MicroRNA signature of central nervous system-infiltrating dendritic cells in an animal model of multiple sclerosis.

Innate immune cells are integral to the pathogenesis of several diseases of the central nervous system (CNS), including multiple sclerosis (MS). Dendritic cells (DCs) are potent CD11c+ antigen-presenting cells that are critical regulators of adaptive immune responses, particularly in autoimmune diseases such as MS. The regulation of DC function in both the periphery and CNS compartment has not been fully elucidated. One limitation to studying the role of CD11c+ DCs in the CNS is that microglia can upregulate CD11c during inflammation, making it challenging to distinguish bone marrow-derived DCs (BMDCs) from microglia. Selective expression of microRNAs (miRNAs) has been shown to distinguish populations of innate cells and regulate their function within the CNS during neuro-inflammation. Using the experimental autoimmune encephalomyelitis (EAE) murine model of MS, we characterized the expression of miRNAs in CD11c+ cells using a non-biased murine array. Several miRNAs, including miR-31, were enriched in CD11c+ cells within the CNS during EAE, but not LysM+ microglia. Moreover, to distinguish CD11c+ DCs from microglia that upregulate CD11c, we generated bone marrow chimeras and found that miR-31 expression was specific to BMDCs. Interestingly, miR-31-binding sites were enriched in mRNAs downregulated in BMDCs that migrated into the CNS, and a subset was confirmed to be regulated by miR-31. Finally, miR-31 was elevated in DCs migrating through an in vitro blood-brain barrier. Our findings suggest miRNAs, including miR-31, may regulate entry of DCs into the CNS during EAE, and could potentially represent therapeutic targets for CNS autoimmune diseases such as MS.

Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes.

The molecular clock maintains energy constancy by producing circadian oscillations of rate-limiting enzymes involved in tissue metabolism across the day and night. During periods of feeding, pancreatic islets secrete insulin to maintain glucose homeostasis, and although rhythmic control of insulin release is recognized to be dysregulated in humans with diabetes, it is not known how the circadian clock may affect this process. Here we show that pancreatic islets possess self-sustained circadian gene and protein oscillations of the transcription factors CLOCK and BMAL1. The phase of oscillation of islet genes involved in growth, glucose metabolism and insulin signalling is delayed in circadian mutant mice, and both Clock and Bmal1 (also called Arntl) mutants show impaired glucose tolerance, reduced insulin secretion and defects in size and proliferation of pancreatic islets that worsen with age. Clock disruption leads to transcriptome-wide alterations in the expression of islet genes involved in growth, survival and synaptic vesicle assembly. Notably, conditional ablation of the pancreatic clock causes diabetes mellitus due to defective beta-cell function at the very latest stage of stimulus-secretion coupling. These results demonstrate a role for the beta-cell clock in coordinating insulin secretion with the sleep-wake cycle, and reveal that ablation of the pancreatic clock can trigger the onset of diabetes mellitus.

Gene expression profiles in febrile children with defined viral and bacterial infection.

Viral infections are common causes of fever without an apparent source in young children. Despite absence of bacterial infection, many febrile children are treated with antibiotics. Virus and bacteria interact with different pattern recognition receptors in circulating blood leukocytes, triggering specific host transcriptional programs mediating immune response. Therefore, unique transcriptional signatures may be defined that discriminate viral from bacterial causes of fever without an apparent source. Gene expression microarray analyses were conducted on blood samples from 30 febrile children positive for adenovirus, human herpesvirus 6, or enterovirus infection or with acute bacterial infection and 22 afebrile controls. Blood leukocyte transcriptional profiles clearly distinguished virus-positive febrile children from both virus-negative afebrile controls and afebrile children with the same viruses present in the febrile children. Virus-specific gene expression profiles could be defined. The IFN signaling pathway was uniquely activated in febrile children with viral infection, whereas the integrin signaling pathway was uniquely activated in children with bacterial infection. Transcriptional profiles classified febrile children with viral or bacterial infection with better accuracy than white blood cell count in the blood. Similarly accurate classification was shown with data from an independent study using different microarray platforms. Our results support the paradigm of using host response to define the etiology of childhood infections. This approach could be an important supplement to highly sensitive tests that detect the presence of a possible pathogen but do not address its pathogenic role in the patient being evaluated.

