pandemrix® narcolepsy (PP-N) cases and 18 age-matched post Pandemrix® controls (PP-C), and 48 recent (≤6 months) early onset narcolepsy (EO-N) cases and 70 age-matched other controls (O-C). Anti-HCRTR2 autoantibodies were detected using three strategies: (1) Human embryonic kidney (HEK) 293T cells with transient expression of HCRTR2 were stained with human sera and then analyzed by flow cytometer; (2) In vitro translation of [35S]-radiolabelled HCRTR2 was incubated with human sera and immune complexes of autoantibody and [35S]-radiolabelled HCRTR2 were quantified using a radioligand-binding assay; (3) Optical density (OD) at 450 nm (OD450) of human serum immunoglobulin G (IgG) binding to HCRTR2 stably expressed in Chinese hamster ovary (CHO)-K1 cell line was measured using an in-cell enzyme-linked immunosorbent assay (ELISA). NP 116M mutations were predominantly present in all batches of Pandemrix®, Arepanrix® and Focetria®. The wild-type NP109-123 (ILYDKEEIRRIWRQA), a mimic to HCRTR234-45 (YDDEEFLRYLWR), was not found to bind to DQ0602. Three or four subjects were found positive for anti-HCRTR2 autoantibodies using two strategies or the third one, respectively. None of the post Pandemrix® narcolepsy cases (0 of 40 sera) was found positive with all three strategies. Anti-HCRTR2 autoantibody is not a significant biological feature of narcolepsy or of post Pandemrix® autoimmune responses.
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Absence of anti-hypocretin receptor 2 autoantibodies in post pandemrix narcolepsy cases
Lin, Ling; Jacob, Louis; Bonvalet, Mélodie; Ambati, Aditya; Plazzi, Giuseppe; Pizza, Fabio; Leib, Ryan; Adams, Christopher M.; Partinen, Markku; Mignot, Emmanuel Jean-Marie
2017-01-01
Background A recent publication suggested molecular mimicry of a nucleoprotein (NP) sequence from A/Puerto Rico/8/1934 (PR8) strain, the backbone used in the construction of the reassortant strain X-179A that was used in Pandemrix® vaccine, and reported on anti-hypocretin (HCRT) receptor 2 (anti-HCRTR2) autoantibodies in narcolepsy, mostly in post Pandemrix® narcolepsy cases (17 of 20 sera). In this study, we re-examined this hypothesis through mass spectrometry (MS) characterization of Pandemrix®, and two other pandemic H1N1 (pH1N1)-2009 vaccines, Arepanrix® and Focetria®, and analyzed anti-HCRTR2 autoantibodies in narcolepsy patients and controls using three independent strategies. Methods MS characterization of Pandemrix® (2 batches), Arepanrix® (4 batches) and Focetria® (1 batch) was conducted with mapping of NP 116I or 116M spectrogram. Two sets of narcolepsy cases and controls were used: 40 post Pandemrix® narcolepsy (PP-N) cases and 18 age-matched post Pandemrix® controls (PP-C), and 48 recent (≤6 months) early onset narcolepsy (EO-N) cases and 70 age-matched other controls (O-C). Anti-HCRTR2 autoantibodies were detected using three strategies: (1) Human embryonic kidney (HEK) 293T cells with transient expression of HCRTR2 were stained with human sera and then analyzed by flow cytometer; (2) In vitro translation of [35S]-radiolabelled HCRTR2 was incubated with human sera and immune complexes of autoantibody and [35S]-radiolabelled HCRTR2 were quantified using a radioligand-binding assay; (3) Optical density (OD) at 450 nm (OD450) of human serum immunoglobulin G (IgG) binding to HCRTR2 stably expressed in Chinese hamster ovary (CHO)-K1 cell line was measured using an in-cell enzyme-linked immunosorbent assay (ELISA). Results NP 116M mutations were predominantly present in all batches of Pandemrix®, Arepanrix® and Focetria®. The wild-type NP109-123 (ILYDKEEIRRIWRQA), a mimic to HCRTR234-45 (YDDEEFLRYLWR), was not found to bind to DQ0602. Three or four subjects were found positive for anti-HCRTR2 autoantibodies using two strategies or the third one, respectively. None of the post Pandemrix® narcolepsy cases (0 of 40 sera) was found positive with all three strategies. Conclusion Anti-HCRTR2 autoantibody is not a significant biological feature of narcolepsy or of post Pandemrix® autoimmune responses. PMID:29220370
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Integrated Platform for Expedited Synthesis–Purification–Testing of Small Molecule Libraries
2017-01-01
The productivity of medicinal chemistry programs can be significantly increased through the introduction of automation, leading to shortened discovery cycle times. Herein, we describe a platform that consolidates synthesis, purification, quantitation, dissolution, and testing of small molecule libraries. The system was validated through the synthesis and testing of two libraries of binders of polycomb protein EED, and excellent correlation of obtained data with results generated through conventional approaches was observed. The fully automated and integrated platform enables batch-supported compound synthesis based on a broad array of chemical transformations with testing in a variety of biochemical assay formats. A library turnaround time of between 24 and 36 h was achieved, and notably, each library synthesis produces sufficient amounts of compounds for further evaluation in secondary assays thereby contributing significantly to the shortening of medicinal chemistry discovery cycles. PMID:28435537
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As(V) and P Competitive Sorption on Soils, By-Products and Waste Materials
Rivas-Pérez, Ivana María; Paradelo-Núñez, Remigio; Nóvoa-Muñoz, Juan Carlos; Arias-Estévez, Manuel; Fernández-Sanjurjo, María José; Álvarez-Rodríguez, Esperanza; Núñez-Delgado, Avelino
2015-01-01
Batch-type experiments were used to study competitive As(V) and P sorption on various soils and sorbent materials. The materials assayed were a forest soil, a vineyard soil, pyritic material, granitic material, coarsely and finely ground mussel shell, calcinated mussel shell ash, pine sawdust and slate processing fines. Competition between As(V) and P was pronounced in the case of both soils, granitic material, slate fines, both shells and pine sawdust, showing more affinity for P. Contrary, the pyritic material and mussel shell ash showed high and similar affinity for As(V) and P. These results could be useful to make a correct use of the soils and materials assayed when focusing on As and P removal in solid or liquid media, in circumstances where both pollutants may compete for sorption sites. PMID:26690456
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Virtual Screening of Receptor Sites for Molecularly Imprinted Polymers.
Bates, Ferdia; Cela-Pérez, María Concepción; Karim, Kal; Piletsky, Sergey; López-Vilariño, José Manuel
2016-08-01
Molecularly Imprinted Polymers (MIPs) are highly advantageous in the field of analytical chemistry. However, interference from secondary molecules can also impede capture of a target by a MIP receptor. This greatly complicates the design process and often requires extensive laboratory screening which is time consuming, costly, and creates substantial waste products. Herein, is presented a new technique for screening of "virtually imprinted receptors" for rebinding of the molecular template as well as secondary structures, correlating the virtual predictions with experimentally acquired data in three case studies. This novel technique is particularly applicable to the evaluation and prediction of MIP receptor specificity and efficiency in complex aqueous systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Chevaliez, Stéphane; Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel
2017-04-01
Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTi m e HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. Copyright © 2017 American Society for Microbiology.
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Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel
2017-01-01
ABSTRACT Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTime HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. PMID:28202793
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Tan, Aimin; Saffaj, Taoufiq; Musuku, Adrien; Awaiye, Kayode; Ihssane, Bouchaib; Jhilal, Fayçal; Sosse, Saad Alaoui; Trabelsi, Fethi
2015-03-01
The current approach in regulated LC-MS bioanalysis, which evaluates the precision and trueness of an assay separately, has long been criticized for inadequate balancing of lab-customer risks. Accordingly, different total error approaches have been proposed. The aims of this research were to evaluate the aforementioned risks in reality and the difference among four common total error approaches (β-expectation, β-content, uncertainty, and risk profile) through retrospective analysis of regulated LC-MS projects. Twenty-eight projects (14 validations and 14 productions) were randomly selected from two GLP bioanalytical laboratories, which represent a wide variety of assays. The results show that the risk of accepting unacceptable batches did exist with the current approach (9% and 4% of the evaluated QC levels failed for validation and production, respectively). The fact that the risk was not wide-spread was only because the precision and bias of modern LC-MS assays are usually much better than the minimum regulatory requirements. Despite minor differences in magnitude, very similar accuracy profiles and/or conclusions were obtained from the four different total error approaches. High correlation was even observed in the width of bias intervals. For example, the mean width of SFSTP's β-expectation is 1.10-fold (CV=7.6%) of that of Saffaj-Ihssane's uncertainty approach, while the latter is 1.13-fold (CV=6.0%) of that of Hoffman-Kringle's β-content approach. To conclude, the risk of accepting unacceptable batches was real with the current approach, suggesting that total error approaches should be used instead. Moreover, any of the four total error approaches may be used because of their overall similarity. Lastly, the difficulties/obstacles associated with the application of total error approaches in routine analysis and their desirable future improvements are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.
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ChiLin: a comprehensive ChIP-seq and DNase-seq quality control and analysis pipeline.
Qin, Qian; Mei, Shenglin; Wu, Qiu; Sun, Hanfei; Li, Lewyn; Taing, Len; Chen, Sujun; Li, Fugen; Liu, Tao; Zang, Chongzhi; Xu, Han; Chen, Yiwen; Meyer, Clifford A; Zhang, Yong; Brown, Myles; Long, Henry W; Liu, X Shirley
2016-10-03
Transcription factor binding, histone modification, and chromatin accessibility studies are important approaches to understanding the biology of gene regulation. ChIP-seq and DNase-seq have become the standard techniques for studying protein-DNA interactions and chromatin accessibility respectively, and comprehensive quality control (QC) and analysis tools are critical to extracting the most value from these assay types. Although many analysis and QC tools have been reported, few combine ChIP-seq and DNase-seq data analysis and quality control in a unified framework with a comprehensive and unbiased reference of data quality metrics. ChiLin is a computational pipeline that automates the quality control and data analyses of ChIP-seq and DNase-seq data. It is developed using a flexible and modular software framework that can be easily extended and modified. ChiLin is ideal for batch processing of many datasets and is well suited for large collaborative projects involving ChIP-seq and DNase-seq from different designs. ChiLin generates comprehensive quality control reports that include comparisons with historical data derived from over 23,677 public ChIP-seq and DNase-seq samples (11,265 datasets) from eight literature-based classified categories. To the best of our knowledge, this atlas represents the most comprehensive ChIP-seq and DNase-seq related quality metric resource currently available. These historical metrics provide useful heuristic quality references for experiment across all commonly used assay types. Using representative datasets, we demonstrate the versatility of the pipeline by applying it to different assay types of ChIP-seq data. The pipeline software is available open source at https://github.com/cfce/chilin . ChiLin is a scalable and powerful tool to process large batches of ChIP-seq and DNase-seq datasets. The analysis output and quality metrics have been structured into user-friendly directories and reports. We have successfully compiled 23,677 profiles into a comprehensive quality atlas with fine classification for users.
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Validation of a new ELISA method for in vitro potency testing of hepatitis A vaccines.
Morgeaux, S; Variot, P; Daas, A; Costanzo, A
2013-01-01
The goal of the project was to standardise a new in vitro method in replacement of the existing standard method for the determination of hepatitis A virus antigen content in hepatitis A vaccines (HAV) marketed in Europe. This became necessary due to issues with the method used previously, requiring the use of commercial test kits. The selected candidate method, not based on commercial kits, had already been used for many years by an Official Medicines Control Laboratory (OMCL) for routine testing and batch release of HAV. After a pre-qualification phase (Phase 1) that showed the suitability of the commercially available critical ELISA reagents for the determination of antigen content in marketed HAV present on the European market, an international collaborative study (Phase 2) was carried out in order to fully validate the method. Eleven laboratories took part in the collaborative study. They performed assays with the candidate standard method and, in parallel, for comparison purposes, with their own in-house validated methods where these were available. The study demonstrated that the new assay provides a more reliable and reproducible method when compared to the existing standard method. A good correlation of the candidate standard method with the in vivo immunogenicity assay in mice was shown previously for both potent and sub-potent (stressed) vaccines. Thus, the new standard method validated during the collaborative study may be implemented readily by manufacturers and OMCLs for routine batch release but also for in-process control or consistency testing. The new method was approved in October 2012 by Group of Experts 15 of the European Pharmacopoeia (Ph. Eur.) as the standard method for in vitro potency testing of HAV. The relevant texts will be revised accordingly. Critical reagents such as coating reagent and detection antibodies have been adopted by the Ph. Eur. Commission and are available from the EDQM as Ph. Eur. Biological Reference Reagents (BRRs).
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Sample preparation and detection device for infectious agents
Miles, Robin R.; Wang, Amy W.; Fuller, Christopher K.; Lemoff, Asuncion V.; Bettencourt, Kerry A.; Yu, June
2003-06-10
A sample preparation and analysis device which incorporates both immunoassays and PCR assays in one compact, field-portable microchip. The device provides new capabilities in fluid and particle control which allows the building of a fluidic chip with no moving parts, thus decreasing fabrication cost and increasing the robustness of the device. The device can operate in a true continuous (not batch) mode. The device incorporates magnetohydrodynamic (MHD) pumps to move the fluid through the system, acoustic mixing and fractionation, dielectropheretic (DEP) sample concentration and purification, and on-chip optical detection capabilities.
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Tuberoso, Carlo I G; Rosa, Antonella; Montoro, Paola; Fenu, Maurizio Antonio; Pizza, Cosimo
2016-05-15
Juices obtained from cold-pressed saffron (Crocus sativus L.) floral by-products were evaluated as a potential source of compounds with antioxidant and cytotoxic activities. Floral by-products were split in two batches for extraction 24 and 48h after flower harvesting, respectively. The in vitro anti-oxidant activity of these extracts was tested using the FRAP and DPPH assays, and two biological models of lipid oxidation (activity in preventing cholesterol degradation and protection against Cu(2+)-mediated degradation of the liposomal unsaturated fatty acids). The cytotoxic activity was evaluated using the MTT assay. The results show that extracts obtained 48h post-harvest contained higher levels of total polar phenols and had the highest antioxidant activity in all of the performed assays. The LC-DAD and LC-ESI-(HR)MS(n) metabolic profiles showed high levels of kaempferol derivatives and anthocyanins. This study suggests that juices from saffron floral by-products could potentially be used to develop new products for the food and health industry. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system.
Hartwell, Supaporn Kradtap; Somprayoon, Duangporn; Kongtawelert, Prachya; Ongchai, Siriwan; Arppornchayanon, Olarn; Ganranoo, Lucksagoon; Lapanantnoppakhun, Somchai; Grudpan, Kate
2007-09-26
Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.
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Darwish, Ibrahim A; Al-Obaid, Abdul-Rahman M; Al-Malaq, Hamoud A
2009-11-15
For the first time, an enzyme-linked immunosorbent assay (ELISA) has been developed and validated for the determination of fluvastatin (FLV) in plasma samples at picogram level. The assay employed a polyclonal antibody that specifically recognizes FLV with high affinity, and FLV conjugate of bovine serum albumin (FLV-BSA) immobilized onto microplate wells as a solid-phase. The assay involved a competitive binding reaction between FLV, in plasma sample, and the immobilized FLV-BSA for the binding sites on a limited amount of the anti-FLV antibody. The bound anti-FLV antibody was quantified with horseradish peroxidase-labeled second anti-rabbit IgG antibody (HRP-IgG) and 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate for the peroxidase enzyme. The concentration of FLV in the sample was quantified by its ability to inhibit the binding of the anti-FLV antibody to the immobilized FLV-BSA and subsequently the color intensity in the assay wells. The conditions for the proposed ELISA were investigated and the optimum conditions were employed in the determination of FLV in plasma samples. The assay limit of detection was 10 pg mL(-1) and the effective working range at relative standard deviations (RSD) of
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Hu, Ting; Bailey, Ruth E; Morrall, Stephen W; Aardema, Marilyn J; Stanley, Lesley A; Skare, Julie A
2009-07-24
To address the provision of the 7th Amendment to the EU Cosmetics Directive banning the use of in vivo genotoxicity assays for testing cosmetic ingredients in 2009, the 3D EpiDerm reconstructed human skin micronucleus assay has been developed. To further characterise the EpiDerm tissue for potential use in genotoxicity testing, we have evaluated the dermal penetration and metabolism of two hair dye ingredients, p-aminophenol (PAP) and p-phenylenediamine (PPD) in this reconstructed epidermis model. When EpiDerm tissue was topically exposed to PAP or PPD for 30 min (typical for a hair dye exposure), the majority (80->90%) of PAP or PPD was excluded from skin tissue and removed by rinsing. After a 23.5h recovery period, the PAP fraction that did penetrate was completely N-acetylated to acetaminophen (APAP). Similarly, 30 min topical application of PPD resulted in the formation of the N-mono- and N,N'-diacetylated metabolites of PPD. These results are consistent with published data on the dermal metabolism of these compounds from other in vitro systems as well as from in vivo studies. When tissue was exposed topically (PAP) or via the culture media (PPD) for 24h, there was good batch-to-batch and donor-to-donor reproducibility in the penetration and metabolism of PAP and PPD. Overall, the results demonstrate that these two aromatic amines are biotransformed in 3D EpiDerm tissue via N-acetylation. Characterising the metabolic capability of EpiDerm tissue is important for the evaluation of this model for use in genotoxicity testing.
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Effect of microalgae storage conditions on methane yields.
Barreiro-Vescovo, Santiago; de Godos, Ignacio; Tomás-Pejó, Elia; Ballesteros, Mercedes; González-Fernández, Cristina
2018-05-01
During the last decade, a lot of research has been focused on identifying the methane yields achievable when using microalgae biomass (fresh and pretreated) as a substrate in anaerobic digestion. Encountered differences are frequently attributed to the different microalgae strains (cell walls and macromolecular profiles) or the different metabolic activities of anaerobic sludge used as inoculum. Nevertheless, under the hypothesis that the state of microalgae upon biomass storage may also play a significant role, this study was designed to evaluate the effect of biomass processing and storage on methane yields and hydrolysis kinetics in batch mode assays. Slight changes in the macromolecular profile distribution of the different tested biomass were observed. Regardless of the time that the biomass was stored, results revealed that frozen biomass doubled the hydrolysis constant and enhanced methane yield by 1.56-fold compared to fresh microalgae biomass (82.4 mL CH 4 g COD in -1 ). Similar enhancement was obtained with the freeze-dried biomass, and slightly lower values were obtained (1.34-fold) for the biomass kept at 4 °C longer than a week. Likewise, the semi-continuously operated reactor fed with microalgae biomass stored for 28 days at 4 °C did not show any effect in terms of methane production, although nitrogen mineralization was higher than expected. Remarkably, the initial stage of the biomass should be carefully considered for comparison purposes with the available literature on batch mode assays. This study highlights the importance of considering how the biomass is stored before the anaerobic digestion process to avoid misleading conclusions.
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The Second International Standard for Penicillin*
Humphrey, J. H.; Mussett, M. V.; Perry, W. L. M.
1953-01-01
In 1950 the Department of Biological Standards, National Institute for Medical Research, London, was authorized by the WHO Expert Committee on Biological Standardization to prepare the Second International Standard for Penicillin. A single batch of specially recrystallized sodium penicillin G was obtained and 11 laboratories in seven different countries were requested to take part in its collaborative assay. 112 assays were carried out, of which 101 were done by cup-plate methods using either Staphylococcus aureus or Bacillus subtilis. The results were subjected to standard methods of analysis, on the basis of which the authors define the Second International Standard for Penicillin as containing 1,670 International Units (IU) per mg, with limits of error (P = 0.05) of 1,666-1,674 IU/mg. The International Unit is therefore redefined as the activity contained in 0.0005988 mg of the Second International Standard for Penicillin. PMID:13082387
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Massiah, Michael A; Wright, Katharine M; Du, Haijuan
2016-04-01
This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay. Copyright © 2016 John Wiley & Sons, Inc.
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Martins, Gabriela V; Marques, Ana C; Fortunato, Elvira; Sales, M Goreti F
2016-12-15
An innovative biosensor assembly relying on a simple and straightforward in-situ construction is presented to monitor urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) down to the pmol/L level. The sensing film of the biosensor consisted of a molecularly imprinted polymer (MIP) layer for 8-OHdG assembled on a gold electrode through electropolymerization of monomer combined with the template. The analytical features of the resulting biosensor were assessed by Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). Some experimental parameters such as the initial concentration of the monomer and the ratio template-monomer were investigated and optimized in order to finely tune the performance of the MIP-based sensor. Under optimal conditions, the developed biosensor was able to rebind 8-OHdG with a linear response against EIS from 0.1 to 100pg/ml 3.5-3500 pM. The interference of coexisting species was tested, also with calibrations on urine samples, and good selectivity towards 8-OHdG was obtained. RAMAN spectroscopy, FTIR and SEM evaluations of the prepared films confirmed the formation of a polyphenol thin-film on the electrode surface. The presence and distribution of the imprinted cavities on the MIP layer was confirmed by confocal microscopy imaging of the film, after a post-treatment with Fluorescein Isothiocyanate (FITC) labeled 8-OHdG antibody. Overall, this label-free biosensor for urinary 8-OHdG detection constitutes a promising low-cost alternative to the conventional immunoassay approaches, due to its simplicity, stability, high sensitivity and selectivity for biological sample assays, opening new doors for other applications. Copyright © 2016 Elsevier B.V. All rights reserved.
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Wilkinson, A; Kennedy, K J
2012-01-01
Thin stillage (CTS) from a dry-grind corn ethanol plant was evaluated as a carbon source for anaerobic digestion (AD) by batch and high rate semi-continuous down-flow stationary fixed film (DSFF) reactors. Biochemical methane potential (BMP) assays were carried out with CTS concentrations ranging from approximately 2,460-27,172 mg total chemical oxygen demand (TCOD) per litre, achieved by diluting CTS with clean water or a combination of clean water and treated effluent. High TCOD, SCOD and volatile solids (VS) removal efficiencies of 85 ± 2, 94 ± 0 and 82 ± 1% were achieved for CTS diluted with only clean water at an organic concentration of 21,177 mg TCOD per litre, with a methane yield of 0.30 L methane per gram TCOD(removed) at standard temperature and pressure (STP, 0 °C and 1 atmosphere). Batch studies investigating the use of treated effluent for dilution showed promising results. Continuous studies employed two mesophilic DSFF anaerobic digesters treating thin stillage, operated at hydraulic retention times (HRT) of 20, 14.3, 8.7, 6.3, 5 and 4.2 d. Successful digestion was achieved up to an organic loading rate (OLR) of approximately 7.4 g TCOD L(-1)d(-1) at a 5 d HRT with a yield of 2.05 LCH(4) L(-1)d(-1) (at STP) and TCOD and VS removal efficiencies of 89 ± 3 and 85 ± 3%, respectively.
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Essers, A J; Jurgens, C M; Nout, M J
1995-07-01
The effect of six individual strains of the dominant microflora in solid substrate fermenting cassava on cyanogen levels was examined. Six out of eight batches of disinfected cassava root pieces were incubated for 72 h after inoculation with either of the fungi Geotrichum candidum, Mucor racemosus, Neurospora sitophila, Rhizopus oryzae and Rhizopus stolonifer, or a Bacillus sp., isolated from on-farm fermented cassava flours from Uganda. One non-inoculated batch was incubated as a reference. Levels of initial and final moisture and cyanogens were assayed. The experiment was done in quadruplicate. Incubation of disinfected root pieces reduced cyanogenic glucoside levels significantly to 62.7% (SD 2.8) of the initial value. Microbial growth resulted in significant additional reduction of the cyanogenic glucoside levels to 29.8% (SD 18.9) of the levels which were obtained after non-inoculated incubation. Among the tested strains, N. sitophila reduced cyanogenic glucoside levels most effectively, followed by R. stolonifer and R. oryzae. Of all fermented samples, both Rhizopus spp. showed highest proportion of residual cyanogens in the cyanohydrin form. Flours showed similar patterns of cyanogens as the batches they were prepared from. Cyanogenic glucoside level reduction was significantly correlated (r = 0.86) with the extent of root softening. It is concluded that both incubation and microbial activity are instrumental in reducing the potential toxicity of cassava during the solid substrate fermentation and that effectiveness varies considerably between the species of microorganisms applied.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ozanich, Rich M.; Antolick, Kathryn C.; Bruckner-Lea, Cindy J.
2007-09-15
Automated devices and methods for biological sample preparation often utilize surface functionalized microbeads (superparamagnetic or non-magnetic) to allow capture, purification and pre-concentration of trace amounts of proteins, cells, or nucleic acids (DNA/RNA) from complex samples. We have developed unique methods and hardware for trapping either magnetic or non-magnetic functionalized beads that allow samples and reagents to be efficiently perfused over a micro-column of beads. This approach yields enhanced mass transport and up to 5-fold improvements in assay sensitivity or speed, dramatically improving assay capability relative to assays conducted in more traditional “batch modes” (i.e., in tubes or microplate wells). Summarymore » results are given that highlight the analytical performance improvements obtained for automated microbead processing systems utilizing novel microbead trap/flow-cells for various applications, including: 1) simultaneous capture of multiple cytokines using an antibody-coupled polystyrene bead assay with subsequent flow cytometry detection; 2) capture of nucleic acids using oligonucleotide coupled polystyrene beads with flow cytometry detection; and 3) capture of Escherichia coli 0157:H7 (E. coli) from 50 mL sample volumes using antibody-coupled superparamagnetic microbeads with subsequent culturing to assess capture efficiency.« less
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Cajado-Carvalho, Daniela; Galvão, Juliana; Kuniyoshi, Alexandre K.; Carneiro, Patrícia dos Santos; Paes Leme, Adriana Franco; Pauletti, Bianca Alves; Marengo, Eliana Blini; Portaro, Fernanda V.
2017-01-01
Scorpion stings are the main cause of human envenomation in Brazil and, for the treatment of victims, the World Health Organization (WHO) recommends the use of antivenoms. The first step to achieve effective antivenom is to use a good quality venom pool and to evaluate it, with LD50 determination as the most accepted procedure. It is, however, time-consuming and requires advanced technical training. Further, there are significant ethical concerns regarding the number of animals required for testing. Hence, we investigated the correspondence between LD50 results, in vitro assays, and a strong correlation with proteolytic activity levels was observed, showing, remarkably, that proteases are potential toxicity markers for Tityus serrulatus venom. The comparison of reversed-phase chromatographic profiles also has a potential application in venoms’ quality control, as there were fewer neurotoxins detected in the venom with high LD50 value. These results were confirmed by mass spectrometry analysis. Therefore, these methods could precede the LD50 assay to evaluate the venom excellence by discriminating—and discarding—poor-quality batches, and, consequently, with a positive impact on the number of animals used. Notably, proposed assays are fast and inexpensive, being technically and economically feasible in Tityus serrulatus venom quality control to produce effective antivenoms. PMID:29168766
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Zhao, Jing; Westerholm, Maria; Qiao, Wei; Yin, Dongmin; Bi, Shaojie; Jiang, Mengmeng; Dong, Renjie
2018-05-01
The present study investigates the conversion of acetate, propionate and hydrogen consumption linked to the microbial community structure and related to temperature and substrate concentration. Biogas reactors were continuously fed with coffee powder (20 g-COD/L) or acetate (20, 40, and 60 g-COD/L) and operated for 193 days at 37 °C or 55 °C conditions. Starting HRT was 23 days which was then reduced to 7 days. The kinetics of acetate and propionate degradation and hydrogen consumption rates were measured in batch assays. At HRT 7 days, the degradation rate of propionate was higher in thermophilic batches, while acetate degradation rate was higher at mesophilic conditions. The gaseous hydrogen consumption in acetate reactors increased proportionally with temperature and substrate concentration, while the dissolved hydrogen was not affected. The relative high abundance of hydrogentrophic methanogens indicated that the methanogenesis was directed towards the syntrophic acetate oxidation pathway at high acetate concentration and high temperature. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Ethanol production from lignocellulosic byproducts of olive oil extraction.
Ballesteros, I; Oliva, J M; Saez, F; Ballesteros, M
2001-01-01
The recent implementation of a new two-step centrifugation process for extracting olive oil in Spain has substantially reduced water consumption, thereby eliminating oil mill wastewater. However, a new high sugar content residue is still generated. In this work the two fractions present in the residue (olive pulp and fragmented stones) were assayed as substrate for ethanol production by the simultaneous saccharification and fermentation (SSF) process. Pretreatment of fragmented olive stones by sulfuric acid-catalyzed steam explosion was the most effective treatment for increasing enzymatic digestibility; however, a pretreatment step was not necessary to bioconvert the olive pulp into ethanol. The olive pulp and fragmented olive stones were tested by the SSF process using a fed-batch procedure. By adding the pulp three times at 24-h intervals, 76% of the theoretical SSF yield was obtained. Experiments with fed-batch pretreated olive stones provided SSF yields significantly lower than those obtained at standard SSF procedure. The preferred SSF conditions to obtain ethanol from olives stones (61% of theoretical yield) were 10% substrate and addition of cellulases at 15 filter paper units/g of substrate.
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Li, Sha; Zhang, Jinliang; Xu, Hong; Feng, Xiaohai
2016-02-10
Gluconobacter oxydans is used to produce xylitol from D-arabitol. This study aims to improve xylitol production by increasing the coenzyme regeneration efficiency of the pentose phosphate pathway in G. oxydans. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were overexpressed in G. oxydans. Real-time PCR and enzyme activity assays revealed that G6PDH/6PGDH activity and coenzyme regeneration efficiency increased in the recombinant G. oxydans strains. Approximately 29.3 g/L xylitol was obtained, with a yield of 73.2%, from 40 g/L d-arabitol in the batch biotransformation with the G. oxydans PZ strain. Moreover, the xylitol productivity (0.62 g/L/h) was 3.26-fold of the wild type strain (0.19 g/L/h). In repetitive batch biotransformation, the G. oxydans PZ cells were used for five cycles without incurring a significant loss in productivity. These results indicate that the recombinant G. oxydans PZ strain is economically feasible for xylitol production in industrial bioconversion.
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2014-01-01
Background Present study deals with the removal of Zn(II) ions from effluent using yeast biofilm formed on gravels. Methods The biofilm forming ability of Candida rugosa and Cryptococcus laurentii was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay and monitored by scanning electron microscopy (SEM), and Confocal laser scanning microscopy (CLSM). Copious amount of extracellular polymeric substances (EPS) produced by yeast species was quantified and characterized by Fourier transform infrared spectroscopy (FT-IR). Results Yeast biofilm formed on gravels by C. rugosa and C. laurentii showed 88% and 74.2% removal of Zn(II) ions respectively in batch mode. In column mode, removal of Zn(II) ions from real effluent was found to be 95.29% by C. rugosa biofilm formed on gravels. Conclusion The results of the present study showed that there is a scope to develop a cost effective method for the efficient removal of Zn(II) from effluent using gravels coated with yeast biofilm. PMID:24397917
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Yan, Shi-Kai; Wu, Yan-Wen; Liu, Run-Hui; Zhang, Wei-Dong
2007-01-01
Major bioactive components in various Calculus Bovis, including natural, artificial and in-vitro cultured Calculus Bovis, were comparatively studied. An approach of high-performance liquid chromatography coupled with ultraviolet and evaporative light scattering detections (HPLC/UV/ELSD) was established to simultaneously determinate six bioactive components thereof, including five bile acids (cholic acid, deoxycholic acid, ursodeoxycholic, chenodeoxycholic acid, hyodeoxycholic acid) and bilirubin. ELSD and UV detector were applied to detect bile acids and bilirubin respectively. The assay was performed on a C(18) column with water-acetonitrile gradient elution and the investigated constituents were authenticated by comparing retention times and mass spectra with those of reference compounds. The proposed method was applied to analyze twenty-one Calculus Bovis extraction samples, and produced data with acceptable linearity, precision, repeatability and accuracy. The result indicated the variations among Calculus Bovis samples under different developmental conditions. Artificial and in-vitro cultured Calculus Bovis, especially in-vitro cultured ones, which contain total bioactive constituents no less than natural products and have the best batch-to-batch uniformity, suffice to be used as substitutes of natural Calculus Bovis.
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Macromolecular crowding impacts on the diffusion and conformation of DNA hairpins
NASA Astrophysics Data System (ADS)
Stiehl, Olivia; Weidner-Hertrampf, Kathrin; Weiss, Matthias
2015-01-01
Biochemical reactions in crowded fluids differ significantly from those in dilute solutions. Both, excluded-volume interactions with surrounding macromolecules ("crowders") and an enhanced rebinding of reaction partners due to crowding-induced viscoelasticity and subdiffusion have been hypothesized to shift chemical equilibria towards the associated state. We have explored the impact of both cues in an experimentally tunable system by monitoring the steady-state fraction of open DNA hairpins in crowded fluids with varying viscoelastic characteristics but similar occupied volume fractions. As a result, we observed an increased fraction of closed DNA hairpins in viscoelastic crowded fluids. Our observations compare favorably to a simple statistical model that considers both facets of crowding, while preferential interactions between crowders and DNA hairpins appear to have little influence.
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Zhang, Yong; Zhou, An; Xie, Xiao-Mei
2013-03-01
A simple and sensitive method has been developed to simultaneously determine betunilic acid, oleanolic acid and ursolic acid in the fruits of Ziziphus jujuba from different regions by HPLC-MS. This HPLC assay was performed on PAH polymeric C18 bonded stationary phase column with mobile phase contained acetonitrile-water (90: 10) and with negative ESI detection mode. The developed approach was characterized by short time consumption for chromatographic separation, high sensitivity and good reliability so as to meet the requirements for rapid analysis of large-batch fruits of Z. jujuba from different habitats.
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Opitz, Sebastian E.W.; Goodman, Bernard A.; Keller, Marco; Smrke, Samo; Wellinger, Marco; Schenker, Stefan
2016-01-01
Abstract Introduction Coffee is a widely consumed beverage containing antioxidant active compounds. During roasting the phytochemical composition of the coffee bean changes dramatically and highly polymeric substances are produced. Besides chlorogenic acids that are already present in green coffee beans, melanoidins show antioxidant capacity as well. Objective To employ post‐column derivatisation by coupling high performance size exclusion chromatography (HPSEC) to an antioxidant assay to investigate the effect of roasting on the properties of antioxidant active compounds in coffee brews. Methodology We have investigated the antioxidant capacity of Coffea arabica (Arabica) and C. canephora (Robusta) beans that were roasted over the full spectrum of roast conditions (four roasting speeds to three roast degrees) by comparing the results from HPSEC coupled on‐line to the ABTS assay with those from two batch assays, Folin Ciocalteu (FC) and oxygen radical absorbance capacity (ORAC) assay. Results The antioxidant capacity showed a general decrease towards slower and darker roasted coffee for all three assays, indicative of heat degradation of active compounds. Hence, low molecular weight (LMW) compounds such as chlorogenic acids (CGAs) decreased progressively already from relatively mild roasting conditions. In contrast, high molecular weight (HMW) compounds (e.g. melanoidins) increased from light to dark roast degrees with lowering magnitude towards slower roasting profiles. Conclusion By coupling HPSEC on‐line to the ABTS assay we were able to separately quantify the contribution of HMW and LMW compounds to the total antioxidant capacity, increasing our understanding of the roast process. © 2016 The Authors. Phytochemical Analysis Published by John Wiley & Sons Ltd. PMID:28008674
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Opitz, Sebastian E W; Goodman, Bernard A; Keller, Marco; Smrke, Samo; Wellinger, Marco; Schenker, Stefan; Yeretzian, Chahan
2017-03-01
Coffee is a widely consumed beverage containing antioxidant active compounds. During roasting the phytochemical composition of the coffee bean changes dramatically and highly polymeric substances are produced. Besides chlorogenic acids that are already present in green coffee beans, melanoidins show antioxidant capacity as well. To employ post-column derivatisation by coupling high performance size exclusion chromatography (HPSEC) to an antioxidant assay to investigate the effect of roasting on the properties of antioxidant active compounds in coffee brews. We have investigated the antioxidant capacity of Coffea arabica (Arabica) and C. canephora (Robusta) beans that were roasted over the full spectrum of roast conditions (four roasting speeds to three roast degrees) by comparing the results from HPSEC coupled on-line to the ABTS assay with those from two batch assays, Folin Ciocalteu (FC) and oxygen radical absorbance capacity (ORAC) assay. The antioxidant capacity showed a general decrease towards slower and darker roasted coffee for all three assays, indicative of heat degradation of active compounds. Hence, low molecular weight (LMW) compounds such as chlorogenic acids (CGAs) decreased progressively already from relatively mild roasting conditions. In contrast, high molecular weight (HMW) compounds (e.g. melanoidins) increased from light to dark roast degrees with lowering magnitude towards slower roasting profiles. By coupling HPSEC on-line to the ABTS assay we were able to separately quantify the contribution of HMW and LMW compounds to the total antioxidant capacity, increasing our understanding of the roast process. © 2016 The Authors. Phytochemical Analysis Published by John Wiley & Sons Ltd. © 2016 The Authors. Phytochemical Analysis Published by John Wiley & Sons Ltd.
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Swathirajan, Chinnambedu Ravichandran; Vignesh, Ramachandran; Boobalan, Jayaseelan; Solomon, Sunil Suhas; Saravanan, Shanmugam; Balakrishnan, Pachamuthu
2017-10-01
Sustainable suppression of HIV replication forms the basis of anti-retroviral therapy (ART) medication. Thus, reliable quantification of HIV viral load has become an essential factor to monitor the effectiveness of the ART. Longer turnaround-time (TAT), batch testing and technical skills are major drawbacks of standard real-time PCR assays. The performance of the point-of-care Xpert HIV-1 viral load assay was evaluated against the Abbott RealTime PCR m2000rt system. A total of 96 plasma specimens ranging from 2.5 log10 copies ml -1 to 4.99 log10 copies ml -1 and proficiency testing panel specimens were used. Precision and accuracy were checked using the Pearson correlation co-efficient test and Bland-Altman analysis. Compared to the Abbott RealTime PCR, the Xpert HIV-1 viral load assay showed a good correlation (Pearson r=0.81; P<0.0001) with a mean difference of 0.27 log10 copies ml -1 (95 % CI, -0.41 to 0.96 log10 copies ml -1 ; sd, 0.35 log10 copies ml -1 ). Reliable and ease of testing individual specimens could make the Xpert HIV-1 viral load assay an efficient alternative method for ART monitoring in clinical management of HIV disease in resource-limited settings. The rapid test results (less than 2 h) could help in making an immediate clinical decision, which further strengthens patient care.
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A high-throughput assay of membrane protein stability.
Postis, Vincent L G; Deacon, Sarah E; Roach, Peter C J; Wright, Gareth S A; Xia, Xiaobing; Ingram, Jean C; Hadden, Jonathan M; Henderson, Peter J F; Phillips, Simon E V; McPherson, Michael J; Baldwin, Stephen A
2008-12-01
The preparation of purified, detergent-solubilized membrane proteins in a monodisperse and stable form is usually a prerequisite for investigation not only of their function but also for structural studies by X-ray crystallography and other approaches. Typically, it is necessary to explore a wide range of conditions, including detergent type, buffer pH, and the presence of additives such as glycerol, in order to identify those optimal for stability. Given the difficulty of expressing and purifying membrane proteins in large amounts, such explorations must ideally be performed on as small a scale as practicable. To achieve this objective in the UK Membrane Protein Structure Initiative, we have developed a rapid, economical, light-scattering assay of membrane protein aggregation that allows the testing of 48 buffer conditions in parallel on 6 protein targets, requiring less than 2 mg protein for each target. Testing of the assay on a number of unrelated membrane transporters has shown that it is of generic applicability. Proteins of sufficient purity for this plate-based assay are first rapidly prepared using simple affinity purification procedures performed in batch mode. Samples are then transferred by microdialysis into each of the conditions to be tested. Finally, attenuance at 340 nm is monitored in a 384-well plate using a plate reader. Optimal conditions for protein stability identified in the assay can then be exploited for the tailored purification of individual targets in as stable a form as possible.
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A practical approach to determination of laboratory GC-MS limits of detection.
Underwood, P J; Kananen, G E; Armitage, E K
1997-01-01
Determination of limit of detection (LOD) values in a forensic laboratory serves a fundamental forensic requirement for assay performance. In addition to demonstrating assay capability, LOD values can also be used to fulfill certification requirements of a high-volume forensic drug laboratory. The LOD was defined as the lowest concentration of drug that the laboratory can detect in a specimen with forensic certainty at a minimum of 85% of the time. Overall batch acceptance criteria included acceptable quantitation of control materials (within 20% of target), acceptable chromatography (symmetry, peak integration, peak shape, peak, and baseline resolution), retention time within +/-1% of the extracted standard, and mass ion ratios within +/-20% of the extracted standard mass ion ratios. Individual specimen acceptance criteria were the same as the batch acceptance criteria excluding the quantitation requirement. Data were collected from all instruments on different runs. A minimum of ten data points was required for each certified instrument, and a minimum of 85% of data points was acceptable. Quantitation within +/-20% of the LOD concentration was not required, but acceptable mass ratios were required. Data points with poor chromatography (internal standard failed mass ratios; interference of the baseline, for example, shoulders; asymmetry; and baseline resolution) was omitted from the acceptable rate calculation. Data points with good chromatography with failed mass ion ratios were included in the acceptable rate calculation. With these criteria, we established the following LODs: 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid, 2 ng/mL; benzoylecgonine, 5 ng/mL; phencyclidine, 2.5 ng/mL; amphetamine, 150 ng/mL; methamphetamine, 100 ng/mL; codeine, 500 ng/mL; and morphine, 1000 ng/mL.
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Zuliani, Thomas; Saiagh, Soraya; Knol, Anne-Chantal; Esbelin, Julie; Dréno, Brigitte
2013-01-01
Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors. PMID:23894651
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Van den Abbeele, Pieter; Marzorati, Massimo; Derde, Melanie; De Weirdt, Rosemarie; Joan, Vermeiren; Possemiers, Sam; Van de Wiele, Tom
2016-01-01
The microbiota that colonises the intestinal mucus may particularly affect human health given its proximity to the epithelium. For instance, the presence of the adherent-invasive Escherichia coli (AIEC) in this mucosal microbiota has been correlated with Crohn’s disease. Using short-term screening assays and a novel long-term dynamic gut model, which comprises a simulated mucosal environment (M-SHIME), we investigated how (potential) pro- and prebiotics may repress colonisation of AIEC from mucus. Despite that during the short-term screening assays, some of the investigated Lactobacillus strains adhered strongly to mucins, none of them competed with AIEC for mucin-adhesion. In contrast, AIEC survival and growth during co-culture batch incubations was decreased by Lactobacillus rhamnosus GG and L. reuteri 1063, which correlated with (undissociated) lactic acid and reuterin levels. Regarding the prebiotics, long-chain arabinoxylans (LC-AX) lowered the initial mucin-adhesion of AIEC, while both inulin (IN) and galacto-oligosaccharides (GOS) limited AIEC survival and growth during batch incubations. L. reuteri 1063, LC-AX and IN were thus retained for a long-term study with the M-SHIME. All treatments repressed AIEC from mucus without affecting AIEC numbers in the luminal content. As a possible explanation, L. reuteri 1063 treatment increased lactobacilli levels in mucus, while LC-AX and IN additionally increased mucosal bifidobacteria levels, thus leading to antimicrobial effects against AIEC in mucus. Overall, this study shows that pro- and prebiotics can beneficially modulate the in vitro mucosal microbiota, thus limiting occurrence of opportunistic pathogens among those mucosal microbes which may directly interact with the host given their proximity to the epithelium. PMID:28721250
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Van den Abbeele, Pieter; Marzorati, Massimo; Derde, Melanie; De Weirdt, Rosemarie; Joan, Vermeiren; Possemiers, Sam; Van de Wiele, Tom
2016-01-01
The microbiota that colonises the intestinal mucus may particularly affect human health given its proximity to the epithelium. For instance, the presence of the adherent-invasive Escherichia coli (AIEC) in this mucosal microbiota has been correlated with Crohn's disease. Using short-term screening assays and a novel long-term dynamic gut model, which comprises a simulated mucosal environment (M-SHIME), we investigated how (potential) pro- and prebiotics may repress colonisation of AIEC from mucus. Despite that during the short-term screening assays, some of the investigated Lactobacillus strains adhered strongly to mucins, none of them competed with AIEC for mucin-adhesion. In contrast, AIEC survival and growth during co-culture batch incubations was decreased by Lactobacillus rhamnosus GG and L. reuteri 1063, which correlated with (undissociated) lactic acid and reuterin levels. Regarding the prebiotics, long-chain arabinoxylans (LC-AX) lowered the initial mucin-adhesion of AIEC, while both inulin (IN) and galacto-oligosaccharides (GOS) limited AIEC survival and growth during batch incubations. L. reuteri 1063, LC-AX and IN were thus retained for a long-term study with the M-SHIME. All treatments repressed AIEC from mucus without affecting AIEC numbers in the luminal content. As a possible explanation, L. reuteri 1063 treatment increased lactobacilli levels in mucus, while LC-AX and IN additionally increased mucosal bifidobacteria levels, thus leading to antimicrobial effects against AIEC in mucus. Overall, this study shows that pro- and prebiotics can beneficially modulate the in vitro mucosal microbiota, thus limiting occurrence of opportunistic pathogens among those mucosal microbes which may directly interact with the host given their proximity to the epithelium.
