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Purpose Obesity and its comorbidities are risk factors for absenteeism and unemployment. Bariatric surgery might help to intervene in the vicious circle of unemployment, social disadvantage and increasing obesity. The most common bariatric procedures are sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB). This survey analyzes the influence of the bariatric procedure on return to work. Methods The data of a German nationwide multicenter registry StuDoQ|MBE from 2015 to 2020 are evaluated. Patients are surveyed who underwent a primary SG or RYGB while being unemployed: 782 patients are included. Primary endpoint is any form of return to work within 1 year after treatment. The surgical procedure acts as the binary main treatment variable. A multivariate binary logistic regression model was performed with age, sex, vocational training and weight loss as third variables so that odds ratios (OR) and adjusted ORs were determined. Results Of the patients, 41.56% received a RYGB, 58.44% a SG. One year after bariatric surgery, 39.39% of the patients with SG and 33.85% with RYGB reached a return to work. The OR for return to work is 1.27 (p = 0.11) non-significant in favor of SG. The adjusted OR is 1.26 (p = 0.15), indicating that there is no significant influence of the difference between the two surgical procedures on the outcome of return to work. Conclusion There is a positive effect regarding return to work in bariatric patients: More than a third of the previously unemployed patients were employed 1 year after surgery. Procedure-specific influences could not be determined. Graphical abstract

NeuroImage 264, 119703 (2022). doi:10.1016/j.neuroimage.2022.119703, Published by Academic Press, Orlando, Fla.

This paper is concerned with the contributions of signal-driven and expectation-driven mechanisms to a general understanding of the phenomenon of prosodic prominence from a cross-linguistic perspective. It serves as an introduction to the concept of prosodic prominence and discusses the eight papers in the Special Issue, which cover a genetically diverse range of languages. These include Djambarrpuyŋu (an Australian Pama-Nyungan language), Samoan (an Austronesian Malayo-Polynesian language), the Indo-European languages English (Germanic), French (Romance), and Russian (Slavic), Korean (Koreanic), Medumba (Bantu), and two Sino-Tibetan languages, Mandarin and Taiwanese Southern Min.

Head motion during MR acquisition reduces image quality and has been shown to bias neuromorphometric analysis. The quantification of head motion, therefore, has both neuroscientific as well as clinical applications, for example, to control for motion in statistical analyses of brain morphology, or as a variable of interest in neurological studies. The accuracy of markerless optical head tracking, however, is largely unexplored. Furthermore, no quantitative analysis of head motion in a general, mostly healthy population cohort exists thus far. In this work, we present a robust registration method for the alignment of depth camera data that sensitively estimates even small head movements of compliant participants. Our method outperforms the vendor-supplied method in three validation experiments: 1. similarity to fMRI motion traces as a low-frequency reference, 2. recovery of the independently acquired breathing signal as a high-frequency reference, and 3. correlation with image-based quality metrics in structural T1-weighted MRI. In addition to the core algorithm, we establish an analysis pipeline that computes average motion scores per time interval or per sequence for inclusion in downstream analyses. We apply the pipeline in the Rhineland Study, a large population cohort study, where we replicate age and body mass index (BMI) as motion correlates and show that head motion significantly increases over the duration of the scan session. We observe weak, yet significant interactions between this within-session increase and age, BMI, and sex. High correlations between fMRI and camera-based motion scores of proceeding sequences further suggest that fMRI motion estimates can be used as a surrogate score in the absence of better measures to control for motion in statistical analyses.

Purpose (1) to improve the comprehension of the topographical position of the talar dome beneath the inferior articular surface of the tibia and, (2) to illustrate the changes of possible access to the articular surface of the talar dome during arthroscopic treatment of talar osteochondral defects in an anatomical model. Methods Twenty matched pairs (n = 40) of anatomical ankle specimen were used. All specimens were mounted in a standardized fashion, 3-dimensional radiography was performed in 4 defined positions (maximum dorsiflexion, neutral position, noninvasive distraction, and maximum plantarflexion). All radiographs were analyzed and statistically compared. Results Anterior accessibility was highest in maximum plantarflexion (medial: 49.20 ± 9.86%, lateral: 48.19 ± 8.85%), followed by non-invasive distraction (medial: 33.60 ± 7.96%, lateral: 31.98 ± 8.30%). Neutral position (medial: 19.34 ± 6.90%, lateral: 17.54 ± 6.63%) and dorsiflexion (medial: 15.36 ± 5.03%, lateral: 13.88 ± 4.33%) were not able to significantly increase accessibility. Posterior accessibility was greatest in maximum dorsiflexion (medial: 56.69 ± 9.65%, lateral: 46.82 ± 8.36%), followed by neutral position of the ankle joint (medial: 40.95 ± 8.28%, lateral: 31.06 ± 6.92%). Noninvasive distraction (medial: 31.41 ± 8.18%, lateral: 22.99 ± 7.63%) was still significantly better than plantarflexion (medial: 14.54 ± 5.10%, lateral: 13.89 ± 3.14%) and slightly increased accessibility to the talar dome. Medially, a central area of 5.89 ± 9.76% was accessible by maximum plantarflexion and maximum dorsiflexion from anterior and posterior, respectively, laterally a central blind spot of 4.99 ± 8.61% was detected. Conclusions From an anatomical point of view, maximum joint positions of the ankle (i.e., plantarflexion and dorsiflexion) allow for better access to the talar dome in anterior and posterior ankle arthroscopy. Noninvasive distraction may increase accessibility in anterior approaches, but has no benefit from posterior. Clinical Relevance This study provides insight into the morphology of the ankle joint in a standardized laboratory setup and illustrates the influence of different surgically relevant ankle joint positions. The presented data allow for better preoperative planning for the arthroscopic treatment of talar osteochondral defects.

Beta (ß)-synuclein (ß-Syn) has long been considered to be an attenuator for the neuropathological effects caused by the Parkinson’s disease-related alpha (α)-synuclein (α-Syn) protein. However, recent studies demonstrated that overabundant ß-Syn can form aggregates and induce neurodegeneration in central nervous system (CNS) neurons in vitro and in vivo, albeit at a slower pace as compared with α-Syn. Here, we demonstrate that ß-Syn mutants V70M, detected in a sporadic case of dementia with Lewy bodies (DLB), and P123H, detected in a familial case of DLB, robustly aggravate the neurotoxic potential of ß-Syn. Intriguingly, the two mutations trigger mutually exclusive pathways. ß-Syn V70M enhances morphological mitochondrial deterioration and degeneration of dopaminergic and non-dopaminergic neurons, but it has no influence on neuronal network activity. Conversely, ß-Syn P123H silences neuronal network activity, but it does not aggravate neurodegeneration. ß-Syn wild type (WT), V70M and P123H formed proteinase K-resistant intracellular fibrils within neurons, albeit with less stable C-termini as compared with α-Syn. Under cell-free conditions, ß-Syn V70M demonstrated a much slower pace of fibril formation as compared with WT ß-Syn, and P123H fibrils present with a unique phenotype characterized by large numbers of short, truncated fibrils. Thus, it is possible that V70M and P123H cause structural alterations in ß-Syn, which are linked to their distinct neuropathological profiles. The extent of the lesions caused by these neuropathological profiles is almost identical to that of overabundant α-Syn and is thus likely to be directly involved into the etiology of DLB. Overall, this study provides insights into distinct disease mechanisms caused by mutations of ß-Syn.

