New technologies for DNA analysis--a review of the READNA Project.

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

Positron emission tomography/computed tomography scanning for the diagnosis of occult sepsis in the critically injured.

BACKGROUND: Although the site of nosocomial sepsis in the critically ill ventilated patient is usually identifi able, it may remain occult, despite numerous investigations. The rapid results and precise anatomical location of the septic source using positron emission tomography (PET) scanning, in combination with computed tomography (CT), has promoted this modality as the diagnostic tool of choice for pyrexias of unknown origin. METHOD: The objective of this study was to report our experience using PET/CT scanning for the localisation of a septic focus in critically injured patients in whom no source could be identifi ed using conventional investigations. RESULTS: Two patients with gunshot wounds and two who had sustained multiple fractures following motor vehicle collisions developed pyrexias of unknown origin during their stay in the trauma intensive care unit. Routine screening for a septic focus was unrewarding, and 18F-fl uorodeoxyglucose PET/CT scanning was used to identify the possible source. PET/CT scanning identifi ed the septic focus in all patients. Abscesses were drained successfully in those with penetrating trauma and in one with blunt polytrauma. Pulmonary tuberculosis, not apparent on initial radiology, was identifi ed using PET/CT in one patient with blunt thoracic trauma. CONCLUSION: PET/CT scanning appears to both confi rm and localise the source of sepsis in a variety of pathologies in critically ill patients who develop pyrexias for which no source can be identifi ed by conventional screening techniques.

Bone mass, bone geometry, and body composition in female-to-male transsexual persons after long-term cross-sex hormonal therapy.

CONTEXT: Female-to-male transsexual persons (transsexual men) undergo extreme hormonal changes due to ovariectomy and testosterone substitution, allowing studies on sex steroid effects on bone geometry and physiology in the adult. OBJECTIVE: The objective of the study was to examine the effects of cross-gender sex steroid exposure on volumetric bone parameters in transsexual men. DESIGN: This was a cross-sectional study. SETTING: Participants were recruited from the Center for Sexology and Gender Problems at the Ghent University Hospital (Ghent, Belgium). PARTICIPANTS: Fifty transsexual men after sex reassignment surgery with 50 age-matched control women and an additional 16 transsexual men before testosterone substitution and sex reassignment surgery with 16 control women participated in the study. MAIN OUTCOME MEASURES: The main outcome measures were areal and volumetric bone parameters using dual-energy X-ray absorptiometry and peripheral quantitative computed tomography, body composition (dual-energy X-ray absorptiometry), sex steroids, markers of bone turnover and grip strength. RESULTS: Before hormonal treatment, transsexual men had similar body composition and bone geometry as female controls. The transsexual men on long-term testosterone therapy, however, demonstrated a higher lean body mass and muscle mass and a greater grip strength as well as a lower body and subcutaneous fat mass and a larger waist and smaller hip circumference compared with female controls (all P < 0.001). We observed a larger radial cortical bone size (P < 0.001) and lower cortical volumetric bone mineral density at the radius and tibia (P < 0.05) in transsexual men on testosterone therapy. CONCLUSIONS: Transsexual men on testosterone substitution therapy present with a different body composition with more muscle mass and strength and less fat mass as well as an altered bone geometry with larger bones compared with female controls.

High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing.

The construction of synthetic biological systems involving millions of nucleotides is limited by the lack of high-quality synthetic DNA. Consequently, the field requires advances in the accuracy and scale of chemical DNA synthesis and in the processing of longer DNA assembled from short fragments. Here we describe a highly parallel and miniaturized method, called megacloning, for obtaining high-quality DNA by using next-generation sequencing (NGS) technology as a preparative tool. We demonstrate our method by processing both chemically synthesized and microarray-derived DNA oligonucleotides with a robotic system for imaging and picking beads directly off of a high-throughput pyrosequencing platform. The method can reduce error rates by a factor of 500 compared to the starting oligonucleotide pool generated by microarray. We use DNA obtained by megacloning to assemble synthetic genes. In principle, millions of DNA fragments can be sequenced, characterized and sorted in a single megacloner run, enabling constructive biology up to the megabase scale.

Deciphering the porcine intestinal microRNA transcriptome.

