Mutation analysis of the muscarinic cholinergic receptor genes in isolated growth hormone deficiency type IB

BACKGROUND: Isolated GH deficiency (IGHD) is familial in 5-30% of patients. The most frequent form (IGHD-IB) has autosomal recessive inheritance, and it is known that it can be caused by mutations in the GHRH receptor (GHRHR) gene or in the GH gene. However, most forms of IGHD-IB have an unknown genetic cause. In normal subjects, muscarinic cholinergic stimulation causes an increase in pituitary GH release, whereas its blockade has the opposite effect, suggesting that a muscarinic acetylcholine receptor (mAchR) is involved in stimulating GH secretion. Five types of mAchR (M(1)-M(5)) exist. A transgenic mouse in which the function of the M(3) receptor was selectively ablated in the central nervous system has isolated GH deficiency similar to animals with defective GHRH or GHRHR gene. OBJECTIVE: We hypothesized that mAchR mutations may cause a subset of familial IGHD. PATIENTS/METHODS: After confirming the expression of M(1)-M(5) receptor mRNA in human hypothalamus, we analyzed the index cases of 39 families with IGHD-IB for mutations in the genes encoding for the five receptors. Coding sequences for each of the five mAchRs were subjected to direct sequencing. RESULTS: In one family, an affected member was homozygous for a M(3) change in codon 65 that replaces valine with isoleucine (V65I). The V65I receptor was expressed in CHO cells where it had normal ability to transmit methacholine signaling. CONCLUSION: mAchR mutations are absent or rare (less than 2.6%) in familial IGHD type IB.

A three-dimensional histological atlas of the human basal ganglia. II. Atlas deformation strategy and evaluation in deep brain stimulation for Parkinson disease

OBJECT: The localization of any given target in the brain has become a challenging issue because of the increased use of deep brain stimulation to treat Parkinson disease, dystonia, and nonmotor diseases (for example, Tourette syndrome, obsessive compulsive disorders, and depression). The aim of this study was to develop an automated method of adapting an atlas of the human basal ganglia to the brains of individual patients. METHODS: Magnetic resonance images of the brain specimen were obtained before extraction from the skull and histological processing. Adaptation of the atlas to individual patient anatomy was performed by reshaping the atlas MR images to the images obtained in the individual patient using a hierarchical registration applied to a region of interest centered on the basal ganglia, and then applying the reshaping matrix to the atlas surfaces. RESULTS: Results were evaluated by direct visual inspection of the structures visible on MR images and atlas anatomy, by comparison with electrophysiological intraoperative data, and with previous atlas studies in patients with Parkinson disease. The method was both robust and accurate, never failing to provide an anatomically reliable atlas to patient registration. The registration obtained did not exceed a 1-mm mismatch with the electrophysiological signatures in the region of the subthalamic nucleus. CONCLUSIONS: This registration method applied to the basal ganglia atlas forms a powerful and reliable method for determining deep brain stimulation targets within the basal ganglia of individual patients.

Calibration and analysis of the 2007 data

he physics program of the NA61/SHINE (SHINE = SPS Heavy Ion and Neutrino Experiment) experiment at the CERN SPS consists of three subjects. In the first stage of data taking (2007-2009) measurements of hadron production in hadron-nucleus interactions needed for neutrino (T2K) and cosmic-ray (Pierre Auger and KASCADE) experiments will be performed. In the second stage (2009-2010) hadron production in proton-proton and proton-nucleus interactions needed as reference data for a better understanding of nucleus-nucleus reactions will be studied. In the third stage (2009-2013) energy dependence of hadron production properties will be measured in p+p, p+Pb interactions and nucleus-nucleus collisions, with the aim to identify the properties of the onset of deconfinement and find evidence for the critical point of strongly interacting matter. The NA61 experiment was approved at CERN in June 2007. The first pilot run was performed during October 2007. Calibrations of all detector components have been performed successfully and preliminary uncorrected spectra have been obtained. High quality of track reconstruction and particle identification similar to NA49 has been achieved. The data and new detailed simulations confirm that the NA61 detector acceptance and particle identification capabilities cover the phase space required by the T2K experiment. This document reports on the progress made in the calibration and analysis of the 2007 data.

Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

Background The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. Methodology/Principal Findings We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. Conclusions Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins.

White matter microstructure alterations of the medial forebrain bundle in melancholic depression

BACKGROUND The medial forebrain bundle (MFB) is a key structure of the reward system and connects the ventral tegmental area (VTA) with the nucleus accumbens (NAcc), the medial and lateral orbitofrontal cortex (mOFC, lOFC) and the dorsolateral prefrontal cortex (dlPFC). Previous diffusion tensor imaging (DTI) studies in major depressive disorder point to white matter alterations of regions which may be incorporated in the MFB. Therefore, it was the aim of our study to probe white matter integrity of the MFB using a DTI-based probabilistic fibre tracking approach. METHODS 22 patients with major depressive disorder (MDD) (12 melancholic-MDD patients, 10 non-melancholic-MDD patients) and 21 healthy controls underwent DTI scans. We used a bilateral probabilistic fibre tracking approach to extract pathways between the VTA and NACC, mOFC, lOFC, dlPFC respectively. Mean fractional anisotropy (FA) values were used to compare structural connectivity between groups. RESULTS Mean-FA did not differ between healthy controls and all MDD patients. Compared to healthy controls melancholic MDD-patients had reduced mean-FA in right VTA-lOFC and VTA-dlPFC connections. Furthermore, melancholic-MDD patients had lower mean-FA than non-melancholic MDD-patients in the right VTA-lOFC connection. Mean-FA of these pathways correlated negatively with depression scale rating scores. LIMITATIONS Due to the small sample size and heterogeneous age group comparisons between melancholic and non-melancholic MDD-patients should be regarded as preliminary. CONCLUSIONS Our results suggest that the melancholic subtype of MDD is characterized by white matter microstructure alterations of the MFB. White matter microstructure is associated with both depression severity and anhedonia.

Assessment of the Matrix Degenerative Effects of MMP-3, ADAMTS-4 and HTRA1 injected into a bovine Intervertebral Disc Organ Culture Model

Study Design. In vitro study to develop an intervertebral disc degeneration (IDD) organ culture model, using coccygeal bovine intervertebral discs (IVDs) and injection of proteolytic enzymes MMP-3, ADAMTS-4 and HTRA1.Objective. This study aimed to develop an in-vitro model of enzyme-mediated IDD to mimic the clinical outcome in humans for investigation of therapeutic treatment options.Summary of Background Data. Bovine IVDs are comparable to human IVDs in terms of cell composition and biomechanical behavior. Researchers injected papain and trypsin into them to create an IDD model with a degenerated nucleus pulposus (NP) area. They achieved macroscopic cavities as well as a loss of glycosaminoglycans (GAGs). However, none of these enzymes are clinically relevant.Methods. Bovine IVDs were harvested maintaining the endplates. Active forms of MMP-3, ADAMTS-4 and HTRA1 were injected at a dose of 10μg/ml each. Phosphate buffered saline (PBS) was injected as a control. Discs were cultured for 8 days and loaded diurnally (day 1 to day 4 with 0.4 MPa for 16 h) and left under free swelling condition from day 4 to day 8 to avoid expected artifacts due to dehydration of the NP. Outcome parameters included disc height, metabolic cell activity, DNA content, glycosaminoglycan (GAG) content, total collagen content, relative gene expression and histological investigation.Results. The mean metabolic cell activity was significantly lower in the NP area of discs injected with ADAMTS-4 compared to the day 0 control discs. Disc height was decreased following injection with HTRA1, and was significantly correlated with changes in GAG/DNA of the NP tissue. Total collagen content tended to be lower in groups injected with ADAMTS4 and MMP-3.Conclusion. MMP-3, ADAMTS-4 and HTRA1 neither provoked visible matrix degradation nor major shifts in gene expression. However, cell activity was significantly reduced and HTRA1 induced loss of disc height which positively correlated with changes in GAG/DNA content. The use of higher doses of these enzymes or a combination thereof may therefore be necessary to induce disc degeneration

Assessment of the wind-up phenomenon in the equine nociceptive trigeminal system.