Multi-platform assessment of transcriptional profiling technologies utilizing a precise probe mapping methodology.

BACKGROUND: The arrival of RNA-seq as a high-throughput method competitive to the established microarray technologies has necessarily driven a need for comparative evaluation. To date, cross-platform comparisons of these technologies have been relatively few in number of platforms analyzed and were typically gene name annotation oriented. Here, we present a more extensive and yet precise assessment to elucidate differences and similarities in performance of numerous aspects including dynamic range, fidelity of raw signal and fold-change with sample titration, and concordance with qRT-PCR (TaqMan). To ensure that these results were not confounded by incompatible comparisons, we introduce the concept of probe mapping directed "transcript pattern". A transcript pattern identifies probe(set)s across platforms that target a common set of transcripts for a specific gene. Thus, three levels of data were examined: entire data sets, data derived from a subset of 15,442 RefSeq genes common across platforms, and data derived from the transcript pattern defined subset of 7,034 RefSeq genes. RESULTS: In general, there were substantial core similarities between all 6 platforms evaluated; but, to varying degrees, the two RNA-seq protocols outperformed three of the four microarray platforms in most categories. Notably, a fourth microarray platform, Agilent with a modified protocol, was comparable, or marginally superior, to the RNA-seq protocols within these same assessments, especially in regards to fold-change evaluation. Furthermore, these 3 platforms (Agilent and two RNA-seq methods) demonstrated over 80% fold-change concordance with the gold standard qRT-PCR (TaqMan). CONCLUSIONS: This study suggests that microarrays can perform on nearly equal footing with RNA-seq, in certain key features, specifically when the dynamic range is comparable. Furthermore, the concept of a transcript pattern has been introduced that may minimize potential confounding factors of multi-platform comparison and may be useful for similar evaluations.

Global expression analysis of the yeast Lachancea (Saccharomyces) kluyveri reveals new URC genes involved in pyrimidine catabolism.

Pyrimidines are important nucleic acid precursors which are constantly synthesized, degraded, and rebuilt in the cell. Four degradation pathways, two of which are found in eukaryotes, have been described. One of them, the URC pathway, has been initially discovered in our laboratory in the yeast Lachancea kluyveri. Here, we present the global changes in gene expression in L. kluyveri in response to different nitrogen sources, including uracil, uridine, dihydrouracil, and ammonia. The expression pattern of the known URC genes, URC1-6, helped to identify nine putative novel URC genes with a similar expression pattern. The microarray analysis provided evidence that both the URC and PYD genes are under nitrogen catabolite repression in L. kluyveri and are induced by uracil or dihydrouracil, respectively. We determined the function of URC8, which was found to catalyze the reduction of malonate semialdehyde to 3-hydroxypropionate, the final degradation product of the pathway. The other eight genes studied were all putative permeases. Our analysis of double deletion strains showed that the L. kluyveri Fui1p protein transported uridine, just like its homolog in Saccharomyces cerevisiae, but we demonstrated that is was not the only uridine transporter in L. kluyveri. We also showed that the L. kluyveri homologs of DUR3 and FUR4 do not have the same function that they have in S. cerevisiae, where they transport urea and uracil, respectively. In L. kluyveri, both of these deletion strains grew normally on uracil and urea.

GLUT10 is required for the development of the cardiovascular system and the notochord and connects mitochondrial function to TGFß signaling.

