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Jaing C Gardner S McLoughlin K Mulakken N Alegria-Hartman M Banda P Williams P Gu P Wagner M Manohar C Slezak T 《PloS one》2008,3(5):e2163
Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. 相似文献
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A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples. 相似文献
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Shea N. Gardner James B. Thissen Kevin S. McLoughlin Tom Slezak Crystal J. Jaing 《Journal of microbiological methods》2013
Microarrays to characterize single nucleotide polymorphisms (SNPs) provide a cost-effective and rapid method (under 24 h) to genotype microbes as an alternative to sequencing. We developed a pipeline for SNP discovery and microarray design that scales to 100's of microbial genomes. Here we tested various SNP probe design strategies against 8 sequenced isolates of Bacillus anthracis to compare sequence and microarray data. The best strategy allowed probe length to vary within 32–40 bp to equalize hybridization free energy. This strategy resulted in a call rate of 99.52% and concordance rate of 99.86% for finished genomes. Other probe design strategies averaged substantially lower call rates (94.65–96.41%) and slightly lower concordance rates (99.64–99.80%). These rates were lower for draft than finished genomes, consistent with higher incidence of sequencing errors and gaps. Highly accurate SNP calls were possible in complex soil and blood backgrounds down to 1000 copies, and moderately accurate SNP calls down to 100 spiked copies. The closest genome to the spiked strain was correctly identified at only 10 spiked copies. Discrepancies between sequence and array data did not alter the SNP-based phylogeny, regardless of the probe design strategy, indicating that SNP arrays can accurately place unsequenced isolates on a phylogeny. 相似文献
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Wen-I Lee Jainn-Jim Lin Meng-Ying Hsieh Syh-Jae Lin Tang-Her Jaing Shih-Hsiang Chen Iou-Jih Hung Chao-Ping Yang Chin-Jung Chen Yhu-Chering Huang Shin-Pai Li Jing-Long Huang 《PloS one》2013,8(10)
Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, virus-triggered immune disease. Hypersensitivity to mosquito bite (HMB), a presentation of Chronic Active Epstein-Barr Virus infection (CAEBV), may progress to HLH. This study aimed to investigate the immunologic difference between the HMB episodes and the HLH episodes associated with EBV infection. Immunologic changes of immunoglobulins, lymphocyte subsets, cytotoxicity, intracellular perforin and granzyme expressions, EBV virus load and known candidate genes for hereditary HLH were evaluated and compared. In 12 HLH episodes (12 patients) and 14 HMB episodes (4 patients), there were both decreased percentages of CD4+ and CD8+ and increased memory CD4+ and activated (CD2+HLADR+) lymphocytes. In contrast to HMB episodes that had higher IgE levels and EBV virus load predominantly in NK cells, those HLH episodes with virus load predominantly in CD3+ lymphocyte had decreased perforin expression and cytotoxicity that were recovered in the convalescence period. However, there was neither significant difference of total virus load in these episodes nor candidate genetic mutations responsible for hereditary HLH. In conclusion, decreased perforin expression in the HLH episodes with predominant-CD3+ EBV virus load is distinct from those HMB episodes with predominant-NK EBV virus load. Whether the presence of non-elevated memory CD4+ cells or activated lymphocytes (CD2+HLADR+) increases the mortality rate in the HLH episodes remains to be further warranted through larger-scale studies. 相似文献
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The 21 kDa apoprotein of LHC IId, a minor light-harvesting antenna component of Photosystem II, has been isolated and subjected to N-terminal protein sequencing. A sequence of 66 residues was obtained which contains regions of considerable homology to both those reported for LHC II and LHC I, but which is obviously distinct from them. The proposed occurrence of an identical 21 kDa LHC subunit in both photosystems I and II is shown to be incorrect. 相似文献
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Joseph G. Victoria Chunlin Wang Morris S. Jones Crystal Jaing Kevin McLoughlin Shea Gardner Eric L. Delwart 《Journal of virology》2010,84(12):6033-6040