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Association between stillbirth and illicit drug use and smoking during pregnancy.
Varner, Michael W; Silver, Robert M; Rowland Hogue, Carol J; Willinger, Marian; Parker, Corette B; Thorsten, Vanessa R; Goldenberg, Robert L; Saade, George R; Dudley, Donald J; Coustan, Donald; Stoll, Barbara; Bukowski, Radek; Koch, Matthew A; Conway, Deborah; Pinar, Halit; Reddy, Uma M
2014-01-01
To compare illicit drug and smoking use in pregnancies with and without stillbirth. The Stillbirth Collaborative Research Network conducted a case-control study from March 2006 to September 2008, covering more than 90% of deliveries to residents of five a priori-defined geographically diverse regions. The study attempted to include all stillbirths and representative liveborn controls. Umbilical cord samples from cases and controls were collected and frozen for subsequent batch analysis. Maternal serum was collected at delivery and batch analyzed for cotinine. For 663 stillbirth deliveries, 418 (63%) had cord homogenate and 579 (87%) had maternal cotinine assays performed. For 1,932 live birth deliveries, 1,050 (54%) had cord homogenate toxicology and 1,545 (80%) had maternal cotinine assays performed. A positive cord homogenate test for any illicit drug was associated with stillbirth (odds ratio [OR] 1.94, 95% confidence interval [CI] 1.16-3.27). The most common individual drug was cannabis (OR 2.34 95% CI 1.13-4.81), although the effect was partially confounded by smoking. Both maternal self-reported smoking history and maternal serum cotinine levels were associated in a dose-response relationship with stillbirth. Positive serum cotinine less than 3 ng/mL and no reported history of smoking (proxy for passive smoke exposure) also were associated with stillbirth (OR 2.06, 95% CI 1.24-3.41). Cannabis use, smoking, illicit drug use, and apparent exposure to second-hand smoke, separately or in combination, during pregnancy were associated with an increased risk of stillbirth. Because cannabis use may be increasing with increased legalization, the relevance of these findings may increase as well. II.
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Decolorization of a reactive copper-phthalocyanine dye under methanogenic conditions.
Beydili, M I; Matthews, R D; Pavlostathis, S G
2001-01-01
The objective of this research was to assess the biological decolorization of the copper-phthalocyanine dye Reactive Blue 7 (RB7) under methanogenic conditions using a mixed, methanogenic culture in a repetitive dye addition batch assay. The initial rate of decolorization was 13.2 mg/L-d and 5.7 mg/L-d for the first and second dye addition, respectively. For an initial RB7 concentration of ca. 300 mg/L, the extent of decolorization remained constant (about 62%) for each repetitive RB7 addition and resulted in a residual color build up. Declining absorbance ratio values (A664/A620) with increasing incubation time confirmed that the observed color removal was due to transformation as opposed to adsorption on the biomass. Chemical decolorization assays using sodium dithionite as the reducing agent resulted in similar absorbance spectra to that obtained after biological decolorization. In addition, in both the chemical and biological decolorization assays, partial oxidation of the reduced dye solution upon exposure to air resulted in higher residual color, indicating that the reduction and decolorization of RB7 are partially reversible. These results also suggest that RB7 reduction and decolorization both chemically and biologically most likely followed a similar reduction mechanism.
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Salar-García, María J; Bernal, Vicente; Pastor, José M; Salvador, Manuel; Argandoña, Montserrat; Nieto, Joaquín J; Vargas, Carmen; Cánovas, Manuel
2017-02-08
The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13 C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect - mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L -1 ) and volumetric ectoine yields (up to 4.21 g L -1 ) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic efficiency of C. salexigens can be improved by careful design of culture feeding schemes. Metabolic shifts observed at low ammonium concentrations were explained by a shift in the energy required for nitrogen assimilation. Carbon-limited fed-batch cultures with reduced ammonium supply were the best conditions for cultivation of C. salexigens, supporting high density growth and maintaining high ectoines production.
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NASA Astrophysics Data System (ADS)
Giordano, Raquel L. C.; Trovati, Joubert; Schmidell, Willibaldo
This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silicaenzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica-enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/1 of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/1 of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/1/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10-4 cm/s.
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Aflatoxin and ochratoxin A content of spices in Hungary.
Fazekas, B; Tar, A; Kovács, M
2005-09-01
In October and November 2004, 91 spice samples (70 ground red pepper, six black pepper, five white pepper, five spice mix and five chilli samples), the majority of which originated from commercial outlets, were analysed for aflatoxins B1, B2, G1 and G2 (AFB1, AFB2, AFG1, AFG2) and ochratoxin A (OTA) content by high-performance liquid chromatography (HPLC) after immunoaffinity column clean-up. Eighteen of the 70 ground red pepper samples contained AFB1, seven of them in a concentration exceeding the 'maximum level' of 5 microg kg(-1) (range 6.1-15.7 microg kg(-1)). Of the other spices assayed, the AFB1 contamination of one chilli sample exceeded 5 microg kg(-1) (8.1 microg kg(-1)). Thirty-two of the 70 ground red pepper samples contained OTA, eight of them in a concentration exceeding the 10 microg kg(-1) 'maximum level' (range 10.6-66.2 microg kg(-1)). One chilli sample was contaminated with OTA at 2.1 microg kg(-1). The AFB1 and OTA contamination of ground red pepper exceeding the 'maximum level' (5 and 10 microg kg(-1), respectively) was obviously the consequence of mixing imported ground red pepper batches heavily contaminated with AFB1 and OTA with red pepper produced in Hungary. This case calls attention to the importance of consistently screening imported batches of ground red pepper for aflatoxin and ochratoxin A content and strictly prohibiting the use of batches containing mycotoxin concentrations exceeding the maximum permitted level.
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Kumar, Manish; Das, Nilotpal; Goswami, Ritusmita; Sarma, Kali Prasad; Bhattacharya, Prosun; Ramanathan, A L
2016-12-01
The present work is an attempt to study As and F+ coevality using laboratory based assays which couples fractionation and batch dissolution experiments. Sequential extraction procedure (SEP) resulting into five "operationally defined phases", was performed on sediment and soil samples collected from the Brahmaputra flood plains, Assam, India. High correlation between the Fe (hydr)oxide fraction and total As content of the soil/sediment sample indicates the involvement of Fe (hydr)oxides as the principal source of As. F - being an anion has high potential to be sorbed onto positively charged surfaces. Findings of the SEP were used to design the batch desorption experiments by controlling the Fe (hydr)oxide content of the soil/sediment. Desorption of As and F - was observed under acidic, neutral and alkaline pH from untreated and Fe (hydr)oxide removed samples. Highest amount of As and F - were found to be released from untreated samples under alkaline pH, while the amount leached from samples with no Fe (hydr)oxide was low. The study showed that the Fe (hydr)oxide fraction commonly found in the soils and sediments, had high affinity for negatively charged species like F - oxyanions of As, AsO 4 3- (arsenate) and AsO 3 3- (arsenite). Fe (hydr)oxide fraction was found to play the major role in co-evolution of As and F - . Two sorption coefficients were proposed based on easily leachable fraction and As present in the groundwater of sampling location for understanding of contamination vulnerability from the leaching. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Giordano, Raquel L C; Trovati, Joubert; Schmidell, Willibaldo
2008-03-01
This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica-enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica-enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 x 10(-4) cm/s.
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Smith, Steven G; Smits, Kaatje; Joosten, Simone A; van Meijgaarden, Krista E; Satti, Iman; Fletcher, Helen A; Caccamo, Nadia; Dieli, Francesco; Mascart, Francoise; McShane, Helen; Dockrell, Hazel M; Ottenhoff, Tom H M
2015-01-01
Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.
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Liang, Jidong; Olivares, Christopher; Field, Jim A; Sierra-Alvarez, Reyes
2013-11-15
2,4-Dinitroanisole (DNAN) is an insensitive munitions compound considered to replace conventional explosives such as 2,4,6-trinitrotoluene (TNT). DNAN undergoes facile microbial reduction to 2-methoxy-5-nitroaniline (MENA) and 2,4-diaminoanisole (DAAN). This study investigated the inhibitory effect of DNAN, MENA, and DAAN toward various microbial targets in anaerobic (acetoclastic methanogens) and aerobic (heterotrophs and nitrifiers) sludge, and the bioluminescent bacterium, Aliivibrio fischeri, used in the Microtox assay. Aerobic heterotrophic and nitrifying batch experiments with DAAN could not be performed because the compound underwent extensive autooxidation in these assays. DNAN severely inhibited methanogens, nitrifying bacteria, and A. fischeri (50% inhibitory concentrations (IC50) ranging 41-57μM), but was notably less inhibitory to aerobic heterotrophs (IC50>390 μM). Reduction of DNAN to MENA and DAAN lead to a marked decrease in methanogenic inhibition (i.e., DNAN>MENA≈DAAN). Reduction of all nitro groups in DNAN also resulted in partial detoxification in assays with A. fischeri. In contrast, reduction of a single nitro group did not alter the inhibitory impact of DNAN toward A. fischeri and nitrifying bacteria given the similar IC50 values determined for MENA and DNAN in these assays. These results indicate that reductive biotransformation could reduce the inhibitory potential of DNAN. Copyright © 2013 Elsevier B.V. All rights reserved.
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Wan, Ying-chun; Ma, Hui-ting; Lu, Bin
2015-01-01
When organic solvent-compatible molecularly imprinted polymers (MIPs) are used in aqueous environment, how to reduce nonspecific binding is a major challenge. By modifying the binding solvents and introducing appropriate washing and elution steps, even relatively hydrophobic MIPs can gain optimal rebinding selectivity in aqueous conditions. Furthermore, water-compatible MIPs that can be used to treat aqueous samples directly have been prepared. The use of hydrophilic co-monomers, the controlled surface modification through controlled radical polymerization, and the new interfacial molecular imprinting methods are different strategies to prepare water-compatible MIPs. By combining MIPs with other techniques, both organic solvent-compatible and water-compatible MIPs can display better functional performances in aqueous conditions. Intensive studies on MIPs in aqueous conditions can provide new MIPs with much-improved compatibilities that will lead to more interesting applications in biomedicine and biotechnology.
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Durán-Álvarez, Juan C; Prado, Blanca; Ferroud, Anouck; Juayerk, Narcedalia; Jiménez-Cisneros, Blanca
2014-03-01
Sorption and leaching potential of ibuprofen, estrone and 17β estradiol were tested in two agricultural soils: one irrigated using municipal wastewater and the other used in rainfed agriculture. Batch sorption-desorption experiments and undisturbed soil column assays were carried out using both soils to which were added a mixture of the target compounds. The three compounds were sorbed to a different extent by both soils: estrone>17β estradiol>ibuprofen. Higher sorption was observed in the irrigated soil, which was attributed to the accumulation of organic matter caused by wastewater irrigation. Desorption of hormones was hysteretic in the irrigated soil, while ibuprofen showed low hysteresis in both soils. Retardation of the compounds' displacement was consistent with the sorption pattern observed in the batch tests. Retardation factor (RF) was similar for the three compounds in the two tested soils, indicating that the target compounds are much more mobile in the soil columns than would be predicted based on their equilibrium sorption parameters. The results obtained in the experiments clarify the role of wastewater irrigated soils as a filter and degradation media for the target micropollutants. Copyright © 2013 Elsevier B.V. All rights reserved.
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Mou, Quanbing; Ma, Yuan; Zhu, Xinyuan; Yan, Deyue
2016-05-28
Targeted drug delivery is a broadly applicable approach for cancer therapy. However, the nanocarrier-based targeted delivery system suffers from batch-to-batch variation, quality concerns and carrier-related toxicity issues. Thus, to develop a carrier-free targeted delivery system with nanoscale characteristics is very attractive. Here, a novel targeting small molecule nanodrug self-delivery system consisting of targeting ligand and chemotherapy drug was constructed, which combined the advantages of small molecules and nano-assemblies together and showed excellent targeting ability and long blood circulation time with well-defined structure, high drug loading ratio and on-demand drug release behavior. As a proof-of-concept, lactose (Lac) and doxorubicin (DOX) were chosen as the targeting ligand and chemotherapy drug, respectively. Lac and DOX were conjugated through a pH-responsive hydrazone group. For its intrinsic amphiphilic property, Lac-DOX conjugate could self-assemble into nanoparticles in water. Both in vitro and in vivo assays indicated that Lac-DOX nanoparticles exhibited enhanced anticancer activity and weak side effects. This novel active targeting nanodrug delivery system shows great potential in cancer therapy. Copyright © 2016 Elsevier B.V. All rights reserved.
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Validation of a 30-year-old process for the manufacture of L-asparaginase from Erwinia chrysanthemi.
Gervais, David; Allison, Nigel; Jennings, Alan; Jones, Shane; Marks, Trevor
2013-04-01
A 30-year-old manufacturing process for the biologic product L-asparaginase from the plant pathogen Erwinia chrysanthemi was rigorously qualified and validated, with a high level of agreement between validation data and the 6-year process database. L-Asparaginase exists in its native state as a tetrameric protein and is used as a chemotherapeutic agent in the treatment regimen for Acute Lymphoblastic Leukaemia (ALL). The manufacturing process involves fermentation of the production organism, extraction and purification of the L-asparaginase to make drug substance (DS), and finally formulation and lyophilisation to generate drug product (DP). The extensive manufacturing experience with the product was used to establish ranges for all process parameters and product quality attributes. The product and in-process intermediates were rigorously characterised, and new assays, such as size-exclusion and reversed-phase UPLC, were developed, validated, and used to analyse several pre-validation batches. Finally, three prospective process validation batches were manufactured and product quality data generated using both the existing and the new analytical methods. These data demonstrated the process to be robust, highly reproducible and consistent, and the validation was successful, contributing to the granting of an FDA product license in November, 2011.
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Strategies to evaluate biodegradability: application to chlorinated herbicides.
Sanchis, S; Polo, A M; Tobajas, M; Rodriguez, J J; Mohedano, A F
2014-01-01
The biodegradability of nitrochlorinated (diuron and atrazine) and chlorophenoxy herbicides (2,4-D and MCPA) has been studied through several bioassays using different testing times and biomass/substrate ratios. A fast biodegradability test using unacclimated activated sludge yielded no biodegradation of the herbicides in 24 h. The inherent biodegradability test gave degradation percentages of around 20-30% for the nitrochlorinated herbicides and almost complete removal of the chlorophenoxy compounds. Long-term biodegradability assays were performed using sequencing batch reactor (SBR) and sequencing batch membrane bioreactor (SB-MBR). Fixed concentrations of each herbicide below the corresponding EC50 value for activated sludge were used (30 mg L(-1) for diuron and atrazine and 50 mg L(-1) for 2,4-D and MCPA). No signs of herbicide degradation appeared before 35 days in the case of diuron and atrazine and 21 days for 2,4-D, whereas MCPA was partially degraded since the early stages. Around 25-36% degradation of the nitrochlorinated herbicides and 53-77% of the chlorophenoxy ones was achieved after 180 and 135 days, respectively, in SBR, whereas complete disappearance of 2,4-D was reached after 80 days in SB-MBR.
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Decoloration of a carpet dye effluent using Trametes versicolor.
Ramsay, Juliana A; Goode, Chris
2004-02-01
Although a non-sterile, undiluted carpet dye effluent (containing two anthraquinone dyes) did not support growth of Trametes versicolor, the pre-grown fungus removed 95% of its color in shake-flasks after 10 h of incubation. After decoloration, the COD of the cell-free supernatant increased and the toxicity was unchanged as determined by the Microtox assay using Vibrio fischeri. Decoloration rates decreased when either glucose alone or Mn2+ and glucose were added. T. versicolor, immobilized on jute twine in a rotating biological contacting reactor, also decolorized four successive batches of the effluent. There was no decoloration in any of the uninoculated, non-sterile controls.
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Determination of emamectin benzoate in medicated fish feed: a multisite study.
Farer, Leslie J
2005-01-01
A new method was developed for the quantitation of emamectin benzoate in medicated fish feed at levels between 1 and 30 ppm. The new procedure, based on a previously reported assay, consists of a wet methanolic extraction of ground feed, followed by solid-phase extraction and injection onto a gradient liquid chromatographic system. A multisite study involving 3 laboratories (the developing laboratory and 2 independent laboratories) was performed to evaluate precision, recovery, linearity, and sensitivity. Mean recove;ries for triplicate analyses at 3 levels, performed by 2 analysts per laboratory, were between 89 and 97%, with coefficients of variation ranging from 1.6 to 8.6%. Coefficients of determination (r2) obtained from the plotted data were > or =0.993. The precision of the method, determined from 6 replicate preparations from the same batch of medicated feed assayed in 3 separate trials per laboratory, was between 0.6 and 5.8%. The quantitation limit was established at 0.5 ppm. Specificity and robustness studies were performed by the developing laboratory.
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Monitoring peroxides generation during model wine fermentation by FOX-1 assay.
Bridi, Raquel; González, Alvaro; Bordeu, Edmundo; López-Alarcón, Camilo; Aspée, Alexis; Diethelm, Benjamin; Lissi, Eduardo; Parpinello, Giuseppina Paola; Versari, Andrea
2015-05-15
The quality of wine is mainly determined during the alcoholic fermentation that gradually transforms the grape juice into wine. Along this process the yeast goes through several stressful stages which can affect its fermentative ability and industrial performance, affecting wine quality. Based on their actual application on industrial winemaking, commercial Saccharomyces cerevisiae strains (EC1118, QA23, VIN7 and VL3) were used. They were inoculated in batch laboratory fermentations in a model wine solution for evaluating the production of reactive oxygen species (ROS) during the yeast's alcoholic fermentation. For first time total hydroperoxides were determined by FOX-1 assay to follow ROS generation. The total hydroperoxides accumulated along the 10 days of fermentation peaked up to 10.0 μM in yeast EC1118, of which 1.3 μM was hydrogen peroxide (H2O2). The FOX-1 based analytical approach herein presented is a valuable tool for the quantification of ROS oxidative damage during winemaking. Copyright © 2015. Published by Elsevier Ltd.
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Integrated bioconversion of syngas into bioethanol and biopolymers.
Lagoa-Costa, Borja; Abubackar, Haris Nalakath; Fernández-Romasanta, María; Kennes, Christian; Veiga, María C
2017-09-01
Syngas bioconversion is a promising method for bioethanol production, but some VFA remains at the end of fermentation. A two-stage process was set-up, including syngas fermentation as first stage under strict anaerobic conditions using C. autoethanogenum as inoculum, with syngas (CO/CO 2 /H 2 /N 2 , 30/10/20/40) as gaseous substrate. The second stage consisted in various fed-batch assays using a highly enriched PHA accumulating biomass as inoculum, where the potential for biopolymer production from the remaining acetic acid at the end of the syngas fermentation was evaluated. All of the acetic acid was consumed and accumulated as biopolymer, while ethanol and 2,3-butanediol remained basically unused. It can be concluded that a high C/N ratio in the effluent from the syngas fermentation stage was responsible for non-consumption of alcohols. A maximum PHA content of 24% was reached at the end of the assay. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Third International Standard for Posterior Pituitary
Bangham, D. R.; Mussett, Marjorie V.
1958-01-01
In October 1955, stocks of the Second International Standard for Posterior Pituitary were running low and the Department of Biological Standards of the National Institute for Medical Research, London, was asked to proceed with the arrangements for an international collaborative assay of material for the Third Standard. A single 142-g batch of posterior-pituitary-lobe powder was obtained and distributed in ampoules, in approximately 30-mg quantities. Samples were sent to 19 laboratories in 10 countries. In all, 185 assays were carried out, 122 for oxytocic activity, 53 for vasopressor activity and 10 for antidiuretic activity. On the basis of the results, which were analysed statistically at the National Institute for Medical Research, it was agreed that the potency of the Third Standard (re-named International Standard for Oxytocic, Vasopressor and Antidiuretic Substances in 1956, in view of the recent synthesis of oxytocin and vasopressin) should be expressed as 2.0 International Units per milligram. The International Unit therefore remains unchanged as 0.5 mg of the dry powder. PMID:13585079
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A practical method for extending the biuret assay to protein determination of corn-based products.
Liu, Zelong; Pan, Junhui
2017-06-01
A modified biuret method suitable for protein determination of corn-based products was developed by introducing a combination of an alkaline reagent with sodium dodecyl sulfate (reagent A) and heat treatments. The method was tested on seven corn-based samples. The results showed mostly good agreement (P>0.05) as compared to the Kjeldahl values. The proposed method was found to enhance the accuracy of prediction on zein content using bovine serum albumin as standard. Reagent A and sample treatment were proved to effectively improve protein solubilization for the thermally-dried corn-based products, e.g. corn gluten meal. The absorbance was stable for at least 1-h. Moreover, the whole measurement of protein content only needs 15-20min more than the traditional biuret assay, and can be performed in batches. The findings suggest that the proposed method could be a timesaving alternative for routine protein analyses in corn processing factories. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Gómez-Sala, Beatriz; Herranz, Carmen; Díaz-Freitas, Belén; Hernández, Pablo E; Sala, Ana; Cintas, Luis M
2016-04-16
In this work we describe the development of a biopreservation strategy for fresh fish based on the use of bacteriocinogenic LAB of marine origin. For this purpose, two multibacteriocinogenic LAB strains, Lactobacillus curvatus BCS35 and Enterococcus faecium BNM58, previously isolated from fish and fish products were selected owing to their capability to inhibit the growth of several fish-spoilage and food-borne pathogenic bacteria. Two commercially important fish species were chosen, young hake (Merluccius merluccius) and megrim (Lepidorhombus boscii), and the specimens were acquired at the Marín (Pontevedra, Spain) retail fish market, after one night in the chilled hold of a near-shore fishing vessel. The biopreservation potential and the application strategies of these two LAB strains were first tested at a laboratory scale, where several batches of fresh fish were inoculated with: (i) the multibacteriocinogenic LAB culture(s) as protective culture(s); and/or (ii) their cell-free culture supernatant(s) as food ingredient(s), and (iii) the lyophilized bacteriocin preparation(s) as lyophilized food ingredient(s). All batches were stored in polystyrene boxes, permanently filled with ice at 0-2 °C, for 14 days. Microbiological analyses, as well as sensorial analyses, were carried out during the biopreservation trials. Subsequently, Lb. curvatus BCS35 was selected to up-scale the trials, and combinations of the three application methods were assayed. For this purpose, this strain was grown in a semi-industrial scale fermentor (150l) in modified MRS broth, and three batches of fresh fish were inoculated with the protective culture and/or food ingredient, and stored on ice in a chilled chamber at 0-2 °C at the Marín retail fish market for 14 days. Microbiological analyses were carried out during the storage period, showing that when Lb. curvatus BCS35 culture or the corresponding cell-free culture supernatant was used as protective culture or food ingredient, respectively, bacterial counts were significantly lower than those of the untreated control batches, both for young hake and megrim. In addition, the presence of Listeria spp. in megrim was inhibited in both analyses. The effect of protective culture or food ingredient on the sensory characteristics of fish was evaluated by an official fish appraiser from the Marín retail fish market, who concluded that all the biopreserved batches were worth a higher price in the fish market than the respective control batches, demonstrating that the multibacteriocinogenic strain of marine origin Lb. curvatus BCS35 may be considered as a suitable candidate for its application as fresh fish biopreservative. Copyright © 2016 Elsevier B.V. All rights reserved.
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Reactivity and dynamics of H2S, NO, and O2 interacting with hemoglobins from Lucina pectinata.
Ramos-Alvarez, Cacimar; Yoo, Byung-Kuk; Pietri, Ruth; Lamarre, Isabelle; Martin, Jean-Louis; Lopez-Garriga, Juan; Negrerie, Michel
2013-10-08
Hemoglobin HbI from the clam Lucina pectinata is involved in H2S transport, whereas homologous heme protein HbII/III is involved in O2 metabolism. Despite similar tertiary structures, HbI and HbII/III exhibit very different reactivity toward heme ligands H2S, O2, and NO. To investigate this reactivity at the heme level, we measured the dynamics of ligand interaction by time-resolved absorption spectroscopy in the picosecond to nanosecond time range. We demonstrated that H2S can be photodissociated from both ferric and ferrous HbI. H2S geminately rebinds to ferric and ferrous out-of-plane iron with time constants (τgem) of 12 and 165 ps, respectively, with very different proportions of photodissociated H2S exiting the protein (24% in ferric and 80% in ferrous HbI). The Gln(E7)His mutation considerably changes H2S dynamics in ferric HbI, indicating the role of Gln(E7) in controling H2S reactivity. In ferric HbI, the rate of diffusion of H2S from the solvent into the heme pocket (kentry) is 0.30 μM(-1) s(-1). For the HbII/III-O2 complex, we observed mainly a six-coordinate vibrationally excited heme-O2 complex with O2 still bound to the iron. This explains the low yield of O2 photodissociation and low koff from HbII/III, compared with those of HbI and Mb. Both isoforms behave very differently with regard to NO and O2 dynamics. Whereas the amplitude of geminate rebinding of O2 to HbI (38.5%) is similar to that of myoglobin (34.5%) in spite of different distal heme sites, it appears to be much larger for HbII/III (77%). The distal Tyr(B10) side chain present in HbII/III increases the energy barrier for ligand escape and participates in the stabilization of bound O2 and NO.
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A Novel Biomimetic Tool for Assessing Vitamin K Status Based on Molecularly Imprinted Polymers.
Eersels, Kasper; Diliën, Hanne; Lowdon, Joseph W; Steen Redeker, Erik; Rogosic, Renato; Heidt, Benjamin; Peeters, Marloes; Cornelis, Peter; Lux, Petra; Reutelingsperger, Chris P; Schurgers, Leon J; Cleij, Thomas J; van Grinsven, Bart
2018-06-11
Vitamin K was originally discovered as a cofactor required to activate clotting factors and has recently been shown to play a key role in the regulation of soft tissue calcification. This property of vitamin K has led to an increased interest in novel methods for accurate vitamin K detection. Molecularly Imprinted Polymers (MIPs) could offer a solution, as they have been used as synthetic receptors in a large variety of biomimetic sensors for the detection of similar molecules over the past few decades, because of their robust nature and remarkable selectivity. In this article, the authors introduce a novel imprinting approach to create a MIP that is able to selectively rebind vitamin K₁. As the native structure of the vitamin does not allow for imprinting, an alternative imprinting strategy was developed, using the synthetic compound menadione (vitamin K₃) as a template. Target rebinding was analyzed by means of UV-visible (UV-VIS) spectroscopy and two custom-made thermal readout techniques. This analysis reveals that the MIP-based sensor reacts to an increasing concentration of both menadione and vitamin K₁. The Limit of Detection (LoD) for both compounds was established at 700 nM for the Heat Transfer Method (HTM), while the optimized readout approach, Thermal Wave Transport Analysis (TWTA), displayed an increased sensitivity with a LoD of 200 nM. The sensor seems to react to a lesser extent to Vitamin E, the analogue under study. To further demonstrate its potential application in biochemical research, the sensor was used to measure the absorption of vitamin K in blood serum after taking vitamin K supplements. By employing a gradual enrichment strategy, the sensor was able to detect the difference between baseline and peak absorption samples and was able to quantify the vitamin K concentration in good agreement with a validation experiment using High-Performance Liquid Chromatography (HPLC). In this way, the authors provide a first proof of principle for a low-cost, straightforward, and label-free vitamin K sensor.
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Kong, Xuan; Gao, Ruixia; He, Xiwen; Chen, Langxing; Zhang, Yukui
2012-07-06
In this study, we present a general method to prepare the core-shell magnetic molecularly imprinted polymers (MIPs) nanoparticles (NPs) for sulfamethazine (SMZ). The resulting Fe₃O₄@MIPs NPs possess a highly improved imprinting effect, fast adsorption kinetics and high adsorption capacity, and can be applied to extract sulfonamide in the poultry feed. In this protocol, the magnetite NPs were synthesized by co-precipitating Fe²⁺ and Fe³⁺ in an ammonia solution first. Silica was then coated on the Fe₃O₄ NPs using a sol-gel method to obtain silica shell magnetic NPs. Subsequently, the vinyl groups were grated onto silica-modified Fe₃O₄ surface by 3-methacryloyloxypropyltrimethoxysilane. Finally, the MIPs films were formed on the surface of Fe₃O₄@SiO₂ by the copolymerization of vinyl end groups with functional monomer, methacrylic acid, cross-linking agent, ethylene glycol dimethacrylate, the initiator azo-bis-isobutyronitrile and template molecule, sulfamethazine. The morphology, magnetic, adsorption and recognition properties of Fe₃O₄@MIPs NPs were characterized using transmission electron microscope (TEM), scanning electron microscope (SEM), Fourier transform infrared (FT-IR) spectrometer, vibrating sample magnetometer (VSM) and re-binding experiments. The results showed that the binding sites of Fe₃O₄@MIPs were good accessibility, fast adsorption rate and the maximum adsorption capacity of Fe₃O₄@MIPs to SMZ was 344.8 μg g⁻¹. The selectivity of the obtained Fe₃O₄@MIPs NPs were elucidated by the different rebinding capability of SMZ and structural related sulfonamides in the mixed solution. The results indicated that the Fe₃O₄@MIPs had high imprinting factor 9.5 and significant selectivity. A method was developed for enrichment and determination of SMZ in the poultry feed samples with recoveries of duck and chicken feed ranging from 63.3 to 76.5% and 68.7 to 74.7%, respectively and the relative standard deviations (RSD) (<6.7%). Copyright © 2012 Elsevier B.V. All rights reserved.
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Ramos, Inês I; Gregório, Bruno J R; Barreiros, Luísa; Magalhães, Luís M; Tóth, Ildikó V; Reis, Salette; Lima, José L F C; Segundo, Marcela A
2016-04-01
An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540 nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5 µM of PGR and 125 mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90 ± 5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8 μM. RSD values were <3.4 and <4.9%, for intra and inter-assay precision, respectively. Compared to previous batch automated ORAC assays, the developed system also accounted for high sampling frequency (29 h(-1)), low operating costs and low generation of waste. Copyright © 2015 Elsevier B.V. All rights reserved.
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Valero-Navarro, Angel; Gómez-Romero, María; Fernández-Sánchez, Jorge F; Cormack, Peter A G; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto
2011-10-14
In the current work, a molecularly imprinted polymer (MIP) has been synthesised and used to enable the extraction of a naturally-occurring antioxidant from complex media. More specifically, we describe the first example of a caffeic acid (CA) MIP which has been synthesised in the form of well-defined polymer microspheres, and its use for the extraction of CA from fruit juice sample. The CA MIP was synthesised by precipitation polymerisation using 4-vinylpyridine as functional monomer, divinylbenzene-80 as crosslinker and acetonitrile:toluene (75/25, v/v) as porogen. The particle sizing and morphological characterisation of the polymers was carried out by means of scanning electron microscopy (narrow particle size distribution; ∼5 and 1.5 μm particle diameters for the MIP and NIP [non-imprinted polymer], respectively) and nitrogen sorption porosimetry (specific surface areas of 340 and 350 m(2)g(-1), and specific pore volumes of 0.17 and 0.19 cm(3)g(-1) for the MIP and NIP, respectively). The polymers were evaluated further by batch rebinding experiments, and from the derived isotherms their binding capacity and binding strength were determined (number of binding sites (N(K))=0.6 and 0.3 mmol g(-1) for the MIP and NIP, respectively, and apparent average adsorption constant (K(N))=10.0 and 1.6L mmol(-1) for the MIP and NIP, respectively). To evaluate the molecular recognition character of the MIP it was packed into a stainless steel column (50 mm × 4.6 mm i.d.) and evaluated as an HPLC-stationary phase. The mobile phase composition, flow rate, and the elution profile were then optimised in order to improve the peak shape without negatively affecting the imprinting factor (IF). Very interesting, promising properties were revealed. The imprinting factor (IF) under the optimised conditions was 11.9. Finally, when the imprinted LC column was used for the selective recognition of CA over eight related compounds, very good selectivity was obtained. This outcome enabled the direct extraction of CA in commercial apple juice samples with recoveries in excess of 81% and, rather significantly, without any need for a clean-up step prior to the extraction. Copyright © 2011 Elsevier B.V. All rights reserved.
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Pizarro, Shelly A; Dinges, Rachel; Adams, Rachel; Sanchez, Ailen; Winter, Charles
2009-10-01
Process analytical technology (PAT) is an initiative from the US FDA combining analytical and statistical tools to improve manufacturing operations and ensure regulatory compliance. This work describes the use of a continuous monitoring system for a protein refolding reaction to provide consistency in product quality and process performance across batches. A small-scale bioreactor (3 L) is used to understand the impact of aeration for refolding recombinant human vascular endothelial growth factor (rhVEGF) in a reducing environment. A reverse-phase HPLC assay is used to assess product quality. The goal in understanding the oxygen needs of the reaction and its impact to quality, is to make a product that is efficiently refolded to its native and active form with minimum oxidative degradation from batch to batch. Because this refolding process is heavily dependent on oxygen, the % dissolved oxygen (DO) profile is explored as a PAT tool to regulate process performance at commercial manufacturing scale. A dynamic gassing out approach using constant mass transfer (k(L)a) is used for scale-up of the aeration parameters to manufacturing scale tanks (2,000 L, 15,000 L). The resulting DO profiles of the refolding reaction show similar trends across scales and these are analyzed using rpHPLC. The desired product quality attributes are then achieved through alternating air and nitrogen sparging triggered by changes in the monitored DO profile. This approach mitigates the impact of differences in equipment or feedstock components between runs, and is directly inline with the key goal of PAT to "actively manage process variability using a knowledge-based approach." (c) 2009 Wiley Periodicals, Inc.
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de Mattos, Katherine Antunes; Navega, Elaine Cristina Azevedo; Silva, Vitor Fernandes; Almeida, Alessandra Santos; da Silva, Cristiane Caldeira; Presgrave, Octavio Augusto França; Junior, Daniel da Silva Guedes; Delgado, Isabella Fernandes
2018-03-01
The need for alternatives to animal use in pyrogen testing has been driven by the Three Rs concept. This has resulted in the inclusion of the monocyte activation test (MAT) in the European Pharmacopoeia, 2010. However, some technical and regulatory obstacles must be overcome to ensure the effective implementation of the MAT by the industry, especially for the testing of biological products. The yellow fever (YF) vaccine (17DD-YFV) was chosen for evaluation in this study, in view of: a) the 2016-2018 outbreak of YF in Brazil; b) the increase in demand for 17DD-YFV doses; c) the complex production process with live attenuated virus; d) the presence of possible test interference factors, such as residual process components (e.g. ovalbumin); and e) the need for the investigation of other pyrogens that are not detectable by the methods prescribed in the YF vaccine monograph. The product-specific testing was carried out by using cryopreserved and fresh whole blood, and IL-6 and IL-1β levels were used as the marker readouts. After assessing the applicability of the MAT on a 1:10 dilution of 17DD-YFV, endotoxin and non-endotoxin pyrogens were quantified in spiked batches, by using the lipopolysaccharide and lipoteichoic acid standards, respectively. The quantitative analysis demonstrated the correlation between the MAT and the Limulus amoebocyte lysate (LAL) assays, with respect to the limits of endotoxin recovery in spiked batches and the detection of no pyrogenic contamination in commercial batches of 17DD-YFV. The data demonstrated the applicability of the MAT for 17DD-YFV pyrogen testing, and as an alternative method that can contribute to biological quality control studies. 2018 FRAME.
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High acetone-butanol-ethanol production in pH-stat co-feeding of acetate and glucose.
Gao, Ming; Tashiro, Yukihiro; Wang, Qunhui; Sakai, Kenji; Sonomoto, Kenji
2016-08-01
We previously reported the metabolic analysis of butanol and acetone production from exogenous acetate by (13)C tracer experiments (Gao et al., RSC Adv., 5, 8486-8495, 2015). To clarify the influence of acetate on acetone-butanol-ethanol (ABE) production, we first performed an enzyme assay in Clostridium saccharoperbutylacetonicum N1-4. Acetate addition was found to drastically increase the activities of key enzymes involved in the acetate uptake (phosphate acetyltransferase and CoA transferase), acetone formation (acetoacetate decarboxylase), and butanol formation (butanol dehydrogenase) pathways. Subsequently, supplementation of acetate during acidogenesis and early solventogenesis resulted in a significant increase in ABE production. To establish an efficient ABE production system using acetate as a co-substrate, several shot strategies were investigated in batch culture. Batch cultures with two substrate shots without pH control produced 14.20 g/L butanol and 23.27 g/L ABE with a maximum specific butanol production rate of 0.26 g/(g h). Furthermore, pH-controlled (at pH 5.5) batch cultures with two substrate shots resulted in not only improved acetate consumption but also a further increase in ABE production. Finally, we obtained 15.13 g/L butanol and 24.37 g/L ABE at the high specific butanol production rate of 0.34 g/(g h) using pH-stat co-feeding method. Thus, in this study, we established a high ABE production system using glucose and acetate as co-substrates in a pH-stat co-feeding system with C. saccharoperbutylacetonicum N1-4. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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Biopharmaceutic Risk Assessment of Brand and Generic Lamotrigine Tablets.
Vaithianathan, Soundarya; Raman, Siddarth; Jiang, Wenlei; Ting, Tricia Y; Kane, Maureen A; Polli, James E
2015-07-06
The therapeutic equivalence of generic and brand name antiepileptic drugs has been questioned by neurologists and the epilepsy community. A potential contributor to such concerns is pharmaceutical quality. The objective was to assess the biopharmaceutic risk of brand name Lamictal 100 mg tablets and generic lamotrigine 100 mg tablets from several manufacturers. Lamotrigine was characterized in terms of the Biopharmaceutics Classification System (BCS), including aqueous solubility and Caco-2 permeability. A panel of pharmaceutical quality tests was also performed on three batches of Lamictal, three batches of Teva generic, and one batch of each of four other generics: appearance, identity, assay, impurity, uniformity of dosage units, disintegration, dissolution, friability, and loss on drying. These market surveillance results indicate that all brand name and generic lamotrigine 100 mg tablets passed all tests and showed acceptable pharmaceutical quality and low biopharmaceutic risk. Lamotrigine was classified as a BCS class IIb drug, exhibiting pH-dependent aqueous solubility and dissolution. At pH 1.2 and 4.5, lamotrigine exhibited high solubility, whereas lamotrigine exhibited low solubility at pH 6.8, including non-sink dissolution. Lamotrigine showed high Caco-2 permeability. The apparent permeability (Papp) of lamotrigine was (73.7 ± 8.7) × 10(-6) cm/s in the apical-to-basolateral (AP-BL) direction and (41.4 ± 1.6) × 10(-6) cm/s in the BL-AP direction, which were higher than metoprolol's AP-BL Papp of (21.2 ± 0.9) × 10(-6) cm/s and BL-AP Papp of (34.6 ± 4.6) × 10(-6) cm/s. Overall, lamotrigine's favorable biopharmaceutics from a drug substance perspective and favorable quality characteristics from a tablet formulation perspective suggest that multisource lamotrigine tablets exhibit a low biopharmaceutic risk.
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Validating a faster method for reconstitution of Crotalidae Polyvalent Immune Fab (ovine).
Gerring, David; King, Thomas R; Branton, Richard
2013-07-01
Reconstitution of CroFab(®) (Crotalidae Polyvalent Immune Fab [ovine]) lyophilized drug product was previously performed using 10 mL sterile water for injection followed by up to 36 min of gentle swirling of the vial. CroFab has been clinically demonstrated to be most effective when administered within 6 h of snake envenomation, and improved clinical outcomes are correlated with quicker timing of administration. An alternate reconstitution method was devised, using 18 mL 0.9% saline with manual inversion, with the goal of shortening reconstitution time while maintaining a high quality, efficacious product. An analytical study was designed to compare the physicochemical properties of 3 separate batches of CroFab when reconstituted using the standard procedure (10 mL WFI with gentle swirling) and a modified rapid procedure using 18 mL 0.9% saline and manual inversion. The physical and chemical characteristics of the same 3 batches were assessed using various analytic methodologies associated with routine quality control release testing. In addition further analytical methodologies were applied in order to elucidate possible structural changes that may be induced by the changed reconstitution procedure. Batches A, B, and C required mean reconstitution times of 25 min 51 s using the label method and 3 min 07 s (a 88.0% mean decrease) using the modified method. Physicochemical characteristics (color and clarity, pH, purity, protein content, potency) were found to be highly comparable. Characterization assays (dynamic light scattering, analytical ultracentrifugation, LC-MS, SDS-PAGE and circular dichroism spectroscopy were also all found to be comparable between methods. When comparing CroFab batches that were reconstituted using the labeled and modified methods, the physicochemical and biological (potency) characteristics of CroFab were not significantly changed when challenged by the various standard analytical methodologies applied in routine quality control analysis. Additionally, no changes in the CroFab molecule regarding degradation, aggregation, purity, structure, or mass were observed. The analyses performed validated the use of the more rapid reconstitution method using 18 mL 0.9% saline in order to allow a significantly reduced time to administration of CroFab to patients in need. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Synthesis and Adsorption Study of BSA Surface Imprinted Polymer on CdS Quantum Dots
NASA Astrophysics Data System (ADS)
Tang, Ping-ping; Cai, Ji-bao; Su, Qing-de
2010-04-01
A new bovine serum albumin (BSA) surface imprinting method was developed by the incorporation of quantum dots (QDs) into molecularly imprinted polymers (MIP), which can offer shape selectivity. Preparation and adsorption conditions were optimized. Physical appearance of the QDs and QDs-MIP particles was illustrated by scanning electron microscope images. Photoluminescence emission of CdS was quenched when rebinding of the template. The quenching of photoluminescence emissions is presumably due to the fluorescence resonance energy transfer between quantum dots and BSA template molecules. The adsorption is compiled with Langmuir isotherm, and chemical adsorption is the rate-controlling step. The maximum adsorption capacity could reach 226.0 mg/g, which is 142.4 mg/g larger than that of undoped BSA MIP. This study demonstrates the validity of QDs coupled with MIP technology for analyzing BSA.
