Evaluation of PCR-restriction profile analysis and IS2404 restriction fragment length polymorphism and amplified fragment length polymorphism fingerprinting for identification and typing of Mycobacterium ulcerans and M. marinum

Mycobacterium ulcerans and M. marinum are emerging necrotizing mycobacterial pathogens that reside in common reservoirs of infection and exhibit striking pathophysiological similarities. Furthermore, the interspecific taxonomic relationship between the two species is not clear as a result of the very high phylogenetic relatedness (i.e., >99.8% 16S rRNA sequence similarity), in contrast to only 25 to 47% DNA relatedness. To help understand the genotypic affiliation between these two closely related species, we performed a comparative analysis including PCR restriction profile analysis (PRPA), IS2404 restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) on a set of M. ulcerans (n = 29) and M. marinum (n = 28) strains recovered from different geographic origins. PRPA was based on a triple restriction of the 3' end region of 16S rRNA, which differentiated M. ulcerans into three types; however, the technique could not distinguish M. marinum from M. ulcerans isolates originating from South America and Southeast Asia. RFLP based on IS2404 produced six M. ulcerans types related to six geographic regions and did not produce any band with M. marinum, confirming the previous findings of Chemlal et al. (K. Chemlal, K. DeRidder, P. A. Fonteyne, W. M. Meyers, J. Swings, and F. Portaels, Am. J. Trop. Med. Hyg. 64:270-273, 2001). AFLP analysis resulted in profiles which grouped M. ulcerans and M. marinum into two separate clusters. The numerical analysis also revealed subgroups among the M. marinum and M. ulcerans isolates. In conclusion, PRPA appears to provide a rapid method for differentiating the African M. ulcerans type from other geographical types but is unsuitable for interspecific differentiation of M. marinum and M. ulcerans. In comparison, whole- genome techniques such as IS 2404-RFLP and AFLP appear to be far more useful in discriminating between M. marinum and M. ulcerans, and may thus be promising molecular tools for the differential diagnosis of infections caused by these two species.     

Identification of a nucleocapsid protein as a specific serological marker of human herpesvirus 6 infection.

Enveloped whole virions and nucleocapsids of human herpesvirus 6 (HHV-6) strain Z29 were purified from supernatant fluids of infected human cord blood lymphocytes by filtration through polyvinylpyrrolidone-treated filters, banding on a Nycondenz step gradient, and centrifugation through two successive continuous sucrose gradients. More than 20 proteins ranging in molecular weight from less than 30,000 to more than 200,000 were identified in preparations of purified whole virions labeled with [35S]methionine and [35S]cysteine. Immunogenic virion proteins of HHV-6 were identified in immunoblot assays with human immune sera, immune sera generated from mice immunized with purified whole virions or purified nucleocapsids, and a monoclonal antibody generated from a mouse immunized with purified nucleocapsids. The sera and the monoclonal antibody reacted strongly with a 101-kilodalton protein in the immunoblots, suggesting that the protein is a component of the nucleocapsid. Human sera lacking HHV-6-specific antibodies and seropositive for one or more of the other human herpesviruses failed to react with this protein, indicating that it is a specific serologic marker for HHV-6 infection.     

A project to reduce the burden of diabetes in the African-American Community: Project DIRECT.

Project DIRECT (Diabetes Interventions Reaching and Educating Communities Together) is the first comprehensive community diabetes demonstration project in the United States in an African-American community. This article describes its intervention components and evaluation design. The development and implementation of Project DIRECT has included the community since the project''s beginning. Interventions are targeted in three areas: health promotion (improving diet and physical activity levels), outreach (improving diabetes awareness, detection of undiagnosed diabetes, and ensuring that persons with diabetes who are not receiving continuing diabetes care are integrated into the health-care system), and diabetes care (improving self-care, increasing access, and improving the quality of diabetes preventive care received within the health-care system). Evaluation will be internal (conducted by Project DIRECT staff to assess process outcomes in persons directly exposed to each specific intervention) and external (review of outcomes to assess the impact of the multi-intervention program at the level of the entire community). Because diabetes exacts a disproportionate toll among African Americans, the findings from this project should aid in developing strategies to lessen the burden of this disorder, particularly among minority populations.     

