Detection of human metapneumovirus and respiratory syncytial virus by duplex real-time RT-PCR assay in comparison with direct fluorescent assay

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are important respiratory pathogens of small children and adults. The present study aimed to design a sensitive real-time RT-PCR assay for the detection of hRSV and hMPV in comparison with direct fluorescent assay (DFA) and to determine the incidence of hMPV and hRSV as causative agents of respiratory infections in a Finnish population. For DFA detection of hMPV antigen, four commercial antibodies were evaluated. The duplex real-time RT-PCR assay achieved a sensitivity of 10³ copies/mL of specimen for hRSV and hMPV type A viruses and 10⁴ copies/mL for type B hMPV. The detection rate of the RT-PCR assay was compared with those for DFA detection of hMPV and hRSV in analyses of 350 nasopharyngeal aspirates sent to HUSLAB, Helsinki University Hospital, for routine virus diagnostics during November 2007 to June 2008. Of the samples analyzed, 43 (12.3%) were positive for hRSV by DFA and an additional 13 specimens (3.7%) were positive for hRSV by RT-PCR. Only four samples (1.1 %) were found to be positive for hMPV RNA by RT-PCR, with two of them also positive by DFA. The duplex real-time RT-PCR assay described in the present study can therefore be applied for efficient identification of hMPV and hRSV in clinical specimens and collection of information on the epidemiology and clinical outcome of these viruses.     

Association between two mu-opioid receptor gene (OPRM1) haplotype blocks and drug or alcohol dependence

We examined 13 single nucleotide polymorphisms (SNPs) spanning the coding region of the mu-opioid receptor gene (OPRM1), among 382 European Americans (EAs) affected with substance dependence [alcohol dependence (AD) and/or drug dependence (DD)] and 338 EA healthy controls. These SNPs delineated two haplotype blocks. Genotype distributions for all SNPs were in Hardy-Weinberg equilibrium (HWE) in controls, but in cases, four SNPs in Block I and three SNPs in Block II showed deviation from HWE. Significant differences were found between cases and controls in allele and/or genotype frequencies for six SNPs in Block I and two SNPs in Block II. Association of SNP4 in Block I with DD (allele: P=0.004), SNP5 in Block I with AD and DD (allele: P< or =0.005 for both) and two SNPs in Block II with AD (SNP11 genotype: P=0.002; SNP12 genotype: P=0.001) were significant after correction for multiple testing. Frequency distributions of haplotypes (constructed by five tag SNPs) differed significantly for cases and controls (P<0.001 for both AD and DD). Logistic regression analyses confirmed the association between OPRM1 variants and substance dependence, when sex and age of subjects and alleles, genotypes, haplotypes or diplotypes of five tag SNPs were considered. Population structure analyses excluded population stratification artifact. Additional supporting evidence for association between OPRM1 and AD was obtained in a smaller Russian sample (247 cases and 100 controls). These findings suggest that OPRM1 intronic variants play a role in susceptibility to AD and DD in populations of European ancestry.     

Serological microarray for detection of HSV-1, HSV-2, VZV, and CMV antibodies

The seroprevalence of human herpesviruses is high and reactivations occur frequently. A microarray was designed and tested for the detection of IgG and IgM antibodies for Puumala hantavirus (PUUV) and IgG antibodies against four herpesviruses. Initially, a microarray platform was set up using an unrelated in-house antigen, PUUV recombinant nucleocapsid protein, to optimize the protocol for the detection of antibodies. Detection of the four herpesviruses was set up in a microarray using the recombinant proteins of herpes simplex virus (HSV) glycoprotein G1 and G2, varicella-zoster virus (VZV) glycoprotein E, and cytomegalovirus (CMV) pp150 phosphoprotein.The results of the PUUV panel were in good agreement with the PUUV IgG immunofluorescent assay and IgM enzyme immunoassay (EIA). Seropositive and negative clinical reference panels were tested for herpesviruses by the serological microarray, and the results were compared to those of individual EIAs used for standard diagnostic purposes.The serologic microarray for HSV, VZV and CMV antibody detection gave good specificities for IgG. However, sensitivities of the assay varied depending on the herpesvirus detected. The serological microarray showed potential for screening purposes. The microarray based analyses were easy to perform, and HSV-1, HSV-2, VZV, and CMV antibodies could be detected on the same microarray.     

