Genome-Wide Maps of Circulating miRNA Biomarkers for Ulcerative Colitis

Inflammatory Bowel Disease – comprised of Crohn's Disease and Ulcerative Colitis (UC) - is a complex, multi-factorial inflammatory disorder of the gastrointestinal tract. In this study we have explored the utility of naturally occurring circulating miRNAs as potential blood-based biomarkers for non-invasive prediction of UC incidences. Whole genome maps of circulating miRNAs in micro-vesicles, Peripheral Blood Mononuclear Cells and platelets have been constructed from a cohort of 20 UC patients and 20 normal individuals. Through Significance Analysis of Microarrays, a signature of 31 differentially expressed platelet-derived miRNAs has been identified and biomarker performance estimated through a non-probabilistic binary linear classification using Support Vector Machines. Through this approach, classifier measurements reveal a predictive score of 92.8% accuracy, 96.2% specificity and 89.5% sensitivity in distinguishing UC patients from normal individuals. Additionally, the platelet-derived biomarker signature can be validated at 88% accuracy through qPCR assays, and a majority of the miRNAs in this panel can be demonstrated to sub-stratify into 4 highly correlated intensity based clusters. Analysis of predicted targets of these biomarkers reveal an enrichment of pathways associated with cytoskeleton assembly, transport, membrane permeability and regulation of transcription factors engaged in a variety of regulatory cascades that are consistent with a cell-mediated immune response model of intestinal inflammation. Interestingly, comparison of the miRNA biomarker panel and genetic loci implicated in IBD through genome-wide association studies identifies a physical linkage between hsa-miR-941 and a UC susceptibility loci located on Chr 20. Taken together, analysis of these expression maps outlines a promising catalog of novel platelet-derived miRNA biomarkers of clinical utility and provides insight into the potential biological function of these candidates in disease pathogenesis

Selective Antibacterial Properties of Lysozyme for Oral Microorganisms

The antibacterial properties of lysozyme were investigated with oral microorganisms representing the seven serotypes ( a through g ) of Streptococcus mutans, Veillonella alcalescens , and the virulent (V) and avirulent (AV) strains of Actinomyces viscosus T14. Growth of bacteria in defined medium was monitored spectrophotometrically after the addition of various amounts (25 μg to 5 mg/ml) of enzyme. No growth inhibition of V. alcalescens was observed. Inhibition of A. viscosus T14(V) and A. viscosus T14(AV) occurred with 160 μg of lysozyme per ml. Of the S. mutans cultures tested, the serotype a and b strains were inhibited with as little as 25 μg of enzyme per ml, whereas e and f strains were most resistant to the bacteriostatic activity of lysozyme. The presence of dl -threonine or sucrose in growth medium did not significantly affect the results. A lysoplate assay was developed to rapidly survey the bacterial cultures for their susceptibility to the lytic ability of the enzyme. Lysis, as a measure of a zone of clearing in agarose plates, occurred for all microorganisms in the presence of lysozyme after the subsequent addition of NaCl or detergent. The bactericidal activity of lysozyme was determined on S. mutans BHT and S. mutans LM-7 by the pour plate technique. Preincubation of S. mutans LM-7 with as much as 1 mg of enzyme for 90 min did not affect viability or growth, whereas preincubation of S. mutans BHT with 1 mg of lysozyme resulted in no recoverable colony-forming units. An antigen containing extract of S. mutans LM-7 blocked the growth inhibitory property of lysozyme. Human lysozyme was a more effective antibacterial factor than hen egg white lysozyme. Total growth inhibition of S. mutans BHT was effected with 40 μg of human enzyme, and as little as 10 μg of human enzyme inhibited growth for greater than 20 h. The data presented indicate that different mechanisms may be responsible for the bacteriostatic, lytic, and bactericidal properties of the enzyme and that lysozyme is a selective but effective antibacterial factor for oral microorganisms. </jats:p

Gene ontology (GO) classification and pathway analysis of predicted mRNA targets of the 31 platelet-derived biomarkers.

