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1.  Decreased DNA Methylations at the Progesterone Receptor Promoter A Induce Functional Progesterone Withdrawal in Human Parturition 
Reproductive Sciences  2014;21(7):898-905.
The functional interaction of progesterone receptor (PR) isoforms PRA and PRB regulates myometrial transition from the resting state to excitation–contraction to initiate parturition. However, the regulatory mechanisms responsible for maintenance and functional alteration of the PRA and PRB expression levels during human pregnancy and term labor, respectively, remain unknown. Therefore, this study was designed to investigate whether and how epigenetic DNA modifications, specifically methylations, at the PRs’ promoter regions contribute to the differential expression of PRA and PRB in laboring term myometrium of humans. Comparative analysis of PRA and PRB messenger RNA (mRNA) expression levels and accompanying changes in their promoters’ methylation status was carried out using human myometrial samples from women undergoing singleton, term deliveries by cesarean section, either in the absence of labor (designated as NIL for not-in-labor) or in active labor (designated as IL for in labor). The PRA gene expression was shown to be elevated significantly during labor, while PRB gene expression was unaltered, and this differential expression was accompanied by decreased DNA methylation at the PRA promoter and not at the PRB promoter. In addition, labor-related decreased mRNA expression of the DNA methyltransferase (DNMT) family members DNMT1 and DNMT3a was found, however whether the increased expression of DNMTs directly supports the functional withdrawal of progesterone needs further investigation. Collectively, these data indicate that DNA methylation might represent an important epigenetic mechanism of labor-related differential expression of PRs, thereby mediating the biological process of functional PR withdrawal at term for parturition.
PMCID: PMC4107562  PMID: 24401475
progesterone receptors; myometrium; labor; DNA methylation; DNMT
2.  Disease Progression in Hemodynamically Stable Patients Presenting to the Emergency Department With Sepsis 
Aggressive diagnosis and treatment of patients presenting to the emergency department (ED) with septic shock has been shown to reduce mortality. To enhance the ability to intervene in patients with lesser illness severity, a better understanding of the natural history of the early progression from simple infection to more severe illness is needed.
The objectives were to 1) describe the clinical presentation of ED sepsis, including types of infection and causative microorganisms, and 2) determine the incidence, patient characteristics, and mortality associated with early progression to septic shock among ED patients with infection.
This was a multicenter study of adult ED patients with sepsis but no evidence of shock. Multivariable logistic regression was used to identify patient factors for early progression to shock and its association with 30-day mortality.
Of 472 patients not in shock at ED presentation (systolic blood pressure > 90 mm Hg and lactate < 4 mmol / L), 84 (17.8%) progressed to shock within 72 hours. Independent factors associated with early progression to shock included older age, female sex, hyperthermia, anemia, comorbid lung disease, and vascular access device infection. Early progression to shock (vs. no progression) was associated with higher 30-day mortality (13.1% vs. 3.1%, odds ratio [OR] = 4.72, 95% confidence interval [CI] = 2.01 to 11.1; p ≤ 0.001). Among 379 patients with uncomplicated sepsis (i.e., no evidence of shock or any end-organ dysfunction), 86 (22.7%) progressed to severe sepsis or shock within 72 hours of hospital admission.
A significant portion of ED patients with less severe sepsis progress to severe sepsis or shock within 72 hours. Additional diagnostic approaches are needed to risk stratify and more effectively treat ED patients with sepsis.
PMCID: PMC4283798  PMID: 20370777
sepsis; outcomes; septic shock; progression; biomarkers
3.  An integrated transcriptome and expressed variant analysis of sepsis survival and death 
Genome Medicine  2014;6(11):111.
Sepsis, a leading cause of morbidity and mortality, is not a homogeneous disease but rather a syndrome encompassing many heterogeneous pathophysiologies. Patient factors including genetics predispose to poor outcomes, though current clinical characterizations fail to identify those at greatest risk of progression and mortality.
