Biomedical subjects

James C Smoot

Publications and source records attributed to James C Smoot.

8 recordsLinked to original sources

PubMed2006

Discrimination of shifts in a soil microbial community associated with TNT-contamination using a functional ANOVA of 16S rRNA hybridized to oligonucleotide microarrays.

A functional ANOVA analysis of the thermal dissociation of RNA hybridized to DNA microarrays was used to improve discrimination between two soil microbial communities. Following hybridization of in vitro transcribed 16S rRNA derived from uncontaminated and 2,4,6-trinitrotoluene contaminated soils to an oligonucleotide microarray containing group- and species-specific perfect match (PM) probes and mismatch (MM) variants, thermal dissociation was used to analyze the nucleic acid bound to each PM-MM probe set. Functional ANOVA of the dissociation curves generally discriminated PM-MM probe sets when Td values (temperature at 50% probe-target dissociation) could not. Maximum discrimination for many PM and MM probes often occurred at temperatures greaterthan the Td. Comparison of signal intensities measured prior to dissociation analysis from hybridizations of the two soil samples revealed significant differences in domain-, group-, and species-specific probes. Functional ANOVA showed significantly different dissociation curves for 11 PM probes when hybridizations from the two soil samples were compared, even though initial signal intensities for 3 of the 11 did not vary.

Analysis of Variance↗

PubMed2006

Technology development to explore the relationship between oral health and the oral microbial community.

The human oral cavity contains a complex microbial community that, until recently, has not been well characterized. Studies using molecular tools have begun to enumerate and quantify the species residing in various niches of the oral cavity; yet, virtually every study has revealed additional new species, and little is known about the structural dynamics of the oral microbial community or how it changes with disease. Current estimates of bacterial diversity in the oral cavity range up to 700 species, although in any single individual this number is much lower. Oral microbes are responsible for common chronic diseases and are suggested to be sentinels of systemic human diseases. Microarrays are now being used to study oral microbiota in a systematic and robust manner. Although this technology is still relatively young, improvements have been made in all aspects of the technology, including advances that provide better discrimination between perfect-match hybridizations from non-specific (and closely-related) hybridizations. This review addresses a core technology using gel-based microarrays and the initial integration of this technology into a single device needed for system-wide studies of complex microbial community structure and for the development of oral diagnostic devices.

Journal Article↗

PubMed2003

Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays.

The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.

Base Pair Mismatch↗

PubMed2002

Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence.

Genome sequences are available for many bacterial strains, but there has been little progress in using these data to understand the molecular basis of pathogen emergence and differences in strain virulence. Serotype M3 strains of group A Streptococcus (GAS) are a common cause of severe invasive infections with unusually high rates of morbidity and mortality. To gain insight into the molecular basis of this high-virulence phenotype, we sequenced the genome of strain MGAS315, an organism isolated from a patient with streptococcal toxic shock syndrome. The genome is composed of 1,900,521 bp, and it shares approximately 1.7 Mb of related genetic material with genomes of serotype M1 and M18 strains. Phage-like elements account for the great majority of variation in gene content relative to the sequenced M1 and M18 strains. Recombination produces chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. Strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A(2) (designated Sla). Infected humans had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are made in vivo. SpeK and SSA were pyrogenic and toxic for rabbits. Serotype M3 strains with the phage-encoded speK and sla genes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phage-mediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS.

Amino Acid Sequence↗

PubMed2002

Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks.

Acute rheumatic fever (ARF), a sequelae of group A Streptococcus (GAS) infection, is the most common cause of preventable childhood heart disease worldwide. The molecular basis of ARF and the subsequent rheumatic heart disease are poorly understood. Serotype M18 GAS strains have been associated for decades with ARF outbreaks in the U.S. As a first step toward gaining new insight into ARF pathogenesis, we sequenced the genome of strain MGAS8232, a serotype M18 organism isolated from a patient with ARF. The genome is a circular chromosome of 1,895,017 bp, and it shares 1.7 Mb of closely related genetic material with strain SF370 (a sequenced serotype M1 strain). Strain MGAS8232 has 178 ORFs absent in SF370. Phages, phage-like elements, and insertion sequences are the major sources of variation between the genomes. The genomes of strain MGAS8232 and SF370 encode many of the same proven or putative virulence factors. Importantly, strain MGAS8232 has genes encoding many additional secreted proteins involved in human-GAS interactions, including streptococcal pyrogenic exotoxin A (scarlet fever toxin) and two uncharacterized pyrogenic exotoxin homologues, all phage-associated. DNA microarray analysis of 36 serotype M18 strains from diverse localities showed that most regions of variation were phages or phage-like elements. Two epidemics of ARF occurring 12 years apart in Salt Lake City, UT, were caused by serotype M18 strains that were genetically identical, or nearly so. Our analysis provides a critical foundation for accelerated research into ARF pathogenesis and a molecular framework to study the plasticity of GAS genomes.

