Supplementary Tables 1-4 from A Transgenic Mouse Model of Plasma Cell Malignancy Shows Phenotypic, Cytogenetic, and Gene Expression Heterogeneity Similar to Human Multiple Myeloma

Supplementary Figure 1 from A Transgenic Mouse Model of Plasma Cell Malignancy Shows Phenotypic, Cytogenetic, and Gene Expression Heterogeneity Similar to Human Multiple Myeloma

As both a commensal and a major cause of healthcare-associated infections in humans, Enterococcus faecalis is a remarkably adaptable organism. We investigated how E. faecalis adapts in a mammalian host as a pathogen by characterizing changes in the transcriptome during infection in a rabbit model of subdermal abscess formation using transcriptional microarrays. The microarray experiments detected 222 and 291 differentially regulated genes in E. faecalis OG1RF at two and eight hours after subdermal chamber inoculation, respectively. The profile of significantly regulated genes at two hours post-inoculation included genes involved in stress response, metabolism, nutrient acquisition, and cell surface components, suggesting genome-wide adaptation to growth in an altered environment. At eight hours post-inoculation, 88% of the differentially expressed genes were down-regulated and matched a transcriptional profile consistent with a (p) ppGpp-mediated stringent response. Subsequent subdermal abscess infections with E. faecalis mutants lacking the (p) ppGpp synthetase/hydrolase RSH, the small synthetase RelQ, or both enzymes, suggest that intracellular (p) ppGpp levels, but not stringent response activation, influence persistence in the model. The ability of cells to synthesize (p) ppGpp was also found to be important for growth in human serum and whole blood. The data presented in this report provide the first genome-wide insights on E. faecalis in vivo gene expression and regulation measured by transcriptional profiling during infection in a mammalian host and show that (p) ppGpp levels affect viability of E. faecalis in multiple conditions relevant to mammalian infection. The subdermal abscess model can serve as a novel experimental system for studying the E. faecalis stringent response in the context of the mammalian immune system.

Introduction Continuous-flow left ventricular assist devices (LVADs) are an established therapy for patients with end-stage heart failure. The short- and long-term impact of these devices on peripheral blood gene expression has not been characterized, and may provide insight into the molecular pathways mediated in response to left ventricular remodeling and an improvement in overall systemic circulation. We performed RNA sequencing to identify genes and pathways influenced by these devices. Methods RNA was extracted from blood of 9 heart failure patients (8 male) prior to LVAD implantation, and at 7 and 180 days postoperatively. Libraries were sequenced on an Illumina HiSeq2000 and sequences mapped to the human Ensembl GRCh37.67 genome assembly. Results A specific set of genes involved in regulating cellular immune response, antigen presentation, and T cell activation and survival were down-regulated 7 days after LVAD placement. 6 months following LVAD placement, the expression levels of these genes were significantly increased; yet importantly, remained significantly lower than age and sex-matched samples from healthy controls. Conclusions In summary, this genomic analysis identified a significant decrease in the expression of genes that promote a healthy immune response in patients with heart failure that was partially restored 6 months following LVAD implant.

Purpose The overall success of modern left ventricular assist devices (LVADs) has been mitigated by the complication of gastrointestinal (GI) bleeding. While evidence suggests that anticoagulation and acquired von Willebrand factor deficiency contribute, these two factors do not fully explain all bleeding events. Methods and Materials To identify new targets contributing to bleeding risk in patients with mechanical support we performed whole blood RNA sequencing on 9 patients before LVAD placement, then 7 days and 180 days postoperatively. A paired t-test and false discovery rate method were used to identify statistically significant changes in expression. Transcripts unique to patients with GI bleeding events were identified by comparing the transcriptome of those with GI bleeding (n = 3) to those without bleeding (n = 6) 7 days after LVAD placement. Results Bleeding events occurred between 12 and 202 days after LVAD implant (median 18 days). Glutaredoxin (GLRX) and glutaredoxin 2 (GLRX2) decreased in patients with bleeding 7 days after LVAD implant, but increased in those without bleeding events at 7 days (p = 0.0004 and p = 0.004, respectively). Furthermore, a family of RAB genes were differentially expressed in patients with bleeding compared to non-bleeders 7 days following LVAD implant. These genes include RAB18, RAB1A, RAB21, RAB11A, and RABEP1. Other genes of interest that were significantly and differentially expressed in bleeders versus non-bleeders included transmembrane protein 54 (TMEM54) and family with sequence similarity 26, member F (FAM26F). Conclusions We have identified novel genes that are differentially expressed in LVAD patients who develop GI bleeding. These findings provide an initial step towards uncovering mechanisms that may affect GI bleeding in patients with LVADs.

