Characterization of a PCB Based Pressure Sensor and Its Joining Methods for the Metal Membrane.

Essential quality features of pressure sensors are, among other accuracy-related factors, measurement range, operating temperature, and long-term stability. In this work, these features are optimized for a capacitive pressure sensor with a measurement range of 10 bars. The sensor consists of a metal membrane, which is connected to a PCB and a digital capacitive readout. To optimize the performance, different methods for the joining process are studied. Transient liquid phase bonding (TLP bonding), reactive joining, silver sintering, and electric resistance welding are compared by measurements of the characteristic curves and long-term measurements at maximum pressure. A scanning electron microscope (SEM) with energy-dispersive X-ray spectroscopy (EDX) analysis was used to examine the quality of the joints. The evaluation of the characteristic curves shows the smallest measurement errors for TLP bonding and sintering. For welding and sintering, no statistically significant long-term drift was measured. In terms of equipment costs, reactive joining and sintering are most favorable. With low material costs and short process times, electric resistance welding offers ideal conditions for mass production.

A high-throughput DNA methylation analysis of a single cell.

In recent years, the field of epigenetics has grown dramatically and has become one of the most dynamic and fast-growing branches of molecular biology. The amount of diseases suspected of being influenced by DNA methylation is rising steadily and includes common diseases such as schizophrenia, bipolar disorder, Alzheimer's disease, diabetes, atherosclerosis, cancer, major psychosis, lupus and Parkinson's disease. Due to cellular heterogeneity of methylation patterns, epigenetic analyses of single cells become a necessity. One rationale is that DNA methylation profiles are highly variable across individual cells, even in the same organ, dependent on the function of the gene, disease state, exposure to environmental factors (e.g. radiation, drugs or nutrition), stochastic fluctuations and various other causes. Using a polymerase chain reaction (PCR)-slide microreaction system, we present here a methylation-sensitive PCR analysis, the restriction enzyme-based single-cell methylation assay (RSMA), in the analysis of DNA methylation patterns in single cells. This method addresses the problems of cell heterogeneity in epigenetics research; it is comparably affordable, avoids complicated microfluidic systems and offers the opportunity for high-throughput screening, as many single cells can be screened in parallel. In addition to this study, critical principles and caveats of single cell methylation analyses are discussed.

An intra-cerebral drug delivery system for freely moving animals.

Microinfusions of drugs directly into the central nervous system of awake animals represent a widely used means of unravelling brain functions related to behaviour. However, current approaches generally use tethered liquid infusion systems and a syringe pump to deliver drugs into the brain, which often interfere with behaviour. We address this shortfall with a miniaturised electronically-controlled drug delivery system (20 × 17.5 × 5 mm³) designed to be skull-mounted in rats. The device features a micropump connected to two 8-mm-long silicon microprobes with a cross section of 250 × 250 µm² and integrated fluid microchannels. Using an external electronic control unit, the device allows infusion of 16 metered doses (0.25 µL each, 8 per silicon shaft). Each dosage requires 3.375 Ws of electrical power making the device additionally compatible with state-of-the-art wireless headstages. A dosage precision of 0.25 ± 0.01 µL was determined in vitro before in vivo tests were carried out in awake rats. No passive leakage from the loaded devices into the brain could be detected using methylene blue dye. Finally, the device was used to investigate the effects of the NMDA-receptor antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid, (R)-CPP, administered directly into the prefrontal cortex of rats during performance on a task to assess visual attention and impulsivity. In agreement with previous findings using conventional tethered infusion systems, acute (R)-CPP administration produced a marked increase in impulsivity.

Epigenomic profiling reveals DNA-methylation changes associated with major psychosis.

