Spectrum of beta-thalassemia mutations in various regions of Punjab and Islamabad, Pakistan: establishment of prenatal diagnosis

We present here an analysis of 888 unrelated beta-thal chromosomes consisting of 444 transfusion dependent children from various regions of Punjab and Islamabad Pakistan. By using Multiplex ARMS- PCR, restriction endonuclease analysis, allele specific oligonucleotide (ASO) hybridization and sequencing, 17 beta-thal mutations and 3 Hb variants were detected in 99.5 % (884/888) of the chromosomes analyzed. First trimester prenatal diagnosis by chorionic villus sampling (CVS) was also carried out in seven pregnancies at risk of beta-thalassemia. Our results indicate that three most common mutations accounted for 86.8% of the beta-thal alleles in this region. These findings have important implications for prevention of beta-thalassemia through genetic counseling and prenatal diagnosis in this part of Pakistan.     

Rapid and accurate detection of monoclonal immunoglobulin heavy chain gene rearrangement by DNA melting curve analysis in the LightCycler System

The detection of immunoglobulin heavy chain gene rearrangement (IgH-R) is a standard tool for distinguishing polyclonal from monoclonal B-cell populations. Current DNA-based polymerase chain reactions (PCR) strategies can diagnose monoclonal IgH-R either by measuring the length of the amplicon or by detecting gel mobility variations owing to sequence-dependent conformational changes. However, amplification and analysis remain sequential operations usually requiring manual transfer. We have developed a novel PCR strategy for detecting monoclonal IgH-R that monitors fluorescence of the specific double-stranded DNA binding dye SYBR Green I during melting curve analysis using the LightCycler System. We compared polyacrylamide gel electrophoresis (PAGE) versus melting curve analysis in 130 clinical DNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues (mostly skin biopsies) of 128 patients. The identical FR3 primers were used to amplify the IgH variable region for both analytic techniques. We detected IgH-R in 24 DNA samples from FFPE tissue of 22 patients. Melting curve analysis, compared to PAGE, revealed no false negative and no false positive results, yielding both sensitivity and specificity equal to 100%. We also compared Southern blot analysis versus melting curve analysis in 23 clinical DNA samples from fresh-frozen lymph nodes of 23 patients. We detected IgH-R by melting curve analysis in 7 DNA samples from fresh-frozen lymph nodes. Melting curve analysis, compared to Southern blot analysis, revealed sensitivity equal to 58.3% (7 of 12) and specificity equal to 100% (11 of 11). We conclude that continuous fluorescence monitoring of PCR products with DNA melting curve analysis can rapidly and reproducibly distinguish polyclonal from monoclonal B-cell populations.     

Bacteriophages fEV-1 and fD1 Infect Yersinia pestis

Bacteriophages vB_YpeM_fEV-1 (fEV-1) and vB_YpeM_fD1 (fD1) were isolated from incoming sewage water samples in Turku, Finland, using Yersinia pestis strains EV76 and KIM D27 as enrichment hosts, respectively. Genomic analysis and transmission electron microscopy established that fEV-1 is a novel type of dwarf myovirus, while fD1 is a T4-like myovirus. The genome sizes are 38 and 167 kb, respectively. To date, the morphology and genome sequences of some dwarf myoviruses have been described; however, a proteome characterization such as the one presented here, has currently been lacking for this group of viruses. Notably, fEV-1 is the first dwarf myovirus described for Y. pestis. The host range of fEV-1 was restricted strictly to Y. pestis strains, while that of fD1 also included other members of Enterobacterales such as Escherichia coli and Yersinia pseudotuberculosis. In this study, we present the life cycles, genomes, and proteomes of two Yersinia myoviruses, fEV-1 and fD1.     

Onset of sexual maturation in female mice as measured in behavior and fertility: Interactions of exposure to males, phytoestrogen content of diet, and ano-genital distance

Age of puberty was examined in female mice through non-invasive behavioral and fertility measures, in relationship to ano-genital distance, phytoestrogen content of diet, and exposure to males post-weaning. Throughout gestation and post-natal development, females were exposed to a regular diet or one that was nutritionally similar but deficient in phytoestrogens. After segregation at weaning on the basis of a short or long ano-genital distance index (AGDI), an indirect measure of in utero androgen exposure, females were housed alone or underneath two outbred adult males for two weeks. Subsequently, an outbred male was placed in the cage of each developing female, and mating behavior, escape attempts, biting gestures, and boxing postures were recorded. Next, females were monitored for the occurrence of a copulatory plug and allowed to bear young, with pregnancy and litters monitored up to weaning. Male-exposed females fed a regular diet were immediately sexually receptive when housed directly with males, and their conceptions occurred earlier than did those of other females. Subjects fed a diet deficient in phytoestrogens were least likely to show sexual receptivity. Male-exposed females with longer AGDI displayed more escape attempts in the presence of males, regardless of diet. Once inseminated, most females carried to term and the majority of pups survived until weaning. These data suggest that phytoestrogens and AGDI interact with exposure to males in determining age at onset of puberty.