DETECTING PORCN MICRODELETIONS IN A LARGE FAMILY WITH FOCAL DERMAL HYPOPLASIA.

Focal dermal hypoplasia (FDH), an X-linked dominant disease with a highly variable phenotype, presents mainly with congenital linear pigmentation of the skin, herniation of fat through the dermal defects and multiple papillomas. PORCNmicrodeletions are identified in a total of 12 FDH patients to date. Routine molecular methods for detecting microdeletions have proven not to be effective, as patients also carry a normal allele. Additionally, methods using copy number estimations are labor-intensive, time-consuming and require expensive equipment. With respect to the molecular diagnosis of FDH, we aimed to investigate the inheritance of maternal disease allele in a three-generation FDH pedigree with seven affected members by using a simple yet efficient method. The strategy used in this study appeared to have the benefit of detecting all PORCN micro-deletions identified for FDH so far. The family with the largest number of related patients reported to date presented an opportunity to evaluate clinical variability, which was high, with the least affected and the most severely affected patients being half-sisters. The extensive intra-familial phenotypic variability observed in this FDH family suggests that genetic counselling should be part of management of this syndrome even in a family with a very mild case. The unique finding of IgA deficiency in the most severe case indicated that the feature could be a new characteristic of FDH.

Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.

Characterization of extracellular beta-lactamases from penicillin G-resistant cells of Streptococcus thermophilus.

In this study, biochemical properties of two extracellular beta-lactamases produced by penicillin-resistant Streptococcus thermophilus cells were investigated. Both beta-lactamases showed specificity for penicillins but not for cephaloridins. The beta-lactamases exhibited different affinities for penicillin G. The one with the higher molecular weight (FI) had a Km value of 3.44 microM and a Vmax value of 8.33 mumol/min/mg of protein, whereas the beta-lactamase with the lower molecular weight (FII) had a Km value of 4.76 microM and a Vmax value of 3.13 mumol/min/mg of protein. Both beta-lactamases were inhibited by iodine, copper sulfate, and iron sulfate but not by EDTA. The optimal pH ranged between 6 and 7, and the optimal temperatures were between 40 and 45 degrees C for both enzymes.

Freeze-Drying Injury of Lactobacillus Acidophilus.

Lactobacillus acidophilus ATCC 4355 cells were more stable to freezing at -20°C after supplementation of the growth medium with 0.1% CaCO3. However, added calcium did not affect viability and injury during lyophilization. Inclusion of 5% glycerol in the suspending medium before freeze-drying resulted in superior survival and decreased injury during the drying process.

Growth kinetics of Streptococcus thermophilus at subbacteriostatic penicillin G concentrations.

Streptococcus thermophilus may be subjected to the effects of penicillin G in contaminated milk used for yogurt production. Sensitivity of this microorganism to penicillin G has been conventionally determined by the help of penicillin G-impregnated disks placed on solid media. It was observed that the bacteriostatic penicillin G concentration was much greater in liquid media than in solid media. The conventional disk method may not be appropriate for antibiotic sensitivity determinations if the microorganisms will be used in liquid culture. A simple mathematical model simulated the growth of S. thermophilus in liquid culture. Numerical values of this model's parameters were regarded as the measure of the antibiotic effect on the culture. In penicillin G containing fresh medium, small concentrations of antibiotic decreased the specific growth rate considerably. Increasing the antibiotic concentration caused only slight additional decline. Antibiotic shock, i.e., rapidly introducing penicillin G into an actively growing antibiotic-free culture, stopped growth of the penicillin G-resistant microorganisms, and no death was observed, but a fraction of the microorganisms were killed in the wild culture. Both the wild and the resistant cultures recovered from the shock in a few hours. Addition of penicillin G-resistant microorganisms together with the antibiotic dosage into the wild culture prevented death.

Cardiovascular risk factors in patients with aortic stenosis predict prevalence of coronary artery disease but not of aortic stenosis: an angiographic pair matched case-control study.

BACKGROUND: Traditional cardiovascular risk factors have been associated with aortic stenosis and coronary artery disease. As these two conditions often co-exist, the association of cardiovascular risk factors with aortic stenosis may reflect confounding. OBJECTIVE: To compare the cardiovascular risk profile in patients with severe aortic stenosis undergoing elective coronary angiography with that of patients without aortic stenosis or calcification undergoing coronary angiography for suspected coronary artery disease. METHODS: 523 patients referred for elective diagnostic left heart catheterisation because of severe aortic stenosis formed the case population; 3925 patients without valve disease referred for elective diagnostic left heart catheterisation formed the base control population. Of the latter, 523 were pair matched to the case population for sex, age, and prevalence of relevant coronary artery disease, forming a pair matched control population. Cardiovascular risk factors (male sex, hypertension, hypercholesterolaemia, smoking, diabetes mellitus, family history of coronary artery disease) were assessed in all the patients. RESULTS: None of the cardiovascular risk factors was more prevalent in patients with aortic stenosis than in the base control population or in the pair matched control population. However, male sex, hypercholesterolaemia, smoking, diabetes mellitus, and a family history of coronary artery disease were significantly associated with the presence of additional coronary artery disease in patients with aortic stenosis. CONCLUSIONS: Cardiovascular risk factors are commonly present in patients with aortic stenosis. However, when compared with controls matched for age, sex, and angiographically defined coronary artery disease, no risk factor was significantly associated with the prevalence of aortic stenosis. Thus other factors are likely to be more important in the pathogenesis of aortic stenosis.