Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.     

Real-time automatic extraction of lumen region and boundary from endoscopic images

A new approach to the automatic extraction of the lumen region and its boundary for gastrointestinal (GI) endoscopic images is presented. First, a quasi region of interest, the darker regions of the image, is segmented using a region splitting scheme termed progressive thresholding. The centre of mass of this segmented region acts as a seed for further processing. Then the lumen region is obtained using a region growing technique called the integrated neighbourhood search (INS). A new quad structure based technique is introduced to enhance the INS speed significantly. A back projection algorithm is suggested to optimise the search for pixels belonging to the lumen region and boundary. A boundary-thinning algorithm is also proposed to remove the redundant pixels from the lumen boundary and to generate a connected single pixel width boundary. The proposed approach does not need a priori knowledge about the image characteristics. The experimental results indicate that the proposed technique enhances the speed of conventional INS by 45.5% to 28.6% based on the lumen size varying from 22,709 pixels to 4947 pixels. The main advantage of the proposed technique is its high-speed response that facilitates real-time analysis of endoscopic images.     

Outcome of surgical treatment for early adenocarcinoma of the esophagus or gastro-esophageal junction

Adenocarcinoma of the esophagus, or GEJ, has a poor prognosis. Early lesions [i.e. high grade dysplasia (HGD) or T1-carcinoma] are potentially curable. Local endoscopic therapies are promising treatment options for superficial lesions; however, for deeper lesions, surgical resection is considered to be the treatment of choice. To contribute to therapeutic decision-making, we retrospectively analysed the outcome of transhiatal esophagectomy in 120 patients with pathologically proven HGD (n=13) or T1-adenocarcinoma (n=107) of the distal esophagus or gastro-esophageal junction (GEJ). Tumors were subdivided into six different depths of invasion (T1-mucosal m1-m3, T1-submucosal sm1-sm3), and the frequency of lymphatic dissemination and time to locoregional and/or distant recurrence were analysed. Only one of the 79 T1m1-3/sm1 tumors (1%) showed lymph node metastases as compared with 18 out of 41 T1sm2-3 tumors (44%). There was a significant difference in recurrence-free period between T1m1-m3/sm1 versus T1sm2-sm3 tumor patients (P log rank <0.0001), with 5-year recurrence-free percentages of 97% and 57%, respectively. In multivariate analysis including age, gender, tumor differentiation grade, N-stage and depth of invasion, only N-stage was an independent prognostic factor for recurrence-free period (hazard rate=5.9, 95% CI 1.7–20.7). However, if N-stage was excluded from analysis, only depth of invasion (T1sm2-3 versus T1m1-m3/sm1) was an independent prognostic factor for recurrence-free period (hazard rate=7.5, 95% CI 2.0–27.7). These data indicate that T1m1-m3/sm1 adenocarcinomas of esophagus or GEJ show a very low risk of lymphatic dissemination and are therefore eligible for local endoscopic therapy. After transhiatal surgical resection, almost half of the patients with T1sm2-sm3 lesions develop recurrent disease within 5 years, and therefore need additional therapy to improve survival.     

Early atherosclerotic lesions in infancy: role of parental cigarette smoking

Cigarette smoking is associated with an increased incidence of atherosclerotic diseases. The aim of this study was to examine the progression of the preatherosclerotic lesions previously observed by us in coronary arteries of fetuses of smoker mothers and in infants with smoker parents. We examined the coronary arteries of 34 infants, aged 1–36 months, and the histological and biological [c-fos, proliferating cell nuclear antigen (PCNA), and apoptosis] features of the early atherosclerotic lesions. In 17 infants (50%), at least one parent smoked, generally more than five cigarettes a day. In 18 cases (53%), we observed variable thickening of the coronary walls from preatherosclerotic lesions to juvenile atherosclerotic plaques, related to parental smoking habit. This morphological progression of the lesions was accompanied by a sequence of biological changes in the smooth muscle cells of the tunica media. We suggest that the oxidants present in the gas phase of the parental cigarette smoke pass through the endothelium and induce at first the c-fos gene activation and subsequently the PCNA positivity, that is, a proliferative process.     

Experimental rat model for therapeutic retinal pigment epithelium transplantation—unequivocal microscopic identification of human donor cells by in situ hybridisation of human-specific Alu sequences

Transplantation of retinal pigment epithelial (RPE) cells is discussed as a possible therapeutic approach for retinal degeneration. Xenogeneic transplantation of human RPE cells in animal models has been studied extensively. Various methods have been used to identify the graft cells, but these methods interfere with cell behaviour so that the monitored physiological post-transplantation course may be influenced. In the present study, we applied a method for an unequivocal identification of the graft cells without interfering cell metabolism or behaviour using in situ hybridisation (ISH) of human specific Alu sequences. Visualisation of the strong extended nuclear signal of Alu sequences was much easier than that of the small nuclear signals of donor-specific sex chromosome probes. With Alu probe, even single graft cells can be identified and their development can be observed in short-term and long-term studies. With this procedure, we could prove that donor cells were injected correctly into the subretinal space by a special injection technique that we developed previously. In combination with immunohistochemistry, donor cells could be clearly discriminated from macrophages, which contained phagocytosed donor cell fragments. Application of these ISH methods for species-specific identification was valuable for follow-up-studies of RPE transplantation.     

An enzyme-linked immunosorbent assay (ELISA) for the major crustacean allergen, tropomyosin, in food

Shellfish are a common cause of food reactions in hypersensitive individuals and are among the eight foods that account for over 90% of food allergies. At present, the only way to prevent these serious consequences of food allergies is to avoid the foods that trigger the reactions. A sandwich-ELISA kit has been developed for the detection of crustacean meat in food, based on the major heat-stable shellfish allergen, tropomyosin. Tropomyosin was purified from whole prawn (Penaeus latisulcatus) and used to immunize rabbits after confirming its identity by MALDI-TOF MS. A sandwich-ELISA based on the rabbit antibodies takes less than 2 h to perform, including the food extraction, and has a detection limit of 1 ppm crustacean (prawn, lobster), without detectable cross-reactivity with fish or mammalian meat.     

Novel protein microarray for the detection of avian influenza virus antibodies and simultaneous distinction of antibodies against H5 and H7 subtypes

ABSTRACT

Avian influenza virus (AIV) can cause serious zoonotic disease, thereby threatening the poultry industry and human health. An efficient and rapid detection approach is crucial to prevent and control the spread of avian influenza. In this study, a novel protein microarray was developed. Haemagglutinin proteins of H5 and H7 subtypes and nucleoprotein (NP) were purified and spotted onto the initiator-integrated poly-(dimethylsiloxane) as antigens. Monoclonal antibodies with inhibition effect were screened and utilized for the synchronous detection of three avian influenza antibodies in different species. In the protein microarray, the cut-off values were 40%, 50% and 30% inhibition for H5 antibody detection; 50%, 50% and 20% for NP antibody detection; 40%, 50% and 40% for H7 antibody detection in chicken, peacock and duck sera, respectively. The 95 serum samples were detected by microarray, and results were compared with the findings of AIV antibody test enzyme-linked immunosorbent assay (ELISA) or haemagglutination inhibition (HI) test. NP antibody detection in the microarray showed 100% (55/55) agreement ratio in chicken using ELISA. Compared with HI, H5 antibody detection in the microarray showed 100% (95/95) agreement ratio in chicken, peacock and duck, whilst those of H7 displayed 98.18% (54/55) agreement in chicken, 100% (20/20) in peacock and 90% (18/20) in duck. In conclusion, this novel protein microarray is a high-throughput and specific method for the detection of AIV antibodies and simultaneous distinction of antibodies against H5 and H7 subtypes. It can be applied to the serological diagnosis and epidemiological investigation of AIV.

RESEARCH HIGHLIGHTS

• A novel protein microarray method has been developed.

• The microarray can detect AIV antibodies and distinguish between H5 and H7 subtypes.

• The study lays the foundation for simultaneous identification of multiple pathogens.