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Journal of Bacteriology  1964;88(6):1798-1804.
Wiebers, Joyce L. (Purdue University, West Lafayette, Ind.), and Harold R. Garner. Use of S-methylcysteine and cystathionine by methionineless Neurospora mutants. J. Bacteriol. 88:1798–1804. 1964.—Radioactive methionine was found in hydrolysates of various strains of Neurospora crassa when either S-methylcysteine (SMC)-C14H3 or SMC-S35 is the sole addition to minimal medium. Isotope product-precursor specific activity ratios are very similar for the two sources of label. Wild-type and methionineless mutants use sulfur from SMC in the biosynthesis of methionine, but not of cysteine, when grown in regular medium. With a medium nearly free from sulfate, SMC served as a source of sulfur for both cysteine and methionine. Suppressed methionineless mutants incorporated sulfur from SMC into cellular cysteine even in the presence of normal amounts of sulfate. SMC as a possible metabolic precursor of methionine was compared to cystathionine in an experiment with wild-type Neurospora. The four sources of label used were: SMC-C14H3, SMC-S35, cystathionine-U-C14, and cystathionine-S35. In each flask, the organism was offered one of the labeled compounds plus an equivalent amount of the other compound without label. The amount of each compound was sufficient for either to supply its contribution to all of the cellular methionine, if it were successful in competing with endogenous sources. To avoid adaptive breakdown of substrates, the compounds were added continuously at a rate consistent with the amount of growth present. The ratio of specific activity of cellular methionine to precursor was determined for each labeled compound. The results show that SMC sulfur and methyl carbon are used equally well. Cystathionine carbon and sulfur appear to be equally utilized also. A preference for cystathionine is indicated.
PMCID: PMC277486  PMID: 14240970
2.  Somatic intronic microsatellite loci differentiate glioblastoma from lower-grade gliomas 
Oncotarget  2014;5(15):6003-6014.
Genomic studies of glioma sub-types have amassed new disease specific mutations, yet these only partially explain how mutations are linked to predisposition or progression. We hypothesized that microsatellite variation could expand the understanding of glioma etiology. Furthermore, germline markers for gliomas are typically undetectable; therefore we also hypothesize that the predictability of cancer-associated microsatellite loci in germline DNA may support the current hypothesis of a glioma cell of origin.
In this study, “normal” germline exome sequenced DNA from the 1000 Genomes Project (n=390) were compared with exome sequences from germlines of subjects with WHO grade II and III lower-grade glioma (LGG, n=136) and WHO grade IV glioblastoma (GBM, n=252) from The Cancer Genome Atlas to identify microsatellite loci non-randomly associated with glioma. From germline data, we identified 48 GBM-specific loci, 42 Lower-grade glioma specific loci and 29 loci that distinguish GBM from LGG (p≤ 0.01). We then attempted to distinguish WHO grade II glioma (n=67) from GBM resulting in 8 informative loci. Significantly, in all glioma grades, comparisons between tumor and matched germline sequences demonstrated no significant differences in these variants (p≥ 0.01). Therefore, these microsatellite loci are considered to be components of grade-specific signatures for glioma which distinguish germline sequences of individuals with cancer from those of individuals that are “normal”. In order to better understand the significance of these loci, we identified biological processes enriched in genes with these variants. Most strikingly, six helicase genes were enriched in the GBM cohort (p≤ 1.0 ×10−3). The preservation of these glioma-specific loci could therefore serve as valuable diagnostic and therapeutic markers; especially since the heterogeneity of tumor cell populations can obscure the identification of mutations preceding a metastatic phenotype.
PMCID: PMC4171608  PMID: 25153720
GBM; microsatellite; glioma; oligodendroglioma; helicase; ubiquitin proteasome system
3.  Nicotine and oxidative stress induced exomic variations are concordant and overrepresented in cancer-associated genes 
Oncotarget  2014;5(13):4788-4798.
