Evidence for a cognitive control network for goal-directed attention in simple sustained attention

The deterioration of performance over time is characteristic for sustained attention tasks. This so-called "performance decrement" is measured by the increase of reaction time (RT) over time. Some behavioural and neurobiological mechanisms of this phenomenon are not yet fully understood. Behaviourally, we examined the increase of RT over time and the inter-individual differences of this performance decrement. On the neurophysiological level, we investigated the task-relevant brain areas where neural activity was modulated by RT and searched for brain areas involved in good performance (i.e. participants with no or moderate performance decrement) as compared to poor performance (i.e. participants with a steep performance decrement). For this purpose, 20 healthy, young subjects performed a carefully designed task for simple sustained attention, namely a low-demanding version of the Rapid Visual Information Processing task. We employed a rapid event-related functional magnetic resonance imaging (fMRI) design. The behavioural results showed a significant increase of RT over time in the whole group, and also revealed that some participants were not as prone to the performance decrement as others. The latter was statistically significant comparing good versus poor performers. Moreover, high BOLD-responses were linked to longer RTs in a task-relevant bilateral fronto-cingulate-insular-parietal network. Among these regions, good performance was associated with significantly higher RT-BOLD correlations in the pre-supplementary motor area (pre-SMA). We concluded that the task-relevant bilateral fronto-cingulate-insular-parietal network was a cognitive control network responsible for goal-directed attention. The pre-SMA in particular might be associated with the performance decrement insofar that good performers could sustain activity in this brain region in order to monitor performance declines and adjust behavioural output.

Investigation on colloidal Te doped CdSe (CdSe:Te) quantum dots suspension in a liquid-core fiber

Here, we demonstrate the use of a colloidal CdSe:Te quantum dots suspension as active liquid-core in a specially designed optical element, based on a double-clad optical fiber structure. The liquid-core fiber was realized by filling the hollow core of a capillary and waveguiding of the core was ensured by using a liquid host that exhibits a larger refractive index than the cladding material of the capillary. Since the used capillary possessed a cladding waveguide structure, we obtained a liquid-core double-clad structure. To seal the liquid-core fiber and e.g. prevent the formation of bubbles, we developed a technique based on SMA connectors. The colloidal CdSe:Te quantum dots were excited by cladding-pumping using a pump laser at 532nm operating in the continuous-wave regime. We investigated the photoluminescence emitted from the colloidal CdSe:Te quantum dots suspension liquid-core and guided by the double-clad fiber structure. We observed a red shift of the (core) emission, that depends on the liquid-core fiber length and the pump power. This shift is due to the absorption of unexcited colloidal quantum dots and due to the waveguiding properties of the core. Here we report a core photoluminescence output power of 79.2μW (with an integrated brightness of ≈ 215.5 W/cm2sr ). Finally, we give an explanation, why lasing could not be observed in our experiments when setup as a liquid-core fiber cavity.

The neurophysiological time pattern of illusionary visual perceptual transitions: a simultaneous EEG and fMRI study

Several divergent cortical mechanisms generating multistability in visual perception have been suggested. Here, we investigated the neurophysiologic time pattern of multistable perceptual changes by means of a simultaneous recording with electroencephalography (EEG) and functional magnetic resonance imaging (fMRI). Volunteers responded to the subjective perception of a sudden change between stable patterns of illusionary motion (multistable transition) during a stroboscopic paradigm. We found a global deceleration of the EEG frequency prior to a transition and an occipital-accentuated acceleration after a transition, as obtained by low-resolution electromagnetic tomography analysis (LORETA) analysis. A decrease in BOLD response was found in the prefrontal cortex before, and an increase after the transitions was observed in the right anterior insula, the MT/V5 regions and the SMA. The thalamus and left superior temporal gyrus showed a pattern of decrease before and increase after transitions. No such temporal course was found in the control condition. The multimodal approach of data acquisition allows us to argue that the top-down control of illusionary visual perception depends on selective attention, and that a diminution of vigilance reduces selective attention. These are necessary conditions to allow for the occurrence of a perception discontinuity in absence of a physical change of the stimulus.