The Community Acquired Pneumonia and Sepsis Outcome Diagnostic study enrolled 1,152 subjects with suspected sepsis. We sequenced peripheral blood RNA of 129 representative subjects with systemic inflammatory response syndrome (SIRS) or sepsis (SIRS due to infection), including 78 sepsis survivors and 28 sepsis non-survivors who had previously undergone plasma proteomic and metabolomic profiling. Gene expression differences were identified between sepsis survivors, sepsis non-survivors, and SIRS followed by gene enrichment pathway analysis. Expressed sequence variants were identified followed by testing for association with sepsis outcomes.
The expression of 338 genes differed between subjects with SIRS and those with sepsis, primarily reflecting immune activation in sepsis. Expression of 1,238 genes differed with sepsis outcome: non-survivors had lower expression of many immune function-related genes. Functional genetic variants associated with sepsis mortality were sought based on a common disease-rare variant hypothesis. VPS9D1, whose expression was increased in sepsis survivors, had a higher burden of missense variants in sepsis survivors. The presence of variants was associated with altered expression of 3,799 genes, primarily reflecting Golgi and endosome biology.
The activation of immune response-related genes seen in sepsis survivors was muted in sepsis non-survivors. The association of sepsis survival with a robust immune response and the presence of missense variants in VPS9D1 warrants replication and further functional studies.
Trial registration NCT00258869. Registered on 23 November 2005.
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-014-0111-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4274761  PMID: 25538794
4.  An integrated clinico-metabolomic model improves prediction of death in sepsis 
Science translational medicine  2013;5(195):195ra95.
Sepsis is a common cause of death, but outcomes in individual patients are difficult to predict. Elucidating the molecular processes that differ between sepsis patients who survive and those who die may permit more appropriate treatments to be deployed. We examined the clinical features, and the plasma metabolome and proteome of patients with and without community-acquired sepsis, upon their arrival at hospital emergency departments and 24 hours later. The metabolomes and proteomes of patients at hospital admittance who would die differed markedly from those who would survive. The different profiles of proteins and metabolites clustered into fatty acid transport and β-oxidation, gluconeogenesis and the citric acid cycle. They differed consistently among several sets of patients, and diverged more as death approached. In contrast, the metabolomes and proteomes of surviving patients with mild sepsis did not differ from survivors with severe sepsis or septic shock. An algorithm derived from clinical features together with measurements of seven metabolites predicted patient survival. This algorithm may help to guide the treatment of individual patients with sepsis.
PMCID: PMC3924586  PMID: 23884467
5.  Discriminative Value of Inflammatory Biomarkers for Suspected Sepsis 
Circulating biomarkers can facilitate sepsis diagnosis enabling early management and improved outcomes. Procalcitonin (PCT) has been suggested to have superior diagnostic utility compared to other biomarkers.
Adults with suspected sepsis in the Emergency Department were enrolled. PCT, CRP, and IL-6 were correlated with infection likelihood, sepsis severity, and septicemia. Multivariable models were constructed for length-of-stay and discharge to a higher level of care.
Of 336 enrolled subjects, 60% had definite infection, 13% possible infection and 27% no infection. Of those with infection, 202 presented with sepsis, 28 with severe sepsis, and 17 with septic shock. Overall, 21% of subjects were septicemic. PCT, IL6, and CRP levels were significantly higher in septicemia (median PCT 2.3 vs. 0.2ng/mL; IL-6 178 vs. 72pg/mL; CRP 106 vs. 62mg/dL, p<0.001). Biomarker concentrations increased with greater likelihood of infection and sepsis severity. Using ROC analysis, PCT best predicted septicemia (0.78 vs. IL-6 0.70 and CRP 0.67) but CRP better identified clinical infection (0.75 vs. PCT 0.71 and IL-6 0.69). A PCT cut-off of 0.5ng/mL had 72.6% sensitivity and 69.5% specificity for bacteremia as well as 40.7% sensitivity and 87.2% specificity for diagnosing infection. A combined clinical-biomarker model revealed that CRP was marginally associated with length-of-stay (p=0.015), but no biomarker independently predicted discharge to a higher level of care.
In adult Emergency Department patients with suspected sepsis, PCT, IL-6, and CRP highly correlate with several infection parameters, but do not meaningfully predict length-of-stay or need for discharge to a higher level of care.