A contribution of α-Synuclein (α-Syn) to etiology of Parkinson´s disease (PD) and Dementia with Lewy bodies (DLB) is currently undisputed, while the impact of the closely related β-Synuclein (β-Syn) on these disorders remains enigmatic. β-Syn has long been considered to be an attenuator of the neurotoxic effects of α-Syn, but in a rodent model of PD β-Syn induced robust neurodegeneration in dopaminergic neurons of the substantia nigra. Given that dopaminergic nigral neurons are selectively vulnerable to neurodegeneration in PD, we now investigated if dopamine can promote the neurodegenerative potential of β-Syn. We show that in cultured rodent and human neurons a dopaminergic neurotransmitter phenotype substantially enhanced β-Syn-induced neurodegeneration, irrespective if dopamine is synthesized within neurons or up-taken from extracellular space. Nuclear magnetic resonance interaction and thioflavin-T incorporation studies demonstrated that dopamine and its oxidized metabolites 3,4-dihydroxyphenylacetaldehyde (DOPAL) and dopaminochrome (DCH) directly interact with β-Syn, thereby enabling structural and functional modifications. Interaction of DCH with β-Syn inhibits its aggregation, which might result in increased levels of neurotoxic oligomeric β-Syn. Since protection of outer mitochondrial membrane integrity prevented the additive neurodegenerative effect of dopamine and β-Syn, such oligomers might act at a mitochondrial level similar to what is suggested for α-Syn. In conclusion, our results suggest that β-Syn can play a significant pathophysiological role in etiology of PD through its interaction with dopamine metabolites and thus should be re-considered as a disease-relevant factor, at least for those symptoms of PD that depend on degeneration of nigral dopaminergic neurons.

Purpose: Accurate estimation of the position and orientation (pose) of surgical instruments is crucial for delicate minimally invasive temporal bone surgery. Current techniques lack in accuracy and/or line-of-sight constraints (conventional tracking systems) or expose the patient to prohibitive ionizing radiation (intra-operative CT). A possible solution is to capture the instrument with a c-arm at irregular intervals and recover the pose from the image. Methods: i3PosNet infers the position and orientation of instruments from images using a pose estimation network. Said framework considers localized patches and outputs pseudo-landmarks. The pose is reconstructed from pseudo-landmarks by geometric considerations. Results: We show i3PosNet reaches errors less than 0.05mm. It outperforms conventional image registration-based approaches reducing average and maximum errors by at least two thirds. i3PosNet trained on synthetic images generalizes to real x-rays without any further adaptation. Conclusion: The translation of Deep Learning based methods to surgical applications is difficult, because large representative datasets for training and testing are not available. This work empirically shows sub-millimeter pose estimation trained solely based on synthetic training data., Accepted at International journal of computer assisted radiology and surgery pending publication

Several studies have investigated if the levels of α-synuclein autoantibodies (α-syn AAb) differ in serum of Parkinson's disease (PD) patients and healthy subjects. Reproducible differences in their levels could serve as a biomarker for PD. The results of previous studies however remain inconclusive. With the largest sample size examined so far, we aimed to validate serum α-syn AAb levels as a biomarker for PD and investigated the presence of AAbs against other synucleins. We performed ELISA and immunoblots to determine synuclein AAb levels in the serum of 295 subjects comprising 157 PD patients from two independent cohorts, 46 healthy subjects, and 92 patients with other neurodegenerative disorders. Although serum α- and β-syn AAb levels were significantly reduced in patients with PD and other neurodegenerative disorders as compared to controls, the AAb levels displayed high inter-and intra-cohort variability. Furthermore, α-syn AAb levels showed no correlation to clinical parameters like age, disease duration, disease severity, and gender, that might also be directed against beta- and gamma-syn. In conclusion, serum synuclein AAb levels do allow the separation of PD from healthy subjects but not from other neurodegenerative disorders. Thus, synuclein AAbs cannot be regarded as a reliable biomarker for PD.

Leading neuroimaging studies have pushed 3T MRI acquisition resolutions below 1.0 mm for improved structure definition and morphometry. Yet, only few, time-intensive automated image analysis pipelines have been validated for high-resolution (HiRes) settings. Efficient deep learning approaches, on the other hand, rarely support more than one fixed resolution (usually 1.0 mm). Furthermore, the lack of a standard submillimeter resolution as well as limited availability of diverse HiRes data with sufficient coverage of scanner, age, diseases, or genetic variance poses additional, unsolved challenges for training HiRes networks. Incorporating resolution-independence into deep learning-based segmentation, i.e., the ability to segment images at their native resolution across a range of different voxel sizes, promises to overcome these challenges, yet no such approach currently exists. We now fill this gap by introducing a Voxelsize Independent Neural Network (VINN) for resolution-independent segmentation tasks and present FastSurferVINN, which (i) establishes and implements resolution-independence for deep learning as the first method simultaneously supporting 0.7-1.0 mm whole brain segmentation, (ii) significantly outperforms state-of-the-art methods across resolutions, and (iii) mitigates the data imbalance problem present in HiRes datasets. Overall, internal resolution-independence mutually benefits both HiRes and 1.0 mm MRI segmentation. With our rigorously validated FastSurferVINN we distribute a rapid tool for morphometric neuroimage analysis. The VINN architecture, furthermore, represents an efficient resolution-independent segmentation method for wider application, accepted at NeuroImage

Exosomal transfers represent an important mode of intercellular communication. Syntenin is a small scaffold protein that, when binding ALIX, can direct endocytosed syndecans and syndecan cargo to budding endosomal membranes, supporting the formation of intraluminal vesicles that compose the source of a major class of exosomes. Syntenin, however, can also support the recycling of these same components to the cell surface. Here, by studying mice and cells with syntenin-knock out, we identify syntenin as part of dedicated machinery that integrates both the production and the uptake of secreted vesicles, supporting viral/exosomal exchanges. This study significantly extends the emerging role of heparan sulfate proteoglycans and syntenin as key components for macromolecular cargo internalization into cells. ispartof: SCIENTIFIC REPORTS vol:11 issue:1 ispartof: location:England status: published

Background: The long-term success of nonsurgical weight reduction programs is variable; thus, predictors of outcome are of major interest. We hypothesized that the intestinal microbiota known to be linked with diet and obesity contain such predictive elements. Methods: Metagenome analysis by shotgun sequencing of stool DNA was performed in a cohort of 15 adults with obesity (mean body mass index 43.1 kg/m2) who underwent a one-year multidisciplinary weight loss program and another year of follow-up. Eight individuals were persistently successful (mean relative weight loss 18.2%), and seven individuals were not successful (0.2%). The relationship between relative abundancies of bacterial genera/species and changes in relative weight loss or body mass index was studied using three different statistical modeling methods. Results: When combining the predictor variables selected by the applied statistical modeling, we identified seven bacterial genera and eight bacterial species as candidates for predicting success of weight loss. By classification of relative weight-loss predictions for each patient using 2–5 term models, 13 or 14 out of 15 individuals were predicted correctly. Conclusions: Our data strongly suggest that gut microbiota patterns allow individual prediction of long-term weight loss success. Prediction accuracy seems to be high but needs confirmation by larger prospective trials.

