Centromere repositioning explains fundamental number variability in the New World monkey genus <Emphasis Type="Italic">Saimiri</Emphasis>

Cytogenetics has historically played a key role in research on squirrel monkey (genus Saimiri) evolutionary biology. Squirrel monkeys have a diploid number of 2n = 44, but vary in fundamental number (FN). Apparently, differences in FN have phylogenetic implications and are correlated with geographic regions. A number of hypothetical mechanisms were proposed to explain difference in FN: translocations, heterochromatin, or, most commonly, pericentric inversions. Recently, an additional mechanism, centromere repositioning, was discovered, which can alter chromosome morphology and FN. Here, we used chromosome banding, chromosome painting, and BAC-FISH to test these hypotheses. We demonstrate that centromere repositioning on chromosomes 5 and 15 is the mechanism that accounts for differences in FN. Current phylogenomic trees of platyrrhines provide a temporal framework for evolutionary new centromeres (ENC) in Saimiri. The X-chromosome ENC could be up to 15 million years (my) old that on chromosome 5 as recent as 0.3 my. The chromosome 15 ENC is intermediate, as young as 2.24 my. All ENC have abundant satellite DNAs indicating that the maturation process was fairly rapid. Callithrix jacchus was used as an outgroup for the BAC-FISH data analysis. Comparison with scaffolds from the S. boliviensis genome revealed an error in the last marmoset genome release. Future research including at the sequence level will provide better understanding of chromosome evolution in Saimiri and other platyrrhines. Probably other cases of differences in chromosome morphology and FN, both within and between taxa, will be shown to be due to centromere repositioning and not pericentric inversions.     

d(CGGTGGT) forms an octameric parallel G-quadruplex via stacking of unusual G(:C):G(:C):G(:C):G(:C) octads

Among non-canonical DNA secondary structures, G-quadruplexes are currently widely studied because of their probable involvement in many pivotal biological roles, and for their potential use in nanotechnology. The overall quadruplex scaffold can exhibit several morphologies through intramolecular or intermolecular organization of G-rich oligodeoxyribonucleic acid strands. In particular, several G-rich strands can form higher order assemblies by multimerization between several G-quadruplex units. Here, we report on the identification of a novel dimerization pathway. Our Nuclear magnetic resonance, circular dichroism, UV, gel electrophoresis and mass spectrometry studies on the DNA sequence dCGGTGGT demonstrate that this sequence forms an octamer when annealed in presence of K(+) or NH(4)(+) ions, through the 5'-5' stacking of two tetramolecular G-quadruplex subunits via unusual G(:C):G(:C):G(:C):G(:C) octads.     

A novel P2RX2 mutation in an Italian family affected by autosomal dominant nonsyndromic hearing loss

Hereditary hearing loss (HHL) is a common disorder accounting for at least 60% of prelingual deafness. It is characterized by a large genetic heterogeneity, and despite the presence of a major gene, still there is a need to search for new causative mutations/genes. Very recently, a mutation within ATP-gated P2X(2) receptor (ligand-gated ion channel, purinergic receptor 2) gene (P2RX2) at DNFA41 locus has been reported leading to a bilateral and symmetrical sensorineural non-syndromic autosomal dominant HHL in two Chinese families. We performed a linkage analysis in a large Italian family with a dominant pattern of inheritance showing a significant 3.31 LOD score in a 2 Mb region overlapping with the DNFA41 locus. Molecular analyses of P2RX2 identified a novel missense mutation (p.Gly353Arg) affecting a residue highly conserved across species. Visual inspection of the protein structure as obtained from comparative modeling suggests that substitution of the small glycine residue with a charged bulky residue such as an arginine that is close to the ‘neck’ of the region responsible for ion channel gating should have a high energetic cost and should lead to a severely destabilization of the fold. The identification of a second most likely causative mutation in P2RX2 gene further supports the possible role of this gene in causing autosomal dominant HHL.     

8,000 years of climate,vegetation, fire and land-use dynamics in the thermo-mediterranean vegetation belt of northern Sardinia (Italy)

Vegetation History and Archaeobotany - Knowledge about the vegetation history of Sardinia, the second largest island of the Mediterranean, is scanty. Here, we present a new sedimentary record...     

