Mesenchymal stem cell responses to bone-mimetic electrospun matrices composed of polycaprolactone, collagen I and nanoparticulate hydroxyapatite

The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications.     

Genetic risk for earlier menarche also influences peripubertal body mass index

It is unclear whether earlier age at menarche is associated with higher body mass index (BMI) because they share a common genetic underpinning. We investigated the impact of single nucleotide polymorphisms (SNPs) influencing menarche timing on peripubertal BMI. For 556 Fels Longitudinal Study children (277 boys/279 girls) born 1928–1992, a genetic risk score (GRS₄₂) was computed as the sum of the number of risk alleles in 42 putative menarche SNPs. Serial BMI Z‐scores within ±6.99 years from each individual's age at peak height velocity (Age@PHV) were grouped into seven time points (?6 years, ?4 years, –2 years, Age@PHV, +2 years, +4years, and +6 years). Heritability of BMI ranged from 0.53 to 0.85 across the time points. The effect of GRS₄₂ on BMI Z‐scores at each time point was modeled using variance components‐based procedures. GRS₄₂ had a significant (P < 0.05) effect at every time point; an increase of one risk allele was associated with an increase of 0.03–0.08 BMI Z‐scores. A separate score (GRS₂₉) was computed that excluded 13 of the menarche SNPs previously documented to also influence adiposity; significant main effects were observed at Age@PHV+4 and +6 years. This finding supports a causal effect of advanced sexual development on post‐Age@PHV BMI. Significant positive GRS₄₂ (or GRS₂₉)‐by‐birth year interactions indicate that some genetic influences on BMI have amplified over the 20th century. This gene‐by‐environment interaction also suggests that children with a genetic predisposition to earlier sexual development might avoid elevated BMI through alteration of their nutritional environment. Am J Phys Anthropol, 2013. © 2012 Wiley Periodicals, Inc.     

Whole Genome SNP Genotyping and Exome Sequencing Reveal Novel Genetic Variants and Putative Causative Genes in Congenital Hyperinsulinism

Congenital hyperinsulinism of infancy (CHI) is a rare disorder characterized by severe hypoglycemia due to inappropriate insulin secretion. The genetic causes of CHI have been found in genes regulating insulin secretion from pancreatic β-cells; recessive inactivating mutations in the ABCC8 and KCNJ11 genes represent the most common events. Despite the advances in understanding the molecular pathogenesis of CHI, specific genetic determinants in about 50 % of the CHI patients remain unknown, suggesting additional locus heterogeneity. In order to search for novel loci contributing to the pathogenesis of CHI, we combined a family-based association study, using the transmission disequilibrium test on 17 CHI patients lacking mutations in ABCC8/KCNJ11, with a whole-exome sequencing analysis performed on 10 probands. This strategy allowed the identification of the potential causative mutations in genes implicated in the regulation of insulin secretion such as transmembrane proteins (CACNA1A, KCNH6, KCNJ10, NOTCH2, RYR3, SCN8A, TRPV3, TRPC5), cytosolic (ACACB, CAMK2D, CDKAL1, GNAS, NOS2, PDE4C, PIK3R3) and mitochondrial enzymes (PC, SLC24A6), and in four genes (CSMD1, SLC37A3, SULF1, TLL1) suggested by TDT family-based association study. Moreover, the exome-sequencing approach resulted to be an efficient diagnostic tool for CHI, allowing the identification of mutations in three causative CHI genes (ABCC8, GLUD1, and HNF1A) in four out of 10 patients. Overall, the present study should be considered as a starting point to design further investigations: our results might indeed contribute to meta-analysis studies, aimed at the identification/confirmation of novel causative or modifier genes.     

First Record of Culicoides oxystoma Kieffer and Diversity of Species within the Schultzei Group of Culicoides Latreille (Diptera: Ceratopogonidae) Biting Midges in Senegal

The Schultzei group of Culicoides Latreille (Diptera: Ceratopogonidae) is distributed throughout Africa to northern Asia and Australasia and includes several potential vector species of livestock pathogens. The taxonomy of the species belonging to this species group is confounded by the wide geographical distribution and morphological variation exhibited by many species. In this work, morphological and molecular approaches were combined to assess the taxonomic validity of the species and morphological variants of the Schultzei group found in Senegal by comparing their genetic diversity with that of specimens from other geographical regions. The species list for Senegal was updated with four species: Culicoides kingi, C. oxystoma, C. enderleini and C. nevilli being recorded. This is the first record of C. oxystoma from Africa south of Sahara, and its genetic relationship with samples from Israel, Japan and Australia is presented. This work provides a basis for ecological studies of the seasonal and spatial dynamics of species of this species group that will contribute to better understanding of the epidemiology of the viruses they transmit.     