Growth factor signaling results in dramatic phenotypic changes in cells, which require commensurate alterations in cellular metabolism. Mutations in SLC2A10/GLUT10, a member of the facilitative glucose transporter family, are associated with altered transforming growth factor-ß (TGFß) signaling in patients with arterial tortuosity syndrome (ATS). The objective of this work was to test whether SLC2A10/GLUT10 can serve as a link between TGFß-related transcriptional regulation and metabolism during development. In zebrafish embryos, knockdown of slc2a10 using antisense morpholino oligonucleotide injection caused a wavy notochord and cardiovascular abnormalities with a reduced heart rate and blood flow, which was coupled with an incomplete and irregular vascular patterning. This was phenocopied by treatment with a small-molecule inhibitor of TGFß receptor (tgfbr1/alk5). Array hybridization showed that the changes at the transcriptome level caused by the two treatments were highly correlated, revealing that a reduced tgfbr1 signaling is a key feature of ATS in early zebrafish development. Interestingly, a large proportion of the genes, which were specifically dysregulated after glut10 depletion gene and not by tgfbr1 inhibition, play a major role in mitochondrial function. Consistent with these results, slc2a10 morphants showed decreased respiration and reduced TGFß reporter gene activity. Finally, co-injection of antisense morpholinos targeting slc2a10 and smad7 (a TGFß inhibitor) resulted in a partial rescue of smad7 morphant phenotypes, suggesting scl2a10/glut10 functions downstream of smads. Taken together, glut10 is essential for cardiovascular development by facilitating both mitochondrial respiration and TGFß signaling.

Somatic mutations affect key pathways in lung adenocarcinoma.

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers--including NF1, APC, RB1 and ATM--and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.

Next-generation sequencing identifies the natural killer cell microRNA transcriptome.

Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g., granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates for mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes have been established, little is known about miRNAs in NK cells. Here, we used two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by quantitative real-time PCR (qRT-PCR) and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 21 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range and exhibit isomiR complexity, and a subset is differentially expressed following cytokine activation. Using these miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine activation. Furthermore, we demonstrate that miR-223 specifically targets the 3' untranslated region of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology.

Transcriptional profiling of CD4 T cells identifies distinct subgroups of HIV-1 elite controllers.

Human immunodeficiency virus type 1 (HIV-1) elite controllers maintain undetectable levels of viral replication in the absence of antiretroviral therapy (ART), but their underlying immunological and virological characteristics may vary. Here, we used a whole-genome transcriptional profiling approach to characterize gene expression signatures of CD4 T cells from an unselected cohort of elite controllers. The transcriptional profiles for the majority of elite controllers were similar to those of ART-treated patients but different from those of HIV-1-negative persons. Yet, a smaller proportion of elite controllers showed an alternative gene expression pattern that was indistinguishable from that of HIV-1-negative persons but different from that of highly active antiretroviral therapy (HAART)-treated individuals. Elite controllers with the latter gene expression signature had significantly higher CD4 T cell counts and lower levels of HIV-1-specific CD8(+) T cell responses but did not significantly differ from other elite controllers in terms of HLA class I alleles, HIV-1 viral loads determined by ultrasensitive single-copy PCR assays, or chemokine receptor polymorphisms. Thus, these data identify a specific subgroup of elite controllers whose immunological and gene expression characteristics approximate those of HIV-1-negative persons.

Effects of doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo.

BACKGROUND: Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi. METHODS: Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles. RESULTS AND DISCUSSION: Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion). CONCLUSIONS: Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.

Notch-deficient skin induces a lethal systemic B-lymphoproliferative disorder by secreting TSLP, a sentinel for epidermal integrity.

Epidermal keratinocytes form a highly organized stratified epithelium and sustain a competent barrier function together with dermal and hematopoietic cells. The Notch signaling pathway is a critical regulator of epidermal integrity. Here, we show that keratinocyte-specific deletion of total Notch signaling triggered a severe systemic B-lymphoproliferative disorder, causing death. RBP-j is the DNA binding partner of Notch, but both RBP-j-dependent and independent Notch signaling were necessary for proper epidermal differentiation and lipid deposition. Loss of both pathways caused a persistent defect in skin differentiation/barrier formation. In response, high levels of thymic stromal lymphopoietin (TSLP) were released into systemic circulation by Notch-deficient keratinocytes that failed to differentiate, starting in utero. Exposure to high TSLP levels during neonatal hematopoiesis resulted in drastic expansion of peripheral pre- and immature B-lymphocytes, causing B-lymphoproliferative disorder associated with major organ infiltration and subsequent death, a previously unappreciated systemic effect of TSLP. These observations demonstrate that local skin perturbations can drive a lethal systemic disease and have important implications for a wide range of humoral and autoimmune diseases with skin manifestations.

