Metabolic profiling of serum using Ultra Performance Liquid Chromatography and the LTQ-Orbitrap mass spectrometry system

Advances in analytical instrumentation can provide significant advantages to the volume and quality of biological knowledge acquired in metabolomic investigations. The interfacing of sub-2mum liquid chromatography (UPLC ACQUITY((R))) and LTQ-Orbitrap mass spectrometry systems provides many theoretical advantages. The applicability of the interfaced systems was investigated using a simple 11-component metabolite mix and a complex mammalian biofluid, serum. Metabolites were detected in the metabolite mix with signals that were linear with their concentration over 2.5-3.5 orders of magnitude, with correlation coefficients greater than 0.993 and limits of detection less than 1mumolL(-1). Reproducibility of retention time (RSD<3%) and chromatographic peak area (RSD<15%) and a high mass accuracy (<2ppm) were observed for 14 QC serum samples interdispersed with other serum samples, analysed over a period of 40h. The evaluation of a single deconvolution software package (XCMS) was performed and showed that two parameters (snthresh and bw) provided significant changes to the number of peaks detected and the peak area reproducibility for the dataset used. The data were used to indicate possible biomarkers of pre-eclampsia and showed both the instruments and XCMS to be applicable to the reproducible and valid detection of disease biomarkers present in serum.     

Identification and Characterization of a New Orthoreovirus from Patients with Acute Respiratory Infections

First discovered in the early 1950s, reoviruses (respiratory enteric orphan viruses) were not associated with any known disease, and hence named orphan viruses. Recently, our group reported the isolation of the Melaka virus from a patient with acute respiratory disease and provided data suggesting that this new orthoreovirus is capable of human-to-human transmission and is probably of bat origin. Here we report yet another Melaka-like reovirus (named Kampar virus) isolated from the throat swab of a 54 year old male patient in Kampar, Perak, Malaysia who was suffering from high fever, acute respiratory disease and vomiting at the time of virus isolation. Serological studies indicated that Kampar virus was transmitted from the index case to at least one other individual and caused respiratory disease in the contact case. Sequence analysis of the four small class genome segments indicated that Kampar and Melaka viruses are closely related. This was confirmed by virus neutralization assay, showing an effective two-way cross neutralization, i.e., the serum against one virus was able to neutralize the other. Although the exact origin of Kampar virus is unknown, epidemiological tracing revealed that the house of the index case is surrounded by fruit trees frequently visited by fruit bats. There is a high probability that Kampar virus originated from bats and was transmitted to humans via bat droppings or contaminated fruits. The discovery of Kampar virus highlights the increasing trend of emergence of bat zoonotic viruses and the need to expand our understanding of bats as a source of many unknown viruses.     

Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells

The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.     

Sequence-function relationships provide new insight into the cleavage site selectivity of the 8-17 RNA-cleaving deoxyribozyme

Many sequence variations of the 8–17 RNA-cleaving deoxyribozyme have been isolated through in vitro selection. In an effort to understand how these sequence variations affect cleavage site selectivity, we systematically mutated the catalytic core of 8–17 and measured the cleavage activity of each mutant deoxyribozyme against all 16 possible chimeric (RNA/DNA) dinucleotide junctions. We observed sequence-function relationships that suggest how the following non-conserved positions in the catalytic core influence selectivity at the dinucleotide (5′ rN₁₈-N_1.1 3′) cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3′ position (N_1.1) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5′ position (rN₁₈) of the cleavage site and (iii) the sequence of the 3-bp intramolecular stem has relatively little influence on cleavage site selectivity. Furthermore, we report for the first time that 8–17 variants have the collective ability to cleave all dinucleotide junctions with rate enhancements of at least 1000-fold over background. Three optimal 8–17 variants, identified from ~75 different sequences that were examined, can collectively cleave 10 of 16 junctions with useful rates of ≥0.1 min⁻¹, and exhibit an overall hierarchy of reactivity towards groups of related junctions according to the order NG > NA > NC > NT.     

Vitamin D content in Alaskan Arctic zooplankton,fishes, and marine mammals

We postulated that dietary ingestion of vitamin D may be used by some Alaskan Arctic marine mammal species in addition to, or instead of, cutaneous production to meet nutritional requirements. Zooplankton (n=5) sampled near Kaktovik, Alaska, contained no measurable vitamin D₂ or D₃, but did contain provitamin D (7‐dehydrocholesterol), the cutaneous precursor for previtamin D₃ in mammals. Fillets and livers from five fish species were sampled near Barrow, Alaska, and evaluated for vitamin D₃ content (no vitamin D₂ was detected). Differences in vitamin D₃ content appeared significant (P≤0.10) among fish livers (Kruskal‐Wallis [H test]=8.25, df=4, P=0.08) and among fish fillets (H=7.80, df=4, P=0.01). We also found significant differences in several pairwise comparisons (Mann‐Whitney U‐test) of vitamin D₃ levels in fillets and livers. Blubber from six species of marine mammals had no detectable vitamin D₂. The H test results for blubber vitamin D₃ concentration were highly significant: 28.12, df=5, P<0.001. There were also significant differences in vitamin D₃ content from blubber in pairwise comparisons of primarily invertebrate feeders (bowhead whale (Balaena mysticetus) [mean=4.20 SD±1.10 ng/g], and Pacific walrus (Odobenus rosmarus divergens) [5.43±2.82 ng/g]) vs. primarily piscivorous feeders (ringed seal (Phoca hispida) [746.57±493.00 ng/g] and beluga whale (Delphinapterus leucas) [426.00±174.92 ng/g]) and a semiaquatic terrestrial carnivore (polar bear (Ursus maritimus) [406.17±311.70 ng/g]). The bearded seal (Erignathus barbatus) had intermediate blubber vitamin D₃ concentration (156.83±139.25 ng/g), which may reflect an intermediate‐type feeding strategy or an artifact of the small sample size. Zoo Biol 23:33–43, 2004. © 2004 Wiley‐Liss, Inc.     

Single-copy gene detection using branched DNA (bDNA) in situ hybridization.

We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)     

A genotype-to-phenotype map of in vitro selected RNA-cleaving DNAzymes: implications for accessing the target phenotype

Herein, we describe a case study into the population dynamics of in vitro selection, using RNA-cleaving DNAzymes as a model system. We sought to understand how the composition of the population can change over time in response to different levels of selection pressure, and how well these changes are correlated with selection of the target phenotype. The model population is composed of 857 DNAzyme clones representing 215 discrete sequence classes, which had previously been identified from two parallel selection experiments, conducted under an increasingly stringent, or permissive and constant selection time pressure. In this report, we determined the principal phenotypic properties (i.e. k_obs, maximum cleavage yield and PCR efficiency) from a sample of 58 clones representing 46 different major and minor sequence classes from various rounds of each selection experiment. Interestingly, a positive correlation between the catalytic rate constant and the corresponding frequency and temporal position of a given DNAzyme was not consistently observed; however, the strength of the correlation was qualitatively higher under conditions of more stringent selection time pressure. These results suggest that the selective sampling paradigm on which in vitro selection is based, may underestimate the true functional capacity of any given random-sequence library.