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Immunological assays employed for the elucidation of an histoplasmosis outbreak in São Paulo, SP
Passos, Angela Noronha; Kohara, Valdelene Sayuri; de Freitas, Roseli Santos; Vicentini, Adriana Pardini
2014-01-01
Several reports showed outbreaks of histoplasmosis acquired while bat-inhabited caves were visited by tourists, miners or researchers. We evaluated the performance of double immunodifusion (DI) and immunoblotting (IB) assays, employed for the histoplasmosis outbreak elucidation occurred in Vale do Paraíba, São Paulo. The existence of epidemiologic link, four patients with clinical signs suggestive of histoplasmosis and mycological confirmation has made that all 35 individuals involved to the cave visit were subjected to serological evaluation. By DI, we observed reactivity against H. capsulatum antigen in a single serum examined nearly 20 days after exposure to fungal propagules. On the other hand, IB showed reactivity against H and M fractions in 50% of samples evaluated. The analysis of the second sample batch, collected two months after the exposure showed that 96.7% were reactive by DI with antibodies titers ranging from 1 to 16 and 100% of reactivity against H and M fractions, by IB, suggesting an acute infection. The analysis of the overall agreement between the methods showed to be reasonable (κ = 0.37). This study confirms the importance and efficacy of more sensitive methodologies, such as IB assay, to early elucidation of disease, especially in cases of patients without mycological information. PMID:25763041
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Immunological assays employed for the elucidation of an histoplasmosis outbreak in São Paulo, SP.
Passos, Angela Noronha; Kohara, Valdelene Sayuri; de Freitas, Roseli Santos; Vicentini, Adriana Pardini
2014-01-01
Several reports showed outbreaks of histoplasmosis acquired while bat-inhabited caves were visited by tourists, miners or researchers. We evaluated the performance of double immunodifusion (DI) and immunoblotting (IB) assays, employed for the histoplasmosis outbreak elucidation occurred in Vale do Paraíba, São Paulo. The existence of epidemiologic link, four patients with clinical signs suggestive of histoplasmosis and mycological confirmation has made that all 35 individuals involved to the cave visit were subjected to serological evaluation. By DI, we observed reactivity against H. capsulatum antigen in a single serum examined nearly 20 days after exposure to fungal propagules. On the other hand, IB showed reactivity against H and M fractions in 50% of samples evaluated. The analysis of the second sample batch, collected two months after the exposure showed that 96.7% were reactive by DI with antibodies titers ranging from 1 to 16 and 100% of reactivity against H and M fractions, by IB, suggesting an acute infection. The analysis of the overall agreement between the methods showed to be reasonable (κ = 0.37). This study confirms the importance and efficacy of more sensitive methodologies, such as IB assay, to early elucidation of disease, especially in cases of patients without mycological information.
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Yao, Chunhe; Carlisi, Cristina; Li, Yuning; Chen, Da; Ding, Jianfu; Feng, Yong-Lai
2016-01-01
Increasing use of single-walled carbon nanotubes (SWCNTs) necessitates a novel method for hazard risk assessment. In this work, we investigated the interaction of several types of commercial SWCNTs with single-stranded (ss) and double-stranded (ds) DNA oligonucleotides (20-mer and 20 bp). Based on the results achieved, we proposed a novel assay that employed the DNA interaction potency to assess the hazard risk of SWCNTs. It was found that SWCNTs in different sizes or different batches of the same product number of SWCNTs showed dramatically different potency of interaction with DNAs. In addition, the same SWCNTs also exerted strikingly different interaction potency with ss- versus ds- DNAs. The interaction rates of SWCNTs with DNAs were investigated, which could be utilized as the indicator of potential hazard for acute exposure. Compared to solid SWCNTs, the SWCNTs dispersed in liquid medium (2% sodium cholate solution) exhibited dramatically different interaction potency with DNAs. This indicates that the exposure medium may greatly influence the subsequent toxicity and hazard risk produced by SWCNTs. Based on the findings of dose-dependences and time-dependences from the interactions between SWCNTs and DNAs, a new chemistry based assay for hazard risk assessment of nanomaterials including SWCNTs has been presented. PMID:27936089
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Yao, Chunhe; Carlisi, Cristina; Li, Yuning; Chen, Da; Ding, Jianfu; Feng, Yong-Lai
2016-01-01
Increasing use of single-walled carbon nanotubes (SWCNTs) necessitates a novel method for hazard risk assessment. In this work, we investigated the interaction of several types of commercial SWCNTs with single-stranded (ss) and double-stranded (ds) DNA oligonucleotides (20-mer and 20 bp). Based on the results achieved, we proposed a novel assay that employed the DNA interaction potency to assess the hazard risk of SWCNTs. It was found that SWCNTs in different sizes or different batches of the same product number of SWCNTs showed dramatically different potency of interaction with DNAs. In addition, the same SWCNTs also exerted strikingly different interaction potency with ss- versus ds- DNAs. The interaction rates of SWCNTs with DNAs were investigated, which could be utilized as the indicator of potential hazard for acute exposure. Compared to solid SWCNTs, the SWCNTs dispersed in liquid medium (2% sodium cholate solution) exhibited dramatically different interaction potency with DNAs. This indicates that the exposure medium may greatly influence the subsequent toxicity and hazard risk produced by SWCNTs. Based on the findings of dose-dependences and time-dependences from the interactions between SWCNTs and DNAs, a new chemistry based assay for hazard risk assessment of nanomaterials including SWCNTs has been presented.
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Mano, Yuji; Takenaka, Osamu; Kusano, Kazutomi
2015-10-01
Perampanel (Fycompa®), a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, is registered for the adjunctive treatment of patients (aged ≥12 years) with refractory partial-onset seizures. To support therapeutic drug monitoring, a simple high-performance liquid chromatography (HPLC) assay with fluorescence detection was developed to determine perampanel concentrations in human plasma and validated to support clinical trials. Human plasma samples (1.0 mL) were processed by liquid extraction using diethyl ether, followed by chromatographic separation on a YMC Pack Pro C18 column (150 × 4.6 mm i.d., 5 µm) with isocratic elution of acetonitrile-water-acetic acid-sodium acetate (840:560:3:1.8, v/v/v/w) at a flow rate of 1.0 mL/min. Column eluent was monitored at excitation and emission wavelengths of 290 and 430 nm, respectively. The assay was linear (range 1.0-500 ng/mL) and this could be extended to 25 µg/mL by 50-fold dilution integrity. No endogenous peaks were detected in the elution of analytes in drug-free blank human plasma from six individuals and no interference was observed with co-medications tested. Intra- and inter-batch reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. Validation data demonstrated that our assay is simple, selective, reproducible and suitable for therapeutic drug monitoring of perampanel. Copyright © 2015 John Wiley & Sons, Ltd.
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Analytical characterization of ch14.18
Kallarakal, Abraham T; Michiel, Dennis; Yang, Xiaoyi; Saptharishi, Nirmala; Jiang, Hengguang; Giardina, Steve; Gilly, John; Mitra, George
2012-01-01
Ch14.18 is a mouse-human chimeric monoclonal antibody to the disialoganglioside (GD2) glycolipid. In the clinic, this antibody has been shown to be effective in the treatment of children with high-risk neuroblastoma, either alone or in combination therapy. Extensive product characterization is a prerequisite to addressing the potential issues of product variability associated with process changes and manufacturing scale-up. Charge heterogeneity, glycosylation profile, molecular state and aggregation, interaction (affinity) with Fcγ receptors and functional or biological activities are a few of the critical characterization assays for assessing product comparability for this antibody. In this article, we describe the in-house development and qualification of imaged capillary isoelectric focusing to assess charge heterogeneity, analytical size exclusion chromatography with online static and dynamic light scattering (DLS), batch mode DLS for aggregate detection, biosensor (surface plasmon resonance)-based Fcγ receptor antibody interaction kinetics, N-glycoprofiling with PNGase F digestion, 2-aminobenzoic acid labeling and high performance liquid chromatography and N-glycan analysis using capillary electrophoresis. In addition, we studied selected biological activity assays, such as complement-dependent cytotoxicity. The consistency and reproducibility of the assays are established by comparing the intra-day and inter-day assay results. Applications of the methodologies to address stability or changes in product characteristics are also reported. The study results reveal that the ch14.18 clinical product formulated in phosphate-buffered saline at a concentration of 5 mg/ml and stored at 2–8°C is stable for more than five years. PMID:22327432
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A one-step multiplex RT-PCR assay for simultaneous detection of four viruses that infect peach.
Yu, Y; Zhao, Z; Jiang, D; Wu, Z; Li, S
2013-10-01
A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed to enable the simultaneous detection and differentiation of four viruses that infect peach, namely Apple chlorotic leaf spot virus (ACLSV), Cherry green ring mottle virus (CGRMV), Prunus necrotic ringspot virus (PNRSV) and Apricot pseudo-chlorotic leaf spot virus (APCLSV). In this study, four pairs of primers, one specific for each virus, were designed; the corresponding PCR products were 632, 439, 346 and 282 bp in length for ACLSV, CGRMV, PNRSV and APCLSV, respectively, and the fragments could be distinguished clearly by agarose gel electrophoresis. The sensitivity and specificity of the method were tested using individual RT-PCR and enzyme-linked immunosorbent assay (ELISA), and the identity of the RT-PCR amplification products was also confirmed by DNA sequencing. The results of RT-PCR and ELISA, along with batch detection using samples collected from peach orchards, revealed that this rapid and simple technique is an effective way to identify the four viruses simultaneously. The mRT-PCR assay described in this study was developed for the simultaneous detection of four peach viruses from infected peach samples is reliable and sensitive. In contrast to conventional uniplex RT-PCR, mRT-PCR is more efficient, reducing costs, time and handling when testing large numbers of samples. This rapid and simple method is useful for large-scale surveys of viruses that infect peach. © 2013 The Society for Applied Microbiology.
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New spectrophotometric assay for pilocarpine.
El-Masry, S; Soliman, R
1980-07-01
A quick method for the determination of pilocarpine in eye drops in the presence of decomposition products is described. The method involves complexation of the alkaloid with bromocresol purple at pH 6. After treatment with 0.1N NaOH, the liberated dye is measured at 580 nm. The method has a relative standard deviation of 1.99%, and has been successfully applied to the analysis of 2 batches of pilocarpine eye drops. The recommended method was also used to monitor the stability of a pilocarpine nitrate solution in 0.05N NaOH at 65 degrees C. The BPC method failed to detect any significant decomposition after 2 h incubation, but the recommended method revealed 87.5% decomposition.
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Medication Waste Reduction in Pediatric Pharmacy Batch Processes
Veltri, Michael A.; Hamrock, Eric; Mollenkopf, Nicole L.; Holt, Kristen; Levin, Scott
2014-01-01
OBJECTIVES: To inform pediatric cart-fill batch scheduling for reductions in pharmaceutical waste using a case study and simulation analysis. METHODS: A pre and post intervention and simulation analysis was conducted during 3 months at a 205-bed children's center. An algorithm was developed to detect wasted medication based on time-stamped computerized provider order entry information. The algorithm was used to quantify pharmaceutical waste and associated costs for both preintervention (1 batch per day) and postintervention (3 batches per day) schedules. Further, simulation was used to systematically test 108 batch schedules outlining general characteristics that have an impact on the likelihood for waste. RESULTS: Switching from a 1-batch-per-day to a 3-batch-per-day schedule resulted in a 31.3% decrease in pharmaceutical waste (28.7% to 19.7%) and annual cost savings of $183,380. Simulation results demonstrate how increasing batch frequency facilitates a more just-in-time process that reduces waste. The most substantial gains are realized by shifting from a schedule of 1 batch per day to at least 2 batches per day. The simulation exhibits how waste reduction is also achievable by avoiding batch preparation during daily time periods where medication administration or medication discontinuations are frequent. Last, the simulation was used to show how reducing batch preparation time per batch provides some, albeit minimal, opportunity to decrease waste. CONCLUSIONS: The case study and simulation analysis demonstrate characteristics of batch scheduling that may support pediatric pharmacy managers in redesign toward minimizing pharmaceutical waste. PMID:25024671
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Medication waste reduction in pediatric pharmacy batch processes.
Toerper, Matthew F; Veltri, Michael A; Hamrock, Eric; Mollenkopf, Nicole L; Holt, Kristen; Levin, Scott
2014-04-01
To inform pediatric cart-fill batch scheduling for reductions in pharmaceutical waste using a case study and simulation analysis. A pre and post intervention and simulation analysis was conducted during 3 months at a 205-bed children's center. An algorithm was developed to detect wasted medication based on time-stamped computerized provider order entry information. The algorithm was used to quantify pharmaceutical waste and associated costs for both preintervention (1 batch per day) and postintervention (3 batches per day) schedules. Further, simulation was used to systematically test 108 batch schedules outlining general characteristics that have an impact on the likelihood for waste. Switching from a 1-batch-per-day to a 3-batch-per-day schedule resulted in a 31.3% decrease in pharmaceutical waste (28.7% to 19.7%) and annual cost savings of $183,380. Simulation results demonstrate how increasing batch frequency facilitates a more just-in-time process that reduces waste. The most substantial gains are realized by shifting from a schedule of 1 batch per day to at least 2 batches per day. The simulation exhibits how waste reduction is also achievable by avoiding batch preparation during daily time periods where medication administration or medication discontinuations are frequent. Last, the simulation was used to show how reducing batch preparation time per batch provides some, albeit minimal, opportunity to decrease waste. The case study and simulation analysis demonstrate characteristics of batch scheduling that may support pediatric pharmacy managers in redesign toward minimizing pharmaceutical waste.
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Chemical and Toxicological Fate of Fumonisin B1 during Extrusion Processing of Corn Grits
USDA-ARS?s Scientific Manuscript database
Two batches of flaking corn grits were prepared by growing Fusarium verticillioides to contain low and high levels of fumonisin B1 (FB1), Batch-1 at 9.7 ppm and Batch-2 at 50 ppm FB1 as determined by HPLC. These two batches were extruded (Batch-1E; Batch-2E) or extruded with 10% w/w glucose supplem...
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Burggraeve, A; Van den Kerkhof, T; Hellings, M; Remon, J P; Vervaet, C; De Beer, T
2011-04-18
Fluid bed granulation is a batch process, which is characterized by the processing of raw materials for a predefined period of time, consisting of a fixed spraying phase and a subsequent drying period. The present study shows the multivariate statistical modeling and control of a fluid bed granulation process based on in-line particle size distribution (PSD) measurements (using spatial filter velocimetry) combined with continuous product temperature registration using a partial least squares (PLS) approach. Via the continuous in-line monitoring of the PSD and product temperature during granulation of various reference batches, a statistical batch model was developed allowing the real-time evaluation and acceptance or rejection of future batches. Continuously monitored PSD and product temperature process data of 10 reference batches (X-data) were used to develop a reference batch PLS model, regressing the X-data versus the batch process time (Y-data). Two PLS components captured 98.8% of the variation in the X-data block. Score control charts in which the average batch trajectory and upper and lower control limits are displayed were developed. Next, these control charts were used to monitor 4 new test batches in real-time and to immediately detect any deviations from the expected batch trajectory. By real-time evaluation of new batches using the developed control charts and by computation of contribution plots of deviating process behavior at a certain time point, batch losses or reprocessing can be prevented. Immediately after batch completion, all PSD and product temperature information (i.e., a batch progress fingerprint) was used to estimate some granule properties (density and flowability) at an early stage, which can improve batch release time. Individual PLS models relating the computed scores (X) of the reference PLS model (based on the 10 reference batches) and the density, respectively, flowabililty as Y-matrix, were developed. The scores of the 4 test batches were used to examine the predictive ability of the model. Copyright © 2011 Elsevier B.V. All rights reserved.
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Physiology and biochemistry of a lignin degrading bacterium Erwinia sp. Cu 3614
DOE Office of Scientific and Technical Information (OSTI.GOV)
Rajan, J.S.
1992-01-01
Previous researchers have reported the isolation of a diphenylether cleaving organism, Erwinia sp., using an enrichment medium containing lignin. A copper and dinitrophenol resistant mutant of this organism, Erwinia sp. Cu3614, has also been reported. To assess the effect of copper on the growth and biochemistry of this organism, continuous cultivation was used employing an apparently optimized medium containing ethanol as carbon source. Upon increasing the concentration of copper sulfate in the medium from 5 [mu]g/ml to 10 [mu]g/ml increases in maximum specific growth rate and growth yield were seen. Also decrease in the values for doubling time and themore » coefficient for maintenance energy were seen. At higher levels of copper sulfate a [open quotes]non competitive[close quotes] inhibition of growth was seen as indicated by the values calculated for substrate affinity constant, and inhibition constant. To assess this organism's ligninolytic ability, an assay for residual lignin was developed. The assay measured a reaction between diazotized sulfanilic acid and lignin in alkaline solution by spectrophotometric monitoring of the resulting adduct. Use of this technique indicated that Erwinia sp. Cu3614 could degrade up to 80% of lignin in batch cultures. Further evidence about the ligninolytic ability of this organism was provided by examination of electron micrographs of lignocellulosic substrates incubated with cell suspensions. An assay for monitoring diphenylether cleaving abilities was also developed using resazurin, a redox dye. In vivo assays with cells obtained from continuous culture studies indicated a linear relationship between the rates of reaction with resazurin and the amount of copper associated with cells. In vitro assays, employing cell free extracts and resazurin, were used to obtain a fraction enriched in diphenylether cleaving activity by a heat treatment procedure.« less
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Papadopoulou, Evanthia; Goodchild, Sarah A; Cleary, David W; Weller, Simon A; Gale, Nittaya; Stubberfield, Michael R; Brown, Tom; Bartlett, Philip N
2015-02-03
The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.
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Potency assays for therapeutic live whole cell cancer vaccines.
Petricciani, John; Egan, William; Vicari, Giuseppe; Furesz, John; Schild, Geoffrey
2007-04-01
Therapeutic cancer vaccines are under development with the goal of enhancing the body's immune response to cancer cells sufficient to arrest cancer cell growth. Among the various approaches being used are those based on whole tumor cells. Developing a suitable measure of the potency of such vaccines presents a significant challenge because neither cellular associated markers nor in vivo biological responses that are correlated with efficacy have been identified; nevertheless, manufacturers and regulatory agencies will need to develop methods to evaluate these products. At this moment, the challenge for manufacturers who are developing whole cell vaccines is to demonstrate batch-to-batch consistency for the vaccine used in clinical studies and to show that comparable vaccine batches have the same capacity to achieve an acceptable level of biological activity that may be related to efficacy. This is particularly challenging in that animal models to test that activity do not exist and direct serological or immunological correlates of clinical protection are not available because protection has not yet been established in clinical trials. In the absence of well-defined biological markers and tests for manufacturing consistency, manufacturers and regulators will need to rely heavily on a highly reproducible manufacturing process--the consistency of the process therefore becomes critical. In developing regulatory approaches to whole cell cancer vaccines, the experience from the field of infectious disease vaccines should be examined for general guidance. A framework that draws heavily on the field of infectious disease vaccines is presented and suggests that at this point in the development of this new class of products, it is reasonable to develop data on quantitative antigen expression as a measure of potency with the expectation that when clinical efficacy has been established it will confirm the appropriateness of this approach. But because this will not be known until the end of a pivotal trial, a bioassay should be considered and run in parallel. Several examples of bioassays are presented along with their advantages and disadvantages. The final selection of a potency assay for use in lot release of a commercializable therapeutic whole cell vaccine ultimately will depend on the totality of the data available at the time of approval by regulatory agencies. Based on information currently available, it is likely that quantitative antigen expression or a bioassay could be used to measure potency. If both are determined to be acceptable, the use of quantitative antigen expression could be considered for routine lot release, while the bioassay could be reserved for use as one of the elements in establishing comparability when manufacturing changes are being considered after approval.
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Nygaard, Vegard; Rødland, Einar Andreas; Hovig, Eivind
2016-01-01
Removal of, or adjustment for, batch effects or center differences is generally required when such effects are present in data. In particular, when preparing microarray gene expression data from multiple cohorts, array platforms, or batches for later analyses, batch effects can have confounding effects, inducing spurious differences between study groups. Many methods and tools exist for removing batch effects from data. However, when study groups are not evenly distributed across batches, actual group differences may induce apparent batch differences, in which case batch adjustments may bias, usually deflate, group differences. Some tools therefore have the option of preserving the difference between study groups, e.g. using a two-way ANOVA model to simultaneously estimate both group and batch effects. Unfortunately, this approach may systematically induce incorrect group differences in downstream analyses when groups are distributed between the batches in an unbalanced manner. The scientific community seems to be largely unaware of how this approach may lead to false discoveries. PMID:26272994
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Co-digestion of pig slaughterhouse waste with sewage sludge.
Borowski, Sebastian; Kubacki, Przemysław
2015-06-01
Slaughterhouse wastes (SHW) are potentially very attractive substrates for biogas production. However, mono-digestion of these wastes creates great technological problems associated with the inhibitory effects of ammonia and fatty acids on methanogens as well as with the foaming in the digesters. In the following study, the co-digestion of slaughterhouse wastes with sewage sludge (SS) was undertaken. Batch and semi-continuous experiments were performed at 35°C with municipal sewage sludge and pig SHW composed of meat tissue, intestines, bristles and post-flotation sludge. In batch assays, meat tissue and intestinal wastes gave the highest methane productions of 976 and 826 dm(3)/kg VS, respectively, whereas the methane yield from the sludge was only 370 dm(3)/kg VS. The co-digestion of sewage sludge with 50% SHW (weight basis) provided the methane yield exceeding 600 dm(3)/kg VS, which was more than twice as high as the methane production from sewage sludge alone. However, when the loading rate exceeded 4 kg VS/m(3) d, a slight inhibition of methanogenesis was observed, without affecting the digester stability. The experiments showed that the co-digestion of sewage sludge with large amount of slaughterhouse wastes is feasible, and the enhanced methane production does not affect the digester stability. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Scaglione, Francesco; Lucini, Valeria; Pannacci, Marilou; Caronno, Alessia; Leone, Claude
2008-01-01
Serenoa repens extract is the phytotherapeutic agent most frequently used for the treatment of the urological symptoms caused by benign prostatic hyperplasia. There are many extracts in the market and each manufacturer uses different extraction processes; for this reason, it's possible that one product is not equivalent to another. The aim of this study was to compare the activity of different extracts of Serenoa repens marketed in Italy. The following extracts were tested on 10 day co-cultured epithelial and fibroblast cells by a 5alpha-reductase activity assay: Permixon, Saba, Serpens, Idiprost, Prostamev, Profluss and Prostil. In order to assess the variability in Serenoa repens products, 2 different batches for each brand were evaluated. All extracts tested, albeit variably, are able to inhibit both isoforms of 5alpha-reductase. However, the potency of the extracts appears to be very different, as well as the potencies of 2 different batches of the same extract. This is probably due to qualitative and quantitative differences in the active ingredients. So, the product of each company must be tested to evaluate the clinical efficacy and bioactivity. Copyright 2008 S. Karger AG, Basel.
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[Antibacterial antibodies in human immunoglobulins and sera: past and present].
Romanov, V A; Kulibin, A Iu; Zaĭtseva, I P
2010-01-01
To measure levels of several types of antibacterial antibodies in preparations of normal human immunoglobulin as well as in samples of donor sera obtained in 1965 and 2009. Five batches of human normal immunoglobulin manufactured in 1965 and five batches manufactured in 2009 as well as 77 and 28 blood serum samples respectively were tested by agglutination assay for the presence of antibodies to enterobacteria, Brucella species, tularemia agent, Rickettsia burnetii, Rickettsia prowazekii, and several species of opportunistic bacteria. Higher antibody titers to Salmonella typhi, Salmonella paratyphi A and B, Salmonella enteritidis, Salmonella typhimurium, Shigella flexneri and Shigella sonnei were revealed in immunoglobulin preparations and donor sera obtained in 1965 compared to that obtained in 2009. There was no difference in antibody titers to Shigella boydii, Salmonella choleraesuis, Escherichia coli O-55, Pseudomonas aeruginosa, Proteus vulgaris, Serratia marcescens and E. coli. Antibodies to Brucella species, tularemia agent, R. burnetii, R. prowazekii were not detected in normal human immunoglobulin. Decrease of antibody levels to several pathogenic enterobacteria in human immunoglobulin preparations as well as in sera of donors for 40 years could be linked with decrease of number of immunized persons, changes in circulation of pathogenic bacteria, decrease of rate of asymptomatic infections. Stability of antibody titers to opportunistic bacteria is a rationale to use them for assessment of humoral immunity function.
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Gaikwad, Dinanath; Shewale, Rajnita; Patil, Vinit; Mali, Dipak; Gaikwad, Uday; Jadhav, Namdeo
2017-11-01
The aim of this work was to prepare pectin-poly (vinyl pyrrolidone) [PVP] based curcumin particulates to enhance the anticancer potential of curcumin, solubility and allow its localized controlled release. Pectin-PVP based curcumin particulates (PECTIN-PVP CUR) were prepared by spray drying technique in different ratios and were evaluated for surface morphology, micromeritics, flowability, particle size, drug content, in vitro dissolution, inhalable fraction, anti-angiogenesis/angiolysis and cytotoxicity. Results of micromeritic properties, Carr's index, Hausner's ratio and angle of repose were satisfactory. The batch CP3 was considered as optimum, due to excellent flowability, acceptable aggregation and enhanced solubility. The particle size and size distribution data of selected batch CP3 showed 90% of curcumin particulates having size less than 2.74μm, which may deposit to lungs. Twin Impinger studies showed that 29% of respirable fraction was generated, which could be directly delivered to lungs. The in vitro dissolution data showed many fold increase in dissolution rate. Angiolytic activity and MTT assay of PECTIN-PVP CUR have demonstrated enhancement in the anti-tumor potential, compared to curcumin alone. Altogether, PECTIN-PVP CUR were found suitable for local delivery and enhance its anticancer potential of curcumin. Copyright © 2017 Elsevier B.V. All rights reserved.
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Jansen, Famke; Dorny, Pierre; Berkvens, Dirk; Van Hul, Anke; Van den Broeck, Nick; Makay, Caroline; Praet, Nicolas; Gabriël, Sarah
2016-08-30
The monoclonal antibody-based circulating antigen detecting ELISA (B158/B60 Ag-ELISA) has been used elaborately in several studies for the diagnosis of human, bovine and porcine cysticercosis. Interpretation of test results requires a good knowledge of the test characteristics, including the repeatability and the effect of the borders of the ELISA plates. Repeatability was tested for 4 antigen-negative and 5 antigen-positive reference bovine serum samples by calculating the Percentage Coefficient of Variation (%CV) within and between plates, within and between runs, overall, for two batches of monoclonal antibodies and by 2 laboratory technicians. All CV values obtained were below 20% (except one: 24.45%), which indicates a good repeatability and a negligible technician error. The value of 24.45% for indicating the variability between batches of monoclonal antibodies for one positive sample is still acceptable for repeatability measures. Border effects were determined by calculating the %CV values between the inner and outer wells of one plate for 2 positive serum samples. Variability is a little more present in the outer wells but this effect is very small and no significant border effect was found. Copyright © 2016 Elsevier B.V. All rights reserved.
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Irregular analytical errors in diagnostic testing - a novel concept.
Vogeser, Michael; Seger, Christoph
2018-02-23
In laboratory medicine, routine periodic analyses for internal and external quality control measurements interpreted by statistical methods are mandatory for batch clearance. Data analysis of these process-oriented measurements allows for insight into random analytical variation and systematic calibration bias over time. However, in such a setting, any individual sample is not under individual quality control. The quality control measurements act only at the batch level. Quantitative or qualitative data derived for many effects and interferences associated with an individual diagnostic sample can compromise any analyte. It is obvious that a process for a quality-control-sample-based approach of quality assurance is not sensitive to such errors. To address the potential causes and nature of such analytical interference in individual samples more systematically, we suggest the introduction of a new term called the irregular (individual) analytical error. Practically, this term can be applied in any analytical assay that is traceable to a reference measurement system. For an individual sample an irregular analytical error is defined as an inaccuracy (which is the deviation from a reference measurement procedure result) of a test result that is so high it cannot be explained by measurement uncertainty of the utilized routine assay operating within the accepted limitations of the associated process quality control measurements. The deviation can be defined as the linear combination of the process measurement uncertainty and the method bias for the reference measurement system. Such errors should be coined irregular analytical errors of the individual sample. The measurement result is compromised either by an irregular effect associated with the individual composition (matrix) of the sample or an individual single sample associated processing error in the analytical process. Currently, the availability of reference measurement procedures is still highly limited, but LC-isotope-dilution mass spectrometry methods are increasingly used for pre-market validation of routine diagnostic assays (these tests also involve substantial sets of clinical validation samples). Based on this definition/terminology, we list recognized causes of irregular analytical error as a risk catalog for clinical chemistry in this article. These issues include reproducible individual analytical errors (e.g. caused by anti-reagent antibodies) and non-reproducible, sporadic errors (e.g. errors due to incorrect pipetting volume due to air bubbles in a sample), which can both lead to inaccurate results and risks for patients.
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Xin, Ting; Jia, Hong; Ding, Jiabo; Li, Pingjun; Yang, Hongjun; Hou, Shaohua; Yuan, Weifeng; Guo, Xiaoyu; Wang, Haichun; Liang, Qianqian; Li, Ming
2013-01-01
Bovine tuberculosis (bTB) is a worldwide zoonosis caused mainly by Mycobacterium bovis. The traditional diagnostic method used often is the tuberculin skin test, which uses bovine purified protein derivatives (PPD-B). However, it is difficult to maintain uniformity of PPD-B from batch to batch, and it shares common antigens with nonpathogenic environmental mycobacteria. To overcome these problems, M. bovis-specific antigens that showed good T cell stimulation, such as CFP-10, ESAT-6, Rv3615c, etc., have been used in the skin test, but there have been no large-scale clinical studies on these antigens. In this study, two combinations (CFP-10/ESAT-6/TB10.4 protein cocktail and CFP-10/ESAT-6/Rv3872/MPT63 protein cocktail) were developed and used as stimuli in the skin test. Cattle were double-blind tested to assess the efficiency of the protein cocktail-based skin tests. The results showed that the CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test can differentiate TB-infected cattle from Mycobacterium avium-infected ones and that it shows a high degree of agreement with the traditional tuberculin skin test (κ = 0.8536) and gamma interferon (IFN-γ) release assay (κ = 0.8154). Compared to the tuberculin skin test, the relative sensitivity and relative specificity of the CFP-10/ESAT-6/TB10.4-based skin test were 87% and 97%, respectively., The relative sensitivity and relative specificity of the CFP-10/ESAT-6/TB10.4-based skin test were 93% and 92%, respectively, on comparison with the IFN-γ release assay. The correlation between the increases in skin thickness observed after the inoculation of stimuli was high (PPD-B versus CFP-10/ESAT-6/TB10.4, Spearman r of 0.8435). The correlation between the optical density at 450 nm (OD450) obtained after blood stimulation with PPD-B and the increase in skin thickness observed after inoculation of the CFP-10/ESAT-6/TB10.4 protein cocktail was high (Spearman r = 0.7335). Therefore, the CFP-10/ESAT-6/TB10.4-based skin test responses correlate to traditional measures of bovine TB evaluation, including skin test and gamma interferon release assay. PMID:23365203
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40 CFR 80.75 - Reporting requirements.
Code of Federal Regulations, 2010 CFR
2010-07-01
... section: (i) The batch number; (ii) The date of production; (iii) The volume of the batch; (iv) The grade... the refinery: (A) Identification of the previously certified gasoline as such; (B) The batch number... batch as commercial or non-commercial grade butane; (C) The batch number of the butane; (D) The date of...
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27 CFR 19.748 - Dump/batch records.
Code of Federal Regulations, 2010 CFR
2010-04-01
... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Dump/batch records. 19.748... OF THE TREASURY LIQUORS DISTILLED SPIRITS PLANTS Records and Reports Processing Account § 19.748 Dump/batch records. (a) Format of dump/batch records. Proprietor's dump/batch records shall contain, as...
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A Model-based B2B (Batch to Batch) Control for An Industrial Batch Polymerization Process
NASA Astrophysics Data System (ADS)
Ogawa, Morimasa
This paper describes overview of a model-based B2B (batch to batch) control for an industrial batch polymerization process. In order to control the reaction temperature precisely, several methods based on the rigorous process dynamics model are employed at all design stage of the B2B control, such as modeling and parameter estimation of the reaction kinetics which is one of the important part of the process dynamics model. The designed B2B control consists of the gain scheduled I-PD/II2-PD control (I-PD with double integral control), the feed-forward compensation at the batch start time, and the model adaptation utilizing the results of the last batch operation. Throughout the actual batch operations, the B2B control provides superior control performance compared with that of conventional control methods.
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NASA Technical Reports Server (NTRS)
Gentry, D. M.; Amador, E. S.; Cable, M. L.; Cantrell, T.; Chaudry, N.; Cullen, T.; Duca, Z.; Kirby, J.; Jacobsen, M.; McCaig, H.;
2017-01-01
Understanding the sensitivity of biomarker assays to the local physicochemical environment, and the underlying spatial distribution of the target biomarkers in 'homogeneous' environments, can increase mission science return. We have conducted four expeditions to Icelandic Mars analogue sites in which an increasingly refined battery of physicochemical measurements and biomarker assays were performed, staggered with scouting of further sites. Completed expeditions took place in 2012 (location scouting and field assay use testing), 2013 (sampling of two major sites with three assays and observational physicochemical measurements), 2015 (repeat sampling of prior sites and one new site, scouting of new sites, three assays and three instruments), and 2016 (preliminary sampling of new sites with analysis of returned samples). Target sites were geologically recent basaltic lava flows, and sample loci were arranged in hierarchically nested grids at 10 cm, 1 m, 10 m, 100 m, and >1 km order scales, subject to field constraints. Assays were intended to represent a diversity of potential biomarker types (cell counting via nucleic acid staining and fluorescence microscopy, ATP quantification via luciferase luminescence, and relative DNA quantification with simple domain-level primers) rather a specific mission science target, and were selected to reduce laboratory overhead, require limited consumables, and allow rapid turnaround. All analytical work was performed in situ or in a field laboratory within a day's travel of the field sites unless otherwise noted. We have demonstrated the feasibility of performing ATP quantification and qPCR analysis in a field-based laboratory with single-day turnaround. The ATP assay was generally robust and reliable and required minimal field equipment and training to produce a large amount of useful data. DNA was successfully extracted from all samples, but the serial-batch nature of qPCR significantly limited the number of primers (hence classifications) and replicates that could be run in a single day. Fluorescence microscopy did not prove feasible under the same constraints, primarily due to the large number of person-hours required to view, analyze, and record results from the images; however, this could be mitigated with higher-quality imaging instruments and appropriate image analysis software.
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Aldrich, Melissa B.; Wang, XueJuan; Hart, Amy; Sampath, Lakshmi; Marshall, Milton V.; Sevick-Muraca, Eva M.
2017-01-01
PURPOSE Recent preclinical and clinical studies show dyes that excite and fluoresce in the near infrared range may be used for tracking and detecting disease targets in vivo. A method for quantifying free dye molecules in antibody conjugate preparations is required for agent batch release and for translation into the clinic. PROCEDURES Herein, we developed and validated a SDS-PAGE method to determine the percentage of free IRDye 800CW in (DTPA)n-trastuzumab—(IRDye 800)m conjugate sample preparations in which HPLC assessment of free dye was not possible. RESULTS The SDS-PAGE assay was accurate and valid for free IRDye 800CW amounts between 38 and 4 molar percent of total dye. Gel sample preparation reagent affected the specificity of the assay, and lower and upper limits of quantitation and detection were determined. CONCLUSION This method may be applicable to other near infrared dye-conjugated antibody-based imaging agents in which HPLC assessment of purity is not feasible. This validated method for quality assurance will facilitate the translation of dual-labeled antibody conjugates for nuclear and optical imaging. PMID:20458634
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Ghosh, Pooja; Thakur, Indu Shekhar
2017-07-01
The study investigates the ability of fungus Phanerochaete sp. ISTL01 for biosorption of color from landfill leachate. Batch mode experiments were conducted to study the effects of pH, temperature, adsorbent dose, contact time and initial leachate concentration on biosorption. Maximum biosorption capacity was determined as 17.73 mg g -1 of biomass. Equilibrium isotherms and kinetics were further studied. The biosorption data were found to fit well to the Freundlich isotherm and pseudo-second-order kinetic model. The value of activation energy suggested that chemisorption mechanism was involved. Biosorption efficiency was also evaluated by the Methyltetrazolium (MTT) assay for cytotoxicity and alkaline comet assay in HepG2 human hepato-carcinoma cells. The fungus reduced toxicity as shown by 1.3-fold increase in MTT EC 50 and 1.5- and 1.1-fold reduction in Tail moment and Olive tail moment, respectively, after 12 h biosorption. The fungus showed good biosorption characteristics in terms of contaminant-level reduction per unit mass of adsorbent, process kinetics and toxicity reduction, envisaging its application in leachate treatment.
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Omoruyi, Iyekhoetin Matthew; Kabiersch, Grit; Pohjanvirta, Raimo
2013-01-01
Processed and packaged food items as well as ready-to-eat snacks are neglected and poorly characterised sources of human exposure to endocrine-disrupting chemicals (EDCs). In this study we investigated the presence of xenoestrogens in commercially processed and packaged Finnish foods, arising from substances deliberately added or inadvertently contaminating the food, substances formed as a result of food processing, or substances leaching from food packaging materials. Samples were obtained in three separate batches of equivalent products from both a supermarket and a local representative of a global chain of hamburger restaurants and extracted by a solid-phase extraction method. Their endocrine-disrupting potential was determined by yeast bioluminescent assay, using two recombinant yeast strains Saccharomyces cerevisiae BMAEREluc/ERα and S. cerevisiae BMA64/luc. In this test system, the majority of samples (both foodstuffs and wrappers) analysed proved negative. However, all batches of industrially prepared hamburgers (but not those obtained from a hamburger restaurant) as well as pepper salami significantly induced luciferase activity in the BMAEREluc/ERα yeast strain indicating the presence of xenoestrogens, with estradiol equivalents of these products ranging from 0.2 to 443 pg g(-1). All three products contained soy-based ingredients, which apparently accounted for, or at least contributed to, their high estrogenic activity, since no signal in the assay was observed with extracts of the packaging material, while two different soy sauces tested yielded an intense signal (28 and 54 pg ml(-1) estradiol-equivalent). These findings imply that by and large chemicals arising in the processing or packaging of foodstuffs in Finland constitute an insignificant source of xenoestrogens to consumers. However, soy-derived ingredients in certain food items might render the entire products highly estrogenic. The estrogenic activity of soy is attributed to isoflavones whose health effects - though widely considered beneficial - are controversial. As hamburgers are a popular type of food among children, the findings are noteworthy and possibly of concern.
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Graphite furnace atomic absorption elemental analysis of ecstasy tablets.
French, Holly E; Went, Michael J; Gibson, Stuart J
2013-09-10
Six metals (copper, magnesium, barium, nickel, chromium and lead) were determined in two separate batches of seized ecstasy tablets by graphite furnace atomic absorption spectroscopy (GFAAS) following digestion with nitric acid and hydrogen peroxide. Large intra-batch variations were found as expected for tablets produced in clandestine laboratories. For example, nickel in batch 1 was present in the range 0.47-13.1 parts per million (ppm) and in batch 2 in the range 0.35-9.06 ppm. Although batch 1 had significantly higher 3,4-methylenedioxy-N-methamphetamine (MDMA) content than batch 2, barium was the only element which discriminated between the two ecstasy seizures (batch 1: 0.19-0.66 ppm, batch 2: 3.77-5.47 ppm). Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
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NASA Astrophysics Data System (ADS)
Yu, Anchi; Ye, Xiong; Ionascu, Dan; Cao, Wenxiang; Champion, Paul M.
2005-11-01
An electronically delayed two-color pump-probe instrument was developed using two synchronized laser systems. The instrument has picosecond time resolution and can perform scans over hundreds of nanoseconds without the beam divergence and walk-off effects that occur using standard spatial delay systems. A unique picosecond Ti :sapphire regenerative amplifier was also constructed without the need for pulse stretching and compressing optics. The picosecond regenerative amplifier has a broad wavelength tuning range, which suggests that it will make a significant contribution to two-color pump-probe experiments. To test this instrument we studied the rotational correlation relaxation of myoglobin (τr=8.2±0.5ns) in water as well as the geminate rebinding kinetics of oxygen to myoglobin (kg1=1.7×1011s-1, kg2=3.4×107s-1). The results are consistent with, and improve upon, previous studies.
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Superdiffusive motion of membrane-targeting C2 domains
NASA Astrophysics Data System (ADS)
Campagnola, Grace; Nepal, Kanti; Schroder, Bryce W.; Peersen, Olve B.; Krapf, Diego
2015-12-01
Membrane-targeting domains play crucial roles in the recruitment of signalling molecules to the plasma membrane. For most peripheral proteins, the protein-to-membrane interaction is transient. After proteins dissociate from the membrane they have been observed to rebind following brief excursions in the bulk solution. Such membrane hops can have broad implications for the efficiency of reactions on membranes. We study the diffusion of membrane-targeting C2 domains using single-molecule tracking in supported lipid bilayers. The ensemble-averaged mean square displacement (MSD) exhibits superdiffusive behaviour. However, traditional time-averaged MSD analysis of individual trajectories remains linear and does not reveal superdiffusion. Our observations are explained in terms of bulk excursions that introduce jumps with a heavy-tail distribution. These hopping events allow proteins to explore large areas in a short time. The experimental results are shown to be consistent with analytical models of bulk-mediated diffusion and numerical simulations.
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[Synthesis and Study on Adsorption Property of Congo Red Molecularly Imprinted Polymer Nanospheres].
Chang, Zi-qiang; Chen, Fu-bin; Zhang, Yu; Shi, Zuo-long; Yang, Chun-yan; Zhang, Zhu-jun
2015-07-01
Molecularly imprinted polymer nanospheres (MIP) were prepared with Congo red as the template, methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross linker, azodiisobutyronitrile (AIBN) as an initiator, and acetonitrile as the porogen by precipitation polymerization. The morphology of MIP was characterized by SEM and TEM which showed that the diameter of MIP was nanometer grade (90 nm) and the shape was homogeneous. The specific surface area and pore volumes of MIP and NIP were examined through Brunauer-Emett-Teller method of nitrogen adsorption experiments. Then, the adsorption and selective recognition ability of MIPs were evaluated using the equilibrium rebinding experiments. The results indicated that the prepared MIP showed a good selectivity recognition ability to its template. It concluded that MIP could be employed as an effective material for removing Congo red from waste water.
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Chang, Limin; Li, Ying; Chu, Jia; Qi, Jingyao; Li, Xin
2010-11-08
In this paper, we demonstrated an efficient and robust route to the preparation of well-defined molecularly imprinted polymer based on reversible addition-fragmentation chain transfer (RAFT) polymerization and click chemistry. The alkyne terminated RAFT chain transfer agent was first synthesized, and then click reaction was used to graft RAFT agent onto the surface of silica particles which was modified by azide. Finally, imprinted thin film was prepared in the presence of 2,4-dichlorophenol as the template. The imprinted beads were demonstrated with a homogeneous polymer films (thickness of about 2.27 nm), and exhibited thermal stability under 255°C. The as-synthesized product showed obvious molecular imprinting effects towards the template, fast template rebinding kinetics and an appreciable selectivity over structurally related compounds. Copyright © 2010 Elsevier B.V. All rights reserved.
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Investigation of vinegar production using a novel shaken repeated batch culture system.
