The use of a multichannel scaling method to detect radioactive species in spread monolayers

This study describes a simple and inexpensive method for monitoring radioactive species spread as monolayers at the air/water interface. The combination of a discriminator and multichannel scalar counter with a personal computer functions to unify all measurements, to simplify the operational process and data acquisition, and to provide a real-time display of the data. Its use is demonstrated by following the hydrolysis of L-alpha-[1-14C]dioleoyl phosphatidylcholine by the enzyme, phospholipase A2, isolated from the porcine pancreas.     

Seeing is believing? A beginners' guide to practical pitfalls in image acquisition

Imaging can be thought of as the most direct of experiments. You see something; you report what you see. If only things were truly this simple. Modern imaging technology has brought about a revolution in the kinds of questions we can approach, but this comes at the price of increasingly complex equipment. Moreover, in an attempt to market competing systems, the microscopes have often been inappropriately described as easy to use and suitable for near-beginners. Insufficient understanding of the experimental manipulations and equipment set-up leads to the introduction of errors during image acquisition. In this feature, I review some of the most common practical pitfalls faced by researchers during image acquisition, and how they can affect the interpretation of the experimental data.This article is targeted neither to the microscopy gurus who push forward the frontiers of imaging technology nor to my imaging specialist colleagues who may wince at the overly simplistic comments and lack of detail. Instead, this is for beginners who gulp with alarm when they hear the word "confocal pinhole" or sigh as they watch their cells fade and die in front of their very eyes time and time again at the microscope. Take heart, beginners, if microscopes were actually so simple then many people (including myself) would suddenly be out of a job!     

神经电生理信号多道同步采集和分析系统

单细胞多点同步记录技术在国内外已经被广泛应用,但在国内仍缺乏与国产或日产细胞电生理记录仪器相匹配的多通道同步生物电信号采集与分析系统。本文介绍了新近研制的可进行双通道甚至晚多通道细胞电生理信号采集的神经细胞电生理信号采集与分析系统,及其关键技术及实现方法和应用实例。     

A combinatorial score to distinguish biological and nonbiological protein-protein interfaces

With the large amount of protein-protein complex structural data available, to understand the key features governing the specificity of protein-protein recognition and to define a suitable scoring function for protein-protein interaction predictions, we have analyzed the protein interfaces from geometric and energetic points of view. Atom-based potential of mean force (PMFScore), packing density, contact size, and geometric complementarity are calculated for crystal contacts in 74 homodimers and 91 monomers, which include real biological interactions in dimers and nonbiological contacts in monomers and dimers. Simple cutoffs were developed for single and combinatorial parameters to distinguish biological and nonbiological contacts. The results show that PMFScore is a better discriminator between biological and nonbiological interfaces comparable in size. The combination of PMFScore and contact size is the most powerful pairwise discriminator. A combinatorial score (CFPScore) based on the four parameters was developed, which gives the success rate of the homodimer discrimination of 96.6% and error rate of the monomer discrimination of 6.0% and 19.8% according to Valdar's and our definition, respectively. Compared with other statistical learning models, the cutoffs for the four parameters and their combinations are directly based on physical models, simple, and can be easily applied to protein-protein interface analysis and docking studies.     

P1 and P7 plasmid partition: ParB protein bound to its partition site makes a separate discriminator contact with the DNA that determines species specificity.

The cis-acting P1 and P7 parS sites are responsible for active partition of P1 and P7 plasmids to daughter cells. The two sites are similar but function only with ParB proteins from the correct species. Using hybrid ParB proteins and hybrid parS sites, we show that specificity is determined by contacts between bases that lie within two parS hexamer boxes and a region in the ParB C-terminus. We refer to these contacts as discriminator contacts. The P7 discriminator contacts were mapped to 3 and 2 bp respectively within the two parS hexamer boxes, and a 10 amino acid region of P7 ParB. Similarly placed residues of different sequence are responsible for the P1 discriminator contact. The discriminator contacts are distinct from previously identified DNA binding contacts which involve different ParB and parS regions. Deletion of the ParB C-terminus that makes the discriminator contact does not diminish in vitro binding but renders it species independent. The discriminator contact is therefore a negative function, interfering with binding of the wrong ParB, but not providing energy for the binding of the correct one. Similar discriminator contacts might be responsible for specificities seen among families of eukaryotic DNA binding proteins that share common binding motifs.     

How could static telepathology improve diagnosis in neuropathology?

