Obtaining reliable information from minute amounts of RNA using cDNA microarrays

Background  

High density cDNA microarray technology provides a powerful tool to survey the activity of thousands of genes in normal and diseased cells, which helps us both to understand the molecular basis of the disease and to identify potential targets for therapeutic intervention. The promise of this technology has been hampered by the large amount of biological material required for the experiments (more than 50 μg of total RNA per array). We have modified an amplification procedure that requires only 1 μg of total RNA. Analyses of the results showed that most genes that were detected as expressed or differentially expressed using the regular protocol were also detected using the amplification protocol. In addition, many genes that were undetected or weakly detected using the regular protocol were clearly detected using the amplification protocol. We have carried out a series of confirmation studies by northern blotting, western blotting, and immunohistochemistry assays.     

Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions

A substantial range of protein-protein interactions can be readily monitored in real time using bioluminescence resonance energy transfer (BRET). The procedure involves heterologous coexpression of fusion proteins, which link proteins of interest to a bioluminescent donor enzyme or acceptor fluorophore. Energy transfer between these proteins is then detected. This protocol encompasses BRET1, BRET2 and the recently described eBRET, including selection of the donor, acceptor and substrate combination, fusion construct generation and validation, cell culture, fluorescence and luminescence detection, BRET detection and data analysis. The protocol is particularly suited to studying protein-protein interactions in live cells (adherent or in suspension), but cell extracts and purified proteins can also be used. Furthermore, although the procedure is illustrated with references to mammalian cell culture conditions, this protocol can be readily used for bacterial or plant studies. Once fusion proteins are generated and validated, the procedure typically takes 48-72 h depending on cell culture requirements.     

Tips on improving the efficiency of electrotransfer of target proteins from Phos‐tag SDS‐PAGE gel

The sensitivity of Western blotting analysis after Phos‐tag SDS‐PAGE is occasionally inferior to that after normal (Phos‐tag‐free) SDS‐PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos‐tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet‐tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos‐tag SDS‐PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos‐tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet‐tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos‐tag gels were similar to those from normal gels. In some cases involving the use of a Phos‐tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.     

Nutraceutical effects of bioactive peptides obtained from Pterophylla beltrani (Bolivar & Bolivar) protein isolates

Edible insects have been important sources of food proteins for human consumption and animal feed. In this study, a protein isolate from Pterophylla beltrani Bolivar & Bolivar, 1942 (Orthoptera: Tettigoniidae) was enzymatically processed and its nutraceutical properties were evaluated. Protein isolates were obtained from an insect flour and then was hydrolyzed for 5 h with a sequential process using pepsin and trypsin-chymotrypsin to simulate the gastric intestinal fluids. To evaluate the effect of peptide molecular size on nutraceutical properties, the peptides obtained from Total hydrolyzed (TH) were fractionated by ultrafiltration (UF) with 10 kDa and 3 kDa UF membranes giving fractions (F) with different molecular size (F < 3KDa, F < 10KDa and F > 10KDa). The inhibition assay of Angiotensin-Converting Enzyme (ACE) showed that the best treatment (P < 0.05) was the (TH) with an IC₅₀ value of 0.5 mg/mL while the F < 3KDa was the lowest (P < 0.05) with an IC50 of 1.44 mg/mL, and the peptide size had no effect. However, an α-amylase inhibition was observed with an increase of the IC50 value between TH, F > 10KDa and F < 10KDa although no significative difference (P > 0.05) was found between the TH and F < 3KDa with IC50 of 0.48 and 0.68 mg/mL, respectively. In antioxidant activity, significant differences (P < 0.05) were observed between TH and UF fractions where the best response was in the F < 3KDa. In conclusion, P. beltrani proteins isolate are a source of bioactive peptide, and these could be considered as potential edible insect and sustainable food with nutraceutical effects.     

A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins

We have developed conditions for the efficient electrotransfer from polyacrylamide gels to nitrocellulose sheets of a broad size range of proteins (Mr 8,000 to Mr greater than 400,000). The important features of this procedure include a two-step electrotransfer, beginning with elution of low-molecular-weight polypeptides at a low current density (approximately 1 mA/cm2) for 1 h, followed by prolonged electrotransfer (16-20 h) at high current density (approximately 3.5-7.5 mA/cm2) in conditions that favor the elution of high-molecular-weight proteins. The transfer buffer includes 0.01% sodium dodecyl sulfate to enhance protein elution, and 20% methanol to improve the retention of proteins on the nitrocellulose sheet. The nitrocellulose is air-dried after transfer is complete to eliminate loss of proteins during subsequent processing. This transfer procedure works well with proteins prepared from many different cell types, and is suitable for use with all polyacrylamide gel systems tested. With little or no modification, our method should also be applicable to transfer membranes other than nitrocellulose.     