Although the connection between cancer and cigarette smoke is well established, nicotine is not characterized as a carcinogen. Here, we used exome sequencing to identify nicotine and oxidative stress-induced somatic mutations in normal human epithelial cells and its correlation with cancer. We identified over 6,400 SNVs, indels and microsatellites in each of the stress exposed cells relative to the control, of which, 2,159 were consistently observed at all nicotine doses. These included 429 nsSNVs including 158 novel and 79 cancer-associated. Over 80% of consistently nicotine induced variants overlap with variations detected in oxidative stressed cells, indicating that nicotine induced genomic alterations could be mediated through oxidative stress. Nicotine induced mutations were distributed across 1,585 genes, of which 49% were associated with cancer. MUC family genes were among the top mutated genes. Analysis of 591 lung carcinoma tumor exomes from The Cancer Genome Atlas (TCGA) revealed that 20% of non-small-cell lung cancer tumors in smokers have mutations in at least one of the MUC4, MUC6 or MUC12 genes in contrast to only 6% in non-smokers. These results indicate that nicotine induces genomic variations, promotes instability potentially mediated by oxidative stress, implicating nicotine in carcinogenesis, and establishes MUC genes as potential targets.
PMCID: PMC4148099  PMID: 24947164
Nicotine; Exome sequencing; MUC4; Biomarker; Mutation targets
4.  The Dynamic Exome: acquired variants as individuals age 
Aging (Albany NY)  2014;6(6):511-521.
A singular genome used for inference into population-based studies is a standard method in genomics. Recent studies show that spontaneous genomic variants can propagate into new generations and these changes can contribute to individual cell aging with environmental and evolutionary elements contributing to cumulative genomic variation. However, the contribution of aging to genomic changes in tissue samples remains uncharacterized. Here, we report the impact of aging on individual human exomes and their implications. We found the human genome to be dynamic, acquiring a varying number of mutations with age (5,000 to 50,000 in 9 to 16 years). This equates to a variation rate of 9.6×10−7 to 8.4×10−6 bp−1 year−1 for nonsynonymous single nucleotide variants and 2.0×10−4 to 1.0×10−3 locus−1 year−1 for microsatellite loci in these individuals. These mutations span across 3,000 to 13,000 genes, which commonly showed association with Wnt signaling and Gonadotropin releasing hormone receptor pathways, and indicated for individuals a specific and significant enrichment for increased risk for diabetes, kidney failure, cancer, Rheumatoid arthritis, and Alzheimer's disease– conditions usually associated with aging. The results suggest that “age” is an important variable while analyzing an individual human genome to extract individual-specific clinically significant information necessary for personalized genomics.
PMCID: PMC4100812  PMID: 25063753
Aging; dynamic exome; exome sequencing; personalized genomics; personalized medicine
5.  Improved variation calling via an iterative backbone remapping and local assembly method for bacterial genomes 
Genomics  2012;100(5):271-276.
Sequencing data analysis remains limiting and problematic, especially for low complexity repeat sequences and transposon elements due to inherent sequencing errors and short sequence read lengths. We have developed a program, ReviSeq, which uses a hybrid method comprised of iterative remapping and local assembly upon a bacterial sequence backbone. Application of this method to six Brucella suis field isolates compared to the newly revised Brucella suis 1330 reference genome identified on average 13, 15, 19 and 9 more variants per sample than STAMPY/SAMtools, BWA/SAMtools, iCORN and BWA/PINDEL pipelines, and excluded on average 4, 2, 3 and 19 variants per sample, respectively. In total, using this iterative approach, we identified on average 87 variants including SNVs, short INDELs and long INDELs per strain when compared to the reference. Our program outperforms other methods especially for long INDEL calling.
The program is available at
PMCID: PMC3483379  PMID: 22967795
Brucella; sequence assembly; resequencing; variant calling; comparative genomics; iterative mapping
6.  Exome-Wide Somatic Microsatellite Variation Is Altered in Cells with DNA Repair Deficiencies 
PLoS ONE  2014;9(11):e110263.
