Substantially improved gene transfer to interneurons with second-generation glutamate receptor-targeted DART-AAV vectors.

BACKGROUND: Adeno-associated viral vectors (AAVs) are a widely used gene transfer platform in neuroscience. Although naturally AAV serotypes can have preferences for certain tissues, selectivity for particular cell types in the CNS does not exist. Towards interneuron targeting, capsid engineering of AAV2 including display of the designed ankyrin repeat protein (DARPin) 2K19 specific for the glutamate receptor subunit 4 (GluA4) at the N-terminus of the VP2 capsid protein has been established. The resulting AAV-VP2N is highly specific for interneurons, but exhibits rather moderate transduction efficiencies. METHODS: Two alternative insertion sites for 2K19 in the GH2/GH3 loop of capsid proteins VP1 (AAV-VP1L) or VP2 (AAV-VP2L) were exploited to yield second generation GluA4-AAVs. Having packaged reporter genes under ubiquitous promoters, the vectors were characterized for biochemical properties as well as gene delivery into cell lines and rat hippocampal slice cultures. Electrophysiological recordings monitored the functional properties of transduced cells. RESULTS: Compared to AAV-VP2N, the second-generation vectors, especially AAV-VP1L, achieved about 2-fold higher genomic titers as well as a substantially improved GluA4 binding. Improvements in gene transfer activities were 18-fold on GluA4-overexpressing A549 cells and five-fold on rat hippocampal organotypic slice cultures reaching approximately 60 % of all parvalbumin positive interneurons upon a single administration. The spiking behaviour of transduced cells was unaltered and characteristic for a heterogeneous group of interneurons. CONCLUSION: The substantially improved gene transfer activity of the second generation GluA4-targeted AAV combined with low toxicity makes this vector an attractive tool for interneuron-directed gene transfer with unrestricted promotor and transgene choice.

Establishment of a reliable in-vivo model of implant-associated infection to investigate innovative treatment options.

The increasing number of implant-associated infections and of multiresistant pathogens is a major problem in the daily routine. In the field of osteomyelitis, it is difficult to manage a valid clinical study because of multiple influencing factors. Therefore, models of osteomyelitis with a simulation of the pathophysiology to evaluate treatment options for implant-associated infections are necessary. The aim of this study is to develop a standardized and reproducible osteomyelitis model in-vivo to improve treatment options. This study analyses the influence of a post-infectious implant exchange one week after infection and the infection progress afterward in combination with a systemic versus a local antibiotic treatment in-vivo. Therefore, the implant exchange, the exchange to a local drug-delivery system with gentamicin, and the implant removal are examined. Furthermore, the influence of an additional systemic antibiotic therapy is evaluated. An in-vivo model concerning the implant exchange is established that analyzes clinic, radiologic, microbiologic, histologic, and immunohistochemical diagnostics to obtain detailed evaluation and clinical reproducibility. Our study shows a clear advantage of the combined local and systemic antibiotic treatment in contrast to the implant removal and to a non-combined antibiotic therapy. Group genta/syst. showed the lowest infection rate with a percentage of 62.5% concerning microbiologic analysis, which is in accordance with the immunohistochemical, cytochemical, histologic, and radiologic analysis. Our in-vivo rat model has shown valid and reproducible results, which will lead to further investigations regarding treatment options and influencing factors concerning the therapy of osteomyelitis and implant-associated infections.

Fabrication of pH-degradable supramacromolecular microgels with tunable size and shape via droplet-based microfluidics.

This study presents a versatile method to synthesize stimuli-responsive microgels with supramolecular cross-links exhibiting tunable size and shape via droplet-based microfluidics. The natural polyphenol tannic acid (TA) is used to cross-link poly(N-vinylcaprolactam) (PVCL) chains in aqueous droplets by the formation of hydrogen bonds and hydrophobic interactions between the phenolic groups of TA and the carbonyl group and the hydrophobic segments of lactam ring of PVCL chains. The obtained microgels exhibit diameters in the range of 130-150µm in swollen state in aqueous solution. Synthesized microgels exhibit pH-responsive behavior: at low pH microgels deswell and shrink due to the protonation of phenolic groups and enhanced hydrophobic interactions; at high pH microgels swell and disintegrate due to the deprotonation of phenolic groups and destruction of hydrogen bonds with PVCL chains. Additionally, we present supramacromolecular microgels in cylindrical shape with different aspect ratios using a new design of microfluidic chip by varying flow rates at high concentration of the prepolymerized precursor combined with rapid pH-triggered on-chip gelation. Furthermore, developed synthesis methodology allows on-chip encapsulation of colloidal objects into large supramacromolecular microgels during the cross-linking step. The complete and fast release of objects by pH-triggered degradation indicates that the pH-responsive supramacromolecular microgels can be used for controlled loading/release of various payloads, like probiotics. Moreover, cell studies of L929 fibroblast clearly show the biocompatibility of the microgels.