Spinal muscular atrophy: SMN2 pre-mRNA splicing corrected by a U7 snRNA derivative carrying a splicing enhancer sequence

Spinal muscular atrophy (SMA) is a lethal hereditary disease caused by homozygous deletion/inactivation of the survival of motoneuron 1 (SMN1) gene. The nearby SMN2 gene, despite its identical coding capacity, is only an incomplete substitute, because a single nucleotide difference impairs the inclusion of its seventh exon in the messenger RNA (mRNA). This splicing defect can be corrected (transiently) by specially designed oligonucleotides. Here we have developed a more permanent correction strategy based on bifunctional U7 small nuclear RNAs (snRNAs). These carry both an antisense sequence that allows specific binding to exon 7 and a splicing enhancer sequence that will improve the recognition of the targeted exon. When expression cassettes for these RNAs are stably introduced into cells, the U7 snRNAs become incorporated into small nuclear ribonucleoprotein (snRNP) particles that will induce a durable splicing correction. We have optimized this strategy to the point that virtually all SMN2 pre-mRNA becomes correctly spliced. In fibroblasts from an SMA patient, this approach induces a prolonged restoration of SMN protein and ensures its correct localization to discrete nuclear foci (gems).

Orthogonal polarization spectroscopy to detect mesenteric hypoperfusion

OBJECTIVE: Orthogonal polarization spectral (OPS) imaging is used to assess mucosal microcirculation. We tested sensitivity and variability of OPS in the assessment of mesenteric blood flow (Q (sma)) reduction. SETTING: University Animal Laboratory. INTERVENTIONS: In eight pigs, Q (sma) was reduced in steps of 15% from baseline; five animals served as controls. Jejunal mucosal microcirculatory blood flow was recorded with OPS and laser Doppler flowmetry at each step. OPS data from each period were collected and randomly ordered. Samples from each period were individually chosen by two blinded investigators and quantified [capillary density (number of vessels crossing predefined lines), number of perfused villi] after agreement on the methodology. MEASUREMENT AND RESULTS: Interobserver coefficient of variation (CV) for capillary density from samples representing the same flow condition was 0.34 (0.04-1.41) and intraobserver CV was 0.10 (0.02-0.61). Only one investigator observed a decrease in capillary density [to 62% (48-82%) of baseline values at 45% Q (sma) reduction; P = 0.011], but comparisons with controls never revealed significant differences. In contrast, reduction in perfused villi was detected by both investigators at 75% of mesenteric blood flow reduction. Laser Doppler flow revealed heterogeneous microcirculatory perfusion. CONCLUSIONS: Assessment of capillary density did not reveal differences between animals with and without Q (sma) reduction, and evaluation of perfused villi revealed blood flow reduction only when Q (sma) was very low. Potential explanations are blood flow redistribution and heterogeneity, and suboptimal contrast of OPS images. Despite agreement on the method of analysis, interobserver differences in the quantification of vessel density on gut mucosa using OPS are high.

Reducing Myofibroblast Arrhythmogeneicity by Pharmacological Targeting of Their Cytoskeleton