BACKGROUND: While more than 700 microRNAs (miRNAs) are known in human, a comparably low number has been identified in swine. Because of the close phylogenetic distance to humans, pigs serve as a suitable model for studying e.g. intestinal development or disease. Recent studies indicate that miRNAs are key regulators of intestinal development and their aberrant expression leads to intestinal malignancy. RESULTS: Here, we present the identification of hundreds of apparently novel miRNAs in the porcine intestine. MiRNAs were first identified by means of deep sequencing followed by miRNA precursor prediction using the miRDeep algorithm as well as searching for conserved miRNAs. Second, the porcine miRNAome along the entire intestine (duodenum, proximal and distal jejunum, ileum, ascending and transverse colon) was unraveled using customized miRNA microarrays based on the identified sequences as well as known porcine and human ones. In total, the expression of 332 intestinal miRNAs was discovered, of which 201 represented assumed novel porcine miRNAs. The identified hairpin forming precursors were in part organized in genomic clusters, and most of the precursors were located on chromosomes 3 and 1, respectively. Hierarchical clustering of the expression data revealed subsets of miRNAs that are specific to distinct parts of the intestine pointing to their impact on cellular signaling networks. CONCLUSIONS: In this study, we have applied a straight forward approach to decipher the porcine intestinal miRNAome for the first time in mammals using a piglet model. The high number of identified novel miRNAs in the porcine intestine points out their crucial role in intestinal function as shown by pathway analysis. On the other hand, the reported miRNAs may share orthologs in other mammals such as human still to be discovered.

A flexible and fully integrated system for amplification, detection and genotyping of genomic DNA targets based on microfluidic oligonucleotide arrays.

A strategy allowing for amplification, detection and genotyping of different genomic DNA targets in a single reaction container is described. The method makes use of primer-directed solution-phase amplification with integrated labeling in a closed, microfluidic oligonucleotide array. Selective array probes allow for subsequent detection and genotyping of generated amplicons by hybridization. The array contains up to 15,624 programmable features that can be designed, de novo synthesized and tested within 24 hours using an automated benchtop microarray synthesizer. This enables rapid prototyping and adaptation of the system to newly emerging targets such as pathogenic bacterial or viral subtypes. The system was evaluated by amplifying and detecting different loci of viral (HPV), bacterial (Bacillus sp.) and eukaryotic (human) genomes. Multiplex PCR and semi-quantitative detection with excellent detection limits of <100 target copies is hereby demonstrated. The high automation grade of the system reduces contamination risk and workload and should enhance safety and reproducibility.

Targeted high throughput sequencing of a cancer-related exome subset by specific sequence capture with a fully automated microarray platform.

Sequence capture methods for targeted next generation sequencing promise to massively reduce cost of genomics projects compared to untargeted sequencing. However, evaluated capture methods specifically dedicated to biologically relevant genomic regions are rare. Whole exome capture has been shown to be a powerful tool to discover the genetic origin of disease and provides a reduction in target size and thus calculative sequencing capacity of >90-fold compared to untargeted whole genome sequencing. For further cost reduction, a valuable complementing approach is the analysis of smaller, relevant gene subsets but involving large cohorts of samples. However, effective adjustment of target sizes and sample numbers is hampered by the limited scalability of enrichment systems. We report a highly scalable and automated method to capture a 480 Kb exome subset of 115 cancer-related genes using microfluidic DNA arrays. The arrays are adaptable from 125 Kb to 1 Mb target size and/or one to eight samples without barcoding strategies, representing a further 26 - 270-fold reduction of calculative sequencing capacity compared to whole exome sequencing. Illumina GAII analysis of a HapMap genome enriched for this exome subset revealed a completeness of >96%. Uniformity was such that >68% of exons had at least half the median depth of coverage. An analysis of reference SNPs revealed a sensitivity of up to 93% and a specificity of 98.2% or higher.

Specific sequence selection and next generation resequencing of 68 E. coli genes using HybSelect.

Recent years have seen tremendous progress in next generation sequencing technologies, allowing genomic sequencing in a highly cost-effective manner. However, sample preparation for these sequencers remains a bottleneck as the human genome is too complex to be routinely resequenced. We present here an in-depth study of HybSelect, a method that can specifically enrich a large number of genes or regions of interest from any chromosomal DNA. The study used Escherichia coli K12 MG1655 as a model organism to test parameters such as method fidelity, capacity or reproducibility as a proof-of-principle.

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing.