The present paper reports our experience with, and our opinion of static telepathology as applied to neuropathology by means of the PHAROS acquisition system and conventional telephone data transmission (modem). The classical procedure of expert consultation based on surface mailing of histological slides is routinely performed, especially in highly specialized fields of pathology. Telepathology is an easy means of sharing scientific expertise at international level and could thus improve diagnosis particularly in neuropathology, where certain tumor types are very rare and complex to diagnose. Dynamic telepathology allows the referring pathologist to capture by himself images supporting their diagnosis. Using static telepathology the pathologist could be limited in diagnosis by problems in fields selection. We devoted a whole year to collecting all the technical parameters characterizing the use of digitized neuropathological data files in order to investigate the feasibility of telepathology and the extent to which its use could improve diagnoses. Our results on a series of 38 histological brain examinations illustrate how we successfully established an international connection between two departments of pathology in Belgium and the USA. The referring pathologists gave diagnoses in 35 cases and deferred only 3. Despite a time-consuming procedure for the telepathology session of a few cases, this tool provides easy access to expert diagnosis and real-time discussion, both of which are of considerable interest and offer significant improvements in neuropathology.     

The role of a commercial radiation dose index monitoring system in establishing local dose reference levels for fluoroscopically guided invasive cardiac procedures

PurposeThe primary goal was to evaluate local dose level for fluoroscopically guided invasive cardiac procedures in a high-volume activity catheterization laboratory, using automatic data registration with minimal impact on operator workload. The secondary goal was to highlight the relationship between dose indices and acquisition parameters, in order to establish an effective strategy for protocols optimization.MethodsFrom September 2016 to December 2018, a dosimetric survey was conducted in the 2 rooms of the catheterization laboratory of our institution. Data collection burden was minimized using a commercial Radiation Dose Index Monitoring System (RDIMs) that analyzes dicom files automatically sent by the x-ray equipment. Data were combined with clinical information extracted from the HIS records reported by the interventional cardiologist. Local dose levels were established for different invasive cardiac procedures.ResultsA total of 3029 procedures performed for 2615 patients were analyzed. Median KAP were 21 Gycm² for invasive coronary angiography (ICA) procedures, 61 Gycm² for percutaneous coronary intervention (PCI) procedures, 59 Gycm² for combined (ICA+PCI) procedures, 87 Gycm² for structural heart intervention (TAVI) procedures. A significant dose reduction (51% for ICA procedures and 58% for PCI procedures) was observed when noise reduction acquisition techniques were applied.ConclusionsRDIMs are effective tools in the establishment of local dose level in interventional cardiology, as they mitigate the burden to collect and register extensive dosimetric data and exposure parameters. Systematic review of data support the multi-disciplinary team in the definition of an effective strategy for protocol management and dose optimization.     

Hypothermic preservation of hepatocytes on gelatin gels

Hypothermic preservation of hepatocytes on gelatin gels allows hepatocytes to be stored for at least 9 days. The procedure is easy, inexpensive and does not require specialised equipment. The cells retain their morphology and are released as separate spherical entities by dissolving the gelatin layer at 37°. The recovered cells have an intact plasma membrane as judged by lactate dehydrogenase activity, attachment efficiency and subsequent monolayer formation in culture. Functional tests show that the cells recover quickly from the storage conditions. Rates of protein synthesis are maintained over a 6 day period and remain at 62% of the initial level on day 9. The liver specific, hormonal induction of the enzyme tyrosine aminotransferase is apparent throughout the culture period. In addition a phenotypic marker enzyme, γ-glutamyl transpeptidase, remained at basal levels.     

Interaction between Escherichia coli RNase P RNA and the discriminator base results in slow product release.

We suggested previously that a purine at the discriminator base position in a tRNA precursor interacts with the well-conserved U294 in M1 RNA, the catalytic subunit of Escherichia coli RNase P. Here we investigated this interaction and its influence on the kinetics of cleavage as well as on cleavage site selection. The discriminator base in precursors to tRNA(Tyr)Su3 and tRNA(Phe) was changed from A to C and cleavage kinetics were studied by wild-type M1 RNA and a mutant M1 RNA carrying the compensatory substitution of a U to a G at position 294 in M1 RNA. Our data suggest that the discriminator base interacts with the residue at position 294 in M1 RNA. Although product release is a rate-limiting step both in the absence and in the presence of this interaction, its presence results in a significant reduction in the rate of product release. In addition, we studied cleavage site selection on various tRNA(His) precursor derivatives. These precursors carry a C at the discriminator base position. The results showed that the mutant M1 RNA harboring a G at position 294 miscleaved a wild-type tRNA(His) precursor and a tRNA(His) precursor carrying an inosine at the cleavage site. The combined data suggest a functional interaction between the discriminator base and the well-conserved U294 in M1 RNA. This interaction is suggested to play an important role in determining the rate of product release during multiple turnover cleavage of tRNA precursors by M1 RNA as well as in cleavage site selection.     

