SPIKEII: an integrate-and-fire AVLSI chip

We present the SPIKEII chip, an integrate-and-fire neural network chip with programmable synapses implemented in analogue VLSI. It is the successor to the SPIKEI chip. We describe the circuitry, and show some results using networks of integrate-and-fire neurons.     

Colorectal cancer DNA methylation marker panel validated with high performance in Non-Hodgkin lymphoma

Genes with altered DNA methylation can be used as biomarkers for cancer detection and assessment of prognosis. Here we analyzed the methylation status of a colorectal cancer biomarker panel (CNRIP1, FBN1, INA, MAL, SNCA, and SPG20) in 97 cancer cell lines, derived from 17 different cancer types. Interestingly, the genes were frequently methylated also in hematological cancer types and were therefore subjected to analyses in primary tumor samples from the major types of non-Hodgkin lymphomas (NHL) and in healthy controls. In total, the genes CNRIP1, FBN1, INA, MAL, SNCA, and SPG20 were methylated in 53%, 23%, 52%, 69%, 97%, and 92% of the tumor samples, respectively, and were unmethylated in all healthy controls. With the exception of a single tumor sample, a correct prediction of lymphoma or normal sample was made in a blinded analysis of the validation series using a combination of SNCA and SPG20. The combined ROC-curve analysis of these genes resulted in an area under the curve of 0.999 (P = 4.2 × 10⁻¹⁸), and a sensitivity and specificity of 98% and 100%, respectively, across the test and validation series. Interestingly, the promoter methylation of CNRIP1 was associated with decreased overall survival in diffuse large B-cell lymphoma (DLBCL) (P = 0.03).   In conclusion, our results demonstrate that SNCA and SPG20 methylation might be suitable for early detection and monitoring of NHL. Furthermore, CNRIP1 could potentially be used as a prognostic factor in DLBCL.     

Colorectal cancer DNA methylation marker panel validated with high performance in Non-Hodgkin lymphoma

Genes with altered DNA methylation can be used as biomarkers for cancer detection and assessment of prognosis. Here we analyzed the methylation status of a colorectal cancer biomarker panel (CNRIP1, FBN1, INA, MAL, SNCA, and SPG20) in 97 cancer cell lines, derived from 17 different cancer types. Interestingly, the genes were frequently methylated also in hematological cancer types and were therefore subjected to analyses in primary tumor samples from the major types of non-Hodgkin lymphomas (NHL) and in healthy controls. In total, the genes CNRIP1, FBN1, INA, MAL, SNCA, and SPG20 were methylated in 53%, 23%, 52%, 69%, 97%, and 92% of the tumor samples, respectively, and were unmethylated in all healthy controls. With the exception of a single tumor sample, a correct prediction of lymphoma or normal sample was made in a blinded analysis of the validation series using a combination of SNCA and SPG20. The combined ROC-curve analysis of these genes resulted in an area under the curve of 0.999 (P = 4.2 × 10^?18), and a sensitivity and specificity of 98% and 100%, respectively, across the test and validation series. Interestingly, the promoter methylation of CNRIP1 was associated with decreased overall survival in diffuse large B-cell lymphoma (DLBCL) (P = 0.03).

In conclusion, our results demonstrate that SNCA and SPG20 methylation might be suitable for early detection and monitoring of NHL. Furthermore, CNRIP1 could potentially be used as a prognostic factor in DLBCL.     

Mitochondrial DNA: an important female contribution to thoroughbred racehorse performance

The mitochondrial DNA (mtDNA) molecule, carrying genes encoding for respiratory chain enzymes, is a primary candidate for demonstrating associations between genotype and athletic performance in mammalian species. In humans, variation at seven protein encoding mitochondrial loci has been implicated in influencing fitness and performance characteristics. Although thoroughbred horses are selected for racing ability, there have not been any previous reported associations between genotypes and racecourse performance. The multi-factorial nature of the inheritance of racing ability is an obvious complicating factor. However, mitochondrial gene variation may represent a measurable component contributing to performance variability. Previous population studies of thoroughbreds have shown the existence of D-loop variation. Importantly, we have observed that there is also independent and extensive functional mitochondrial gene variation in the current thoroughbred racehorse population and that significant associations exist between mtDNA haplotype, as defined by functional genes, and aspects of racing performance.     