Alpha-synuclein (aSyn) is a central player in Parkinson’s disease (PD) but the precise molecular mechanisms underlying its pathogenicity remain unclear. It has recently been suggested that nuclear aSyn may modulate gene expression, possibly via interactions with DNA. However, the biological behavior of aSyn in the nucleus and the factors affecting its transcriptional role are not known. Here, we investigated the mechanisms underlying aSyn-mediated transcription deregulation by assessing its effects in the nucleus and the impact of phosphorylation in these dynamics. We found that aSyn induced severe transcriptional deregulation, including the downregulation of important cell cycle-related genes. Importantly, transcriptional deregulation was concomitant with reduced binding of aSyn to DNA. By forcing the nuclear presence of aSyn in the nucleus (aSyn-NLS), we found the accumulation of high molecular weight aSyn species altered gene expression and reduced toxicity when compared with the wild-type or exclusively cytosolic protein. Interestingly, nuclear localization of aSyn, and the effect on gene expression and cytotoxicity, was also modulated by phosphorylation on serine 129. Thus, we hypothesize that the role of aSyn on gene expression and, ultimately, toxicity, may be modulated by the phosphorylation status and nuclear presence of different aSyn species. Our findings shed new light onto the subcellular dynamics of aSyn and unveil an intricate interplay between subcellular location, phosphorylation and toxicity, opening novel avenues for the design of future strategies for therapeutic intervention in PD and other synucleinopathies.

Background We report a rare case of Mammalian orthoreovirus (MRV) infection in a child with a primary immunodeficiency (PID). Infections with Mammalian orthoreovirus are very rare and probably of zoonotic origin. Only a few cases have been described so far, including one with similar pathogenesis as in our case. Case presentation The patient, age 11, presented with flu-like symptoms and persistent severe diarrhea. Enterovirus has been detected over several months, however, exact typing of a positive cell culture remained inconclusive. Unbiased metagenomic sequencing then detected MRV in stool samples from several time points. The sequencing approach further revealed co-infection with a recombinant Coxsackievirus and Adenovirus. MRV-specific antibodies detected by immunofluorescence proved that the patient seroconverted. Conclusion This case highlights the potential of unbiased metagenomic sequencing in supplementing routine diagnostic methods, especially in situations of chronic infection with multiple viruses as seen here in an immunocompromised host. The origin, transmission routes and implications of MRV infection in humans merit further investigation. Electronic supplementary material The online version of this article (10.1186/s12879-018-2946-7) contains supplementary material, which is available to authorized users.

International journal of computer assisted radiology and surgery (2020). doi:10.1007/s11548-020-02189-w, Published by Springer, Heidelberg [u.a.]

A major hallmark of Parkinson's disease is loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The pathophysiological mechanisms causing this relatively selective neurodegeneration are poorly understood, and thus experimental systems allowing to study dopaminergic neuron dysfunction are needed. Induced pluripotent stem cells (iPSCs) differentiated toward a dopaminergic neuronal phenotype offer a valuable source to generate human dopaminergic neurons. However, currently available protocols result in a highly variable yield of dopaminergic neurons depending on the source of hiPSCs. We have now developed a protocol based on HBA promoter-driven transient expression of transcription factors by means of adeno-associated viral (AAV) vectors, that allowed to generate very consistent numbers of dopaminergic neurons from four different human iPSC lines. We also demonstrate that AAV vectors expressing reporter genes from a neuron-specific hSyn1 promoter can serve as surrogate markers for maturation of hiPSC-derived dopaminergic neurons. Dopaminergic neurons differentiated by transcription factor expression showed aggravated neurodegeneration through α-synuclein overexpression, but were not sensitive to γ-synuclein overexpression, suggesting that these neurons are well suited to study neurodegeneration in the context of Parkinson’s disease.

Non-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood–brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

Neurons with a desired neurotransmitter phenotype can be differentiated from induced pluripotent stem cells or from somatic cells only through tedious protocols with relatively low yield. Readily available cortical neurons isolated from embryonic rat brain, which have already undergone a complete neuronal differentiation process, might serve as alternative template source. These cultures consist of 85% glutamatergic and 15% GABAergic neurons, and we attempted to trans-differentiate them into dopaminergic neurons. Transcription factors Nurr1, Lmx1A and Pitx3, essential determinants of a dopaminergic cell fate during CNS development, were not sufficient to induce tyrosine hydroxylase expression in a significant number of cells. Combining Nurr1 with the generic neuronal differentiator and re-programming factor Ascl1, however, resulted in generation of neurons which express dopaminergic markers TH, AADC, VMAT2 and DAT. Only neurons of GABAergic phenotype could be trans-differentiated towards a dopaminergic neurotransmitter phenotype, while for glutamatergic neurons, this process proved to be neurotoxic. Intriguingly, GABAergic neurons isolated from embryonal midbrain could not be trans-differentiated into dopaminergic neurons by Ascl1 and Nurr1. Thus, in principle, post-mitotic embryonal neurons can serve as templates for neurons with a desired neurotransmitter phenotype. However, neurotransmitter phenotype plasticity critically depends on the differentiation history of the template neurons, which can result in relatively low yields of dopaminergic neurons.

TAU protein aggregation is the main characteristic of neurodegenerative diseases known as tauopathies. Low-grade chronic inflammation is also another hallmark that indicates crosstalk between damaged neurons and glial cells. Previously, we have demonstrated that neurons overexpressing TAUP301L release CX3CL1, which activates the transcription factor NRF2 signalling to limit over-activation in microglial cells in vitro and in vivo. However, the connection between CX3CL1/CX3CR1 and NRF2 system and its functional implications in microglia are poorly described. We evaluated CX3CR1/NRF2 axis in the context of tauopathies and its implication in neuroinflammation. Regarding the molecular mechanisms that connect CX3CL1/CX3CR1 and NRF2 systems, we observed that in primary microglia from Cx3cr1-/- mice the mRNA levels of Nrf2 and its related genes were significantly decreased, establishing a direct linking between both systems. To determine functional relevance of CX3CR1, migration and phagocytosis assays were evaluated. CX3CR1-deficient microglia showed impaired cell migration and deficiency of phagocytosis, as previously described for NRF2-deficient microglia, reinforcing the idea of the relevance of the CX3CL1/CX3CR1 axis in these events. The importance of these findings was evident in a tauopathy mouse model where the effects of sulforaphane (SFN), an NRF2 inducer, were examined on neuroinflammation in Cx3cr1+/+ and Cx3cr1-/- mice. Interestingly, the treatment with SFN was able to modulate astrogliosis but failed to reduce microgliosis in Cx3cr1-/- mice. These findings suggest an essential role of the CX3CR1/NRF2 axis in microglial function and in tauopathies. Therefore, polymorphisms with loss of function in CX3CR1 or NRF2 have to be taken into account for the development of therapeutic strategies., This work was supported by a Spanish Ministry of Economy and Competitiveness (Grants refs. SAF2016-76520-R).

Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and regeneration. Though of prime interest, their potentially protective role on neutrophil-induced tissue damage, associated with high morbidity and mortality, has not been explored in sufficient detail. Here we report the therapeutic skill of MSCs to suppress unrestrained neutrophil activation and to attenuate severe tissue damage in a murine immune-complex mediated vasculitis model of unbalanced neutrophil activation. MSC-mediated neutrophil suppression was due to intercellular adhesion molecule 1-dependent engulfment of neutrophils by MSCs, decreasing overall neutrophil numbers. Similar to MSCs in their endogenous niche of murine and human vasculitis, therapeutically injected MSCs via upregulation of the extracellular superoxide dismutase (SOD3), reduced superoxide anion concentrations and consequently prevented neutrophil death, neutrophil extracellular trap formation and spillage of matrix degrading neutrophil elastase, gelatinase and myeloperoxidase. SOD3-silenced MSCs did not exert tissue protective effects. Thus, MSCs hold substantial therapeutic promise to counteract tissue damage in conditions with unrestrained neutrophil activation.

Navigation in high-precision minimally invasive surgery (HP-MIS) demands high tracking accuracy in the absence of line of sight (LOS). Currently, no tracking technology can satisfy this requirement. Electromagnetic tracking (EMT) is the best tracking paradigm in the absence of LOS despite limited accuracy and robustness. Novel evaluation protocols are needed to ensure high-precision and robust EMT for navigation in HP-MIS. We introduce a novel protocol for EMT measurement evaluation featuring a high-accuracy phantom based on LEGO $$^{\circledR }$$ , which is calibrated by a coordinate measuring machine to ensure accuracy. Our protocol includes relative sequential positions and an uncertainty estimation of positioning. We show effects on distortion compensation using a learned interpolation model. Our high-precision protocol clarifies properties of errors and uncertainties of EMT for high-precision use cases. For EMT errors reaching clinically relevant 0.2 mm, our design is 5–10 times more accurate than previous protocols with 95% confidence margins of 0.02 mm. This high-precision protocol ensures the performance improvement in compensated EMT by 0.05 mm. Our protocol improves the reliability of EMT evaluations because of significantly lower protocol-inherent uncertainties. To reduce patient risk in HP-MIS and to evaluate magnetic field distortion compensation, more high-accuracy protocols such as the one proposed here are required.

We compared a published computational model of the action potential of a specific type of human induced pluripotent stem cell -derived cardiomyocytes (hiPSC-CM) with experimental field potential data with regard to their inter-beat interval and the duration of repolarization. In particular, concomitant changes in inter-beat interval and duration of repolarization were calculated after reduction and/or augmentation of specific ion channel conductances as a surrogate for pharmacological manipulation. The observed mismatches between calculations and experimental data indicate that there is information missing about the cellular test system. Based on our results we hypothesize that, among other currents, the actual If ("funny current") may deviate from the prediction. We show that replacement of the If formulation by alternative equations causes the model predictions to change qualitatively, however, none of the available formulations is actually achieving a satisfactory match with experimental data. We suggest a strategy to clarify whether the mismatch can be completely resolved at all using single cell models and, if yes, how this goal could be reached.

NeuroLNC, a previously unknown long ncRNA, regulates neuronal activity by interacting with the neurodegeneration protein TDP-43., The cellular and the molecular mechanisms by which long noncoding RNAs (lncRNAs) may regulate presynaptic function and neuronal activity are largely unexplored. Here, we established an integrated screening strategy to discover lncRNAs implicated in neurotransmitter and synaptic vesicle release. With this approach, we identified neuroLNC, a neuron-specific nuclear lncRNA conserved from rodents to humans. NeuroLNC is tuned by synaptic activity and influences several other essential aspects of neuronal development including calcium influx, neuritogenesis, and neuronal migration in vivo. We defined the molecular interactors of neuroLNC in detail using chromatin isolation by RNA purification, RNA interactome analysis, and protein mass spectrometry. We found that the effects of neuroLNC on synaptic vesicle release require interaction with the RNA-binding protein TDP-43 (TAR DNA binding protein-43) and the selective stabilization of mRNAs encoding for presynaptic proteins. These results provide the first proof of an lncRNA that orchestrates neuronal excitability by influencing presynaptic function.

Early afterdepolarizations (EADs) are pathological voltage oscillations during the repolarization phase of cardiac action potentials. They are considered as potential precursors to cardiac arrhythmias and have recently gained much attention in the context of preclinical drug safety testing under the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. From the viewpoint of multiple time scales theory, the onset of EADs has previously been studied by means of mathematical action potential models with one slow ion channel gating variable. In this article, we for the first time associate EADs with mixed mode oscillations in dynamical systems with two slow gating variables and present a folded node singularity of the slow flow as a novel mechanism for EADs genesis. We derive regions of the pharmacology parameter space in which EADs occur using both the folded node analysis and a full system bifurcation analysis, and we suggest the normal distance to the boundary of the EADs region as a mechanism-based risk metric to computationally estimate a drug's proarrhythmic liability.

The grey matter is a central target of pathological processes in neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. The grey matter is often also affected in multiple sclerosis, an autoimmune disease of the central nervous system. The mechanisms that underlie grey matter inflammation and degeneration in multiple sclerosis are not well understood. Here we show that, in Lewis rats, T cells directed against the neuronal protein β-synuclein specifically invade the grey matter and that this is accompanied by the presentation of multifaceted clinical disease. The expression pattern of β-synuclein induces the local activation of these T cells and, therefore, determined inflammatory priming of the tissue and targeted recruitment of immune cells. The resulting inflammation led to significant changes in the grey matter, which ranged from gliosis and neuronal destruction to brain atrophy. In humans, β-synuclein-specific T cells were enriched in patients with chronic-progressive multiple sclerosis. These findings reveal a previously unrecognized role of β-synuclein in provoking T-cell-mediated pathology of the central nervous system. In a rat model of multiple sclerosis, β-synuclein-specific T cells induce inflammation and pathological changes in the grey matter of the central nervous system; these cells were also found in higher numbers in patients with multiple sclerosis, particularly those with a chronic progressive course.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF's neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF's effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.