Schlepütz, Tino; Büchs, Jochen
2013-01-01
Nowadays, bioprocesses are developed or optimized on small scale. Also, vinegar industry is motivated to reinvestigate the established repeated batch fermentation process. As yet, there is no small-scale culture system for optimizing fermentation conditions for repeated batch bioprocesses. Thus, the aim of this study is to propose a new shaken culture system for parallel repeated batch vinegar fermentation. A new operation mode - the flushing repeated batch - was developed. Parallel repeated batch vinegar production could be established in shaken overflow vessels in a completely automated operation with only one pump per vessel. This flushing repeated batch was first theoretically investigated and then empirically tested. The ethanol concentration was online monitored during repeated batch fermentation by semiconductor gas sensors. It was shown that the switch from one ethanol substrate quality to different ethanol substrate qualities resulted in prolonged lag phases and durations of the first batches. In the subsequent batches the length of the fermentations decreased considerably. This decrease in the respective lag phases indicates an adaptation of the acetic acid bacteria mixed culture to the specific ethanol substrate quality. Consequently, flushing repeated batch fermentations on small scale are valuable for screening fermentation conditions and, thereby, improving industrial-scale bioprocesses such as vinegar production in terms of process robustness, stability, and productivity. Copyright © 2013 American Institute of Chemical Engineers.
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Tada, Shiori; Katakura, Yoshio; Ninomiya, Kazuaki; Shioya, Suteaki
2007-06-01
In a batch coculture of kefiran-producing lactic acid bacteria Lactobacillus kefiranofaciens and lactate-assimilating yeast Saccharomyces cerevisiae, lactate accumulation in the medium was observed, which inhibited kefiran production. To enhance kefiran productivity by preventing lactate accumulation, we conducted lactose-feeding batch operation with feedforward/feedback control during the coculture, so that the lactate production rate of L. kefiranofaciens was balanced with the lactate consumption rate of S. cerevisiae. The lactate concentration was maintained at less than 6 g l(-1) throughout the fed-batch coculture using a 5 l jar fermentor, although the concentration reached 33 g l(-1) in the batch coculture. Kefiran production was increased to 6.3 g in 102 h in the fed-batch coculture, whereas 4.5 g kefiran was produced in 97 h in the batch coculture. The kefiran yield on lactose basis was increased up to 0.033 g g(-1) in the fed-batch coculture, whereas that in the batch coculture was 0.027 g g(-1).
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Soman, Gopalan; Kallarakal, Abraham T; Michiel, Dennis; Yang, Xiaoyi; Saptharish, Nirmala; Jiang, Hengguang; Giardina, Steve; Gilly, John; Mitra, George
2012-01-01
Ch14.18 is a mouse-human chimeric monoclonal antibody to the disialoganglioside (GD2) glycolipid. In the clinic, this antibody has been shown to be effective in the treatment of children with high-risk neuroblastoma, either alone or in combination therapy. Extensive product characterization is a prerequisite to addressing the potential issues of product variability associated with process changes and manufacturing scale-up. Charge heterogeneity, glycosylation profile, molecular state and aggregation, interaction (affinity) with Fcγ receptors and functional or biological activities are a few of the critical characterization assays for assessing product comparability for this antibody. In this article, we describe the in-house development and qualification of imaged capillary isoelectric focusing to assess charge heterogeneity, analytical size exclusion chromatography with online static and dynamic light scattering (DLS), batch mode DLS for aggregate detection, biosensor (surface plasmon resonance)-based Fcγ receptor antibody interaction kinetics, N-glycoprofiling with PNGase F digestion, 2-aminobenzoic acid labeling and high performance liquid chromatography and N-glycan analysis using capillary electrophoresis. In addition, we studied selected biological activity assays, such as complement-dependent cytotoxicity. The consistency and reproducibility of the assays are established by comparing the intra-day and inter-day assay results. Applications of the methodologies to address stability or changes in product characteristics are also reported. The study results reveal that the ch14.18 clinical product formulated in phosphate-buffered saline at a concentration of 5 mg/ml and stored at 2-8°C is stable for more than five years.
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27 CFR 19.598 - Dump/batch records.
Code of Federal Regulations, 2014 CFR
2014-04-01
... ingredients used; (j) Formula number; (k) Quantity of ingredients used in the batch that have been previously... product transferred; (q) Batch gain or loss; and (r) For each batch to be tax determined in accordance...
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27 CFR 19.598 - Dump/batch records.
Code of Federal Regulations, 2013 CFR
2013-04-01
... ingredients used; (j) Formula number; (k) Quantity of ingredients used in the batch that have been previously... product transferred; (q) Batch gain or loss; and (r) For each batch to be tax determined in accordance...
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27 CFR 19.598 - Dump/batch records.
Code of Federal Regulations, 2011 CFR
2011-04-01
... ingredients used; (j) Formula number; (k) Quantity of ingredients used in the batch that have been previously... product transferred; (q) Batch gain or loss; and (r) For each batch to be tax determined in accordance...
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27 CFR 19.598 - Dump/batch records.
Code of Federal Regulations, 2012 CFR
2012-04-01
... ingredients used; (j) Formula number; (k) Quantity of ingredients used in the batch that have been previously... product transferred; (q) Batch gain or loss; and (r) For each batch to be tax determined in accordance...
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Ball, Nicholas; Cagen, Stuart; Carrillo, Juan-Carlos; Certa, Hans; Eigler, Dorothea; Emter, Roger; Faulhammer, Frank; Garcia, Christine; Graham, Cynthia; Haux, Carl; Kolle, Susanne N; Kreiling, Reinhard; Natsch, Andreas; Mehling, Annette
2011-08-01
An integral part of hazard and safety assessments is the estimation of a chemical's potential to cause skin sensitization. Currently, only animal tests (OECD 406 and 429) are accepted in a regulatory context. Nonanimal test methods are being developed and formally validated. In order to gain more insight into the responses induced by eight exemplary surfactants, a battery of in vivo and in vitro tests were conducted using the same batch of chemicals. In general, the surfactants were negative in the GPMT, KeratinoSens and hCLAT assays and none formed covalent adducts with test peptides. In contrast, all but one was positive in the LLNA. Most were rated as being irritants by the EpiSkin assay with the additional endpoint, IL1-alpha. The weight of evidence based on this comprehensive testing indicates that, with one exception, they are non-sensitizing skin irritants, confirming that the LLNA tends to overestimate the sensitization potential of surfactants. As results obtained from LLNAs are considered as the gold standard for the development of new nonanimal alternative test methods, results such as these highlight the necessity to carefully evaluate the applicability domains of test methods in order to develop reliable nonanimal alternative testing strategies for sensitization testing. Copyright © 2011 Elsevier Inc. All rights reserved.
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Solano, Gabriela; Gómez, Aarón; León, Guillermo
2015-10-01
Snake antivenoms are parenterally administered; therefore, endotoxin content must be strictly controlled. Following international indications to calculate endotoxin limits, it was determined that antivenom doses between 20 mL and 120 mL should not exceed 17.5 Endotoxin Units per milliliter (EU/mL) and 2.9 EU/mL, respectively. The rabbit pyrogen test (RPT) has been used to evaluate endotoxin contamination in antivenoms, but some laboratories have recently implemented the LAL assay. We compared the capability of both tests to evaluate endotoxin contamination in antivenoms, and we found that both methods can detect all endotoxin concentrations in the range of the antivenom specifications. The acceptance criteria of RPT and LAL must be harmonized by calculating the endotoxin limit as the quotient of the threshold pyrogenic dose and the therapeutic dose and the dose administered to rabbits as the quotient of the threshold pyrogenic dose and the endotoxin limit. Since endotoxins from Gram-negative bacteria exert different pyrogenicity, if contamination occurred, antivenom batches that induce pyrogenic reactions may be found in spite of passing LAL specifications. Although LAL assay can be used to assess endotoxin content throughout the antivenom manufacturing process, we recommend that the release of final products be based on the results of both methods. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Yang, Xing-Xin; Zhang, Xiao-Xia; Chang, Rui-Miao; Wang, Yan-Wei; Li, Xiao-Ni
2011-01-01
A simple and reliable high performance liquid chromatography (HPLC) method has been developed for the simultaneous quantification of five major bioactive components in ‘Shu-Jin-Zhi-Tong’ capsules (SJZTC), for the purposes of quality control of this commonly prescribed traditional Chinese medicine. Under the optimum conditions, excellent separation was achieved, and the assay was fully validated in terms of linearity, precision, repeatability, stability and accuracy. The validated method was applied successfully to the determination of the five compounds in SJZTC samples from different production batches. The HPLC method can be used as a valid analytical method to evaluate the intrinsic quality of SJZTC. PMID:29403711
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Collaborative study for the establishment of the 4(th) International Standard for Streptomycin.
Jorajuria, S; Raphalen, C; Dujardin, V; Daas, A
2015-01-01
An international collaborative study was organised to establish the 4(th) World Health Organization (WHO) International Standard (IS) for Streptomycin. Fourteen laboratories from different countries participated. Potencies of the candidate material were estimated by microbiological assays with sensitive micro-organisms. To ensure continuity between consecutive batches, the 3(rd) IS for Streptomycin was used as a reference. Based on the results of the study, the 4(th) IS for Streptomycin was adopted at the meeting of the WHO Expert Committee for Biological Standardization (ECBS) in 2015 with an assigned potency of 76 000 International Units (IU) per vial. The 4(th) IS for Streptomycin is available from the European Directorate for the Quality of Medicines & HealthCare (EDQM).
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Comparison of the release of constituents from granular materials under batch and column testing.
Lopez Meza, Sarynna; Garrabrants, Andrew C; van der Sloot, Hans; Kosson, David S
2008-01-01
Column leaching testing can be considered a better basis for assessing field impact data than any other available batch test method and thus provides a fundamental basis from which to estimate constituent release under a variety of field conditions. However, column testing is time-intensive compared to the more simplified batch testing, and may not always be a viable option when making decisions for material reuse. Batch tests are used most frequently as a simple tool for compliance or quality control reasons. Therefore, it is important to compare the release that occurs under batch and column testing, and establish conservative interpretation protocols for extrapolation from batch data when column data are not available. Five different materials (concrete, construction debris, aluminum recycling residue, coal fly ash and bottom ash) were evaluated via batch and column testing, including different column flow regimes (continuously saturated and intermittent unsaturated flow). Constituent release data from batch and column tests were compared. Results showed no significant difference between the column flow regimes when constituent release data from batch and column tests were compared. In most cases batch and column testing agreed when presented in the form of cumulative release. For arsenic in carbonated materials, however, batch testing underestimates the column constituent release for most LS ratios and also on a cumulative basis. For cases when As is a constituent of concern, column testing may be required.
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DETAIL VIEW OF BATCH CAR, BUILT BY ATLAS CAR & ...
DETAIL VIEW OF BATCH CAR, BUILT BY ATLAS CAR & MANUFACTURING COMPANY. BATCH STORAGE SILOS IN BACKGROUND - Chambers Window Glass Company, Batch Plant, North of Drey (Nineteenth) Street, West of Constitution Boulevard, Arnold, Westmoreland County, PA
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Angulo, Jesús; Enríquez-Navas, Pedro M; Nieto, Pedro M
2010-07-12
The direct evaluation of dissociation constants (K(D)) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor-ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K(D), as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor-ligand dissociation constants (K(D)) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein-ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein-ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein-ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein-ligand systems: the binding of the saccharides GlcNAc and GlcNAcbeta1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the experimental K(D) measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein-ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.
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Role of distal arginine in early sensing intermediates in the heme domain of the oxygen sensor FixL.
Jasaitis, Audrius; Hola, Klara; Bouzhir-Sima, Latifa; Lambry, Jean-Christophe; Balland, Veronique; Vos, Marten H; Liebl, Ursula
2006-05-16
FixL is a bacterial heme-based oxygen sensor, in which release of oxygen from the sensing PAS domain leads to activation of an associated kinase domain. Static structural studies have suggested an important role of the conserved residue arginine 220 in signal transmission at the level of the heme domain. To assess the role of this residue in the dynamics and properties of the initial intermediates in ligand release, we have investigated the effects of R220X (X = I, Q, E, H, or A) mutations in the FixLH heme domain on the dynamics and spectral properties of the heme upon photolysis of O(2), NO, and CO using femtosecond transient absorption spectroscopy. Comparison of transient spectra for CO and NO dissociation with steady-state spectra indicated less strain on the heme in the ligand dissociation species for all mutants compared to the wild type (WT). For CO and NO, the kinetics were similar to those of the wild type, with the exception of (1) a relatively low yield of picosecond NO rebinding to R220A, presumably related to the increase in the free volume of the heme pocket, and (2) substantial pH-dependent picosecond to nanosecond rebinding of CO to R220H, related to formation of a hydrogen bond between CO and histidine 220. Upon excitation of the complex bound with the physiological sensor ligand O(2), a 5-8 ps decay phase and a nondecaying (>4 ns) phase were observed for WT and all mutants. The strong distortion of the spectrum associated with the decay phase in WT is substantially diminished in all mutant proteins, indicating an R220-induced role of the heme in the primary intermediate in signal transmission. Furthermore, the yield of dissociated oxygen after this phase ( approximately 10% in WT) is increased in all mutants, up to almost unity in R220A, indicating a key role of R220 in caging the oxygen near the heme through hydrogen bonding. Molecular dynamics simulations corroborate these findings and suggest motions of O(2) and arginine 220 away from the heme pocket as a second step in the signal pathway on the 50 ps time scale.
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INTERIOR VIEW OF MIXER LOCATED ON SECOND FLOOR OF BATCH ...
INTERIOR VIEW OF MIXER LOCATED ON SECOND FLOOR OF BATCH PLANT. RECENTLY PURCHASED TO REPLACE OLD MIXER. USED TO MIX THE BATCH - Chambers-McKee Window Glass Company, Batch Plant, Clay Avenue Extension, Jeannette, Westmoreland County, PA
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Loman, Abdullah Al; Islam, S M Mahfuzul; Li, Qian; Ju, Lu-Kwang
2017-10-01
Despite having high protein and carbohydrate, soybean flour utilization is limited to partial replacement of animal feed to date. Enzymatic process can be exploited to increase its value by enriching protein content and separating carbohydrate for utilization as fermentation feedstock. Enzyme hydrolysis with fed-batch and recycle designs were evaluated here for achieving this goal with high productivities. Fed-batch process improved carbohydrate conversion, particularly at high substrate loadings of 250-375g/L. In recycle process, hydrolysate retained a significant portion of the limiting enzyme α-galactosidase to accelerate carbohydrate monomerization rate. At single-pass retention time of 6h and recycle rate of 62.5%, reducing sugar concentration reached up to 120g/L using 4ml/g enzyme. When compared with batch and fed-batch processes, the recycle process increased the volumetric productivity of reducing sugar by 36% (vs. fed-batch) to 57% (vs. batch) and that of protein product by 280% (vs. fed-batch) to 300% (vs. batch). Copyright © 2017 Elsevier Ltd. All rights reserved.
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Nieć, Dawid; Kunicki, Paweł K
2015-10-01
Measurements of plasma concentrations of free normetanephrine (NMN), metanephrine (MN) and methoxytyramine (MTY) constitute the most diagnostically accurate screening test for pheochromocytomas and paragangliomas. The aim of this article is to present the results from a validation of an analytical method utilizing high performance liquid chromatography with coulometric detection (HPLC-CD) for quantifying plasma free NMN, MN and MTY. Additionally, peak integration by height and area and the use of one calibration curve for all batches or individual calibration curve for each batch of samples was explored as to determine the optimal approach with regard to accuracy and precision. The method was validated using charcoal stripped plasma spiked with solutions of NMN, MN, MTY and internal standard (4-hydroxy-3-methoxybenzylamine) with the exception of selectivity which was evaluated by analysis of real plasma samples. Calibration curve performance, accuracy, precision and recovery were determined following both peak-area and peak-height measurements and the obtained results were compared. The most accurate and precise method of calibration was evaluated by analyzing quality control samples at three concentration levels in 30 analytical runs. The detector response was linear over the entire tested concentration range from 10 to 2000pg/mL with R(2)≥0.9988. The LLOQ was 10pg/mL for each analyte of interest. To improve accuracy for measurements at low concentrations, a weighted (1/amount) linear regression model was employed, which resulted in inaccuracies of -2.48 to 9.78% and 0.22 to 7.81% following peak-area and peak-height integration, respectively. The imprecisions ranged from 1.07 to 15.45% and from 0.70 to 11.65% for peak-area and peak-height measurements, respectively. The optimal approach to calibration was the one utilizing an individual calibration curve for each batch of samples and peak-height measurements. It was characterized by inaccuracies ranging from -3.39 to +3.27% and imprecisions from 2.17 to 13.57%. The established HPLC-CD method enables accurate and precise measurements of plasma free NMN, MN and MTY with reasonable selectivity. Preparing calibration curve based on peak-height measurements for each batch of samples yields optimal accuracy and precision. Copyright © 2015. Published by Elsevier B.V.
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Gdowski, Andrew; Johnson, Kaitlyn; Shah, Sunil; Gryczynski, Ignacy; Vishwanatha, Jamboor; Ranjan, Amalendu
2018-02-12
The process of optimization and fabrication of nanoparticle synthesis for preclinical studies can be challenging and time consuming. Traditional small scale laboratory synthesis techniques suffer from batch to batch variability. Additionally, the parameters used in the original formulation must be re-optimized due to differences in fabrication techniques for clinical production. Several low flow microfluidic synthesis processes have been reported in recent years for developing nanoparticles that are a hybrid between polymeric nanoparticles and liposomes. However, use of high flow microfluidic synthetic techniques has not been described for this type of nanoparticle system, which we will term as nanolipomer. In this manuscript, we describe the successful optimization and functional assessment of nanolipomers fabricated using a microfluidic synthesis method under high flow parameters. The optimal total flow rate for synthesis of these nanolipomers was found to be 12 ml/min and flow rate ratio 1:1 (organic phase: aqueous phase). The PLGA polymer concentration of 10 mg/ml and a DSPE-PEG lipid concentration of 10% w/v provided optimal size, PDI and stability. Drug loading and encapsulation of a representative hydrophobic small molecule drug, curcumin, was optimized and found that high encapsulation efficiency of 58.8% and drug loading of 4.4% was achieved at 7.5% w/w initial concentration of curcumin/PLGA polymer. The final size and polydispersity index of the optimized nanolipomer was 102.11 nm and 0.126, respectively. Functional assessment of uptake of the nanolipomers in C4-2B prostate cancer cells showed uptake at 1 h and increased uptake at 24 h. The nanolipomer was more effective in the cell viability assay compared to free drug. Finally, assessment of in vivo retention in mice of these nanolipomers revealed retention for up to 2 h and were completely cleared at 24 h. In this study, we have demonstrated that a nanolipomer formulation can be successfully synthesized and easily scaled up through a high flow microfluidic system with optimal characteristics. The process of developing nanolipomers using this methodology is significant as the same optimized parameters used for small batches could be translated into manufacturing large scale batches for clinical trials through parallel flow systems.
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EMISSIONS REDUCTION OF COMMERCIAL GLASSMAKING USING SELECTIVE BATCHING
The vertical bubble populations of selectively batched melts were compared to the vertical bubble populations of conventionally batched melts. “Conventional” refers to the use of a powdered batch. Bubble position and diameter measurements were taken on 24 crucibles...
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Energy efficiency of batch and semi-batch (CCRO) reverse osmosis desalination.
Warsinger, David M; Tow, Emily W; Nayar, Kishor G; Maswadeh, Laith A; Lienhard V, John H
2016-12-01
As reverse osmosis (RO) desalination capacity increases worldwide, the need to reduce its specific energy consumption becomes more urgent. In addition to the incremental changes attainable with improved components such as membranes and pumps, more significant reduction of energy consumption can be achieved through time-varying RO processes including semi-batch processes such as closed-circuit reverse osmosis (CCRO) and fully-batch processes that have not yet been commercialized or modelled in detail. In this study, numerical models of the energy consumption of batch RO (BRO), CCRO, and the standard continuous RO process are detailed. Two new energy-efficient configurations of batch RO are analyzed. Batch systems use significantly less energy than continuous RO over a wide range of recovery ratios and source water salinities. Relative to continuous RO, models predict that CCRO and batch RO demonstrate up to 37% and 64% energy savings, respectively, for brackish water desalination at high water recovery. For batch RO and CCRO, the primary reductions in energy use stem from atmospheric pressure brine discharge and reduced streamwise variation in driving pressure. Fully-batch systems further reduce energy consumption by not mixing streams of different concentrations, which CCRO does. These results demonstrate that time-varying processes can significantly raise RO energy efficiency. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Singh, Chandra K; Ojha, Abhishek; Kachru, Devendra N
2007-01-01
To comply with international labeling regulations for genetically modified (GM) crops and food, and to enable proper identification of GM organisms (GMOs), effective methodologies and reliable approaches are needed. The spurious and unapproved GM planting has contributed to crop failures and commercial losses. To ensure effective and genuine GM cultivation, a methodology is needed to detect and identify the trait of interest and concurrently evaluate the structural and functional stability of the transgene insert. A multiple polymerase chain reaction (PCR) approach was developed for detection, identification, and gene stability confirmation of cry1Ac transgene construct in Bt cotton. As many as 9 samples of Bt cotton hybrid seeds comprising 3 approved Bt hybrids, MECH-12Bt, MECH-162Bt, MECH-184Bt, and a batch of 6 nonapproved Bt hybrids were tested. Initially, single standard PCR assays were run to amplify predominant GM DNA sequences (CaMV 35S promoter, nos terminator, and npt-II marker gene); a housekeeping gene, Gossypium hirsutum fiber-specific acyl carrier protein gene (acp1); a trait-specific transgene (cry1Ac); and a sequence of 7S 3' transcription terminator which specifically borders with 3' region of cry1Ac transgene cassette. The concurrent amplification of all sequences of the entire cassette was performed by 3 assays, duplex, triplex, and quadruplex multiplex PCR assays, under common assay conditions. The identity of amplicons was reconfirmed by restriction endonuclease digestion profile. The 2 distinct transgene cassettes, cry1Ac and npt-II, of the Bt cotton were amplified using the respective forward primer of promoter and reverse primer of terminator. The resultant amplicons were excised, eluted, and purified. The purified amplicons served as template for nested PCR assays. The nested PCR runs confirmed the transgene construct orientation and identity. The limit of detection as established by our assay for GM trait (cry1Ac) was 0.1%. This approach can be adopted as a standard procedure for complete molecular characterization of Bt cotton. These assays will be of interest and use to importers, breeders, research laboratories, safety regulators, and food processors for detection of cry1Ac bearing GMOs.
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Humbert, María Victoria; Christodoulides, Myron
2018-05-23
Neisseria meningitidis (Nm) and N. gonorrhoeae (Ng) express a Macrophage Infectivity Potentiator (MIP, NMB1567/NEIS1487) protein in their outer membrane (OM). In this study, we prepared independent batches of liposomes (n = 3) and liposomes + MonoPhosphoryl Lipid A (MPLA) (n = 3) containing recombinant truncated Nm-MIP protein encoded by Allele 2 (rT-Nm-MIP, amino acids 22-142), and used these to immunize mice. We tested the hypothesis that independent vaccine batches showed similar antigenicity, and that antisera could recognise both meningococcal and gonococcal MIP and induce cross-species bactericidal activity. The different batches of M2 rT-Nm-MIP-liposomes ± MPLA showed no significant (P > 0.05) batch-to-batch variation in antigenicity. Anti-rT-Nm-MIP sera reacted equally and specifically with Nm-MIP and Ng-MIP in OM and on live bacterial cell surfaces. Specificity was shown by no antiserum reactivity with Δmip bacteria. Using human complement/serum bactericidal assays, anti-M2 rT-Nm-MIP sera killed homologous meningococcal serogroup B (MenB) strains (median titres of 32-64 for anti-rT-Nm-MIP-liposome sera; 128-256 for anti-rT-Nm-MIP-liposome + MPLA sera) and heterologous M1 protein-expressing MenB strains (titres of 64 for anti rT-Nm-MIP-liposome sera; 128-256 for anti-rT-Nm-MIP-liposome + MPLA sera). Low-level killing (P < 0.05) was observed for a MenB isolate expressing M7 protein (titres 4-8), but MenB strains expressing M6 protein were not killed (titre < 4-8). Killing (P < 0.05) was observed against MenC and MenW bacteria expressing homologous M2 protein (titres of 8-16) but not against MenA or MenY bacteria (titres < 4-8). Antisera to M2 rT-Nm-MIP showed significant (P < 0.05) cross-bactericidal activity against gonococcal strain P9-17 (expressing M35 Ng-MIP, titres of 64-512) and strain 12CFX_T_003 (expressing M10 Ng-MIP, titres 8-16) but not against FA1090 (expressing M8 Ng-MIP). As an alternative to producing recombinant protein, we engineered successfully the Nm-OM to express M2 Truncated-Nm-MIP, but lipooligosaccharide-extraction with Na-DOC was contra-indicated. Our data suggest that a multi-component vaccine containing a select number of Nm- and Ng-MIP type proteins would be required to provide broad coverage of both pathogens. Copyright © 2018. Published by Elsevier Ltd.
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Müller, Christian; Schillert, Arne; Röthemeier, Caroline; Trégouët, David-Alexandre; Proust, Carole; Binder, Harald; Pfeiffer, Norbert; Beutel, Manfred; Lackner, Karl J.; Schnabel, Renate B.; Tiret, Laurence; Wild, Philipp S.; Blankenberg, Stefan
2016-01-01
Technical variation plays an important role in microarray-based gene expression studies, and batch effects explain a large proportion of this noise. It is therefore mandatory to eliminate technical variation while maintaining biological variability. Several strategies have been proposed for the removal of batch effects, although they have not been evaluated in large-scale longitudinal gene expression data. In this study, we aimed at identifying a suitable method for batch effect removal in a large study of microarray-based longitudinal gene expression. Monocytic gene expression was measured in 1092 participants of the Gutenberg Health Study at baseline and 5-year follow up. Replicates of selected samples were measured at both time points to identify technical variability. Deming regression, Passing-Bablok regression, linear mixed models, non-linear models as well as ReplicateRUV and ComBat were applied to eliminate batch effects between replicates. In a second step, quantile normalization prior to batch effect correction was performed for each method. Technical variation between batches was evaluated by principal component analysis. Associations between body mass index and transcriptomes were calculated before and after batch removal. Results from association analyses were compared to evaluate maintenance of biological variability. Quantile normalization, separately performed in each batch, combined with ComBat successfully reduced batch effects and maintained biological variability. ReplicateRUV performed perfectly in the replicate data subset of the study, but failed when applied to all samples. All other methods did not substantially reduce batch effects in the replicate data subset. Quantile normalization plus ComBat appears to be a valuable approach for batch correction in longitudinal gene expression data. PMID:27272489
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Multi-stage high cell continuous fermentation for high productivity and titer.
Chang, Ho Nam; Kim, Nag-Jong; Kang, Jongwon; Jeong, Chang Moon; Choi, Jin-dal-rae; Fei, Qiang; Kim, Byoung Jin; Kwon, Sunhoon; Lee, Sang Yup; Kim, Jungbae
2011-05-01
We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.
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NASA Astrophysics Data System (ADS)
Bakar, Khomsaton Abu; Selambakkannu, Sarala; Ting, Teo Ming; Shariff, Jamaliah
2012-09-01
The combination of irradiation and biological technique was used to study COD, BOD5 and colour removal of textiles effluent in the presence of food industry wastewater at two different ratios. Two biological treatment system, the first consisting a mix of unirradiated textile and food industry wastewater and the second a mix of irradiated textile wastewater and food industry wastewater were operated in parallel. The experiment was conducted by batch. For the first batch the ratio was use for textile wastewater and food industry wastewater in biological treatment was 1:1. Meanwhile, for the second batch the ratio used for textile wastewater and food industry wastewater in biological treatment was 1:2. The results obtained for the first and second batch varies from each other. After irradiation, COD reduce in textile wastewater for the both batches are roughly 29% - 33% from the unirradiated wastewater. But after undergoing the biological treatment the percentage of COD reduction for first batch and second batch was 62.1% and 80.7% respectively. After irradiation the BOD5 of textile wastewater reduced by 22.2% for the first batch and 55.1% for the second batch. But after biological treatment, the BOD5 value for the first batch was same as its initial, 36mg/l and 40.4mg/l for the second batch. Colour had decreased from 899.5 ADMI to 379.3 ADMI after irradiation and decrease to 109.3 after undergoes biological treatment for the first batch. Meantime for the batch two, colour had decreased from 1000.44 ADMI to 363.40 ADMI after irradiation and dropped to 79.20 ADMI after biological treatment. The experiment show that 1:2 ratio show better reduction on COD, BOD5 and colour, compared to the ratio of 1:1.
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Request queues for interactive clients in a shared file system of a parallel computing system
DOE Office of Scientific and Technical Information (OSTI.GOV)
Bent, John M.; Faibish, Sorin
Interactive requests are processed from users of log-in nodes. A metadata server node is provided for use in a file system shared by one or more interactive nodes and one or more batch nodes. The interactive nodes comprise interactive clients to execute interactive tasks and the batch nodes execute batch jobs for one or more batch clients. The metadata server node comprises a virtual machine monitor; an interactive client proxy to store metadata requests from the interactive clients in an interactive client queue; a batch client proxy to store metadata requests from the batch clients in a batch client queue;more » and a metadata server to store the metadata requests from the interactive client queue and the batch client queue in a metadata queue based on an allocation of resources by the virtual machine monitor. The metadata requests can be prioritized, for example, based on one or more of a predefined policy and predefined rules.« less
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NASA Astrophysics Data System (ADS)
Yusriski, R.; Sukoyo; Samadhi, T. M. A. A.; Halim, A. H.
2016-02-01
In the manufacturing industry, several identical parts can be processed in batches, and setup time is needed between two consecutive batches. Since the processing times of batches are not always fixed during a scheduling period due to learning and deterioration effects, this research deals with batch scheduling problems with simultaneous learning and deterioration effects. The objective is to minimize total actual flow time, defined as a time interval between the arrival of all parts at the shop and their common due date. The decision variables are the number of batches, integer batch sizes, and the sequence of the resulting batches. This research proposes a heuristic algorithm based on the Lagrange Relaxation. The effectiveness of the proposed algorithm is determined by comparing the resulting solutions of the algorithm to the respective optimal solution obtained from the enumeration method. Numerical experience results show that the average of difference among the solutions is 0.05%.
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Batch and fed-batch production of butyric acid by Clostridium butyricum ZJUCB
He, Guo-qing; Kong, Qing; Chen, Qi-he; Ruan, Hui
2005-01-01
The production of butyric acid by Clostridium butyricum ZJUCB at various pH values was investigated. In order to study the effect of pH on cell growth, butyric acid biosynthesis and reducing sugar consumption, different cultivation pH values ranging from 6.0 to 7.5 were evaluated in 5-L bioreactor. In controlled pH batch fermentation, the optimum pH for cell growth and butyric acid production was 6.5 with a cell yield of 3.65 g/L and butyric acid yield of 12.25 g/L. Based on these results, this study then compared batch and fed-batch fermentation of butyric acid production at pH 6.5. Maximum value (16.74 g/L) of butyric acid concentration was obtained in fed-batch fermentation compared to 12.25 g/L in batch fermentation. It was concluded that cultivation under fed-batch fermentation mode could enhance butyric acid production significantly (P<0.01) by C. butyricum ZJUCB. PMID:16252341
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Production of nattokinase by batch and fed-batch culture of Bacillus subtilis.
Cho, Young-Han; Song, Jae Yong; Kim, Kyung Mi; Kim, Mi Kyoung; Lee, In Young; Kim, Sang Bum; Kim, Hyeon Shup; Han, Nam Soo; Lee, Bong Hee; Kim, Beom Soo
2010-09-30
Nattokinase was produced by batch and fed-batch culture of Bacillus subtilis in flask and fermentor. Effect of supplementing complex media (peptone, yeast extract, or tryptone) was investigated on the production of nattokinase. In flask culture, the highest cell growth and nattokinase activity were obtained with 50 g/L of peptone supplementation. In this condition, nattokinase activity was 630 unit/ml at 12 h. In batch culture of B. subtilis in fermentor, the highest nattokinase activity of 3400 unit/ml was obtained at 10h with 50 g/L of peptone supplementation. From the batch kinetics data, it was shown that nattokinase production was growth-associated and culture should be harvested before stationary phase for maximum nattokinase production. In fed-batch culture of B. subtilis using pH-stat feeding strategy, cell growth (optical density monitored at 600 nm) increased to ca. 100 at 22 h, which was 2.5 times higher than that in batch culture. The highest nattokinase activity was 7100 unit/ml at 19 h, which was also 2.1 times higher than that in batch culture. Copyright 2010 Elsevier B.V. All rights reserved.
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Zhao, Xuebing; Dong, Lei; Chen, Liang; Liu, Dehua
2013-05-01
Formiline pretreatment pertains to a biomass fractionation process. In the present work, Formiline-pretreated sugarcane bagasse was hydrolyzed with cellulases by batch and multi-step fed-batch processes at 20% solid loading. For wet pulp, after 144 h incubation with cellulase loading of 10 FPU/g dry solid, fed-batch process obtained ~150 g/L glucose and ~80% glucan conversion, while batch process obtained ~130 g/L glucose with corresponding ~70% glucan conversion. Solid loading could be further increased to 30% for the acetone-dried pulp. By fed-batch hydrolysis of the dried pulp in pH 4.8 buffer solution, glucose concentration could be 247.3±1.6 g/L with corresponding 86.1±0.6% glucan conversion. The enzymatic hydrolyzates could be well converted to ethanol by a subsequent fermentation using Saccharomices cerevisiae with ethanol titer of 60-70 g/L. Batch and fed-batch SSF indicated that Formiline-pretreated substrate showed excellent fermentability. The final ethanol concentration was 80 g/L with corresponding 82.7% of theoretical yield. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Fontes, Pedro Ribeiro; Souza, Paula Monteiro; William Fagg, Christopher; Neves Silva Guerra, Eliete; de Medeiros Nóbrega, Yanna Karla; Silveira, Damaris; Fonseca-Bazzo, Yris; Simeoni, Luiz Alberto; Homem-de-Mello, Maurício; Oliveira Magalhães, Pérola
2016-01-01
Melanogenesis is a process responsible for melanin production, which is stored in melanocytes containing tyrosinase. Inhibition of this enzyme is a target in the cosmetics industry, since it controls undesirable skin conditions such as hyperpigmentation due to the overproduction of melanin. Species of the Morus genus are known for the beneficial uses offered in different parts of its plants, including tyrosinase inhibition. Thus, this project aimed to study the inhibitory activity of tyrosinase by extracts from Morus nigra leaves as well as the characterization of its chromatographic profile and cytotoxicity in order to become a new therapeutic option from a natural source. M. nigra leaves were collected, pulverized, equally divided into five batches and the standardized extract was obtained by passive maceration. There was no significant difference between batches for total solids content, yield and moisture content, which shows good reproducibility of the extraction process. Tyrosinase enzymatic activity was determined for each batch, providing the percentage of enzyme inhibition and IC50 values obtained by constructing dose-response curves and compared to kojic acid, a well-known tyrosinase inhibitor. High inhibition of tyrosinase activity was observed (above 90% at 15.625 μg/mL). The obtained IC50 values ranged from 5.00 μg/mL ± 0.23 to 8.49 μg/mL ± 0.59 and were compared to kojic acid (3.37 μg/mL ± 0.65). High Performance Liquid Chromatography analysis revealed the presence of chlorogenic acid, rutin and, its major compound, isoquercitrin. The chromatographic method employed was validated according to ICH guidelines and the extract was standardized using these polyphenols as markers. Cytotoxicity, assessed by MTT assay, was not observed on murine melanomas, human keratinocytes and mouse fibroblasts in tyrosinase IC50 values. This study demonstrated the potential of M. nigra leaf extract as a promising whitening agent of natural source against skin hyperpigmentation. PMID:27655047
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Coexistence of nitrifying, anammox and denitrifying bacteria in a sequencing batch reactor
Langone, Michela; Yan, Jia; Haaijer, Suzanne C. M.; Op den Camp, Huub J. M.; Jetten, Mike S. M.; Andreottola, Gianni
2014-01-01
Elevated nitrogen removal efficiencies from ammonium-rich wastewaters have been demonstrated by several applications, that combine nitritation and anammox processes. Denitrification will occur simultaneously when organic carbon is also present. In this study, the activity of aerobic ammonia oxidizing, anammox and denitrifying bacteria in a full scale sequencing batch reactor, treating digester supernatants, was studied by means of batch-assays. AOB and anammox activities were maximum at pH of 8.0 and 7.8–8.0, respectively. Short term effect of nitrite on anammox activity was studied, showing nitrite up to 42 mg/L did not result in inhibition. Both denitrification via nitrate and nitrite were measured. To reduce nitrite-oxidizing activity, high NH3-N (1.9–10 mg NH3-N/L) and low nitrite (3–8 mg TNN/L) are required conditions during the whole SBR cycle. Molecular analysis showed the nitritation-anammox sludge harbored a high microbial diversity, where each microorganism has a specific role. Using ammonia monooxygenase α–subunit (amoA) gene as a marker, our analyses suggested different macro- and micro-environments in the reactor strongly affect the AOB community, allowing the development of different AOB species, such as N. europaea/eutropha and N. oligotropha groups, which improve the stability of nitritation process. A specific PCR primer set, used to target the 16S rRNA gene of anammox bacteria, confirmed the presence of the “Ca. Brocadia fulgida” type, able to grow in presence of organic matter and to tolerate high nitrite concentrations. The diversity of denitrifiers was assessed by using dissimilatory nitrite reductase (nirS) gene-based analyses, who showed denitifiers were related to different betaproteobacterial genera, such as Thauera, Pseudomonas, Dechloromonas and Aromatoleum, able to assist in forming microbial aggregates. Concerning possible secondary processes, no n-damo bacteria were found while NOB from the genus Nitrobacter was detected. PMID:24550899
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40 CFR 63.1326 - Batch process vents-recordkeeping provisions.
Code of Federal Regulations, 2013 CFR
2013-07-01
... (CONTINUED) National Emission Standards for Hazardous Air Pollutant Emissions: Group IV Polymers and Resins § 63.1326 Batch process vents—recordkeeping provisions. (a) Group determination records for batch... this section for each batch process vent subject to the group determination procedures of § 63.1323...
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7 CFR 58.728 - Cooking the batch.
Code of Federal Regulations, 2010 CFR
2010-01-01
... 7 Agriculture 3 2010-01-01 2010-01-01 false Cooking the batch. 58.728 Section 58.728 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Procedures § 58.728 Cooking the batch. Each batch of cheese within the cooker, including the optional...
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Parker, David; Belaud-Rotureau, Marc-Antoine
2014-01-01
Break-apart fluorescence in situ hybridization (FISH) is the gold standard test for anaplastic lymphoma kinase (ALK) gene rearrangement. However, this methodology often is assumed to be expensive and potentially cost-prohibitive given the low prevalence of ALK-positive non-small cell lung cancer (NSCLC) cases. To more accurately estimate the cost of ALK testing by FISH, we developed a micro-cost model that accounts for all cost elements of the assay, including laboratory reagents, supplies, capital equipment, technical and pathologist labor, and the acquisition cost of the commercial test and associated reagent kits and controls. By applying a set of real-world base-case parameter values, we determined that the cost of a single ALK break-apart FISH test result is $278.01. Sensitivity analysis on the parameters of batch size, testing efficiency, and the cost of the commercial diagnostic testing products revealed that the cost per result is highly sensitive to batch size, but much less so to efficiency or product cost. This implies that ALK testing by FISH will be most cost effective when performed in high-volume centers. Our results indicate that testing cost may not be the primary determinant of crizotinib (Xalkori(®)) treatment cost effectiveness, and suggest that testing cost is an insufficient reason to limit the use of FISH testing for ALK rearrangement.
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Studies on antibacterial activity of ZnO nanoparticles by ROS induced lipid peroxidation.
Dutta, R K; Nenavathu, Bhavani P; Gangishetty, Mahesh K; Reddy, A V R
2012-06-01
Recent studies indicated the role of ROS toward antibacterial activity. In our study we report ROS mediated membrane lipid oxidation of Escherichia coli treated with ZnO nanoparticles (NPs) as supported by detection and spectrophotometric measurement of malondialdehyde (MDA) by TBARS (thiobarbituric acid-reactive species) assay. The antibacterial effects of ZnO NPs were studied by measuring the growth curve of E. coli, which showed concentration dependent bacteriostatic and bacteriocidal effects of ZnO NPs. The antibacterial effects were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Further, antibacterial effect of ZnO NPs was found to decrease by introducing histidine to the culture medium treated with ZnO NPs. The ROS scavenging action of histidine was confirmed by treating histidine to the batch of Escherichia coli+ZnO NPs at the end of the lag phase of the growth curve (Set-I) and during inoculation (Set-II). A moderate bacteriostatic effect (lag in the E. coli growth) was observed in Set-II batch while Set-I showed no bacteriostatic effect. From these evidences we confirmed that the antibacterial effect of bare as well as TG capped ZnO NPs were due to membrane lipid peroxidation caused by the ROS generated during ZnO NPs interaction in culture medium. Copyright © 2012 Elsevier B.V. All rights reserved.
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Le Dimna, M; Vrancken, R; Koenen, F; Bougeard, S; Mesplède, A; Hutet, E; Kuntz-Simon, G; Le Potier, M F
2008-01-01
Two real-time RT-PCR kits, developed by LSI (TaqVet CSF) and ADIAGENE (Adiavet CSF), obtained an agreement to be commercialised in France, subject to conditions, defined by the French Classical Swine Fever (CSF) National Reference Laboratory. The producers were asked to introduce an internal control to check the RNA extraction efficacy. The different criteria assessed were sensitivity, "pestivirus specificity", reproducibility and ease of handling, using 189 different samples. These samples were either CSFV inactivated strains or blood/serum/organs collected from CSFV experimentally infected pigs or naturally infected wild boars. The reproducibility of the assays was confirmed by the analysis of a batch-to-batch panel control that was used for inter-laboratory tests involving nine laboratories. The two kits were also tested for the use in mass diagnostics and the results proved the kits to be suited using pools of blood, serum and tonsils. Moreover, a field evaluation, carried out on spleen samples collected from the CSF surveillance of wild boars in an area known to be infected and from domestic pigs at a slaughterhouse, confirmed the high sensitivity and specificity of the two kits. This step-by-step evaluation procedure confirmed that the two commercial CSF real-time RT-PCR kits have a higher predictive value than the current diagnostic standard, Virus Isolation.
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Bonertz, A; Roberts, G; Slater, J E; Bridgewater, J; Rabin, R L; Hoefnagel, M; Timon, M; Pini, C; Pfaar, O; Sheikh, A; Ryan, D; Akdis, C; Goldstein, J; Poulsen, L K; van Ree, R; Rhyner, C; Barber, D; Palomares, O; Pawankar, R; Hamerlijnk, D; Klimek, L; Agache, I; Angier, E; Casale, T; Fernandez-Rivas, M; Halken, S; Jutel, M; Lau, S; Pajno, G; Sturm, G; Varga, E M; Gerth van Wijk, R; Bonini, S; Muraro, A; Vieths, S
2018-04-01
Adequate quality is essential for any medicinal product to be eligible for marketing. Quality includes verification of the identity, content and purity of a medicinal product in combination with a specified production process and its control. Allergen products derived from natural sources require particular considerations to ensure adequate quality. Here, we describe key aspects of the documentation on manufacturing and quality aspects for allergen immunotherapy products in the European Union and the United States. In some key parts, requirements in these areas are harmonized while other fields are regulated separately between both regions. Essential differences are found in the use of Reference Preparations, or the requirement to apply standardized assays for potency determination. As the types of products available are different in specific regions, regulatory guidance for such products may also be available in one specific region only, such as for allergoids in the European Union. Region-specific issues and priorities are a result of this. As allergen products derived from natural sources are inherently variable in their qualitative and quantitative composition, these products present special challenges to balance the variability and ensuring batch-to-batch consistency. Advancements in scientific knowledge on specific allergens and their role in allergic disease will consequentially find representation in future regulatory guidelines. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.