SnoRNA microarray analysis reveals changes in H/ACA and C/D RNA levels caused by dyskerin ablation in mouse liver.

snoRNAs (small nucleolar RNAs) are key components of snoRNP (small nucleolar ribonucleoprotein) particles involved in modifying specific residues of ribosomal and other RNAs by pseudouridylation (H/ACA snoRNAs) or methylation (C/D snoRNAs). They are encoded within the introns of host genes, which tend to be genes whose products are involved in ribosome biogenesis or function. Although snoRNPs are abundant, ubiquitous and their components highly conserved, information concerning their expression during development or how their expression is altered in diseased states is sparse. To facilitate these studies we have developed a snoRNA microarray platform for the analysis of the abundance of snoRNAs in different RNA samples. In the present study we show that the microarray is sensitive and specific for the detection of snoRNAs. A mouse snoRNA microarray was used to monitor changes in abundance of snoRNAs after ablation of dyskerin, an H/ACA snoRNA protein component, from mouse liver, which causes a decrease in ribosome production. H/ACA snoRNAs were decreased in abundance in these livers while, unexpectedly, C/D snoRNAs were increased. The increase in C/D snoRNAs corresponded with an increase in the abundance of the mRNAs transcribed from snoRNA host genes, suggesting the increase may be part of a cellular response to defective ribosome synthesis.

Herpes simplex virus virion host shutoff attenuates establishment of the antiviral state.

Herpes simplex virus mutants lacking the vhs protein are severely attenuated in animal models of pathogenesis and exhibit reduced growth in primary cell culture. As a result of these properties, viruses with vhs deleted have been proposed as live-attenuated vaccines. Despite these findings and their implications for vaccines, the mechanisms by which vhs promotes infection in cell culture and in vivo are not understood. In this study we demonstrate that vhs-deficient viruses replicate to reduced levels in interferon (IFN)-primed cells and that this deficit has both IFN-dependent and IFN-independent components. Furthermore, vhs-defective viruses induce increased and physiologically active levels of IFN, increased amounts of IFN-stimulated transcripts, and more phosphorylated eIF2alpha. In addition, we demonstrate greater accumulation of viral RNAs following infection with a vhs-deficient virus. This leads to the hypothesis that attenuation of viruses lacking vhs may be attributed to increased levels of double-stranded RNA, a potent pathogen-associated molecular pattern. Together these data show that vhs likely functions to reduce innate immune responses and thereby acts as a critical determinant of viral pathogenesis.

Human cardiac-specific cDNA array for idiopathic dilated cardiomyopathy: sex-related differences.

Idiopathic dilated cardiomyopathy (IDCM) constitutes a large portion of patients with heart failure of unknown etiology. Up to 50% of all transplant recipients carry this clinical diagnosis. Female-specific gene expression in IDCM has not been explored. We report sex-related differences in the gene expression profile of ventricular myocardium from patients undergoing cardiac transplantation. We produced and sequenced subtractive cDNA libraries, using human left ventricular myocardium obtained from male transplant recipients with IDCM and nonfailing human heart donors. With the resulting sequence data, we generated a custom human heart failure microarray for IDCM containing 1,145 cardiac-specific oligonucleotide probes. This array was used to characterize RNA samples from female IDCM transplant recipients. We identified a female gene expression pattern that consists of 37 upregulated genes and 18 downregulated genes associated with IDCM. Upon functional analysis of the gene expression pattern, deregulated genes unique to female IDCM were those that are involved in energy metabolism and regulation of transcription and translation. For male patients we found deregulation of genes related to muscular contraction. These data suggest that 1) the gene expression pattern we have detected for IDCM may be specific for this disease and 2) there is a sex-specific profile to IDCM. Our observations further suggest for the first time ever novel targets for treatment of IDCM in women and men.

Fingerprint profile of alcohol-associated heart failure in human hearts.