The lack of efficient high-throughput methods for enrichment of specific sequences from genomic DNA represents a key bottleneck in exploiting the enormous potential of next-generation sequencers. Such methods would allow for a systematic and targeted analysis of relevant genomic regions. Recent studies reported sequence enrichment using a hybridization step to specific DNA capture probes as a possible solution to the problem. However, so far no method has provided sufficient depths of coverage for reliable base calling over the entire target regions. We report a strategy to multiply the enrichment performance and consequently improve depth and breadth of coverage for desired target sequences by applying two iterative cycles of hybridization with microfluidic Geniom biochips. Using this strategy, we enriched and then sequenced the cancer-related genes BRCA1 and TP53 and a set of 1000 individual dbSNP regions of 500 bp using Illumina technology. We achieved overall enrichment factors of up to 1062-fold and average coverage depths of 470-fold. Combined with high coverage uniformity, this resulted in nearly complete consensus coverages with >86% of target region covered at 20-fold or higher. Analysis of SNP calling accuracies after enrichment revealed excellent concordance, with the reference sequence closely mirroring the previously reported performance of Illumina sequencing conducted without sequence enrichment.

Targeted next-generation sequencing by specific capture of multiple genomic loci using low-volume microfluidic DNA arrays.

We report a flexible method for selective capture of sequence fragments from complex, eukaryotic genome libraries for next-generation sequencing based on hybridization to DNA microarrays. Using microfluidic array architecture and integrated hardware, the process is amenable to complete automation and does not introduce amplification steps into the standard library preparation workflow, thereby avoiding bias of sequence distribution and fragment lengths. We captured a discontiguous human genomic target region of 185 kb using a tiling design with 50mer probes. Analysis by high-throughput sequencing using an Illumina/Solexa 1G Genome Analyzer revealed 2150-fold enrichment with mean per base coverage between 4.6 and 107.5-fold for the individual target regions. This method represents a flexible and cost-effective approach for large-scale resequencing of complex genomes.

Microfluidic-based enzymatic on-chip labeling of miRNAs.

Small noncoding RNAs (sncRNAs) have moved from oddity to recognized important players in gene regulation. Next generation sequencing approaches discover more and more such molecules from a variety of different groups, but flexible tools translating this sequence information into affordable high-throughput assays are missing. Here we describe a microfluidic primer extension assay (MPEA) for the detection of sncRNAs on highly flexible microfluidic microarrays which combines several beneficial parameters: it can effortless incorporate any new sequence information; it is sensitive enough to work with as little as 20ng of total RNA and has a high level of specificity owing to a combination of a conventional hybridization assay and an enzymatic elongation step. Importantly, no labeling step is needed before hybridization and - because of its high sensitivity - no amplification is required. Both aspects ensure that no bias is introduced by such processes. Although the assay is exemplified with miRNAs, the flexibility of the technology platform allows the analysis of any type of sncRNA, such as piRNAs.

Molecular epidemiology of ovine herpesvirus type 2 infection in Kashmir, India.

The aims of this investigation were to determine the prevalence of ovine herpesvirus type 2 (OvHV-2) (the causative agent of malignant catarrhal fever) infection in cattle, the carrier status of sheep and goats, and to define the pattern of acquisition of OvHV-2 in lambs under natural flock conditions in Kashmir, India. None of the buffy coat samples from 21 lambs contained OvHV-2 DNA sequences up to 28 days after birth, only one lamb had sequences of OvHV-2 DNA as early as 29 days after birth, and they were detected in the other 20 lambs when they were between 43 and 94 days of age. Sequences of OvHV-2 DNA were detected in buffy coat samples from 28 (85 per cent) of 33 adult sheep and in 16 (61 per cent) of 26 samples from adult goats by hemi-nested PCR. Seventeen (31 per cent) of 55 cattle with malignant catarrhal fever-like clinical signs had sequences of OvHV-2 DNA in their blood, and nine of the 17 died, all of them during the months of April to November, between November 2002 and March 2004. No clinical cases of sheep-associated malignant catarrhal fever was recorded during the months of December to March. The overall prevalence of OvHV-2 infection in the cattle in the region was estimated to be less than 1 per cent.

An improved intravital microscopy system.

The use of intravital microscopy as a tool for studying the microcirculation has increased greatly over the last several decades. Early microscopes provided the first pictures of the microcirculation, but were cumbersome to use and subjected the tissue to a high light intensity, a problem which has recently become the subject of much discussion. The goal of this project was therefore to build a more ergodynamic microscope which minimizes the light exposure to the tissue. The automation of the microscope controls provides a platform on which other options can be built into the microscope, such as an autofocus feature. Furthermore, the use of the Optimas software also opens the possibility for on-line data processing.