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Archibald, F S; DeVoe, I W
1978-01-01
A simple defined medium (neisseria defined medium) was devised that does not require iron extraction to produce iron-limited growth of Neisseria meningitidis (SDIC). Comparison of this medium to Mueller-Hinton broth and agar showed nearly identical growth rates and yields. The defined medium was used in batch cultures to determine the disappearance of iron from the medium and its uptake by cells. To avoid a number of problems inherent in batch culture, continuous culture, in which iron and dissolved oxygen were varied independently, was used. Most of the cellular iron was found to be nonheme and associated with the particulate fraction in sonically disrupted cells. Nonheme and catalase-heme iron were reduced by iron starvation far more than cytochromes b and c and N,N,N',N'-tetramethylphenylenediamine-oxidase. The respiration rate and efficiency also decreased under iron limitation, whereas generation times increased. The iron-starved meningococcus took up iron by an energy-independent system operating in the first minute after an iron pulse and a slower energy-dependent system inhibited by respiratory poisons and an uncoupler. The energy-dependent system showed saturation kinetics and was stimulated nearly fourfold by iron privation. In addition, to determine the availability to the meningococcus of the iron in selected compounds, a sensitive assay was devised in which an iron-limited continuous culture was pulsed with the iron-containing compound. PMID:101516
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Parker, David; Belaud-Rotureau, Marc-Antoine
2014-01-01
Break-apart fluorescence in situ hybridization (FISH) is the gold standard test for anaplastic lymphoma kinase (ALK) gene rearrangement. However, this methodology often is assumed to be expensive and potentially cost-prohibitive given the low prevalence of ALK-positive non-small cell lung cancer (NSCLC) cases. To more accurately estimate the cost of ALK testing by FISH, we developed a micro-cost model that accounts for all cost elements of the assay, including laboratory reagents, supplies, capital equipment, technical and pathologist labor, and the acquisition cost of the commercial test and associated reagent kits and controls. By applying a set of real-world base-case parameter values, we determined that the cost of a single ALK break-apart FISH test result is $278.01. Sensitivity analysis on the parameters of batch size, testing efficiency, and the cost of the commercial diagnostic testing products revealed that the cost per result is highly sensitive to batch size, but much less so to efficiency or product cost. This implies that ALK testing by FISH will be most cost effective when performed in high-volume centers. Our results indicate that testing cost may not be the primary determinant of crizotinib (Xalkori®) treatment cost effectiveness, and suggest that testing cost is an insufficient reason to limit the use of FISH testing for ALK rearrangement. PMID:25520569
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CSF Aβ1-42 - an excellent but complicated Alzheimer's biomarker - a route to standardisation.
Kuhlmann, Julia; Andreasson, Ulf; Pannee, Josef; Bjerke, Maria; Portelius, Erik; Leinenbach, Andreas; Bittner, Tobias; Korecka, Magdalena; Jenkins, Rand G; Vanderstichele, Hugo; Stoops, Erik; Lewczuk, Piotr; Shaw, Leslie M; Zegers, Ingrid; Schimmel, Heinz; Zetterberg, Henrik; Blennow, Kaj
2017-04-01
The 42 amino acid form of amyloid β (Aβ 1 - 42 ) in cerebrospinal fluid (CSF) has been widely accepted as a central biomarker for Alzheimer's disease. Several immunoassays for CSF Aβ 1-42 are commercially available, but can suffer from between laboratory and batch-to-batch variability as well as lack of standardisation across assays. As a consequence, no general cut-off values have been established for a specific context of use (e.g., clinical diagnostics) and selection of individuals for enrolment in clinical trials (patient stratification) remains challenging. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has initiated a working group for CSF proteins (WG-CSF) to facilitate standardisation of CSF Aβ 1-42 measurement results. The efforts of the IFCC WG-CSF include the development of certified reference materials (CRMs) and reference measurement procedures (RMPs) for key biomarkers. Two candidate RMPs for quantification of Aβ 1-42 in CSF based on liquid chromatography tandem mass spectrometry have been developed and tested in two ring trials. Furthermore, two commutability studies including native CSF pools, artificial CSF and spiked materials have been completed. On the basis of these studies, human CSF pools containing only endogenous Aβ 1-42 at three concentrations were selected as the format for future CRMs that are now being processed. Copyright © 2016 Elsevier B.V. All rights reserved.
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Harbers, Gregory M.; Emoto, Kazunori; Greef, Charles; Metzger, Steven W.; Woodward, Heather N.; Mascali, James J.; Grainger, David W.; Lochhead, Michael J.
2008-01-01
This paper describes a new bioassay surface chemistry that effectively inhibits non-specific biomolecular and cell binding interactions, while providing a capacity for specific immobilization of desired biomolecules. Poly(ethylene glycol) (PEG) as the primary component in nonfouling film chemistry is well-established, but the multicomponent formulation described here is unique in that it (1) is applied in a single, reproducible, solution-based coating step; (2) can be applied to diverse substrate materials without the use of special primers; and (3) is readily functionalized to provide specific attachment chemistries. Surface analysis data are presented, detailing surface roughness, polymer film thickness, and film chemistry. Protein non-specific binding assays demonstrate significant inhibition of serum, fibrinogen, and lysozyme adsorption to coated glass, indium tin oxide, and tissue culture polystyrene dishes. Inhibition of S. aureus and K. pneumoniae microbial adhesion in a microfluidic flow cell, and inhibition of fibroblast cell adhesion from serum-based cell culture is shown. Effective functionalization of the coating is demonstrated by directing fibroblast adhesion to polymer surfaces activated with an RGD peptide. Batch-to-batch reproducibility data are included. The in situ cross-linked PEG-based coating chemistry is unique in its formulation, and its surface properties are attractive for a broad range of in vitro bioassay applications. PMID:18815622
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Melamine milk powder and infant formula sold in East Africa.
Schoder, Dagmar
2010-09-01
This is the first study proving the existence of melamine in milk powder and infant formula exported to the African market. A total of 49 milk powder batches were collected in Dar-es-Salaam (Tanzania, East Africa), the center of international trade in East Africa, which serves as a commercial bottleneck and shipment hub for sub-Saharan, Central, and East Africa. Two categories of samples were collected between October and December 2008, immediately after the melamine contamination of Chinese products became public: (i) market brands of all international companies supplying the East African market and (ii) illegally sold products from informal channels. Melamine concentration was determined with the AgraQuant Melamine Sensitive Assay. Despite the national import prohibition of Chinese milk products and unlabeled milk powder in Tanzania, 11% (22 of 200) of inspected microretailers sold milk powder on the local black market. Manufacturers could be identified for only 55% (27) of the 49 investigated batches. Six percent (3 of 49) of all samples and 11% (3 of 27) of all international brand name products tested revealed melamine concentrations up to 5.5 mg/kg of milk powder. This amount represents about twice the tolerable daily intake as suggested by the U.S Food and Drug Administration. Based on our study, we can assume that the number of affected children in Africa is substantial.
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Analytical and functional similarity of Amgen biosimilar ABP 215 to bevacizumab.
Seo, Neungseon; Polozova, Alla; Zhang, Mingxuan; Yates, Zachary; Cao, Shawn; Li, Huimin; Kuhns, Scott; Maher, Gwendolyn; McBride, Helen J; Liu, Jennifer
ABP 215 is a biosimilar product to bevacizumab. Bevacizumab acts by binding to vascular endothelial growth factor A, inhibiting endothelial cell proliferation and new blood vessel formation, thereby leading to tumor vasculature normalization. The ABP 215 analytical similarity assessment was designed to assess the structural and functional similarity of ABP 215 and bevacizumab sourced from both the United States (US) and the European Union (EU). Similarity assessment was also made between the US- and EU-sourced bevacizumab to assess the similarity between the two products. The physicochemical properties and structural similarity of ABP 215 and bevacizumab were characterized using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. ABP 215 has the same amino acid sequence and exhibits similar post-translational modification profiles compared to bevacizumab. The functional similarity assessment employed orthogonal assays designed to interrogate all expected biological activities, including those known to affect the mechanisms of action for ABP 215 and bevacizumab. More than 20 batches of bevacizumab (US) and bevacizumab (EU), and 13 batches of ABP 215 representing unique drug substance lots were assessed for similarity. The large dataset allows meaningful comparisons and garners confidence in the overall conclusion for the analytical similarity assessment of ABP 215 to both US- and EU-sourced bevacizumab. The structural and purity attributes, and biological properties of ABP 215 are demonstrated to be highly similar to those of bevacizumab.
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Wang, Zong-Min; Lu, Zhen-Ming; Yu, Yong-Jian; Li, Guo-Quan; Shi, Jin-Song; Xu, Zheng-Hong
2015-09-01
Solid-state fermentation of traditional Chinese vinegar is a mixed-culture refreshment process that proceeds for many centuries without spoilage. Here, we investigated bacterial community succession and flavor formation in three batches of Zhenjiang aromatic vinegar using pyrosequencing and metabolomics approaches. Temporal patterns of bacterial succession in the Pei (solid-state vinegar culture) showed no significant difference (P > 0.05) among three batches of fermentation. In all the batches investigated, the average number of community operational taxonomic units (OTUs) decreased dramatically from 119 ± 11 on day 1 to 48 ± 16 on day 3, and then maintained in the range of 61 ± 9 from day 5 to the end of fermentation. We confirmed that, within a batch of fermentation process, the patterns of bacterial diversity between the starter (took from the last batch of vinegar culture on day 7) and the Pei on day 7 were similar (90%). The relative abundance dynamics of two dominant members, Lactobacillus and Acetobacter, showed high correlation (coefficient as 0.90 and 0.98 respectively) among different batches. Furthermore, statistical analysis revealed dynamics of 16 main flavor metabolites were stable among different batches. The findings validate the batch-to-batch uniformity of bacterial community succession and flavor formation accounts for the quality of Zhenjiang aromatic vinegar. Based on our understanding, this is the first study helps to explain the rationality of age-old artistry from a scientific perspective. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Bhaisare, Roshan; Kamble, Bhavna
2016-07-01
Note taking while attending a PPT requires high activity of memory and writing process which ultimately leads to what is called "death by power point" referring to boredom and fatigue. To overcome this we planned to evaluate the impact of utilisation of uncompleted handouts given prior to PPT presentations. Final year MBBS students were divided in 2 batches, batch A and batch B. For a set of lectures one batch was provided with handouts before lecture while the other batch was given lectures only. Crossover was done to avoid bias, all the lectures being given by the same presenter. At the end of each lecture, a short questionnaire of 10 Multiple Choice Question (MCQ) was provided to the students. Mean scores were calculated for lectures with handouts and without handouts. For a set of lectures, when batch A was provided with handouts, the mean score was 28.2; for batch B to which no handouts were given the mean score was 23.4. Similarly, for batch B when provided with handouts the mean score was 29.1, for batch A which was not provided with handouts the mean score was 24. There was an average increase of 4.2 marks. Actual gain when handouts were provided was 1.2 marks per lecture. It was more for the batch comprising of repeater students as compared to the batch of fresher students. Increase in attendance was also noted. Providing uncompleted handouts before a didactic lecture definitely results in increase in knowledge gain; repeater students benefit more with uncompleted handouts.
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40 CFR 63.1406 - Reactor batch process vent provisions.
Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 11 2011-07-01 2011-07-01 false Reactor batch process vent provisions... § 63.1406 Reactor batch process vent provisions. (a) Emission standards. Owners or operators of reactor... reactor batch process vent located at a new affected source shall control organic HAP emissions by...
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40 CFR 63.1406 - Reactor batch process vent provisions.
Code of Federal Regulations, 2010 CFR
2010-07-01
... 40 Protection of Environment 11 2010-07-01 2010-07-01 true Reactor batch process vent provisions... § 63.1406 Reactor batch process vent provisions. (a) Emission standards. Owners or operators of reactor... reactor batch process vent located at a new affected source shall control organic HAP emissions by...
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40 CFR 63.1321 - Batch process vents provisions.
Code of Federal Regulations, 2010 CFR
2010-07-01
... Standards for Hazardous Air Pollutant Emissions: Group IV Polymers and Resins § 63.1321 Batch process vents..., owners and operators of new and existing affected sources with batch process vents shall comply with the... applicable reporting requirements in § 63.1327. (b) New SAN batch affected sources. Owners and operators of...
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40 CFR 63.1322 - Batch process vents-reference control technology.
Code of Federal Regulations, 2013 CFR
2013-07-01
... 40 Protection of Environment 12 2013-07-01 2013-07-01 false Batch process vents-reference control... (CONTINUED) National Emission Standards for Hazardous Air Pollutant Emissions: Group IV Polymers and Resins § 63.1322 Batch process vents—reference control technology. (a) Batch process vents. The owner or...
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40 CFR 63.1322 - Batch process vents-reference control technology.
Code of Federal Regulations, 2012 CFR
2012-07-01
... 40 Protection of Environment 12 2012-07-01 2011-07-01 true Batch process vents-reference control technology. 63.1322 Section 63.1322 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... Batch process vents—reference control technology. (a) Batch process vents. The owner or operator of a...
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40 CFR 63.1322 - Batch process vents-reference control technology.
Code of Federal Regulations, 2014 CFR
2014-07-01
... 40 Protection of Environment 12 2014-07-01 2014-07-01 false Batch process vents-reference control technology. 63.1322 Section 63.1322 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... § 63.1322 Batch process vents—reference control technology. (a) Batch process vents. The owner or...
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40 CFR 63.1322 - Batch process vents-reference control technology.
Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 11 2011-07-01 2011-07-01 false Batch process vents-reference control technology. 63.1322 Section 63.1322 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... Batch process vents—reference control technology. (a) Batch process vents. The owner or operator of a...
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A Semi-Batch Reactor Experiment for the Undergraduate Laboratory
ERIC Educational Resources Information Center
Derevjanik, Mario; Badri, Solmaz; Barat, Robert
2011-01-01
This experiment and analysis offer an economic yet challenging semi-batch reactor experience. Household bleach is pumped at a controlled rate into a batch reactor containing pharmaceutical hydrogen peroxide solution. Batch temperature, product molecular oxygen, and the overall change in solution conductivity are metered. The reactor simulation…
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40 CFR 63.1322 - Batch process vents-reference control technology.
Code of Federal Regulations, 2010 CFR
2010-07-01
... 40 Protection of Environment 11 2010-07-01 2010-07-01 true Batch process vents-reference control technology. 63.1322 Section 63.1322 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... Batch process vents—reference control technology. (a) Batch process vents. The owner or operator of a...
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Lin, Jian-Ping; Wei, Lian; Xia, Li-Ming; Cen, Pei-Lin
2003-01-01
The production of laccase by Coriolus versicolor was studied. The effect of cultivation conditions on laccase production by Coriolus versicolor was examined to obtain optimal medium and cultivation conditions. Both batch and repeated-batch processes were performed for laccase production. In repeated-batch fermentation with self-immobilized mycelia, total of 14 cycles were performed with laccase activity in the range between 3.4 and 14.8 U/ml.
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A neural network strategy for end-point optimization of batch processes.
Krothapally, M; Palanki, S
1999-01-01
The traditional way of operating batch processes has been to utilize an open-loop "golden recipe". However, there can be substantial batch to batch variation in process conditions and this open-loop strategy can lead to non-optimal operation. In this paper, a new approach is presented for end-point optimization of batch processes by utilizing neural networks. This strategy involves the training of two neural networks; one to predict switching times and the other to predict the input profile in the singular region. This approach alleviates the computational problems associated with the classical Pontryagin's approach and the nonlinear programming approach. The efficacy of this scheme is illustrated via simulation of a fed-batch fermentation.
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Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors.
Haghverdi, Laleh; Lun, Aaron T L; Morgan, Michael D; Marioni, John C
2018-06-01
Large-scale single-cell RNA sequencing (scRNA-seq) data sets that are produced in different laboratories and at different times contain batch effects that may compromise the integration and interpretation of the data. Existing scRNA-seq analysis methods incorrectly assume that the composition of cell populations is either known or identical across batches. We present a strategy for batch correction based on the detection of mutual nearest neighbors (MNNs) in the high-dimensional expression space. Our approach does not rely on predefined or equal population compositions across batches; instead, it requires only that a subset of the population be shared between batches. We demonstrate the superiority of our approach compared with existing methods by using both simulated and real scRNA-seq data sets. Using multiple droplet-based scRNA-seq data sets, we demonstrate that our MNN batch-effect-correction method can be scaled to large numbers of cells.
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Prezoto, B C; Tanaka-Azevedo, A M; Marcelino, J R; Tashima, A K; Nishiduka, E S; Kapronezai, J; Mota, J O; Rocha, M M T; Serino-Silva, C; Oguiura, N
2018-06-15
The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED 50 and LD 50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a β-neurotoxin phospholipase A 2 -like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED 50 ) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro- or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Friedrich, Ricarda; Rappold, Elfriede; Bogdan, Christian; Held, Jürgen
2018-06-13
Background (1→3)-β-D-glucan (BDG) is a biomarker for invasive fungal disease. Until now, all BDG-data in the western hemisphere were obtained using the Fungitell® assay (FA). How it compares to the Wako β-Glucan Test (GT), that was recently launched in Europe, is largely unknown. Methods We conducted a case-control study to compare both assays in serum samples of 120 candidemia and 63 Pneumocystis jirovecii pneumonia (PCP) patients. 200 patients with bacteremia or negative blood cultures served as candidemia control group. Results In patients with candidemia the median BDG values of the FA and the GT were 351 and 8.4 pg/ml, respectively. With both assays, the BDG levels in candidemia were significantly higher than those measured in the control group (p<0.001). The sensitivity, specificity, positive and negative predictive values for the diagnosis of candidemia were 86.7%, 85.0%, 6.0% and 99.8% for the FA and 42.5%, 98.0%, 19.0% and 99.4% for the GT, respectively.In PCP patients the median BDG values of the FA and the GT were 963 and 57.7 pg/ml, respectively. The sensitivity for PCP diagnosis was 100% for the FA and 88.9% for the GT. In practical terms, the GT proved to be robust and applicable for testing single samples, whereas for economic reasons the FA required the samples to be tested in batch. Conclusions The sensitivity of the FA is superior to that of the GT. However, the GT is a valuable alternative to the FA especially in patients with suspected PCP and in laboratories with low sample throughput. Copyright © 2018 American Society for Microbiology.
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Tsai, Yun-Long; Wang, Han-Ching; Lo, Chu-Fang; Tang-Nelson, Kathy; Lightner, Donald; Ou, Bor-Rung; Hour, Ai-Ling; Tsai, Chuan-Fu; Yen, Cheng-Chi; Chang, Hsiao-Fen Grace; Teng, Ping-Hua; Lee, Pei-Yu
2014-01-01
Timely pond-side detection of white spot syndrome virus (WSSV) plays a critical role in the implementation of bio-security measures to help minimize economic losses caused by white spot syndrome disease, an important threat to shrimp aquaculture industry worldwide. A portable device, namely POCKIT™, became available recently to complete fluorescent probe-based insulated isothermal PCR (iiPCR), and automatic data detection and interpretation within one hour. Taking advantage of this platform, the IQ Plus™ WSSV Kit with POCKIT system was established to allow simple and easy WSSV detection for on-site users. The assay was first evaluated for its analytical sensitivity and specificity performance. The 95% limit of detection (LOD) of the assay was 17 copies of WSSV genomic DNA per reaction (95% confidence interval [CI], 13 to 24 copies per reaction). The established assay has detection sensitivity similar to that of OIE-registered IQ2000™ WSSV Detection and Protection System with serial dilutions of WSSV-positive Litopenaeus vannamei DNA. No cross-reaction signals were generated from infectious hypodermal and haematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), and hepatopancreatic parvovirus (HPV) positive samples. Accuracy analysis using700 L. vannamei of known WSSV infection status shows that the established assayhassensitivity93.5% (95% CI: 90.61–95.56%) and specificity 97% (95% CI: 94.31–98.50%). Furthermore, no discrepancy was found between the two assays when 100 random L. vannamei samples were tested in parallel. Finally, excellent correlation was observed among test results of three batches of reagents with 64 samples analyzed in three different laboratories. Working in a portable device, IQ Plus™ WSSV Kit with POCKIT system allows reliable, sensitive and specific on-site detection of WSSV in L. vannamei. PMID:24625894
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40 CFR 63.1407 - Non-reactor batch process vent provisions.
Code of Federal Regulations, 2010 CFR
2010-07-01
... 40 Protection of Environment 11 2010-07-01 2010-07-01 true Non-reactor batch process vent... § 63.1407 Non-reactor batch process vent provisions. (a) Emission standards. (1) Owners or operators of non-reactor batch process vents located at new or existing affected sources with 0.25 tons per year (0...
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40 CFR 63.1407 - Non-reactor batch process vent provisions.
Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 11 2011-07-01 2011-07-01 false Non-reactor batch process vent... § 63.1407 Non-reactor batch process vent provisions. (a) Emission standards. (1) Owners or operators of non-reactor batch process vents located at new or existing affected sources with 0.25 tons per year (0...
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40 CFR 63.487 - Batch front-end process vents-reference control technology.
Code of Federal Regulations, 2010 CFR
2010-07-01
... § 63.487 Batch front-end process vents—reference control technology. (a) Batch front-end process vents... 40 Protection of Environment 9 2010-07-01 2010-07-01 false Batch front-end process vents-reference control technology. 63.487 Section 63.487 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... properties may vary with time. For a unit operation operated in a batch mode (i.e., batch unit operation... means a unit operation operated in a batch mode. Block means the time period that comprises a single batch cycle. Combustion device burner means a device designed to mix and ignite fuel and air to provide...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... properties may vary with time. For a unit operation operated in a batch mode (i.e., batch unit operation... means a unit operation operated in a batch mode. Block means the time period that comprises a single batch cycle. Combustion device burner means a device designed to mix and ignite fuel and air to provide...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... properties may vary with time. For a unit operation operated in a batch mode (i.e., batch unit operation... means a unit operation operated in a batch mode. Block means the time period that comprises a single batch cycle. Combustion device burner means a device designed to mix and ignite fuel and air to provide...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... properties may vary with time. For a unit operation operated in a batch mode (i.e., batch unit operation... means a unit operation operated in a batch mode. Block means the time period that comprises a single batch cycle. Combustion device burner means a device designed to mix and ignite fuel and air to provide...
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Code of Federal Regulations, 2010 CFR
2010-07-01
... properties may vary with time. For a unit operation operated in a batch mode (i.e., batch unit operation... means a unit operation operated in a batch mode. Block means the time period that comprises a single batch cycle. Combustion device burner means a device designed to mix and ignite fuel and air to provide...
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Commeau, Natalie; Cornu, Marie; Albert, Isabelle; Denis, Jean-Baptiste; Parent, Eric
2012-03-01
Assessing within-batch and between-batch variability is of major interest for risk assessors and risk managers in the context of microbiological contamination of food. For example, the ratio between the within-batch variability and the between-batch variability has a large impact on the results of a sampling plan. Here, we designed hierarchical Bayesian models to represent such variability. Compatible priors were built mathematically to obtain sound model comparisons. A numeric criterion is proposed to assess the contamination structure comparing the ability of the models to replicate grouped data at the batch level using a posterior predictive loss approach. Models were applied to two case studies: contamination by Listeria monocytogenes of pork breast used to produce diced bacon and contamination by the same microorganism on cold smoked salmon at the end of the process. In the first case study, a contamination structure clearly exists and is located at the batch level, that is, between batches variability is relatively strong, whereas in the second a structure also exists but is less marked. © 2012 Society for Risk Analysis.
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Rácz, Norbert; Kormány, Róbert; Fekete, Jenő; Molnár, Imre
2015-04-10
Column technology needs further improvement even today. To get information of batch-to-batch repeatability, intelligent modeling software was applied. Twelve columns from the same production process, but from different batches were compared in this work. In this paper, the retention parameters of these columns with real life sample solutes were studied. The following parameters were selected for measurements: gradient time, temperature and pH. Based on calculated results, batch-to-batch repeatability of BEH columns was evaluated. Two parallel measurements on two columns from the same batch were performed to obtain information about the quality of packing. Calculating the average of individual working points at the highest critical resolution (R(s,crit)) it was found that the robustness, calculated with a newly released robustness module, had a success rate >98% among the predicted 3(6) = 729 experiments for all 12 columns. With the help of retention modeling all substances could be separated independently from the batch and/or packing, using the same conditions, having high robustness of the experiments. Copyright © 2015 Elsevier B.V. All rights reserved.
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[Batch release of immunoglobulin and monoclonal antibody products].
Gross, S
2014-10-01
The Paul-Ehrlich Institute (PEI) is an independent institution of the Federal Republic of Germany responsible for performing official experimental batch testing of sera. The institute decides about the release of each batch and performs experimental research in the field. The experimental quality control ensures the potency of the product and also the absence of harmful impurities. For release of an immunoglobulin batch the marketing authorization holder has to submit the documentation of the manufacture and the results of quality control measures together with samples of the batch to the PEI. Experimental testing is performed according to the approved specifications regarding the efficacy and safety. Since implementation of the 15th German drug law amendment, the source of antibody is not defined anymore. According to § 32 German drug law, all batches of sera need to be released by an official control laboratory. Sera are medicinal products, which contain antibodies, antibody fragments or fusion proteins with a functional antibody portion. Therefore, all batches of monoclonal antibodies and derivatives must also be released by the PEI and the marketing authorization holder has to submit a batch release application. Under certain circumstances a waiver for certain products can be issued with regard to batch release. The conditions for such a waiver apply to the majority of monoclonal antibodies.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Gardner, Christopher J.
Equations were derived for the transverse emittance growth of a single batch of protons passing turn-by-turn through the H-minus stripping foil in Booster. It was assumed there that all particles in the batch go the same number of turns around the machine and pass through the foil the same number of times. The equations therefore apply to the case in which the duration of the pulse of beam injected into Booster is less than one revolution period. We consider here the case in which the incoming beam consists of a series of batches injected one after another into the machine.more » Each batch is assumed to contain the same number of particles. The leading edge of an incoming batch is assumed to be exactly one revolution period behind the leading edge of the batch in front of it. The batch length is assumed to be less than one revolution period and is assumed to be the same for all batches. By taking the batch length to be less than but close to one revolution period, we can approximate, as closely as we like, the case of a continuous ribbon of beam injected into the machine over a period of several turns.« less
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NASA Astrophysics Data System (ADS)
Hakim Halim, Abdul; Ernawati; Hidayat, Nita P. A.
2018-03-01
This paper deals with a model of batch scheduling for a single batch processor on which a number of parts of a single items are to be processed. The process needs two kinds of setups, i. e., main setups required before processing any batches, and additional setups required repeatedly after the batch processor completes a certain number of batches. The parts to be processed arrive at the shop floor at the times coinciding with their respective starting times of processing, and the completed parts are to be delivered at multiple due dates. The objective adopted for the model is that of minimizing total inventory holding cost consisting of holding cost per unit time for a part in completed batches, and that in in-process batches. The formulation of total inventory holding cost is derived from the so-called actual flow time defined as the interval between arrival times of parts at the production line and delivery times of the completed parts. The actual flow time satisfies not only minimum inventory but also arrival and delivery just in times. An algorithm to solve the model is proposed and a numerical example is shown.
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BHAISARE, ROSHAN; KAMBLE, BHAVNA
2016-01-01
Introduction Note taking while attending a PPT requires high activity of memory and writing process which ultimately leads to what is called “death by power point” referring to boredom and fatigue. To overcome this we planned to evaluate the impact of utilisation of uncompleted handouts given prior to PPT presentations. Methods Final year MBBS students were divided in 2 batches, batch A and batch B. For a set of lectures one batch was provided with handouts before lecture while the other batch was given lectures only. Crossover was done to avoid bias, all the lectures being given by the same presenter. At the end of each lecture, a short questionnaire of 10 Multiple Choice Question (MCQ) was provided to the students. Mean scores were calculated for lectures with handouts and without handouts. Results For a set of lectures, when batch A was provided with handouts, the mean score was 28.2; for batch B to which no handouts were given the mean score was 23.4. Similarly, for batch B when provided with handouts the mean score was 29.1, for batch A which was not provided with handouts the mean score was 24. There was an average increase of 4.2 marks. Actual gain when handouts were provided was 1.2 marks per lecture. It was more for the batch comprising of repeater students as compared to the batch of fresher students. Increase in attendance was also noted. Conclusion Providing uncompleted handouts before a didactic lecture definitely results in increase in knowledge gain; repeater students benefit more with uncompleted handouts, PMID:27382583
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George, S; Gill, L; Braithwaite, R A
2000-05-01
A simple and rapid high-performance liquid chromatographic method has been developed for the determination of vigabatrin concentrations in plasma or serum. The assay uses only 100 microL of specimen and has been found to be linear over a concentration range of 1 to 50 mg/L. The limit of detection has been determined as 1 mg/L, and the between-batch coefficient of variation for the two internal quality controls routinely analysed (n = 33) has been found to be less than 5%. There was no evidence of interferences in the assay from other commonly prescribed anti-epileptic drugs. This method has been applied to routine clinical specimens to determine the concentration of vigabatrin in 47 patient specimens over a 12-month period. It was found that only 63% of the male group and 53% of the female group were within the proposed target concentration of 5 to 35 mg/L. In addition, it was found that 26% of the male group and 36% of the female group were found to have concentrations below 5 mg/L, which may indicate lack of compliance and/or lack of therapeutic efficacy of treatment.
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Xiang, Yun; Koomen, John M.
2012-01-01
Protein quantification with liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has emerged as a powerful platform for assessing panels of biomarkers. In this study, direct infusion, using automated, chip-based nanoelectrospray ionization, coupled with MRM (DI-MRM) is used for protein quantification. Removal of the LC separation step increases the importance of evaluating the ratios between the transitions. Therefore, the effects of solvent composition, analyte concentration, spray voltage, and quadrupole resolution settings on fragmentation patterns have been studied using peptide and protein standards. After DI-MRM quantification was evaluated for standards, quantitative assays for the expression of heat shock proteins (HSPs) were translated from LC-MRM to DI-MRM for implementation in cell line models of multiple myeloma. Requirements for DI-MRM assay development are described. Then, the two methods are compared; criteria for effective DI-MRM analysis are reported based on the analysis of HSP expression in digests of whole cell lysates. The increased throughput of DI-MRM analysis is useful for rapid analysis of large batches of similar samples, such as time course measurements of cellular responses to therapy. PMID:22293045
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Hirtz, Christophe; Vialaret, Jérôme; Gabelle, Audrey; Nowak, Nora; Dauvilliers, Yves; Lehmann, Sylvain
2016-01-01
I125 radioimmunoassay (RIA) is currently the standard technique for quantifying cerebrospinal fluid (CSF) orexin-A/hypocretin-1, a biomarker used to diagnose narcolepsy type 1. However, orexin-A RIA is liable to undergo cross-reactions with matrix constituents generating interference, high variability between batches, low precision and accuracy, and requires special radioactivity precautions. Here we developed the first quantitative mass spectrometry assay of orexin-A based on a multiple reaction monitoring (MRM) approach. This method was tested in keeping with the Clinical and Laboratory Standards Institute (CLSI) guidelines and its clinical relevance was confirmed by comparing patients with narcolepsy type 1 versus patients with other neurological conditions. The results obtained using MRM and RIA methods were highly correlated, and Bland–Altman analysis established their interchangeability. However, the MRM values had a wider distribution and were 2.5 time lower than the RIA findings. In conclusion, this method of assay provides a useful alternative to RIA to quantify orexin-A, and may well replace it not only in narcolepsy type 1, but also in the increasing number of pathologies in which the quantification of this analyte is relevant. PMID:27165941
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Quintero, Catherine; Kariv, Ilona
2009-06-01
To meet the needs of the increasingly rapid and parallelized lead optimization process, a fully integrated local compound storage and liquid handling system was designed and implemented to automate the generation of assay-ready plates directly from newly submitted and cherry-picked compounds. A key feature of the system is the ability to create project- or assay-specific compound-handling methods, which provide flexibility for any combination of plate types, layouts, and plate bar-codes. Project-specific workflows can be created by linking methods for processing new and cherry-picked compounds and control additions to produce a complete compound set for both biological testing and local storage in one uninterrupted workflow. A flexible cherry-pick approach allows for multiple, user-defined strategies to select the most appropriate replicate of a compound for retesting. Examples of custom selection parameters include available volume, compound batch, and number of freeze/thaw cycles. This adaptable and integrated combination of software and hardware provides a basis for reducing cycle time, fully automating compound processing, and ultimately increasing the rate at which accurate, biologically relevant results can be produced for compounds of interest in the lead optimization process.
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Ramis, Joana M; Calvo, Javier; Matas, Aina; Corbillo, Cristina; Gayà, Antoni; Monjo, Marta
2018-06-28
Osteoinductive capacity of demineralized bone matrix (DBM) is sometimes insufficient or shows high variability between different batches of DBM. Here, we tried to improve its osteoinductive activity by alkali-urea or trypsin treatment but this strategy was unsuccessful. Then, we tested the enrichment of DBM with a bone protein extract (BPE) containing osteogenic growth factors comparing two sources: cortical bone powder and DBM. The osteoinductive capacity (alkaline phosphatase activity) of the obtained BPEs was evaluated in vitro in C2C12 cells. Specific protein levels present in the different BPE was determined by enzyme-linked immunosorbent assay or by a multiplex assay. BPE from cortical bone powder showed a lack of osteoinductive effect, in agreement with the low content on osteoinductive factors. In contrast, BPE from DBM showed osteoinductive activity but also high variability among donors. Thus, we decided to enrich DBM with BPE obtained from a pool of DBM from different donors. Following this strategy, we achieved increased osteoinductive activity and lower variability among donors. In conclusion, the use of a BPE obtained from a pool of demineralized bone to enrich DBM could be used to increase its osteoinductive effect and normalize the differences between donors.
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Bioplastic production using wood mill effluents as feedstock.
Ben, M; Mato, T; Lopez, A; Vila, M; Kennes, C; Veiga, M C
2011-01-01
Fibreboard production is one of the most important industrial activities in Galicia (Spain). Great amounts of wastewater are generated, with properties depending on the type of wood, treatment process, final product and water reusing, among others. These effluents are characterized by a high chemical oxygen demand, low pH and nutrients limitation. Although anaerobic digestion is one of the most suitable processes for the treatment, lately bioplastics production (mainly polyhydroxyalkanoates) from wastewaters with mixed cultures is being evaluated. Substrate requirements for these processes consist of high organic matter content and low nutrient concentration. Therefore, wood mill effluents could be a suitable feedstock. In this work, the possibility of producing bioplastics from to wood mill effluents is evaluated. First, wood mill effluent was converted to volatile fatty acids in an acidogenic reactor operated at two different hydraulic retention times of 1 and 1.5 d. The acidification percentage obtained was 37% and 42%, respectively. Then, aerobic batch assays were performed using fermented wood mill effluents obtained at different hydraulic retention times. Assays were developed using different cultures as inoculums. The maximum storage yield of 0.57 Cmmol/Cmmol was obtained when when the culture was enriched on a synthetic media.
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Inorganic fouling mitigation by salinity cycling in batch reverse osmosis.
Warsinger, David M; Tow, Emily W; Maswadeh, Laith A; Connors, Grace B; Swaminathan, Jaichander; Lienhard V, John H
2018-06-15
Enhanced fouling resistance has been observed in recent variants of reverse osmosis (RO) desalination which use time-varying batch or semi-batch processes, such as closed-circuit RO (CCRO) and pulse flow RO (PFRO). However, the mechanisms of batch processes' fouling resistance are not well-understood, and models have not been developed for prediction of their fouling performance. Here, a framework for predicting reverse osmosis fouling is developed by comparing the fluid residence time in batch and continuous (conventional) reverse osmosis systems to the nucleation induction times for crystallization of sparingly soluble salts. This study considers the inorganic foulants calcium sulfate (gypsum), calcium carbonate (calcite), and silica, and the work predicts maximum recovery ratios for the treatment of typical water sources using batch reverse osmosis (BRO) and continuous reverse osmosis. The prediction method is validated through comparisons to the measured time delay for CaSO 4 membrane scaling in a bench-scale, recirculating reverse osmosis unit. The maximum recovery ratio for each salt solution (CaCO 3 , CaSO 4 ) is individually predicted as a function of inlet salinity, as shown in contour plots. Next, the maximum recovery ratios of batch and conventional RO are compared across several water sources, including seawater, brackish groundwater, and RO brine. Batch RO's shorter residence times, associated with cycling from low to high salinity during each batch, enable significantly higher recovery ratios and higher salinity than in continuous RO for all cases examined. Finally, representative brackish RO brine samples were analyzed to determine the maximum possible recovery with batch RO. Overall, the induction time modeling methodology provided here can be used to allow batch RO to operate at high salinity and high recovery, while controlling scaling. The results show that, in addition to its known energy efficiency improvement, batch RO has superior inorganic fouling resistance relative to conventional RO. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Vereecken, H; Vanderborght, J; Kasteel, R; Spiteller, M; Schäffer, A; Close, M
2011-01-01
In this study, we analyzed sorption parameters for pesticides that were derived from batch and column or batch and field experiments. The batch experiments analyzed in this study were run with the same pesticide and soil as in the column and field experiments. We analyzed the relationship between the pore water velocity of the column and field experiments, solute residence times, and sorption parameters, such as the organic carbon normalized distribution coefficient ( ) and the mass exchange coefficient in kinetic models, as well as the predictability of sorption parameters from basic soil properties. The batch/column analysis included 38 studies with a total of 139 observations. The batch/field analysis included five studies, resulting in a dataset of 24 observations. For the batch/column data, power law relationships between pore water velocity, residence time, and sorption constants were derived. The unexplained variability in these equations was reduced, taking into account the saturation status and the packing status (disturbed-undisturbed) of the soil sample. A new regression equation was derived that allows estimating the values derived from column experiments using organic matter and bulk density with an value of 0.56. Regression analysis of the batch/column data showed that the relationship between batch- and column-derived values depends on the saturation status and packing of the soil column. Analysis of the batch/field data showed that as the batch-derived value becomes larger, field-derived values tend to be lower than the corresponding batch-derived values, and vice versa. The present dataset also showed that the variability in the ratio of batch- to column-derived value increases with increasing pore water velocity, with a maximum value approaching 3.5. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hunn, John D.; Helmreich, Grant W.; Dyer, John A.
Coated particle batches J52O-16-93172B and J52O-16-93173B were produced by Babcock and Wilcox Technologies (BWXT) as part of the production campaign for the Advanced Gas Reactor Fuel Development and Qualification (AGR) Program’s AGR-5/6/7 irradiation test in the Idaho National Laboratory (INL) Advanced Test Reactor (ATR), but were not used in the final fuel composite. However, these batches may be used as demonstration production-scale coated particle fuel for other experiments. Each batch was coated in a 150-mm-diameter production-scale fluidized-bed chemical vapor deposition (CVD) furnace. Tristructural isotropic (TRISO) coatings were deposited on 425-μm-nominal-diameter spherical kernels from BWXT lot J52R-16-69317 containing a mixture ofmore » 15.5%-enriched uranium carbide and uranium oxide (UCO). The TRISO coatings consisted of four consecutive CVD layers: a ~50% dense carbon buffer layer with 100-μm-nominal thickness, a dense inner pyrolytic carbon (IPyC) layer with 40-μm-nominal thickness, a silicon carbide (SiC) layer with 35-μm-nominal thickness, and a dense outer pyrolytic carbon (OPyC) layer with 40-μm-nominal thickness. The TRISO-coated particle batches were sieved to upgrade the particles by removing over-sized and under-sized material, and the upgraded batches were designated by appending the letter A to the end of the batch number (e.g., 93172A). Secondary upgrading by sieving was performed on the A-designated batches to remove particles with missing or very-thin buffer layers that were identified during previous analysis of the individual batches for defective IPyC, as reported in the acceptance test data report for the AGR-5/6/7 production batches [Hunn et al. 2017b]. The additionally-upgraded batches were designated by appending the letter B to the end of the batch number (e.g., 93172B).« less
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Lokshina, L Y; Vavilin, V A; Salminen, E; Rintala, J
2003-01-01
The anaerobic bioconversion of solid poultry slaughterhouse wastes was kinetically investigated. The modified version of
simulation model was applied for description of experimental data in mesophilic laboratory digester and assays. Additionally, stages of formation and consumption of long chain fatty acids (LCFA) were included in the model. Batch data on volatile solids, ammonium, acetate, butyrate, propionate, LCFA concentrations, pH level, cumulative volume, and methane partial pressure were used for model calibration. As a reference, the model was used to describe digestion of solid sorted household waste. Simulation results showed that an inhibition of polymer hydrolysis by volatile fatty acids and acetogenesis by NH3 or LCFA could be responsible for the complex system dynamics during degradation of lipid- and protein-rich wastes. -
Jorajuria, S; Raphalen, C; Dujardin, V; Daas, A
2015-01-01
Organization (WHO) International Standard (IS) for bleomycin complex A2/B2. Eight laboratories from different countries participated. Potencies of the candidate material were estimated by microbiological assays with sensitive micro-organisms. To ensure continuity between consecutive batches, the 1(st) IS for bleomycin complex A2/B2 was used as a reference. Based on the results of the study, the 2(nd) IS for bleomycin complex A2/B2 was adopted at the meeting of the WHO Expert Committee for Biological Standardization (ECBS) in 2014 with an assigned potency of 12 500 International Units (IU) per vial. The 2(nd) IS for bleomycin complex A2/B2 is available from the European Directorate for the Quality of Medicines & HealthCare (EDQM).
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Yazdi, Soroush H; Giles, Kristen L; White, Ian M
2013-11-05
We demonstrate sensitive and multiplexed detection of DNA sequences through a surface enhanced resonance Raman spectroscopy (SERRS)-based competitive displacement assay in an integrated microsystem. The use of the competitive displacement scheme, in which the target DNA sequence displaces a Raman-labeled reporter sequence that has lower affinity for the immobilized probe, enables detection of unlabeled target DNA sequences with a simple single-step procedure. In our implementation, the displacement reaction occurs in a microporous packed column of silica beads prefunctionalized with probe-reporter pairs. The use of a functionalized packed-bead column in a microfluidic channel provides two major advantages: (i) immobilization surface chemistry can be performed as a batch process instead of on a chip-by-chip basis, and (ii) the microporous network eliminates the diffusion limitations of a typical biological assay, which increases the sensitivity. Packed silica beads are also leveraged to improve the SERRS detection of the Raman-labeled reporter. Following displacement, the reporter adsorbs onto aggregated silver nanoparticles in a microfluidic mixer; the nanoparticle-reporter conjugates are then trapped and concentrated in the silica bead matrix, which leads to a significant increase in plasmonic nanoparticles and adsorbed Raman reporters within the detection volume as compared to an open microfluidic channel. The experimental results reported here demonstrate detection down to 100 pM of the target DNA sequence, and the experiments are shown to be specific, repeatable, and quantitative. Furthermore, we illustrate the advantage of using SERRS by demonstrating multiplexed detection. The sensitivity of the assay, combined with the advantages of multiplexed detection and single-step operation with unlabeled target sequences makes this method attractive for practical applications. Importantly, while we illustrate DNA sequence detection, the SERRS-based competitive displacement assay is applicable to detection of a variety of biological macromolecules, including proteins and proteolytic enzymes.
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1984-02-01
have been described previously (2]. The actual batch used was designated Batch D and was identical to that referred to as Batch C in Reference [2...Tetrazene was type RD1357 prepared at Materials Research Laboratories. The batch used was designated Batch 10/83(A). Lead Azide was type RD1343 and was...Preparation of Experimental Detonators Eperimental detonators were prepared in mild steel tubes, 6 mm o.d., 3.2 mm i.d., length 6 mm, prepared from
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Habijanic, Jozica; Berovic, Marin; Boh, Bojana; Wraber, Branka; Petravic-Tominac, Vlatka
2013-01-01
Submerged batch and repeated fed-batch cultivation techniques were used for mycelia cultivation and polysaccharide production of the Lingzhi or Reishi medicinal mushroom Ganoderma lucidum. Although most publications use various Asiatic G. lucidum strains, the growth of the strain Ga.l 4 (Biotechnical Faculty Strain Collection, Ljubljana, Slovenia), originally isolated from the Slovenian forest, is much faster. The results between the batch and repeated fed-batch cultivation are compared with the polysaccharide production in batch cultivation. From the aspect of biomass production, the best results were obtained in repeated fed-batch after 44 days, where 12.4 g/L of dry fungal biomass was obtained.