Optimal time for laparoscopic intrauterine insemination performed on ewes detected in natural heat

The aim of this study was to optimize the laparoscopic intrauterine insemination (LAI) methodology by testing different time intervals between the natural heat detection and ewe insemination. Three experiments were performed in the breeding conditions of Southern Kazakhstan. Ewes (n = 243) were exposed for one hour to direct contact with the teaser rams (once a day, morning or evening). Ewes expressing behavioral symptoms of heat were detected and inseminated with the use of LAI during 210–1290 min (3.5–21.5 hrs) after heat detection. Reproductive traits of lambing rate (LR) and litter size (LS) were recorded according to the births registered at 137 to 152 days post insemination. Our statistical model showed significance only for the effects of ewe age category and the time interval from heat detection to LAI on the LR attribute. The highest LR (38.8%) was detected in ewes at 2.5–3.5 years of age. Corrected least-square means of LR indicated 18.5 hrs. as an optimal time for LAI of ewes in natural heat. In the present study, the percentage value of lambing rate obtained at 18.5 h interval was 70.7%. Therefore, our study suggested an effective methodology to spread valuable genetic information in the sheep population in the regions of extensive farming where heat cycle synchronization is not usually performed. Importantly, our study is among the first ones that follow the European strategy to eliminate the occurrence of hormones in livestock production and the environment.     

Differential proteomic analysis on the effects of 2-methoxy-1,4-naphthoquinone towards MDA-MB-231 cell line

BackgroundWe have previously reported the anti-metastatic effects of 2-methoxy-1,4-naphthoquinone (MNQ) against MDA-MB-231 cell line.PurposeTo investigate the molecular mechanism underlying the anti-metastatic effects of MNQ towards MDA-MB-231 cell line via the comparative proteomic approach.Study design/methodsDifferentially expressed proteins in MNQ-treated MDA-MB-231 cells were identified by using two-dimensional gel electrophoresis coupled with tandem mass spectrometry. Proteins and signalling pathways associated with the identified MNQ-altered proteins were studied by using Western blotting.ResultsSignificant modulation of MDA-MB-231 cell proteome was observed upon treatment with MNQ in which the expressions of 19 proteins were found to be downregulated whereas another eight were upregulated (>1.5 fold, p < 0.05). The altered proteins were mainly related to cytoskeletal functions and regulations, mRNA processing, protein modifications and oxidative stress response. Notably, two of the downregulated proteins, protein S100-A4 (S100A4) and laminin-binding protein (RPSA) are known to play key roles in driving metastasis and were verified using Western blotting. Further investigation using Western blotting also revealed that MNQ decreased the activations of pro-metastatic ERK1/2 and NF-κB signalling pathways. Moreover, MNQ was shown to stimulate the expression of the metastatic suppressor, E-cadherin.ConclusionThis study reports a proposed mechanism by which MNQ exerts its anti-metastatic effects against MDA-MB-231 cell line. The findings from this study offer new insights on the potential of MNQ to be developed as a novel anti-metastatic agent.     

Cell-based assay for the detection of chemically induced cellular stress by immortalized untransformed transgenic hepatocytes

Background  

Primary hepatocytes, one of the most widely used cell types for toxicological studies, have a very limited life span and must be freshly derived from mice or even humans. Attempts to use stable cell lines maintaining the enzymatic pattern of liver cells have been so far unsatisfactory. Stress proteins (heat shock proteins, HSPs) have been proposed as general markers of cellular injury and their use for environmental monitoring has been suggested. The aim of this work is to develop a bi-transgenic hepatocyte cell line in order to evaluate the ability of various organic and inorganic chemicals to induce the expression of the HSP70 driven reporter gene.     