BACKGROUND: Excessive alcohol consumption is recognized as a cause of left ventricular dysfunction and leads often to alcohol-induced heart failure. It is thought that 36% of all cases of dilated cardiomyopathy are due to excessive alcohol intake. In addition, since chronic alcohol-consumption is a social behavior that is not always clearly self-reported clinically, it has been difficult to diagnose alcohol-induced heart failure versus heart failure due to idiopathic dilated cardiomyopathy (IDCM). Interestingly, both diseases are associated with left ventricular dysfunction and congestive heart failure. METHODS: We have created a human heart failure cDNA array for IDCM from nonfailing and failing human hearts. The array contains 1,143 heart specific oligonucleotide probes. This array was used to screen RNA samples from transplant recipients and organ donors with alcohol-related heart failure. RESULTS: Our study shows that alcohol-induced heart failure has a "specific fingerprint" profile of de-regulated genes. This profile can differentiate patients with pure alcohol-induced heart failure from patients with heart failure from IDCM with alcohol as a complicating or contributing factor. Furthermore, the pattern of gene de-regulation suggests a pivotal role for changes in matrix, cytoskeletal, and structural proteins in the development of clinical heart failure resulting from excessive alcohol consumption. CONCLUSIONS: We report for the first time a genomic "fingerprint" profile of de-regulated genes associated with human alcohol-induced heart failure. We conclude that the pathogenesis of alcohol-induced heart failure in humans is likely related to changes in architectural (e.g. cytoskeletal), matrix, and/or structural proteins. The reversibility of the disease upon cessation of alcohol consumption makes this a likely pathogenetic mechanism. Nevertheless, there is a point at which extracellular as well as cellular changes result in irreversible heart failure.

Brugia malayi: whole genome amplification for genomic characterization of filarial parasites.

Genetic characterization of field isolates and clinical specimens of filarial nematodes is often limited by a shortage of DNA; therefore, we evaluated a multiple displacement amplification (MDA) based whole genome amplification method. The quality of amplified DNA was examined by conventional PCR, real-time PCR, and DNA hybridization. MDA of 5.0 ng of adult Brugia malayi DNA and one-fifteenth of the DNA isolated from a single microfilaria resulted in 6.3 and 4.2 microg of amplified DNA, respectively. Amplified DNA was equivalent to native genomic DNA for hybridization to B. malayi BAC library clones or to an oligonucleotide microarray with approximately 18,000 filarial DNA sequences. MDA is useful for whole genome amplification of filarial DNA from very small amounts of starting material. This technology will permit detailed studies of genetic diversity that were not previously feasible.

A multi-amplicon 16S rRNA sequencing and analysis method for improved taxonomic profiling of bacterial communities.

Metagenomic sequencing of bacterial samples has become the gold standard for profiling microbial populations, but 16S rRNA profiling remains widely used due to advantages in sample throughput, cost, and sensitivity even though the approach is hampered by primer bias and lack of specificity. We hypothesized that a hybrid approach, that combined targeted PCR amplification with high-throughput sequencing of multiple regions of the genome, would capture many of the advantages of both approaches. We developed a method that identifies and quantifies members of bacterial communities through simultaneous analysis of multiple variable regions of the bacterial 16S rRNA gene. The method combines high-throughput microfluidics for PCR amplification, short read DNA sequencing, and a custom algorithm named MVRSION (Multiple 16S Variable Region Species-Level IdentificatiON) for optimizing taxonomic assignment. MVRSION performance was compared to single variable region analyses (V3 or V4) of five synthetic mixtures of human gut bacterial strains using existing software (QIIME), and the results of community profiling by shotgun sequencing (COPRO-Seq) of fecal DNA samples collected from gnotobiotic mice colonized with a defined, phylogenetically diverse consortium of human gut bacterial strains. Positive predictive values for MVRSION ranged from 65%-91% versus 44%-61% for single region QIIME analyses (p < .01, p < .001), while the abundance estimate r2 for MVRSION compared to COPRO-Seq was 0.77 vs. 0.46 and 0.45 for V3-QIIME and V4-QIIME, respectively. MVRSION represents a generally applicable tool for taxonomic classification that is superior to single-region 16S rRNA methods, resource efficient, highly scalable for assessing the microbial composition of up to thousands of samples concurrently, with multiple applications ranging from whole community profiling to targeted tracking of organisms of interest in diverse habitats as a function of specified variables/perturbations.

