The first genomewide interaction and locus-heterogeneity linkage scan in bipolar affective disorder: strong evidence of epistatic effects between loci on chromosomes 2q and 6q

We present the first genomewide interaction and locus-heterogeneity linkage scan in bipolar affective disorder (BPAD), using a large linkage data set (52 families of European descent; 448 participants and 259 affected individuals). Our results provide the strongest interaction evidence between BPAD genes on chromosomes 2q22-q24 and 6q23-q24, which was observed symmetrically in both directions (nonparametric LOD [NPL] scores of 7.55 on 2q and 7.63 on 6q; P<.0001 and P=.0001, respectively, after a genomewide permutation procedure). The second-best BPAD interaction evidence was observed between chromosomes 2q22-q24 and 15q26. Here, we also observed a symmetrical interaction (NPL scores of 6.26 on 2q and 4.59 on 15q; P=.0057 and .0022, respectively). We covered the implicated regions by genotyping additional marker sets and performed a detailed interaction linkage analysis, which narrowed the susceptibility intervals. Although the heterogeneity analysis produced less impressive results (highest NPL score of 3.32) and a less consistent picture, we achieved evidence of locus heterogeneity at chromosomes 2q, 6p, 11p, 13q, and 22q, which was supported by adjacent markers within each region and by previously reported BPAD linkage findings. Our results provide systematic insights in the framework of BPAD epistasis and locus heterogeneity, which should facilitate gene identification by the use of more-comprehensive cloning strategies.     

Human adipose tissue-derived multipotent stem cells differentiate in vitro and in vivo into osteocyte-like cells

Cell-based therapies are used to treat bone defects. We recently described that human multipotent adipose-derived stem (hMADS) cells, which exhibit a normal karyotype, self renewal, and the maintenance of their differentiation properties, are able to differentiate into different lineages. Herein, we show that hMADS cells can differentiate into osteocyte-like cells. In the presence of a low amount of serum and EGF, hMADS cells express specific molecular markers, among which alkaline phosphatase, CBFA-1, osteocalcin, DMP1, PHEX, and podoplanin and develop functional gap-junctions. When loaded on a hardening injectable bone substitute (HIBS) biomaterial and injected subcutaneously into nude mice, hMADS cells develop mineralized woven bone 4 weeks after implantation. Thus hMADS cells represent a valuable tool for pharmacological and biological studies of osteoblast differentiation in vitro and bone development in vivo.     

Increased heart rate variability in mice overexpressing the Cu/Zn superoxide dismutase

Cu/Zn superoxide dismutase (SOD1) is implicated in various pathological conditions including Down's syndrome, neurodegenerative diseases, and afflictions of the autonomic nervous system (ANS). To assess the SOD1 contribution to ANS dysfunction, especially its influence on cardiac regulation, we studied the heart rate variability (HRV) and cardiac arrhythmias in conscious 12-month-old male and female transgenic mice for the human SOD1 gene (TghSOD1). TghSOD1 mice presented heart rate reduction as compared with control FVB/N individuals. All HRV parameters reflecting parasympathetic activity were increased in TghSOD1. Pharmacological studies confirmed that the parasympathetic tone was exacerbated and the sympathetic pathway was functional in TghSOD1 mice. A high frequency of atrioventricular block and premature ventricular contractions was observed in TghSOD1. By biochemical assays we found that SOD1 activities were multiplied by 9 and 4 respectively in the heart and brainstem of transgenic mice. A twofold decrease in cholinesterase activity was observed in the heart but not in the brainstem. We demonstrate that SOD1 overexpression induces an ANS dysfunction by an exacerbated vagal tone that may be related to impaired cardiac activity of the cholinesterases and may explain the high occurrence of arrhythmias.     

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

Introduction

Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC₅₀ values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.     

In vivo preclinical low field MRI monitoring of tumor growth following a suicide gene therapy in an orthotopic mice model of human glioblastoma

Purpose

The aim of this study was to monitor in vivo with low field MRI growth of a murine orthotopic glioma model following a suicide gene therapy.

Methods

The gene therapy consisted in the stereotactic injection in the mice brain of a modified vaccinia virus Ankara (MVA) vector encoding for a suicide gene (FCU1) that transforms a non toxic prodrug 5-fluorocytosine (5-FC) to its highly cytotoxic derivatives 5-fluorouracil (5-FU) and 5’-fluorouridine-5’monophosphate (5’-FUMP). Using a warmed-up imaging cell, sequential 3D T1 and T2 0.1T MRI brain examinations were performed on 16 Swiss female nu/nu mice bearing orthotopic human glioblastoma (U87-MG cells). The 6-week in vivo MRI follow-up consisted in a weekly measurement of the intracerebral tumor volume leading to a total of 65 examinations. Mice were divided in four groups: sham group (n = 4), sham group treated with 5-FC only (n = 4), sham group with injection of MVA-FCU1 vector only (n = 4), therapy group administered with MVA-FCU1 vector and 5-FC (n = 4). Measurements of tumor volumes were obtained after manual segmentation of T1- and T2-weighted images.