Introduction: Slow conduction and ectopic activity are key elements of cardiac arrhythmogenesis. Both anomalies can be caused by myofibroblasts (MFBs) following establishment of heterocellular gap junctional coupling with cardiomyocytes. Because MFBs are characterized by the expression of {alpha}-smooth muscle actin ({alpha}-SMA) containing stress fibers, we investigated whether pharmacological interference with stress fiber formation might affect myofibroblast arrhythmogenicity. Methods: Experiments were done with patterned growth strands of neonatal rat ventricular cardiomyocytes coated with cardiac MFBs. Impulse propagation characteristics were measured optically using voltage sensitive dyes. Electrophysiological characteristics of single MFBs were assessed using patch clamp techniques. Actin polymerization was inhibited by latrunculin B (LtB). Data are given as mean±S.D. (n=5 to 22). Results: As assessed by immunocytochemistry, exposure of MFBs to LtB (0.3–10 µmol/L) profoundly disrupted stress fiber formation. This led, within minutes, to a dramatic change in cell morphology with MFBs assuming an astrocyte-like shape. In pure cardiomyocyte preparations, LtB had negligible effects on impulse conduction velocity ({theta}) and maximal action potential upstroke velocities (dV/dtmax). In contrast, LtB applied to MFB coated cardiomyocyte strands substantially increased {theta} from 247±32 to 371±26 mm/s and dV/dtmax from 40±7 to 81±1 %APA/ms, i.e., to values similar to those of pure cardiomyocyte strands (342±13 mm/s; 82±1 %APA/ms). Moreover, LtB at 1 µmol/L completely abolished MFB induced ectopic activity. LtB induced normalization of electrophysiologic parameters can be explained by the finding that LtB hyperpolarized MFBs from –25 mV to –50 mV, thus limiting their depolarizing effect on cardiomyocytes which was shown before to cause slow conduction and ectopic activity. Conclusions: Pharmacological interference with the cytoskeleton of cardiac MFBs alters their electrophysiological phenotype to such an extent that detrimental effects on cardiomyocyte electrophysiology are completely abolished. This observation might form a basis for the development of therapeutic strategies aimed at limiting the arrhythmogenic potential of MFBs.

Modification of Actin Fibers Changes the Electrical Phenotype of Cardiac Myofibroblasts

Background: Slow conduction and ectopic activity are major determinants of cardiac arrhythmogenesis. Both of these conditions can be elicited by myofibroblasts (MFBs) following establishment of heterocellular gap junctional coupling with cardiomyocytes. MFBs appear during structural remodeling of the heart and are characterized by the expression of α-smooth muscle actin (α-SMA) containing stress fibers. In this study, we investigated whether pharmacological interference with the actin cytoskeleton affects myofibroblast arrhythmogeneicity. Methods: Experiments were performed with patterned growth strands of neonatal rat ventricular cardiomyocytes coated with cardiac MFBs. Impulse conduction velocity (θ) and maximal upstroke velocities of propagated action potentials (dV/dtmax), expressed as % action potential amplitude change (%APA) per ms, were measured optically using voltage sensitive dyes. Actin was destabilized by latrunculin B (LtB) and cytochalasin D and stabilized with jasplakinolide. Data are given as mean ± S.D. (n = 5-22). Single cell electrophysiology was assessed using standard patch-clamp techniques. Results: As revealed by immunocytochemistry, exposure of MFBs to LtB (0.01-10 μmol/L) profoundly disrupted stress fibers which led to drastic changes in cell morphology with MFBs assuming an astrocyte-like shape. In control cardiomyocyte strands (no MFB coat), LtB had negligible effects on θ and dV/dtmax. In contrast, LtB applied to MFB-coated strands increased θ dose-dependently from 197 ± 35 mm/s to 344 ± 26 mm/s and dV/dtmax from 38 ± 5 to 78 ± 3% APA/ms, i.e., to values virtually identical to those of cardiomyocyte control strands (339 ± 24 mm/s; 77 ± 3% APA/ms). Highly similar results were obtained when exposing the preparations to cytochalasin D. In contrast, stabilization of actin with increasing concentrations of jasplakinolide exerted no significant effects on impulse conduction characteristics in MFB-coated strands. Whole-cell patch-clamp experiments showed that LtB hyperpolarized MFBs from -25 mV to -50 mV, thus limiting their depolarizing effect on cardiomyocytes which was shown before to cause arrhythmogenic slow conduction and ectopic activity. Conclusion: Pharmacological interference with the actin cytoskeleton of cardiac MFBs affects their electrophysiological phenotype to such an extent that they loose their detrimental effects on cardiomyocyte electrophysiology. This result might form a basis for the development of therapeutic strategies aimed at limiting the arrhythmogenic potential of MFBs.