Effect of human trabecular bone composition on its electrical properties

Mechanical properties of bone are determined not only by bone mineral density (BMD), but also by tissue trabecular structure and organic composition. Impedance spectroscopy has shown potential to diagnose trabecular bone BMD and strength, however, the relationships between organic composition and electrical and dielectric properties have not been systematically investigated. To investigate these issues organic composition of 26 human trabecular bone samples harvested from the distal femur and proximal tibia was determined and compared with relative permittivity, loss factor, conductivity, phase angle, specific impedance and dissipation factor measured at wide range (50 Hz to 5 MHz) of frequencies. A strong linear correlation was found between the relative permittivity at 1.2 MHz and trabecular bone fat content (r = -0.85, p<0.01, n=26). On the other hand, relative permittivity measured at 200 Hz served as a good predictor of water content (r = 0.83). Phase angle, specific impedance and especially conductivity were strongly related to the trabecular bone dry density and water content (|r| > or = 0.69). Variation in bone tissue collagen content was strongly related to the relative permittivity measured at 1.2 MHz (r = 0.64), but only moderately to other parameters. Glycosaminoglycan content showed no significant relations with any investigated electrical parameters. The present study indicates that if the trabecular bone composition is known, the relationships presented in this study could facilitate calculation of current field distribution, e.g. during electrical stimulation of osteogenesis. On the other hand, our results suggest that permittivity measured at low (<1 kHz) or high (>100 kHz) frequencies could be used, e.g. during implant surgery, for prediction of trabecular bone water or fat contents, respectively.     

Ethylene–Propylene Copolymerisations: Effect of Metallocene Structure on Termination Reactions and Polymer Microstructure

Summary: Ethylene–propylene (EP) copolymerisations were performed with two sterically different metallocenes activated by methylaluminoxane (MAO) in an attempt to better understand the effect of catalyst structure on termination reactions and polymer microstructure. The metallocene precursors under investigation were rac‐dimethylsilylbis(2‐methyl‐4‐phenyl‐1‐indenyl)zirconium dichloride ( 1 ) and a more sterically hindered counterpart rac‐dimethylsilylbis(2‐isopropyl‐4‐[3,5‐dimethylphenyl]indenyl) zirconium dichloride ( 2 ). For both catalyst systems, the most common termination mechanism was chain transfer to aluminium. In addition, for polymer samples polymerised with 1 /MAO, chain growth was terminated by chain transfer to Zr metal in propylene‐rich polymerisations and by chain transfer to ethylene monomer in ethylene‐rich polymerisations. The steric hindrance of 2 was able to suppress the chain transfer to the ethylene monomer, and chain transfer to Zr metal was also found in the ethylene‐rich polymerisations. The greater steric hindrance of 2 also affected the EP copolymer microstructure: regioregularity in the propylene‐rich copolymers was greater and isotacticity less with 2 /MAO than with 1 /MAO.

The catalyst precursors used: rac‐dimethylsilylbis(2‐methyl‐4‐phenyl‐1‐indenyl)zirconium dichloride ( 1 ) and rac‐dimethylsilylbis(2‐isopropyl‐4‐[3,5‐dimethylphenyl]indenyl) zirconium dichloride ( 2 ).     

