Amplification protocols introduce systematic but reproducible errors into gene expression studies

The desire to perform microarray experiments with small starting amounts of RNA has led to the development of a variety of protocols for preparing and amplifying mRNA. This has consequences not only for the standardization of experimental design, but also for reproducibility and comparability between experiments. Here we investigate the differences between the Affymetrix standard and small sample protocols and address the data analysis issues that arise when comparing samples and experiments that have been processed in different ways. We show that data generated on the same platform using different protocols are not directly comparable. Further, protocols introduce systematic biases that can be largely accounted for by using the correct data analysis techniques.     

Sequencing of double-stranded PCR products

Very often the experimental step following PCR is sequencing of the amplified fragment. Two protocols that allow direct sequencing of a double-stranded PCR product are described. The first involves removal of one strand of the PCR product using an Ml3 single-stranded DNA clone, allowing the second strand to be sequenced. The second protocol involves Maxam-Gilbert chemical sequencing after PCR amplification with one labeled primer. The advantages and disadvantages of the two protocols are compared, but both yield DNA sequence without cloning of the PCR product.     

Anatomic and anesthetic considerations in experimental cardiopulmonary surgery in swine

We have used immature commercial swine (13-25 kg) successfully in a variety of experimental cardiopulmonary surgical procedures in our laboratories since 1981. Multiple drug anesthetic protocols using barbiturates, narcotics, paralytic and antiarrhythmic agents have been employed in over 400 procedures per year. Complications, including fatal cardiac arrhythmias, have been greatly reduced by anesthetic protocols and surgical procedures developed through experience.     

Role of the blood service in cellular therapy

Cellular therapy is a novel form of medical or surgical treatment using cells in place of or in addition to traditional chemical drugs. The preparation of cellular products - called advanced therapy medicinal products - ATMP in Europe, requires compliance with good manufacturing practices (GMP). Based on long-term experience in blood component manufacturing, product traceability and hemovigilance, selected blood services may represent ideal settings for the development and experimental use of ATMP. International harmonization of the protocols and procedures for the preparation of ATMP is of paramount importance to facilitate the development of multicenter clinical trials with adequate sample size, which are urgently needed to determine the clinical efficacy of ATMP. This article describes European regulations on cellular therapy and summarizes the activities of the 'Franco Calori' Cell Factory, a GMP unit belonging to the department of regenerative medicine of a large public university hospital, which acquired a certification for the GMP production of ATMP in 2007 and developed nine experimental clinical protocols during 2003-2011.     

Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation.

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.     

Myoelectric activity along human gastrocnemius medialis: Different spatial distributions of postural and electrically elicited surface potentials

It has recently been shown that motor units in human medial gastrocnemius (MG), activated during standing, occupy relatively small territories along the muscle’s longitudinal axis. Such organisation provides potential for different motor tasks to produce differing regional patterns of activity. Here, we investigate whether postural control and nerve electrical stimulation produce equal longitudinal activation patterns in MG. Myoelectric activity, at different proximal–distal locations of MG, was recorded using a linear electrode array. To ensure differences in signal amplitude between channels did not result from local, morphological factors two experimental protocols were completed: (i) quiet standing; (ii) electrical stimulation of the tibial nerve. Averaged, rectified values (ARVs) were calculated for each channel in each condition. The distribution of signals along electrode channels was described using linear regression and differences between protocols at each channel determined as the ratio between mean ARV from standing: stimulation protocols. Ratio values changed systematically across electrode channels in seven (of eight) participants, with larger values in distal channels. The distribution of ARV along MG therefore differed between experimental conditions. Compared to fibres of units activated during MG nerve stimulation, units activated during standing may have a tendency to be more highly represented in the distal muscle portion.     