Gene-based identification of bacterial colonies with an electric chip

A method for the identification of bacterial colonies based on their content of specific genes is presented. This method does not depend on DNA separation or DNA amplification. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin was identified with this method. It is based on target DNA hybridization to a capturing probe immobilized on magnetic beads, followed by enzymatic labeling and measurement of the enzyme product with a silicon-based chip. An hblC-positive colony containing 10(7) cells could be assayed in 30 min after ultrasonication and centrifugation. The importance of optimizing the ultrasonication is illustrated by analysis of cell disruption kinetics and DNA fragmentation. An early endpoint PCR analysis was used to characterize the DNA fragmentation as a function of ultrasonication time. The first minutes of sonication increased the signal due to both increased DNA release and increased DNA fragmentation. The latter is assumed to increase the signal due to improved diffusion and faster hybridization of the target DNA. Too long sonication decreased the signal, presumably due to loss of hybridization sites on the targets caused by extensive DNA fragmentation. The results form a basis for rational design of an ultrasound cell disruption system integrated with analysis on chip that will move nucleic acid-based detection through real-time analysis closer to reality.     

Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria

Different factors influencing chip array-based electrical detection of DNA for analysis of pathogenic bacteria were examined. Both rehydration of capture probe layer of functionalized chip arrays and efficient hybridization of targets irrespective of their length resulted in signal enhancement when high-ionic phosphate-buffered saline (i.e., 600 mM sodium chloride and 40 mM disodium hydrogen phosphate) was used. Similarly, placement of two adjacent capture and detection probe-binding sites at a terminal part of the target strand resulted in significant signal increase. Moreover, 10-min ultrasonic fragmentation of targets amplified the signals up to twofold for longer DNA strands (i.e., >300 bp). No obvious effects on signals were visible for shorter than 400-bp PCR amplicons subjected to ultrasonication. For DNA strands of all sizes, more than 10 min ultrasonication diminished the specific electrical responses. Our results also demonstrate that target analytes are detected with discrimination against mismatches even for single nucleotide sequence alteration. The mismatch detection appeared in order of ease of recognition as follows: triple random > quintuple middle > triple middle > single middle mismatch. Among the three variants of one-base mismatches, a sequence variation was most remarkable for adenine. On the other hand, no benefits in assay sensitivity were recognized by the use of longer capture probe linkers as the 6-C linker.     

Ultrasensitive DNA chip: gene expression profile analysis without RNA amplification

We have developed a new DNA chip whose substrate has a unique minute columnar array structure made of plastic. The DNA chip exhibits ultrahigh sensitivity, up to 100-fold higher than that of reference DNA chips, which makes it possible to monitor gene expression profiles even with very small amounts of RNA (0.1-0.01 microg of total RNA) without amplification. Differential expression ratios obtained with the new DNA chip were validated against those obtained with quantitative real-time PCR assays. This novel microarray technology would be a powerful tool for monitoring gene expression profiles, especially for clinical diagnosis.     

Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency.

We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (approximately 10(4) base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk-ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.     

Development of multisample detection system using a tag insertion primer and an electrochemical DNA chip

We have developed a novel multisample detection system by employing a technology combining a tag insertion primer and an electrochemical DNA chip. In the first application, Helicobacter species-infected mouse samples were detected. The primers that insert a different tag sequence in each sample were prepared, and loop-mediated isothermal amplification (LAMP) reaction was carried out. Then amplification products in which a part of the sequence was different in each sample could be obtained. The target sample in which these amplification products were mixed was injected into a cassette that included the DNA chip with immobilized probes. After the cassette was set in the DNA detection system, Genelyzer, the processes of hybridization, washing, and detection were performed by the system automatically. The positive and negative concordance rates of the existing nested polymerase chain reaction (PCR) method and this method were 100% (40/40 samples) and 97.3% (117/120 samples), respectively. This is a simple high-throughput method. Moreover, the cost per sample can be drastically lowered. Therefore, it is expected to contribute to the diagnosis of infectious agents in humans and animals.     

CPORT: a consensus interface predictor and its performance in prediction-driven docking with HADDOCK

Background

Macromolecular complexes are the molecular machines of the cell. Knowledge at the atomic level is essential to understand and influence their function. However, their number is huge and a significant fraction is extremely difficult to study using classical structural methods such as NMR and X-ray crystallography. Therefore, the importance of large-scale computational approaches in structural biology is evident. This study combines two of these computational approaches, interface prediction and docking, to obtain atomic-level structures of protein-protein complexes, starting from their unbound components.

Methodology/Principal Findings

Here we combine six interface prediction web servers into a consensus method called CPORT (Consensus Prediction Of interface Residues in Transient complexes). We show that CPORT gives more stable and reliable predictions than each of the individual predictors on its own. A protocol was developed to integrate CPORT predictions into our data-driven docking program HADDOCK. For cases where experimental information is limited, this prediction-driven docking protocol presents an alternative to ab initio docking, the docking of complexes without the use of any information. Prediction-driven docking was performed on a large and diverse set of protein-protein complexes in a blind manner. Our results indicate that the performance of the HADDOCK-CPORT combination is competitive with ZDOCK-ZRANK, a state-of-the-art ab initio docking/scoring combination. Finally, the original interface predictions could be further improved by interface post-prediction (contact analysis of the docking solutions).