Graft remodeling during growth following anterior cruciate ligament reconstruction in skeletally immature sheep

INTRODUCTION: Ruptures of the anterior cruciate ligament are being diagnosed with increasing frequency in skeletally immature individuals. It was our aim to investigate the graft remodelling process following an autologous, transphyseal reconstruction of the anterior cruciate ligament (ACL) in skeletally immature sheep. We hypothesized that the ligamentisation process in immature sheep is quicker and more complete when compared to adult sheep. MATERIALS AND METHODS: Skeletally immature sheep with an age of 4 months underwent a fully transphyseal ACL reconstruction using an autologous tendon. The animals were subsequently sacrificed at 3, 6, 12 and 24 weeks following surgery. Each group was characterised histomorphometrically, by immunostaining (VEGF, SMA), by transmission electron microscopy (TEM) and biomechanically (UFS Roboter). RESULTS: The histomorphometric analysis and presence of VEGF and SMA positive cells demonstrated a rapid return to a ligament like structure. The biomechanical analysis revealed an anteroposterior translation that was still increased even 6 months following surgery. CONCLUSION: As in adult sheep models, the remodeling of a soft tissue graft used for ACL reconstruction results in a biomechanically inferior substitute. However, the immature tissue seems to remodel faster and more complete when compared to adults.

Neonatal steroids induce a down-regulation of tenascin-C and elastin and cause a deceleration of the first phase and an acceleration of the second phase of lung alveolarization

Pre- and postnatal corticosteroids are often used in perinatal medicine to improve pulmonary function in preterm infants. To mimic this clinical situation, newborn rats were treated systemically with dexamethasone (Dex), 0.1-0.01 mg/kg/day on days P1-P4. We hypothesized that postnatal Dex may have an impact on alveolarization by interfering with extracellular matrix proteins and cellular differentiation. Morphological alterations were observed on 3D images obtained by high-resolution synchrotron radiation X-ray tomographic microscopy. Alveolarization was quantified stereologically by estimating the formation of new septa between days P4 and P60. The parenchymal expression of tenascin-C (TNC), smooth muscle actin (SMA), and elastin was measured by immunofluorescence and gene expression for TNC by qRT-PCR. After Dex treatment, the first phase of alveolarization was significantly delayed between days P6 and P10, whereas the second phase was accelerated. Elastin and SMA expressions were delayed by Dex treatment, whereas TNC expression was delayed and prolonged. A short course of neonatal steroids impairs the first phase of alveolarization, most likely by altering the TNC and elastin expression. Due to an overshooting catch-up during the second phase of alveolarization, the differences disappear when the animals reach adulthood.

3D scaffolds co-seeded with human endothelial progenitor and mesenchymal stem cells: evidence of prevascularisation within 7 days

Blood supply is a critical issue in most tissue engineering approaches for large defect healing. As vessel ingrowth from surrounding tissues is proven to be insufficient, current strategies are focusing on the neo-vascularisation process. In the present study, we developed an in vitro pre-vascularised construct using 3D polyurethane (PU) scaffolds, based on the association of human Endothelial Progenitor Cells (EPC, CD34+ and CD133+) with human Mesenchymal Stem Cells (MSC). We showed the formation of luminal tubular structures in the co-seeded scaffolds as early as day 7 in culture. These tubular structures were proven positive for endothelial markers von Willebrand Factor and PECAM-1. Of special significance in our constructs is the presence of CD146-positive cells, as a part of the neovasculature scaffolding. These cells, coming from the mesenchymal stem cells population (MSC or EPC-depleted MSC), also expressed other markers of pericyte cells (NG2 and αSMA) that are known to play a pivotal function in the stabilisation of newly formed pre-vascular networks. In parallel, in co-cultures, osteogenic differentiation of MSCs occurred earlier when compared to MSCs monocultures, suggesting the close cooperation between the two cell populations. The presence of angiogenic factors (from autologous platelet lysates) in association with osteogenic factors seems to be crucial for both cell populations' cooperation. These results are promising for future clinical applications, as all components (cells, growth factors) can be prepared in an autologous way.

