Inhibition of GSK-3β Rescues the Impairments in Bone Formation and Mechanical Properties Associated with Fracture Healing in Osteoblast Selective Connexin 43 Deficient Mice

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/ Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.     

Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Red-breasted goose colonies on the Taimyr Peninsula: Factors responsible for the proximity of goose nests to nests of peregrine falcons,rough-legged buzzards,and snowy owls

Red-breasted goose colonies have been studied near Medusa Bay (73°21′N, 80°32′E), on the northwestern Taimyr Peninsula, and along the Agapa River (70°11′N, 86°15′E) down to its mouth (71°26′N, 89° 13′E), in the central Taimyr Peninsula. Red-breasted geese nesting near peregrine falcons are protected by the falcons from arctic foxes; however, they are sometimes attacked by the falcons themselves. In the colonies near peregrine falcon nests, the vast majority of goose nests were situated no farther than 100 m from the falcon nest. When food is abundant, falcons protect a larger area around their nest. The distance between the falcon nest and the surrounding goose nests is inversely related to the falcon’s activity. In years of higher falcon activity, falcons prevent red-breasted geese from nesting as close to their nest as in years of lower falcon activity. Additional stimuli are required for red-breasted geese to form colonies near rough-legged buzzard nests. The distance between snowy owl nests and red-breasted goose nests was smaller when arctic foxes were abundant than when they were scarce.     

Properties of carboxypeptidase A-treated chicken gizzard tropomyosin

Chicken gizzard tropomyosin was digested with carboxypeptidase A at the weight ratios of enzyme to substrate 1:200 and 1:50. Removal of about 16 C-terminal amino acid residues per tropomyosin molecule, at lower enzyme concentration, caused reversion of the effect on skeletal actomyosin ATPase activity from activating to inhibiting without an influence on polymerizability and actin-binding ability. Removal of about 26 C-terminal amino acid residues per molecule, at higher enzyme concentration, resulted in loss of polymerizability and actin binding ability. Digestion of gizzard tropomyosin with carboxypeptidase A has no dramatic effect on its binding to troponin T. The results show that not only the existence of head-to-tail overlapping regions but also their length is important for the functional properties of chicken gizzard tropomyosin.     

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c₁. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we noticed a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.     

Sex-dependent effect of melatonin on the secretory activity of rat and hamster adrenal gland in vitro

Adrenal quarters from adult male or female hamsters were incubated in the presence of melatonin (10(-7) or 10(-4)M), and cortisol concentration in the incubation medium was assayed by RIA. Melatonin did not change cortisol output by adrenals obtained from the male hamsters, while a slight stimulatory effect was observed in female glands, the lower concentration of melatonin being more effective than the higher one. At both concentrations tested, melatonin notably stimulated corticosterone output by isolated rat adrenocortical cells derived from the males, and lowered corticosterone secretion by the cells obtained from the female glands only at a concentration of 10(-7) M. The lower concentration of melatonin increased ACTH (0.1 mU.ml-1)-stimulated corticosterone output by the cells of male and female rat adrenals. The pineal hormone was ineffective at a concentration of 10(-4) M, as well as in the presence of a higher dose of ACTH (1.0 mU.ml-1). These findings indicate a distinct sex-dependent effect of melatonin on in vitro cortisol and corticosterone production, and demonstrate that the modulatory effect of melatonin of the secretion of steroid hormones is more effective at lower concentrations.     

NINJA-OPS: Fast Accurate Marker Gene Alignment Using Concatenated Ribosomes

The explosion of bioinformatics technologies in the form of next generation sequencing (NGS) has facilitated a massive influx of genomics data in the form of short reads. Short read mapping is therefore a fundamental component of next generation sequencing pipelines which routinely match these short reads against reference genomes for contig assembly. However, such techniques have seldom been applied to microbial marker gene sequencing studies, which have mostly relied on novel heuristic approaches. We propose NINJA Is Not Just Another OTU-Picking Solution (NINJA-OPS, or NINJA for short), a fast and highly accurate novel method enabling reference-based marker gene matching (picking Operational Taxonomic Units, or OTUs). NINJA takes advantage of the Burrows-Wheeler (BW) alignment using an artificial reference chromosome composed of concatenated reference sequences, the “concatesome,” as the BW input. Other features include automatic support for paired-end reads with arbitrary insert sizes. NINJA is also free and open source and implements several pre-filtering methods that elicit substantial speedup when coupled with existing tools. We applied NINJA to several published microbiome studies, obtaining accuracy similar to or better than previous reference-based OTU-picking methods while achieving an order of magnitude or more speedup and using a fraction of the memory footprint. NINJA is a complete pipeline that takes a FASTA-formatted input file and outputs a QIIME-formatted taxonomy-annotated BIOM file for an entire MiSeq run of human gut microbiome 16S genes in under 10 minutes on a dual-core laptop.