Aggregation and fibril formation of human alpha-Synuclein (αS) are neuropathological hallmarks of Parkinson's disease and other synucleinopathies. The molecular mechanisms of αS aggregation and fibrillogenesis are largely unknown. Several studies suggested a sequence of events from αS dimerization via oligomerization and pre-fibrillar aggregation to αS fibril formation. In contrast to αS, little evidence suggests that γS can form protein aggregates in the brain, and for βS its neurotoxic properties and aggregation propensities are controversially discussed. These apparent differences in aggregation behavior prompted us to investigate the first step in Synuclein aggregation, i.e. the formation of dimers or oligomers, by Bimolecular Fluorescence Complementation in cells. This assay showed some Synuclein-specific limitations, questioning its performance on a single cell level. Nevertheless, we unequivocally demonstrate that all Synucleins can interact with each other in a very similar way. Given the divergent aggregation properties of the three Synucleins this suggests that formation of dimers is not predictive for the aggregation of αS, βS or γS in the aged or diseased brain.

Background and Purpose Human ether‐a‐go‐go‐related gene (hERG; Kv11.1) channel inhibition is a widely accepted predictor of cardiac arrhythmia. hERG channel inhibition alone is often insufficient to predict pro‐arrhythmic drug effects. This study used a library of dofetilide derivatives to investigate the relationship between standard measures of hERG current block in an expression system and changes in action potential duration (APD) in human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs). The interference from accompanying block of Cav1.2 and Nav1.5 channels was investigated along with an in silico AP model. Experimental Approach Drug‐induced changes in APD were assessed in hiPSC‐CMs using voltage‐sensitive dyes. The IC50 values for dofetilide and 13 derivatives on hERG current were estimated in an HEK293 expression system. The relative potency of each drug on APD was estimated by calculating the dose (D150) required to prolong the APD at 90% (APD90) repolarization by 50%. Key Results The D150 in hiPSC‐CMs was linearly correlated with IC50 of hERG current. In silico simulations supported this finding. Three derivatives inhibited hERG without prolonging APD, and these compounds also inhibited Cav1.2 and/or Nav1.5 in a channel state‐dependent manner. Adding Cav1.2 and Nav1.2 block to the in silico model recapitulated the direction but not the extent of the APD change. Conclusions and Implications Potency of hERG current inhibition correlates linearly with an index of APD in hiPSC‐CMs. The compounds that do not correlate have additional effects including concomitant block of Cav1.2 and/or Nav1.5 channels. In silico simulations of hiPSC‐CMs APs confirm the principle of the multiple ion channel effects.

BACKGROUND: Adeno-associated virus (AAV) vectors are used to deliver potentially therapeutic genes in clinical trials in Parkinson's disease (PD). Pre-existing immunity to AAV and a local neuroinflammatory response might negatively affect the efficacy of such AAV-mediated gene delivery. METHODS: We pre-immunized rats with wild-type AAV-2. Three months later, we created PD-like lesions by intrastriatal injections of 6-hydroxydopamine (6-OHDA) in 50% of the animals. One month later, we injected AAV2 vector expressing enhanced green fluorescent protein (eGFP) in the striatum. Using immunohistochemistry, we assessed eGFP expression, microglia activation and CD8 T cell infiltration. We also measured AAV-2 specific neutralizing antibody titers in the serum. RESULTS: The number of striatal cells transduced with AAV2 vector expressing eGFP was reduced by 71% in rats pre-immunized with wild-type AAV2 compared to non-immunized animals. We detected elevated numbers of OX6(+) activated microglia in the striatum and circulating AAV2-specific neutralizing antibodies in pre-immunized rats. We also observed that the intrastriatal 6-OHDA injection promoted CD8(+) T cell infiltration and enhanced microglia activation. Nevertheless, the 6-OHDA lesion did not alter AAV2-mediated expression of eGFP in either pre-immunized or non-immunized rats. CONCLUSIONS: Our findings indicate that intracerebral AAV2-based gene therapy is compromised in rats with pre-existing immunity to AAV2. By contrast, a local neuroinflammatory response, caused by intrastriatal a 6-OHDA injection, does not affect viral vector-mediated transgene expression. Our results emphasize the importance of monitoring circulating AAV-specific neutralizing antibodies in patients undergoing intracerebral gene therapy using AAV vectors. Copyright © 2014 John Wiley & Sons, Ltd. peerReviewed

The chemokine fractalkine modulates microglial responses in neurodegenerative diseases, including tauopathies, but the mechanistic processes and their relevance in human brain pathologies is not yet known. Here, we show that hippocampal HT22 cells expressing human TAU(P301L) mutant protein produce fractalkine, which in microglia activates AKT, inhibits glycogen synthase kinase-3β and upregulates the transcription factor NRF2/NFE2L2 and its target genes including heme oxygenase 1. In a mouse model of tauopathy based on stereotaxic delivery in hippocampus of an adeno-associated viral vector for expression of TAU(P301L), we confirmed that tau-injured neurons express fractalkine. NRF2- and fractalkine receptor-knockout mice did not express heme oxygenase 1 in microglia and exhibited increased microgliosis and astrogliosis in response to neuronal TAU(P301L) expression, demonstrating a crucial role of the fractalkine/NRF2/heme oxygenase 1 pathway in attenuation of the pro-inflammatory phenotype. The hippocampus of patients with Alzheimer's disease also exhibits increased expression of fractalkine in TAU-injured neurons that recruit microglia. These events correlated with increased levels of NRF2 and heme oxygenase 1 proteins, suggesting an attempt of the diseased brain to limit microgliosis. Our combined results indicate that fractalkine mobilizes NRF2 to limit over-activation of microglia and identify this new target to control unremitting neuroinflammation in tauopathies.

Voltage sensors (VSs) initiate the pore opening and closure in voltage-gated ion channels. Here, we propose a technique for estimation of the equilibrium constant of the up- and downward VS movements and rate constants of pore transitions from macroscopic current kinetics. Bell-shaped voltage dependence of the activation/deactivation time constants and Bolzmann distributions of CaV1.2 activation were analyzed in terms of a circular four-state (rest, activated, open, deactivated) channel model: both dependencies uniquely constrain the model parameters. Neutralization of gating charges in IS4 or IIS4 only slightly affects the equilibrium constant of VS transition while affecting simultaneously the rate constants of pore opening and closure. The application of our technique revealed that pore mutations on IS6–IVS6 segments induce pronounced shifts of the VS equilibrium between the resting (down) and activated (up) position. Analyzing a channelopathy mutation highlighted that the leftward shift of the activation curve induced by I781T on IIS6 is only partially (35 %) caused by a destabilization of the channel pore but predominantly (65 %) by a shifted VS equilibrium towards activation. The algorithm proposed for CaV1.2 may be applicable for calculating rate constants from macroscopic current kinetics in other voltage-gated ion channels. Electronic supplementary material The online version of this article (doi:10.1007/s00424-013-1319-8) contains supplementary material, which is available to authorized users.