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Androgen receptors in the pelvic diaphragm muscles of dogs with and without perineal hernia.
Mann, F A; Nonneman, D J; Pope, E R; Boothe, H W; Welshons, W V; Ganjam, V K
1995-01-01
Levator ani and coccygeus muscle estrogen and androgen receptors were measured in 6, healthy, > or = 5-year-old, noncastrated, male Beagles (controls) and in 24 dogs with perineal hernia. Estrogen and androgen receptor analyses were performed on levator ani and coccygeus muscle specimens obtained from control dogs at the time of castration; contralateral levator ani and coccygeus muscle specimens were assayed 2 months after castration. During herniorrhaphy of dogs with perineal hernia, levator ani (non-castrated, n = 12; castrated, n = 7) and/or coccygeus (noncastrated, n = 5; castrated, n = 4) muscle biopsy specimens were obtained for estrogen and androgen receptor analyses. For estrogen and androgen receptor assays, each muscle biopsy specimen was homogenized in Tris-EDTA-glycerol buffer, and centrifuged at 30,000 x g; extracts were used for binding with ligands: [3H]methyltrienolone (3HR1881) for androgen receptors, and [3H]estradiol-17 beta for estrogen receptors. Extracts were incubated overnight at 0 to 4 C. Nonspecific binding was estimated, using 100-fold concentration of cold ligands. Bound and free hormones were separated, using hydroxylapatite batch assay. Receptor numbers for each tissue were calculated as femtomoles (fmol) per milligram of protein. Quantified data were compared between precastration and postcastration controls, using a paired t-test. One-way ANOVA and Bonferroni post-hoc test were used to compare values for precastration controls, postcastration controls, castrated dogs with perineal hernia, and noncastrated dogs with perineal hernia. Significance was set at P < 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)
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Ziels, Ryan M; Beck, David A C; Martí, Magalí; Gough, Heidi L; Stensel, H David; Svensson, Bo H
2015-04-01
The ecophysiology of long-chain fatty acid-degrading syntrophic β-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic β-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated. Microbial community dynamics were followed using qPCR and Illumina-based high-throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid. With repeated oleic acid feeding, the initial specific methane production rate significantly increased along with the relative abundances of Syntrophomonas and methanogenic archaea in the bioreactor communities. The novel qPCR assays showed that Syntrophomonas increased from 7 to 31% of the bacterial community 16S rRNA gene concentration, whereas that of Syntrophus decreased from 0.02 to less than 0.005%. High-throughput amplicon sequencing also revealed that Syntrophomonas became the dominant genus within the bioreactor microbiomes. These results suggest that increased specific mineralization rates of oleic acid were attributed to quantitative shifts within the microbial communities toward higher abundances of syntrophic β-oxidizing bacteria and methanogenic archaea. The novel qPCR assays targeting syntrophic β-oxidizing bacteria may thus serve as monitoring tools to indicate the fatty acid β-oxidization potential of anaerobic digester communities. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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The International Standard for Aureomycin
Humphrey, J. H.; Lightbown, J. W.; Mussett, M. V.; Perry, W. L. M.
1953-01-01
In 1950, the Department of Biological Standards, National Institute for Medical Research, London, was authorized by the WHO Expert Committee on Biological Standardization to proceed with the establishment of an International Standard for Aureomycin. A 100-g batch of aureomycin was obtained and was compared with the Standard Preparation of Aureomycin of the United States Food and Drug Administration (FDA) in a collaborative assay in which six laboratories in five countries participated. In all, 30 assays were carried out; 26 of these were done by biological methods, using Sarcina lutea, Bacillus pumilus, Staphylococcus aureus, or Bacillus cereus, and the remaining four by physicochemical methods. The results were subjected to standard methods of analysis, and the overall weighted mean potency (calculated from the biological assays only) was 1.0139, with limits of error of 99.5% to 100.5%. Since the International Standard is 1.39% more potent than the FDA Standard Preparation, it is probable that the latter contains a small amount of inert material; it is also possible that the International Standard itself is not 100% pure. For most practical purposes, however, both preparations may be regarded as substantially pure, and it is considered that to alter the present practice of quoting aureomycin dosage in metric units of weight would be inadvisable. Nevertheless, since the International Standard may not be a pure substance, a unit notation—for use where required in bioassays—is desirable, and the International Unit of Aureomycin has therefore been defined as the activity contained in one microgram of the International Standard. PMID:13141137
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Kuddus, Md Ruhul; Rumi, Farhana; Tsutsumi, Motosuke; Takahashi, Rika; Yamano, Megumi; Kamiya, Masakatsu; Kikukawa, Takashi; Demura, Makoto; Aizawa, Tomoyasu
2016-06-01
Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum). Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris. A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and (1)H NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active SN-1 in P. pastoris. Copyright © 2016 Elsevier Inc. All rights reserved.
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Wagner, L; Isbrucker, R; Locht, C; Arciniega, J; Costanzo, A; McFarland, R; Oh, H; Hoonakker, M; Descamps, J; Andersen, S R; Gupta, R K; Markey, K; Chapsal, J M; Lidster, K; Casey, W; Allen, D
2016-01-01
The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: In Search of Acceptable Alternatives to the Murine Histamine Sensitization Test (HIST): What is Possible and Practical?' was held on 4 and 5 March 2015 in London, United Kingdom. Participants discussed the results of the data generated from an international collaborative study (BSP114 Phase 2) sponsored by the European Directorate for the Quality of Medicines & Health Care (EDQM) to determine if a modified Chinese hamster ovary (CHO) cell-based clustering assay is a suitable alternative to replace HIST. Workshop participants agreed that protocol transferability demonstrated in the collaborative study indicates that a standardised CHO cell assay is adequate for measuring pure PTx in reference preparations. However, vaccine manufacturers would still need to demonstrate that the method is valid to detect or measure residual PTx in their specific adjuvanted products. The 2 modified CHO cell protocols included in the study (the Direct and the Indirect Methods) deserve further consideration as alternatives to HIST. Using the CHO cell assay, an in vitro alternative, for acellular pertussis (aP) vaccine batch release testing would reduce the number of animals used for aP vaccine safety testing. A strategic, stepwise adoption plan was proposed, in which the alternative test would be used for release purposes first, and then, once sufficient confidence in its suitable performance has been gained, its use would be extended to stability testing.
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Escobedo-Bonilla, César Marcial; Rangel, José Luis Ibarra
2014-01-01
Abstract The present study evaluated the susceptibility of three different batches of whiteleg shrimp Litopenaeus vannamei from Mexico to an inoculum of infectious hypodermal and haematopoietic necrosis virus (IHHNV). Each of the three shrimp batches came from a different hatchery. Because of their origin, it was possible that the genetic makeup of these batches was different among each other. The three batches tested showed differences in IHHNV susceptibility. Here, susceptibility is defined as the capacity of the host to become infected, and it can be measured by the infectivity titer. Susceptibility to IHHNV was observed in decreasing order in shrimp from batch 1 (hatchery from El Rosario, Sinaloa), batch 3 (hatchery from Nayarit) and batch 2 (hatchery from El Walamo, Sinaloa), respectively. The largest susceptibility difference between batches was 5012 times, and that between early and late juveniles from the same batch was 25 times. These results indicate that within a species, susceptibility to a pathogen such as IHHNV can have large differences. Susceptibility to pathogens is an important trait to consider before performing studies on pathogenesis. It may influence virological parameters such as speed of replication, pathogenicity and virus titer. In order to evaluate the potential use of IHHNV as a natural control agent against white spot syndrome virus (WSSV), it is necessary to know host susceptibility and the kinetics of IHHNV infection. These features can help to determine the conditions in which IHHNV could be used as antagonist in a WSSV infection. PMID:25561847
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Liu, Xiaoyan; Lv, Jinshun; Zhang, Tong; Deng, Yuanfang
2015-01-01
In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH4)2SO4, KH2PO4, or Mg(2+) were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the batch cultivation (3.9 g/L), and only 1.6 g/L of reducing sugar was left in the medium at the end of fed-batch cultivation, suggesting that most of the added carbon was used in the cultivation.
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Sun, Guoxiang; Zhang, Jingxian
2009-05-01
The three wavelength fusion high performance liquid chromatographic fingerprin (TWFFP) of Longdanxiegan pill (LDXGP) was established to identify the quality of LDXGP by the systematic quantified fingerprint method. The chromatographic fingerprints (CFPs) of the 12 batches of LDXGP were determined by reversed-phase high performance liquid chromatography. The technique of multi-wavelength fusion fingerprint was applied during processing the fingerprints. The TWFFPs containing 63 co-possessing peaks were obtained when choosing baicalin peak as the referential peak. The 12 batches of LDXGP were identified with hierarchical clustering analysis by using macro qualitative similarity (S(m)) as the variable. According to the results of classification, the referential fingerprint (RFP) was synthesized from 10 batches of LDXGP. Taking the RFP for the qualified model, all the 12 batches of LDXGP were evaluated by the systematic quantified fingerprint method. Among the 12 batches of LDXGP, 9 batches were completely qualified, the contents of 1 batch were obviously higher while the chemical constituents quantity and distributed proportion in 2 batches were not qualified. The systematic quantified fingerprint method based on the technique of multi-wavelength fusion fingerprint ca effectively identify the authentic quality of traditional Chinese medicine.
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Burmeister Getz, E; Carroll, K J; Mielke, J; Benet, L Z; Jones, B
2017-03-01
We previously demonstrated pharmacokinetic differences among manufacturing batches of a US Food and Drug Administration (FDA)-approved dry powder inhalation product (Advair Diskus 100/50) large enough to establish between-batch bio-inequivalence. Here, we provide independent confirmation of pharmacokinetic bio-inequivalence among Advair Diskus 100/50 batches, and quantify residual and between-batch variance component magnitudes. These variance estimates are used to consider the type I error rate of the FDA's current two-way crossover design recommendation. When between-batch pharmacokinetic variability is substantial, the conventional two-way crossover design cannot accomplish the objectives of FDA's statistical bioequivalence test (i.e., cannot accurately estimate the test/reference ratio and associated confidence interval). The two-way crossover, which ignores between-batch pharmacokinetic variability, yields an artificially narrow confidence interval on the product comparison. The unavoidable consequence is type I error rate inflation, to ∼25%, when between-batch pharmacokinetic variability is nonzero. This risk of a false bioequivalence conclusion is substantially higher than asserted by regulators as acceptable consumer risk (5%). © 2016 The Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of The American Society for Clinical Pharmacology and Therapeutics.
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Automated data processing and radioassays.
Samols, E; Barrows, G H
1978-04-01
Radioassays include (1) radioimmunoassays, (2) competitive protein-binding assays based on competition for limited antibody or specific binding protein, (3) immunoradiometric assay, based on competition for excess labeled antibody, and (4) radioreceptor assays. Most mathematical models describing the relationship between labeled ligand binding and unlabeled ligand concentration have been based on the law of mass action or the isotope dilution principle. These models provide useful data reduction programs, but are theoretically unfactory because competitive radioassay usually is not based on classical dilution principles, labeled and unlabeled ligand do not have to be identical, antibodies (or receptors) are frequently heterogenous, equilibrium usually is not reached, and there is probably steric and cooperative influence on binding. An alternative, more flexible mathematical model based on the probability or binding collisions being restricted by the surface area of reactive divalent sites on antibody and on univalent antigen has been derived. Application of these models to automated data reduction allows standard curves to be fitted by a mathematical expression, and unknown values are calculated from binding data. The vitrues and pitfalls are presented of point-to-point data reduction, linear transformations, and curvilinear fitting approaches. A third-order polynomial using the square root of concentration closely approximates the mathematical model based on probability, and in our experience this method provides the most acceptable results with all varieties of radioassays. With this curvilinear system, linear point connection should be used between the zero standard and the beginning of significant dose response, and also towards saturation. The importance is stressed of limiting the range of reported automated assay results to that portion of the standard curve that delivers optimal sensitivity. Published methods for automated data reduction of Scatchard plots for radioreceptor assay are limited by calculation of a single mean K value. The quality of the input data is generally the limiting factor in achieving good precision with automated as it is with manual data reduction. The major advantages of computerized curve fitting include: (1) handling large amounts of data rapidly and without computational error; (2) providing useful quality-control data; (3) indicating within-batch variance of the test results; (4) providing ongoing quality-control charts and between assay variance.
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A uniform bacterial growth potential assay for different water types.
Farhat, Nadia; Hammes, Frederik; Prest, Emmanuelle; Vrouwenvelder, Johannes
2018-06-06
The bacterial growth potential is important to understand and manage bacterial regrowth-related water quality concerns. Bacterial growth potential depends on growth promoting/limiting compounds, therefore, nutrient availability is the key factor governing bacterial growth potential. Selecting proper tools for bacterial growth measurement is essential for routine implementation of the growth potential measurement. This study proposes a growth potential assay that is universal and can be used for different water types and soil extract without restrictions of pure culture or cultivability of the bacterial strain. The proposed assay measures the sample bacterial growth potential by using the indigenous community as inocula. Flow cytometry (FCM) and adenosine tri-phosphate (ATP) were used to evaluate the growth potential of six different microbial communities indigenous to the sample being analyzed, with increasing carbon concentrations. Bottled mineral water, non-chlorinated tap water, seawater, river water, wastewater effluent and a soil organic carbon extract were analyzed. Results showed that indigenous bacterial communities followed normal batch growth kinetics when grown on naturally present organic carbon. Indigenous bacterial growth could detect spiked organic carbon concentrations as low as 10 μg/L. The indigenous community in all samples responded proportionally to the increase in acetate-carbon and proportional growth could be measured with both FCM and ATP. Bacterial growth was proportional to the carbon concentration but not the same proportion factor for the different water samples tested. The effect of inoculating the same water with different indigenous microbial communities on the growth potential was also examined. The FCM results showed that the highest increase in total bacterial cell concentration was obtained with bacteria indigenous to the water sample. The growth potential assay using indigenous bacterial community revealed consistent results of bacterial growth in all the different samples tested and therefore providing a fast, more stable, and accurate approach for monitoring the biological stability of waters compared to the previously developed assays. The growth potential assay can be used to aid in detecting growth limitations by compounds other than organic carbon. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Efficacy of rabies immunoglobulins in an experimental post-exposure prophylaxis rodent model.
Servat, Alexandre; Lutsch, Charles; Delore, Valentine; Lang, Jean; Veitch, Keith; Cliquet, Florence
2003-12-12
In a recently published Syrian hamster animal challenge study [Vaccine 19 (2001) 2273], a highly purified, heat-treated equine rabies immunoglobulin (pERIG HT, Favirab) did not elicit satisfactory protection. The efficacies of this batch, a second stage pERIG HT batch and reference RIG preparations (Imorab, Imogam Rage pasteurised, Berna antiserum) were compared in mice challenged with either Ariana canine field strain or CVS strain. Survival rates against Ariana challenge with the second pERIG HT batch were indistinguishable from those of other licensed preparations (83-90% survival), but the deficient batch did not provide satisfactory protection (53%). These data confirm the inadequate response to a first stage pERIG HT batch, but a current batch provides equivalent protection to that afforded by licensed HRIG and ERIG preparations.
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Bacteriophage PRD1 batch experiments to study attachment, detachment and inactivation processes
NASA Astrophysics Data System (ADS)
Sadeghi, Gholamreza; Schijven, Jack F.; Behrends, Thilo; Hassanizadeh, S. Majid; van Genuchten, Martinus Th.
2013-09-01
Knowledge of virus removal in subsurface environments is pivotal for assessing the risk of viral contamination of water resources and developing appropriate protection measures. Columns packed with sand are frequently used to quantify attachment, detachment and inactivation rates of viruses. Since column transport experiments are very laborious, a common alternative is to perform batch experiments where usually one or two measurements are done assuming equilibrium is reached. It is also possible to perform kinetic batch experiments. In that case, however, it is necessary to monitor changes in the concentration with time. This means that kinetic batch experiments will be almost as laborious as column experiments. Moreover, attachment and detachment rate coefficients derived from batch experiments may differ from those determined using column experiments. The aim of this study was to determine the utility of kinetic batch experiments and investigate the effects of different designs of the batch experiments on estimated attachment, detachment and inactivation rate coefficients. The experiments involved various combinations of container size, sand-water ratio, and mixing method (i.e., rolling or tumbling by pivoting the tubes around their horizontal or vertical axes, respectively). Batch experiments were conducted with clean quartz sand, water at pH 7 and ionic strength of 20 mM, and using the bacteriophage PRD1 as a model virus. Values of attachment, detachment and inactivation rate coefficients were found by fitting an analytical solution of the kinetic model equations to the data. Attachment rate coefficients were found to be systematically higher under tumbling than under rolling conditions because of better mixing and more efficient contact of phages with the surfaces of the sand grains. In both mixing methods, more sand in the container yielded higher attachment rate coefficients. A linear increase in the detachment rate coefficient was observed with increased solid-water ratio using tumbling method. Given the differences in the attachment rate coefficients, and assuming the same sticking efficiencies since chemical conditions of the batch and column experiments were the same, our results show that collision efficiencies of batch experiments are not the same as those of column experiments. Upscaling of the attachment rate from batch to column experiments hence requires proper understanding of the mixing conditions. Because batch experiments, in which the kinetics are monitored, are as laborious as column experiments, there seems to be no major advantage in performing batch instead of column experiments.
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Plastic antibody for the recognition of chemical warfare agent sulphur mustard.
Boopathi, M; Suryanarayana, M V S; Nigam, Anil Kumar; Pandey, Pratibha; Ganesan, K; Singh, Beer; Sekhar, K
2006-06-15
Molecularly imprinted polymers (MIPs) known as plastic antibodies (PAs) represent a new class of materials possessing high selectivity and affinity for the target molecule. Since their discovery, PAs have attracted considerable interest from bio- and chemical laboratories to pharmaceutical institutes. PAs are becoming an important class of synthetic materials mimicking molecular recognition by natural receptors. In addition, they have been utilized as catalysts, sorbents for solid-phase extraction, stationary phase for liquid chromatography and mimics of enzymes. In this paper, first time we report the preparation and characterization of a PA for the recognition of blistering chemical warfare agent sulphur mustard (SM). The SM imprinted PA exhibited more surface area when compared to the control non-imprinted polymer (NIP). In addition, SEM image showed an ordered nano-pattern for the PA of SM that is entirely different from the image of NIP. The imprinting also enhanced SM rebinding ability to the PA when compared to the NIP with an imprinting efficiency (alpha) of 1.3.
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Ince, Gozde Ozaydin; Armagan, Efe; Erdogan, Hakan; Buyukserin, Fatih; Uzun, Lokman; Demirel, Gokhan
2013-07-24
Molecular imprinting is a powerful, generic, and cost-effective technique; however, challenges still remain related to the fabrication and development of these systems involving nonhomogeneous binding sites, insufficient template removing, incompatibility with aqueous media, low rebinding capacity, and slow mass transfer. The vapor-phase deposition of polymers is a unique technique because of the conformal nature of coating and offers new possibilities in a number of applications including sensors, microfluidics, coating, and bioaffinity platforms. Herein, we demonstrated a simple but versatile concept to generate one-dimensional surface-imprinted polymeric nanotubes within anodic aluminum oxide (AAO) membranes based on initiated chemical vapor deposition (iCVD) technique for biorecognition of immunoglobulin G (IgG). It is reported that the fabricated surface-imprinted nanotubes showed high binding capacity and significant specific recognition ability toward target molecules compared with the nonimprinted forms. Given its simplicity and universality, the iCVD method can offer new possibilities in the field of molecular imprinting.
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Tang, Yiwei; Gao, Jingwen; Liu, Xiuying; Lan, Jianxing; Gao, Xue; Ma, Yong; Li, Min; Li, Jianrong
2016-06-15
A new magnetic molecularly imprinted polymers (MMIPs) for separation and concentration of ractopamine (RAC) were prepared using surface molecular imprinting technique with methacryloyl chloride as functional monomer and RAC as template. The MMIPs were characterized using transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and vibrating sample magnetometer. The results of re-binding experiments indicated that the MMIPs had fast adsorption kinetics and could reach binding equilibrium within 20 min, and the adsorption capacity of the MMIPs was 2.87-fold higher than that of the corresponding non-imprinted polymer. The selectivity of the MMIPs was evaluated according to its recognition to RAC and its analogues. The synthesized MMIPs were successfully applied to extraction, followed by high performance liquid chromatography to determine RAC in real food samples. Spiked recoveries ranged from 73.60% to 94.5%, with relative standard deviations of <11.17%. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Phospholipid imprinted polymers as selective endotoxin scavengers
NASA Astrophysics Data System (ADS)
Sulc, Robert; Szekely, Gyorgy; Shinde, Sudhirkumar; Wierzbicka, Celina; Vilela, Filipe; Bauer, David; Sellergren, Börje
2017-03-01
Herein we explore phospholipid imprinting as a means to design receptors for complex glycolipids comprising the toxic lipopolysaccharide endotoxin. A series of polymerizable bis-imidazolium and urea hosts were evaluated as cationic and neutral hosts for phosphates and phosphonates, the latter used as mimics of the phospholipid head groups. The bis-imidazolium hosts interacted with the guests in a cooperative manner leading to the presence of tight and well defined 1:2 ternary complexes. Optimized monomer combinations were subsequently used for imprinting of phosphatidic acid as an endotoxin dummy template. Presence of the aforementioned ternary complexes during polymerization resulted in imprinting of lipid dimers - the latter believed to crudely mimic the endotoxin Lipid A motif. The polymers were characterized with respect to template rebinding, binding affinity, capacity and common structural properties, leading to the identification of polymers which were thereafter subjected to an industrially validated endotoxin removal test. Two of the polymers were capable of removing endotoxin down to levels well below the accepted threshold (0.005 EU/mg API) in pharmaceutical production.
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Hung, Chih-Chang; Yabushita, Atsushi; Kobayashi, Takayoshi; Chen, Pei-Feng; Liang, Keng S
2016-01-01
Ultrafast transient absorption spectroscopy of endothelial NOS oxygenase domain (eNOS-oxy) was performed to study dynamics of ligand or substrate interaction under Soret band excitation. Photo-excitation dissociates imidazole ligand in <300fs, then followed by vibrational cooling and recombination within 2ps. Such impulsive bond breaking and late rebinding generate proteinquakes, which relaxes in several tens of picoseconds. The photo excited dynamics of eNOS-oxy with L-arginine substrate mainly occurs at the local site of heme, including ultrafast internal conversion within 400fs, vibrational cooling, charge transfer, and complete ground-state recovery within 1.4ps. The eNOS-oxy without additive is partially bound with water molecule, thus its photoexcited dynamics also shows ligand dissociation in <800fs. Then it followed by vibrational cooling coupled with charge transfer in 4.8ps, and recombination of ligand to distal side of heme in 12ps. Copyright © 2016 Elsevier B.V. All rights reserved.
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Recognition of Conformational Changes in β-Lactoglobulin by Molecularly Imprinted Thin Films
Turner, Nicholas W.; Liu, Xiao; Piletsky, Sergey A.; Hlady, Vladimir; Britt, David W.
2008-01-01
Pathogenesis in protein conformational diseases is initiated by changes in protein secondary structure. This molecular restructuring presents an opportunity for novel shape-based detection approaches, as protein molecular weight and chemistry are otherwise unaltered. Here we apply molecular imprinting to discriminate between distinct conformations of the model protein β-lactoglobulin (BLG). Thermal- and fluoro-alcohol-induced BLG isoforms were imprinted in thin films of 3-aminophenylboronic acid on quartz crystal microbalance chips. Enhanced rebinding of the template isoform was observed in all cases when compared to the binding of nontemplate isoforms over the concentration range of 1–100 µg mL−1. Furthermore, it was observed that the greater the changes in the secondary structure of the template protein the lower the binding of native BLG challenges to the imprint, suggesting a strong steric influence in the recognition system. This feasibility study is a first demonstration of molecular imprints for recognition of distinct conformations of the same protein. PMID:17665947
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Recognition of conformational changes in beta-lactoglobulin by molecularly imprinted thin films.
Turner, Nicholas W; Liu, Xiao; Piletsky, Sergey A; Hlady, Vladimir; Britt, David W
2007-09-01
Pathogenesis in protein conformational diseases is initiated by changes in protein secondary structure. This molecular restructuring presents an opportunity for novel shape-based detection approaches, as protein molecular weight and chemistry are otherwise unaltered. Here we apply molecular imprinting to discriminate between distinct conformations of the model protein beta-lactoglobulin (BLG). Thermal- and fluoro-alcohol-induced BLG isoforms were imprinted in thin films of 3-aminophenylboronic acid on quartz crystal microbalance chips. Enhanced rebinding of the template isoform was observed in all cases when compared to the binding of nontemplate isoforms over the concentration range of 1-100 microg mL(-1). Furthermore, it was observed that the greater the changes in the secondary structure of the template protein the lower the binding of native BLG challenges to the imprint, suggesting a strong steric influence in the recognition system. This feasibility study is a first demonstration of molecular imprints for recognition of distinct conformations of the same protein.
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CTCF and cohesin regulate chromatin loop stability with distinct dynamics
Hansen, Anders S; Pustova, Iryna; Cattoglio, Claudia; Tjian, Robert; Darzacq, Xavier
2017-01-01
Folding of mammalian genomes into spatial domains is critical for gene regulation. The insulator protein CTCF and cohesin control domain location by folding domains into loop structures, which are widely thought to be stable. Combining genomic and biochemical approaches we show that CTCF and cohesin co-occupy the same sites and physically interact as a biochemically stable complex. However, using single-molecule imaging we find that CTCF binds chromatin much more dynamically than cohesin (~1–2 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin for which the search process is long (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging 'dynamic complex' rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops are dynamic and frequently break and reform throughout the cell cycle. DOI: http://dx.doi.org/10.7554/eLife.25776.001 PMID:28467304
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Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin.
Zhou, Yongjun; Murphy, Annabel C; Samborskyy, Markiyan; Prediger, Patricia; Dias, Luiz Carlos; Leadlay, Peter F
2015-06-18
Conglobatin is an unusual C2-symmetrical macrodiolide from the bacterium Streptomyces conglobatus with promising antitumor activity. Insights into the genes and enzymes that govern both the assembly-line production of the conglobatin polyketide and its dimerization are essential to allow rational alterations to be made to the conglobatin structure. We have used a rapid, direct in vitro cloning method to obtain the entire cluster on a 41-kbp fragment, encoding a modular polyketide synthase assembly line. The cloned cluster directs conglobatin biosynthesis in a heterologous host strain. Using a model substrate to mimic the conglobatin monomer, we also show that the conglobatin cyclase/thioesterase acts iteratively, ligating two monomers head-to-tail then re-binding the dimer product and cyclizing it. Incubation of two different monomers with the cyclase produces hybrid dimers and trimers, providing the first evidence that conglobatin analogs may in future become accessible through engineering of the polyketide synthase. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
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Stuttering Min oscillations within E. coli bacteria: a stochastic polymerization model
NASA Astrophysics Data System (ADS)
Sengupta, Supratim; Derr, Julien; Sain, Anirban; Rutenberg, Andrew D.
2012-10-01
We have developed a 3D off-lattice stochastic polymerization model to study the subcellular oscillation of Min proteins in the bacteria Escherichia coli, and used it to investigate the experimental phenomenon of Min oscillation stuttering. Stuttering was affected by the rate of immediate rebinding of MinE released from depolymerizing filament tips (processivity), protection of depolymerizing filament tips from MinD binding and fragmentation of MinD filaments due to MinE. Processivity, protection and fragmentation each reduce stuttering, speed oscillations and MinD filament lengths. Neither processivity nor tip protection were, on their own, sufficient to produce fast stutter-free oscillations. While filament fragmentation could, on its own, lead to fast oscillations with infrequent stuttering; high levels of fragmentation degraded oscillations. The infrequent stuttering observed in standard Min oscillations is consistent with short filaments of MinD, while we expect that mutants that exhibit higher stuttering frequencies will exhibit longer MinD filaments. Increased stuttering rate may be a useful diagnostic to find observable MinD polymerization under experimental conditions.
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Ryan, Michael C; Zeeberg, Barry R; Caplen, Natasha J; Cleland, James A; Kahn, Ari B; Liu, Hongfang; Weinstein, John N
2008-01-01
Background Over 60% of protein-coding genes in vertebrates express mRNAs that undergo alternative splicing. The resulting collection of transcript isoforms poses significant challenges for contemporary biological assays. For example, RT-PCR validation of gene expression microarray results may be unsuccessful if the two technologies target different splice variants. Effective use of sequence-based technologies requires knowledge of the specific splice variant(s) that are targeted. In addition, the critical roles of alternative splice forms in biological function and in disease suggest that assay results may be more informative if analyzed in the context of the targeted splice variant. Results A number of contemporary technologies are used for analyzing transcripts or proteins. To enable investigation of the impact of splice variation on the interpretation of data derived from those technologies, we have developed SpliceCenter. SpliceCenter is a suite of user-friendly, web-based applications that includes programs for analysis of RT-PCR primer/probe sets, effectors of RNAi, microarrays, and protein-targeting technologies. Both interactive and high-throughput implementations of the tools are provided. The interactive versions of SpliceCenter tools provide visualizations of a gene's alternative transcripts and probe target positions, enabling the user to identify which splice variants are or are not targeted. The high-throughput batch versions accept user query files and provide results in tabular form. When, for example, we used SpliceCenter's batch siRNA-Check to process the Cancer Genome Anatomy Project's large-scale shRNA library, we found that only 59% of the 50,766 shRNAs in the library target all known splice variants of the target gene, 32% target some but not all, and 9% do not target any currently annotated transcript. Conclusion SpliceCenter provides unique, user-friendly applications for assessing the impact of transcript variation on the design and interpretation of RT-PCR, RNAi, gene expression microarrays, antibody-based detection, and mass spectrometry proteomics. The tools are intended for use by bench biologists as well as bioinformaticists. PMID:18638396
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The bacterial biota of laboratory-reared edible mealworms (Tenebrio molitor L.): From feed to frass.
Osimani, Andrea; Milanović, Vesna; Cardinali, Federica; Garofalo, Cristiana; Clementi, Francesca; Pasquini, Marina; Riolo, Paola; Ruschioni, Sara; Isidoro, Nunzio; Loreto, Nino; Franciosi, Elena; Tuohy, Kieran; Petruzzelli, Annalisa; Foglini, Martina; Gabucci, Claudia; Tonucci, Franco; Aquilanti, Lucia
2018-05-02
Tenebrio molitor represents one of the most popular species used for the large-scale conversion of plant biomass into protein and is characterized by high nutritional value. In the present laboratory study, the bacterial biota characterizing a pilot production chain of fresh T. molitor larvae was investigated. To this end, different batches of fresh mealworm larvae, their feeding substrate (wheatmeal) and frass were analyzed by viable microbial counts, PCR-DGGE and Illumina sequencing. Moreover, the occurrence of Coxiella burnetii, Pseudomonas aeruginosa and Shiga toxin-producing E. coli (STEC) was assessed through qualitative real-time PCR assays. Microbial viable counts highlighted low microbial contamination of the wheatmeal, whereas larvae and frass were characterized by high loads of Enterobacteriaceae, lactic acid bacteria, and several species of mesophilic aerobes. Spore-forming bacteria were detected to a lesser extent in all the samples. The combined molecular approach used to profile the microbiota confirmed the low microbial contamination of wheatmeal and allowed the detection of Enterobacter spp., Erwinia spp., Enterococcus spp. and Lactococcus spp. as dominant genera in both larvae and frass. Moreover, Klebsiella spp., Pantoea spp., and Xenorhabdus spp. were found to be in the minority. Entomoplasmatales (including Spiroplasma spp.) constituted a major fraction of the microbiota of one batch of larvae. From the real-time PCR assays, no sample was positive for either C. burnetii or STEC, whereas P. aeruginosa was detected in one sample of frass. Based on the overall results, two sources of microbial contamination were hypothesized, namely feeding with wheatmeal and vertical transmission of microorganisms from mother to offspring. Since mealworms are expected to be eaten as a whole, the overall outcomes collected in this laboratory study discourage the consumption of fresh mealworm larvae. Moreover, microbial loads and the absence of potential pathogens known to be associated with this insect species should be carefully assessed in order to reduce the minimum risk for consumers, by identifying the most opportune processing methods (e.g., boiling, frying, drying, etc.). Copyright © 2018 Elsevier B.V. All rights reserved.
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Production Methods in Industrial Microbiology.
ERIC Educational Resources Information Center
Gaden, Elmer L., Jr.
1981-01-01
Compares two methods (batch and continuous) in which microorganisms are used to produce industrial chemicals. Describes batch and continuous stirred-tank reactors and offers reasons why the batch method may be preferred. (JN)
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40 CFR 63.9520 - What procedures must I use to demonstrate initial compliance?
Code of Federal Regulations, 2010 CFR
2010-07-01
... the emission limitations in § 63.9500(a) and (b). (1) Record the date and time of each mix batch. (2) Record the identity of each mix batch using a unique batch ID, as defined in § 63.9565. (3) Measure and record the weight of HAP solvent loaded into the solvent mixer for each mix batch. (4) Measure and record...
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Jongenburger, I; Reij, M W; Boer, E P J; Gorris, L G M; Zwietering, M H
2011-11-15
The actual spatial distribution of microorganisms within a batch of food influences the results of sampling for microbiological testing when this distribution is non-homogeneous. In the case of pathogens being non-homogeneously distributed, it markedly influences public health risk. This study investigated the spatial distribution of Cronobacter spp. in powdered infant formula (PIF) on industrial batch-scale for both a recalled batch as well a reference batch. Additionally, local spatial occurrence of clusters of Cronobacter cells was assessed, as well as the performance of typical sampling strategies to determine the presence of the microorganisms. The concentration of Cronobacter spp. was assessed in the course of the filling time of each batch, by taking samples of 333 g using the most probable number (MPN) enrichment technique. The occurrence of clusters of Cronobacter spp. cells was investigated by plate counting. From the recalled batch, 415 MPN samples were drawn. The expected heterogeneous distribution of Cronobacter spp. could be quantified from these samples, which showed no detectable level (detection limit of -2.52 log CFU/g) in 58% of samples, whilst in the remainder concentrations were found to be between -2.52 and 2.75 log CFU/g. The estimated average concentration in the recalled batch was -2.78 log CFU/g and a standard deviation of 1.10 log CFU/g. The estimated average concentration in the reference batch was -4.41 log CFU/g, with 99% of the 93 samples being below the detection limit. In the recalled batch, clusters of cells occurred sporadically in 8 out of 2290 samples of 1g taken. The two largest clusters contained 123 (2.09 log CFU/g) and 560 (2.75 log CFU/g) cells. Various sampling strategies were evaluated for the recalled batch. Taking more and smaller samples and keeping the total sampling weight constant, considerably improved the performance of the sampling plans to detect such a type of contaminated batch. Compared to random sampling, stratified random sampling improved the probability to detect the heterogeneous contamination. Copyright © 2011 Elsevier B.V. All rights reserved.
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Kirwan, J A; Broadhurst, D I; Davidson, R L; Viant, M R
2013-06-01
Direct infusion mass spectrometry (DIMS)-based untargeted metabolomics measures many hundreds of metabolites in a single experiment. While every effort is made to reduce within-experiment analytical variation in untargeted metabolomics, unavoidable sources of measurement error are introduced. This is particularly true for large-scale multi-batch experiments, necessitating the development of robust workflows that minimise batch-to-batch variation. Here, we conducted a purpose-designed, eight-batch DIMS metabolomics study using nanoelectrospray (nESI) Fourier transform ion cyclotron resonance mass spectrometric analyses of mammalian heart extracts. First, we characterised the intrinsic analytical variation of this approach to determine whether our existing workflows are fit for purpose when applied to a multi-batch investigation. Batch-to-batch variation was readily observed across the 7-day experiment, both in terms of its absolute measurement using quality control (QC) and biological replicate samples, as well as its adverse impact on our ability to discover significant metabolic information within the data. Subsequently, we developed and implemented a computational workflow that includes total-ion-current filtering, QC-robust spline batch correction and spectral cleaning, and provide conclusive evidence that this workflow reduces analytical variation and increases the proportion of significant peaks. We report an overall analytical precision of 15.9%, measured as the median relative standard deviation (RSD) for the technical replicates of the biological samples, across eight batches and 7 days of measurements. When compared against the FDA guidelines for biomarker studies, which specify an RSD of <20% as an acceptable level of precision, we conclude that our new workflows are fit for purpose for large-scale, high-throughput nESI DIMS metabolomics studies.
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Are allergen batch differences and the use of double skin prick test important?
Thomsen, Gert F; Schlünssen, Vivi; Skadhauge, Lars R; Malling, Tine Halsen; Sherson, David L; Omland, Øyvind; Sigsgaard, Torben
2015-04-09
Skin prick tests (SPT) are widely used both in clinical diagnostics and in research. The standardization of allergen extracts is well documented to be crucial for the validity of SPT, whereas less emphasis has been placed on reproducibility and the SPT procedure itself. The objectives of this study are to clarify how the double skin prick test procedure influence the sensitivity and specificity of the test and to analyse the differences in weal size in skin prick tests between two batches of allergen extracts from the same vendor. The association between rhinitis and SPT was assessed among 1135 persons from a general population sample. SPT was performed twice with 10 common aeroallergens. In a subsample of 90 persons SPT was performed simultaneously with five of the allergens using different batches. Thirty percent had at least one positive SPT. Among asthmatics this number was 62%. Only minor differences were seen between the sizes of two weals from the same batch. A second SPT with the same batch did not change the association between rhinitis and sensitization. When performing SPT with two different batches disagreement was observed in 2% (Birch) to 11% (Cat) of the subjects. Performing SPT twice with the same allergen batch does not enhance the validity of the test, and value of double testing can be questioned. Considerable differences in SPT response with different batches from the same manufacturer were observed. Thus inter batch differences in allergen extracts might be a source of variability.
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El-Hawaz, Rabia F; Bridges, William C; Adelberg, Jeffrey W
2015-01-01
Plant density was varied with P, Ca, Mg, and KNO3 in a multifactor experiment to improve Curcuma longa L. micropropagation, biomass and microrhizome development in fed-batch liquid culture. The experiment had two paired D-optimal designs, testing sucrose fed-batch and nutrient sucrose fed-batch techniques. When sucrose became depleted, volume was restored to 5% m/v sucrose in 200 ml of modified liquid MS medium by adding sucrose solutions. Similarly, nutrient sucrose fed-batch was restored to set points with double concentration of treatments' macronutrient and MS micronutrient solutions, along with sucrose solutions. Changes in the amounts of water and sucrose supplementations were driven by the interaction of P and KNO3 concentrations. Increasing P from 1.25 to 6.25 mM increased both multiplication and biomass. The multiplication ratio was greatest in the nutrient sucrose fed-batch technique with the highest level of P, 6 buds/vessel, and the lowest level of Ca and KNO3. The highest density (18 buds/vessel) produced the highest fresh biomass at the highest concentrations of KNO3 and P with nutrient sucrose fed-batch, and moderate Ca and Mg concentrations. However, maximal rhizome dry biomass required highest P, sucrose fed-batch, and a moderate plant density. Different media formulations and fed-batch techniques were identified to maximize the propagation and storage organ responses. A single experimental design was used to optimize these dual purposes.
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El-Hawaz, Rabia F.; Bridges, William C.; Adelberg, Jeffrey W.
2015-01-01
Plant density was varied with P, Ca, Mg, and KNO3 in a multifactor experiment to improve Curcuma longa L. micropropagation, biomass and microrhizome development in fed-batch liquid culture. The experiment had two paired D-optimal designs, testing sucrose fed-batch and nutrient sucrose fed-batch techniques. When sucrose became depleted, volume was restored to 5% m/v sucrose in 200 ml of modified liquid MS medium by adding sucrose solutions. Similarly, nutrient sucrose fed-batch was restored to set points with double concentration of treatments’ macronutrient and MS micronutrient solutions, along with sucrose solutions. Changes in the amounts of water and sucrose supplementations were driven by the interaction of P and KNO3 concentrations. Increasing P from 1.25 to 6.25 mM increased both multiplication and biomass. The multiplication ratio was greatest in the nutrient sucrose fed-batch technique with the highest level of P, 6 buds/vessel, and the lowest level of Ca and KNO3. The highest density (18 buds/vessel) produced the highest fresh biomass at the highest concentrations of KNO3 and P with nutrient sucrose fed-batch, and moderate Ca and Mg concentrations. However, maximal rhizome dry biomass required highest P, sucrose fed-batch, and a moderate plant density. Different media formulations and fed-batch techniques were identified to maximize the propagation and storage organ responses. A single experimental design was used to optimize these dual purposes. PMID:25830292
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Wang, Lu; Zeng, Shanshan; Chen, Teng; Qu, Haibin
2014-03-01
A promising process analytical technology (PAT) tool has been introduced for batch processes monitoring. Direct analysis in real time mass spectrometry (DART-MS), a means of rapid fingerprint analysis, was applied to a percolation process with multi-constituent substances for an anti-cancer botanical preparation. Fifteen batches were carried out, including ten normal operations and five abnormal batches with artificial variations. The obtained multivariate data were analyzed by a multi-way partial least squares (MPLS) model. Control trajectories were derived from eight normal batches, and the qualification was tested by R(2) and Q(2). Accuracy and diagnosis capability of the batch model were then validated by the remaining batches. Assisted with high performance liquid chromatography (HPLC) determination, process faults were explained by corresponding variable contributions. Furthermore, a batch level model was developed to compare and assess the model performance. The present study has demonstrated that DART-MS is very promising in process monitoring in botanical manufacturing. Compared with general PAT tools, DART-MS offers a particular account on effective compositions and can be potentially used to improve batch quality and process consistency of samples in complex matrices. Copyright © 2014 Elsevier B.V. All rights reserved.
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Xu, Jian; Chen, Yang-Qiu; Zhang, Hong-Jian; Tang, Lei; Wang, Ke; Zhang, Jian-Hua; Chen, Xu-Sheng; Mao, Zhong-Gui
2014-08-01
In order to solve the problem of extraction wastewater pollution in citric acid industry, an integrated citric acid-methane fermentation process is proposed in this study. Extraction wastewater was treated by mesophilic anaerobic digestion and then used to make mash for the next batch of citric acid fermentation. The recycling process was done for seven batches. Citric acid production (82.4 g/L on average) decreased by 34.1 % in the recycling batches (2nd-7th) compared with the first batch. And the residual reducing sugar exceeded 40 g/L on average in the recycling batches. Pigment substances, acetic acid, ammonium, and metal ions in anaerobic digestion effluent (ADE) were considered to be the inhibitors, and their effects on the fermentation were studied. Results indicated that ammonium, Na(+) and K(+) in the ADE significantly inhibited citric acid fermentation. Therefore, the ADE was treated by acidic cation exchange resin prior to reuse to make mash for citric acid fermentation. The recycling process was performed for ten batches, and citric acid productions in the recycling batches were 126.6 g/L on average, increasing by 1.7 % compared with the first batch. This process could eliminate extraction wastewater discharge and reduce water resource consumption.