Metagenomics and a panmicrobial microarray were used to examine eight live-attenuated viral vaccines. Viral nucleic acids in trivalent oral poliovirus (OPV), rubella, measles, yellow fever, varicella-zoster, multivalent measles/mumps/rubella, and two rotavirus live vaccines were partially purified, randomly amplified, and pyrosequenced. Over half a million sequence reads were generated covering from 20 to 99% of the attenuated viral genomes at depths reaching up to 8,000 reads per nucleotides. Mutations and minority variants, relative to vaccine strains, not known to affect attenuation were detected in OPV, mumps virus, and varicella-zoster virus. The anticipated detection of endogenous retroviral sequences from the producer avian and primate cells was confirmed. Avian leukosis virus (ALV), previously shown to be noninfectious for humans, was present as RNA in viral particles, while simian retrovirus (SRV) was present as genetically defective DNA. Rotarix, an orally administered rotavirus vaccine, contained porcine circovirus-1 (PCV1), a highly prevalent nonpathogenic pig virus, which has not been shown to be infectious in humans. Hybridization of vaccine nucleic acids to a panmicrobial microarray confirmed the presence of endogenous retroviral and PCV1 nucleic acids. Deep sequencing and microarrays can therefore detect attenuated virus sequence changes, minority variants, and adventitious viruses and help maintain the current safety record of live-attenuated viral vaccines.Highly effective, safe, and relatively inexpensive, live-attenuated viruses protect against numerous human and animal viral infections. Attenuation is achieved by genetically adapting viruses for replication in a different host species or under nonphysiological conditions, such that viruses lose their pathogenic potential in their original host species while remaining sufficiently antigenic to induce lasting protective immunity. Live-attenuated vaccines are highly efficacious due to the physiologic presentation of native antigen to the host''s immune system and include the earliest human vaccine developed by serial passages of rabies virus in rabbits. In very rare instances, one attenuated viral vaccine, the oral poliovirus vaccine (OPV), can accumulate mutations as well as recombine with other coinfecting enteroviruses and revert to a pathogenic state (18, 24). Attenuated live vaccines also carry a potential risk of contamination with adventitious viruses introduced during the attenuation process, from the cell lines used, and/or from the animal sera or other biologics often used in cell cultures. Very early Theiler''s yellow fever attenuated virus was once “stabilized” with human plasma thought to contain hepatitis B virus, resulting in many cases of hepatitis (5, 28). Some early Sabin poliovirus vaccines were contaminated with the simian virus 40 (SV40) polyomavirus from the monkey cells used to amplify polioviruses. While carcinogenic in rodents, SV40 has no epidemiologic association with human cancers (10). Avian leukosis virus (ALV) and endogenous avian virus (AEV) have been reported in attenuated vaccines grown in chicken embryo fibroblasts (CEF), but extensive testing has also ruled out human infections (14, 15). Vaccine-associated ALV and AEV are thought to originate from endogenous retroviruses in the chicken germ line (14, 15, 17).Because the chemical inactivation used in the manufacture of killed-virus vaccines is also likely to inactivate adventitious viruses, we focused on eight live-attenuated viruses, OPV (Biopolio), rubella (Meruvax-II), measles (Attenuvax), yellow fever (YF-Vax), human herpesvirus 3 (HHV-3) (Varivax), rotavirus (Rotarix and Rotateq), and multivalent measles/mumps/rubella (MMR-II), to resequence the attenuated viruses and test for the presence of adventitious viruses after viral particle purification, massively parallel pyrosequencing, and viral sequence similarity searches. Vaccine nucleic acids were also analyzed using a panmicrobial microarray. 相似文献
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Shea N. Gardner Kevin McLoughlin Nicholas A. Be Jonathan Allen Scott C. Weaver Naomi Forrester Mathilde Guerbois Crystal Jaing 《PloS one》2016,11(4)
Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broad panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Finally, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype. 相似文献
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Allogeneic hematopoietic stem cell transplantation is a deterministic curative procedure for various hematologic disorders and congenital immunodeficiency. Despite its increased use, the mortality rate for patients undergoing this proce dure remains high, mainly due to the perceived risk of exacerbating graft-versus-host disease (GVHD). However, even with immunosuppressive agents, some patients still develop GVHD. Advanced mesenchymal stem/stromal cell (MSC) strategies have been proposed to achieve better therapeutic outcomes, given their immunosuppressive potential. However, the efficacy and trial designs have varied among the studies, and some research findings appear contradictory due to the challenges in characterizing the in vivo effects of MSCs. This review aims to provide real insights into this clinical entity, emphasizing diagnostic, and therapeutic considerations and generating pathophysiology hypotheses to identify research avenues. The indications and timing for the clinical application of MSCs are still subject to debate. 相似文献
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