Metabolism of [U‐13C]glucose in human brain tumors in vivo

Glioblastomas and brain metastases demonstrate avid uptake of 2‐[¹⁸F]fluoro‐2‐deoxyglucose by positron emission tomography and display perturbations of intracellular metabolite pools by ¹H MRS. These observations suggest that metabolic reprogramming contributes to brain tumor growth in vivo. The Warburg effect, excess metabolism of glucose to lactate in the presence of oxygen, is a hallmark of cancer cells in culture. 2‐[¹⁸F]Fluoro‐2‐deoxyglucose‐positive tumors are assumed to metabolize glucose in a similar manner, with high rates of lactate formation relative to mitochondrial glucose oxidation, but few studies have specifically examined the metabolic fates of glucose in vivo. In particular, the capacity of human brain cancers to oxidize glucose in the tricarboxylic acid cycle is unknown. Here, we studied the metabolism of human brain tumors in situ. [U‐¹³ C]Glucose (uniformly labeled glucose, i.e. d ‐glucose labeled with ¹³ C in all six carbons) was infused during surgical resection, and tumor samples were subsequently subjected to ¹³C NMR spectroscopy. The analysis of tumor metabolites revealed lactate production, as expected. We also determined that pyruvate dehydrogenase, turnover of the tricarboxylic acid cycle, anaplerosis and de novo glutamine and glycine synthesis contributed significantly to the ultimate disposition of glucose carbon. Surprisingly, less than 50% of the acetyl‐coenzyme A pool was derived from blood‐borne glucose, suggesting that additional substrates contribute to tumor bioenergetics. This study illustrates a convenient approach that capitalizes on the high information content of ¹³C NMR spectroscopy and enables the analysis of intermediary metabolism in diverse cancers growing in their native microenvironment. Copyright © 2012 John Wiley & Sons, Ltd.     

IL-1beta disrupts postnatal lung morphogenesis in the mouse

Pulmonary inflammation and increased production of the inflammatory cytokine IL-1beta are associated with the development of bronchopulmonary dysplasia (BPD) in premature infants. To study the actions of IL-1beta in the fetal and newborn lung in vivo, we developed a bitransgenic mouse in which IL-1beta is expressed under conditional control in airway epithelial cells. Perinatal pulmonary expression of IL-1beta caused respiratory insufficiency that was associated with increased postnatal mortality. While intrauterine growth of IL-1beta-expressing mice was normal, their postnatal growth was impaired. IL-1beta disrupted alveolar septation and caused abnormalities in alpha-smooth muscle actin and elastin deposition in the septa of distal airspaces. IL-1beta disturbed capillary development and inhibited the production of vascular endothelial growth factor in the lungs of infant mice. IL-1beta induced the expression of CXC chemokines KC (CXCL1) and macrophage inflammatory protein-2 (CXCL2) and of CC chemokines monocyte chemotactic protein (MCP)-1 (CCL2) and MCP-3 (CCL7), consistent with neutrophilic and monocytic infiltration of the lungs. IL-1beta caused goblet cell metaplasia and bronchial smooth muscle hyperplasia. Perinatal expression of IL-1beta in epithelial cells of the lung caused a lung disease that was clinically and histologically similar to BPD.     

Genotypic analysis of varicella-zoster virus and its seroprevalence in Finland

We evaluated the seroprevalence of varicella-zoster virus (VZV) in the Finnish population among various age groups and genetically characterized VZV strains from documented cases of varicella and zoster. VZV-specific immunoglobulin G was measured in 2,842 serum samples that had been submitted for virological studies to the Department of Virology, University of Helsinki, from 1995 to 1996. Specimens for VZV genotyping were obtained from vesicular lesions from two pediatric patients and 26 adult patients. Seroprevalence to VZV varied markedly by age: 45% in children aged < or = 2 months, 12.5% in children aged 6 to 8 months, and > 90% in children near 10 years of age, plateauing thereafter into advanced age. The seroprevalence rates indicate that in Finland, as in other countries with temperate climates, primary VZV infection usually occurs during the first decade of life. Twenty-eight VZV DNA-positive specimens were analyzed to identify VZV vaccine and wild-type genotypes. All analyzed specimens were wild type and the European (E) genotype.