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.

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.

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.

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).

Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury.

Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls.

Enhanced sphingosine-1-phosphate receptor 2 expression underlies female CNS autoimmunity susceptibility.

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is characterized by BBB dysfunction and has a much higher incidence in females. Compared with other strains of mice, EAE in the SJL mouse strain models multiple features of MS, including an enhanced sensitivity of female mice to disease; however, the molecular mechanisms that underlie the sex- and strain-dependent differences in disease susceptibility have not been described. We identified sphingosine-1-phosphate receptor 2 (S1PR2) as a sex- and strain-specific, disease-modifying molecule that regulates BBB permeability by destabilizing adherens junctions.

Validation of a next-generation sequencing assay for clinical molecular oncology.

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci.

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.

Serum from humans on long-term calorie restriction enhances stress resistance in cell culture.

Calorie restriction (CR) without malnutrition is the most robust intervention to slow aging and extend healthy lifespan in experimental model organisms. Several metabolic and molecular adaptations have been hypothesized to play a role in mediating the anti-aging effects of CR, including enhanced stress resistance, reduced oxidative stress and several neuroendocrine modifications. However, little is known about the independent effect of circulating factors in modulating key molecular pathways.

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.

Calorie restriction in humans inhibits the PI3K/AKT pathway and induces a younger transcription profile.

Caloric restriction (CR) and down-regulation of the insulin/IGF pathway are the most robust interventions known to increase longevity in lower organisms. However, little is known about the molecular adaptations induced by CR in humans. Here, we report that long-term CR in humans inhibits the IGF-1/insulin pathway in skeletal muscle, a key metabolic tissue.

Serum response factor controls transcriptional network regulating epidermal function and hair follicle morphogenesis.

Serum response factor (SRF) is a transcription factor that regulates the expression of growth-related immediate-early, cytoskeletal, and muscle-specific genes to control growth, differentiation, and cytoskeletal integrity in different cell types. To investigate the role for SRF in epidermal development and homeostasis, we conditionally knocked out SRF in epidermal keratinocytes. We report that SRF deletion disrupted epidermal barrier function leading to early postnatal lethality.

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.

Genome-Wide Identification and Quantification of cis- and trans-Regulated Genes Responding to Marek's Disease Virus Infection via Analysis of Allele-Specific Expression.

Marek's disease (MD) is a commercially important neoplastic disease of chickens caused by Marek's disease virus (MDV), a naturally occurring oncogenic alphaherpesvirus. Selecting for increased genetic resistance to MD is a control strategy that can augment vaccinal control measures. To identify high-confidence candidate MD resistance genes, we conducted a genome-wide screen for allele-specific expression (ASE) amongst F(1) progeny of two inbred chicken lines that differ substantially in MD resistance.

GLUT10 is required for the development of the cardiovascular system and the notochord and connects mitochondrial function to TGFβ 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-β (TGFβ) 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 TGFβ-related transcriptional regulation and metabolism during development.

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.

Loss of Nix in Pdx1-deficient mice prevents apoptotic and necrotic β cell death and diabetes.

Mutations in pancreatic duodenal homeobox (PDX1) are linked to human type 2 diabetes and maturity-onset diabetes of the young type 4. Consistent with this, Pdx1-haploinsufficient mice develop diabetes. Both apoptosis and necrosis of β cells are mechanistically implicated in diabetes in these mice, but a molecular link between Pdx1 and these 2 forms of cell death has not been defined.

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.

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.

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.

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.

'Calling Cards' method for high-throughput identification of targets of yeast DNA-binding proteins.

We present a protocol for a novel method for identifying the targets of DNA-binding proteins in the genome of the yeast Saccharomyces cerevisiae. This is accomplished by engineering a DNA-binding protein so that it leaves behind in the genome a permanent mark -- a 'calling card' -- that provides a record of that protein's visit to that region of the genome. The calling card is the yeast Ty5 retrotransposon, whose integrase interacts with the Sir4 protein.

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.

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.

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.