Surfactant protein A enhances Mycobacterium avium ingestion but not killing by rat macrophages

Mycobacterium avium complex (MAC) is a significant cause of opportunistic infection in patients with acquired immunodeficiency syndrome. Although the major route of entry of MAC is via the gastrointestinal tract, MAC can infect humans through the respiratory tract and eventually encounter alveolar macrophages within the lung. Once in the lung, MAC can potentially interact with surfactant protein A (SP-A), an important component of the pulmonary innate-immune response. Previous work on other pulmonary pathogens including Mycobacterium bovis Bacillus Calmette-Guerin (BCG) suggests that SP-A participates in promoting efficient clearance of these organisms by alveolar macrophages. In the present study, we investigated the role of SP-A in clearance of MAC by cultured rat macrophages. SP-A bound to MAC organisms and enhanced the ingestion of the mycobacteria by macrophages. Infection of macrophages with SP-A-MAC complexes induced the production of nitric oxide (NO) and tumor necrosis factor-alpha. However, intracellular survival of MAC was not altered by preopsonization with SP-A. In addition, inhibitors of inducible NO synthase did not alter MAC clearance. These results suggest that SP-A can bind to and enhance the uptake of MAC by alveolar macrophages, similar to previous findings with BCG and Mycobacterium tuberculosis.However, unlike BCG and other pulmonary pathogens that are cleared effectively in the presence of SP-A via a NO-dependent pathway, macrophage-mediated clearance of MAC is not enhanced by SP-A.     

Assessment of the COBAS Amplicor HBV Monitor Test for quantitation of serum hepatitis B virus DNA levels

Treatment of patients with chronic hepatitis B virus (HBV) infections with potent antiviral therapy often results in dramatic reductions in the levels of viremia to very low levels. Monitoring of serum HBV DNA levels is a consistent method for the assessment of antiviral potency; however, widely used hybridization assays for the monitoring of HBV DNA levels have limited sensitivities and are not effective for the monitoring of patients whose serum HBV DNA levels have decreased to below approximately 700,000 HBV genomes/ml. The objective of the present study was to assess a PCR-based assay (the COBAS-AM assay) for quantitation of serum HBV DNA levels and to compare the results of the COBAS-AM assay with those of a solution hybridization assay with a radiolabeled probe. The precision and accuracy of the assay were determined with low-positive and high-positive controls consisting of a plasmid DNA molecule containing HBV-specific primer binding regions, and the sensitivity of the assay was determined by using serial dilutions of sera from subjects with chronic HBV infection. HBV DNA levels were quantitated in 1,695 serum samples from subjects with chronic HBV infection who were enrolled in clinical trials of lamivudine in North America or Asia. The COBAS-AM assay demonstrated high levels of inter- and intra-assay precision and accuracy, and the linear range of the COBAS-AM assay was greater than that of the solution hybridization assay. The assay is linear over a 3-log(10) range and is able to quantitate serum HBV DNA at levels 3 log(10) lower than those that can be detected by the solution hybridization assay. We found that the COBAS-AM assay is an accurate PCR-based assay for quantitation of serum HBV DNA levels in subjects with chronic HBV infection.     