Model building of biochemical reaction networks typically involves experiments in which changes in the behavior due to natural or experimental perturbations are observed. Computational models of reaction networks are also used in a systems biology approach to study how transitions from a healthy to a diseased state result from changes in genetic or environmental conditions. In this paper we consider the nonlinear inverse problem of inferring information about the Jacobian of a Langevin type network model from covariance data of steady state concentrations associated to two different experimental conditions. Under idealized assumptions on the Langevin fluctuation matrices we prove that relative alterations in the network Jacobian can be uniquely identified when comparing the two data sets. Based on this result and the premise that alteration is locally confined to separable parts due to network modularity we suggest a computational approach using hybrid stochastic-deterministic optimization for the detection of perturbations in the network Jacobian using the sparsity promoting effect of $$\ell _p$$ -penalization. Our approach is illustrated by means of published metabolomic and signaling reaction networks.

This article is a follow-up study of Féry and Kügler (2008. Pitch accent scaling on given, new and focused constituents in German. Journal of Phonetics, 36, 680–703). It reports on an experiment of the F0 height of potential pitch accents in the postfocal region of German sentences and addresses in this way an aspect of the influence of information structure on the intonation of sentences that was left open in the previous article. The results of the experiment showed that, when several constituents are located in this position, they are often in a downstep relation, but are rarely upstepped. In 37% of the cases, the pitch accents are only realized dynamically and there is no down- or upstepping. We interpret these results as evidence that postfocal constituents are phrased independently. The data examined speak against a model of postfocal intonation in which postfocal phrasing is eliminated and all accents are reduced to zero. Instead, the pitch accents are often present, although reduced. Moreover, the facts support the existence of prosodic phrasing of the postfocal constituents; the postfocal position implies an extremely compressed register, but no dephrasing or systematic complete deaccentuation of all pitch accents. We propose adopting a model of German intonation in which prosodic phrasing is determined by syntactic structure and cannot be changed by information structure. The role of information structure in prosody is limited to changes in the register relationship of the different parts of the sentence. Prefocally, there is no or only little register compression because of givenness. Postfocally, register compression is the rule. A model of intonation must take this asymmetry into account.

Peripheral immune responses can be modified by associative learning procedures. Less is known, however, whether and to what extent neuroendocrine parameters can be classically conditioned. In this randomized double-blind study, we modified an established paradigm to behaviorally condition endocrine responses in humans. Thirty-one healthy male participants received a distinctively flavored green drink as the conditioned stimulus (CS) and intravenous injections of corticotropin-releasing hormone (CRH) (CRH group, N = 17) or NaCl (placebo group, N = 14) as the unconditioned stimulus (US) during two subsequent acquisition trials. Plasma levels of cortisol and noradrenaline, heart rate, and psychological parameters were analyzed before and 15, 30, 60, 120, and 180 min after injection. The two acquisition trials were followed by two evocation trials, during which participants underwent the same procedure but now receiving NaCl injections. CRH administration induced pronounced increases in cortisol and noradrenaline plasma concentrations, heart rate, and anxiety levels. However, re-exposure to the CS during evocations trials did not provoke conditioned increases in neuroendocrine parameters. Median split of the CRH group based on the cortisol baseline level into “cort-high” and “cort-low” subgroups showed that the “cort-high” subgroup displayed a significantly increased cortisol production on evocation days compared to the “cort-low” subgroup and the placebo group. This taste-endocrine paradigm employing CRH injection as the US in healthy male volunteers failed to induce a behaviorally conditioned cortisol release as a learned endocrine response. Future studies should clarify a possible role of higher baseline cortisol levels in perhaps facilitating a conditioned cortisol response.

The antibody species that patrol in a patient's blood are an invaluable part of the immune system. While most of them shield us from life-threatening infections, some of them do harm in autoimmune diseases. If we knew exactly all the antigens that elicited all the antibody species within a group of patients, we could learn which ones correlate with immune protection, are irrelevant, or do harm. Here, we demonstrate an approach to this question: First, we use a plethora of phage-displayed peptides to identify many different serum antibody binding peptides. Next, we synthesize identified peptides in the array format and rescreen the serum used for phage panning to validate antibody binding peptides. Finally, we systematically vary the sequence of validated antibody binding peptides to identify those amino acids within the peptides that are crucial for binding "their" antibody species. The resulting immune fingerprints can then be used to trace them back to potential antigens. We investigated the serum of an individual in this pipeline, which led to the identification of 73 antibody fingerprints. Some fingerprints could be traced back to their most likely antigen, for example the immunodominant capsid protein VP1 of enteroviruses, most likely elicited by the ubiquitous poliovirus vaccination. Thus, with our approach, it is possible, to pinpoint those antibody species that correlate with a certain antigen, without any pre-information. This can help to unravel hitherto enigmatic diseases.

Based on the observation that microRNA (miRNA) 133b enhances regeneration after spinal cord injury in the adult zebrafish, we investigated whether this miRNA would be beneficial in a mammalian system in vitro and in vivo. We found that infection of cultured neurons with miR-133b promotes neurite outgrowth in vitro on an inhibitory substrate consisting of mixed chondroitin sulfate proteoglycans, when compared to infection with green fluorescent protein (GFP) for control. In vivo, viral infection of the injured adult mouse spinal cord at the time of injury at and in the vicinity of the lesion site enhanced expression of miR-133b. Measurements of locomotor recovery by Basso Mouse Scale (BMS) showed improvement of recovery starting at 4 weeks after injury and virus injection. This improvement was associated with downregulation of the expression levels of Ras homolog gene family member A (RhoA), chondroitin sulfate proteoglycans, and microglia/macrophage marker in the spinal cord as assayed 6 weeks after injury. Potential inhibitory molecules carrying consensus sequences for binding of miR-133b were identified in silico and verified in a reporter assay in vitro showing reductions in expression of RhoA, xylosyltransferase 1 (Xylt1), ephrin receptor A7 (Epha7), and purinergic receptor P2X ligand-gated ion channel 4 (P2RX4). These results encourage targeting miR-133 for therapy.