Distortion-sensitive insight into the pretransitional behavior of 4- n -octyloxy-4′-cyanobiphenyl (8OCB)

Results of studies of the static and dynamic dielectric properties in rod-like 4-n-octyloxy-4'-cyanobiphenyl (8OCB) with isotropic (I)–nematic (N)–smectic A (SmA)–crystal (Cr) mesomorphism, combined with measurements of the low-frequency nonlinear dielectric effect and heat capacity are presented. The analysis is supported by the derivative-based and distortion-sensitive transformation of experimental data. Evidence for the I–N and N–SmA pretransitional anomalies, indicating the influence of tricritical behavior, is shown. It has also been found that neither the N phase nor the SmA phase are uniform and hallmarks of fluid–fluid crossovers can be detected. The dynamics, tested via the evolution of the primary relaxation time, is clearly non-Arrhenius and described via τ(T) = τc(T−TC)−phgr. In the immediate vicinity of the I–N transition a novel anomaly has been found: Δτ ∝ 1/(T − T*), where T* is the temperature of the virtual continuous transition and Δτ is the excess over the 'background behavior'. Experimental results are confronted with the comprehensive Landau–de Gennes theory based modeling.

The craniosacral progression of muscle development influences the emergence of neuromuscular junction alterations in a severe murine model for spinal muscular atrophy.

AIMS As 4-day-old mice of the severe spinal muscular atrophy (SMA) model (dying at 5-8 days) display pronounced neuromuscular changes in the diaphragm but not the soleus muscle, we wanted to gain more insight into the relationship between muscle development and the emergence of pathological changes and additionally to analyse intercostal muscles which are affected in human SMA. METHODS Structures of muscle fibres and neuromuscular junctions (NMJs) of the diaphragm, intercostal and calf muscles of prenatal (E21) and postnatal (P0 and P4) healthy and SMA mice were analysed by light and transmission electron microscopy. NMJ innervation was studied by whole mount immunofluorescence in diaphragms of P4 mice. RESULTS During this period, the investigated muscles still show a significant neck-to-tail developmental gradient. The diaphragm and calf muscles are most and least advanced, respectively, with respect to muscle fibre fusion and differentiation. The number and depth of subsynaptic folds increases, and perisynaptic Schwann cells (PSCs) acquire a basal lamina on their outer surface. Subsynaptic folds are connected to an extensive network of tubules and beaded caveolae, reminiscent of the T system in adult muscle. Interestingly, intercostal muscles from P4 SMA mice show weaker pathological involvement (that is, vacuolization of PSCs and perineurial cells) than those previously described by us for the diaphragm, whereas calf muscles show no pathological changes. CONCLUSION SMA-related alterations appear to occur only when the muscles have reached a certain developmental maturity. Moreover, glial cells, in particular PSCs, play an important role in SMA pathogenesis.

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the reclamation of unassembled snRNP components.

Genetic Engineering of Induced Pluripotent Stem Cells and Reprogramming to Motor Neurons to Elucidate Selective Motor Neuron Death in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease, fatal within 1 to 5 years after onset of symptoms. About 3 out of 100’000 persons are diagnosed with ALS and there is still no cure available [1, 2]. 95% of all cases occur sporadically and the aetiology remains largely unknown [XXXX]. However, up to now 16 genes were identified to play a role in the development of familial ALS. One of these genes is FUS that encodes for the protein fused in sarcoma/translocated in liposarcoma (FUS/TLS). Mutations in this gene are responsible for some cases of sporadic as well as of inherited ALS [3]. FUS belongs to the family of heterogeneous nuclear ribonucleoproteins and is predicted to be involved in several cellular functions like transcription regulation [4], RNA splicing [5, 6], mRNA transport in neurons [7] and microRNA processing [8]. Aberrant accumulation of mutated FUS has been found in the cytoplasm of motor neurons from ALS patients [9]. The mislocalization of FUS is based on a mutation in the nuclear localization signal of FUS [10]. However, it is still unclear if the cytoplasmic localization of FUS leads to a toxic gain of cytoplasmic function and/or a loss of nuclear function that might be crucial in the course of ALS. The goal of this project is to characterize the impact of ALS-associated FUS mutations on in vitro differentiated motor neurons. To this end, we edit the genome of induced pluripotent stem cells (iPSC) using transcription activator-like effector nucleases (TALENs) [11,12] to create three isogenic cell lines, each carrying an ALS-associated FUS mutation (G156E, R244C and P525L). These iPSC’s will then be differentiated to motor neurons according to a recently establishe protocol (Ref Wichterle) and serve to study alterations in the transcriptome, proteome and metabolome upon the expression of ALS-associated FUS. With this approach, we hope to unravel the molecular mechanism leading to FUS-associated ALS and to provide new insight into the emerging connection between misregulation of RNA metabolism and neurodegeneration, a connection that is currently implied in a variety of additional neurological diseases, including spinocerebellar ataxia 2 (SCA-2), spinal muscular atrophy (SMA), fragile X syndrome, and myotonic dystrophy.

Genetic Engineering of Induced Pluripotent Stem Cells and Reprogramming to Motor Neurons to Elucidate Selective Motor Neuron Death in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease, fatal within 1 to 5 years after onset of symptoms. About 3 out of 100’000 persons are diagnosed with ALS and there is still no cure available [1, 2]. 95% of all cases occur sporadically and the aetiology remains largely unknown [3]. However, up to now 16 genes were identified to play a role in the development of familial ALS. One of these genes is FUS that encodes for the protein fused in sarcoma (FUS). Mutations in this gene are responsible for some cases of sporadic as well as of inherited ALS [4]. FUS belongs to the family of heterogeneous nuclear ribonucleoproteins and is predicted to be involved in several cellular functions like transcription regulation, RNA splicing, mRNA transport in neurons and microRNA processing [5] Aberrant accumulation of mutated FUS has been found in the cytoplasm of motor neurons from ALS patients [6]. The mislocalization of FUS is based on a mutation in the nuclear localization signal of FUS [7]. However, it is still unclear if the cytoplasmic localization of FUS leads to a toxic gain of cytoplasmic function and/or a loss of nuclear function that might be crucial in the course of ALS. The goal of this project is to characterize the impact of ALS-associated FUS mutations on in vitro differentiated motor neurons. To this end, we edit the genome of induced pluripotent stem cells (iPSC) using transcription activator-like effector nucleases (TALENs) [8,9] to create three isogenic cell lines, each carrying an ALS-associated FUS mutation (G156E, R244C and P525L). These iPSC’s will then be differentiated to motor neurons according to a recently established protocol [10] and serve to study alterations in the transcriptome, proteome and metabolome upon the expression of ALS-associated FUS. With this approach, we hope to unravel the molecular mechanism leading to FUS-associated ALS and to provide new insight into the emerging connection between misregulation of RNA metabolism and neurodegeneration, a connection that is currently implied in a variety of additional neurological diseases, including spinocerebellar ataxia 2 (SCA-2), spinal muscular atrophy (SMA), fragile X syndrome, and myotonic dystrophy. [1] Cleveland, D.W. et al. (2001) Nat Rev Neurosci 2(11): 806-819 [2] Sathasivam, S. (2010) Singapore Med J 51(5): 367-372 [3] Schymick, J.C. et al. (2007) Hum Mol Genet Vol 16: 233-242 [4] Pratt, A.J. et al. (2012). Degener Neurol Neuromuscul Dis 2012(2): 1-14 [5] Lagier-Tourenne, C. Hum Mol Genet, 2010. 19(R1): p. R46-64 [6] Mochizuki, Y. et al. (2012) J Neurol Sci 323(1-2): 85-92 [7] Dormann, D. et al. (2010) EMBO J 29(16): 2841-2857 [8] Hockemeyer, D. et al. (2011) Nat Biotech 29(8): 731-734 [9] Joung, J.K. and J.D. Sander (2013) Nat Rev Mol Cell Biol 14(1): 49-55 [10]Amoroso, M.W. et al. (2013) J Neurosci 33(2): 574-586.