Results

Intra-observer and inter-observer tumor volume measurements show no significant differences. No differences were found between T1 and T2 volume tumor doubling times between the three sham groups. A significant statistical difference (p < 0.05) in T1 and T2 volume tumor doubling times between the three sham groups and the animals treated with the intratumoral injection of MVA-FCU1 vector in combination with 2 weeks per os 5-FC administration was demonstrated.

Conclusion

Preclinical low field MRI was able to monitor efficacy of suicide gene therapy in delaying the tumor growth in an in vivo mouse model of orthotopic glioblastoma.     

VCD study of α‐methylbenzyl amine derivatives: Detection of the unchanged chiral motif

Chiral α‐methylbenzyl amine is a well known and often used chiral auxiliary, e.g., in the resolution of racemates or asymmetric catalysis. In this work, α‐methylbenzyl amine and its derivatives N,α‐dimethylbenzyl amine, N,N,α‐trimethylbenzyl amine, and bis[α‐methylbenzyl] amine were investigated by vibrational circular dichroism (VCD) spectroscopy and density functional theory (DFT). For all compounds, stable low energy conformers were obtained by the DFT calculations and based on those, the theoretical vibrational absorption (VA) and VCD spectra were calculated and compared with experimental spectra. Hence, the absolute configurations and conformational preferences were determined. A qualitative comparison of all the experimental VCD spectra of the investigated chiral molecules supported by the calculated ones is given which clearly shows similarities between the spectra of the different chiral amines. These can be assigned to vibrations of the unchanged chiral center. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

1H, 13C and 15N resonance assignments of the Calmodulin-Munc13-1 peptide complex

Ca²⁺-Calmodulin binding to the variable N-terminal region of the diacylglycerol/phorbol ester-binding UNC13/Munc13 family of proteins modulates the short-term synaptic plasticity characteristics in neurons. Here, we report the sequential backbone and side chain resonance assignment of the Ca²⁺-Calmodulin/Munc13-1^458–492 peptide complex at pH 6.8 and 35°C (BMRB No. 15470).     

Northern glacial refugia for the pygmy shrew Sorex minutus in Europe revealed by phylogeographic analyses and species distribution modelling

The southern European peninsulas (Iberian, Italian and Balkan) are traditionally recognized as glacial refugia from where many species colonized central and northern Europe after the Last Glacial Maximum (LGM). However, evidence that some species had more northerly refugia is accumulating from phylogeographic, palaeontological and palynological studies, and more recently from species distribution modelling (SDM), but further studies are needed to test the idea of northern refugia in Europe. Here, we take a rarely implemented multidisciplinary approach to assess if the pygmy shrew Sorex minutus, a widespread Eurasian mammal species, had northern refugia during the LGM, and if these influenced its postglacial geographic distribution. First, we evaluated the phylogeographic and population expansion patterns using mtDNA sequence data from 123 pygmy shrews. Then, we used SDM to predict present and past (LGM) potential distributions using two different training data sets, two different algorithms (Maxent and GARP) and climate reconstructions for the LGM with two different general circulation models. An LGM distribution in the southern peninsulas was predicted by the SDM approaches, in line with the occurrence of lineages of S. minutus in these areas. The phylogeographic analyses also indicated a widespread and strictly northern‐central European lineage, not derived from southern peninsulas, and with a postglacial population expansion signature. This was consistent with the SDM predictions of suitable LGM conditions for S. minutus occurring across central and eastern Europe, from unglaciated parts of the British Isles to much of the eastern European Plain. Hence, S. minutus likely persisted in parts of central and eastern Europe during the LGM, from where it colonized other northern areas during the late‐glacial and postglacial periods. Our results provide new insights into the glacial and postglacial colonization history of the European mammal fauna, notably supporting glacial refugia further north than traditionally recognized.     

Ionizing radiation‐induced long‐term expression of senescence markers in mice is independent of p53 and immune status

Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR‐induced senescence markers could persist long‐term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analyzed) DNA damage foci and increased p16^INK4a expression, two hallmarks of cellular senescence and aging. BrdU‐labeling experiments revealed that IR‐induced damaged cells are preferentially eliminated, at least partially, in a tissue‐dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild‐type and Rag2^?/? γC^?/? mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long‐term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors.