Improved DOP-PCR-based representational whole-genome amplification using quantitative real-time PCR.

In many cases, only a minute amount of partially degraded genomic DNA can be extracted from archived clinical samples. Diverse whole-genome amplification methods are applied to provide sufficient amount of DNA for comparative genome hybridization, single-nucleotide polymorphism, and microsatellite analyses. In these applications, the reliability of the amplification techniques is particularly important. In PCR-based approaches, the plateau effect can seriously alter the original relative copy number of certain chromosomal regions. To eliminate this distorting effect, we improved the standard degenerate oligonucleotide-primed PCR (DOP-PCR) technique by following the amplification status with quantitative real-time PCR (QRT-PCR). With real-time detection of the products, we could eliminate DNA overamplification. Probes were prepared from 10 different tumor samples: primary and metastatic melanoma tissues, epidermoid and bronchioloalveolar lung carcinomas, 2 renal cell carcinomas, 2 colorectal carcinomas, and a Conn and Cushing adenoma. Probes were generated by using nonamplified and amplified genomic DNA with DOP-PCR and DOP-PCR combined with QRT-PCR. To demonstrate the reliability of the QRT-PCR based amplification protocol, altogether 152 relative copy number changes of 44 regions were determined. There was 85.6% concordance in copy number alterations between the QRT-PCR protocol and the nonamplified samples, whereas this value was only 63.8% for the traditional DOP-PCR. Our results demonstrate that our protocol preserves the original copy number of different chromosomal regions in amplified genomic DNA than standard DOP-PCR techniques more accurately.     

Genetic,household and spatial clustering of leprosy on an island in Indonesia: a population-based study

Background  

It is generally accepted that genetic factors play a role in susceptibility to both leprosy per se and leprosy type, but only few studies have tempted to quantify this. Estimating the contribution of genetic factors to clustering of leprosy within families is difficult since these persons often share the same environment. The first aim of this study was to test which correlation structure (genetic, household or spatial) gives the best explanation for the distribution of leprosy patients and seropositive persons and second to quantify the role of genetic factors in the occurrence of leprosy and seropositivity.     

Postextrasystolic regulation patterns of blood pressure and heart rate in patients with idiopathic dilated cardiomyopathy

Assessment of fluctuations in heart rate (HR) following a premature ventricular complex (PVC) is valuable for identifying patients at high risk of sudden cardiac death. We hypothesised that postextrasystolic potentiation is the main determinant of the regulation patterns of blood pressure (BP) and HR following a PVC. Twelve patients with idiopathic dilated cardiomyopathy (IDC) and 13 control subjects with single PVCs (comparable coupling intervals) were investigated. Non-invasive finger arterial BP and ECGs were analysed. Regulation patterns following a single PVC were quantified using the indices postextrasystolic amplitude potentiation (PEAP) and maximum turbulence slope of five consecutive mean BP values (MBP-TS), and compared with the HR turbulence parameters turbulence slope (HR-TS) and turbulence onset (HR-TO). PEAP was significantly higher in IDC patients compared to controls (48.7 ± 32.6 vs. 9.8 ± 5.4 %, P < 0.01), whereas MBP-TS was lower (0.97 ± 0.60 vs. 2.07 ± 1.04 mmHg BBI⁻¹ (BBI, beat-to-beat interval), P < 0.05), as was HR-TS (8.46 ± 7.90 vs. 30.73 ± 22.90 ms BBI⁻¹, P < 0.01). HR-TO was significantly higher in IDC patients (−0.56 ± 2.19 vs. −5.52 ± 4.13 %, P < 0.01). In addition, the regulation patterns of BP and HR following a single PVC differed significantly between IDC patients and controls. Specifically, we observed pronounced PEAPs in IDC patients. The baroreflex response initiated by the low pressure amplitude of the PVC was suppressed in IDC patients due to the augmented potentiation of the first postextrasystolic blood pressure. Furthermore, IDC patients displayed impressive postextrasystolic pulsus alternans phenomena, whereas healthy subjects exhibited a typical baroreflex pattern. The pulsus alternans phenomenon seems to be triggered by a PVC.     