Nanoscale characterization coupled to multi-parametric optimization of Hi5 cell transient gene expression

Polyethylenimine (PEI)-based transient gene expression (TGE) is nowadays a well-established methodology for rapid protein production in mammalian cells, but it has been used to a much lower extent in insect cell lines. A fast and robust TGE methodology for suspension Hi5 (Trichoplusia ni) cells is presented. Significant differences in size and morphology of DNA:PEI polyplexes were observed in the different incubation solutions tested. Moreover, minimal complexing time (&lt; 1 min) between DNA and PEI in 150 mM NaCl solution provided the highest transfection efficiency. Nanoscopic characterization by means of cryo-EM revealed that DNA:PEI polyplexes up to 300–400 nm were the most efficient for transfection. TGE optimization was performed using eGFP as model protein by means of the combination of advanced statistical designs. A global optimal condition of 1.5 × 10⁶ cell/mL, 2.1 μg/mL of DNA, and 9.3 μg/mL PEI was achieved through weighted-based optimization of transfection, production, and viability responses. Under these conditions, a 60% transfection and 0.8 μg/10⁶ transfected cell·day specific productivity were achieved. The TGE protocol developed for Hi5 cells provides a promising baculovirus-free and worthwhile approach to produce a wide variety of recombinant proteins in a short period of time.

     

Synthesis and application of photoaffinity probe containing an intact isoprenoid chain

Two novel chemical probes each carrying an intact isoprenoid chain, a biotin tag and a benzophenone moiety were synthesized. Photoaffinity labeling of the Saccharomyces cerevisiae cell lysate revealed that these probes could selectively trap some proteins, and proteins with molecular weight of ～70 KDa appeared as a major band upon Streptavidin blot analysis.     

Binding Kinetics of Histone Chaperone Chz1 and Variant Histone H2A.Z-H2B by Relaxation Dispersion NMR Spectroscopy

The genome of eukaryotic cells is packed into a compact structure called chromatin that consists of DNA as well as histone and non-histone proteins. Histone chaperones associate with histone proteins and play important roles in the assembly of chromatin structure and transport of histones in the cell. The recently discovered histone chaperone Chz1 associates with the variant histone H2A.Z of budding yeast and plays a critical role in the exchange of the canonical histone pair H2A-H2B for the variant H2A.Z-H2B. Here, we present an NMR approach that provides accurate estimates for the rates of association and dissociation of Chz1 and H2A.Z-H2B. The methodology exploits the fact that in a 1:1 mixture of Chz1 and H2A.Z-H2B, the small amounts of unbound proteins that are invisible in spectra produce line broadening of signals from the complex that can be quantified in terms of the thermodynamics and kinetics of the exchange process. The dissociation rate constant measured, 22 ± 2 s^− 1, provides an upper bound for the rate of transfer of H2A.Z-H2B to the chromatin remodeling complex, and the faster-than-diffusion association rate, 10⁸ ± 10⁷ M^− 1 s^− 1, establishes the importance of attractive electrostatic interactions that form the chaperone-histone complex.     

Comparison of three multi-cryoprotectant loading protocols for vitrification of porcine articular cartilage

Vitrification is a cryopreservation technique for the long-term storage of viable tissue, but the success of this technique relies on multiple factors. In 2012, our group published a working vitrification protocol for intact human articular cartilage and reported promising chondrocyte recovery after using a four-step multi-cryoprotectant (CPA) loading method that required 570 min. However, this protocol requires further optimization for clinical practice. Herein, we compared three multi-step CPA loading protocols to investigate their impact on chondrocyte recovery after vitrification of porcine articular cartilage on a bone base, including our previous four-step protocol (original: 570 min), and two shorter three-step protocols (optimized: 420 min, and minimally vitrifiable: 310 min). Four different CPAs were used including glycerol, dimethyl sulfoxide, ethylene glycol and propylene glycol. As vitrification containers, two conical tubes (50 ml and 15 ml) were evaluated for their heat transfer impact on chondrocyte recovery after vitrification. Osteochondral dowels were cored into two diameters of 10.0 mm and 6.9 mm with an approximately 10-mm thick bone base, and then allocated into the twelve experimental groups based on CPA loading protocol, osteochondral dowel size, and vitrification container size. After vitrification at −196 °C and tissue warming and CPA removal, samples in all groups were assessed for both chondrocyte viability and metabolic activity. The optimized protocol proposed based on mathematical modelling resulted in similar chondrocyte recovery to our original protocol and it was 150 min shorter. Furthermore, this study illustrated the role of CPA permeation (dowel size) and heat transfer (container size) on vitrification protocol outcome.     