Organizing research data

Research relies on ever larger amounts of data from experiments, automated production equipment, questionnaries, times series such as weather records, and so on. A major task in science is to combine, process and analyse such data to obtain evidence of patterns and correlations.Most research data are on digital form, which in principle ensures easy processing and analysis, easy long-term preservation, and easy reuse in future research, perhaps in entirely unanticipated ways. However, in practice, obstacles such as incompatible or undocumented data formats, poor data quality and lack of familiarity with current technology prevent researchers from making full use of available data.This paper argues that relational databases are excellent tools for veterinary research and animal production; provides a small example to introduce basic database concepts; and points out some concerns that must be addressed when organizing data for research purposes.     

Influence of the GCGC discriminator motif introduced into the ribosomal RNA P2- and tac promoter on growth-rate control and stringent sensitivity.

The synthesis of stable RNA in bacteria is known to be regulated by a stringent control mechanism. Characteristic of stringent-regulated promoters, all ribosomal RNA promoters P1, but not P2, contain a GC-rich discriminator sequence assumed to be important for such a control. Using site-directed mutagenesis we have altered both the rrnB P2 and the synthetic tac promoter to the consensus GCGC discriminator motif. The modified promoters were placed upstream of the structural gene encoding the chloramphenicol acetyltransferase. The response of the modified promoters to amino acid starvation, changes in the growth rate or differences in the basal level of guanosine tetraphosphate (ppGpp) were determined in vivo. The results clearly show, that the discriminator motif is sufficient to convert the ribosomal RNA promoter P2 to a stringent, as well as growth-rate regulated, promoter. By contrast, the same discriminator sequence linked to the synthetic tac promoter does not convert this promoter to either stringency or growth-rate regulation. Finally, the results presented in this study reinforce the view that stringent and growth-rate regulation utilize the same mechanism, with ppGpp being the common mediator.     

General purpose, field-portable cell-based biosensor platform.

There are several groups of researchers developing cell-based biosensors for chemical and biological warfare agents based on electrophysiologic monitoring of cells. In order to transition such sensors from the laboratory to the field, a general-purpose hardware and software platform is required. This paper describes the design, implementation, and field-testing of such a system, consisting of cell-transport and data acquisition instruments. The cell-transport module is a self-contained, battery-powered instrument that allows various types of cell-based modules to be maintained at a preset temperature and ambient CO(2) level while in transit or in the field. The data acquisition module provides 32 channels of action potential amplification, filtering, and real-time data streaming to a laptop computer. At present, detailed analysis of the data acquired is carried out off-line, but sufficient computing power is available in the data acquisition module to enable the most useful algorithms to eventually be run real-time in the field. Both modules have sufficient internal power to permit realistic field-testing, such as the example presented in this paper.     

FRET imaging and in silico simulation: analysis of the signaling network of nerve growth factor-induced neuritogenesis

Genetically encoded probes based on Förster resonance energy transfer (FRET) enable us to decipher spatiotemporal information encoded in complex tissues such as the brain. Firstly, this review focuses on FRET probes wherein both the donor and acceptor are fluorescence proteins and are incorporated into a single molecule, i.e. unimolecular probes. Advantages of these probes lie in their easy loading into cells, the simple acquisition of FRET images, and the clear evaluation of data. Next, we introduce our recent study which encompasses FRET imaging and in silico simulation. In nerve growth factor-induced neurite outgrowth in PC12 cells, we found positive and negative signaling feedback loops. We propose that these feedback loops determine neurite-budding sites. We would like to emphasize that it is now time to accelerate crossover research in neuroscience, optics, and computational biology.     

Nonparametric methods for identifying differentially expressed genes in microarray data

MOTIVATION: Gene expression experiments provide a fast and systematic way to identify disease markers relevant to clinical care. In this study, we address the problem of robust identification of differentially expressed genes from microarray data. Differentially expressed genes, or discriminator genes, are genes with significantly different expression in two user-defined groups of microarray experiments. We compare three model-free approaches: (1). nonparametric t-test, (2). Wilcoxon (or Mann-Whitney) rank sum test, and (3). a heuristic method based on high Pearson correlation to a perfectly differentiating gene ('ideal discriminator method'). We systematically assess the performance of each method based on simulated and biological data under varying noise levels and p-value cutoffs. RESULTS: All methods exhibit very low false positive rates and identify a large fraction of the differentially expressed genes in simulated data sets with noise level similar to that of actual data. Overall, the rank sum test appears most conservative, which may be advantageous when the computationally identified genes need to be tested biologically. However, if a more inclusive list of markers is desired, a higher p-value cutoff or the nonparametric t-test may be appropriate. When applied to data from lung tumor and lymphoma data sets, the methods identify biologically relevant differentially expressed genes that allow clear separation of groups in question. Thus the methods described and evaluated here provide a convenient and robust way to identify differentially expressed genes for further biological and clinical analysis.     

FLIM on a wide field fluorescence microscope

Summary FLIM (Fluorescence Lifetime Imaging Microscopy) is a new tool to detect interaction between proteins. The proteins under investigation are fused with fluorescent donor and acceptor molecules. Interaction between the two proteins is accompanied by direct energy transfer from donor to acceptor (FRET), resulting in a shorter lifetime of the fluorescence emitted by the donor molecule. This change in lifetime is detected by FLIM. Fluorescence lifetime imaging can now be done on a widefield fluorescence microscope by using an attachment that is easy to install and simple to operate. The new LIFA attachment is equipped to use different excitation sources. High brightness modulated LEDs as well as lasers modulated by an Accousto Optical Modulator can be used as excitation light source. A modulated image intensifier with digital camera is used as a detector. Power supplies and signal generator are integrated in one control unit that is connected to the light source, detector and computer. All parameters for image acquisition, processing and viewing are easy accessible in the user interface of the software package that uses a modular structure. Lifetime images showing FRET in MCF7 cells with ErbB1-GFP as donor and Py72/Cy3 as acceptor that were taken at EMBL, Heidelberg are shown.     

MITICS (MALDI Imaging Team Imaging Computing System): a new open source mass spectrometry imaging software

MITICS is a new software developed for MALDI imaging. We tried to render this software compatible with all types of instruments. MITICS is divided in two parts: MITICS control for data acquisition and MITICS Image for data processing and images reconstruction. MITICS control is available for Applied BioSystems MALDI-TOF instruments and MITICS Image for both Applied BioSystems and Bruker Daltonics ones. MITICS Control provides an interface to the user for setting the acquisition parameters for the imaging sequence, namely set instruments acquisition parameters, create the raster of acquisition and control post-acquisition data processing, and provide this settings to the automatic acquisition software of the MALDI instrument. MITICS Image ensures image reconstruction, files are first converted to XML files before being loaded in a database. In MITICS image we have chosen to implement different data representations and calculations for image reconstruction. MITICS Image uses three different representations that have shown to ease extraction of information from the whole data set. It also offers image reconstruction base either on the maximum peak intensity or the peak area. Image reconstruction is possible for single ions but also by summing signals of different ions. MITICS was validated on biological cases.     

Microcomputer-based low-cost method for measurement of spatial and temporal parameters of gait

The instantaneous velocity of the forward-going foot is measured throughout the swing phase during several successive gait cycles using punched paper tapes attached to the feet. The velocity profiles thus obtained show clearly any asymmetry or departure from the normal walking pattern. The data are processed to provide the spatial and temporal parameters of gait, i.e. step lengths, stride times, double-support times, cadence and walking speed. The equipment is low-cost, easy to set up and use, and the results, which are presented on the VDU or as hard copy, are readily interpreted and related to the underlying pathology.     

Chiral separation of barbiturates and hydantoins by reversed-phase high-performance liquid chromatography using a 25 or 50 mm short ODS cartridge column via β-cyclodextrin inclusion complexes

A high-performance liquid chromatographic method on a 25 or 50 mm short ODS cartridge column has been developed for the resolution of the enantiomers of some optically active barbiturates and hydantoins in human serum. β-Cyclodextrin was used in the mobile phase. This method also seems to be an easy and effective way to test whether β-cyclodextrin would be a useful chiral discriminator for a particular racemate.