Mitochondrial genetics of Coprinus: Recombination of mitochondrial genomes

Summary The formation of the sexual mycelium or dikaryon in the basidiomycete Coprinus cinereus involves exchange and migration of nuclei without accompanying exchange of mitochondria. The dikaryotic growth which appears around the periphery of mated monokaryons has exclusively the mitochondrial genome of the recipient cells. Recombination of mitochondrial genomes is not, however, precluded during dikaryosis. Using monokaryons with different mitochondrial gene mutations, [acu-10] causing cytochrome aa ₃ deficiency and[cap-1.1] conferring resistance to chloramphenicol, it was shown that recombinant mitochondria arise in the zone of contact of mated monokaryons.     

Combined MEK Inhibition and BMP2 Treatment Promotes Osteoblast Differentiation and Bone Healing in Nf1Osx−/− Mice

Neurofibromatosis type I (NF1) is an autosomal dominant disease with an incidence of 1/3000, caused by mutations in the NF1 gene, which encodes the RAS/GTPase‐activating protein neurofibromin. Non‐bone union after fracture (pseudarthrosis) in children with NF1 remains a challenging orthopedic condition to treat. Recent progress in understanding the biology of neurofibromin suggested that NF1 pseudarthrosis stems primarily from defects in the bone mesenchymal lineage and hypersensitivity of hematopoietic cells to TGFβ. However, clinically relevant pharmacological approaches to augment bone union in these patients remain limited. In this study, we report the generation of a novel conditional mutant mouse line used to model NF1 pseudoarthrosis, in which Nf1 can be ablated in an inducible fashion in osteoprogenitors of postnatal mice, thus circumventing the dwarfism associated with previous mouse models where Nf1 is ablated in embryonic mesenchymal cell lineages. An ex vivo–based cell culture approach based on the use of Nf1^flox/flox bone marrow stromal cells showed that loss of Nf1 impairs osteoprogenitor cell differentiation in a cell‐autonomous manner, independent of developmental growth plate–derived or paracrine/hormonal influences. In addition, in vitro gene expression and differentiation assays indicated that chronic ERK activation in Nf1‐deficient osteoprogenitors blunts the pro‐osteogenic property of BMP2, based on the observation that only combination treatment with BMP2 and MEK inhibition promoted the differentiation of Nf1‐deficient osteoprogenitors. The in vivo preclinical relevance of these findings was confirmed by the improved bone healing and callus strength observed in Nf1_osx^?/? mice receiving Trametinib (a MEK inhibitor) and BMP2 released locally at the fracture site via a novel nanoparticle and polyglycidol‐based delivery method. Collectively, these results provide novel evidence for a cell‐autonomous role of neurofibromin in osteoprogenitor cells and insights about a novel targeted approach for the treatment of NF1 pseudoarthrosis. © 2014 American Society for Bone and Mineral Research.     

Reversible skeletal abnormalities in gamma-glutamyl transpeptidase-deficient mice

Gamma-glutamyl transpeptidase (GGT) is a widely distributed ectopeptidase responsible for the degradation of glutathione in the gamma-glutamyl cycle. This cycle is implicated in the metabolism of cysteine, and absence of GGT causes a severe intracellular decrease in this amino acid. GGT-deficient (GGT-/-) mice have multiple metabolic abnormalities and are dwarf. We show here that this latter phenotype is due to a decreased of the growth plate cartilage total height resulting from a proliferative defect of chondrocytes. In addition, analysis of vertebrae and tibiae of GGT-/- mice revealed a severe osteopenia. Histomorphometric studies showed that this low bone mass phenotype results from an increased osteoclast number and activity as well as from a marked decrease in osteoblast activity. Interestingly, neither osteoblasts, osteoclasts, nor chondrocytes express GGT, suggesting that the observed defects are secondary to other abnormalities. N-acetylcysteine supplementation has been shown to reverse the metabolic abnormalities of the GGT-/- mice and in particular to restore the level of IGF-1 and sex steroids in these mice. Consistent with these previous observations, N-acetylcysteine treatment of GGT-/- mice ameliorates their skeletal abnormalities by normalizing chondrocytes proliferation and osteoblastic function. In contrast, resorbtion parameters are only partially normalized in GGT-/- N-acetylcysteine-treated mice, suggesting that GGT regulates osteoclast biology at least partly independently of these hormones. These results establish the importance of cysteine metabolism for the regulation of bone remodeling and longitudinal growth.     

Bone mass regulation by leptin: a hypothalamic control of bone formation

Bone mass is maintained constant between puberty and menopause by the balance between osteoblasts and osteoclasts activity. The existence of a hormonal control of osteoblast activity has been speculated for years by analogy to osteoclast biology. Through the search for such humoral signal(s) regulating bone formation, leptin has been identified as a powerful inhibitor of bone formation. Furthermore, by means of intracerebroventricular infusion of leptin, it has been shown that the effect of this adipocyte-derived hormone on bone is mediated via a brain relay, like all its other functions. Subsequent studies have led to the identification of hypothalamic neurons involved in leptin's antiosteogenic function. In addition, it has been shown that those neurons or neuronal pathways are distinct from neurons responsible for the regulation of energy metabolism. Finally, the peripheral mediator of leptin's antiosteogenic function has been identified as being the sympathetic nervous system. Catecholamine-deficient mice have a high bone mass and sympathomimetics administered to mice decreased bone formation and bone mass. Conversely, beta-blockers increased bone formation and bone mass and blunt the bone loss induced by ovariectomy.     

