Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

Osteomyelitis caused by Enterobacter cancerogenus infection following a traumatic injury: case report and review of the literature

We report a case of osteomyelitis caused by Enterobacter cancerogenus resistant to aminopenicillins in a 56-year-old male who had a motorcycle accident and suffered from multiple bone fractures with abundant environmental exposure. E. cancerogenus has rarely been associated with human infections, and its clinical significance remains unclear.     

Validation of a pXO2-A PCR assay to explore diversity among Italian isolates of Bacillus anthracis strains closely related to the live, attenuated Carbosap vaccine

Several circulating Bacillus anthracis strains isolated in Italy and belonging to the A1.a cluster, genotype 3 (A1.a-3) are genotypically indistinguishable from Carbosap, a live attenuated vaccine strain, containing both pXO1 and pXO2 plasmids. The genotype was assessed by using eight-locus multilocus variable-number tandem repeat analysis. We describe here the use of a ninth locus able to explore variability among strains that have the same genotype. It is important to be able to genotype the wild isolate of B. anthracis strains from outbreaks of anthrax in areas where Carbosap vaccination of cattle and sheep is common practice. A total of 27 representative field strains isolated in Italy and four vaccinal strains, namely, Carbosap, Sterne, Pasteur I, and Pasteur II, were characterized by a ninth marker, called pXO2-A. Twenty-three field strains were genotype 3 and therefore identical to Carbosap. The marker was in the pXO2 plasmid and is based on the polymorphism of the already-known VX2-3 locus. Detection was obtained by PCR with fluorescence-labeled forward primers in order to produce appropriate fragments for capillary electrophoresis with an ABI 310 genetic analyzer. Genetic relationships showed heterogeneity in all of the examined samples. Interestingly, with respect to genotype 3, samples grouped into eight different subtypes, A to H, and the subtype G, had only two samples indistinguishable from Carbosap. The results of the present study confirm the validity of a hierarchical progressive protocol for discrimination among closely related isolates.     

Characterization of Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis.

We initially used 25 different random primers in order to test their ability to generate random amplified polymorphic DNA fragments from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. From the tested primers we chose five to distinguish between seven isolates of this microorganism. The DNA amplification patterns allowed clear differentiation of the seven isolates into two distinct groups with only 35% genomic identity. One of these groups contained two subgroups with 81% genetic similarity. The random amplified polymorphic DNA analysis method proved to be a good tool for analyzing and comparing different genomes of P. brasiliensis isolates.     

Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation.

The in vivo virulence patterns of promastigote populations defined on the basis of agglutination by the lectin peanut agglutinin (PNA) were studied for various cloned lines of Leishmania major. Promastigotes derived from logarithmic-phase cultures, which were routinely 100% agglutinated at 100 micrograms of PNA per ml, were relatively avirulent for BALB/c mice. The relative virulence of stationary-phase promastigotes appeared to be attributable to the proportion of nonagglutinable (PNA-) promastigotes contained within these populations. Purification of PNA- organisms from stationary cultures provided for each clone the most virulent inoculum, supporting the view that this change in lectin binding accurately reflects the development of infective metacyclic stage promastigotes. By studying this marker, we found that there was considerable variation in the degree to which different strains and clones underwent metacyclogenesis during growth. Examination of a reportedly avirulent L. major clone revealed that metacyclogenesis was unusually delayed and inefficient for this clone, but that those PNA- promastigotes which could be recovered from late-stationary-phase cultures were virulent for BALB/c mice. The loss of virulence associated with frequent subculture could also be attributed to a drastic diminution in metacyclogenesis potential over time. A clone which yielded over 90% PNA- promastigotes during growth within passage 1 generated fewer than 10% PNA- promastigotes during growth by passage 94. Subcloning of late-passage attenuated promastigotes yielded a clone for which no PNA- promastigotes could be generated during growth, and an infective population could not be derived from this clone. Thus, metacyclogenesis does not appear to be stable for even cloned lines of Leishmania promastigotes, and virulence comparisons between different strains and clones can be meaningfully made only if the metacyclic populations contained within the respective inocula are determined.     

T cell subpopulations in myocardial inflammatory infiltrates detected by confocal microscopy: dose dependence in mice treated with cyclophosphamide during acute Trypanosoma cruzi infection

In this article, we have characterized cell subpopulations found in the hearts of mice presenting acute Chagas' disease by immunocytochemistry and subjected to different schedules of an immunosuppressive therapy with cyclophosphamide (CY). In this comparative study, CY treatment with different doses was carried out before or after infection with Trypanosoma cruzi Y strain trypomastigotes, enabling us to discriminate the parasitemic kinetics and inflammatory processes in the heart, 12 d after infection. Animals treated with 200 mg/kg of CY 2 d before infection presented high parasitaemia as well as heavy inflammation and low parasite loads in the heart. Mice treated 5 d after infection with the same dose, developed the same parasitaemic peak but were not able to control it. Their heart did not present inflammation, but a high number of parasites could be seen. Animals treated with five 3 mg/kg doses of CY every other day presented heavy inflammatory reaction and low parasitaemia. In this group, as well as the one treated before infection, immunocytochemistry studies have shown predominance of CD8(+) T cells in the myocardium. On the other hand, mice treated with 200 mg/kg of CY 5 d after infection, presented small amounts of CD4(+) T cells while no CD8(+) could be found. These results have confirmed the dose dependence influence of this drug on the T cell populations in the inflammatory infiltrates as well as the importance of the schedule employed.     

Identification and Subcellular Localization of Neuronal Calcium Sensor-1 (NCS-1) in Human Neutrophils and HL-60 Cells

Secretion in neutrophils is thought to be regulated in different ways for the different granule types. Specific granules are endowed with proteins which are related to docking and fusion events and are absent on azurophilic granules. Furthermore, even if secretion of content from all neutrophil granules is a Ca(2+)-dependent process, a higher concentration of cytosolic calcium is required for azurophilic than for specific granule secretion. In this paper we show that human neutrophils and promyelocitic cells express neuronal calcium sensor-1 (NCS-1), a calcium binding protein involved in exocytosis in various cell types. Both mRNA and protein were found in mature cells and precursors. NCS-1 is shown to be mainly associated with azurophilic granules and, therefore could play an instrumental role in the calcium-dependent secretion of azurophilic granules.