Autologous chondrocyte implantation (ACI) for cartilage defects of the knee: A guideline by the working group "Clinical Tissue Regeneration" of the German Society of Orthopaedics and Trauma (DGOU).

BACKGROUND: Autologous chondrocyte implantation (ACI) is an established and well-accepted procedure for the treatment of localised full-thickness cartilage defects of the knee. METHODS: The present review of the working group "Clinical Tissue Regeneration" of the German Society of Orthopaedics and Trauma (DGOU) describes the biology and function of healthy articular cartilage, the present state of knowledge concerning therapeutic consequences of primary cartilage lesions and the suitable indication for ACI. RESULTS: Based on best available scientific evidence, an indication for ACI is given for symptomatic cartilage defects starting from defect sizes of more than three to four square centimetres; in the case of young and active sports patients at 2.5cm(2), while advanced degenerative joint disease needs to be considered as the most important contraindication. CONCLUSION: The present review gives a concise overview on important scientific background and the results of clinical studies and discusses the advantages and disadvantages of ACI. LEVEL OF EVIDENCE: Non-systematic Review.

Skin edema in first trimester fetuses.

PURPOSE: The aim of this study was to prove an association between generalized skin edema and nuchal translucency (NT) thickness and its predictive value for aneuploidy and structural anomalies. MATERIALS AND METHODS: In this retrospective study fetuses with and without skin edema in the first trimester with an NT above 2.2 mm were compared. Statistical significance was calculated with the Chi-square test (p < 0.05). RESULTS: 237 fetuses were included in this study (median NT of 3.0; IQR: 2.5 - 3.9 mm, median CRL 68.8; IQR: 58.9 - 74.9 mm). 17.3 % presented with skin edema. The rate of skin edema was 1.3 % in the group with an NT < 95th percentile, 2.7 % in the group with an NT between the 95th and 99th percentile, 17.5 % with an NT of 3.5 - 4.4 mm, 36.4 % with an NT of 4.5 - 5.4 mm, 54.5 % with an NT of 5.5 - 6.4 mm and 95.5 % with an NT above 6.5 mm. 19 % had chromosomal disorders. The rate of aneuploidy was 61.0 % (25/41) in the group with skin edema which was significantly higher than the rate of 10.2 % (20/196) in those without skin edema (p < 0.0001). 12 % had structural anomalies in euploid fetuses. The rate of anomalies was 43.8 % (7/16) in the group with skin edema and significantly higher compared to 9.1 % (16/176) in those without skin edema (p < 0.0005). CONCLUSION: Our data show a clear association between the thickness of NT and the rate of skin edema. Skin edema has a high predictive value for aneuploidy or structural malformations. Therefore, fetuses with skin edema should have early malformation scans in case of normal karyotype.

[Autologous chondrocyte implantation (ACI) for cartilage defects of the knee: a guideline by the working group "Tissue Regeneration" of the German Society of Orthopaedic Surgery and Traumatology (DGOU)]. / Stellenwert der autologen Chondrozytentransplantation (ACT) in der Behandlung von Knorpelschäden des Kniegelenks - Empfehlungen der AG Klinische Geweberegeneration der DGOU.

Autologous chondrocyte transplantation/implantation (ACT/ACI) is an established and recognised procedure for the treatment of localised full-thickness cartilage defects of the knee. The present review of the working group "Clinical Tissue Regeneration" of the German Society of Orthopaedics and Traumatology (DGOU) describes the biology and function of healthy articular cartilage, the present state of knowledge concerning potential consequences of primary cartilage lesions and the suitable indication for ACI. Based on current evidence, an indication for ACI is given for symptomatic cartilage defects starting from defect sizes of more than 3-4 cm2; in the case of young and active sports patients at 2.5 cm2. Advanced degenerative joint disease is the single most important contraindication. The review gives a concise overview on important scientific background, the results of clinical studies and discusses advantages and disadvantages of ACI.

Evaluation strategies for isotope ratio measurements of single particles by LA-MC-ICPMS.