Conclusions/Significance

The current study shows that blind, prediction-driven docking using CPORT and HADDOCK is competitive with ab initio docking methods. This is encouraging since prediction-driven docking represents the absolute bottom line for data-driven docking: any additional biological knowledge will greatly improve the results obtained by prediction-driven docking alone. Finally, the fact that original interface predictions could be further improved by interface post-prediction suggests that prediction-driven docking has not yet been pushed to the limit. A web server for CPORT is freely available at http://haddock.chem.uu.nl/services/CPORT.     

Producing single-stranded DNA probes with the Taq DNA polymerase: a high yield protocol

We report an efficient procedure to synthesize either single- or double-stranded probes labeled with biotin-11-dUTP, biotin-21-dUTP or digoxigenin-11-dUTP. To produce the single-stranded probe, only a single primer is utilized in a Taq polymerase amplification of 55 cycles. A cytomegalovirus probe is presented. This procedure allows easy production of nonradioactively labeled pure single-stranded probes of any desired length and specificity.     

Real time image processing with an analog vision chip system

A linear analog network model is proposed to characterize the function of the outer retinal circuit in terms of the standard regularization theory. Inspired by the function and the architecture of the model, a vision chip has been designed using analog CMOS Very Large Scale Integrated circuit technology. In the chip, sample/hold amplifier circuits are incorporated to compensate for statistic transistor mismatches. Accordingly, extremely low noise outputs were obtained from the chip. Using the chip and a zero-crossing detector, edges of given images were effectively extracted in indoor illumination.     

DNA sequences amplified in cancer cells: an interface between tumor biology and human genome analysis.

There is growing evidence that amplification of specific genes is associated with tumor progression. While several proto-oncogenes are known to be activated by amplification, it is clear that not all the genes involved in DNA amplification in human tumors have been discovered. Our approach to the identification of such genes is based on the 'reverse genetics' methodology. Anonymous amplified DNA fragments are cloned by virtue of their amplification in a given tumor. These sequences are mapped in the normal genome and hence define a new genetic locus. The amplified domain is isolated by long-range cloning and analyzed along three lines of investigation: new genes are sought that can explain the biological significance of the amplification; the structure of the domain is studied in normal cells and in the amplification unit in the cancer cell; attempts are made to identify molecular probes of diagnostic value within the amplified domain. This application of genome technology to cancer biology is demonstrated in our study of a new genomic domain at chromosome 10q26 which is amplified specifically in human gastric carcinomas.     

SpeedHap: an accurate heuristic for the single individual SNP haplotyping problem with many gaps, high reading error rate and low coverage

Single nucleotide polymorphism (SNP) is the most frequent form of DNA variation. The set of SNP's present in a chromosome (called the em haplotype) is of interest in a wide area of applications in molecular biology and biomedicine, including diagnostic and medical therapy. In this paper we propose a new heuristic method for the problem of haplotype reconstruction for (portions of) a pair of homologous human chromosomes from a single individual (SIH). The problem is well known in literature and exact algorithms have been proposed for the case when no (or few) gaps are allowed in the input fragments. These algorithms, though exact and of polynomial complexity, are slow in practice. When gaps are considered no exact method of polynomial complexity is known. The problem is also hard to approximate with guarantees. Therefore fast heuristics have been proposed. In this paper we describe SpeedHap, a new heuristic method that is able to tackle the case of many gapped fragments and retains its effectiveness even when the input fragments have high rate of reading errors (up to 20%) and low coverage (as low as 3). We test SpeedHap on real data from the HapMap Project.     

Living with DNA breaks is an everyday reality for cells adapted to high NaCl

A rapid, coordinated response to DNA breaks, including activation of cell cycle checkpoints and initiation of accurate DNA repair is believed to be necessary to maintain genomic integrity and prevent accumulation of mutations. That is why it was so unexpected to discover recently that in the mouse renal inner medulla the otherwise healthy cells contain numerous DNA breaks, yet they survive and function adequately. The DNA breaks in the renal inner medulla are caused by the high NaCl concentrations to which the cells are constantly exposed as a consequence of the urinary concentrating mechanism. Cells adapted to high NaCl in cell culture also contain many DNA breaks. The DNA breaks do not trigger cell cycle arrest or cause apoptosis, and the cells safely proliferate rapidly despite their presence. Further, high NaCl inhibits the activity of key components of the classical DNA damage response such as Mre11, chk1 and H2AX. In order to explain why the DNA breaks do not cause disabling mutations, oncogenic transformations and/or apoptosis we speculate that in the presence of high NaCl there might be alternative DNA damage response pathways or special ways of coping with DNA damage.