ABSTRACT Enterococcus faecalis is a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades. E. faecalis must be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-based in vivo expression technology (RIVET) to identify promoters on the E. faecalis OG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putative in vivo -activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected in in vitro - and in vivo -grown cells, providing the first evidence of in vivo -expressed antisense RNAs in E. faecalis . Deletions in the in vivo -activated genes that encode glutamate 5-kinase ( proB [EF0038]), the transcriptional regulator EbrA ( ebrA [EF1809]), and the membrane metalloprotease Eep ( eep [EF2380]) did not hinder biofilm formation in in vitro assays. In a rabbit model of endocarditis, the Δ ebrA strain was fully virulent, the Δ proB strain was slightly attenuated, and the Δ eep strain was severely attenuated. The Δ eep virulence defect could be complemented by the expression of the wild-type gene in trans . Microscopic analysis of early Δ eep biofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.

Sprouty1 (Spry1) is a conserved antagonist of FGF signaling. The goal of this study was to further explore the downstream mechanisms governing Spry1 inhibition of endothelial cell proliferation. Up-regulation of Spry1 in HUVECs inhibited tube formation on Matrigel (n = 6, P < 0.001). This was associated with decreased proliferation as measured by BrdU incorporation (n = 6, P < 0.001) and increased protein expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A), p21 and cyclin-dependent kinase inhibitor 1B (CDKN1B), p27. A transcriptional analysis using a targeted human angiogenesis array following up-regulation of Spry1 demonstrated a >2-fold increase in an anti-angiogenic factor, serpin peptidase inhibitor, clad F (Serpinf1), and a >2-fold decrease in pro-angiogenic factors fms-related tyrosine kinase 1 (FLT1), angiopoietin2 (Ang-2), and placental growth factor (PGF) (n = 2). To define upstream mechanisms that may regulate endogenous Spry1, we performed a search for responsive elements upstream of the promoter region. This search resulted in the identification of multiple degenerate hypoxia responsive elements. Exposure to hypoxia resulted in a significant increase in Spry1 expression (n = 8, P < 0.01). These findings shed new light on downstream signaling pathways associated with Spry1 anti-proliferative responses, and provide new evidence that hypoxia stimulates Spry1 expression.

ABSTRACT The ability of Enterococcus faecalis to form robust biofilms on host tissues and on abiotic surfaces such as catheters likely plays a major role in the pathogenesis of opportunistic antibiotic-resistant E. faecalis infections and in the transfer of antibiotic resistance genes. We have carried out a comprehensive analysis of genetic determinants of biofilm formation in the core genome of E. faecalis. Here we describe 68 genetic loci predicted to be involved in biofilm formation that were identified by recombinase in vivo expression technology (RIVET); most of these genes have not been studied previously. Differential expression of a number of these determinants during biofilm growth was confirmed by quantitative reverse transcription-PCR, and genetic complementation studies verified a role in biofilm formation for several candidate genes. Of particular interest was genetic locus EF1809, predicted to encode a regulatory protein of the GntR family. We isolated 14 independent nonsibling clones containing the putative promoter region for this gene in the RIVET screen; EF1809 also showed the largest increase in expression during biofilm growth of any of the genes tested. Since an in-frame deletion of EF1809 resulted in a severe biofilm defect that could be complemented by the cloned wild-type gene, we have designated EF1809 ebrA (enterococcal biofilm regulator). Most of the novel genetic loci identified in our studies are highly conserved in gram-positive bacterial pathogens and may thus constitute a pool of uncharacterized genes involved in biofilm formation that may be useful targets for drug discovery.

Background— We report the first comprehensive analysis of gene expression differences by sex and age in left ventricular samples from 102 patients with dilated cardiomyopathy. Methods and Results— Gene expression data (HG-U133A gene chip, Affymetrix) were analyzed from 30 females and 72 males from 3 separate centers. More than 1800 genes displayed sexual dimorphism in the heart (adjusted P value <0.05). A significant number of these genes were highly represented in gene ontology pathways involved in ion transport and G-protein-coupled receptor signaling. Localization of these genes revealed enrichment on both the sex chromosomes as well as chromosomes 3, 4, and 14. The second goal of this study was to determine the effect of age on gene expression. Within the female cohort, >140 genes were differentially expressed in the <55 years age group compared with the >55 years age group. These genes were highly represented in gene ontology pathways involved in DNA damage. In contrast, zero genes in the male cohort <55 years met statistical significance when compared with the >55 years age group. Conclusions— Gene expression in dilated cardiomyopathy displayed evidence of sexual dimorphism similar to other somatic tissues and age dimorphism within the female cohort.