Data evaluation is a crucial step when it comes to the determination of accurate and precise isotope ratios computed from transient signals measured by multi-collector-inductively coupled plasma mass spectrometry (MC-ICPMS) coupled to, for example, laser ablation (LA). In the present study, the applicability of different data evaluation strategies (i.e. 'point-by-point', 'integration' and 'linear regression slope' method) for the computation of (235)U/(238)U isotope ratios measured in single particles by LA-MC-ICPMS was investigated. The analyzed uranium oxide particles (i.e. 9073-01-B, CRM U010 and NUSIMEP-7 test samples), having sizes down to the sub-micrometre range, are certified with respect to their (235)U/(238)U isotopic signature, which enabled evaluation of the applied strategies with respect to precision and accuracy. The different strategies were also compared with respect to their expanded uncertainties. Even though the 'point-by-point' method proved to be superior, the other methods are advantageous, as they take weighted signal intensities into account. For the first time, the use of a 'finite mixture model' is presented for the determination of an unknown number of different U isotopic compositions of single particles present on the same planchet. The model uses an algorithm that determines the number of isotopic signatures by attributing individual data points to computed clusters. The (235)U/(238)U isotope ratios are then determined by means of the slopes of linear regressions estimated for each cluster. The model was successfully applied for the accurate determination of different (235)U/(238)U isotope ratios of particles deposited on the NUSIMEP-7 test samples.

Aortic dissecting aneurysms--histopathological findings.

Acute aortic dissection is a life-threatening disease with a high rate of mortality. At the Institute of Legal Medicine of the Hanover Medical School, 30 cases with aortic dissections were found during autopsy and examined histologically between 2006 and 2009. The grade of medial alterations in the form of cystic medial necrosis, elastin fragmentation, fibrosis and medionecrosis were estimated semi-quantitatively. In order to assess the normal aging process, samples of the aortic wall of 25 decedents without dissecting aneurysms were analyzed histologically. This study demonstrates that there are partly quantitative differences, particularly with a statistically significant increase in cystic medial necrosis (p<0.001) and elastin fragmentation (p<0.001), between aortas from dissecting aneurysms and the normal aging aorta, which may help to identify genetically predisposed relatives of patients with a dissection of the aorta.

Tunable fragmentation of organic molecules in laser ablation glow discharge time-of-flight mass spectrometry.

A DC-pulsed glow discharge (GD) has distinct temporal regimes which are characterized by "softer" or "harder" ionization of analytes introduced into the discharge. It is thus possible to obtain both molecular weight and structural fragment information from the same spectra. In order to extend the capabilities of this technique a laser ablation (LA) sampling system was coupled to a DC-pulsed GD and to a time-of-flight (TOF) mass spectrometer (MS) for characterizing organic samples such as oleic acid, reserpine, two different peptides, and a polymer. Both hard and soft ionization regimes were studied. These LAGD-TOFMS results were compared to matrix-assisted laser desorption ionization (MALDI) spectra using the same compounds (i.e., analytes, concentration, and matrix). It was found that LAGD offers tunable ionization and provides a reduced matrix dependence. However, the sensitivity achieved by the prototype LAGD-TOFMS was significantly lower when compared with commercially available MALDI-TOFMS instrumentation. Since LAGD-TOFMS is rather new, some technical details to increase its sensitivity are discussed.

Critical assessment of the elemental composition of Corning archeological reference glasses by LA-ICP-MS.

Corning archeological reference glasses A, B, C, and D have been made to simulate different historic technologies of glass production and are used as standards in historic glass investigations. In this work, nanoseconds (193, 266 nm) and femtosecond (800 nm) laser ablation were used to study the elemental composition of Corning glasses using laser ablation inductively coupled plasma mass spectrometry. The determined concentrations of 26 oxides (Li(2)O, B(2)O(3), Na(2)O, MgO, Al(2)O(3), SiO(2), P(2)O(5), K(2)O, CaO, TiO(2), V(2)O(5), Cr(2)O(3), MnO, Fe(2)O(3), CoO, NiO, CuO, ZnO, Rb(2)O, SrO, ZrO(2), SnO(2), Sb(2)O(5), BaO, PbO, Bi(2)O(3)) are compared with values reported in the literature. Results show variable discrepancies between the data, with the largest differences found for Cr(2)O(3) in Corning A; Li(2)O, B(2)O(3), and Cr(2)O(3) in Corning B; and MnO, Sb(2)O(5), Cr(2)O(3), and Bi(2)O(3) in Corning C. The best agreement between the measured and literature values was found for Corning D. However, even for this reference, glass re-evaluation of the data was necessary and new values for PbO, BaO, and Bi(2)O(3) are proposed.

Plasma noradrenaline and state anxiety levels predict placebo response in learned immunosuppression.