Pregnancy with frozen-thawed and fresh testicular biopsy after motile and immotile sperm microinjection, using the mechanical touch technique to assess viability

BACKGROUND: There are few reports of pregnancy using immotile sperm, and none using a purely mechanical assessment of viability. METHODS: In this pilot study, we retrospectively analysed 66 cycles in 61 patients with determinant male factor, recording rates of fertilization, implantation, normal pregnancy and take-home babies achieved with ICSI. Sperm selection was based on morphologically normal appearance under the inverted microscope. Viability of immotile spermatozoa was assessed by the mechanical touch technique to observe tail flexibility and tail shape recovery. RESULTS: Of 17 ICSI cycles using frozen-thawed testicular sperm, six microinjected with immotile and 11 with motile sperm, we achieved fertilization rates of 65.7 and 74.3%, respectively, and five pregnancies (two and three, respectively). Of 49 ICSI cycles using fresh testicular sperm, 10 microinjected with immotile and 39 with motile sperm, we achieved fertilization rates of 73.4 and 64.4%, respectively, and 12 pregnancies (three and nine, respectively). CONCLUSIONS: Immotile (fresh and frozen-thawed) testicular sperm of normal morphological appearance can be used to achieve clinical pregnancy with ICSI. Our results strongly suggest that immotile sperm viability can be assessed by the mechanical touch technique.     

Adaptive Wiener Filtering for Improved Acquisition of Distortion Product Otoacoustic Emissions

An innovative acoustic noise canceling method using adaptive Wiener filtering (AWF) was developed for improved acquisition of distortion product otoacoustic emissions (DPOAEs). The system used one microphone placed in the test ear for the primary signal. Noise reference signals were obtained from three different sources: (a) pre-stimulus response from the test ear microphone, (b) post-stimulus response from a microphone placed near the head of the subject and (c) post-stimulus response obtained from a microphone placed in the subjects nontest ear. In order to improve spectral estimation, block averaging of a different number of single sweep responses was used. DPOAE data were obtained from 11 ears of healthy newborns in a well-baby nursery of a hospital under typical noise conditions. Simultaneously obtained recordings from all three microphones were digitized, stored and processed off-line to evaluate the effects of AWF with respect to DPOAE detection and signal-to-noise ratio (SNR) improvement. Results show that compared to standard DPOAE processing, AWF improved signal detection and improved SNR. © 1998 Biomedical Engineering Society. PAC98: 4364Jb, 4360-c, 8790+y     

Multiscale structure of sheet nacre

This work was conducted on Pinctada maxima nacre (mother of pearl) in order to understand its multiscale ordering and the role of the organic matrix in its structure. Intermittent-contact atomic force microscopy with phase detection imaging reveals a nanostructure within the tablet. A continuous organic framework divides each tablet into nanograins. Their shape is supposed to be flat with a mean extension of 45nm. TEM performed in the darkfield mode evidences that at least part of the intracrystalline matrix is crystallized and responds like a 'single crystal'. The tablet is a 'hybrid composite'. The organic matrix is continuous. The mineral phase is thus finely divided still behaving as a single crystal. It is proposed that each tablet results from the coherent aggregation of nanograins keeping strictly the same crystallographic orientation thanks to a hetero-epitaxy mechanism. Finally, high-resolution TEM performed on bridges from one tablet to the next, in the overlying row, did not permit to evidence a mineral lattice but crystallized organic bridges. The same organic bridges were evidenced by SEM in the interlaminar sequence.     

Alteration of the LRP1B gene region is associated with high grade of urothelial cancer

We have delineated regions of interest at chromosome 2q21.2, 2q36.3, and 2q37.1 by deletion mapping of 114 urothelial cancers (UC). Altogether, 17%, 18%, and 63% of the G1, G2, and G3 tumors displayed loss of heterozygosity at chromosome 2q, respectively, The region at 2q21.2 was narrowed down to the LRP1B gene (NT_005129.6). Hemi- and homozygous deletion at the LRP1B gene region was seen in 31 of 114 UCs. Only 8% of the UCs with G1 and none with G2 tumors showed loss of heterozygosity at the LRP1B gene, whereas 49% of the G3 UCs had allelic loss at this region. RT-PCR analysis of the LRP1B gene showed the lack of expression of several exons in 2 of 9 cases analyzed. Our analysis suggests that the LRP1B gene is a candidate tumor suppressor gene in UCs.