Current gene therapy approaches for Parkinson’s disease (PD) deliver neurotrophic factors like glial cell line-derived neurotrophic factor (GDNF) or neurturin via neuronal transgene expression. Since these potent signaling-inducing neurotrophic factors can be distributed through long-distance neuronal projections to unaffected brain sites, this mode of delivery may eventually cause side effects. To explore a localized and thus potentially safer alternative for gene therapy of PD, we expressed GDNF exclusively in astrocytes and evaluated the efficacy of this approach in the mouse 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) and rat 6- hydroxy-dopamine (6-OHDA) models of PD. In terms of protection of dopaminergic cell bodies and projections, dopamine (DA) synthesis and behaviour, astrocyte- derived GDNF demonstrated the same efficacy as neuron-derived GDNF. In terms of safety, unilateral striatal GDNF expression in astrocytes did not result in delivery of bio-active GDNF to the contralateral hemispheres (potential off-target sites) as happened when GDNF was expressed in neurons. Thus, astrocytic GDNF expression represents a localized but efficient alternative to current gene therapeutic strategies for the treatment of PD, especially if viral vectors with enhanced tissue penetration are considered. Astrocytic neurotrophic factor expression may open new venues for neurotrophic factor-based gene therapy targeting severe diseases of the brain. peerReviewed

Sumoylation of α-synuclein decreases its rate of aggregation and its deleterious effects in vitro and in vivo., Posttranslational modification of proteins by attachment of small ubiquitin-related modifier (SUMO) contributes to numerous cellular phenomena. Sumoylation sometimes creates and abolishes binding interfaces, but increasing evidence points to another role for sumoylation in promoting the solubility of aggregation-prone proteins. Using purified α-synuclein, an aggregation-prone protein implicated in Parkinson’s disease that was previously reported to be sumoylated upon overexpression, we compared the aggregation kinetics of unmodified and modified α-synuclein. Whereas unmodified α-synuclein formed fibrils, modified α-synuclein remained soluble. The presence of as little as 10% sumoylated α-synuclein was sufficient to delay aggregation significantly in vitro. We mapped SUMO acceptor sites in α-synuclein and showed that simultaneous mutation of lysines 96 and 102 to arginine significantly impaired α-synuclein sumoylation in vitro and in cells. Importantly, this double mutant showed increased propensity for aggregation and cytotoxicity in a cell-based assay and increased cytotoxicity in dopaminergic neurons of the substantia nigra in vivo. These findings strongly support the model that sumoylation promotes protein solubility and suggest that defects in sumoylation may contribute to aggregation-induced diseases.

Adeno-associated virus (AAV)-mediated expression of wild-type or mutant P301L protein tau produces massive degeneration of pyramidal neurons without protein tau aggregation. We probed this novel model for genetic and structural factors and early parameters of pyramidal neurodegeneration. In yellow fluorescent protein expressing transgenic mice, intracerebral injection of AAV-tauP301L revealed early damage to apical dendrites of CA1 pyramidal neurons, whereas their somata remained normal. Ultrastructurally, more and enlarged autophagic vacuoles were contained in degenerating dendrites and manifested as dark, discontinuous, vacuolated processes surrounded by activated astrocytes. Dendritic spines were lost in AAV-tauP301L-injected yellow fluorescent protein-expressing transgenic mice, and ultrastructurally, spines appeared dark and degenerating. In CX3CR1(EGFP/EGFP)-deficient mice, microglia were recruited early to neurons expressing human tau. The inflammatory response was accompanied by extravasation of plasma immunoglobulins alpha 2-Macroglobulin, but neither albumin nor transferrin, became lodged in the brain parenchyma. Large proteins, but not Evans blue, entered the brain of mice injected with AAV-tauP301L. Ultrastructurally, brain capillaries were constricted and surrounded by swollen astrocytes with extensions that contacted degenerating dendrites and axons. Together, these data corroborate the hypothesis that neuroinflammation participates essentially in tau-mediated neurodegeneration, and the model recapitulates early dendritic defects reminiscent of "dendritic amputation" in Alzheimer's disease. (Am J Pathol 2011, 179:2001-2015; DOI: 10.1016/j.ajpath.2011.06.025)

Alzheimer's dementia is developing ever more as a complex syndrome with various unknown genetic and epigenetic contributions. These are compounded on and exacerbating the underlying amyloid and tau pathology that remain the basis of the pathological definition of Alzheimer's disease. Here, we present a selection of aspects of recent bigenic and virus-based mouse strains, developed as pre-clinical models for Alzheimer's disease. We discuss newer features in the context of the characteristics defined in previously validated transgenic models. We focus on specific aspects of single and multiple transgenic mouse models for Alzheimer's disease and for tauopathies, rather than providing an exhaustive list of all available models. We concentrate on the content of information related to neurodegeneration and disease mechanisms. We pay attention to aspects and defects that are predicted by the models and can be tested in humans. We discuss implications that help translate the fundamental knowledge into clinical, diagnostic and therapeutic applications. We elaborate on the increasing knowledge extracted from transgenic models and from newer adeno-associated viral models. We advocate this combination as a valuable strategy to study molecular, cellular and system-related pathogenic mechanisms in AD and tauopathies. We believe that innovative animal models remain needed to critically test current views, to identify and validate therapeutic targets, to allow testing of compounds, to help understand and eventually treat tauopathies, including Alzheimer's disease.

Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R–A–O and a deactivation O–D–R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A–O) and deactivation (D–R) are described by rate constants α and β, and γ and δ, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 CaV1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the “pore-releasing” sensor movement ((x(V)) was much weaker than the voltage dependence of “pore-locking” sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening CaV1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.

A recombinant immunotoxin was constructed by fusing a single chain fragment variable antibody fragment, specific for the melanoma-associated chondroitin sulfate proteoglycan (MCSP), to a truncated variant of Pseudomonas exotoxin A (ETA'), carrying a C-terminal KDEL-peptide for improved retrograde intracellular transport. The resulting immunotoxin MCSP-ETA' was periplasmatically expressed in Escherichia coli and purified under native conditions by affinity chromatography resulting in a yield of approximately 30 mug/l bacterial culture. This immunotoxin induced antigen-specific apoptosis in the cultured human melanoma-derived cell lines A2058 and A375M, and treatment with a single dose of the agent eliminated up to 80% of these cells within 72 h. The dose needed for half-maximum killing (EC50) was approximately 1 nmol/l for both cell lines. MCSP-ETA' also displayed cytotoxic activity against cultured primary melanoma cells from patients with advanced disease (pathologic stages IIIC and IV), with net cell death reaching up to 70% within 96 h after treatment with a single dose of 14 nmol/l. MCSP-ETA' induced cell death synergistically with cyclosporin A, both in established human melanoma cell lines and cultured primary melanoma cells. The distinctive antigen-restricted induction of apoptosis and the synergy with cyclosporin A justify further evaluation of this novel agent with regard to its potential application for the treatment of malignant melanoma.

Neuropsychological and neuroimaging data have shown a functional lateralization of the medial temporal lobes for verbal and non-verbal memory material respectively. We hypothesized that this lateralization is related to language lateralization. Using fMRI we conducted three memory tasks with different memory material (words, faces, landscape images) alongside with a paradigm for the determination of language dominance in 44 healthy subjects. We included left as well as right-handed subjects because persons with atypical language dominance are known to be overrepresented in the group of left-handers. Lateralization indices for the BOLD activation in the medial temporal lobes from the memory tasks were correlated with those for the language task. We show that the material-specific lateralization is related to language dominance such that verbal encoding shows strong positive relation to language dominance whereas face encoding shows the opposite effect. Our data provide first fMRI evidence for a relation between language dominance and material specificity of the medial temporal lobes for memory functions. We suggest that the language-dominant hemisphere is more strongly engaged in memory processing of verbal material. These data provide grounds for the investigation of pathological changes in this relationship due to cortical dysfunctions.