Local low‐dose lovastatin delivery improves the bone‐healing defect caused by Nf1 loss of function in osteoblasts

Postfracture tibial nonunion (pseudoarthrosis) leads to lifelong disability in patients with neurofibromatosis type I (NF1), a disorder caused by mutations in the NF1 gene. To determine the contribution of NF1 in bone healing, we assessed bone healing in the Nf1 conditional mouse model lacking Nf1 specifically in osteoblasts. A closed distal tibia fracture protocol and a longitudinal study design were used. During the 21‐ to 28‐day postfracture period, callus volume, as expected, decreased in wild‐type but not in Nf1 mice, suggesting delayed healing. At these two time points, bone volume (BV/TV) and volumetric bone mineral density (vBMD) measured by 3D micro–computed tomography were decreased in Nf1 callus‐bridging cortices and trabecular compartments compared with wild‐type controls. Histomorphometric analyses revealed the presence of cartilaginous remnants, a high amount of osteoid, and increased osteoclast surfaces in Nf1 calluses 21 days after fracture, which was accompanied by increased expression of osteopontin, Rankl, and Tgfβ. Callus strength measured by three‐point bending 28 days after fracture was reduced in Nf1 versus wild‐type calluses. Importantly, from a clinical point of view, this defect of callus maturation and strength could be ameliorated by local delivery of low‐dose lovastatin microparticles, which successfully decreased osteoid volume and cartilaginous remnant number and increased callus BV/TV and strength in mutant mice. These results thus indicate that the dysfunctions caused by loss of Nf1 in osteoblasts impair callus maturation and weaken callus mechanical properties and suggest that local delivery of low‐dose lovastatin may improve bone healing in NF1 patients. © 2010 American Society for Bone and Mineral Research     

International quality assurance study for characterization of Streptococcus pyogenes

Surveillance of group A streptococcal (GAS) infections was undertaken as a major component of the European Commission-funded project on severe GAS disease in Europe (strep-EURO). One aim of strep-EURO was to improve the quality of GAS characterization by standardization of methods. An external quality assurance study (EQA) was therefore carried out to evaluate current global performance. Eleven strep-EURO and seven other streptococcal reference centers received a panel of 20 coded GAS isolates for typing. Conventional phenotypic typing (based on cell surface T and M protein antigens and opacity factor [OF] production) and molecular methods (emm gene typing) were used either as single or combined approaches to GAS typing. T typing was performed by 16 centers; 12 centers found one or more of the 20 strains nontypeable (typeability, 89%), and 11 centers reported at least one incorrect result (concordance, 93%). The 10 centers that tested for OF production achieved 96% concordance. Limited availability of antisera resulted in poor typeability values from the four centers that performed phenotypic M typing (41%), three of which also performed anti-OF typing (typeability, 63%); however, concordance was high for both M (100%) and anti-OF (94%) typing. In contrast, the 15 centers that performed emm gene sequencing achieved excellent typeability (97%) and concordance (98%), although comparison of the performance between centers yielded typeability rates from 65 to 100% and concordance values from 83 to 100%. With the rapid expansion and use of molecular genotypic methods to characterize GAS, continuation of EQA is essential in order to achieve international standardization and comparison of type distributions.     

The ortho-nitrophenol (ONPG) test and acid from lactose in Gram-negative genera

The results are given of the ortho-nitrophenol test for beta-galactosidase production (ONPG test) on 588 strains of 123 aerobic species of bacteria, representing 30 genera. Apart from some strains of Erwinia herbicola (synonym Chromobacterium typhiflavum) and of Yersinia spp, these strains were not members of the Enterobacteriaceae, in which family the ONPG test is widely used and well documented. The strains were also tested for acid production from 1, 5, and 10% lactose and the findings are discussed in relation to the ONPG test.     

Epidemiology of severe Streptococcus pyogenes disease in Europe

The past 2 decades have brought worrying increases in severe Streptococcus pyogenes diseases globally. To investigate and compare the epidemiological patterns of these diseases within Europe, data were collected through a European Union FP-5-funded program (Strep-EURO). Prospective population-based surveillance of severe S. pyogenes infection diagnosed during 2003 and 2004 was undertaken in 11 countries across Europe (Cyprus, the Czech Republic, Denmark, Finland, France, Germany, Greece, Italy, Romania, Sweden, and the United Kingdom) using a standardized case definition. A total of 5,522 cases were identified across the 11 countries during this period. Rates of reported infection varied, reaching 3/100,000 population in the northern European countries. Seasonal patterns of infection showed remarkable congruence between countries. The risk of infection was highest among the elderly, and rates were higher in males than in females in most countries. Skin lesions/wounds were the most common predisposing factor, reported in 25% of cases; 21% had no predisposing factors reported. Skin and soft tissue were the most common foci of infection, with 32% of patients having cellulitis and 8% necrotizing fasciitis. The overall 7-day case fatality rate was 19%; it was 44% among patients who developed streptococcal toxic shock syndrome. The findings from Strep-EURO confirm a high incidence of severe S. pyogenes disease in Europe. Furthermore, these results have identified targets for public health intervention, as well as raising awareness of severe S. pyogenes disease across Europe.     

Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of Corynebacterium diphtheriae Strains

Sixty-six Corynebacterium diphtheriae strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian C. diphtheriae strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of C. diphtheriae (PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of C. diphtheriae strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates.