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.     

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.     

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.     

Insulin and orthovanadate stimulate multiple phosphotyrosine-containing serine kinases

Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transfected with a human insulin receptor cDNA construct, we have studied an insulin-sensitive serine kinase activity. This activity is rapidly stimulated by insulin (maximum within 5 min) and also by orthovanadate. During cell extract preparation, paranitrophenylphosphate and phosphotyrosine are able to preserve the enzyme activity, while phosphothreonine and phosphoserine fail to do so. Using antiphosphotyrosine antibodies, specific immunoprecipitation of this insulin- and orthovanadate-sensitive serine kinase was obtained. We then analysed by gel filtration chromatography eluates containing tyrosine-phosphorylated proteins obtained from unstimulated, insulin- and vanadate-treated cells. We found that several activities, with molecular weights estimated to be 30 kDa and smaller, are stimulated by both, insulin and orthovanadate. As a whole, our data indicate that insulin and orthovanadate enhance the cytosolic content in at least 2 or 3 phosphotyrosine-containing serine kinase activities.Abbreviations EGF Epidermal Growth Factor - IGF I Insulin-like Growth Factor I - PDGF Platelet-Derived Growth Factor - DMEM Dulbecco's Modified Eagle's Medium - FCS Fetal Calf Serum - PBS Phosphate Buffered Saline - PNPP Para-nitrophenylphosphate - BSA Bovine Serum Albumin - -Tyr Antiphosphotyrosine Antibodies - MAP 2 Microtubule-Associated Protein 2 - Hepes N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - EDTA Ethylenediamine Tetraacetic Acid - DTT Dithiothreitol - SDS-PAGE Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis - EGTA [Ethylenebis(oxyethylenenitrilo)] Tetraacetic Acid - TRIS Tris(hydroxymethyl)-Aminoethane - IRSK Insulin Receptor-Associated Serine Kinase - KIK Kemptide Insulin-stimulated Kinase     

Rhinovirus 3C protease catalyzes efficient cleavage of a fluorescein-labeled peptide affording a rapid and robust assay.

The 3C protease encoded by human rhinovirus type 2 catalyzes with equal efficiency cleavage of a peptide substrate with or without a fluorescein label attached to the amino acid at the P7' position. Substrates Ac-MEALFQGPLQYKDL-NH2 and MEALFQGPLQYKE(fluorescein)L are hydrolyzed with values of Vmax/KM of 970 M-1 s-1 and 1100 M-1 s-1, respectively. With the labeled substrate, HPLC achieves separation of substrate and product in 2.5 min. Separation in as little as 12 s is feasible. Fluorescein was derivatized so that it could be incorporated into peptides using automated solid-phase peptide synthesis.     

Ecological comparison of the pre- and post-impoundment macrophyte flora of the river Niger and Lake Kainji,Nigeria

The floristic composition of the macrophyte vegetation of Lake Kainji was investigated and compared with the vegetation of the river Niger before impoundment.Of 18 hydatophytes, 40 tenagophytes and 57 trichophytes listed for the Niger, 18 tenagophytes, 12 trichophytes and 7 hydatophytes were found growing in the lake 17 years after impoundment. Fourteen new tenagophytes, 3 new hydatophytes and 1 new trichophyte were recorded. The hydatophytes which were expected to colonize the lake are limited in number and range.The failure of the hydatophytes to colonize the lake, as predicted by Cook (1968), is discussed in relation to the inflow-to-volume ratio of the lake.Nomenclature follows Hutchinson & Dalziel (1952–1972). Flora of West Tropical Africa.     

Traditional preparation and uses of cassava in Nigeria

Various methods of cassava preparation are practised by different ethnic groups in Nigeria. These methods involve peeling cassava roots, soaking roots in streams, grating cassava, and pressing grated cassava. Other methods include heating sieved, grated cassava, boiling peeled cassava roots, and pounding boiled or dried cassava roots. The traditional, cassava-based products aregari, fufu, akpu, cassava flour, edible starch, and tapioca. Detoxification of fresh cassava roots is partly achieved through cell rupture during cutting and grating, soaking in running or standing water in earthen pots for 3–5 days, heating, drying, and boiling.     

Separate Branches of the uvr Gene-Dependent Excision Repair Process in Ultraviolet-Irradiated Escherichia coli K-12 Cells; Their Dependence upon Growth Medium and the polA, recA, recB, and exrA Genes

The extent of repair of single-strand breaks (incision breaks) induced in the deoxyribonucleic acid (DNA) of Escherichia coli K-12 cells by the uvr gene-dependent excision repair process after ultraviolet (UV) radiation was determined in the wild-type, polA1, recA56, recB21, and exrA strains. The wild-type strain repaired all incision breaks after incident doses of UV radiation (254 nm) of approximately 60 J m(-2) or less when incubated in growth medium, or approximately 15 J m(-2) or less when incubated in buffer. The polA1 strain repaired the incision breaks completely after incident doses of approximately 12 J m(-2) or less when incubated in growth medium, or after approximately 4 J m(-2) when incubated in buffer. The recA13, recB21, and exrA strains showed essentially complete repair after incident doses of 10 to 15 J m(-2) whether the cells were incubated in buffer or growth medium. These results suggest that the uvr gene-dependent excision repair process may be divided into two branches, one which is dependent on the presence of growth medium and also the rec(+)exr(+) genotype, and a second which can occur in buffer (growth medium-independent) and is largely dependent on DNA polymerase I. The presence of chloramphenicol in the growth medium resulted in an inhibition of the growth medium-dependent repair occurring in wild-type and polA1 cells and had little or no effect on the extent of repair observed in recA56, recB21, or exrA cells. The similarities between the growth medium-dependent and -independent branches of excision repair and two known processes for the repair of X-ray-induced single-strand breaks are discussed.