Epigenetic misregulation is consistent with various non-Mendelian features of schizophrenia and bipolar disorder. To date, however, few studies have investigated the role of DNA methylation in major psychosis, and none have taken a genome-wide epigenomic approach. In this study we used CpG-island microarrays to identify DNA-methylation changes in the frontal cortex and germline associated with schizophrenia and bipolar disorder. In the frontal cortex we find evidence for psychosis-associated DNA-methylation differences in numerous loci, including several involved in glutamatergic and GABAergic neurotransmission, brain development, and other processes functionally linked to disease etiology. DNA-methylation changes in a significant proportion of these loci correspond to reported changes of steady-state mRNA level associated with psychosis. Gene-ontology analysis highlighted epigenetic disruption to loci involved in mitochondrial function, brain development, and stress response. Methylome network analysis uncovered decreased epigenetic modularity in both the brain and the germline of affected individuals, suggesting that systemic epigenetic dysfunction may be associated with major psychosis. We also report evidence for a strong correlation between DNA methylation in the MEK1 gene promoter region and lifetime antipsychotic use in schizophrenia patients. Finally, we observe that frontal-cortex DNA methylation in the BDNF gene is correlated with genotype at a nearby nonsynonymous SNP that has been previously associated with major psychosis. Our data are consistent with the epigenetic theory of major psychosis and suggest that DNA-methylation changes are important to the etiology of schizophrenia and bipolar disorder.

Improved Mounting of Strain Sensors by Reactive Bonding.

Aim of this work is to improve the bond between a strain sensor and a device on which the strain shall be determined. As strain sensor, a CMOS-integrated chip featuring piezoresistive sensor elements was used which is capable of wireless energy and data transmission. The sensor chip was mounted on a standardized tensile test specimen of stainless steel by a bonding process using reactive multilayer systems (RMS). RMS provide a well-defined amount of heat within a very short reaction time of a few milliseconds and are placed in-between two bonding partners. RMS were combined with layers of solder which melt during the bonding process. Epoxy adhesive films were used as a reference bonding process. Under mechanical tensile loading, the sensor bonded with RMS shows a linear strain sensitivity in the whole range of tested forces whereas the adhesive-bonded sensor has slightly nonlinear behavior for low forces. Compared to the adhesive-bonded chips, the sensitivity of the reactively bonded chips is increased by a factor of about 2.5. This indicates a stronger mechanical coupling by reactive bonding as compared to adhesive bonding.

Drug delivery from the oral cavity: focus on a novel mechatronic delivery device.

Dental drug delivery systems have been used for a long time, in particular for the local therapy of diseases affecting the oral cavity. Research today concentrates on the design of formulations to increase their retention time. Even today, however, prosthetic devices incorporating drug delivery are rarely used. Mainly, they are focused on prophylaxis and the release of antibacterial agents. However, as buccal delivery, because of its undeniable advantages, has become popular for systemic drug delivery, and prolonged well-controlled release has been identified as beneficial, especially for chronic diseases, a new class of delivery systems is evolving: highly miniaturized computerized delivery systems, integrated into a dental appliance. Dental delivery systems today are used in two ways: the main application is the local treatment of diseases affecting the oral cavity itself like periodontitis or fungal infections. The second is for systemic drug delivery.

Application of microarrays for DNA methylation profiling.

Comprehensive analyses of the human epigenome may be of critical importance in understanding the molecular mechanisms of complex diseases, development, aging, tissue specificity, parental origin effects, and sex differences, among other systemic aspects of human biology. However, traditional DNA methylation methods allowed for screening of only relatively short DNA fragments. The advent of microarrays has provided new possibilities in DNA methylation analysis, because this technology is able to interrogate a very large number of loci in a highly parallel fashion. There are several permutations of the microarray application in DNA methylation profiling, and such include microarray analysis of bisulfite modified DNA and also the enriched unmethylated or hypermethylated DNA fractions using methylation-sensitive restriction enzymes or antibodies against methylated cytosines. The method described in detail here is based on the analysis of the enriched unmethylated DNA fraction, using a series of treatments with methylation-sensitive restriction enzymes, adaptor ligation, PCR amplification, and quantitative mapping of unmethylated DNA sequences using microarrays. The key advantages of this approach are the ability to investigate DNA methylation patterns using very small DNA amounts and relatively high informativeness in comparison to the other restriction-enzyme- based strategies for DNA methylation profiling [1].

Microarray-based DNA methylation profiling: technology and applications.