Skeletal muscle microvascular and tissue injury after varying durations of ischemia.

The purpose of this study was to examine the effect of varying durations of ischemia on several microvascular parameters in the awake hamster chamber model. The goal was to characterize the microvascular damage that occurs in skeletal muscle as a result of ischemia and reperfusion. The chamber tissues were subjected to 1-5 h of ischemia, and then the following parameters were measured: vessel diameter, endothelial thickness, macromolecular leakage, red blood cell velocity, adherent leukocytes, rolling leukocytes, freely flowing leukocytes, functional capillary density, and propidium iodide-positive cell nuclei. In control animals there was no significant difference in any parameters over the entire observation period. After 1 or 2 h of ischemia an increase in rolling and adherent leukocytes was measured. After 3 h of ischemia there was a significant increase in the mean endothelial thickness and in the number of nonviable cells. After 4 h of ischemia a significant difference in the extent of macromolecular leakage and the functional capillary density was additionally observed. After 5 h of ischemia this damage was more pronounced and often so severe that approximately 50% of the vessels demonstrated no reflow.

31P-spectroscopy of head and neck tumors--surface coil technique.

Comparative phosphorus-31 magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) of 15 patients with superficial masses such as sarcoma, carcinoma, lymphoma, adenoma, and tuberculosis revealed significant increased concentrations of phosphomonoester, phosphodiester, and inorganic phosphorus in the lesion, whereas the concentration of the phosphocreatine was lower in comparison to muscle tissue. In nearly all masses, pH showed a slight alkaline shift. Existence of necrotic regions detected by MRI was marked by an increase of inorganic phosphorous in the spectra. Tumor growth was characterized by raised concentrations of phosphomonoester. Follow-up studies in a case of lymphoma showed a six-fold decrease of the tumor, while the spectra indicated a gradual transition of tumor values to muscle values. A follow-up study during irradiation of a squamous cell carcinoma revealed a considerable decrease of inorganic phosphate and a subsequent increase of phosphodiester.

[In vivo 31P magnetic resonance spectroscopy and MRI in patients with superficial tumors]. / In vivo 31P Magnetresonanz Spektroskopie und MRI bei Patienten mit oberflächlich gelegenen Tumoren.

Simultaneous 31P-MR spectroscopy (MRS) and MR imaging (MRI) of 10 patients suffering from superficial tumours like carcinoma, lymphoma and adenoma, revealed significantly enhanced concentrations of phosphomonoester, phosphodiester and inorganic phosphorus in the tumour, whereas the concentration of phosphocreatine was lower in comparison to muscle tissue. In all tumours the pH showed a slight alkaline shift. The existing of necrotic regions detected by MRI was accompanied by an increase of inorganic phosphorus in the spectra. A follow-up study of a patient with a lymphoma during chemotherapy showed a tumour regression, whereas the spectra indicated a continuous approach of tumour values to the muscle values.

[Diseases of the lymphatic system of the head and neck region. A comparative study of MRI and CT]. / Erkrankungen des lymphatischen Systems im Kopf-Hals-Bereich. Vergleichsstudie KST und CT.

The diagnostic possibilities of magnetic resonance imaging compared with computed tomography in lymphomas and pathological enlargement of the lymph nodes in the head and neck region are presented. Whereas plain MRI examinations showed the same diagnostic sensitivity as CT, application of the paramagnetic contrast medium Gd-DTPA in 50 of 87 patients clearly increased diagnostic accuracy. Signal intensities of T1- or T2-weighted images before therapy (operation, chemotherapy, radiotherapy) and after administration of Gd-DTPA were enhanced, compared with posttherapeutic and plain examinations. An increase after therapy in two patients signalled a relapse or residual tumour tissue; a decrease in three cases was evaluated as response to therapy. Other differential diagnostic processes such as lipomas, neurinomas, glomus tumours etc. were differentiated with the help of signal intensity curves after administration of contrast media and the use of a gradient echo sequence TR/TE = 30/12 msec with a flip angle of 30 degrees. Differentiation of tissue based on morphological criteria such as homogeneity of tumour tissue or the delineation against surrounding tissue structures showed in the case of Hodgkin's disease and inflammatory diseases mainly homogeneous elements without ring-shaped structure. Non-Hodgkin lymphomas had a predominantly homogeneous structure. In squamous cell carcinomas MR revealed in two cases a ring-shaped enhancement.