<p>Overlap^# refers to the number of gene annotations intersecting with genome annotations. The top ranking GO terms, % overlaps and the counts of overlapping entities ranked by the most significant p-values are listed.</p

Class-Sparing Regimens for Initial Treatment of HIV-1 Infection

BACKGROUND: The use of either efavirenz or lopinavir–ritonavir plus two nucleoside reverse-transcriptase inhibitors (NRTIs) is recommended for initial therapy for patients with human immunodeficiency virus type 1 (HIV-1) infection, but which of the two regimens has greater efficacy is not known. The alternative regimen of lopinavir–ritonavir plus efavirenz may prevent toxic effects associated with NRTIs. METHODS: In an open-label study, we compared three regimens for initial therapy: efavirenz plus two NRTIs (efavirenz group), lopinavir–ritonavir plus two NRTIs (lopinavir–ritonavir group), and lopinavir–ritonavir plus efavirenz (NRTI-sparing group). We randomly assigned 757 patients with a median CD4 count of 191 cells per cubic millimeter and a median HIV-1 RNA level of 4.8 log(10) copies per milliliter to the three groups. RESULTS: At a median follow-up of 112 weeks, the time to virologic failure was longer in the efavirenz group than in the lopinavir–ritonavir group (P = 0.006) but was not significantly different in the NRTI-sparing group from the time in either of the other two groups. At week 96, the proportion of patients with fewer than 50 copies of plasma HIV-1 RNA per milliliter was 89% in the efavirenz group, 77% in the lopinavir–ritonavir group, and 83% in the NRTI-sparing group (P = 0.003 for the comparison between the efavirenz group and the lopinavir–ritonavir group). The groups did not differ significantly in the time to discontinuation because of toxic effects. At virologic failure, antiretroviral resistance mutations were more frequent in the NRTI-sparing group than in the other two groups. CONCLUSIONS: Virologic failure was less likely in the efavirenz group than in the lopinavir–ritonavir group. The virologic efficacy of the NRTI-sparing regimen was similar to that of the efavirenz regimen but was more likely to be associated with drug resistance. (ClinicalTrials.gov number, NCT00050895.

Comparison of expression levels of the platelet-derived miRNA biomarkers in the patient and control cohorts.

<p>Unsupervised hierarchical clustering of samples (controls in blue: C1–C20 and patients in red: P2–P21) based on summarized intensity values from the 31 differentially expressed miRNA biomarkers. The log₂ intensity values are shown in the Color Key bar scale.</p

Characteristics and performance measures of biomarkers derived from Platelets, Micro-vesicles or Platelet/Micro-vesicle combined fraction.

<p>(A) Cumulative distribution frequency (CDF) plots of biomarkers derived from the different fractions. The dotted line represents the 90% cut-off frequency across the study population (B) Counts and overlaps of miRNA biomarkers derived from the platelet, micro-vesicular and combined fraction (C) Performance measures of biomarkers from each fraction selected at the 90% frequency threshold.</p

Demographics of individuals recruited for this study.

<p>Gender is designated as either M (Male) or F (Female). Disorders are designated as GI (Gastro-intestinal) or CD (Crohn's Disease).</p

Sub-clusters of platelet-derived miRNA biomarkers.

<p>Unsupervised hierarchical clustering of miRNA biomarkers based on the Pearson correlation coefficients among the miRNA expression profiles from 20 patients and 20 controls. The coefficient values are shown in the bar scale. Each of the 4 main clusters with significant correlation values are indicated in boxes.</p

Validation of expression levels of miRNA biomarkers derived from the platelet fraction.

<p>miRNA expression levels of 7 biomarkers (n = 4, *P values<0.001) were validated by qPCR. The p values are calculated based on a Student's t-test of the replicate 2∧ (−ΔCt) values for each miRNA in the control group (normal individuals) and test groups (patients).</p