Global Profiling of PknG Interactions Using a Human Proteome Microarray Reveals Novel Connections with CypA.

Mycobacterium tuberculosis (Mtb) serine/threonine kinase PknG plays an important role in the Mtb-host interaction by facilitating the survival of Mtb in macrophages. However, the human proteins with which the PknG interacts, and the underlying molecular mechanisms are still largely unknown. In this study, a HuProt array is been applied to globally identify the host proteins to which PknG binds. In this way, 125 interactors are discovered, including a cyclophilin protein, CypA. This interaction between PknG and CypA is validated both in vitro and in vivo, and functional studies show that PknG significantly reduces the protein levels of CypA through phosphorylation, which consequently inhibit the inflammatory response through downregulation of NF-κB and ERK1/2 pathways. Phenotypically, overexpression of PknG reduces cytokine levels and promotes the survival of Mycobacterium smegmatis (Msm) in macrophages. Overall, it is expected that the PknG interactors identified in this study will serve as a useful resource for further systematic studies of the roles that PknG plays in the Mtb-host interactions.

Prevention and Control of Pathogens Based on Big-Data Mining and Visualization Analysis.

Morbidity and mortality caused by infectious diseases rank first among all human illnesses. Many pathogenic mechanisms remain unclear, while misuse of antibiotics has led to the emergence of drug-resistant strains. Infectious diseases spread rapidly and pathogens mutate quickly, posing new threats to human health. However, with the increasing use of high-throughput screening of pathogen genomes, research based on big data mining and visualization analysis has gradually become a hot topic for studies of infectious disease prevention and control. In this paper, the framework was performed on four infectious pathogens (Fusobacterium, Streptococcus, Neisseria, and Streptococcus salivarius) through five functions: 1) genome annotation, 2) phylogeny analysis based on core genome, 3) analysis of structure differences between genomes, 4) prediction of virulence genes/factors with their pathogenic mechanisms, and 5) prediction of resistance genes/factors with their signaling pathways. The experiments were carried out from three angles: phylogeny (macro perspective), structure differences of genomes (micro perspective), and virulence and drug-resistance characteristics (prediction perspective). Therefore, the framework can not only provide evidence to support the rapid identification of new or unknown pathogens and thus plays a role in the prevention and control of infectious diseases, but also help to recommend the most appropriate strains for clinical and scientific research. This paper presented a new genome information visualization analysis process framework based on big data mining technology with the accommodation of the depth and breadth of pathogens in molecular level research.

Construction of a multiplex allele-specific PCR-based universal array (ASPUA) and its application to hearing loss screening.

We demonstrate a new method, using a universal array approach termed multiplex allele-specific PCR-based universal array (ASPUA), and applied it to the mutation detection of hereditary hearing loss. Mutations in many different genes may be the cause of hereditary hearing loss, a sensory defect disorder. Effective methods for genetic diagnosis are clearly needed to provide clinical management. Owing to the broad genetic basis of this condition, clinical assay of such a highly heterogeneous disorder is expensive and time consuming. In ASPUA, the allele discrimination reaction is carried out in solution by multiplex allele-specific PCR and a universal solid phase array with different tag probes is used to display the PCR result. The purpose of developing the ASPUA platform was to utilize the rapidity and simplicity of the amplification refractory mutation system (ARMS) with the detection power of microarray hybridization. This is the first report of the combination of these two technologies, which allow for the completion of allele-specific detection of 11 of the most frequent mutations causing hereditary hearing loss in under 5 hr. The ASPUA platform was validated by accurately analyzing 141 patient samples that had been previously genotyped for GJB2, GJB3, SLC26A4, and MTRNR1. In addition, we also developed a simplified assay by using streptavidin-coated magnetic beads instead of fluorescence for signal display that can be assessed through a conventional light microscope. We demonstrate that the ASPUA platform is rapid, cost-effective, and easily-used, and is especially appropriate for mutation detection in clinical genetic diagnostics.

[The building of chip-based capillary electrophoresis platform and application].

OBJECTIVE: To detect the point mutations by chip-based capillary electrophoresis and to provide a rapid and sensitive technique detection for beta-thalassemia. METHODS: Multiplex primer-extension reaction was used to amplify the common loci of the samples for beta-thalassemia. The reaction products were detected by the chip-based capillary electrophoresis and the genotypes of the samples were discrened. RESULTS: A system was constructed to detect the point mutations of beta-thalassemia by chip-based capillary electrophoresis, and the technology was ralidated by the patients' samples and the results coincided with those of detection kit. CONCLUSION: Beta-thalassemia can be detected by chip-based capillary electrophoresis rapidly with a small amount of samples. It would be a new detection method of the genetic disorders.

The design and application of DNA chips for early detection of SARS-CoV from clinical samples.

BACKGROUND: SARS coronavirus has been identified as the cause of severe acute respiratory syndrome (SARS). Few tests allow confirmation or exclusion of SARS within the first few days of infection. A gene chip is a useful tool for the study of microbial infections mainly for its capability of performing multi-target analysis in a single test. OBJECTIVES: Investigate the possibility of early detection of SARS virus from clinical samples using the gene chip-based method. STUDY DESIGN: We purified RNA from SARS-CoV obtained from routinely collected peripheral blood and sputum samples of 34 patients who had been identified as probable SARS patients by following the interim U.S. case definition. Four segments of the SARS-CoV were amplified using reverse transcription-nested PCR and the products examined using the 70-mer gene chips for SARS-CoV detection. RESULTS: A blind-test of both peripheral blood and sputum specimens lead to the positive detection of SARS-CoV in 31 out of 34 patients. SARS-CoV was not found in peripheral blood or sputum specimens from three patients. Two of the 34 patients were only 3 days post-onset of symptoms and were subsequently confirmed to be SARS positive. Our results indicate that the gene chip-based molecular test is specific for SARS-CoV and allows early detection of patients with SARS with detection rate about 8% higher than the single PCR test when the sputum sample is available.

Blocking oligo--a novel approach for improving chip-based DNA hybridization efficiency.

For most of the commonly used DNA chips, the probes are usually single-stranded oligonucleotides and the targets are double-stranded DNAs (dsDNAs). Only one strand of the DNA serves as the target while the other competes with the probes immobilized on the chip for the target and therefore is regarded as the interfering strand. In this report, a novel technique was developed for improving the hybridization efficiency on DNA chips by using blocking oligos, which is complimentary to the target interfering strand to reduce the influence of the interfering strand. The hybridization efficiency of dsDNA was much lower than that of single-stranded DNA (ssDNA) when synthesized DNA targets were tested on the DNA chip. Blocking oligos can improve the hybridization efficiency of dsDNA to about 2/3 that of ssDNA. Blocking oligos have also been applied to PCR products of different lengths for hybridization. The hybridization efficiency with blocking oligos is about three times higher than that without blocking oligos. We have tested PCR products of 1054 and 435 bp using our blocking procedure, and the results are consistent.