PMCID: PMC3740117  PMID: 22056545
Sepsis; Procalcitonin; Interleukin-6; C-Reactive Protein; Sensitivity and Specificity
6.  Hepatitis B Virus Induces IL-23 Production in Antigen Presenting Cells and Causes Liver Damage via the IL-23/IL-17 Axis 
PLoS Pathogens  2013;9(6):e1003410.
IL-23 regulates myriad processes in the innate and adaptive immune systems, and is a critical mediator of the proinflammatory effects exerted by Th17 cells in many diseases. In this study, we investigated whether and how hepatitis B virus (HBV) causes liver damage directly through the IL-23 signaling pathway. In biopsied liver tissues from HBV-infected patients, expression of both IL-23 and IL-23R was remarkably elevated. In vivo observations also indicated that the main sources of IL-23 were myeloid dendritic cells (mDCs) and macrophages. Analysis of in vitro differentiated immature DCs and macrophages isolated from healthy donors revealed that the HBV surface antigen (HBsAg) efficiently induces IL-23 secretion in a mannose receptor (MR)-dependent manner. Culture with an endosomal acidification inhibitor and the dynamin inhibitor showed that, upon binding to the MR, the HBsAg is taken up by mDCs and macrophages through an endocytosis mechanism. In contrast, although the HBV core antigen (HBcAg) can also stimulate IL-23 secretion from mDCs, the process was MR- and endocytosis-independent. In addition, IL-23 was shown to be indispensible for HBsAg-stimulated differentiation of naïve CD4+ T cells into Th17 cells, which were determined to be the primary source of IL-17 in HBV-infected livers. The cognate receptor, IL-17R, was found to exist on the hepatic stellate cells and mDCs, both of which might represent the potential target cells of IL-17 in hepatitis B disease. These data provide novel insights into a yet unrecognized mechanism of HBV-induced hepatitis, by which increases in IL-23 expression, through an MR/endocytosis-dependent or -independent manner, produce liver damage through the IL-23/IL-17 axis.
Author Summary
While it is known that IL-23 plays a pivotal role in maintenance of the Th17 phenotype and their production of the IL-17 cytokine, the mechanisms by which HBV induces particular immune cell types to produce IL-23 remain unknown. In the study of human hepatitis B described herein, we demonstrated that IL-23 is principally derived from the liver myeloid dendritic cells (mDCs) and macrophages. In vitro assay showed that mDCs produce large amounts of IL-23 upon stimulation with HBV surface antigen (HBsAg) through the mannose receptor (MR) and an endocytosis mechanism. In contrast, although the HBV core antigen (HBcAg) was also capable of stimulating IL-23 secretion from mDCs, the process occurs in an MR- and endocytosis-independent manner. IL-23 was also shown to efficiently stimulate the differentiation of naïve CD4+ T cells into Th17 cells in the presence of HBsAg or HBcAg; furthermore, the Th17 cells were shown to be the primary source of IL-17. The results also indicated that both hepatic satellite cells and mDCs might be the potential target cells of IL-17 in hepatitis B disease. Therefore, our study not only provides further insights into the mechanisms underlying HBV pathogenesis, but also suggests the potential intervening targets for patient treatment.
PMCID: PMC3694858  PMID: 23825942
7.  Gene Expression-Based Classifiers Identify Staphylococcus aureus Infection in Mice and Humans 
PLoS ONE  2013;8(1):e48979.
Staphylococcus aureus causes a spectrum of human infection. Diagnostic delays and uncertainty lead to treatment delays and inappropriate antibiotic use. A growing literature suggests the host’s inflammatory response to the pathogen represents a potential tool to improve upon current diagnostics. The hypothesis of this study is that the host responds differently to S. aureus than to E. coli infection in a quantifiable way, providing a new diagnostic avenue. This study uses Bayesian sparse factor modeling and penalized binary regression to define peripheral blood gene-expression classifiers of murine and human S. aureus infection. The murine-derived classifier distinguished S. aureus infection from healthy controls and Escherichia coli-infected mice across a range of conditions (mouse and bacterial strain, time post infection) and was validated in outbred mice (AUC>0.97). A S. aureus classifier derived from a cohort of 94 human subjects distinguished S. aureus blood stream infection (BSI) from healthy subjects (AUC 0.99) and E. coli BSI (AUC 0.84). Murine and human responses to S. aureus infection share common biological pathways, allowing the murine model to classify S. aureus BSI in humans (AUC 0.84). Both murine and human S. aureus classifiers were validated in an independent human cohort (AUC 0.95 and 0.92, respectively). The approach described here lends insight into the conserved and disparate pathways utilized by mice and humans in response to these infections. Furthermore, this study advances our understanding of S. aureus infection; the host response to it; and identifies new diagnostic and therapeutic avenues.