This work is dedicated to the development of a technology for unbiased, high-throughput DNA methylation profiling of large genomic regions. In this method, unmethylated and methylated DNA fractions are enriched using a series of treatments with methylation sensitive restriction enzymes, and interrogated on microarrays. We have investigated various aspects of the technology including its replicability, informativeness, sensitivity and optimal PCR conditions using microarrays containing oligonucleotides representing 100 kb of genomic DNA derived from the chromosome 22 COMT region in addition to 12 192 element CpG island microarrays. Several new aspects of methylation profiling are provided, including the parallel identification of confounding effects of DNA sequence variation, the description of the principles of microarray design for epigenomic studies and the optimal choice of methylation sensitive restriction enzymes. We also demonstrate the advantages of using the unmethylated DNA fraction versus the methylated one, which substantially improve the chances of detecting DNA methylation differences. We applied this methodology for fine-mapping of methylation patterns of chromosomes 21 and 22 in eight individuals using tiling microarrays consisting of over 340 000 oligonucleotide probe pairs. The principles developed in this work will help to make epigenetic profiling of the entire human genome a routine procedure.

Effect of ex vivo storage and Cyto-Chex on the expression of P-selectin glycoprotein ligand-1 (PSGL-1) on human peripheral leukocytes.

P-selectin glycoprotein ligand-1 (PSGL-1) is expressed on leukocytes and its expression is elevated in asthma and chronic obstructive pulmonary disease. This study assessed whether the cell stabilisation solution Cyto-Chex is able to preserve such quantitative differences for delayed testing. Peripheral blood was mixed with Cyto-Chex and stored refrigerated or at ambient temperature. Aliquots were tested by flow cytometry 2 to 168 h after collection for PSGL-1, CD3, CD4, CD8, and CD16 expression and marker-positive cell counts. Compared with controls a marked reduction of staining intensities is seen at all time points for all markers, irrespective of the storage temperature. This loss of bright staining did not or only moderately decrease marker-positive cell counts except for PSGL-1+ lymphocytes which declined in parallel with staining intensity. These findings indicate that Cyto-Chex is not able to preserve the expression or affinity to antibodies of surface markers to allow delayed determination of quantitative differences or detection of weakly staining cells. Immunophenotyping is mostly possible for 7 days after collection, however, this has to be tested for each marker and cell type of interest.

Influence of in vitro manipulation on the stability of methylation patterns in the Snurf/Snrpn-imprinting region in mouse embryonic stem cells.

Recent work on embryonic stem (ES) cells showed that stem cell-derived tissues and embryos, cloned from ES cell nuclei, often fail to maintain epigenetic states of imprinted genes. This deregulation is frequently associated with in vitro manipulations and culture conditions which might affect the cells potential to develop into normal fetuses. Usually, epigenetic instability is reported in differentially methylated regions of mostly growth-related imprinted genes. However, little is known about the epigenetic stability of genes that function late in organogenesis. Hence, we set out to investigate the epigenetic stability of neuronal genes and analyzed DNA methylation patterns in the Snurf/Snrpn imprinted cluster in several cultured mouse ES cell lines. We also determined the effects of in vitro stress factors such as consecutive passaging, trypsination, mechanical handling, single cell cloning, centrifugation, staurosporine-induced neurogenesis and the insertion of viral (foreign) DNA into the host genome. Intriguingly, none of these in vitro manipulations interfered with the stability of the methylation patterns in the analyzed neuronal genes. These data imply that, in contrast to growth-related genes like Igf2, H19, Igf2r or Grb10, the methylation imprints of the analyzed neuronal genes in the Snurf/Snrpn cluster may be particularly stable in manipulated ES cells.

Infection of cells with replication deficient adenovirus induces cell cycle alterations and leads to downregulation of E2F-1.

