Search

Silica column-based RNA purification procedures have widespread use in mRNA profiling for body fluid identification in forensic samples. Also, automated RNA purification systems employing magnetic bead technology have recently become available. In this preliminary study, to ascertain which RNA purification technology is more suitable for the identification of body fluids by real-time reverse transcription polymerase chain reaction (RT-PCR), comparative analyses of the yield and quality of total RNA were performed between automated purification using an EZ1 Advanced Instrument and manual purification using an RNeasy Mini Kit. The yield and size distribution of total RNA were compared by gene expression analysis of two different sized fragments of the β-actin gene. In addition, the relative amounts of several target genes were compared between the purification methods, and the integrity of total RNA was determined by chip-based electrophoresis. The results of this study suggest that RNeasy can purify higher-quality RNA as compared with automated purification using EZ1. The sensitivity of the RT-PCR analysis, however, was higher in the EZ1-purified samples, likely due to the relative efficiency of EZ1 in extracting short-length RNA from degraded samples. We also show that the quantification of relative levels of body fluid-specific genes could be influenced by the purification procedure. Our results indicate that although use of high-quality RNA is generally required for reproducible results in gene expression analysis, the forensic relevance of short RNA fragments in highly degraded samples cannot be ruled out. Furthermore, our results suggest that automated purification procedures as well as silica column-based manual purification procedures can be used for mRNA-based body fluid identification in forensic samples. [ABSTRACT FROM AUTHOR]

A X-prolyl-dipeptidyl aminopeptidase (PepX, EC 3.4.14.11) from Lactobacillus helveticus ATCC 12046 was automatically purified to homogeneity after cultivation in MRS broth. The purification of PepX lasted only 6 h and the PepX activity yield was 56%. The specific activity was determined at 370 nkat H-Ala-Pro- p NA mg −1 and the purification factor was 162-fold. A gas chromatographic assay was established for the determination of the PepX activity using original tripeptide substrates. The kinetic parameters of purified PepX were analyzed using different original tripeptide substrates with the structure Xxx-Pro-Yyy. Alanine at position P 2 (Xxx) or P 1 ′ (Yyy) was retained, while the corresponding amino acid at P 1 ′ or P 2 (serine, tyrosine, aspartic acid, or arginine) was varied. We analyzed the influence of the position of the different amino acids (P 2 or P 1 ′) and the side chain group (serine: polar and uncharged; tyrosine: hydrophobic; aspartic acid: negatively charged; arginine: positively charged) on the PepX activity and the kinetic parameters. Significant differences were observed by comparing the kinetic parameters of PepX using original tripeptides as substrates with the chromogenic peptide H-Ala-Pro- p NA as a substrate; for instance, a K m -value of 0.54 mM and a V max -value of 4.76 nkat mL −1 were determined using the tripeptide Ser-Pro-Ala as a substrate. The kinetic parameters for the chromogenic peptide H-Ala-Pro- p NA were 1.53 mM and 2.61 nkat mL −1 for K m and V max , respectively. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we investigated the suppressive effect of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (DL-PCBs) transfer from the feed to the eggs of laying hens by using activated carbon as a feed additive. Four groups of six hens (White Leghorn egg-layers; age, 11weeks) were housed as two control groups and two exposure groups for a period of 20weeks. Two control groups were fed with either the basal feed “Control” or basal feed additing activated carbon “Control+C”. Another two exposure groups were fed with feed contaminated (about 6ng TEQ kg−1 feed) by standard solutions of PCDDs/PCDFs and DL-PCBs “Exposure” alone and contaminated feed adding activated carbon “Exposure+C”. There was no significant effect on each groups for the growth rate, biochemical blood components, and egg production: these were around the standard levels for poultry in general. Moreover the results in this study showed the availability of activated carbon as a feed additive owing to the reduction in the risk of food pollution by PCDDs/PCDFs and DL-PCBs. The concentration in the eggs of the Exposure group gradually increased following the start of egg-laying but reached a steady state after about 1month. In contrast, the concentration for the Exposure+C group was stationary and below the maximum EU level (6pgTEQg−1 fat). In comparison to the Exposure group, the Exposure+C group showed a significant decline in the percentage of bioaccumulation into the egg. This reduction due to activated carbon was also observed in the muscle and abdominal fat. The reductions were compound- and congener-dependent for DL-PCBs as follows: PCDDs/PCDFs, non-ortho-PCBs, and mono-ortho-PCBs were more than 90%, 80%, and 50%, respectively, irrespective of the type of tissues. Fat soluble vitamin concentrations in the eggs of the Exposure+C group showed lower trends than the Exposure group. The γ-tocopherol and α-tocopherol concentrations in eggs of Exposure+C group showed a significant reduction of about 40%. However, the addition of activated carbon into animal feed could obviate the remote potential for accidents causing unintentional food pollution with PCDDs/PCDFs and DL-PCBs. [Copyright &y& Elsevier]