PMCID: PMC3541361  PMID: 23326304
8.  Global Mapping of H3K4me1 and H3K4me3 Reveals the Chromatin State-Based Cell Type-Specific Gene Regulation in Human Treg Cells 
PLoS ONE  2011;6(11):e27770.
Regulatory T cells (Treg) contribute to the crucial immunological processes of self-tolerance and immune homeostasis. Genomic mechanisms that regulate cell fate decisions leading to Treg or conventional T cells (Tconv) lineages and those underlying Treg function remain to be fully elucidated, especially at the histone modification level. We generated high-resolution genome-wide distribution maps of monomethylated histone H3 lysine 4 (H3K4me1) and trimethylated H3K4 (H3K4me3) in human CD4+CD25+FOXP3+ Tregs and CD4+CD25+FOXP3− activated (a)Tconv cells by DNA sequencing-by-synthesis. 2115 H3K4me3 regions corresponded to proximal promoters; in Tregs, the genes associated with these regions included the master regulator FOXP3 and the chemokine (C-C motif) receptor 7 (CCR7). 41024 Treg-specific H3K4me1 regions were identified. The majority of the H3K4me1 regions differing between Treg and aTconv cells were located at promoter-distal sites, and in vitro reporter gene assays were used to evaluate and identify novel enhancer activity. We provide for the first time a comprehensive genome-wide dataset of lineage-specific H3K4me1 and H3K4me3 patterns in Treg and aTconv cells, which may control cell type-specific gene regulation. This basic principle is likely not restricted to the two closely-related T cell populations, but may apply generally to somatic cell lineages in adult organisms.
PMCID: PMC3223197  PMID: 22132139
9.  Genome, epigenome and RNA sequences of monozygotic twins discordant for multiple sclerosis 
Nature  2010;464(7293):1351-1356.
Monozygotic (MZ) or “identical” twins have been widely studied to dissect the relative contributions of genetics and environment in human diseases. In multiple sclerosis (MS), an autoimmune demyelinating disease and common cause of neurodegeneration and disability in young adults, disease discordance in MZ twins has been interpreted to indicate environmental importance in its pathogenesis1–8. However, genetic and epigenetic differences between MZ twins have been described, challenging the accepted experimental paradigm in disambiguating effects of nature and nurture.9–12 Here, we report the genome sequences of one MS-discordant MZ twin pair and messenger RNA (mRNA) transcriptome and epigenome sequences of CD4+ lymphocytes from three MS-discordant, MZ twin pairs. No reproducible differences were detected between co-twins among ~3.6 million single nucleotide polymorphisms (SNPs) or ~0.2 million insertion-deletion polymorphisms (indels). Nor were any reproducible differences observed between siblings of the three twin pairs in HLA haplotypes, confirmed MS-susceptibility SNPs, copy number variations, mRNA and genomic SNP and indel genotypes, or expression of ~19,000 genes in CD4+ T cells. Only two to 176 differences in methylation of ~2 million CpG dinucleotides were detected between siblings of the three twin pairs, in contrast to ~800 methylation differences between T cells of unrelated individuals and several thousand differences between tissues or normal and cancerous tissues. In the first systematic effort to estimate sequence variation among MZ co-twins, we did not find evidence for genetic, epigenetic or transcriptome differences that explained disease discordance. These are the first female, twin and autoimmune disease individual genome sequences reported.