Repeated sub-threshold nociceptive electrical stimulation resulting in temporal summation of the limb nociceptive withdrawal reflex is a well-established non-invasive model to investigate the wind-up phenomenon in horses. Due to structural similarities of the trigeminal sensory nucleus to the dorsal horn of the spinal cord, temporal summation should be evoked by repeated transcutaneous electrical stimulation of trigeminal afferents. To evaluate this hypothesis repeated transcutaneous electrical stimulation was applied to the supraorbital and infraorbital nerves of 10 horses. Stimulation intensities varied between 0.5 and 1.3 times the trigemino-cervical reflex threshold defined for single stimulation. Evoked electromyographic activity of the orbicularis oculi, splenius and cleidomastoideus muscles was recorded and the signals analysed in the previously established epochs typical to the early and late component of the blink reflex and to the trigemino-cervical reflex. Behavioural reactions were evaluated with the aid of numerical rating scale. The nociceptive late component and the trigemino-cervical reflex were not elicited by sub-threshold intensity repeated transcutaneous electrical stimulation. Furthermore, the median reflex amplitude for the 10 horses showed a tendency to decline over the stimulation train so temporal summation of afferent trigeminal inputs could not be observed. Therefore, the modulation of trigeminal nociceptive processing attributable to repeated Aδ fibre stimulations seems to differ from spinal processing of similar inputs as it seems to have an inhibitory rather than facilitatory effect. Further evaluation is necessary to highlight the underlying mechanism.

Molecular cloning and characterization of NcROP2Fam-1, a member of the ROP2 family of rhoptry proteins in Neospora caninum that is targeted by antibodies neutralizing host cell invasion in vitro.

Recent publications demonstrated that a fragment of a Neospora caninum ROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein, N. caninum ROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of 'cysts' produced in vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasion in vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

White matter pathway organization of the reward system is related to positive and negative symptoms in schizophrenia

The reward systemin schizophrenia has been linked to the emergence of delusions on the one hand and to negative symptoms such as affective flattening on the other hand. Previous Diffusion Tensor Imaging (DTI) studies reported white matter microstructure alterations of regions related to the reward system. The present study aimed at extending these findings by specifically investigating connection pathways of the reward system in schizophrenia. Therefore, 24 patients with schizophrenia and 22 healthy controls matched for age and gender underwent DTI-scans. Using a probabilistic fiber tracking approachwe bilaterally extracted pathways connecting the ventral tegmental area (VTA) with the nucleus accumbens (NAcc), themedial and lateral orbitofrontal cortices (mOFC, lOFC), the dorsolateral prefrontal cortex (dlPFC) and the amygdala; as well as pathways connecting NAcc with mOFC, lOFC, dlPFC and amygdala resulting in a total of 18 connections. Probability indices forming part of a bundle of interest (PIBI) were compared between groups using independent t-tests. In 6 connection pathways PIBI-valueswere increased in schizophrenia. In 3 of these pathways the spatial extension of connection pathways was decreased. In schizophrenia patients, there was a negative correlation of PIBI-values and PANSS negative scores in the left VTA–amygdala and in the left NAcc–mOFC connection. A sum score of delusions and hallucinations correlated positively with PIBI-values of the left amygdala–NAcc connection. Structural organization of specific segments ofwhite matter pathways of the reward systemin schizophrenia may contribute to the emergence of delusions and negative symptoms in schizophrenia.

Non-Hebbian Long-Term Potentiation of Inhibitory Synapses in the Thalamus

The thalamus integrates and transmits sensory information to the neocortex. The activity of thalamocortical relay (TC) cells is modulated by specific inhibitory circuits. Although this inhibition plays a crucial role in regulating thalamic activity, little is known about long-term changes in synaptic strength at these inhibitory synapses. Therefore, we studied long-term plasticity of inhibitory inputs to TC cells in the posterior medial nucleus of the thalamus by combining patch-clamp recordings with two-photon fluorescence microscopy in rat brain slices. We found that specific activity patterns in the postsynaptic TC cell induced inhibitory long-term potentiation (iLTP). This iLTP was non-Hebbian because it did not depend on the timing between presynaptic and postsynaptic activity, but it could be induced by postsynaptic burst activity alone. iLTP required postsynaptic dendritic Ca2+ influx evoked by low-threshold Ca2+ spikes. In contrast, tonic postsynaptic spiking from a depolarized membrane potential (−50 mV), which suppressed these low-threshold Ca2+ spikes, induced no plasticity. The postsynaptic dendritic Ca2+ increase triggered the synthesis of nitric oxide that retrogradely activated presynaptic guanylyl cyclase, resulting in the presynaptic expression of iLTP. The dependence of iLTP on the membrane potential and therefore on the postsynaptic discharge mode suggests that this form of iLTP might occur during sleep, when TC cells discharge in bursts. Therefore, iLTP might be involved in sleep state-dependent modulation of thalamic information processing and thalamic oscillations.

Extended duration of torsional loading reduced the survival of the NP cells of the intervertebral disc

Introduction Previous studies on the influence of torsion and combined torsion-compression loading revealed a positive effect on the cell viability when a repetitive short-term torsion was applied at a physiological magnitude to intervertebral disc organ culture.1 However, after an extended period (8 hours) of combined torsion-compression loading, substantial cell death was detected in the nucleus pulposus (NP).2 In this follow-up study, we aimed to investigate the relationship, if any, between the duration of torsion applied to the intervertebral disc (IVD) and the level of NP cell viability. Materials and Methods Bovine caudal discs were harvested and cultured in a custom-built multiaxis dynamic loading bioreactor.2 Torsion (± 2 degrees) was applied to the samples at a frequency of 0.2 Hz. Torsion was applied for durations of 0, 1, 4, and 8 h/d, repeated over 7 days. After the last day of loading, disc tissue was dissected for analysis of cell viability and gene expression. Results Disc NP cell viability remained above 85% after torsional loading for 0, 1, or 4 h/d. Viability was statistical significantly reduced to below 70% when torsion was applied for 8 h/d (p = 0.03) (Table 1). The daily duration of torsional loading did not affect the AF cell viability (> 80% for all loading durations). The trend of collagen 2 gene upregulation and matrix metalloproteases 13 downregulation with an increasing duration of torsion was observed in both NP and AF (Fig. 1).Conclusion We have demonstrated that an extended duration of torsion could inhibit the survival of NP cells within the IVD in organ culture. Acknowledgments Funds from the Orthopedic Department of the Insel University Hospital of Bern and a private donation from Prof. Dr. Paul Heini, Spine Surgeon, Sonnenhof Clinic Bern were received to support this work. Disclosure of Interest None declared References References 1 Chan SC, Ferguson SJ, Wuertz K, Gantenbein-Ritter B. Biological response of the intervertebral disc to repetitive short-term cyclic torsion. Spine 2011;36(24):2021–2030 2 Chan SC, Walser J, Käppeli P, Shamsollahi MJ, Ferguson SJ, Gantenbein-Ritter B. Region specific response of intervertebral disc cells to complex dynamic loading: an organ culture study using a dynamic torsion-compression bioreactor. PLoS ONE 2013;8(8):e72489

Human DMTF1β antagonizes DMTF1α regulation of the p14(ARF) tumor suppressor and promotes cellular proliferation

The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, β and γ, arise from the human gene. We now show that DMP1α, β and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1β and γ did not activate the ARF promoter, whereas only β resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1β reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1β- and γ-isoforms share domains necessary for the inhibitory function of the β-isoform. That DMP1β may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/β in the nucleus. Cells stably expressing DMP1β, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and β-ratios are tightly regulated in hematopoietic cells and DMP1β antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.