Summary Encephalopathies and neurological disorders are sometimes associated with respiratory tract infections caused by Bordetella pertussis. For these complications to occur cerebral barriers have to be compromised. Therefore, the influence of pertussis toxin (PT), a decisive virulence determinant of B. pertussis, on endothelial barrier integrity was investigated. Human brain microvascular endothelial cells cultured on Transwell filter devices were used as model for the blood brain barrier. PT, but not its B-oligomer, induced a reduction of the transendothelial resistance and enhanced the permeability for the protein marker horseradish peroxidase. Moreover, transmigration of human monocytes was also elevated suggesting a PT-associated enhancement of the diapedesis of blood leucocytes. Uptake and trafficking of PT was followed by electron microscopy via clathrin-coated pits and accumulation in lysosomes and microvesicular bodies. The breach in barrier integrity was accompanied by a transient disintegration of Golgi structures. Interestingly, PT-induced effects were only transient and restoration of barrier function was observed after 24 h. In summary, intoxication by PT causes a transient destruction of the cellular organization in human brain-derived endothelial cells resulting in a transient disruption of barrier functions. We suggest that these findings reflect early steps in the development of neurological disorders associated with pertussis disease.

Molybdenum cofactor (MoCo) deficiency is a progressive neurological disorder that inevitably leads to early childhood death because of the lack of any effective therapy. In a mouse model of MoCo deficiency type A, the most frequent form of this autosomal recessively inherited disease, the affected animals show the biochemical characteristics of sulphite and xanthine intoxication and do not survive2 wk after birth. We have constructed a recombinant-expression cassette for the gene MOCS1, which, via alternative splicing, facilitates the expression of the proteins MOCS1A and MOCS1B, both of which are necessary for the formation of a first intermediate, cyclic pyranopterin monophosphate (cPMP), within the biosynthetic pathway leading to active MoCo. A recombinant adeno-associated virus (AAV) vector was used to express the artificial MOCS1 minigene, in an attempt to cure the lethal MOCS1-deficient phenotype. The vector was used to transduce Mocs1-deficient mice at both 1 and 4 d after birth or, after a pretreatment with purified cPMP, at 40 d after birth. We report here that all Mocs1-deficient animals injected with a control AAV-enhanced green fluorescent protein vector died approximately 8 d after birth or after withdrawal of cPMP supplementation, whereas AAV-MOCS1-transduced animals show significantly increased longevity. A single intrahepatic injection of AAV-MOCS1 resulted in fertile adult animals without any pathological phenotypes.

This chapter outlines some general principles of transcriptional targeting approaches using viral vectors in the central nervous system. Transcriptional targeting is first discussed in the context of vector tropism and appropriate delivery. Then, some of our own attempts to restrict expression of therapeutic factors to distinct brain cell populations are discussed, followed by a detailed description of the setscrews that are available for these experiments. A critical discussion of current stumbling blocks and necessary developments to achieve clinical applicability of advanced targeted vector systems is provided.

Background Ticks act as vectors for a large number of different pathogens, perhaps most notably Borrelia burgdorferi, the causative agent of Lyme disease. The most prominent tick vector in the United States is the blacklegged tick, Ixodes scapularis. Tick bites are of special public health concern since there are no vaccines available against most tick-transmitted pathogens. Based on the observation that certain non-natural host animals such as guinea pigs or humans can develop adaptive immune responses to tick bites, anti-tick vaccination is a potential approach to tackle health risks associated with tick bites. Results The aim of this study was to use an oligopeptide phage display strategy to identify immunogenic salivary gland proteins from I. scapularis that are recognized by human immune sera. Oligopeptide libraries were generated from salivary gland mRNA of 18 h fed nymphal I. scapularis. Eight immunogenic oligopeptides were selected using human immune sera. Three selected immunogenic oligopeptides were cloned and produced as recombinant proteins. The immunogenic character of an identified metalloprotease (MP1) was validated with human sera. This enzyme has been described previously and was hypothesized as immunogenic which was confirmed in this study. Interestingly, it also has close homologs in other Ixodes species. Conclusion An immunogenic protein of I. scapularis was identified by oligopeptide phage display. MP1 is a potential candidate for vaccine development.

Machado-Joseph disease (MJD) is a fatal, dominantly inherited neurodegenerative disorder associated with an expanded polyglutamine tract within the ataxin-3 protein, and characterized by progressive impairment of motor coordination, associated with neurodegeneration of specific brain regions, including cerebellum and striatum. The currently available therapies do not allow modification of disease progression. Neuropeptide Y (NPY) has been shown to exert potent neuroprotective effects by multiple pathways associated with the MJD mechanisms of disease. Thus, we evaluated NPY levels in MJD and investigated whether raising NPY by gene transfer would alleviate neuropathological and behavioural deficits in cerebellar and striatal mouse models of the disease. For that, a cerebellar transgenic and a striatal lentiviral-based models of MJD were used. NPY overexpression in the affected brain regions in these two mouse models was obtained by stereotaxic injection of adeno-associated viral vectors encoding NPY. Up to 8 weeks after viral injection, balance and motor coordination behaviour and neuropathology were analysed. We observed that NPY levels were decreased in two MJD patients' cerebella and in striata and cerebella of disease mouse models. Furthermore, overexpression of NPY alleviated the motor coordination impairments and attenuated the related neuropathological parameters, preserving cerebellar volume and granular layer thickness, reducing striatal lesion and decreasing mutant ataxin-3 aggregation. Additionally, NPY mediated increase of brain-derived neurotrophic factor levels and decreased neuroinflammation markers. Our data suggest that NPY is a potential therapeutic strategy for MJD.

Spinal fusion surgery causes severe postoperative pain, hampering reconvalescense. We investigated the efficacy of patient-controlled epidural analgesia (PCEA) in a prospective, double-blind, randomized, controlled comparison with patient-controlled IV analgesia (PCIA).After lumbar anterior-posterior fusion receiving an epidural catheter intraoperatively, 72 patients were given either PCEA (ropivacaine 0.125% and sufentanil 1.0 microg/mL at 14 mL/h; bolus: 5 mL; lockout time: 15 min) and IV placebo or PCIA (morphine 2.0 mg/mL; bolus: 3 mg; lockout time: 15 min) and epidural placebo. Pain levels (visual analog scale 0-10), functional capabilities (turning in bed, standing, and walking), analgesic consumption, and side effects were evaluated until 72 h after surgery.Fourteen patients were excluded by predetermined criteria, leaving 58 patients for data analysis. Pain levels at rest and during mobilization were significantly lower in the PCEA when compared with that in the PCIA group throughout the study period (P0.0001 in all cases). Time until able to turn in bed was achieved earlier in the PCEA group (P0.05). Patients in the PCEA group were significantly more satisfied with pain therapy (P0.01).We conclude that PCEA with ropivacaine and sufentanil, using intraoperatively placed epidural catheters, provides superior analgesia and higher patient satisfaction when compared with PCIA after spinal fusion surgery.