Microsatellites (MST), tandem repeats of 1–6 nucleotide motifs, are mutational hot-spots with a bias for insertions and deletions (INDELs) rather than single nucleotide polymorphisms (SNPs). The majority of MST instability studies are limited to a small number of loci, the Bethesda markers, which are only informative for a subset of colorectal cancers. In this paper we evaluate non-haplotype alleles present within next-gen sequencing data to evaluate somatic MST variation (SMV) within DNA repair proficient and DNA repair defective cell lines. We confirm that alleles present within next-gen data that do not contribute to the haplotype can be reliably quantified and utilized to evaluate the SMV without requiring comparisons of matched samples. We observed that SMV patterns found in DNA repair proficient cell lines without DNA repair defects, MCF10A, HEK293 and PD20 RV:D2, had consistent patterns among samples. Further, we were able to confirm that changes in SMV patterns in cell lines lacking functional BRCA2, FANCD2 and mismatch repair were consistent with the different pathways perturbed. Using this new exome sequencing analysis approach we show that DNA instability can be identified in a sample and that patterns of instability vary depending on the impaired DNA repair mechanism, and that genes harboring minor alleles are strongly associated with cancer pathways. The MST Minor Allele Caller used for this study is available at
PMCID: PMC4234249  PMID: 25402475
7.  Complete Genome Sequence of Brucella suis VBI22, Isolated from Bovine Milk 
Journal of Bacteriology  2012;194(4):910.
Brucella suis is the causative agent of swine brucellosis and is known to be able to infect several different hosts, including cattle, dogs, and horses, without causing disease symptoms. Here we report the complete genome sequence of Brucella suis VBI22, which was isolated from raw milk from an infected cow.
PMCID: PMC3272945  PMID: 22275106
8.  Sporadic Breast Cancer Patients' Germline DNA Exhibit an AT-Rich Microsatellite Signature 
Genes, chromosomes & cancer  2011;50(4):275-283.
Using a custom CGH-like oligonucleotide array to measure the global microsatellite content in the genomes of 72 cancer, cancer-free, and high risk patient and cell line samples (56 germline DNA and 16 in tumor or tumor cell line DNA) we found a unique, reproducible, and statistically significant pattern of 18 motif-specific microsatellite families (out of 962 possible 1-6 mer repeats) in breast cancer patient germline and tumor DNA, but not in germline DNA of cancer-free volunteer controls or in breast cancer patients with BRCA1/2 mutations. These high-similarity A/T rich repetitive motifs were also more pronounced in the germlines and tumors of colon cancer tumor patients (3/6 samples) and microsatellite unstable colon cancer cell lines; however, germline DNA of sporadic breast cancer patients exhibited the largest global content shift for those motifs with extreme AT/GC ratios. These results indicate that global microsatellite variability is complex, suggest the existence of a previously unknown genomic destabilization mechanism in breast cancer patients' germline DNA, and warrant further testing of such microsatellite variability as a predictor of future breast cancer development.
PMCID: PMC3107400  PMID: 21319262
9.  ReviSTER: an automated pipeline to revise misaligned reads to simple tandem repeats 
Bioinformatics  2013;29(14):1734-1741.
Motivation: Simple tandem repeats are highly variable genetic elements and widespread in genomes of many organisms. Next-generation sequencing technologies have enabled a robust comparison of large numbers of simple tandem repeat loci; however, analysis of their variation using traditional sequence analysis approaches still remains limiting and problematic due to variants occurring in repeat sequences confusing alignment programs into mapping sequence reads to incorrect loci when the sequence reads are significantly different from the reference sequence.
Results: We have developed a program, ReviSTER, which is an automated pipeline using a ‘local mapping reference reconstruction method’ to revise mismapped or partially misaligned reads at simple tandem repeat loci. RevisSTER estimates alleles of repeat loci using a local alignment method and creates temporary local mapping reference sequences, and finally remaps reads to the local mapping references. Using this approach, ReviSTER was able to successfully revise reads misaligned to repeat loci from both simulated data and real data.
Availability: ReviSTER is open-source software available at
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3702252  PMID: 23677944
10.  EvoCor: a platform for predicting functionally related genes using phylogenetic and expression profiles 
Nucleic Acids Research  2014;42(Web Server issue):W72-W75.
The wealth of publicly available gene expression and genomic data provides unique opportunities for computational inference to discover groups of genes that function to control specific cellular processes. Such genes are likely to have co-evolved and be expressed in the same tissues and cells. Unfortunately, the expertise and computational resources required to compare tens of genomes and gene expression data sets make this type of analysis difficult for the average end-user. Here, we describe the implementation of a web server that predicts genes involved in affecting specific cellular processes together with a gene of interest. We termed the server ‘EvoCor’, to denote that it detects functional relationships among genes through evolutionary analysis and gene expression correlation. This web server integrates profiles of sequence divergence derived by a Hidden Markov Model (HMM) and tissue-wide gene expression patterns to determine putative functional linkages between pairs of genes. This server is easy to use and freely available at
PMCID: PMC4086105  PMID: 24848012
11.  Life cycle of an n-globin pseudogene microsatellite locus 
Frontiers in Genetics  2013;4:267.
PMCID: PMC3849843  PMID: 24363661
microsatellites; n-globin; evolution; microsatellite life cycle; gorilla
12.  Characterizing the Genetic Basis for Nicotine Induced Cancer Development: A Transcriptome Sequencing Study 
PLoS ONE  2013;8(6):e67252.
Nicotine is a known risk factor for cancer development and has been shown to alter gene expression in cells and tissue upon exposure. We used Illumina® Next Generation Sequencing (NGS) technology to gain unbiased biological insight into the transcriptome of normal epithelial cells (MCF-10A) to nicotine exposure. We generated expression data from 54,699 transcripts using triplicates of control and nicotine stressed cells. As a result, we identified 138 differentially expressed transcripts, including 39 uncharacterized genes. Additionally, 173 transcripts that are primarily associated with DNA replication, recombination, and repair showed evidence for alternative splicing. We discovered the greatest nicotine stress response by HPCAL4 (up-regulated by 4.71 fold) and NPAS3 (down-regulated by -2.73 fold); both are genes that have not been previously implicated in nicotine exposure but are linked to cancer. We also discovered significant down-regulation (-2.3 fold) and alternative splicing of NEAT1 (lncRNA) that may have an important, yet undiscovered regulatory role. Gene ontology analysis revealed nicotine exposure influenced genes involved in cellular and metabolic processes. This study reveals previously unknown consequences of nicotine stress on the transcriptome of normal breast epithelial cells and provides insight into the underlying biological influence of nicotine on normal cells, marking the foundation for future studies.
PMCID: PMC3688980  PMID: 23825647
13.  Molecular Profiling of Individual Tumor Cells by Hyperspectral Microscopic Imaging 
Translational Research  2011;159(5):366-375.
We have developed a hyperspectral microscopic imaging (HMI) platform that can precisely identify and quantify 10 molecular markers in individual cancer cells in a single pass. Exploitation of an improved separation of circulating tumor cells and the application of HMI has provided an opportunity to identify molecular changes in these cells, the recognition of co-expression of these markers, and poses an important opportunity for non-invasive diagnosis, and the use of targeted therapy. We have balanced the intensity of 10 fluorochromes bound to 10 different antibodies, each specific to a particular tumor marker, so that the intensity of each fluorochrome can be discerned from overlapping emissions. Using 2 touch preps from each primary breast cancer, the average molecular marker-intensities of 25 tumor cells gave a representative molecular signature for the tumor despite some cellular heterogeneity. The intensities determined by the HMI correlate well with the conventional 0-3+ analysis by experts in cellular pathology. Since additional multiplexes can be developed using the same fluorochromes but different antibodies, this analysis allows quantification of a large number of molecular markers on individual tumor cells. HMI can be completely automated and, eventually, could allow standardization of protein biomarkers and improve reproducibility among clinical pathology laboratories.
PMCID: PMC3337082  PMID: 22500509
15.  Transcriptional profile of the intracellular pathogen Brucella melitensis following HeLa cells infection 
Microbial pathogenesis  2011;51(5):338-344.