Conductometric immunoassay for interleukin-6 in human serum based on organic/inorganic hybrid membrane-functionalized interface

Various sensor-based immunoassay methods have been extensively developed for the detection of interleukin-6 (IL6), but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. The aim of this work is to develop a simple and sensitive conductometric immunoassay for IL6 in human serum by using an organic/inorganic hybrid membrane-functionalized interface. Initially, thionine-bound 3,4,9,10-perylenetetracarboxylic acid was doped into colloidal alumina, then nanogold particles were immobilized onto the thionine surface, and then horseradish peroxidase-labeled anti-IL6 antibodies were conjugated on the nanogold surface. The organic/inorganic hybrid membrane provides a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The performance and factors influencing the performance of the immunosensor were evaluated. The detection is based on the change in local conductivity before and after the antigen-antibody interaction in 0.02 M phosphate buffer solution (pH 6.8) containing 50 μM H₂O₂, 0.01 M KI and 0.15 M NaC1. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 25 to 400 pg/ml towards IL6 with a relatively low detection limit of 5 pg/ml (S/N = 3). The stability, reproducibility and precision of the immunosensor were acceptable. 37 serum specimens were assayed by the developed immunosensor and standard enzyme-linked immunosorbent assay, respectively, and the results obtained were almost consistent. More importantly, the detection methodology provides a promising approach for other proteins or biosecuritys.     

Scale-up of cell culture bioreactors using biomechatronic design

Scale-up of cell culture bioreactors is a challenging engineering work that requires wide competence in cell biology, mechanical engineering and bioprocess design. In this article, a new approach for cell culture bioreactor scale-up is suggested that is based on biomechatronic design methodology. The approach differs from traditional biochemical engineering methodology by applying a sequential design procedure where the needs of the users and alternative design solutions are systematically analysed. The procedure is based on the biological and technical functions of the scaled-up bioreactor that are derived in functional maps, concept generation charts and scoring and interaction matrices. Basic reactor engineering properties, such as mass and heat transfer and kinetics are integrated in the procedure. The methodology results in the generation of alternative design solutions that are thoroughly ranked with help of the user needs. Examples from monoclonal antibodies and recombinant protein production illuminate the steps of the procedure. The methodology provides engineering teams with additional tools that can significantly facilitate the design of new production methods for cell culture processes.     

A rapid screening method for cell lines producing singly-tagged recombinant proteins using the “TARGET tag” system

Recombinant production of extracellular glycoproteins in stable mammalian cell lines is an ideal technique for obtaining a large quantity of high-quality proteins. In most cases, however, current methodologies do not allow for sufficiently rapid cell line development and protein purification. Here, we describe a 21-residue peptide tag (designated as TARGET tag) and its use for rapid stable cell line development and purification. The ability of the anti-tag antibody P20.1 to withstand repetitive regeneration cycles has enabled the development of a sensitive surface plasmon resonance-based screening format that requires only 20 µl of cell culture supernatants. Direct and semi-quantitative screening at the 96-well culture scale eliminated the need for a second screening, re-cloning, or sorting, thereby minimizing culture pre-production time. Using this system, “high producer” cell lines were established in less than a month, and milligram quantities of target proteins could be purified with a standardized protocol.     

A statistical approach to improve compound screening in cell culture media

The Chinese hamster ovary (CHO) cell line is widely used for the production of recombinant proteins due to its high growing capacity and productivity, as well as other cell lines derived later than CHO. Adapting cell culture media for each specific cell line is a key to exploit these features for cost effective and fast product generation. Media supplementation is generally addressed by means of one‐factor‐at‐a‐time or classical design of experiments approaches but these techniques may not be efficient enough in preliminary screening phases. In this study, a novel strategy consisting in folding over the Plackett–Burman design was used to increase cell growth and trastuzumab production of different CHO cell lines through supplementation with nonanimal recombinant compounds. Synergies between compounds could be detected with a reduced number of experiments by using this methodology in comparison to more conventional fractional factorial designs. In the particular case reported here, the sequential use of this modified Plackett–Burman in combination with a Box‐Behnken design led to a 1.5‐fold increase in cell growth (10 × 10⁶ cells/mL) and a two‐fold in trastuzumab titer (122 mg/L) in suspension batch culture.     

Use of a Western blotting technique in the purification of a cysteine proteinase inhibitor

In Western blotting procedures, proteins are resolved in sodium dodecyl sulfate-polyacrylamide gels with subsequent electrophoretic transfer onto nitrocellulose membranes. Although this procedure is generally employed as an analytical technique for assessing interactions of proteins with antibodies, the present report describes the use of Western blotting as a preparative procedure in the purification of a biologically active proteinase inhibitor from the cellular slime mold, Dictyostelium discoideum. The feasibility of using Western blotting for inhibitor purification depended upon the unique stability properties of the inhibitor under denaturing conditions.