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Yan, Bin-jun; Qu, Hai-bin
2013-01-01
The efficacy of traditional Chinese medicine (TCM) is based on the combined effects of its constituents. Variation in chemical composition between batches of TCM has always been the deterring factor in achieving consistency in efficacy. The batch mixing process can significantly reduce the batch-to-batch quality variation in TCM extracts by mixing them in a well-designed proportion. However, reducing the quality variation without sacrificing too much of the production efficiency is one of the challenges. Accordingly, an innovative and practical batch mixing method aimed at providing acceptable efficiency for industrial production of TCM products is proposed in this work, which uses a minimum number of batches of extracts to meet the content limits. The important factors affecting the utilization ratio of the extracts (URE) were studied by simulations. The results have shown that URE was affected by the correlation between the contents of constituents, and URE decreased with the increase in the number of targets and the relative standard deviations of the contents. URE could be increased by increasing the number of storage tanks. The results have provided a reference for designing the batch mixing process. The proposed method has possible application value in reducing the quality variation in TCM and providing acceptable production efficiency simultaneously. PMID:24190450
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Yan, Bin-jun; Qu, Hai-bin
2013-11-01
The efficacy of traditional Chinese medicine (TCM) is based on the combined effects of its constituents. Variation in chemical composition between batches of TCM has always been the deterring factor in achieving consistency in efficacy. The batch mixing process can significantly reduce the batch-to-batch quality variation in TCM extracts by mixing them in a well-designed proportion. However, reducing the quality variation without sacrificing too much of the production efficiency is one of the challenges. Accordingly, an innovative and practical batch mixing method aimed at providing acceptable efficiency for industrial production of TCM products is proposed in this work, which uses a minimum number of batches of extracts to meet the content limits. The important factors affecting the utilization ratio of the extracts (URE) were studied by simulations. The results have shown that URE was affected by the correlation between the contents of constituents, and URE decreased with the increase in the number of targets and the relative standard deviations of the contents. URE could be increased by increasing the number of storage tanks. The results have provided a reference for designing the batch mixing process. The proposed method has possible application value in reducing the quality variation in TCM and providing acceptable production efficiency simultaneously.
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Xu, Li-Jian; Liu, Yuan-Shuai; Zhou, Li-Gang; Wu, Jian-Yong
2011-09-01
Beauvericin (BEA) is a cyclic hexadepsipeptide mycotoxin with notable phytotoxic and insecticidal activities. Fusarium redolens Dzf2 is a highly BEA-producing fungus isolated from a medicinal plant. The aim of the current study was to develop a simple and valid kinetic model for F. redolens Dzf2 mycelial growth and the optimal fed-batch operation for efficient BEA production. A modified Monod model with substrate (glucose) and product (BEA) inhibition was constructed based on the culture characteristics of F. redolens Dzf2 mycelia in a liquid medium. Model parameters were derived by simulation of the experimental data from batch culture. The model fitted closely with the experimental data over 20-50 g l(-1) glucose concentration range in batch fermentation. The kinetic model together with the stoichiometric relationships for biomass, substrate and product was applied to predict the optimal feeding scheme for fed-batch fermentation, leading to 54% higher BEA yield (299 mg l(-1)) than in the batch culture (194 mg l(-1)). The modified Monod model incorporating substrate and product inhibition was proven adequate for describing the growth kinetics of F. redolens Dzf2 mycelial culture at suitable but not excessive initial glucose levels in batch and fed-batch cultures.
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Combined effects of presalted prerigor and postrigor batter mixtures on chicken breast gelation.
Choi, Yun-Sang; Kim, Hyun-Wook; Hwang, Ko-Eun; Song, Dong-Hun; Jeong, Tae-Jun; Jeon, Ki-Hong; Kim, Young-Boong; Kim, Cheon-Jei
2015-04-01
We examined the combined effects of prerigor and postrigor batter mixtures on protein gelation. The postrigor batter was prepared with 2% salt, whereas the prerigor meat at 5 min postmortem was used to prepare postrigor batters at different salt levels. For 5 treatments, prerigor batters were mixed with postrigor batter that had 2% salt (control) as follows: T1: ground presalted (1%) hot-boned breast with 1% salt for 50% total batch; T2: ground presalted (2%) hot-boned breast for 50% total batch; T3: ground presalted (3%) hot-boned breast for 30% total batch that was mixed with cold-boned batter for 50% total batch; T4: ground presalted (4%) hot-boned breast for 25% total batch that was mixed with cold-boned batter for 50% total batch; and T5: ground presalted (5%) hot-boned breast for 20% total batch that was mixed with cold-boned batter for 50% total batch. Treatments with both presalted prerigor and postrigor muscle showed less cooking loss and lower emulsion stability than the control, except T5. The protein solubility and apparent viscosity of the control was the lowest. Thus, presalted hot-boned muscle combined with cold-boned muscle positively affected physicochemical properties. © 2015 Poultry Science Association Inc.
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Fault detection and diagnosis in an industrial fed-batch cell culture process.
Gunther, Jon C; Conner, Jeremy S; Seborg, Dale E
2007-01-01
A flexible process monitoring method was applied to industrial pilot plant cell culture data for the purpose of fault detection and diagnosis. Data from 23 batches, 20 normal operating conditions (NOC) and three abnormal, were available. A principal component analysis (PCA) model was constructed from 19 NOC batches, and the remaining NOC batch was used for model validation. Subsequently, the model was used to successfully detect (both offline and online) abnormal process conditions and to diagnose the root causes. This research demonstrates that data from a relatively small number of batches (approximately 20) can still be used to monitor for a wide range of process faults.
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T'jollyn, Huybrecht; Snoeys, Jan; Van Bocxlaer, Jan; De Bock, Lies; Annaert, Pieter; Van Peer, Achiel; Allegaert, Karel; Mannens, Geert; Vermeulen, An; Boussery, Koen
2017-06-01
Although the measurement of cytochrome P450 (CYP) contributions in metabolism assays is straightforward, determination of actual in vivo contributions might be challenging. How representative are in vitro for in vivo CYP contributions? This article proposes an improved strategy for the determination of in vivo CYP enzyme-specific metabolic contributions, based on in vitro data, using an in vitro-in vivo extrapolation (IVIVE) approach. Approaches are exemplified using tramadol as model compound, and CYP2D6 and CYP3A4 as involved enzymes. Metabolism data for tramadol and for the probe substrates midazolam (CYP3A4) and dextromethorphan (CYP2D6) were gathered in human liver microsomes (HLM) and recombinant human enzyme systems (rhCYP). From these probe substrates, an activity-adjustment factor (AAF) was calculated per CYP enzyme, for the determination of correct hepatic clearance contributions. As a reference, tramadol CYP contributions were scaled-back from in vivo data (retrograde approach) and were compared with the ones derived in vitro. In this view, the AAF is an enzyme-specific factor, calculated from reference probe activity measurements in vitro and in vivo, that allows appropriate scaling of a test drug's in vitro activity to the 'healthy volunteer' population level. Calculation of an AAF, thus accounts for any 'experimental' or 'batch-specific' activity difference between in vitro HLM and in vivo derived activity. In this specific HLM batch, for CYP3A4 and CYP2D6, an AAF of 0.91 and 1.97 was calculated, respectively. This implies that, in this batch, the in vitro CYP3A4 activity is 1.10-fold higher and the CYP2D6 activity 1.97-fold lower, compared to in vivo derived CYP activities. This study shows that, in cases where the HLM pool does not represent the typical mean population CYP activities, AAF correction of in vitro metabolism data, optimizes CYP contributions in the prediction of hepatic clearance. Therefore, in vitro parameters for any test compound, obtained in a particular batch, should be corrected with the AAF for the respective enzymes. In the current study, especially the CYP2D6 contribution was found, to better reflect the average in vivo situation. It is recommended that this novel approach is further evaluated using a broader range of compounds.
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Results of Hg speciation testing on DWPF SMECT-8, OGCT-1, AND OGCT-2 samples
DOE Office of Scientific and Technical Information (OSTI.GOV)
Bannochie, C.
2016-02-22
The Savannah River National Laboratory (SRNL) was tasked with preparing and shipping samples for Hg speciation by Eurofins Frontier Global Sciences, Inc. in Seattle, WA on behalf of the Savannah River Remediation (SRR) Mercury Task Team. The sixteenth shipment of samples was designated to include a Defense Waste Processing Facility (DWPF) Slurry Mix Evaporator Condensate Tank (SMECT) sample from Sludge Receipt and Adjustment Tank (SRAT) Batch 738 processing and two Off-Gas Condensate Tank (OGCT) samples, one following Batch 736 and one following Batch 738. The DWPF sample designations for the three samples analyzed are provided. The Batch 738 ‘End ofmore » SME Cycle’ SMECT sample was taken at the conclusion of Slurry Mix Evaporator (SME) operations for this batch and represents the fourth SMECT sample examined from Batch 738. Batch 738 experienced a sludge slurry carryover event, which introduced sludge solids to the SMECT that were particularly evident in the SMECT-5 sample, but less evident in the ‘End of SME Cycle’ SMECT-8 sample.« less
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Han, Xiaolong; Song, Wenxia; Liu, Guodong; Li, Zhonghai; Yang, Piao; Qu, Yinbo
2017-03-01
Medium optimization and repeated fed-batch fermentation were performed to improve the cellulase productivity by P. oxalicum RE-10 in submerged fermentation. First, Plackett-Burman design (PBD) and central composite design (CCD) were used to optimize the medium for cellulase production. PBD demonstrated wheat bran and NaNO 3 had significant influences on cellulase production. The CCD results showed the maximum filter paper activity (FPA) production of 8.61U/mL could be achieved in Erlenmeyer flasks. The maximal FPA reached 12.69U/mL by submerged batch fermentation in a 7.5-L stirred tank, 1.76-fold higher than that on the original medium. Then, the repeated fed-batch fermentation strategy was performed successfully for increasing the cellulase productivity from 105.75U/L/h in batch fermentation to 158.38U/L/h. The cellulase activity and the glucan conversion of delignined corn cob residue hydrolysis had no significant difference between the enzymes sampled from different cycles of the repeated fed-batch fermentation and that from batch culture. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Gahlawat, Geeta; Srivastava, Ashok K
2013-06-01
In the present investigation, batch cultivation of Azohydromonas australica DSM 1124 was carried out in a bioreactor for growth associated PHB production. The observed batch PHB production kinetics data was then used for the development of a mathematical model which adequately described the substrate limitation and inhibition during the cultivation. The statistical validity test demonstrated that the proposed mathematical model predictions were significant at 99% confidence level. The model was thereafter extrapolated to fed-batch to identify various nutrients feeding regimes during the bioreactor cultivation to improve the PHB accumulation. The distinct capability of the mathematical model to predict highly dynamic fed-batch cultivation strategies was demonstrated by experimental implementation of two fed-batch cultivation strategies. A significantly high PHB concentration of 22.65 g/L & an overall PHB content of 76% was achieved during constant feed rate fed-batch cultivation which is the highest PHB content reported so far using A. australica. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Ötes, Ozan; Flato, Hendrik; Winderl, Johannes; Hubbuch, Jürgen; Capito, Florian
2017-10-10
The protein A capture step is the main cost-driver in downstream processing, with high attrition costs especially when using protein A resin not until end of resin lifetime. Here we describe a feasibility study, transferring a batch downstream process to a hybrid process, aimed at replacing batch protein A capture chromatography with a continuous capture step, while leaving the polishing steps unchanged to minimize required process adaptations compared to a batch process. 35g of antibody were purified using the hybrid approach, resulting in comparable product quality and step yield compared to the batch process. Productivity for the protein A step could be increased up to 420%, reducing buffer amounts by 30-40% and showing robustness for at least 48h continuous run time. Additionally, to enable its potential application in a clinical trial manufacturing environment cost of goods were compared for the protein A step between hybrid process and batch process, showing a 300% cost reduction, depending on processed volumes and batch cycles. Copyright © 2017 Elsevier B.V. All rights reserved.
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Choi, Minsung; Al-Zahrani, Saeed M; Lee, Sang Yup
2014-06-01
Arabic date is overproduced in Arabic countries such as Saudi Arabia and Iraq and is mostly composed of sugars (70-80 wt%). Here we developed a fed-batch fermentation process by using a kinetic model for the efficient production of lactic acid to a high concentration from Arabic date juice. First, a kinetic model of Lactobacillus rhamnosus grown on date juice in batch fermentation was constructed in EXCEL so that the estimation of parameters and simulation of the model can be easily performed. Then, several fed-batch fermentations were conducted by employing different feeding strategies including pulsed feeding, exponential feeding, and modified exponential feeding. Based on the results of fed-batch fermentations, the kinetic model for fed-batch fermentation was also developed. This new model was used to perform feed-forward controlled fed-batch fermentation, which resulted in the production of 171.79 g l(-1) of lactic acid with the productivity and yield of 1.58 and 0.87 g l(-1) h(-1), respectively.
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Flow Cytometry: Evolution of Microbiological Methods for Probiotics Enumeration.
Pane, Marco; Allesina, Serena; Amoruso, Angela; Nicola, Stefania; Deidda, Francesca; Mogna, Luca
2018-05-14
The purpose of this trial was to verify that the analytical method ISO 19344:2015 (E)-IDF 232:2015 (E) is valid and reliable for quantifying the concentration of the probiotic Lactobacillus rhamnosus GG (ATCC 53103) in a finished product formulation. Flow cytometry assay is emerging as an alternative rapid method for microbial detection, enumeration, and population profiling. The use of flow cytometry not only permits the determination of viable cell counts but also allows for enumeration of damaged and dead cell subpopulations. Results are expressed as TFU (Total Fluorescent Units) and AFU (Active Fluorescent Units). In December 2015, the International Standard ISO 19344-IDF 232 "Milk and milk products-Starter cultures, probiotics and fermented products-Quantification of lactic acid bacteria by flow cytometry" was published. This particular ISO can be applied universally and regardless of the species of interest. Analytical method validation was conducted on 3 different industrial batches of L. rhamnosus GG according to USP39<1225>/ICH Q2R1 in term of: accuracy, precision (repeatability), intermediate precision (ruggedness), specificity, limit of quantification, linearity, range, robustness. The data obtained on the 3 batches of finished product have significantly demonstrated the validity and robustness of the cytofluorimetric analysis. On the basis of the results obtained, the ISO 19344:2015 (E)-IDF 232:2015 (E) "Quantification of lactic acid bacteria by flow cytometry" can be used for the enumeration of L. rhamnosus GG in a finished product formulation.
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Karray, Raida; Karray, Fatma; Loukil, Slim; Mhiri, Najla; Sayadi, Sami
2017-03-01
Ulva rigida is a green macroalgae, abundantly available in the Mediterranean which offers a promising source for the production of valuable biomaterials, including methane. In this study, anaerobic digestion assays in a batch mode was performed to investigate the effects of various inocula as a mixture of fresh algae, bacteria, fungi and sediment collected from the coast of Sfax, on biogas production from Ulva rigida. The results revealed that the best inoculum to produce biogas and feed an anaerobic reactor is obtained through mixing decomposed macroalgae with anaerobic sludge and water, yielding into 408mL of biogas. The process was then investigated in a sequencing batch reactor (SBR) which led to an overall biogas production of 375mL with 40% of methane. Further co-digestion studies were performed in an anaerobic up-flow bioreactor using sugar wastewater as a co-substrate. A high biogas production yield of 114mL g -1 VS added was obtained with 75% of methane. The co-digestion proposed in this work allowed the recovery of natural methane, providing a promising alternative to conventional anaerobic microbial fermentation using Tunisian green macroalgae. Finally, in order to identify the microbial diversity present in the reactor during anaerobic digestion of Ulva rigida, the prokaryotic diversity was investigated in this bioreactor by the denaturing gradient gel electrophoresis (DGGE) method targeting the 16S rRNA gene. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Expectoration of Flaviviruses during sugar feeding by mosquitoes (Diptera: Culicidae).
van den Hurk, Andrew F; Johnson, Petrina H; Hall-Mendelin, Sonja; Northill, Judy A; Simmons, Russell J; Jansen, Cassie C; Frances, Stephen P; Smith, Greg A; Ritchie, Scott A
2007-09-01
Biological transmission of arboviruses to a vertebrate host occurs when virions are expelled along with saliva during blood feeding by a hematophagous arthropod. We undertook experiments to determine whether mosquitoes expectorate flaviviruses in their saliva while sugar feeding. Batches of Culex annulirostris Skuse and Culex gelidus Theobald (Diptera: Culicidae) were orally infected with Japanese encephalitis (family Flaviviridae, genus Flavivirus, JEV), Kunjin (family Flaviviridae, genus Flavivirus, KUNV; a subtype of West Nile virus), and Murray Valley encephalitis (family Flaviviridae, genus Flavivirus, MVEV) viruses. After a 7-d extrinsic incubation, these mosquitoes were offered sucrose meals via cotton pledgets, which were removed daily and processed for viral RNA by using real-time TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) assays. JEV, MVEV, and KUNV RNA was detected in all pledgets removed from batches of Cx. gelidus on days 7-14 postexposure. In contrast, detection rates were variable for Cx. annulirostris, with KUNV detected in 0.3 M sucrose pledgets on all days postexposure, and JEV and MVEV detected on 57 and 50% of days postexposure, respectively. Higher concentrations of sucrose in the pledget did not increase virus detection rates. When individual JEV-infected Cx. gelidus were exposed to the sucrose pledget, 73% of mosquitoes expectorated virus with titers that were detectable by TaqMan RT-PCR. These results clearly show that flaviviruses are expectorated by infected mosquitoes during the process of sugar feeding on artificial pledgets. Potential applications of the method for arboviral bioassays and field surveillance are discussed.
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Analytical and functional similarity of Amgen biosimilar ABP 215 to bevacizumab
Seo, Neungseon; Polozova, Alla; Zhang, Mingxuan; Yates, Zachary; Cao, Shawn; Li, Huimin; Kuhns, Scott; Maher, Gwendolyn; McBride, Helen J.; Liu, Jennifer
2018-01-01
ABSTRACT ABP 215 is a biosimilar product to bevacizumab. Bevacizumab acts by binding to vascular endothelial growth factor A, inhibiting endothelial cell proliferation and new blood vessel formation, thereby leading to tumor vasculature normalization. The ABP 215 analytical similarity assessment was designed to assess the structural and functional similarity of ABP 215 and bevacizumab sourced from both the United States (US) and the European Union (EU). Similarity assessment was also made between the US- and EU-sourced bevacizumab to assess the similarity between the two products. The physicochemical properties and structural similarity of ABP 215 and bevacizumab were characterized using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. ABP 215 has the same amino acid sequence and exhibits similar post-translational modification profiles compared to bevacizumab. The functional similarity assessment employed orthogonal assays designed to interrogate all expected biological activities, including those known to affect the mechanisms of action for ABP 215 and bevacizumab. More than 20 batches of bevacizumab (US) and bevacizumab (EU), and 13 batches of ABP 215 representing unique drug substance lots were assessed for similarity. The large dataset allows meaningful comparisons and garners confidence in the overall conclusion for the analytical similarity assessment of ABP 215 to both US- and EU-sourced bevacizumab. The structural and purity attributes, and biological properties of ABP 215 are demonstrated to be highly similar to those of bevacizumab. PMID:29553864
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Code of Federal Regulations, 2012 CFR
2012-04-01
... 21 Food and Drugs 4 2012-04-01 2012-04-01 false What actions must I take if a batch of PET drug... actions must I take if a batch of PET drug product does not conform to specifications? (a) Rejection of nonconforming product. You must reject a batch of a PET drug product that does not conform to specifications...
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Code of Federal Regulations, 2014 CFR
2014-04-01
... 21 Food and Drugs 4 2014-04-01 2014-04-01 false What actions must I take if a batch of PET drug... actions must I take if a batch of PET drug product does not conform to specifications? (a) Rejection of nonconforming product. You must reject a batch of a PET drug product that does not conform to specifications...
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Code of Federal Regulations, 2011 CFR
2011-04-01
... 21 Food and Drugs 4 2011-04-01 2011-04-01 false What actions must I take if a batch of PET drug... actions must I take if a batch of PET drug product does not conform to specifications? (a) Rejection of nonconforming product. You must reject a batch of a PET drug product that does not conform to specifications...
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Code of Federal Regulations, 2013 CFR
2013-04-01
... 21 Food and Drugs 4 2013-04-01 2013-04-01 false What actions must I take if a batch of PET drug... actions must I take if a batch of PET drug product does not conform to specifications? (a) Rejection of nonconforming product. You must reject a batch of a PET drug product that does not conform to specifications...
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Laboratory Evaluation of Expedient Low-Temperature Admixtures for Runway Craters in Cold Weather
2014-10-01
ignores typical concerns, such as long-term durability, aesthetics, and corrosion , that are of minimal importance in this expedient field- use ...those identified to be in common use although it was acknowledged that the chloride based compounds caused corrosion problems (Korhonen 1990...batch to batch when using less than a 2000 lb batch, our ability to model and predict results with each batch is degraded. How- ever, when we control
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Correcting for batch effects in case-control microbiome studies
Gibbons, Sean M.; Duvallet, Claire
2018-01-01
High-throughput data generation platforms, like mass-spectrometry, microarrays, and second-generation sequencing are susceptible to batch effects due to run-to-run variation in reagents, equipment, protocols, or personnel. Currently, batch correction methods are not commonly applied to microbiome sequencing datasets. In this paper, we compare different batch-correction methods applied to microbiome case-control studies. We introduce a model-free normalization procedure where features (i.e. bacterial taxa) in case samples are converted to percentiles of the equivalent features in control samples within a study prior to pooling data across studies. We look at how this percentile-normalization method compares to traditional meta-analysis methods for combining independent p-values and to limma and ComBat, widely used batch-correction models developed for RNA microarray data. Overall, we show that percentile-normalization is a simple, non-parametric approach for correcting batch effects and improving sensitivity in case-control meta-analyses. PMID:29684016
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Li, Xin; Zhou, Jin; Ouyang, Shuiping; Ouyang, Jia; Yong, Qiang
2017-02-01
Production of fumaric acid from alkali-pretreated corncob (APC) at high solids loading was investigated using a combination of separated hydrolysis and fermentation (SHF) and fed-batch simultaneous saccharification and fermentation (SSF) by Rhizopus oryzae. Four different fermentation modes were tested to maximize fumaric acid concentration at high solids loading. The highest concentration of 41.32 g/L fumaric acid was obtained from 20 % (w/v) APC at 38 °C in the combined SHF and fed-batch SSF process, compared with 19.13 g/L fumaric acid in batch SSF alone. The results indicated that a combination of SHF and fed-batch SSF significantly improved production of fumaric acid from lignocellulose by R. oryzae than that achieved with batch SSF at high solids loading.
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Data-driven monitoring for stochastic systems and its application on batch process
NASA Astrophysics Data System (ADS)
Yin, Shen; Ding, Steven X.; Haghani Abandan Sari, Adel; Hao, Haiyang
2013-07-01
Batch processes are characterised by a prescribed processing of raw materials into final products for a finite duration and play an important role in many industrial sectors due to the low-volume and high-value products. Process dynamics and stochastic disturbances are inherent characteristics of batch processes, which cause monitoring of batch processes a challenging problem in practice. To solve this problem, a subspace-aided data-driven approach is presented in this article for batch process monitoring. The advantages of the proposed approach lie in its simple form and its abilities to deal with stochastic disturbances and process dynamics existing in the process. The kernel density estimation, which serves as a non-parametric way of estimating the probability density function, is utilised for threshold calculation. An industrial benchmark of fed-batch penicillin production is finally utilised to verify the effectiveness of the proposed approach.
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Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose
DiScipio, Richard G.; Liddington, Robert C.; Schraufstatter, Ingrid U.
2016-01-01
A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315
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Jiang, Jianlan; Zhang, Huan; Li, Zidan; Zhang, Xiaohang; Su, Xin; Li, Yan; Qiao, Bin; Yuan, Yingjin
2013-08-01
We investigated the fingerprints of 48 batches of turmeric total extracts (TTE) by HPLC-MS-MS and GC-MS analyses and 43 characteristic peaks (22 constituents from HPLC-MS-MS; 21 from GC-MS) were analyzed qualitatively and quantitatively. An MTT {3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide} assay was implemented to measure the cytotoxicity of the TTE against HeLa cells. Then we utilized orthogonal partial least squares analysis, which correlated the chemical composition of the TTE to its cytotoxic activity, to identify potential cytotoxic constituents from turmeric. The result showed that 19 constituents contributed significantly to the cytotoxicity. The obtained result was verified by canonical correlation analysis. Comparison with previous reports also indicated some interaction between the curcuminoids and sesquiterpenoids in turmeric.
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Jagannathan, Bharat; Golbeck, John H
2008-12-01
The photosynthetic reaction center from the green sulfur bacterium Chlorobium tepidum (CbRC) was solubilized from membranes using Triton X-100 and isolated by sucrose density ultra-centrifugation. The CbRC complexes were subsequently treated with 0.5 M NaCl and ultrafiltered over a 100 kDa cutoff membrane. The resulting CbRC cores did not exhibit the low-temperature EPR resonances from FA- and FB- and were unable to reduce NADP+. SDS-PAGE and mass spectrometric analysis showed that the PscB subunit, which harbors the FA and FB clusters, had become dissociated, and was now present in the filtrate. Attempts to rebind PscB onto CbRC cores were unsuccessful. Mössbauer spectroscopy showed that recombinant PscB contains a heterogeneous mixture of [4Fe-4S]2+,1+ and other types of Fe/S clusters tentatively identified as [2Fe-2S]2+,1+ clusters and rubredoxin-like Fe3+,2+ centers, and that the [4Fe-4S]2+,1+ clusters which were present were degraded at high ionic strength. Quantitative analysis confirmed that the amount of iron and sulfide in the recombinant protein was sub-stoichiometric. A heme-staining assay indicated that cytochrome c551 remained firmly attached to the CbRC cores. Low-temperature EPR spectroscopy of photoaccumulated CbRC complexes and CbRC cores showed resonances between g=5.4 and 4.4 assigned to a S=3/2 ground spin state [4Fe-4S]1+ cluster and at g=1.77 assigned to a S=1/2 ground spin state [4Fe-4S]1+ cluster, both from FX-. These results unify the properties of the acceptor side of the Type I homodimeric reaction centers found in green sulfur bacteria and heliobacteria: in both, the FA and FB iron-sulfur clusters are present on a salt-dissociable subunit, and FX is present as an interpolypeptide [4Fe-4S]2+,1+ cluster with a significant population in a S=3/2 ground spin state.
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Clayton, R N; Shakespear, R A; Duncan, J A; Marshall, J C
1979-05-01
Inactivation of LHRH by purified bovine pituitary plasma membranes was studied in vitro. After incubation of [125I]iodo-LHRH with plasma membranes, the amount of tracer bound to the pellet was measured, and the integrity of the unbound tracer in the supernatant was assessed. Reduction in ability to bind to anti-LHRH serum and to rebind to plasma membranes together with altered electrophoretic mobility on polyacrylamide gels showed that the unbound [125I]iodo-LHRH was inactivated. LHRH inactivation occurred rapidly and was dependent upon membrane concentration and incubation temperature. These results indicate that hormone inactivation must be taken into account in the interpretation of LHRH-receptor interactions. During 37 C incubations, the apparent absence of specific LHRH binding can be explained by inactivation of tracer hormone. Significant LHRH inactivation also occurred at 0 C, which in part explains the insensitivity of LHRH receptor assays. Assessment of LHRH inactivation by different particulate subcellular fractions of pituitary tissue showed that the inactivating enzyme was associated with the plasma membranes; other organelles did not alter LHRH. The enzyme appeared to be an integral part of the plasma membrane structure, since enzymic activity could not be removed by washing without reducing specific LHRH binding. Additionally, reduction of LHRH inactivation by the inhibitors Bacitracin and Trasylol and by magnesium was also accompanied by reduced LHRH binding. Previous studies have shown that the majority of LHRH binding to pituitary plasma membranes is to the low affinity site (approximately 10(-6) M), but the significance of this binding has been uncertain. Our findings indicate that low affinity binding probably represents binding of LHRH to the inactivating enzyme. The LHRH analog, D-Ser6(TBu), des Gly10, ethylamide, has greater biological activity than LHRH and is not inactivated to a significant extent by pituitary plasma membranes. The enhanced biological activity of the analog, therefore, may be due to its resistance to inactivation by enzymes on the pituitary cell surface. The membrane-associated inactivating enzyme could play an important role in vivo in determining the concentration of intact LHRH available at the receptor site which initiates gonadotropin release.
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Bacteriophage PRD1 batch experiments to study attachment, detachment and inactivation processes.
Sadeghi, Gholamreza; Schijven, Jack F; Behrends, Thilo; Hassanizadeh, S Majid; van Genuchten, Martinus Th
2013-09-01
Knowledge of virus removal in subsurface environments is pivotal for assessing the risk of viral contamination of water resources and developing appropriate protection measures. Columns packed with sand are frequently used to quantify attachment, detachment and inactivation rates of viruses. Since column transport experiments are very laborious, a common alternative is to perform batch experiments where usually one or two measurements are done assuming equilibrium is reached. It is also possible to perform kinetic batch experiments. In that case, however, it is necessary to monitor changes in the concentration with time. This means that kinetic batch experiments will be almost as laborious as column experiments. Moreover, attachment and detachment rate coefficients derived from batch experiments may differ from those determined using column experiments. The aim of this study was to determine the utility of kinetic batch experiments and investigate the effects of different designs of the batch experiments on estimated attachment, detachment and inactivation rate coefficients. The experiments involved various combinations of container size, sand-water ratio, and mixing method (i.e., rolling or tumbling by pivoting the tubes around their horizontal or vertical axes, respectively). Batch experiments were conducted with clean quartz sand, water at pH 7 and ionic strength of 20 mM, and using the bacteriophage PRD1 as a model virus. Values of attachment, detachment and inactivation rate coefficients were found by fitting an analytical solution of the kinetic model equations to the data. Attachment rate coefficients were found to be systematically higher under tumbling than under rolling conditions because of better mixing and more efficient contact of phages with the surfaces of the sand grains. In both mixing methods, more sand in the container yielded higher attachment rate coefficients. A linear increase in the detachment rate coefficient was observed with increased solid-water ratio using tumbling method. Given the differences in the attachment rate coefficients, and assuming the same sticking efficiencies since chemical conditions of the batch and column experiments were the same, our results show that collision efficiencies of batch experiments are not the same as those of column experiments. Upscaling of the attachment rate from batch to column experiments hence requires proper understanding of the mixing conditions. Because batch experiments, in which the kinetics are monitored, are as laborious as column experiments, there seems to be no major advantage in performing batch instead of column experiments. Copyright © 2013 Elsevier B.V. All rights reserved.
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Costas Malvido, Mónica; Alonso González, Elisa; Pérez Guerra, Nelson
2016-09-01
Nisin production by Lactococcus lactis CECT 539 was followed in batch cultures in whey supplemented with different concentrations of glucose and in two realkalized fed-batch fermentations in unsupplemented whey, which were fed, respectively, with concentrated solutions of lactose and glucose. In the batch fermentations, supplementation of whey with glucose inhibited both the growth and bacteriocin production. However, fed-batch cultures were characterized with high productions of biomass (1.34 and 1.51 g l(-1)) and nisin (50.6 and 60.3 BU ml(-1)) in comparison to the batch fermentations in unsupplemented whey (0.48 g l(-1) and 22.5 BU ml(-1)) and MRS broth (1.59 g l(-1) and 50.0 BU ml(-1)). In the two realkalized fed-batch fermentations, the increase in bacteriocin production parallels both the biomass production and pH drop generated in each realkalization and feeding cycle, suggesting that nisin was synthesized as a pH-dependent primary metabolite. A shift from homolactic to heterolactic fermentation was observed at the 108 h of incubation, and other metabolites (acetic acid and butane-2,3-diol) in addition to lactic acid accumulated in the medium. On the other hand, the feeding with glucose improved the efficiencies in glucose, nitrogen, and phosphorus consumption as compared to the batch cultures. The realkalized fed-batch fermentations showed to be an effective strategy to enhance nisin production in whey by using an appropriate feeding strategy to avoid the substrate inhibition.
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7. NORTHWEST VIEW OF FLUX CONVEYORS FEEDING BATCH HOPPERS ON ...
7. NORTHWEST VIEW OF FLUX CONVEYORS FEEDING BATCH HOPPERS ON THE BATCHING FLOOR OF THE FURNACE AISLE IN THE BOP SHOP. - U.S. Steel Duquesne Works, Basic Oxygen Steelmaking Plant, Along Monongahela River, Duquesne, Allegheny County, PA
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A comparison of abundance estimates from extended batch-marking and Jolly–Seber-type experiments
Cowen, Laura L E; Besbeas, Panagiotis; Morgan, Byron J T; Schwarz, Carl J
2014-01-01
Little attention has been paid to the use of multi-sample batch-marking studies, as it is generally assumed that an individual's capture history is necessary for fully efficient estimates. However, recently, Huggins et al. (2010) present a pseudo-likelihood for a multi-sample batch-marking study where they used estimating equations to solve for survival and capture probabilities and then derived abundance estimates using a Horvitz–Thompson-type estimator. We have developed and maximized the likelihood for batch-marking studies. We use data simulated from a Jolly–Seber-type study and convert this to what would have been obtained from an extended batch-marking study. We compare our abundance estimates obtained from the Crosbie–Manly–Arnason–Schwarz (CMAS) model with those of the extended batch-marking model to determine the efficiency of collecting and analyzing batch-marking data. We found that estimates of abundance were similar for all three estimators: CMAS, Huggins, and our likelihood. Gains are made when using unique identifiers and employing the CMAS model in terms of precision; however, the likelihood typically had lower mean square error than the pseudo-likelihood method of Huggins et al. (2010). When faced with designing a batch-marking study, researchers can be confident in obtaining unbiased abundance estimators. Furthermore, they can design studies in order to reduce mean square error by manipulating capture probabilities and sample size. PMID:24558576
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Helmreich, Grant W.; Hunn, John D.; Skitt, Darren J.
Coated particle composite J52R-16-98005 was produced by Babcock and Wilcox Technologies (BWXT) as fuel for the Advanced Gas Reactor Fuel Development and Qualification (AGR) Program’s AGR-5/6/7 irradiation test in the Idaho National Laboratory (INL) Advanced Test Reactor (ATR). This composite was comprised of four coated particle fuel batches J52O-16-93165B (26%), 93168B (26%), 93169B (24%), and 93170B (24%), chosen based on the Quality Control (QC) data acquired for each individual candidate AGR-5/6/7 batch. Each batch was coated in a 150-mm-diameter production-scale fluidized-bed chemical vapor deposition (CVD) furnace. Tristructural isotropic (TRISO) coatings were deposited on 425-μm-nominal-diameter spherical kernels from BWXT Lot J52R-16-69317more » containing a mixture of 15.5%-enriched uranium carbide and uranium oxide (UCO). The TRISO coatings consisted of four consecutive CVD layers: a ~50% dense carbon buffer layer with 100-μm-nominal thickness, a dense inner pyrolytic carbon (IPyC) layer with 40-μm-nominal thickness, a silicon carbide (SiC) layer with 35-μm-nominal thickness, and a dense outer pyrolytic carbon (OPyC) layer with 40-μm-nominal thickness. The TRISO-coated particle batches were sieved to upgrade the particles by removing over-sized and under-sized material, and the upgraded batches were designated by appending the letter A to the end of the batch number (e.g., 93165A). Secondary upgrading by sieving was performed on the A-designated batches to remove particles with missing or very-thin buffer layers that were identified during previous analysis of the individual batches for defective IPyC, as reported in the acceptance test data report for the AGR-5/6/7 production batches [Hunn et al. 2017]. The additionally-upgraded batches were designated by appending the letter B to the end of the batch number (e.g., 93165B).« less
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40 CFR 80.825 - How is the refinery or importer annual average toxics value determined?
Code of Federal Regulations, 2010 CFR
2010-07-01
... number of batches of gasoline produced or imported during the averaging period. i = Individual batch of... toxics value, Ti, of each batch of gasoline is determined using the Phase II Complex Model specified at...
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Using Forensics to Untangle Batch Effects in TCGA Data - TCGA
Rehan Akbani, Ph.D., and colleagues at the University of Texas MD Anderson Cancer Center developed a tool called MBatch to detect, diagnose, and correct batch effects in TCGA data. Read more about batch effects in this Case Study.
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Code of Federal Regulations, 2012 CFR
2012-07-01
... subpart that references this subpart. Batch process means a process in which the equipment is fed... generally emptied. Examples of industries that use batch processes include pharmaceutical production and pesticide production. Batch product-process equipment train means the collection of equipment (e.g...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... subpart that references this subpart. Batch process means a process in which the equipment is fed... generally emptied. Examples of industries that use batch processes include pharmaceutical production and pesticide production. Batch product-process equipment train means the collection of equipment (e.g...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... subpart that references this subpart. Batch process means a process in which the equipment is fed... generally emptied. Examples of industries that use batch processes include pharmaceutical production and pesticide production. Batch product-process equipment train means the collection of equipment (e.g...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... subpart that references this subpart. Batch process means a process in which the equipment is fed... generally emptied. Examples of industries that use batch processes include pharmaceutical production and pesticide production. Batch product-process equipment train means the collection of equipment (e.g...
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3. INSIDE BATCH FURNACE BUILDING, VIEW LOOKING NORTH AT REGENERATIVE ...
3. INSIDE BATCH FURNACE BUILDING, VIEW LOOKING NORTH AT REGENERATIVE BATCH FURNACES ON LEFT AND 5 TON CAPACITY CHARGING MACHINE ON RIGHT. - U.S. Steel Duquesne Works, 22-Inch Bar Mill, Along Monongahela River, Duquesne, Allegheny County, PA
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Identifying and mitigating batch effects in whole genome sequencing data.
Tom, Jennifer A; Reeder, Jens; Forrest, William F; Graham, Robert R; Hunkapiller, Julie; Behrens, Timothy W; Bhangale, Tushar R
2017-07-24
Large sample sets of whole genome sequencing with deep coverage are being generated, however assembling datasets from different sources inevitably introduces batch effects. These batch effects are not well understood and can be due to changes in the sequencing protocol or bioinformatics tools used to process the data. No systematic algorithms or heuristics exist to detect and filter batch effects or remove associations impacted by batch effects in whole genome sequencing data. We describe key quality metrics, provide a freely available software package to compute them, and demonstrate that identification of batch effects is aided by principal components analysis of these metrics. To mitigate batch effects, we developed new site-specific filters that identified and removed variants that falsely associated with the phenotype due to batch effect. These include filtering based on: a haplotype based genotype correction, a differential genotype quality test, and removing sites with missing genotype rate greater than 30% after setting genotypes with quality scores less than 20 to missing. This method removed 96.1% of unconfirmed genome-wide significant SNP associations and 97.6% of unconfirmed genome-wide significant indel associations. We performed analyses to demonstrate that: 1) These filters impacted variants known to be disease associated as 2 out of 16 confirmed associations in an AMD candidate SNP analysis were filtered, representing a reduction in power of 12.5%, 2) In the absence of batch effects, these filters removed only a small proportion of variants across the genome (type I error rate of 3%), and 3) in an independent dataset, the method removed 90.2% of unconfirmed genome-wide SNP associations and 89.8% of unconfirmed genome-wide indel associations. Researchers currently do not have effective tools to identify and mitigate batch effects in whole genome sequencing data. We developed and validated methods and filters to address this deficiency.
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Accelerating deep neural network training with inconsistent stochastic gradient descent.
Wang, Linnan; Yang, Yi; Min, Renqiang; Chakradhar, Srimat
2017-09-01
Stochastic Gradient Descent (SGD) updates Convolutional Neural Network (CNN) with a noisy gradient computed from a random batch, and each batch evenly updates the network once in an epoch. This model applies the same training effort to each batch, but it overlooks the fact that the gradient variance, induced by Sampling Bias and Intrinsic Image Difference, renders different training dynamics on batches. In this paper, we develop a new training strategy for SGD, referred to as Inconsistent Stochastic Gradient Descent (ISGD) to address this problem. The core concept of ISGD is the inconsistent training, which dynamically adjusts the training effort w.r.t the loss. ISGD models the training as a stochastic process that gradually reduces down the mean of batch's loss, and it utilizes a dynamic upper control limit to identify a large loss batch on the fly. ISGD stays on the identified batch to accelerate the training with additional gradient updates, and it also has a constraint to penalize drastic parameter changes. ISGD is straightforward, computationally efficient and without requiring auxiliary memories. A series of empirical evaluations on real world datasets and networks demonstrate the promising performance of inconsistent training. Copyright © 2017 Elsevier Ltd. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Mukerjee, S.; Thurston, T.R.; Jisrawi, N.M.
The authors describe synchrotron based X-ray diffraction techniques and issues related to in situ studies of intercalation processes in battery electrodes. They then demonstrate the utility of this technique, through a study of two batches of Li{sub x}Mn{sub 2}O{sub 4} cathode materials. The structural evolution of these spinel materials was monitored in situ during the initial charge of these electrodes in actual battery cells. Significant differences were observed in the two batches, particularly in the intercalation range of x = 0.45 to 0.20. The first-order structural transitions in this region indicated coexistence of two cubic phases in the batch 2more » material, whereas the batch 1 material showed suppressed two-phase coexistence. Batch 2 cells also indicated structural evolution in the low-potential region below 3.0 V in contrast to the batch 1 material. Differences in structural evolution between batches of Li{sub x}Mn{sub 2}O{sub 4} could have important ramifications in their cycle life and stability characteristics.« less
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NASA Astrophysics Data System (ADS)
Carter, Alex B.; Davies, Campbell R.; Mapstone, Bruce D.; Russ, Garry R.; Tobin, Andrew J.; Williams, Ashley J.
2014-09-01
Batch fecundity of female Plectropomus leopardus, a coral reef fish targeted by commercial and recreational fishing, was compared between reefs open to fishing and reefs within no-take marine reserves within three regions of the Great Barrier Reef (GBR), Australia. Length, weight, and age had positive effects on batch fecundity of spawners from northern and central reefs but negligible effects on spawners from southern reefs. Females were least fecund for a given length, weight, and age in the southern GBR. Batch fecundity of a 500-mm fork length female was 430 % greater on central reefs and 207 % greater on northern reefs than on southern reefs. The effects of length and age on batch fecundity did not differ significantly between reserve and fished reefs in any region, but weight-specific fecundity was 100 % greater for large 2.0 kg females on reserve reefs compared with fished reefs in the central GBR. We hypothesize that regional variation in batch fecundity is likely driven by water temperature and prey availability. Significant regional variation in batch fecundity highlights the need for understanding spatial variation in reproductive output where single conservation or fishery management strategies cover large, potentially diverse, spatial scales.
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Park, Seonghwan; Kim, Jeongmi; Park, Younghyun; Son, Suyoung; Cho, Sunja; Kim, Changwon; Lee, Taeho
2017-06-01
Two competitive strategies, fed-batch and sequencing-batch cultivation, were compared in cost-effective biomass production of a high lipid microalgae, Micractinium inermum NLP-F014 using a blended wastewater medium. For fed-batch cultivations, additional nutrient was supplemented at day 2 (FB1) or consecutively added at day 2 and 4 (FB2). Through inoculum size test, 1.0g-DCWL -1 was selected for the sequencing-batch cultivation (SB) where about 65% of culture was replaced with fresh medium every 2days. Both fed-batch cultivations showed the maximum biomass productivity of 0.95g-DCWL -1 d -1 , while average biomass productivity in SB was slightly higher as 0.96±0.08g-DCWL -1 d -1 . Furthermore, remained concentrations of organics (426mg-CODL -1 ), total nitrogen (15.4mg-NL -1 ) and phosphorus (0.6mg-PL -1 ) in SB were much lower than those of fed-batch conditions. The results suggested that SB could be a promising strategy to cultivate M. inermum NLP-F014 with the blended wastewater medium. Copyright © 2017 Elsevier Ltd. All rights reserved.