Large interindividual differences exist in the presence and extent of placebo responses in both experimental and clinical studies, but little is known about possible predictors of these responses. We employed a behaviorally conditioned immunosuppression paradigm in healthy men to analyze predictors of learned placebo responses. During acquisition, the subjects received either the immunosuppressant cyclosporin A (n = 32) or a placebo (n = 14) (unconditioned stimuli (US)) together with a novel-tasting drink (conditioned stimulus (CS)). During evocation, the subjects were reexposed to the CS alone. In responders (n = 15), the CS alone caused a significant inhibition of interleukin (IL)-2 production by anti-CD3-stimulated peripheral blood T cells, closely mimicking the drug effect. Nonresponders (n = 17) did not show responses different from those of the controls. Multiple-regression analyses showed that baseline IL-2, plasma noradrenaline, and state anxiety predicted nearly 60% of the variance in the conditioned IL-2 response. These data provide first evidence for putative biological and psychological predictors of learned placebo responses.

Quantitative chemical imaging of element diffusion into heterogeneous media using laser ablation inductively coupled plasma mass spectrometry, synchrotron micro-X-ray fluorescence, and extended X-ray absorption fine structure spectroscopy.

Quantitative chemical imaging of trace elements in heterogeneous media is important for the fundamental understanding of a broad range of chemical and physical processes. The primary aim of this study was to develop an analytical methodology for quantitative high spatial resolution chemical imaging based on the complementary use of independent microanalytical techniques. The selected scientific case study is focused on high spatially resolved quantitative imaging of major elements, minor elements, and a trace element (Cs) in Opalinus clay, which has been proposed as the host rock for high-level radioactive waste repositories. Laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS), providing quantitative chemical information, and synchrotron radiation based micro-X-ray fluorescence (SR-microXRF), providing high spatial resolution images, were applied to study Cs migration into Opalinus clay rock. The results indicate that combining the outputs achievable by the two independent techniques enhances the imaging capabilities significantly. The qualitative high resolution image of SR-microXRF is in good agreement with the quantitative image recorded with lower spatial resolution by LA-ICPMS. Combining both techniques, it was possible to determine that the Opalinus clay sample contains two distinct domains: (i) a clay mineral rich domain and (ii) a calcium carbonate dominated domain. The two domains are separated by sharp boundaries. The spatial Cs distribution is highly correlated to the distribution of the clay. Furthermore, extended X-ray absorption fine structure analysis indicates that the trace element Cs preferentially migrates into clay interlayers rather than into the calcite domain, which complements the results acquired by LA-ICPMS and SR-microXRF. By using complementary techniques, the quantification robustness was improved to quantitative micrometer spatial resolution. Such quantitative, microscale chemical images allow a more detailed understanding of the chemical reactive transport process into and within heterogeneous media to be gained.

[Clusters of respiratory tract infections and alert strategy in nursing homes]. / Cas groupés d'infections respiratoires aiguës et stratégie d'alerte en institutions de personnes âgées.

OBJECTIVE: Outbreaks of acute respiratory infections (ARI) are common in institutions for elderly people. We had for objective to investigate clusters of cases (lower respiratory tract infection and influenza-like illness [LRTI/ILI]) in order to improve and validate alert strategies in these institutions. METHODOLOGY: Prospective surveillance for LRTI/ILI was implemented in 11 institutions in Alsace, over five years. Clinical criteria were used to identify infected residents and clusters. Nasopharyngeal swabs were collected and rapid tests (Immunoassay) were performed to identify the influenza virus. RESULTS: The three week periods were analyzed if three cases or more were recorded during the first week. This analysis demonstrated an important risk of epidemic when this number of cases was reached in healthcare units. The influenza virus (10 clusters) and respiratory syncytial virus ([RSV], two clusters) were identified. CONCLUSION: The authors confirmed and emphasized the importance of adequate surveillance for clusters of respiratory tract infection cases. Early identification of an outbreak (three cases) is an important point to prevent transmission, especially during epidemic periods and if a virus is identified in the unit or institution.

[The unrecognized myocardial infarction]. / Der nicht erkannte Myokardinfarkt.