Combining membrane potential imaging with L-glutamate or GABA photorelease

Combining membrane potential imaging using voltage sensitive dyes with photolysis of L-glutamate or GABA allows the monitoring of electrical activity elicited by the neurotransmitter at different sub-cellular sites. Here we describe a simple system and some basic experimental protocols to achieve these measurements. We show how to apply the neurotransmitter and how to vary the dimension of the area of photolysis. We assess the localisation of photolysis and of the recorded membrane potential changes by depolarising the dendrites of cerebellar Purkinje neurons with L-glutamate photorelease using different experimental protocols. We further show in the apical dendrites of CA1 hippocampal pyramidal neurons how L-glutamate photorelease can be used to calibrate fluorescence changes from voltage sensitive dyes in terms of membrane potential changes (in mV) and how GABA photorelease can be used to investigate the phenomenon of shunting inhibition. We also show how GABA photorelease can be used to measure chloride-mediated changes of membrane potential under physiological conditions originating from different regions of a neuron, providing important information on the local intracellular chloride concentrations. The method and the proof of principle reported here open the gateway to a variety of important applications where the advantages of this approach are necessary.     

Modeling and simulation of cancer immunoprevention vaccine

We present an in silico model that simulates the immune system responses to tumor cells in naive and vaccinated mice. We have demonstrated the ability of this model to accurately reproduce the experimental results. MOTIVATION: In vivo experiments on HER-2/neu mice have shown the effectiveness of Triplex vaccine in the protection of mice from mammary carcinoma. Full protection was conferred using chronic (prophylactic) vaccination protocol while therapeutic vaccination was less efficient. Our in silico model was able to closely reproduce the effects of various vaccination protocols. This model is the first step towards the development of in silico experiments searching for optimal vaccination protocols. RESULTS: In silico experiments carried out on two large statistical samples of virtual mice showed very good agreements with in vivo experiments for all experimental vaccination protocols. They also show, as supported by in vivo experiments, that the humoral response is fundamental in controlling the tumor growth and therefore suggest the selection and timing of experiments for measuring the activity of T cells. CONTACT: francesco@dmi.unict.it SUPPLEMENTARY INFORMATION: http://www.dmi.unict.it/CIG/suppdata_bioinf.html.     

A nonlinear anisotropic inverse method for computational dissection of inhomogeneous planar tissues

Quantification of the mechanical behavior of soft tissues is challenging due to their anisotropic, heterogeneous, and nonlinear nature. We present a method for the ‘computational dissection’ of a tissue, by which we mean the use of computational tools both to identify and to analyze regions within a tissue sample that have different mechanical properties. The approach employs an inverse technique applied to a series of planar biaxial experimental protocols. The aggregated data from multiple protocols provide the basis for (1) segmentation of the tissue into regions of similar properties, (2) linear analysis for the small-strain behavior, assuming uniform, linear, anisotropic behavior within each region, (3) subsequent nonlinear analysis following each individual experimental protocol path and using local linear properties, and (4) construction of a strain energy data set W(E) at every point in the material by integrating the differential stress–strain functions along each strain path. The approach has been applied to simulated data and captures not only the general nonlinear behavior but also the regional differences introduced into the simulated tissue sample.     

Cost-efficacy in measuring farmland biodiversity – lessons from the Farm Scale Evaluations of genetically modified herbicide-tolerant crops

Measuring farmland biodiversity is time‐consuming and costly. Operational data from the Farm Scale Evaluation of genetically modified crops project were collated to identify the financial and time costs of each of the 14 protocols used. A subset of 113 of the 266 experimental sites was used. The mean overall cost per site was £19 453 (£ of 2002). Laboratory time was almost 2.5 times that in the field. The most costly protocol was soil surface invertebrates because it required species level identification. The ‘bees and butterflies’ protocol at £418 per site was particularly cost‐effective. The six vegetation protocols accounted for 65% and the six arthropod protocols accounted for 29% of the total costs. The recommended reduction from 12 to 3 transects would have saved £1356 per site, 6% of total budget. A minimalist approach using the single‐season seedbank protocol would cost £3437 per site. The effect of geographical spread of sites on cost was small because of clustering of sites and the large number of protocols. Careful selection of ecological indicators can save considerable resources.     

Fast energy-aware OLSR routing in VANETs by means of a parallel evolutionary algorithm

This work tackles the problem of reducing the power consumption of the OLSR routing protocol in vehicular networks. Nowadays, energy-aware and green communication protocols are important research topics, specially when deploying wireless mobile networks. This article introduces a fast automatic methodology to search for energy-efficient OLSR configurations by using a parallel evolutionary algorithm. The experimental analysis demonstrates that significant improvements over the standard configuration can be attained in terms of power consumption, with no noteworthy loss in the QoS.     