Acute Disease↗

PubMed2002

Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains.

Several human pathogens (e.g., Bacillus anthracis, Yersinia pestis, Bordetella pertussis, Plasmodium falciparum, and Mycobacterium tuberculosis) have very restricted unselected allelic variation in structural genes, which hinders study of the genetic relationships among strains and strain-trait correlations. To address this problem in a representative pathogen, 432 M. tuberculosis complex strains from global sources were genotyped on the basis of 230 synonymous (silent) single nucleotide polymorphisms (sSNPs) identified by comparison of four genome sequences. Eight major clusters of related genotypes were identified in M. tuberculosis sensu stricto, including a single cluster representing organisms responsible for several large outbreaks in the United States and Asia. All M. tuberculosis sensu stricto isolates of previously unknown phylogenetic position could be rapidly and unambiguously assigned to one of the eight major clusters, thus providing a facile strategy for identifying organisms that are clonally related by descent. Common clones of M. tuberculosis sensu stricto and M. bovis are distinct, deeply branching genotypic complexes whose extant members did not emerge directly from one another in the recent past. sSNP genotyping rapidly delineates relationships among closely related strains of pathogenic microbes and allows construction of genetic frameworks for examining the distribution of biomedically relevant traits such as virulence, transmissibility, and host range.

Alleles↗

PubMed2002

Characterization of two novel pyrogenic toxin superantigens made by an acute rheumatic fever clone of Streptococcus pyogenes associated with multiple disease outbreaks.

The pathogenesis of acute rheumatic fever (ARF) is poorly understood. We identified two contiguous bacteriophage genes, designated speL and speM, encoding novel inferred superantigens in the genome sequence of an ARF strain of serotype M18 group A streptococcus (GAS). speL and speM were located at the same genomic site in 33 serotype M18 isolates, and no nucleotide sequence diversity was observed in the 33 strains analyzed. Furthermore, the genes were absent in 13 non-M18 strains tested. These data indicate a recent acquisition event by a distinct clone of serotype M18 GAS. speL and speM were transcribed in vitro and upregulated in the exponential phase of growth. Purified SpeL and SpeM were pyrogenic and mitogenic for rabbit splenocytes and human peripheral blood mononuclear cells in picogram amounts. SpeL preferentially expanded human T cells expressing T-cell receptors Vbeta1, Vbeta5.1, and Vbeta23, and SpeM had specificity for Vbeta1 and Vbeta23 subsets, indicating that both proteins had superantigen activity. SpeL was lethal in two animal models of streptococcal toxic shock, and SpeM was lethal in one model. Serologic studies indicated that ARF patients were exposed to serotype M18 GAS, SpeL, and SpeM. The data demonstrate that SpeL and SpeM are pyrogenic toxin superantigens and suggest that they may participate in the host-pathogen interactions in some ARF patients.

Acute Disease↗

PubMed2002

Molecular analysis of group A Streptococcus type emm18 isolates temporally associated with acute rheumatic fever outbreaks in Salt Lake City, Utah.

Acute rheumatic fever (ARF) and subsequent rheumatic heart disease are rare but serious sequelae of group A Streptococcus (GAS) infections in most western countries. Salt Lake City (SLC), Utah, and the surrounding intermountain region experienced a resurgence of ARF in 1985 which has persisted. The largest numbers of cases were encountered in 1985-1986 and in 1997-1998. Organisms with a mucoid colony phenotype when grown on blood agar plates were temporally associated with the higher incidence of ARF. To develop an understanding of the molecular population genetic structure of GAS strains associated with ARF in the SLC region, 964 mucoid and nonmucoid pharyngeal isolates recovered in SLC from 1984 to 1999 were studied by sequencing the emm gene. Isolates with an emm18 allele were further characterized by sequencing the spa, covR, and covS genes. Peak periods of ARF were associated with GAS isolates possessing an emm18 allele encoding the protein found in serotype M18 isolates. Among the serotype M18 isolates, the difference in the number of C repeats produced three size variants. Variation was limited in spa, a gene that encodes a streptococcal protective antigen, and covR and covS, genes that encode a two-component regulatory system that, when inactivated, results in a mucoid phenotype and enhanced virulence in mouse infection models. Pulsed-field gel electrophoresis showed a single restriction profile for serotype M18 organisms isolated during both peak periods of ARF. In SLC, the incidence of ARF coresurged with the occurrence of GAS serotype M18 isolates that have very restricted genetic variation.

Acute Disease↗