Template-dependent multiple displacement amplification for profiling human circulating RNA.

Multiple displacement amplification (MDA) is widely used in whole-genome/transcriptome amplification. However, template-independent amplification (TIA) in MDA is a commonly observed phenomenon, particularly when using high concentrations of random hexamer primers and extended incubation times. Here, we demonstrate that the use of random pentamer primers with 5´ ends blocked by a C18 spacer results in MDA solely in a template-dependent manner, a technique we have named tdMDA. Together with an optimized procedure for the removal of residual genomic DNA during RNA extraction, tdMDA was used to profile circulating RNA from 0.2 mL of patient sera. In comparison to regular MDA, tdMDA demonstrated a lack of quantifiable DNA amplification in the negative control, a remarkable reduction of unmapped reads from Illumina sequencing (7 ± 10.9% versus 58.6 ± 39%, P = 0.006), and increased mapping rates of the serum transcriptome (26.9 ± 7.9% versus 5.8 ± 8.2%, P = 3.8 × 10-4). Transcriptome profiles could be used to separate patients with chronic hepatitis C virus (HCV) infection from those with HCV-associated hepatocellular carcinoma (HCC). We conclude that tdMDA should facilitate RNA-based liquid biopsy, as well as other genome studies with biological specimens having ultralow amounts of genetic material.

Synthesis from caffeine of a mixed N-heterocyclic carbene-silver acetate complex active against resistant respiratory pathogens.

The bis(N-heterocyclic carbene) (NHC) silver complex, 3, with a methyl carbonate anion was formed from the reaction of the iodide salt of methylated caffeine, 1, with silver (I) oxide in methanol. Attempts to crystallize this complex from a mixture of common alcohols and ethyl acetate led to the formation of an NHC-silver acetate complex, 4. The more direct synthesis of 4 was accomplished by the in-situ deprotonation of 1 by silver acetate in methanol. Complex 4 demonstrated antimicrobial activity against numerous resistant respiratory pathogens from the lungs of cystic fibrosis (CF) patients including members of the Burkholderia cepacia complex that cause a high rate of mortality in patients with cystic fibrosis (CF). Application of this NHC silver complex to primary cultures of murine respiratory epithelial cells followed by microarray analysis showed minimal gene expression changes at the concentrations effective against respiratory pathogens. Furthermore, methylated caffeine without silver showed some antibacterial and antifungal activity.

Profiling of gender-regulated gene transcripts in the filarial nematode Brugia malayi by cDNA oligonucleotide array analysis.

Microarray technology permits high-throughput comparisons of gene expression in different parasite stages or sexes and has been used widely. We report the first use of this technology for analysis of gene expression in filarial male and female worms. The slide array (comprised of 65-mer oligos representing 3569 EST clusters) was spotted with sequences selected from the extensive Brugia malayi EST database (). Arrays were hybridized with Cy dye labeled male and female cDNA. The experimental design included both biological and technical (dye-flip) replicates. The data were normalized for background and probe intensity, and the relative abundance of hybridized cDNA for each spot was determined. Genes showing two-fold or greater differences with P<0.05 were considered gender-regulated candidates. One thousand one hundred and seventy of 2443 clusters (48%) with signals above threshold in at least one sex were considered as gender-regulated gene candidates. This included 520 and 650 clusters up-regulated in male and female worms, respectively. Fifty of 53 (94%) gender-regulated candidate genes identified by microarray analysis were confirmed by real-time RT-PCR. Approximately 61% of gender-regulated genes had significant similarity to known genes in other organisms such as Caenorhabditis elegans. Many C. elegans homologues of these genes have been reported to have reproductive phenotypes (sterility or abnormal embryo development) by RNA interference. This study has provided the first broad view of gender-regulated gene expression in B. malayi; this should lead to improved understanding of reproduction in filarial nematodes. More generally, this approach holds great promise as a means of studying stage-specific or tissue-specific gene expression in parasitic nematodes.