ST6Gal-I regulates macrophage apoptosis via α2-6 sialylation of the TNFR1 death receptor

Macrophages play a central role in innate immunity, however mechanisms regulating macrophage survival are not fully understood. Herein we describe a novel apoptotic pathway involving α2-6 sialylation of the TNFR1 death receptor by the ST6Gal-I sialyltransferase. Variant glycosylation of TNFR1 has not previously been implicated in TNFR1 function, and little is known regarding the TNFR1 glycan composition. To study sialylation in macrophages, we treated U937 monocytic cells with PMA, which stimulates both macrophage differentiation and apoptosis. Interestingly, macrophage differentiation induces ST6Gal-I down-regulation, leading to reduced α2-6 sialylation of selected receptors. To prevent loss of α2-6 sialylation, we forced constitutive expression of ST6Gal-I, and found that this strongly inhibited PMA-induced apoptosis. Given that PMA-mediated apoptosis is thought to result from up-regulation of TNFα, which then activates TNFR1, we next evaluated the α2-6 sialylation of TNFR1. U937 cells with forced ST6Gal-I displayed TNFR1 with elevated α2-6 sialylation, and this was associated with diminished TNFα-stimulated apoptosis. Correspondingly, removal of α2-6 sialylation from TNFR1 through either neuraminidase treatment or expression of ST6Gal-I shRNA markedly enhanced TNFα-mediated apoptosis. To confirm the physiologic importance of TNFR1 sialylation, we generated overexpressing ST6Gal-I transgenic mice. Peritoneal macrophages from transgenic lines displayed TNFR1 with elevated α2-6 sialylation, and these cells were significantly protected against TNFα-stimulated apoptosis. Moreover, greater numbers of thioglycollate-induced peritoneal cells were observed in transgenic mice. These collective results highlight a new mechanism of TNFR1 regulation, and further intimate that loss of α2-6 sialylation during macrophage differentiation may limit macrophage lifespan by sensitizing cells to TNFα-stimulated apoptosis.     

Syntheses, crystal structure and theoretical investigation of novel heteroleptic complexes of nickel(II) with N-R-sulfonyldithiocarbimate and phosphine ligands

Five new complexes of general formula: [Ni(RSO₂NCS₂)(dppe)], where R = C₆H₅ (1), 4-ClC₆H₄ (2), 4-BrC₆H₄ (3), 4-IC₆H₄ (4) and dppe = 1,2-bis(diphenylphosphino)ethane and [Ni(4-IC₆H₄SO₂NCS₂)(PPh₃)₂] (5), where PPh₃ = triphenylphosphine, were obtained in crystalline form by the reaction of the appropriate potassium N-R-sulfonyldithiocarbimate K₂(RSO₂NCS₂) and dppe or PPh₃ with nickel(II) chloride in ethanol/water. The elemental analyses and the IR, ¹H NMR, ¹³C NMR and ³¹P NMR spectra are consistent with the formation of the square planar nickel(II) complexes with mixed ligands. All complexes were also characterized by X-ray diffraction techniques and present a distorted cis-NiS₂P₂ square-planar configuration around the Ni atom. Quantum chemical calculations reproduced the crystallographic structures and are in accord with the spectroscopic data. Rare C-H···Ni intramolecular short contact interactions were observed in the complexes 1-5.     

Clonothrix fusca Roze 1896, a filamentous, sheathed, methanotrophic gamma-proteobacterium

Crenothrix polyspora Cohn 1870 and Clonothrix fusca Roze 1896 are two filamentous, sheathed microorganisms exhibiting complex morphological differentiation, whose phylogeny and physiology have been obscure for a long time due to the inability to cultivate them. Very recently, DNA sequencing data from uncultured C. polyspora-enriched material have suggested that Crenothrix is a methane-oxidizing gamma-proteobacterium (39). In contrast, the possible ecological function of C. fusca, originally considered a developmental stage of C. polyspora, is unknown. In this study, temporal succession of two filamentous, sheathed microorganisms resembling Cohn's Crenothrix and Roze's Clonothrix was observed by analyzing the microbial community of an artesian well by optical microscopy. Combined culture-based and culture-independent approaches enabled us to assign C. fusca to a novel subgroup of methane-oxidizing gamma-proteobacteria distinct from that of C. polyspora. This assignment was supported by (i) methane uptake and assimilation experiments, (ii) ultrastructural data showing the presence in C. fusca cytoplasm of an elaborate membrane system resembling that of methanotrophic gamma-proteobacteria, and (iii) sequencing data demonstrating the presence in its genome of a methanol dehydrogenase alpha subunit-encoding gene (mxaF) and a conventional particulate methane mono-oxygenase alpha subunit-encoding gene (pmoA) that is different from the unusual pmoA (u-pmoA) of C. polyspora.