Brucella spp. infect hosts primarily by adhering and penetrating mucosal surfaces; however the initial molecular phenomena of this host:pathogen interaction remain poorly understood. Using cDNA microarray analysis, we characterized the transcriptional profile of the intracellular pathogen Brucella melitensis at 4 h (adaptational period) and 12 h (replicative phase) following HeLa cells infection. The intracellular pathogen transcriptome was determined using initially enriched and then amplified B. melitensis RNA from total RNA of B. melitensis-infected HeLa cells. Analysis of microarray results identified 161 and 115 pathogen genes differentially expressed at 4 and 12 h p.i., respectively. In concordance with phenotypic studies, most of the genes expressed were involved in pathogen growth and metabolism, and were down-regulated at the earliest time point (78%), but up-regulated at 12 h p.i. (75%). Further characterization of specific genes identified in this study will elucidate biological processes and pathways to help understand how both host and Brucella interact during the early infectious process to the eventual benefit of the pathogen and to the detriment of the naïve host.
PMCID: PMC3170451  PMID: 21798337
Brucella melitensis; Gene expression; Microarray; Bacterial pathogenesis
16.  Parallel assessment of CpG methylation by two-color hybridization with oligonucleotide arrays 
Analytical biochemistry  2002;309(2):301-310.
We have developed a method for the parallel analysis of multiple CpG sites in genomic DNA for their state of methylation. Hypermethylation of CpG islands within the promoters and 5′ exons of genes has been found to be a mechanism of transcriptional inactivation associated with a variety of tumors. The method that we developed relies on the differential reactivity of methylated and unmethylated cytosines with sodium bisulfite, which exclusively converts unmethylated cytosines to deoxyuracils. The resulting sequence changes are determined with single-nucleotide resolution by hybridization to an oligonucleotide array. Cohybridization with a reference sample containing a different label provides an internal standard for assessment of methylation state. This method provides advantages in parallelism over existing methods of methylation analysis. We have demonstrated this technique with a region from the promoter of the tumor suppressor gene p16, which is hypermethylated in many cancers.
PMCID: PMC3484840  PMID: 12413464
Hypermethylation; CpG island; Oligonucleotide array; Sodium bisulfite; Tumor suppressor
17.  Systems Biology Analysis of Gene Expression during In Vivo Mycobacterium avium paratuberculosis Enteric Colonization Reveals Role for Immune Tolerance 
PLoS ONE  2012;7(8):e42127.
Survival and persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in the intestinal mucosa is associated with host immune tolerance. However, the initial events during MAP interaction with its host that lead to pathogen survival, granulomatous inflammation, and clinical disease progression are poorly defined. We hypothesize that immune tolerance is initiated upon initial contact of MAP with the intestinal Peyer's patch. To test our hypothesis, ligated ileal loops in neonatal calves were infected with MAP. Intestinal tissue RNAs were collected (0.5, 1, 2, 4, 8 and 12 hrs post-infection), processed, and hybridized to bovine gene expression microarrays. By comparing the gene transcription responses of calves infected with the MAP, informative complex patterns of expression were clearly visible. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis, and genes were grouped into the specific pathways and gene ontology categories to create a holistic model. This model revealed three different phases of responses: i) early (30 min and 1 hr post-infection), ii) intermediate (2, 4 and 8 hrs post-infection), and iii) late (12 hrs post-infection). We describe here the data that include expression profiles for perturbed pathways, as well as, mechanistic genes (genes predicted to have regulatory influence) that are associated with immune tolerance. In the Early Phase of MAP infection, multiple pathways were initiated in response to MAP invasion via receptor mediated endocytosis and changes in intestinal permeability. During the Intermediate Phase, perturbed pathways involved the inflammatory responses, cytokine-cytokine receptor interaction, and cell-cell signaling. During the Late Phase of infection, gene responses associated with immune tolerance were initiated at the level of T-cell signaling. Our study provides evidence that MAP infection resulted in differentially regulated genes, perturbed pathways and specifically modified mechanistic genes contributing to the colonization of Peyer's patch.
PMCID: PMC3422314  PMID: 22912686
18.  Revised Genome Sequence of Brucella suis 1330 
Journal of Bacteriology  2011;193(22):6410.