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A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis
Cortina, María E.; Balzano, Rodrigo E.; Rey Serantes, Diego A.; Caillava, Ana J.; Elena, Sebastián; Ferreira, A. C.; Nicola, Ana M.; Ugalde, Juan E.
2016-01-01
Brucellosis is a highly zoonotic disease that affects animals and human beings. Brucella suis is the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide–protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glyco-iELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected with B. suis biovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of “smooth” brucellosis in animals and humans. PMID:26984975
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Protocols and programs for high-throughput growth and aging phenotyping in yeast.
Jung, Paul P; Christian, Nils; Kay, Daniel P; Skupin, Alexander; Linster, Carole L
2015-01-01
In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting "Colony Forming Units". To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.
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Detection of rabbit and hare processed material in compound feeds by TaqMan real-time PCR.
Pegels, N; López-Calleja, I; García, T; Martín, R; González, I
2013-01-01
Food and feed traceability has become a priority for governments due to consumer demand for comprehensive and integrated safety policies. In the present work, a TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for specific detection of rabbit and hare material in animal feeds and pet foods. The technique is based on the use of three species-specific primer/probe detection systems targeting three 12S rRNA gene fragments: one from rabbit species, another one from hare species and a third fragment common to rabbit and hare (62, 102 and 75 bp length, respectively). A nuclear 18S rRNA PCR system, detecting a 77-bp amplicon, was used as positive amplification control. Assay performance and sensitivity were assessed through the analysis of a batch of laboratory-scale feeds treated at 133°C at 3 bar for 20 min to reproduce feed processing conditions dictated by European regulations. Successful detection of highly degraded rabbit and hare material was achieved at the lowest target concentration assayed (0.1%). Furthermore, the method was applied to 96 processed commercial pet food products to determine whether correct labelling had been used at the market level. The reported real-time PCR technique detected the presence of rabbit tissues in 80 of the 96 samples analysed (83.3%), indicating a possible labelling fraud in some pet foods. The real-time PCR method reported may be a useful tool for traceability purposes within the framework of feed control.
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Code of Federal Regulations, 2010 CFR
2010-04-01
... necessary to assure that all batches of the same drug product meet an appropriate in vitro test, he shall include in the bioequivalence requirement a requirement for manufacturers to submit samples of each batch...
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Code of Federal Regulations, 2011 CFR
2011-04-01
... necessary to assure that all batches of the same drug product meet an appropriate in vitro test, he shall include in the bioequivalence requirement a requirement for manufacturers to submit samples of each batch...
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Cadmium removal using Cladophora in batch, semi-batch and flow reactors.
Sternberg, Steven P K; Dorn, Ryan W
2002-02-01
This study presents the results of using viable algae to remove cadmium from a synthetic wastewater. In batch and semi-batch tests, a local strain of Cladophora algae removed 80-94% of the cadmium introduced. The flow experiments that followed were conducted using non-local Cladophora parriaudii. Results showed that the alga removed only 12.7(+/-6.4)% of the cadmium introduced into the reactor. Limited removal was the result of insufficient algal quantities and poor contact between the algae and cadmium solution.
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Batch Proving and Proof Scripting in PVS
NASA Technical Reports Server (NTRS)
Munoz, Cesar A.
2007-01-01
The batch execution modes of PVS are powerful, but highly technical, features of the system that are mostly accessible to expert users. This paper presents a PVS tool, called ProofLite, that extends the theorem prover interface with a batch proving utility and a proof scripting notation. ProofLite enables a semi-literate proving style where specification and proof scripts reside in the same file. The goal of ProofLite is to provide batch proving and proof scripting capabilities to regular, non-expert, users of PVS.
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Design of penicillin fermentation process simulation system
NASA Astrophysics Data System (ADS)
Qi, Xiaoyu; Yuan, Zhonghu; Qi, Xiaoxuan; Zhang, Wenqi
2011-10-01
Real-time monitoring for batch process attracts increasing attention. It can ensure safety and provide products with consistent quality. The design of simulation system of batch process fault diagnosis is of great significance. In this paper, penicillin fermentation, a typical non-linear, dynamic, multi-stage batch production process, is taken as the research object. A visual human-machine interactive simulation software system based on Windows operation system is developed. The simulation system can provide an effective platform for the research of batch process fault diagnosis.
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Reese, Sarah E; Archer, Kellie J; Therneau, Terry M; Atkinson, Elizabeth J; Vachon, Celine M; de Andrade, Mariza; Kocher, Jean-Pierre A; Eckel-Passow, Jeanette E
2013-11-15
Batch effects are due to probe-specific systematic variation between groups of samples (batches) resulting from experimental features that are not of biological interest. Principal component analysis (PCA) is commonly used as a visual tool to determine whether batch effects exist after applying a global normalization method. However, PCA yields linear combinations of the variables that contribute maximum variance and thus will not necessarily detect batch effects if they are not the largest source of variability in the data. We present an extension of PCA to quantify the existence of batch effects, called guided PCA (gPCA). We describe a test statistic that uses gPCA to test whether a batch effect exists. We apply our proposed test statistic derived using gPCA to simulated data and to two copy number variation case studies: the first study consisted of 614 samples from a breast cancer family study using Illumina Human 660 bead-chip arrays, whereas the second case study consisted of 703 samples from a family blood pressure study that used Affymetrix SNP Array 6.0. We demonstrate that our statistic has good statistical properties and is able to identify significant batch effects in two copy number variation case studies. We developed a new statistic that uses gPCA to identify whether batch effects exist in high-throughput genomic data. Although our examples pertain to copy number data, gPCA is general and can be used on other data types as well. The gPCA R package (Available via CRAN) provides functionality and data to perform the methods in this article. reesese@vcu.edu
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Ogunshe, Adenike A O; Adepoju, Adedayo A; Oladimeji, Modupe E
2011-01-01
This study aimed at evaluating the in vitro efficacy and health implications of inconsistencies in different production batches of antimycotic drugs. in vitro susceptibility profiles of 36 Candida spp. - C. albicans (19.4%), C. glabrata (30.6%), C. tropicalis (33.3%), and C. pseudotropicalis (16.7%) - obtained from human endocervical and high vaginal swabs (ECS/HVS) to two different batches (B1 and B2) of six antimycotic drugs (clotrimazole, doxycycline, iconazole, itraconazole, metronidazole and nystatin) was determined using modified agar well-diffusion method. None of the Candida strains had entirely the same (100%) susceptibility / resistance profiles in both batches of corresponding antimycotic drugs; while, different multiple antifungal susceptibility (MAS) rates were also recorded in batches 1 and 2 for corresponding antifungals. Only 14.3%, 27.3%, 16.7-33.3%, and 8.3-25.0% of C. albicans, C. glabrata, C. pseudotropicalis, and C. tropicalis strains, respectively, had similar susceptibility/resistance profiles toward coressponding antifungal agents in both batches; while up to 57.1% of C. albicans, 45.5% of C. glabrata, 66.7% of C. pseudotropicalis, and 50.0% of C. tropicalis strains were susceptible to one batch of antifungals but resistant to corresponding antifungals in the second batch. As high as 71.4% (C. albicans), 73.0% (C. glabrata), 50.0% (C. pseudotropicalis), and 66.74% (C. tropicalis) strains had differences of ≥ 10.0 mm among corresponding antimycotic agents. Candida strains exhibited different in vitro susceptibility / resistance patterns toward two batches of corresponding antimycotic agents, which has clinical implications on the efficacy of the drugs and treatment of patients. The findings of the present study will be of benefit in providing additional information in support of submission of drugs for registration to appropriate regulatory agencies.
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Liu, Ya-Juan; André, Silvère; Saint Cristau, Lydia; Lagresle, Sylvain; Hannas, Zahia; Calvosa, Éric; Devos, Olivier; Duponchel, Ludovic
2017-02-01
Multivariate statistical process control (MSPC) is increasingly popular as the challenge provided by large multivariate datasets from analytical instruments such as Raman spectroscopy for the monitoring of complex cell cultures in the biopharmaceutical industry. However, Raman spectroscopy for in-line monitoring often produces unsynchronized data sets, resulting in time-varying batches. Moreover, unsynchronized data sets are common for cell culture monitoring because spectroscopic measurements are generally recorded in an alternate way, with more than one optical probe parallelly connecting to the same spectrometer. Synchronized batches are prerequisite for the application of multivariate analysis such as multi-way principal component analysis (MPCA) for the MSPC monitoring. Correlation optimized warping (COW) is a popular method for data alignment with satisfactory performance; however, it has never been applied to synchronize acquisition time of spectroscopic datasets in MSPC application before. In this paper we propose, for the first time, to use the method of COW to synchronize batches with varying durations analyzed with Raman spectroscopy. In a second step, we developed MPCA models at different time intervals based on the normal operation condition (NOC) batches synchronized by COW. New batches are finally projected considering the corresponding MPCA model. We monitored the evolution of the batches using two multivariate control charts based on Hotelling's T 2 and Q. As illustrated with results, the MSPC model was able to identify abnormal operation condition including contaminated batches which is of prime importance in cell culture monitoring We proved that Raman-based MSPC monitoring can be used to diagnose batches deviating from the normal condition, with higher efficacy than traditional diagnosis, which would save time and money in the biopharmaceutical industry. Copyright © 2016 Elsevier B.V. All rights reserved.
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Zhu, Linjiang; Fan, Zihao; Kuai, Hui; Li, Qi
2017-09-01
During natural fermentation processes, a characteristic microbial community structure (MCS) is naturally formed, and it is interesting to know about its batch-batch stability. This issue was explored in a traditional semi-solid-state fermentation process of huyumei, a Chinese broad bean paste product. The results showed that this MCS mainly contained four aerobic Bacillus species (8 log CFU per g), including B. subtilis, B. amyloliquefaciens, B. methylotrophicus, and B. tequilensis, and the facultative anaerobe B. cereus with a low concentration (4 log CFU per g), besides a very small amount of the yeast Zygosaccharomyces rouxii (2 log CFU per g). The dynamic change of the MCS in the brine fermentation process showed that the abundance of dominant species varied within a small range, and in the beginning of process the growth of lactic acid bacteria was inhibited and Staphylococcus spp. lost its viability. Also, the MCS and its dynamic change were proved to be highly reproducible among seven batches of fermentation. Therefore, the MCS naturally and stably forms between different batches of the traditional semi-solid-state fermentation of huyumei. Revealing microbial community structure and its batch-batch stability is helpful for understanding the mechanisms of community formation and flavour production in a traditional fermentation. This issue in a traditional semi-solid-state fermentation of huyumei broad bean paste was firstly explored. This fermentation process was revealed to be dominated by a high concentration of four aerobic species of Bacillus, a low concentration of B. cereus and a small amount of Zygosaccharomyces rouxii. Lactic acid bacteria and Staphylococcus spp. lost its viability at the beginning of fermentation. Such the community structure was proved to be highly reproducible among seven batches. © 2017 The Society for Applied Microbiology.
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Lack of genotoxicity in vivo for food color additive Allura Red AC.
Bastaki, Maria; Farrell, Thomas; Bhusari, Sachin; Pant, Kamala; Kulkarni, Rohan
2017-07-01
Allura Red AC is an approved food color additive internationally with INS number 129, in the United States as food color subject to batch certification "Food, Drug, and Cosmetic" (FD&C) Red No. 40, and in Europe as food color additive with E number 129. In their evaluation of the color (2013), the European Food Safety Authority (EFSA) expressed concerns of potential genotoxicity, based primarily on one genotoxicity study that was not conducted according to Guidelines. The present in vivo genotoxicity study was conducted according to OECD Guidelines in response to EFSA's request for additional data. The animal species and strain, and the tissues examined were selected specifically to address the previously reported findings. The results show clear absence of genotoxic activity for Allura Red AC, in the bone marrow micronucleus assay and the Comet assay in the liver, stomach, and colon. These data addressed EFSA's concerns for genotoxicity. The Joint WHO/FAO Committee on Food Additives (JECFA) (2016) also reviewed the study and concluded that there is no genotoxicity concern for Allura Red AC. Negative findings in parallel genotoxicity studies on Tartrazine and Ponceau 4R (published separately) are consistent with lack of genotoxicity for azo dyes used as food colors. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Lack of genotoxicity in vivo for food color additive Tartrazine.
Bastaki, Maria; Farrell, Thomas; Bhusari, Sachin; Pant, Kamala; Kulkarni, Rohan
2017-07-01
Tartrazine is approved as a food color additive internationally with INS number 102, in the United States as food color subject to batch certification "Food, Drug, and Cosmetic" (FD&C) Yellow No. 5, and in Europe as food color additive with E number 102. In their evaluation of the color (2013), the European Food Safety Authority (EFSA) expressed concerns of potential genotoxicity, based primarily on one genotoxicity study that was not conducted according to Guidelines. The present in vivo genotoxicity study was conducted according to OECD Guidelines in response to EFSA's request for additional data. The animal species and strain, and the tissues examined were selected specifically to address the previously reported findings. The results of this study show clear absence of genotoxic activity for Tartrazine, in the bone marrow micronucleus assay and the Comet assay in the liver, stomach, and colon. These data addressed EFSA's concerns for genotoxicity. The Joint WHO/FAO Committee on Food Additives (JECFA) (2016) also reviewed these data and concluded that there is no genotoxicity concern for Tartrazine. Negative findings in parallel genotoxicity studies on Allura Red AC and Ponceau 4R (published separately) are consistent with lack of genotoxicity for azo dyes used as food colors. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Nale, Janet Y.; Redgwell, Tamsin A.; Millard, Andrew; Clokie, Martha R. J.
2018-01-01
Clostridium difficile infection (CDI) is a major cause of infectious diarrhea. Conventional antibiotics are not universally effective for all ribotypes, and can trigger dysbiosis, resistance and recurrent infection. Thus, novel therapeutics are needed to replace and/or supplement the current antibiotics. Here, we describe the activity of an optimised 4-phage cocktail to clear cultures of a clinical ribotype 014/020 strain in fermentation vessels spiked with combined fecal slurries from four healthy volunteers. After 5 h, we observed ~6-log reductions in C. difficile abundance in the prophylaxis regimen and complete C. difficile eradication after 24 h following prophylactic or remedial regimens. Viability assays revealed that commensal enterococci, bifidobacteria, lactobacilli, total anaerobes, and enterobacteria were not affected by either regimens, but a ~2-log increase in the enterobacteria, lactobacilli, and total anaerobe abundance was seen in the phage-only-treated vessel compared to other treatments. The impact of the phage treatments on components of the microbiota was further assayed using metagenomic analysis. Together, our data supports the therapeutic application of our optimised phage cocktail to treat CDI. Also, the increase in specific commensals observed in the phage-treated control could prevent further colonisation of C. difficile, and thus provide protection from infection being able to establish. PMID:29438355
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Evaluation of a Hypocrea jecorina enzyme preparation for hydrolysis of Tifton 85 bermudagrass.
Ximenes, E A; Brandon, S K; Doran-Peterson, J
2008-03-01
Tifton 85 bermudagrass, developed at the ARS-USDA in Tifton, GA, is grown on over ten million acres in the USA for hay and forage. Of the bermudagrass cultivars, Tifton 85 exhibits improved digestibility because the ratio of ether- to ester-linked phenolic acids has been lowered using traditional plant breeding techniques. A previously developed pressurized batch hot water (PBHW) method was used to treat Tifton 85 bermudagrass for enzymatic hydrolysis. Native grass (untreated) and PBHW-pretreated material were compared as substrates for fungal cultivation to produce enzymes. Cellulase activity, measured via the filter paper assay, was higher for fungi cultivated on PBHW-pretreated grass, whereas the other nine enzyme assays produced higher activities for the untreated grass. Ferulic acid and vanillin levels increased significantly for the enzyme preparations produced using PBHW-pretreated grass and the release of these phenolic compounds may have contributed to the observed reduction in enzyme activities. Culture supernatant from Tifton 85 bermudagrass-grown fungi were combined with two commercial enzyme preparations and the enzyme activity profiles are reported. The amount of reducing sugar liberated by the enzyme mixture from Hypocrea jecorina (after 192 h incubation with untreated bermudagrass) individually or in combination with feruloyl esterase was 72.1 and 84.8%, respectively, of the commercial cellulase preparation analyzed under the same conditions.
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Levin, D; Jonak, J; Harris, T N
1977-01-01
Dinitrophenyl-bovine albumin was coupled at room temperature to sheep red blood cells in a procedure which minimized spontaneous lysis and allowed the preparation of large batches and their use for at least 3 weeks. The modified erythrocytes were used as a substrate for detecting local hemolytic plaques in agar by myeloma MOPC 315 cells, which secrete a paraprotein IgA with high affinity for dinitrophenyl ligand. Conditions maximizing the number of plaques formed by a given number of tumor cells were found to include coupling the erythrocytes at 1 mg/ml dinitrophenyl-bovine albumin with a molar ratio of about 50, and incubation with an amino-to-carboxy cross-linking agent, 1-ethyl-3(3 dimethyl aminopropyl) carbodiimide, at 2 mg/ml for 50 min. The method thus developed was employed to measure cellular and antibody-dependent immune reactions against the MOPC 315 cells. The experimental results show comparisons of the plaque technique with other measurements of tumor cell injury. The nature of the assay, which requires only 500 cells per plating, and which tests the synthetic capacity of single cells, suggests its use in experiments which limit the number of target cells, and in immune reactions causing injury, but not necessarily lysis, of the target cells.
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Veenuttranon, Kornautchaya; Nguyen, Loc Thai
2018-08-15
The aim of this work was to develop a programmable flow system for rapid assessment of total antioxidant capacity (TAC). Novel features of the prototype include a single pressure-driven source, versatile manipulation of fluid flows, ability to adapt to different TAC assays, and compatibility with microfluidic design. Antioxidant activity was determined by electrochemical measurement of residual 2,2-diphenyl-2-picrylhydrayl hydrate (DPPH•) free radicals in the solution. The overall performance of the device was validated by the spectrophotometric method. The results indicated that dosing of reagents and samples could be controlled by pressure (R 2 = 0.992) and time (R 2 = 0.999) with high accuracy, and the mixing uniformity of the device was equivalent to that of the batch mixing (R 2 = 0.994). TAC assays of a standard antioxidant, ascorbic acid, as well as selected samples such as orange and pomegranate juices, white wine, and green tea by the device were comparable to traditional measurement techniques. Due to the short incubation time, the approach may be more suitable for fast, rather than slow reacting antioxidant compounds. The developed system could be used for rapid TAC screening of food and biological samples. Copyright © 2018 Elsevier B.V. All rights reserved.
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Combined subcritical water and enzymatic hydrolysis for reducing sugar production from coconut husk
NASA Astrophysics Data System (ADS)
Muharja, Maktum; Junianti, Fitri; Nurtono, Tantular; Widjaja, Arief
2017-05-01
Coconut husk wastes are abundantly available in Indonesia. It has a potential to be used into alternative renewable energy sources such as hydrogen using enzymatic hydrolysis followed by a fermentation process. Unfortunately, enzymatic hydrolysis is hampered by the complex structure of lignocellulose, so the cellulose component is hard to degrade. In this study, Combined Subcritical Water (SCW) and enzymatic hydrolysis are applied to enhance fermentable, thereby reducing production of sugar from coconut husk. There were two steps in this study, the first step was coconut husk pretreated by SCW in batch reactor at 80 bar and 150-200°C for 60 minutes reaction time. Secondly, solid fraction from the results of SCW was hydrolyzed using the mixture of pure cellulose and xylanase enzymes. Analysis was conducted on untreated and SCW-treated by gravimetric assay, liquid fraction after SCW and solid fraction after enzymatic hydrolysis using DNS assay. The maximum yield of reducing sugar (including xylose, arabinose glucose, galactose, mannose) was 1.254 gr per 6 gr raw material, representing 53.95% of total sugar in coconut husk biomass which was obtained at 150°C 80 bar for 60 minutes reaction time of SCW-treated and 6 hour of enzymatic hydrolysis using mixture of pure cellulose and xylanase enzymes (18.6 U /gram of coconut husk).
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Evaluation of a Hypocrea jecorina Enzyme Preparation for Hydrolysis of Tifton 85 Bermudagrass
NASA Astrophysics Data System (ADS)
Ximenes, E. A.; Brandon, S. K.; Doran-Peterson, J.
Tifton 85 bermudagrass, developed at the ARS-USDA in Tifton, GA, is grown on over ten million acres in the USA for hay and forage. Of the bermudagrass cultivars, Tifton 85 exhibits improved digestibility because the ratio of ether- to ester-linked phenolic acids has been lowered using traditional plant breeding techniques. A previously developed pressurized batch hot water (PBHW) method was used to treat Tifton 85 bermudagrass for enzymatic hydrolysis. Native grass (untreated) and PBHW-pretreated material were compared as substrates for fungal cultivation to produce enzymes. Cellulase activity, measured via the filter paper assay, was higher for fungi cultivated on PBHW-pretreated grass, whereas the other nine enzyme assays produced higher activities for the untreated grass. Ferulic acid and vanillin levels increased significantly for the enzyme preparations produced using PBHW-pretreated grass and the release of these phenolic compounds may have contributed to the observed reduction in enzyme activities. Culture supernatant from Tifton 85 bermudagrass-grown fungi were combined with two commercial enzyme preparations and the enzyme activity profiles are reported. The amount of reducing sugar liberated by the enzyme mixture from Hypocrea jecorina (after 192 h incubation with untreated bermudagrass) individually or in combination with feruloyl esterase was 72.1 and 84.8%, respectively, of the commercial cellulase preparation analyzed under the same conditions.
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G-protein based ELISA as a potency test for rabies vaccines.
Chabaud-Riou, Martine; Moreno, Nadège; Guinchard, Fabien; Nicolai, Marie Claire; Niogret-Siohan, Elisabeth; Sève, Nicolas; Manin, Catherine; Guinet-Morlot, Françoise; Riou, Patrice
2017-03-01
The NIH test is currently used to assess the potency of rabies vaccine, a key criterion for vaccine release. This test is based on mice immunization followed by intracerebral viral challenge. As part of global efforts to reduce animal experimentation and in the framework of the development of Sanofi Pasteur next generation, highly-purified vaccine, produced without any material of human or animal origin, we developed an ELISA as an alternative to the NIH test. This ELISA is based on monoclonal antibodies recognizing specifically the native form of the viral G-protein, the major antigen that induces neutralizing antibody response to rabies virus. We show here that our ELISA is able to distinguish between potent and different types of sub-potent vaccine lots. Satisfactory agreement was observed between the ELISA and the NIH test in the determination of the vaccine titer and their capacity to discern conform from non-conform batches. Our ELISA meets the criteria for a stability-indicating assay and has been successfully used to develop the new generation of rabies vaccine candidates. After an EPAA international pre-collaborative study, this ELISA was selected as the assay of choice for the EDQM collaborative study aimed at replacing the rabies vaccine NIH in vivo potency test. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.
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Characterization of allergoids.
Himly, Martin; Carnés, Jerónimo; Fernández-Caldas, Enrique; Briza, Peter; Ferreira, Fatima
2009-01-01
So far it has not been possible to measure the amount of major allergens in the complexes after chemical modification. Furthermore, the presence of minor allergens remained obscure, unless antibodies were successfully generated by animal immunization with allergoids and shown to be reactive with purified natural or recombinant allergens in immunological assays. Thus, we adapted and employed a set of physicochemical methods with the aim of elucidating the molecular size and allergen composition of allergoids. Using online-HPSEC light scattering and DLS, it was shown that two thirds of depigmented allergoid prepared from birch pollen extracts adopted a MW between 1000 and 2000 kDa. The question of reproducibility of the polymerization reaction was addressed by investigating four batches of P. pratense allergoid. Three out of the four batches contained 73 to 77% of polymerized molecules in the above-mentioned range of molecular sizes. One batch showed a significantly higher content of molecules with a MW exceeding 2 MDa. Analysis of allergen composition in B. alba allergoids revealed the presence of all relevant Bet v 1 isoforms and minor allergens except for Bet v 3 and Bet v 4, which was in good agreement with the allergens detected in the native extracts. It should be noted that Bet v 3 has not been detected at the protein level before. Similarly, good agreement in allergen composition between allergoid and native extract was also found for D. pteronyssinus. Presently, the European Directorate for Quality of Medicines and Healthcare (EDQM) is committed to the application of the 3R principles (i. e. replace, reduce, refine the use of animals) for the quality control of medicines wherever possible. This is reflected by the regular review of the monographs of the European Pharmacopoeia and the introduction of alternative tests. For instance, recently it was decided to replace the rabbit pyrogen test by an in vitro test. Furthermore, through the Biological Standardisation Programme the EDQM develops, validates, and establishes alternative test methods in the field of quality control of biologicals (personal communication with Karl-Heinz Buchheit, EDQM). Therefore, the approach presented here for the characterization of allergoids relying on physicochemical methods shall also serve the growing needs for alternative methods to animal testing.
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2012-01-01
Background Second generation hydrogen fermentation technologies using organic agricultural and forestry wastes are emerging. The efficient microbial fermentation of hexoses and pentoses resulting from the pretreatment of lingocellulosic materials is essential for the success of these processes. Results Conversion of arabinose and glucose to hydrogen, by extreme thermophilic, anaerobic, mixed cultures was studied in continuous (70°C, pH 5.5) and batch (70°C, pH 5.5 and pH 7) assays. Two expanded granular sludge bed (EGSB) reactors, Rarab and Rgluc, were continuously fed with arabinose and glucose, respectively. No significant differences in reactor performance were observed for arabinose and glucose organic loading rates (OLR) ranging from 4.3 to 7.1 kgCOD m-3 d-1. However, for an OLR of 14.2 kgCOD m-3 d-1, hydrogen production rate and hydrogen yield were higher in Rarab than in Rgluc (average hydrogen production rate of 3.2 and 2.0 LH2 L-1 d-1 and hydrogen yield of 1.10 and 0.75 molH2 mol-1substrate for Rarab and Rgluc, respectively). Lower hydrogen production in Rgluc was associated with higher lactate production. Denaturing gradient gel electrophoresis (DGGE) results revealed no significant difference on the bacterial community composition between operational periods and between the reactors. Increased hydrogen production was observed in batch experiments when hydrogen partial pressure was kept low, both with arabinose and glucose as substrate. Sugars were completely consumed and hydrogen production stimulated (62% higher) when pH 7 was used instead of pH 5.5. Conclusions Continuous hydrogen production rate from arabinose was significantly higher than from glucose, when higher organic loading rate was used. The effect of hydrogen partial pressure on hydrogen production from glucose in batch mode was related to the extent of sugar utilization and not to the efficiency of substrate conversion to hydrogen. Furthermore, at pH 7.0, sugars uptake, hydrogen production and yield were higher than at pH 5.5, with both arabinose and glucose as substrates. PMID:22330180
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López, Pío; Arguedas Mohs, Adriano; Abdelnour Vásquez, Arturo; Consuelo-Miranda, Maria; Feroldi, Emmanuel; Noriega, Fernando; Jordanov, Emilia; B Chir, Siham; Zambrano, Betzana
2017-11-01
Hexavalent diphtheria-tetanus-acellular pertussis-inactivated poliovirus-hepatitis B-Haemophilus influenzae type b (DTaP-IPV-HB-PRP-T)-containing vaccines are increasingly the standard of care. This study evaluated the primary series (NCT01177722) and booster (NCT01444781) of a fully liquid DTaP-IPV-HB-PRP-T vaccine in Latin America. Infants (N = 1375) received hepatitis B vaccine at birth and were randomized to one of 3 batches of the investigational DTaP-IPV-HB-PRP-T or licensed control vaccine (DTaP-HB-IPV//PRP-T) at 2-4 to 6 months of age, coadministered with 7-valent pneumococcal conjugate vaccine (PCV7) (2-4-6 months) and rotavirus vaccine (2-4 months). A booster of either DTaP-IPV-HB-PRP-T or control was given at 12-24 months, coadministered with PCV7. Immunogenicity was assessed by validated assays and safety from parental reports. Primary series seroprotection and vaccine response rates were equivalent for DTaP-IPV-HB-PRP-T batches. For pooled batches, noninferiority to the control vaccine was demonstrated for each antigen. There were no descriptive differences in antibody persistence or booster response between DTaP-IPV-HB-PRP-T and the control. The booster responses to either vaccine following DTaP-IPV-HB-PRP-T primary series or to DTaP-IPV-HB-PRP-T following a control vaccine primary series were similar. The anti-aP component (filamentous hemagglutinin [FHA] and pertussis toxin [PT]) vaccine response and anti-Haemophilus influenzae type b (PRP) series seroprotection (≥0.15 µg/mL) rates were ≥73.0% after 2 primary series doses. Antipyretics had no effect on the immune response, and an extra (oral) polio vaccination had no effect on the antipolio booster response. Responses to PCV7 and rotavirus vaccine were similar for each coadministration. There were no safety concerns observed with any vaccine. These results confirm the suitability of the fully liquid DTaP-IPV-HB-PRP-T vaccine for primary and booster vaccination of infants.
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Knibbe, Carole; Schneider, Dominique; Beslon, Guillaume
2017-01-01
Metabolic cross-feeding interactions between microbial strains are common in nature, and emerge during evolution experiments in the laboratory, even in homogeneous environments providing a single carbon source. In sympatry, when the environment is well-mixed, the reasons why emerging cross-feeding interactions may sometimes become stable and lead to monophyletic genotypic clusters occupying specific niches, named ecotypes, remain unclear. As an alternative to evolution experiments in the laboratory, we developed Evo2Sim, a multi-scale model of in silico experimental evolution, equipped with the whole tool case of experimental setups, competition assays, phylogenetic analysis, and, most importantly, allowing for evolvable ecological interactions. Digital organisms with an evolvable genome structure encoding an evolvable metabolic network evolved for tens of thousands of generations in environments mimicking the dynamics of real controlled environments, including chemostat or batch culture providing a single limiting resource. We show here that the evolution of stable cross-feeding interactions requires seasonal batch conditions. In this case, adaptive diversification events result in two stably co-existing ecotypes, with one feeding on the primary resource and the other on by-products. We show that the regularity of serial transfers is essential for the maintenance of the polymorphism, as it allows for at least two stable seasons and thus two temporal niches. A first season is externally generated by the transfer into fresh medium, while a second one is internally generated by niche construction as the provided nutrient is replaced by secreted by-products derived from bacterial growth. In chemostat conditions, even if cross-feeding interactions emerge, they are not stable on the long-term because fitter mutants eventually invade the whole population. We also show that the long-term evolution of the two stable ecotypes leads to character displacement, at the level of the metabolic network but also of the genome structure. This difference of genome structure between both ecotypes impacts the stability of the cross-feeding interaction, when the population is propagated in chemostat conditions. This study shows the crucial role played by seasonality in temporal niche partitioning and in promoting cross-feeding subgroups into stable ecotypes, a premise to sympatric speciation. PMID:28358919
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Code of Federal Regulations, 2011 CFR
2011-07-01
... shall have the meaning given them in the Act and in this section. Batch process means a process in which... which the equipment is generally emptied. Examples of industries that use batch processes include pharmaceutical production and pesticide production. Batch product-process equipment train means the collection of...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... shall have the meaning given them in the Act and in this section. Batch process means a process in which... which the equipment is generally emptied. Examples of industries that use batch processes include pharmaceutical production and pesticide production. Batch product-process equipment train means the collection of...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... shall have the meaning given them in the Act and in this section. Batch process means a process in which... which the equipment is generally emptied. Examples of industries that use batch processes include pharmaceutical production and pesticide production. Batch product-process equipment train means the collection of...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... shall have the meaning given them in the Act and in this section. Batch process means a process in which... which the equipment is generally emptied. Examples of industries that use batch processes include pharmaceutical production and pesticide production. Batch product-process equipment train means the collection of...
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40 CFR 63.1326 - Batch process vents-recordkeeping provisions.
Code of Federal Regulations, 2014 CFR
2014-07-01
... PROGRAMS (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS FOR SOURCE CATEGORIES (CONTINUED) National Emission Standards for Hazardous Air Pollutant Emissions: Group IV Polymers and Resins... requirements for Group 2 batch process vents that are exempt from the batch mass input limitation provisions...
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Different cultivation methods to acclimatise ammonia-tolerant methanogenic consortia.
Tian, Hailin; Fotidis, Ioannis A; Mancini, Enrico; Angelidaki, Irini
2017-05-01
Bioaugmentation with ammonia tolerant-methanogenic consortia was proposed as a solution to overcome ammonia inhibition during anaerobic digestion process recently. However, appropriate technology to generate ammonia tolerant methanogenic consortia is still lacking. In this study, three basic reactors (i.e. batch, fed-batch and continuous stirred-tank reactors (CSTR)) operated at mesophilic (37°C) and thermophilic (55°C) conditions were assessed, based on methane production efficiency, incubation time, TAN/FAN (total ammonium nitrogen/free ammonia nitrogen) levels and maximum methanogenic activity. Overall, fed-batch cultivation was clearly the most efficient method compared to batch and CSTR. Specifically, by saving incubation time up to 150%, fed-batch reactors were acclimatised to nearly 2-fold higher FAN levels with a 37%-153% methanogenic activity improvement, compared to batch method. Meanwhile, CSTR reactors were inhibited at lower ammonia levels. Finally, specific methanogenic activity test showed that hydrogenotrophic methanogens were more active than aceticlastic methanogens in all FAN levels above 540mgNH 3 -NL -1 . Copyright © 2017 Elsevier Ltd. All rights reserved.
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Integrated production of lactic acid and biomass on distillery stillage.
Djukić-Vuković, Aleksandra P; Mojović, Ljiljana V; Vukašinović-Sekulić, Maja S; Nikolić, Svetlana B; Pejin, Jelena D
2013-09-01
The possibilities of parallel lactic acid and biomass production in batch and fed-batch fermentation on distillery stillage from bioethanol production were studied. The highest lactic acid yield and productivity of 92.3 % and 1.49 g L(-1) h(-1) were achieved in batch fermentation with initial sugar concentration of 55 g L(-1). A significant improvement of the process was achieved in fed-batch fermentation where the concentration of lactic acid was increased to 47.6 % and volumetric productivity for 21 % over the batch process. A high number of Lactobacillus rhamnosus ATCC 7469 viable cells of 10(9) CFU ml(-1) was attained at the end of fed-batch fermentation. The survival of 92.9 % of L. rhamnosus cells after 3 h of incubation at pH 2.5 validated that the fermentation media remained after lactic acid removal could be used as a biomass-enriched animal feed thus making an additional value to the process.
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Zhao, Bo; Wang, Limin; Li, Fengsong; Hua, Dongliang; Ma, Cuiqing; Ma, Yanhe; Xu, Ping
2010-08-01
D-lactic acid was produced by Sporolactobacillus sp. strain CASD in repeated batch fermentation with one- and two-reactor systems. The strain showed relatively high energy consumption in its growth-related metabolism in comparison with other lactic acid producers. When the fermentation was repeated with 10% (v/v) of previous culture to start a new batch, D-lactic acid production shifted from being cell-maintenance-dependent to cell-growth-dependent. In comparison with the one-reactor system, D-lactic acid production increased approximately 9% in the fourth batch of the two-reactor system. Strain CASD is an efficient D-lactic acid producer with increased growth rate at the early stage of repeated cycles, which explains the strain's physiological adaptation to repeated batch culture and improved performance in the two-reactor fermentation system. From a kinetic point of view, two-reactor fermentation system was shown to be an alternative for conventional one-reactor repeated batch operation. Copyright 2010 Elsevier Ltd. All rights reserved.
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Ji, Xiao-Jun; Zhang, Ai-Hui; Nie, Zhi-Kui; Wu, Wen-Jia; Ren, Lu-Jing; Huang, He
2014-10-01
Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a long fermentation period needed process due to the low growth rate of the filamentous fungus used. This causes the low productivity of ARA-rich oil and hinders its industrial mass scale production. In the present study, different fed-batch strategies were conducted to shorten the fermentation period. The result showed that compared with the batch culture, the fermentation period was shortened from 7days to 5days with the productivity of ARA-rich oil increased from 0.9g/(L·d) to 1.3g/(L·d) by using the fed-batch fermentation strategy. Furthermore, repeated fed-batch fermentation strategy was adopted to achieve the purpose of continuous production. By using this strategy, the fermentation period was shortened from 40days to 26days in a four cycle repeated fed-batch fermentation. This strategy proved to be convenient and economical for ARA-rich oil commercial production process. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Lipid Content and Cryotolerance of Bakers' Yeast in Frozen Doughs †
Gélinas, Pierre; Fiset, Gisèle; Willemot, Claude; Goulet, Jacques
1991-01-01
The relationship between lipid content and tolerance to freezing at −50°C was studied in Saccharomyces cerevisiae grown under batch or fed-batch mode and various aeration and temperature conditions. A higher free-sterol-to-phospholipid ratio as well as higher free sterol and phospholipid contents correlated with the superior cryoresistance in dough or in water of the fed-batch-grown compared with the batch-grown cells. For both growth modes, the presence of excess dissolved oxygen in the culture medium greatly improved yeast cryoresistance and trehalose content (P. Gélinas, G. Fiset, A. LeDuy, and J. Goulet, Appl. Environ. Microbiol. 26:2453-2459, 1989) without significantly changing the lipid profile. Under the batch or fed-batch modes, no correlation was found between the cryotolerance of bakers' yeast and the total cellular lipid content, the total sterol content, the phospholipid unsaturation index, the phosphate or crude protein content, or the yeast cell morphology (volume and roundness). PMID:16348412
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Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs
Gélinas, Pierre; Fiset, Gisèle; LeDuy, Anh; Goulet, Jacques
1989-01-01
The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min−1. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h−1 compared with 0.117 h−1) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium. PMID:16348024
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Lee, Sang Myeon; Park, Kwang Hyun; Jung, Seungon; Park, Hyesung; Yang, Changduk
2018-05-14
For a given π-conjugated polymer, the batch-to-batch variations in molecular weight (M w ) and polydispersity index (Ð) can lead to inconsistent process-dependent material properties and consequent performance variations in the device application. Using a stepwise-heating protocol in the Stille polycondensation in conjunction with optimized processing, we obtained an ultrahigh-quality PTB7 polymer having high M w and very narrow Ð. The resulting ultrahigh-quality polymer-based solar cells demonstrate up to 9.97% power conversion efficiencies (PCEs), which is over 24% enhancement from the control devices fabricated with commercially available PTB7. Moreover, we observe almost negligible batch-to-batch variations in the overall PCE values from ultrahigh-quality polymer-based devices. The proposed stepwise polymerization demonstrates a facile and effective strategy for synthesizing high-quality semiconducting polymers that can significantly improve device yield in polymer-based solar cells, an important factor for the commercialization of organic solar cells, by mitigating device-to-device variations.
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NITRATE CONVERSION OF HB-LINE REILLEXTM HPQ RESIN
DOE Office of Scientific and Technical Information (OSTI.GOV)
Steimke, J.; Williams, M.; Steeper, T.
Reillex{trademark} HPQ ion exchange resin is used by HB Line to remove plutonium from aqueous streams. Reillex{trademark} HPQ resin currently available from Vertellus Specialties LLC is a chloride ionic form, which can cause stress corrosion cracking in stainless steels. Therefore, HB Line Engineering requested that Savannah River National Laboratory (SRNL) convert resin from chloride form to nitrate form in the Engineering Development Laboratory (EDL). To perform this task, SRNL treated two batches of resin in 2012. The first batch of resin from Reilly Industries Batch 80302MA was initially treated at SRNL in 2001 to remove chloride. This batch of resin,more » nominally 30 liters, has been stored wet in carboys since that time until being retreated in 2012. The second batch of resin from Batch 23408 consisted of 50 kg of new resin purchased from Vertellus Specialties in 2012. Both batches were treated in a column designed to convert resin using downflow of 1.0 M sodium nitrate solution through the resin bed followed by rinsing with deionized water. Both batches were analyzed for chloride concentration, before and after treatment, using Neutron Activation Analysis (NAA). The resin specification [Werling, 2003] states the total chlorine and chloride concentration shall be less than 250 ppm. The resin condition for measuring this concentration is not specified; however, in service the resin would always be fully wet. Measurements in SRNL showed that changing from oven dry resin to fully wet resin, with liquid in the particle interstices but no supernatant, increases the total weight by a factor of at least three. Therefore, concentration of chlorine or chloride expressed as parts per million (ppm) decreases by a factor of three. Therefore, SRNL recommends measuring chlorine concentration on an oven dry basis, then dividing by three to estimate chloride concentration in the fully wet condition. Chloride concentration in the first batch (No.80302MA) was nearly the same before the current treatment (759 ppm dry) and after treatment (745 ppm dry or {approx}248 ppm wet). Treatment of the second batch of resin (No.23408) was very successful. Chloride concentration decreased from 120,000 ppm dry to an average of 44 ppm dry or {approx}15ppm wet, which easily passes the 250 ppm wet criterion. Per guidance from HB Line Engineering, SRNL blended Batch 80302 resin with Batch P9059 resin which had been treated previously by ResinTech to remove chloride. The chloride concentrations for the two drums of Batch P9059 were 248 ppm dry ({approx}83 ppm wet) {+-}22.8% and 583 ppm dry ({approx}194 ppm wet) {+-} 11.8%. The blended resin was packaged in five gallon buckets.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Lee, S. Y.; Hyder, L. K.; Baxter, P. M.
1989-07-01
One objective of the Sedimentary Rock Program at the Oak Ridge National Laboratory has been to examine end-member shales to develop a data base that will aid in evaluations if shales are ever considered as a repository host rock. Five end-member shales were selected for comprehensive characterization: the Chattanooga Shale from Fentress County, Tennessee; the Pierre Shale from Gregory County, South Dakota; the Green River Formation from Garfield County, Colorado; and the Nolichucky Shale and Pumpkin Valley Shale from Roane County, Tennessee. Detailed micromorphological and mineralogical characterizations of the shales were completed by Lee et al. (1987) in ORNL/TM-10567. Thismore » report is a supplemental characterization study that was necessary because second batches of the shale samples were needed for additional studies. Selected physical, chemical, and mineralogical properties were determined for the second batches; and their properties were compared with the results from the first batches. Physical characterization indicated that the second-batch and first-batch samples had a noticeable difference in apparent-size distributions but had similar primary-particle-size distributions. There were some differences in chemical composition between the batches, but these differences were not considered important in comparison with the differences among the end-member shales. The results of x-ray diffraction analyses showed that the second batches had mineralogical compositions very similar to the first batches. 9 refs., 9 figs., 4 tabs.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Helmreich, Grant W.; Hunn, John D.; Skitt, Darren J.
2017-03-01
Coated particle fuel batches J52O-16-93165, 93166, 93168, 93169, 93170, and 93172 were produced by Babcock and Wilcox Technologies (BWXT) for possible selection as fuel for the Advanced Gas Reactor Fuel Development and Qualification (AGR) Program’s AGR-5/6/7 irradiation test in the Idaho National Laboratory (INL) Advanced Test Reactor (ATR). Some of these batches may alternately be used as demonstration coated particle fuel for other experiments. Each batch was coated in a 150-mm-diameter production-scale fluidized-bed chemical vapor deposition (CVD) furnace. Tristructural isotropic (TRISO) coatings were deposited on 425-μm-nominal-diameter spherical kernels from BWXT lot J52R-16-69317 containing a mixture of 15.5%-enriched uranium carbide andmore » uranium oxide (UCO). The TRISO coatings consisted of four consecutive CVD layers: a ~50% dense carbon buffer layer with 100-μm-nominal thickness, a dense inner pyrolytic carbon (IPyC) layer with 40-μm-nominal thickness, a silicon carbide (SiC) layer with 35-μm-nominal thickness, and a dense outer pyrolytic carbon (OPyC) layer with 40-μmnominal thickness. The TRISO-coated particle batches were sieved to upgrade the particles by removing over-sized and under-sized material, and the upgraded batches were designated by appending the letter A to the end of the batch number (e.g., 93165A).« less