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, most of the genetic analysis of these organisms has focused on mobile genetic elements, and existing tools for manipulation and analysis of the core E. faecalis chromosome are limited. We are interested in a comprehensive analysis of the genetic determinants for biofilm formation encoded within the core E. faecalis genome. To identify such determinants, we developed a substantially improved system for transposon mutagenesis in E. faecalis based on a mini-mariner transposable element. Mutagenesis of wild-type E. faecalis with this element yielded predominantly mutants carrying a single copy of the transposable element, and insertions were distributed around the entire chromosome in an apparently random fashion. We constructed a library of E. faecalis transposon insertion mutants and screened this library to identify mutants exhibiting a defect in biofilm formation. Biofilm-defective mutants were found to carry transposon insertions both in genes that were previously known to play a role in biofilm formation and in new genes lacking any known function; for several genes identified in the screen, complementation analysis confirmed a direct role in biofilm formation. These results provide significant new information about the genetics of enterococcal biofilm formation and demonstrate the general utility of our transposon system for functional genomic analysis of E. faecalis.

Background Anaplasma phagocytophilum ( Ap ) is an obligate intracellular bacterium and the agent of human granulocytic anaplasmosis, an emerging tick-borne disease. Ap alternately infects ticks and mammals and a variety of cell types within each. Understanding the biology behind such versatile cellular parasitism may be derived through the use of tiling microarrays to establish high resolution, genome-wide transcription profiles of the organism as it infects cell lines representative of its life cycle (tick; ISE6) and pathogenesis (human; HL-60 and HMEC-1). Results Detailed, host cell specific transcriptional behavior was revealed. There was extensive differential Ap gene transcription between the tick (ISE6) and the human (HL-60 and HMEC-1) cell lines, with far fewer differentially transcribed genes between the human cell lines, and all disproportionately represented by membrane or surface proteins. There were Ap genes exclusively transcribed in each cell line, apparent human- and tick-specific operons and paralogs, and anti-sense transcripts that suggest novel expression regulation processes. Seven virB2 paralogs (of the bacterial type IV secretion system) showed human or tick cell dependent transcription. Previously unrecognized genes and coding sequences were identified, as were the expressed p44/msp2 (major surface proteins) paralogs (of 114 total), through elevated signal produced to the unique hypervariable region of each – 2/114 in HL-60, 3/114 in HMEC-1, and none in ISE6. Conclusion Using these methods, whole genome transcription profiles can likely be generated for Ap , as well as other obligate intracellular organisms, in any host cells and for all stages of the cell infection process. Visual representation of comprehensive transcription data alongside an annotated map of the genome renders complex transcription into discernable patterns.

Adequate cell-based repair of adult myocardium remains an elusive goal because most cells that are used cannot generate mature myocardium sufficient to promote large functional improvements. Embryonic stem cells can generate both mature cardiocytes and vasculature, but their use is hampered by associated teratoma formation and the need for an allogeneic source. The detection of sca-1+, c-kit+, or isl-1+ cardiac precursors and the creation of cardiospheres from adult heart tissues suggest that a persistent population of immature progenitor cells is present in the mature myocardium. These cell populations probably represent stages along a continuum of cardiac stem cell development and differentiation. We report isolation from ventricle of uncommitted cardiac progenitor cells, which appear to resemble the more immature, common pool of embryonic lateral plate mesoderm progenitors that yield both myocardial and endocardial cells during normal cardiac development. Under controlled in vitro conditions and in vivo, these cells can differentiate into endothelial, smooth muscle, and cardiomyocyte lineages and can be isolated and expanded to clinically relevant numbers from adult rat myocardial tissue. In this article, we discuss the potential for autologous repair or even cardiac regeneration with cells that follow a developmental pathway similar to embryonic cardiac precursors but without the inherent limitations associated with undifferentiated embryonic stem cells.