Brucella suis is a causative agent of porcine brucellosis. We report the resequencing of the original sample upon which the published sequence of Brucella suis 1330 is based and describe the differences between the published assembly and our assembly at 12 loci.
PMCID: PMC3209196  PMID: 22038969
19.  Inhibitory Role of Notch1 in Calcific Aortic Valve Disease 
PLoS ONE  2011;6(11):e27743.
Aortic valve calcification is the most common form of valvular heart disease, but the mechanisms of calcific aortic valve disease (CAVD) are unknown. NOTCH1 mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood. The aim of this study was to investigate the molecular changes that occur with inhibition of Notch signaling in the aortic valve. Notch signaling pathway members are expressed in adult aortic valve cusps, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs). We found significant downregulation of Sox9 along with several cartilage-specific genes that were direct targets of the transcription factor, Sox9. Loss of Sox9 expression has been published to be associated with aortic valve calcification. Utilizing an in vitro porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process. Finally, the addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition. In conclusion, loss of Notch signaling contributes to aortic valve calcification via a Sox9-dependent mechanism.
PMCID: PMC3218038  PMID: 22110751
20.  Role of SPI-1 Secreted Effectors in Acute Bovine Response to Salmonella enterica Serovar Typhimurium: A Systems Biology Analysis Approach 
PLoS ONE  2011;6(11):e26869.
Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis with diarrhea and polymorphonuclear cell (PMN) influx into the intestinal mucosa in humans and calves. The Salmonella Type III Secretion System (T3SS) encoded at Pathogenicity Island I translocates Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into epithelial cells and is required for induction of diarrhea. These effector proteins act together to induce intestinal fluid secretion and transcription of C-X-C chemokines, recruiting PMNs to the infection site. While individual molecular interactions of the effectors with cultured host cells have been characterized, their combined role in intestinal fluid secretion and inflammation is less understood. We hypothesized that comparison of the bovine intestinal mucosal response to wild type Salmonella and a SipA, SopABDE2 effector mutant relative to uninfected bovine ileum would reveal heretofore unidentified diarrhea-associated host cellular pathways. To determine the coordinated effects of these virulence factors, a bovine ligated ileal loop model was used to measure responses to wild type S. Typhimurium (WT) and a ΔsipA, sopABDE2 mutant (MUT) across 12 hours of infection using a bovine microarray. Data were analyzed using standard microarray analysis and a dynamic Bayesian network modeling approach (DBN). Both analytical methods confirmed increased expression of immune response genes to Salmonella infection and novel gene expression. Gene expression changes mapped to 219 molecular interaction pathways and 1620 gene ontology groups. Bayesian network modeling identified effects of infection on several interrelated signaling pathways including MAPK, Phosphatidylinositol, mTOR, Calcium, Toll-like Receptor, CCR3, Wnt, TGF-β, and Regulation of Actin Cytoskeleton and Apoptosis that were used to model of host-pathogen interactions. Comparison of WT and MUT demonstrated significantly different patterns of host response at early time points of infection (15 minutes, 30 minutes and one hour) within phosphatidylinositol, CCR3, Wnt, and TGF-β signaling pathways and the regulation of actin cytoskeleton pathway.
PMCID: PMC3214023  PMID: 22096503
21.  A species independent universal bio-detection microarray for pathogen forensics and phylogenetic classification of unknown microorganisms 
BMC Microbiology  2011;11:132.
The ability to differentiate a bioterrorist attack or an accidental release of a research pathogen from a naturally occurring pandemic or disease event is crucial to the safety and security of this nation by enabling an appropriate and rapid response. It is critical in samples from an infected patient, the environment, or a laboratory to quickly and accurately identify the precise pathogen including natural or engineered variants and to classify new pathogens in relation to those that are known. Current approaches for pathogen detection rely on prior genomic sequence information. Given the enormous spectrum of genetic possibilities, a field deployable, robust technology, such as a universal (any species) microarray has near-term potential to address these needs.