G-quadruplex-forming oligonucleotide conjugated to magnetic nanoparticles: synthesis, characterization, and enzymatic stability assays

In the present work, we report the conjugation of superparamagnetic nanoparticles to a fluorescently labeled oligodeoxyribonucleotide (ODN) able to fold into stable unimolecular guanine quadruple helix under proper ion conditions by means of its thrombin-binding aptamer (TBA) sequence. The novel modified ODN, which contained a fluorescent dU(Py) unit at 3'-end and a 12-amino-dodecyl spacer (C(12)-NH(2)) at 5' terminus, was characterized by ESI-MS and optical spectroscopy (UV, CD, fluorescence), and analyzed by RP-HPLC chromatography and electrophoresis. From CD and fluorescence experiments, we verified that dU(Py) and C(12)-NH(2) incorporation does not interfere with the conformational stability of the G-quadruplex. Subsequently, the conjugation of the pyrene-labeled ODN with the magnetite particles was performed, and the ODN-conjugated nanoparticles were studied through optical spectroscopy (UV, CD, fluorescence) and by enzymatic and chemical assays. We found that the nanoparticles enhanced the stability of the TBA ODN to enzymatic degradation. Finally, we evaluated the amount of the TBA-conjugated nanoparticles immobilized on a magnetic separator in view of the potential use of the nanosystem for the magnetic capture of thrombin from complex mixtures.     

Differentiation of mesenchymal stem cells derived from pancreatic islets and bone marrow into islet-like cell phenotype

Background

Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself.

Methodology/Principal Findings

In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin.

Conclusions/Significance

Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes.     

Post-natal heart adaptation in a knock-in mouse model of calsequestrin 2-linked recessive catecholaminergic polymorphic ventricular tachycardia

Cardiac calsequestrin (CASQ2) contributes to intracellular Ca²⁺ homeostasis by virtue of its low-affinity/high-capacity Ca²⁺ binding properties, maintains sarcoplasmic reticulum (SR) architecture and regulates excitation–contraction coupling, especially or exclusively upon β-adrenergic stimulation. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease associated with cardiac arrest in children or young adults. Recessive CPVT variants are due to mutations in the CASQ2 gene. Molecular and ultra-structural properties were studied in hearts of CASQ2^R33Q/R33Q and of CASQ2^−/− mice from post-natal day 2 to week 8. The drastic reduction of CASQ2-R33Q is an early developmental event and is accompanied by down-regulation of triadin and junctin, and morphological changes of jSR and of SR-transverse-tubule junctions. Although endoplasmic reticulum stress is activated, no signs of either apoptosis or autophagy are detected. The other model of recessive CPVT, the CASQ2^−/− mouse, does not display the same adaptive pattern. Expression of CASQ2-R33Q influences molecular and ultra-structural heart development; post-natal, adaptive changes appear capable of ensuring until adulthood a new pathophysiological equilibrium.     

A long winter for the Red Queen: rethinking the evolution of seasonal migration

This paper advances an hypothesis that the primary adaptive driver of seasonal migration is maintenance of site fidelity to familiar breeding locations. We argue that seasonal migration is therefore principally an adaptation for geographic persistence when confronted with seasonality – analogous to hibernation, freeze tolerance, or other organismal adaptations to cyclically fluctuating environments. These ideas stand in contrast to traditional views that bird migration evolved as an adaptive dispersal strategy for exploiting new breeding areas and avoiding competitors. Our synthesis is supported by a large body of research on avian breeding biology that demonstrates the reproductive benefits of breeding‐site fidelity. Conceptualizing migration as an adaptation for persistence places new emphasis on understanding the evolutionary trade‐offs between migratory behaviour and other adaptations to fluctuating environments both within and across species. Seasonality‐induced departures from breeding areas, coupled with the reproductive benefits of maintaining breeding‐site fidelity, also provide a mechanism for explaining the evolution of migration that is agnostic to the geographic origin of migratory lineages (i.e. temperate or tropical). Thus, our framework reconciles much of the conflict in previous research on the historical biogeography of migratory species. Although migratory behaviour and geographic range change fluidly and rapidly in many populations, we argue that the loss of plasticity for migration via canalization is an overlooked aspect of the evolutionary dynamics of migration and helps explain the idiosyncratic distributions and migratory routes of long‐distance migrants. Our synthesis, which revolves around the insight that migratory organisms travel long distances simply to stay in the same place, provides a necessary evolutionary context for understanding historical biogeographic patterns in migratory lineages as well as the ecological dynamics of migratory connectivity between breeding and non‐breeding locations.     

OX40 ligand-transduced tumor cell vaccine synergizes with GM-CSF and requires CD40-Apc signaling to boost the host T cell antitumor response

Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-gamma or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.