COMPARISON OF THE STATISTICAL ANALYSIS OF HEDONIC DATA USING ANALYSIS OF VARIANCE AND MULTIPLE COMPARISONS VERSUS AN R-INDEX ANALYSIS OF THE RANKED DATA

Consumers rated a set of toothbrushes and a set of potato chips on 9‐point and 21‐point hedonic scales under two experimental protocols: a traditional approach and rank‐rating. The hedonic data were analyzed in the usual way by using anova and multiple comparisons and also by ranking the data and using an R‐index analysis. The hypothesis that the latter analysis would elicit fewer significant differences was not confirmed.     

Effects of 50 Hz magnetic field exposure on tumor experimental models

The aim of this study was to investigate the interaction between a 50 Hz, 2 mT magnetic field (MF) exposure and cell growth of mammary murine adenocarcinoma, injected into experimental mice. Six different experimental protocols were performed over 2 years; several different protocols of timing of exposure were tested. X-ray radiation was adopted as the positive control. Tumor incidence and the tumor development time were calculated. No effect was observed in any experiment, and there was no statistically significant difference related to time courses among the protocols used. Neither the time of tumor cell injection nor the time of exposure produced differences between unexposed, sham, and exposed mice. When X-ray radiation was applied, the cytotoxic effect of ionizing radiation was clear, but was not increased or modified by MF exposure. Finally, the study revealed how the host-tumor system has shown a distinctive variability, unmodified by MF exposure.     

Acute insulin responses to intravenous glucose and GLP-1 are independent of preceding high-frequency insulin pulse-defects induced by glucose entrainment in healthy humans.

The objective of this study was to test the hypothesis that high-frequency oscillations in insulin release is a part of the mechanistic basis of a prompt and adequate insulin response to iv-glucose and GLP-1 exposure. In ten healthy subjects, five different insulin release patterns were induced for 360 min using computer-based glucose infusion (glucose delivered in a constant, a regular pulsatile, an irregular pulse frequency, an irregular pulse amplitude or a regular but very fast-pulsatile manner) in healthy subjects. The amount of glucose infused was identical in all five protocols (24 mg/kg/h). After 360 min, insulin secretion was assessed by means of a first-phase insulin secretion test (25 g glucose) and injection of GLP-1 (9 microg). By frequent blood sampling and analysis of insulin concentration, glucose-induced entrainment was evident in all protocols except in the constant infusion and the very fast-pulse protocol. The first-phase insulin release to glucose and GLP-1-induced insulin release were, however, comparable in the protocols. We therefore conclude from this short-term experimental setting in healthy subjects that beta-cell response to either iv-glucose or GLP-1 is independent of the preceding regularity of oscillations in insulin release.     

A dilution-filtration system for removing cryoprotective agents

In most cryopreservation applications, the final concentrations of cryoprotective agents (CPAs) must be reduced to biocompatible levels. However, traditional methods for removing CPAs usually have disadvantages of operation complexity, time consumption, and ease of contamination, especially for the applications involving large volumes of cell suspensions. A dilution-filtration system, which involves pure ultrafiltration for separation, was developed for continuous, automatic, and closed process of removing CPAs. To predict the optimal protocols under given experimental conditions, a theoretical model was established first. Cell-free experiments were then conducted to investigate the variation in CPA concentration during the process, and the experimental data were compared with the theoretical values for the validation of the model. Finally, ten units (212.9?ml/unit±9.5?ml/unit) of thawed human red blood cells (cryopreserved with 40% (w/v) glycerol) were deglycerolized using the theoretically optimal operation protocols to further validate the effectiveness and advantage of the system. In the cell-free experiments, glycerol was continuously removed and the concentration variations fitted the simulated results quite well. In the in-vitro experiments, glycerol concentration in RBC suspension was reduced to 5.57?g/l±2.81?g/l within an hour, and the cell count recovery rate was 91.19%±3.57%, (n=10), which proves that the system is not only safe for removing CPAs, but also particularly efficient for processing large-scale samples. However, the operation parameters must be carefully controlled and the optimal protocols should be specialized and various from case to case. The presented theoretical model provides an effective approach to find out the optimal operation protocols under given experimental conditions and constrains.