Comparison of 3D kinetic and hydrodynamic models to ROSINA-COPS measurements of the neutral coma of 67P/Churyumov-Gerasimenko

67P/Churyumov-Gerasimenko (67P) is a Jupiter-family comet and the object of investigation of the European Space Agency mission Rosetta. This report presents the first full 3D simulation results of 67P’s neutral gas coma. In this study we include results from a direct simulation Monte Carlo method, a hydrodynamic code, and a purely geometric calculation which computes the total illuminated surface area on the nucleus. All models include the triangulated 3D shape model of 67P as well as realistic illumination and shadowing conditions. The basic concept is the assumption that these illumination conditions on the nucleus are the main driver for the gas activity of the comet. As a consequence, the total production rate of 67P varies as a function of solar insolation. The best agreement between the model and the data is achieved when gas fluxes on the night side are in the range of 7% to 10% of the maximum flux, accounting for contributions from the most volatile components. To validate the output of our numerical simulations we compare the results of all three models to in situ gas number density measurements from the ROSINA COPS instrument. We are able to reproduce the overall features of these local neutral number density measurements of ROSINA COPS for the time period between early August 2014 and January 1 2015 with all three models. Some details in the measurements are not reproduced and warrant further investigation and refinement of the models. However, the overall assumption that illumination conditions on the nucleus are at least an important driver of the gas activity is validated by the models. According to our simulation results we find the total production rate of 67P to be constant between August and November 2014 with a value of about 1 × 10²⁶ molecules s⁻¹.

Geomorphology of the Imhotep region on comet 67P/Churyumov-Gerasimenko from OSIRIS observations

Context. Since August 2014, the OSIRIS Narrow Angle Camera (NAC) onboard the Rosetta spacecraft has acquired high spatial resolution images of the nucleus of comet 67P/Churyumov-Gerasimenko, down to the decimeter scale. This paper focuses on the Imhotep region, located on the largest lobe of the nucleus, near the equator. Aims. We map, inventory, and describe the geomorphology of the Imhotep region. We propose and discuss some processes to explain the formation and ongoing evolution of this region. Methods. We used OSIRIS NAC images, gravitational heights and slopes, and digital terrain models to map and measure the morphologies of Imhotep. Results. The Imhotep region presents a wide variety of terrains and morphologies: smooth and rocky terrains, bright areas, linear features, roundish features, and boulders. Gravity processes such as mass wasting and collapse play a significant role in the geomorphological evolution of this region. Cometary processes initiate erosion and are responsible for the formation of degassing conduits that are revealed by elevated roundish features on the surface. We also propose a scenario for the formation and evolution of the Imhotep region; this implies the presence of large primordial voids inside the nucleus, resulting from its formation process.

Permittivity measurements of porous matter in support of investigations of the surface and interior of 67P/Churyumov-Gerasimenko

Aims. Permittivity measurements on porous samples of volcanic origin have been performed in the 0.05-190 GHz range under laboratory conditions in support of the Rosetta mission to comet 67P/Churyumov-Gerasimenko, specifically with the MIRO radiometric experiment and CONSERT radar experiment. Methods. The samples were split into several subsamples with different size ranges covering a few mu m to 500 mu m. Bulk densities of the subsamples were estimated to be in the 800 to 1500 kg/m(3) range. The porosities were in the range of 48% to 65%. From 50 MHz to 6 GHz and at 190 GHz, permittivity has been determined with a coaxial cell and with a quasi-optical bench, respectively. Results. Without taking into account the volume-scattering effect at 190 GHz, the real part of the permittivity, normalized by the bulk density, is in the range of 2.1 to 2.6. The results suggest that the real part of the permittivity of an ice-free dust mantle covering the nucleus is in the 1.5-2.2 range at 190 GHz. From these values, a lower limit for the absorption length for the millimeter receiver of MIRO has been estimated to be between 0.6 and 2 cm, in agreement with results obtained from MIRO in September 2014. At frequencies of interest for CONSERT experiment, the real part of the permittivity of a suspected ice-free dust mantle should be below 2.2. It may be in the range of 1.2 to 1.7 for the nucleus, in agreement with first CONSERT results, taking into account a mean temperature of 110 K and different values for the dust-to-ice volumetric ratio. Estimations of contributions of the different parameters to the permittivity variation may indicate that the porosity is the main parameter.