An Algorithm for the Selection of Probes for Specific Detection of Human Disease Pathogens Using the DNA Microarray Technology.

The aim of the study was to develop an algorithm for the selection of discriminating probes to identify a wide range of causative agents of human infectious diseases. Materials and Methods: The algorithm for selecting the probes was implemented in the form of the disprose (DIScrimination PRObe SElection) computer program written in the R language. Additionally, third-party software was used: the BLAST+ and ViennaRNA Package programs. The developed algorithm was tested by selecting specific probes for detecting Chlamydophila (Chlamydia) pneumoniae - an atypical bacterial pathogen causing community-acquired pneumonia (CAP). Nucleotide sequences for analysis were downloaded from the NCBI databank. Results: An algorithm for the selection of specific probes capable of detecting human infectious pathogens has been developed. The algorithm is implemented in the form of the disprose modular program, which allows for performing all stages of the probe selection process: loading the nucleotide sequences and their metadata from available databanks, creating local databases, forming a pool of probes, calculating their physicochemical parameters, aligning the probes and sequences contained in local databases, processing and evaluating the alignment results. The algorithm was successfully tested and its performance was confirmed by selecting a set of probes for the specific detection of Chlamydophila pneumoniae. The specificity of the selected probes calculated in silico indicated a low risk of their nonspecific binding and a high potential of using them as molecular genetic diagnostic tools (DNA microarrays, PCR). Conclusion: An algorithm for the selection of specific probes detecting a wide range of human pathogens in clinical biomaterial has been developed and implemented in the form of the disprose modular program. The probes selected using this program can serve as the functional basis of DNA-oriented microarrays able to identify causative agents of polyetiological diseases, such as CAP. Due to the flexibility and openness of the program, the scope of its application can be expanded.

M probe comes first: Impact of initial probe choice on diagnostic performance of vibration controlled transient elastography.

BACKGROUND & AIMS: Probe choice (M or XL) in transient elastography can be made by the user's own measure of skin-to-liver-capsule distance (SCD) or with an automated tool (AUTO). We studied how AUTO depends on initial probe choice. METHODS: Three fictive clinics were considered: The "M-first clinic" uses AUTO from the M probe, the "XL-first clinic" uses AUTO from the XL probe and a "reference clinic" measures SCD independently. Agreement and discrepancies to the reference clinic were measured. RESULTS: 200 patients with chronic liver disease were prospectively included (58% female, 56 years, BMI 28.1 kg/m²). Fleiss' kappa for agreement in probe selection was 0.11 (95% CI -0.09 to 0.31), but accuracy was above 0.8 for both. Probe failure occurred for 16 (M-first clinic), 4 (XL-first clinic) and 3 patients (reference clinic). Use of XL probe given M probe failure improved performance of the M-first approach. The odds ratio for discrepancy in the XL-first vs M-first clinic is 2.4 (95% CI 1.2 to 5.2, p = 0.012) for liver fibrosis and 4.8 (95% CI 1.8 to 16.1, p < 0.001) for steatosis. CONCLUSIONS: Agreement in AUTO between M and XL probes is poor although each has acceptable accuracy. The M-first approach leads to fewer discrepancies and should be adopted as a standard.

COMBO-FISH enables high precision localization microscopy as a prerequisite for nanostructure analysis of genome loci.

With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization) technique called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15-30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs)-synthetic DNA analogues with a neutral backbone-which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488(®) showed reversible photobleaching (blinking between dark and bright state) a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy) a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10-20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome.

Atomic Force Microscopy of Proteins.

Atomic force microscopy (AFM) enables imaging of surface-deposited proteins and protein structures under physiological conditions, which is a benefit compared to ultra-high vacuum techniques such as electron microscopy. AFM also has the potential to provide more information from the phase in tapping mode or from functional AFM modes. The sample preparation, probe selection, and imaging conditions are crucial for successful imaging of proteins. Here we give a detailed account of the steps toward imaging of soft samples in both air and liquid along with the basic theory underpinning these details.

Selecting Reliable mRNA Expression Measurements Across Platforms Improves Downstream Analysis.

With increasing use of publicly available gene expression data sets, the quality of the expression data is a critical issue for downstream analysis, gene signature development, and cross-validation of data sets. Thus, identifying reliable expression measurements by leveraging multiple mRNA expression platforms is an important analytical task. In this study, we propose a statistical framework for selecting reliable measurements between platforms by modeling the correlations of mRNA expression levels using a beta-mixture model. The model-based selection provides an effective and objective way to separate good probes from probes with low quality, thereby improving the efficiency and accuracy of the analysis. The proposed method can be used to compare two microarray technologies or microarray and RNA sequencing measurements. We tested the approach in two matched profiling data sets, using microarray gene expression measurements from the same samples profiled on both Affymetrix and Illumina platforms. We also applied the algorithm to mRNA expression data to compare Affymetrix microarray data with RNA sequencing measurements. The algorithm successfully identified probes/genes with reliable measurements. Removing the unreliable measurements resulted in significant improvements for gene signature development and functional annotations.

Pigeons: A Novel GUI Software for Analysing and Parsing High Density Heterologous Oligonucleotide Microarray Probe Level Data.

Genomic DNA-based probe selection by using high density oligonucleotide arrays has recently been applied to heterologous species (Xspecies). With the advent of this new approach, researchers are able to study the genome and transcriptome of a non-model or an underutilised crop species through current state-of-the-art microarray platforms. However, a software package with a graphical user interface (GUI) to analyse and parse the oligonucleotide probe pair level data is still lacking when an experiment is designed on the basis of this cross species approach. A novel computer program called Pigeons has been developed for customised array data analysis to allow the user to import and analyse Affymetrix GeneChip(®) probe level data through XSpecies. One can determine empirical boundaries for removing poor probes based on genomic hybridisation of the test species to the Xspecies array, followed by making a species-specific Chip Description File (CDF) file for transcriptomics in the heterologous species, or Pigeons can be used to examine an experimental design to identify potential Single-Feature Polymorphisms (SFPs) at the DNA or RNA level. Pigeons is also focused around visualization and interactive analysis of the datasets. The software with its manual (the current release number version 1.2.1) is freely available at the website of the Nottingham Arabidopsis Stock Centre (NASC).

Space pruning monotonic search for the non-unique probe selection problem.

Identification of targets, generally viruses or bacteria, in a biological sample is a relevant problem in medicine. Biologists can use hybridisation experiments to determine whether a specific DNA fragment, that represents the virus, is presented in a DNA solution. A probe is a segment of DNA or RNA, labelled with a radioactive isotope, dye or enzyme, used to find a specific target sequence on a DNA molecule by hybridisation. Selecting unique probes through hybridisation experiments is a difficult task, especially when targets have a high degree of similarity, for instance in a case of closely related viruses. After preliminary experiments, performed by a canonical Monte Carlo method with Heuristic Reduction (MCHR), a new combinatorial optimisation approach, the Space Pruning Monotonic Search (SPMS) method, is introduced. The experiments show that SPMS provides high quality solutions and outperforms the current state-of-the-art algorithms.

Ultrasound: Basic understanding and learning the language.

Ultrasound (US) use has rapidly entered the field of acute pain medicine and regional anesthesia and interventional pain medicine over the last decade, and it may even become the standard of practice. The advantages of US guidance over conventional techniques include the ability to both view the targeted structure and visualize, in real time, the distribution of the injected medication, and the capacity to control its distribution by readjusting the needle position, if needed. US guidance should plausibly improve the success rate of the procedures, their safety and speed. This article provides basic information on musculoskeletal US techniques, with an emphasis on the principles and practical aspects. We stress that for the best use of US, one should venture beyond the "pattern recognition" mode to the more advanced systematic approach and use US as a tool to visualize structures beyond the skin (sonoanatomy mode). We discuss the sonographic appearance of different tissues, introduce the reader to commonly used US-related terminology, cover basic machine "knobology" and fundamentals of US probe selection and manipulation. At the end, we discuss US-guided needle advancement. We only briefly touch on topics dealing with physics, artifacts, or sonopathology, which are available elsewhere in the medical literature.