A fundamental challenge in high-throughput (HT) expression screening is to rapidly identify the appropriate expression system for many targets in parallel. Known or unknown open reading frames (ORFs) are typically amplified by PCR and then cloned into a variety of vectors, producing recombinants used to direct target protein expression in Escherichia coli, insect cells, mammalian cells, or yeast. To facilitate rapid expression and purification in Spodoptera insect cells ( Sf9), we developed transient expression vectors that include an enterokinase cleavage site immediately upstream of a ligation-independent cloning site (Ek/LIC). We also developed a high-efficiency insect cell transfection reagent, and automation-compatible fusion protein purification system for insect cells to facilitate expression screening and protein production. Positive clones identified from the small-scale screening were subjected to a larger scale production. Using this InsectDirectTM approach, we successfully expressed milligram quantities of different human proteins including heat shock proteins, phospholipases, and protein kinases. [ABSTRACT FROM AUTHOR]

Different techniques can be applied for the automated production of small and large compound collections. Large libraries that are often generated and tested during the lead-finding stage of a project are typically produced by solid-phase chemistry. Libraries that are significantly smaller in size are often synthesized in solution. Chemistry in solution is rather versatile, offers numerous advantages and is therefore often the method of choice for generating small libraries during a lead optimization process. Fast and reliable purification procedures are required to yield compounds of high quality that can be immediately used in biological as well as pharmacological assays. Solution-phase chemistry combined with automated purification was applied to optimize initial lead inhibitors for the two human orexin receptors OX1 and OX2. Starting from a submicro-molar OX1 selective lead compound, low nanomolar analogues with improved physico-chemical properties were synthesized that antagonize either one or both orexin receptors.

We have constructed a dual expression vector for the production of recombinant proteins in both Escherichia coli and insect cells. In this vector, the baculoviral polyhedrin promoter was positioned upstream of the bacteriophage T7 promoter and the lac operator. This vector, designated pBEV, was specifically designed to exploit the advantages that both hosts would provide. This vector also facilitates one-stop cloning, thereby simplifying the expression process for automation, and the development of a high-throughput method for protein expression. Utilizing the multi-system vector pBEV, a high-throughput process was developed with expression in deep-well blocks and purification in micro-titer plates enabling the identification of expression and solubility in both E. coli and insect cells. In this study, using pBEV, we have successfully expressed and purified multiple human kinases produced in E. coli and insect cells. Our results validate expression screening as a strategy to rapidly triage proteins identifying the optimum expression system and conditions for production. [Copyright &y& Elsevier]

MagneSil™ Paramagnetic Particles are silica-paramagnetic particles with an affinity for nucleic acids under defined conditions. Particle structure and solution composition can be altered to selectively adsorb nucleic acids based upon type and size. These properties have been used to develop purification methods based on a three-step bind, wash, and elute process. The MagneSil™ technology is readily adaptable to robotic platforms, allowing complete automation of the purification process in either 96- or 384- well plate formats. This article introduces the basic physical and performance characteristics of the MagneSil™ Paramagnetic Particles. [Copyright &y& Elsevier]

Lung PET/CT is a promising imaging modality for regional lung function assessment. Our aim was to develop and validate a fast, simple, and fully automated GMP compliant [ 68 Ga]Ga-MAA labeling procedure, using a commercially available [ 99m Tc]Tc-MAA kit, a direct gallium-68 eluate and including a purification of the [ 68 Ga]Ga-MAA. Method: The synthesis parameters (pH, heating temperature) were manually determined. Automated 68 Ga-labeling of MAA was then developed on a miniAIO (Trasis®, Ans, Belgium) module. An innovative automated process was developed for the purification. The process was then optimized and adapted to automate both the [ 68 Ga]Ga-MAA synthesis and the isolation of gallium-68 eluate required for the pulmonary ventilation PET/CT. Results: The 15-min process demonstrated high reliability and reproducibility, with high synthesis yield (>95 %). Mean [ 68 Ga]Ga-MAA radiochemical purity was 99 % ± 0.6 %. The 68 Ga-labeled MAA particles size and morphology remained unchanged. Conclusion: A fast, user friendly, and fully automated process to produce GMP [ 68 Ga]Ga-MAA for clinical use was developed. This automated process combining the advantages of using a non-modified MAA commercial kit, a gallium-68 eluate without pre-purification and an efficient final purification of the [ 68 Ga]Ga-MAA may facilitate the implementation of lung PET/CT imaging in nuclear medicine departments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Blanc-Béguin, Masset, Robin, Tripier, Hennebicq, Guilloux, Vriamont, Warnier, Cogulet, Eu, Salaün and Le Roux.)

Flavourzyme is sold as a peptidase preparation from Aspergillus oryzae. The enzyme preparation is widely and diversely used for protein hydrolysis in industrial and research applications. However, detailed information about the composition of this mixture is still missing due to the complexity. The present study identified eight key enzymes by mass spectrometry and partially by activity staining on native polyacrylamide gels or gel zymography. The eight enzymes identified were two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one α-amylase from the A. oryzae strain ATCC 42149/RIB 40 (yellow koji mold). Various specific marker substrates for these Flavourzyme enzymes were ascertained. An automated, time-saving nine-step protocol for the purification of all eight enzymes within 7 h was designed. Finally, the purified Flavourzyme enzymes were biochemically characterized with regard to pH and temperature profiles and molecular sizes.