A new and comprehensive sequence-independent array (Universal Bio-Signature Detection Array) was designed with approximately 373,000 probes. The main feature of this array is that the probes are computationally derived and sequence independent. There is one probe for each possible 9-mer sequence, thus 49 (262,144) probes. Each genome hybridized on this array has a unique pattern of signal intensities corresponding to each of these probes. These signal intensities were used to generate an un-biased cluster analysis of signal intensity hybridization patterns that can easily distinguish species into accepted and known phylogenomic relationships. Within limits, the array is highly sensitive and is able to detect synthetically mixed pathogens. Examples of unique hybridization signal intensity patterns are presented for different Brucella species as well as relevant host species and other pathogens. These results demonstrate the utility of the UBDA array as a diagnostic tool in pathogen forensics.
This pathogen detection system is fast, accurate and can be applied to any species. Hybridization patterns are unique to a specific genome and these can be used to decipher the identity of a mixed pathogen sample and can separate hosts and pathogens into their respective phylogenomic relationships. This technology can also differentiate between different species and classify genomes into their known clades. The development of this technology will result in the creation of an integrated biomarker-specific bio-signature, multiple select agent specific detection system.
PMCID: PMC3130645  PMID: 21672191
22.  Selective amplification of Brucella melitensis mRNA from a mixed host-pathogen total RNA 
BMC Research Notes  2010;3:244.
Brucellosis is a worldwide anthropozoonotic disease caused by an in vivo intracellular pathogen belonging to genus Brucella. The characterization of brucelae transcriptome's during host-pathogen interaction has been limited due to the difficulty of obtaining an adequate quantity of good quality eukaryotic RNA-free pathogen RNA for downstream applications.
Here, we describe a combined protocol to prepare RNA from intracellular B. melitensis in a quantity and quality suitable for pathogen gene expression analysis. Initially, B. melitensis total RNA was enriched from a host:pathogen mixed RNA sample by reducing the eukaryotic RNA..Then, to increase the Brucella RNA concentration and simultaneously minimize the contaminated host RNA in the mixed sample, a specific primer set designed to anneal to all B. melitensis ORF allows the selective linear amplification of sense-strand prokaryotic transcripts in a previously enriched RNA sample.
The novelty of the method we present here allows analysis of the gene expression profile of B. melitensis when limited amounts of pathogen RNA are present, and is potentially applicable to both in vivo and in vitro models of infection, even at early infection time points.
PMCID: PMC2954846  PMID: 20920187
23.  Carbamazepine alone and in combination with doxycycline attenuates isoproterenol-induced cardiac hypertrophy 
Heart International  2010;5(1):e7.
β-adrenergic signaling is involved in the development of cardiac hypertrophy (CH), justifying the use of β-blockers as a therapy to minimize and postpone the consequences of this disease. Evidence suggests that adenylate cyclase, a downstream effector of the β-adrenergic pathway, might be a therapeutic target. We examined the effects of the anti-epileptic drug carbamazepine (CBZ), an inhibitor of adenylate cyclase. In a murine cardiac hypertrophy model, carbamazepine significantly attenuates isoproteronol (ISO)-induced cardiac hypertrophy. Carbamazepine also has an effect in transverse aortic banding induced cardiac hypertrophy (TAB) (P=0.07). When carbamazepine was given in combination with the antibiotic doxycycline (DOX), which inhibits matrix metalloproteinases (MMPs), therapeutic outcome measured by heart weight-to-body weight and heart weight-to-tibia length ratios was improved compared to either drug alone. Additionally, the combination therapy resulted in an increase in the survival rate over a 56-day period compared to that of untreated mice with cardiac hypertrophy or either drug used alone. Moreover, in support of a role for carbamaze -pine as a β-adrenergic antagonist via cAMP inhibition, a lower heart rate and a lower level of the activated phosphorylated form of the cAMP Response Element-Binding (CREB) were observed in heart extracts from mice treated with carbamazepine. Gene expression analysis identified 19 genes whose expression is significantly altered in treated animals and might be responsible for the added benefit provided by the combination therapy. These results suggest that carbamazepine acts as a β-adrenergic antagonist. Carbamazepine and doxycycline are approved by the US Food and Drug Administration (FDA) as drugs that might complement medications for cardiac hypertrophy or serve as an alternative therapy to traditional β-blockers. Furthermore, these agents reproducibly impact the expression of genes that may serve as additional therapeutic targets in the management of cardiac hypertrophy.