BACKGROUND AND OBJECTIVES: despite the improved quality of diagnostic technology, myocardial infarction still belongs to the diseases that are most frequently overlooked. Especially asymptomatic patients or patients with atypical symptoms are more common to be misdiagnosed. Failure to correctly diagnose is the leading cause of malpractice claims. It was the aim of this study to evaluate the records of patients who died from myocardial infarction and to analyse whether medical malpractice had occured in those who had undergone a medical examination shortly before their death. METHODS: in the years 2008 and 2009 myocardial infarction had been diagnosed in 109 auotopsies performed at the Institute of Legal Medicine of the Hanover Medical School. The records of these patients who had died from myocardial infarction were retrospectively analysed with particular emphasis on an antemortem medical consultation, reported symptoms and diagnostic measures. RESULTS: in 38 persons (34.9 %) an antemortem medical consultation or hospitalisation has taken place, whereby in five persons the diagnosis of myocardial infarction was suspected. In 33 persons, a myocardial infarction could not be diagnosed antemortem. In two cases an additionally cardiologic assessment was recommended to estimate if medical malpractice was present and in another two cases with insufficient diagnostic measures medical malpractice was reproached from forensic pathologists. CONCLUSION: autopsy give the most accurate diagnostic information. On the other hand, it may provide an effective defence against medical malpractice litigation.

LA-ICP-MS analysis of waste polymer materials.

Waste polymer materials were analyzed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The concentrations of 35 elements were determined by using different types of external standards, namely glass and polyethylene (PE) based. Prior to the LA-ICP-MS analysis of determined elements, Na and/or Zn were used as internal standards. The investigations concentrated mainly on the detection of Cr, As, Cd, Sn, Sb, Hg, and Pb. Using PE-based calibration standards, the measured concentrations in the waste polymers were within 49% of the wet chemical data. The determined deviation was up to 102% when using the glass standards. Trace concentration of As and Hg (and also of S) could be determined with a concentration below 1 mg/kg. However, Hg provided very low intensity with a high relative standard deviation (RSD) and was therefore not further evaluated. Cryomilling of polymers was applied to reduce the particle size of the material and improved the precision and accuracy of LA-ICP-MS analysis. On average, the LA-ICP-MS results showed a deviation from the wet chemical reference analysis of 38% and an RSD of 56% for pressed polymer powder samples prepared by cryomilling. In general, waste pellets without sample preparation (i.e., use of pellets as delivered) are too heterogeneous, not suitable for micro-beam techniques, and showed a strong matrix dependence. With homogeneous pellets that appear similar to each other agreement in the determined concentrations was found for some elements.

Increased short circuit current in organic photovoltaic using high-surface area electrode based on ZnO nanowires decorated with CdTe quantum dots.

A photosensitized high-surface area transparent electrode has been employed to increase the short circuit current of a photovoltaic device with a blend of poly(3-hexylthiophene) (P3HT) and (6,6)-phenyl C61 butyric acid methyl ester (PCBM) as the active layer. This is achieved by directly growing ZnO nanowires on indium tin oxide (ITO) film via a physical vapor method. The nanowire surface is then decorated with CdTe quantum dots by pulsed electron-beam deposition (PED). The nanowires alone provided a 20-fold increase in the short circuit current under visible light illumination. This was further increased by a factor of approximately 1.5 by the photosensitization effect of CdTe, which has an optical absorption of up to 820 nm.

Meticillin-resistant Staphylococcus aureus contamination of healthcare workers' uniforms in long-term care facilities.

Meticillin-resistant Staphylococcus aureus (MRSA) and other multiply resistant bacteria are frequently isolated in long-term care facilities (LTCFs). This study evaluated the contamination of staff clothing in three LTCFs. Over 500 samples were taken from uniforms and their pockets and these samples showed a high level of MRSA contamination. Wearing plastic aprons and managing pocket contents improved the contamination rate. Our results highlight the continued importance of hand hygiene, since staff have frequent contact with their uniforms and could potentially contaminate their hands before care.

[Immunoassay rapid test for influenza diagnosis in institutions for elderly people: a four-year surveillance with the GROG Géronto-Alsace]. / Tests rapides de surveillance (TRS) de la grippe en établissements accueillant des personnes âgées: évaluation de quatre années d'utilisation dans le cadre du réseau GROG Géronto-Alsace.

OBJECTIVE: Outbreaks of acute respiratory infections (ARI) are common in institutions for elderly people. The objective of our study was the assessment of immunoassay rapid test used for influenza diagnosis in institutions for elderly people. METHODOLOGY: Prospective surveillance for ARI was conducted in 11 institutions in Alsace over a four-year period. Clinical case definitions are used to identify the infected residents. For the identification of influenza virus, nasopharyngeal swabs are obtained and rapid tests (immunoassay) are performed. RESULTS: Influenza virus was identified with immunoassay rapid test. Then, prophylaxis according to the Conseil supérieur d'hygiène publique de France guidelines was implemented. Nevertheless, the use of the rapid test was not frequent in the individual institution and the information recorded at the GROG Géronto-Alsace level could be use to inform the institutions when it is important to perform these rapid tests. CONCLUSION: Ours findings show the value of the rapid test used in the influenza surveillance and how the networks could help to improve their uses.