PMCID: PMC2862593  PMID: 20428171
10.  Multiplex PCR To Diagnose Bloodstream Infections in Patients Admitted from the Emergency Department with Sepsis ▿  
Sepsis is caused by a heterogeneous group of infectious etiologies. Early diagnosis and the provision of appropriate antimicrobial therapy correlate with positive clinical outcomes. Current microbiological techniques are limited in their diagnostic capacities and timeliness. Multiplex PCR has the potential to rapidly identify bloodstream infections and fill this diagnostic gap. We identified patients from two large academic hospital emergency departments with suspected sepsis. The results of a multiplex PCR that could detect 25 bacterial and fungal pathogens were compared to those of blood culture. The results were analyzed with respect to the likelihood of infection, sepsis severity, the site of infection, and the effect of prior antibiotic therapy. We enrolled 306 subjects with suspected sepsis. Of these, 43 were later determined not to have infectious etiologies. Of the remaining 263 subjects, 70% had sepsis, 16% had severe sepsis, and 14% had septic shock. The majority had a definite infection (41.5%) or a probable infection (30.7%). Blood culture and PCR performed similarly with samples from patients with clinically defined infections (areas under the receiver operating characteristic curves, 0.64 and 0.60, respectively). However, blood culture identified more cases of septicemia than PCR among patients with an identified infectious etiology (66 and 46, respectively; P = 0.0004). The two tests performed similarly when the results were stratified by sepsis severity or infection site. Blood culture tended to detect infections more frequently among patients who had previously received antibiotics (P = 0.06). Conversely, PCR identified an additional 24 organisms that blood culture failed to detect. Real-time multiplex PCR has the potential to serve as an adjunct to conventional blood culture, adding diagnostic yield and shortening the time to pathogen identification.
PMCID: PMC2812289  PMID: 19846634
11.  Genomic Convergence Analysis of Schizophrenia: mRNA Sequencing Reveals Altered Synaptic Vesicular Transport in Post-Mortem Cerebellum 
PLoS ONE  2008;3(11):e3625.
Schizophrenia (SCZ) is a common, disabling mental illness with high heritability but complex, poorly understood genetic etiology. As the first phase of a genomic convergence analysis of SCZ, we generated 16.7 billion nucleotides of short read, shotgun sequences of cDNA from post-mortem cerebellar cortices of 14 patients and six, matched controls. A rigorous analysis pipeline was developed for analysis of digital gene expression studies. Sequences aligned to approximately 33,200 transcripts in each sample, with average coverage of 450 reads per gene. Following adjustments for confounding clinical, sample and experimental sources of variation, 215 genes differed significantly in expression between cases and controls. Golgi apparatus, vesicular transport, membrane association, Zinc binding and regulation of transcription were over-represented among differentially expressed genes. Twenty three genes with altered expression and involvement in presynaptic vesicular transport, Golgi function and GABAergic neurotransmission define a unifying molecular hypothesis for dysfunction in cerebellar cortex in SCZ.
PMCID: PMC2576459  PMID: 18985160
12.  Salmonella enterica Serovar Typhimurium Resistance to Bile: Identification and Characterization of the tolQRA Cluster 
Journal of Bacteriology  2002;184(5):1270-1276.
Salmonella enterica serovar Typhimurium is resistant to the action of bile salts, and resistance to bile is enhanced in strains in which the PhoP-PhoQ (PhoPQ) two-component regulatory system has been activated. To identify genes necessary for bile resistance, MudJ transposon mutagenesis was performed on a strain containing a phoP mutation that results in constitutive expression of PhoP-activated genes. After screening >10,000 mutants for the loss of growth on Luria-Bertani broth-bile plates, 14 bile-sensitive mutants were identified. Of these 14 mutants, 3 were found to retain the bile sensitivity phenotype upon P22 transduction, to possess wild-type growth characteristics, and to contain a smooth lipopolysaccharide. Southern hybridization experiments showed that all three strains contained unique insertions. DNA sequencing of the transposon-chromosomal-DNA fusion junctions of these strains showed all to be linked to the putative Salmonella orf1-tolQRA operon, with insertions in tolQ, orf1, and a gene upstream of the orf1-tolQRA operon not previously associated with Tol function (orfX). Through the use of transcriptional fusions, none of the putative tol (or tol-associated) genes were shown to be regulated by PhoPQ, bile, or the RcsC-RcsB two-component system; however, all of the genes (orfX, orf1, tolQRA) are predicted to be cotranscribed. This is the first identification of Salmonella serovar Typhimurium Tol homologs and the first demonstration of their role in bile resistance in this organism. In addition, the observed regulation, operon arrangement, and phenotypes associated with these tol genes demonstrate significant differences from their Escherichia coli homologs.