PMCID: PMC3184704  PMID: 21977292
cardiac hypertrophy; gene expression; drug repurposing; FDA approved.
24.  Brucella melitensis VjbR and C12-HSL regulons: contributions of the N-dodecanoyl homoserine lactone signaling molecule and LuxR homologue VjbR to gene expression 
BMC Microbiology  2010;10:167.
Quorum sensing is a communication system that regulates gene expression in response to population density and often regulates virulence determinants. Deletion of the luxR homologue vjbR highly attenuates intracellular survival of Brucella melitensis and has been interpreted to be an indication of a role for QS in Brucella infection. Confirmation for such a role was suggested, but not confirmed, by the demonstrated in vitro synthesis of an auto-inducer (AI) by Brucella cultures. In an effort to further delineate the role of VjbR to virulence and survival, gene expression under the control of VjbR and AI was characterized using microarray analysis.
Analyses of wildtype B. melitensis and isogenic ΔvjbR transciptomes, grown in the presence and absence of exogenous N-dodecanoyl homoserine lactone (C12-HSL), revealed a temporal pattern of gene regulation with variances detected at exponential and stationary growth phases. Comparison of VjbR and C12-HSL transcriptomes indicated the shared regulation of 127 genes with all but 3 genes inversely regulated, suggesting that C12-HSL functions via VjbR in this case to reverse gene expression at these loci. Additional analysis using a ΔvjbR mutant revealed that AHL also altered gene expression in the absence of VjbR, up-regulating expression of 48 genes and a luxR homologue blxR 93-fold at stationary growth phase. Gene expression alterations include previously un-described adhesins, proteases, antibiotic and toxin resistance genes, stress survival aids, transporters, membrane biogenesis genes, amino acid metabolism and transport, transcriptional regulators, energy production genes, and the previously reported fliF and virB operons.
VjbR and C12-HSL regulate expression of a large and diverse number of genes. Many genes identified as virulence factors in other bacterial pathogens were found to be differently expressed, suggesting an important contribution to intracellular survival of Brucella. From these data, we conclude that VjbR and C12-HSL contribute to virulence and survival by regulating expression of virulence mechanisms and thus controlling the ability of the bacteria to survive within the host cell. A likely scenario occurs via QS, however, operation of such a mechanism remains to be demonstrated.
PMCID: PMC2898763  PMID: 20529360
25.  Comparative Global Gene Expression Profiles of Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant at Flea and Human Body Temperatures 
Braun/murein lipoprotein (Lpp) is involved in inflammatory responses and septic shock. We previously characterized a Δlpp mutant of Yersinia pestis CO92 and found that this mutant was defective in surviving in macrophages and was attenuated in a mouse inhalation model of plague when compared to the highly virulent wild-type (WT) bacterium. We performed global transcriptional profiling of WT Y. pestis and its Δlpp mutant using microarrays. The organisms were cultured at 26 and 37 degrees Celsius to simulate the flea vector and mammalian host environments, respectively. Our data revealed vastly different effects of lpp mutation on the transcriptomes of Y. pestis grown at 37 versus 26°C. While the absence of Lpp resulted mainly in the downregulation of metabolic genes at 26°C, the Y. pestis Δlpp mutant cultured at 37°C exhibited profound alterations in stress response and virulence genes, compared to WT bacteria. We investigated one of the stress-related genes (htrA) downregulated in the Δlpp mutant relative to WT Y. pestis. Indeed, complementation of the Δlpp mutant with the htrA gene restored intracellular survival of the Y. pestis Δlpp mutant. Our results support a role for Lpp in Y. pestis adaptation to the host environment, possibly via transcriptional activation of htrA.
PMCID: PMC2873655  PMID: 20508723

Results 1-25 (44)