Background: The 10.9x genomic sequence of Candida albicans, the most important human fungal pathogen, was published in 2004. Assembly 19 consisted of 412 supercontigs, of which 266 were a haploid set, since this fungus is diploid and contains an extensive degree of heterozygosity but lacks a complete sexual cycle. However, sequences of specific chromosomes were not determined.Results: Supercontigs from Assembly 19 ( 183, representing 98.4% of the sequence) were assigned to individual chromosomes purified by pulse-field gel electrophoresis and hybridized to DNA microarrays. Nine Assembly 19 supercontigs were found to contain markers from two different chromosomes. Assembly 21 contains the sequence of each of the eight chromosomes and was determined using a synteny analysis with preliminary versions of the Candida dubliniensis genome assembly, bioinformatics, a sequence tagged site ( STS) map of overlapping fosmid clones, and an optical map. The orientation and order of the contigs on each chromosome, repeat regions too large to be covered by a sequence run, such as the ribosomal DNA cluster and the major repeat sequence, and telomere placement were determined using the STS map. Sequence gaps were closed by PCR and sequencing of the products. The overall assembly was compared to an optical map; this identified some misassembled contigs and gave a size estimate for each chromosome.Conclusion: Assembly 21 reveals an ancient chromosome fusion, a number of small internal duplications followed by inversions, and a subtelomeric arrangement, including a new gene family, the TLO genes. Correlations of position with relatedness of gene families imply a novel method of dispersion. The sequence of the individual chromosomes of C. albicans raises interesting biological questions about gene family creation and dispersion, subtelomere organization, and chromosome evolution.

While there are certain common clinical features in myeloma, the disease shows significant heterogeneity with regard to disease progression, and responses to therapy, affecting both survival and toxicities. Heritable variations in a wide variety of genes and pathways affecting cellular functions and drug responses likely impact patient outcomes. In the Bank On A Cure (BOAC) program we have developed a custom chip that assesses 3,404 SNPs representing variations in cellular functions and pathways that may be involved in myeloma progression and response. The chip has gone through rigorous quality controls checks for high call rates, accuracy, and reproducibility that will be presented. Using the BOAC chip, we have conducted studies to look for SNPs that may identify biologic variations that are associated with good or poor response across a variety of treatments. In this study we looked for SNPs that may distinguish short term and long term survivors in two phase III clinical trials: ECOG E9486 and intergroup trial S9321. E9487 patients were treated with VBMCP followed by randomization to no further treatment, IFN-alpha, or cylcophosphamide; and, although there was variation in survival, no significant differences in survival were noted among the 3 arms of the trial. Patients included in this SNP study from S9321 received VAD induction followed by randomization to VBMCP or high dose melphalan + TBI. SNP profiles were obtained for patients with less than 1 year EFS (n=20 in E9487; n=50 in S9321) and patients showing greater than 3 years EFS (n=32 in E9486; n=41 in S9321). Statistical approaches were performed to identify single and groups of SNPs that best discriminated the survival groups. Previous studies have suggested genetic variations in drug metabolism genes, p-glycoprotein transport, and DNA repair genes may influence survival outcomes. Our results show significant survival associations of genetic variations in genes within these functional categories (eg. GST, XRCC, ABCB, and CYP genes). Although genetic variations were found that were uniquely associated with each clinical trial, several of these genetic variations show survival associations that increase in significance when the two trials were examined as a conglomerate data set. Grouping genetic variations through common pathway approaches using gene set enrichment analysis, as well as clustering or partitioning algorithms, further improve the value of the SNPs as potential prognostic markers of survival outcomes. These results and statistical approaches will be presented, and represent steps toward identifying patient variations in biologic mechanisms important in predicting therapeutic outcomes.

Background— Combination therapy consisting of mechanical unloading using a left ventricular assist device (LVAD) and pharmacological intervention can promote recovery from end-stage heart failure, but the mechanism is unknown. Preliminary microarray analysis revealed a significant and unexpected decrease in myocardial arginine:glycine amidinotransferase (AGAT) gene expression during recovery in these patients. The aim of this study was to evaluate the expression and role of AGAT expression in heart failure and recovery. Methods and Results— We used quantitative real time (TaqMan) polymerase chain reaction to examine myocardial AGAT mRNA expression in implant and explant samples from recovering patients after combination therapy (n=12), end-stage heart failure (ESHF) samples from stable patients undergoing transplantation without LVAD support (n=10), and donor hearts with normal hemodynamic function (n=8). AGAT mRNA expression was significantly elevated in all heart failure patients relative to donors (4.3-fold [ P <0.001] and 2.7-fold [ P <0.005] in LVAD and ESHF relative to donors, respectively) and returned to normal levels after recovery. AGAT enzyme activity was detectable in both human and rat myocardia and was elevated in heart failure. Conclusions— Our data highlight local and potentially regulated expression of AGAT activity in the myocardium and suggest a specific response to heart failure involving elevated local creatine synthesis. These findings have implications both for the management of recovery patients undergoing combination therapy and for heart failure in general.