PMCID: PMC134864  PMID: 11844755
13.  Genetic and Functional Analysis of a PmrA-PmrB-Regulated Locus Necessary for Lipopolysaccharide Modification, Antimicrobial Peptide Resistance, and Oral Virulence of Salmonella enterica Serovar Typhimurium 
Infection and Immunity  2000;68(11):6139-6146.
The two-component regulatory system PmrA-PmrB confers resistance of Salmonella spp. to cationic antimicrobial peptides (AP) such as polymyxin (PM), bactericidal/permeability-increasing protein, and azurocidin. This resistance occurs by transcriptional activation of two loci termed pmrE and pmrHFIJKLM. Both pmrE and pmrHFIJKLM produce products required for the biosynthesis of lipid A with 4-aminoarabinose (Ara4N). Ara4N addition creates a more positively charged lipopolysaccharide (LPS) and thus reduces cationic AP binding. Experiments were conducted to further analyze the regulation of the pmrHFIJKLM operon and the role of this operon and the surrounding genomic region in LPS modification and antimicrobial peptide resistance. The pmrHFIJKLM genes are cotranscribed and over 3,000-fold regulated by PmrA-PmrB. The pmrHFIJKLM promoter bound PmrA, as determined by gel shift analysis, as did a 40-bp region of the PmrA-PmrB-regulated pmrCAB promoter. Construction of nonpolar mutations in the pmrHFIJKLM genes showed that all except pmrM were necessary for the Ara4N addition to lipid A and PM resistance. The flanking genes of the operon (pmrG and pmrD) were not necessary for PM resistance, but pmrD was shown to be regulated by the PhoP-PhoQ regulatory system. BALB/c mice inoculated with pmrA and pmrHFIJKLM mutant strains demonstrated virulence attenuation when the strains were administered orally but not when they were administered intraperitoneally, indicating that Ara4N addition may be important for resistance to host innate defenses within intestinal tissues.
PMCID: PMC97691  PMID: 11035717
14.  PhoP-PhoQ-Regulated Loci Are Required for Enhanced Bile Resistance in Salmonella spp. 
Infection and Immunity  1999;67(4):1614-1622.
As enteric pathogens, Salmonella spp. are resistant to the actions of bile. Salmonella typhimurium and Salmonella typhi strains were examined to better define the bile resistance phenotype. The MICs of bile for wild-type S. typhimurium and S. typhi were 18 and 12%, respectively, and pretreatment of log-phase S. typhimurium with 15% bile dramatically increased bile resistance. Mutant strains of S. typhimurium and S. typhi lacking the virulence regulator PhoP-PhoQ were killed at significantly lower bile concentrations than wild-type strains, while strains with constitutively active PhoP were able to survive prolonged incubation with bile at concentrations of >60%. PhoP-PhoQ was shown to mediate resistance specifically to the bile components deoxycholate and conjugated forms of chenodeoxycholate, and the protective effect was not generalized to other membrane-active agents. Growth of both S. typhimurium and S. typhi in bile and in deoxycholate resulted in the induction or repression of a number of proteins, many of which appeared identical to PhoP-PhoQ-activated or -repressed products. The PhoP-PhoQ regulon was not induced by bile, nor did any of the 21 PhoP-activated or -repressed genes tested play a role in bile resistance. However, of the PhoP-activated or -repressed genes tested, two (prgC and prgH) were transcriptionally repressed by bile in the medium independent of PhoP-PhoQ. These data suggest that salmonellae can sense and respond to bile to increase resistance and that this response likely includes proteins that are members of the PhoP regulon. These bile- and PhoP-PhoQ-regulated products may play an important role in the survival of Salmonella spp. in the intestine or gallbladder.
PMCID: PMC96504  PMID: 10084994

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