A differential efficiency of adenovirus-mediated in vivo gene transfer into skeletal muscle cells of different maturity

High titre (10¹¹–10¹² pfu/ml) suspensions of autonomouslyreplication-defective type 5 human adenovirus (AV) recombinantswith different reporter gene inserts (CMV-Luciferase (Lux),CMV-ß-galactosidase (Lac Z), RSV-Lux and RSV-Lac Z)were injected Into intact quadrlceps muscles of 1–5 dayold (Group 1) or 35–45 day old (Group 2) normal mice,as well as regenerating adult mouse muscles (Group 3) and 35day old mdx muscles (Group 4). The expression of the reportergenes was quantitated 10 days and 2 months later. At 10 dayspostinjection all reporter gene expression was very high inthe neonatally injected (Group 1) muscles. In Group 2 musclesthe transduction was markedly less. In Group 3 muscles the geneexpression was significantly better than in the Group 2 muscles.In adult mdx muscles (Group 4) where spontaneous regenerationis usually present, the results were similar to those in Group3 animals. At 2 months post-injection in Group 1 animals, theRSV-Lux expression was even higher than at 10 days postinjection.The cell surface density of     

Measurement of functional ability and health status in the arthritic patient

Chronic arthritis may have great impact on the patient but also on his or her family, relatives and friends. The assessment of the consequences of chronic arthritis and the effect of therapy not only in terms of physical, but also psychological and social dimensions deserves more attention. Functional ability and health status can be measured using a questionnaire or ‘instrument’, high-lighting important aspects not quantified with more traditional measurements. In this paper, arguments to apply such instruments more frequently are given. Health status instruments can be used not only to assess beneficial but also deleterious (side-)effects of therapeutic interventions. The properties are summarized of the most frequently used instruments assessing functional ability and health status. Many of these instruments have been evaluated sufficiently for validity and reliability; their sensitivity to detect change seems to be satisfactory. Therefore it is advisable to choose an internationally accepted, frequently used instrument, reflecting the area of interest.     

Possible Relation of p53 and mdm-2 Oncoprotein Expression in Thyroid Carcinoma: A Molecular-Pathological and Immunohistochemical Study on Paraffin-Embedded Tissue

Routinely processed tissues from a series of benign and malignant thyroid lesions were immunohistochemically investigated with antibodies against p53 and mdm-2. p53 was immunolocalized in <10% of nuclei in 2/80 nodular goiters, 2/60 follicular adenomas, 26/68 follicular carcinomas, 7/40 papillary carcinomas, 3/10 “insular” carcinomas, and 10/31 anaplastic carcinomas. More than 10% positively stained nuclei were found in 2 widely invasive follicular, 2 insular, and 15 anaplastic carcinomas. All p53-positive cases showed a concomitant immunohistochemical mdm-2 expression; an immunohistochemical colocalization on serial section was demonstrated in 12 anaplastic carcinomas. Screening by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of these 12 cases revealed no relevant mutations in the coding regions of exons 2–11 of the p53 gene. Additionally, 1 follicular adenoma, 6 follicular carcinomas (4 minimally and 2 widely invasive), 1 papillary, and 2 poorly differentiated insular carcinomas were mdm-2 positive without immunohistochemically detectable p53 expression. These results provide evidence that wild-type p53 expression in thyroid carcinomas may be associated with mdm-2 induced formation of stable complexes. However, the role of p53 mutations and p53 protein inactivation owing to other factors (e.g., mdm-2) in the progression of thyroid carcinomas is still poorly understood.     

Occurrence of hlyA and sheA genes in extraintestinal Escherichia coli strains

The association of a hemolytic phenotype with the carriage of the alpha-hemolysin gene (hlyA) and/or the silent hemolysin gene (sheA or clyA) among 540 extraintestinal clinical isolates of Escherichia coli and 110 fecal isolates from healthy individuals was investigated. Though HlyA is an important virulence factor in extraintestinal E. coli infection, the role of SheA is not completely clarified. Two hemolytic sheA+ E. coli strains that lacked hlyA and possessed no other hemolysin genes were identified. No hlyA+ sheA+ strains were identified, suggesting that there is possible incompatibility between hlyA and sheA in the chromosome of E. coli.