Gene products of recombinant replication-deficient adenovirus vectors of the first generation (Ad vector) can induce cell cycle dysregulation and apoptosis after infection in eukaryotic cells. The mechanisms underlying this complex process are largely unknown. Therefore, we investigated the regulation of the pRb/E2F-1 complex, which controls transition from G(0)/G(1) to S phase of the cell cycle. As Ad vector infection results in a decrease in the number of cells in G(0)/G(1) phase of the cell cycle, we observed a decline of the pRb protein level and, surprisingly, also a decrease of the E2F-1 protein and mRNA level in infected cell lines. Furthermore, in contrast to the reduction of cells in the G(0)/G(1) phase we observed increased protein levels of p53 and p21 proteins. However, as experiments in p53 deficient cell lines indicated, the decrease of pRb and E2F-1 is independent of p53 and p21 expression. Moreover, results obtained with Rb deficient cell lines indicated that the reduced E2F-1 expression is independent of pRb. These results suggest that Ad vector-induced cell cycle dysregulation is associated with a specific downregulation of E2F-1 independent of Rb and p53 genomic status of cells.

Effects of inhaled HFA beclomethasone on pulmonary function and symptoms in patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease is characterized by progressive airflow limitation and pulmonary inflammation. Inhaled corticosteroids (ICS) have been shown to be effective in the reduction of the number of exacerbations and the rate of deterioration in health status in patients with more advanced chronic obstructive pulmonary disease (COPD). Therefore current international guidelines recommend ICS for patients with severe COPD (FEV1 < 50%) with at least one exacerbation within the last year. We determined the short-term effect of the inhaled corticosteroid beclomethasone in HFA 134 formulation (Ventolair) on Health related Quality of Life (HRQOL), pulmonary function and the release of the cytokines Interleukin-10 (IL-10), Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF), Interferon-gamma (IFN-gamma) and Macrophage Inflammatory Protein-1alpha (MIP-1alpha) from peripheral blood monocytes of patients with COPD (n = 11) in a 12 week double blind cross over placebo-controlled study. Baseline lung function and the St. George Respiratory Questionnaire (SGRQ) were performed at the start and the end of each treatment phase. Monocytes were separated from blood at the end of each treatment phase. The treatment with Ventolair) resulted in an increase of PEF from 4.92 to 5.53 l/s and a decrease of RV% TLC (% predicted) values from 144.52 to 131.36. Inhaled HFA beclomethasone did not affect the cytokine release of IL-10, IFN-gamma, GM-CSF and MIP-1alpha. All cytokines were measured using commercially available Enzyme Linked Immun Sorbent Assay (ELISA) kits. The symptom score of the St. George Respiratory Questionnaire significantly decreased from 55.12 to 47.77 units in the active period compared to the placebo period after the treatment with HFA beclomethasone. The present study shows that a short-term treatment with inhaled steroid beclomethasone in fine particle HFA formulation decreases the hyperinflation and improves the PEF and the COPD symptoms.

Adenovirus-mediated gene transfer of P16INK4/CDKN2 into bax-negative colon cancer cells induces apoptosis and tumor regression in vivo.

The tumor-suppressor gene p16INK4/CDKN2 (p16) is a cyclin-dependent kinase (cdk) inhibitor and important cell cycle regulator. Here, we show that adenovirus-mediated gene transfer of p16 (AdCMV.p16) into colon cancer cells induces uncoupling of S phase and mitosis and subsequently apoptosis. Flow cytometric analysis revealed that cells infected with AdCMV.p16 showed an initial G2-like arrest followed by S phase without intervening mitosis (DNA >4N). Using microscopic analysis, deformed polyploid cells were detectable only in cells infected with AdCMV.p16 but not in control-infected cells. Subsequently, AdCMV.p16-infected polyploid cells underwent apoptosis, as assessed by AnnexinV staining and DNA fragmentation, suggesting that cell cycle dysregulation is upstream of the onset of apoptosis. Treatment of mice with subcutaneously transplanted tumors of colorectal cancer cells with AdCMV.p16 but not AdCMV.p53 resulted in significantly reduced tumor volume and prolonged survival. Using an orthotopic model of liver metastasis, we observed both reduced local tumor growth and secondary intrahepatic metastasis after AdCMV.p16 treatment. Importantly, induction of apoptosis in vitro and reduction of tumor growth in vivo by p16 was p53- as well as bax-independent because identical results were obtained using cancer cells, either wild type or mutant for p53 or bax. The studies suggest that an AdCMV.p16-based treatment may be especially effective in patients with bax-negative colon cancer where overexpression of p53 appears not to be of therapeutic value.

A collaborative approach to develop a multi-omics data analytics platform for translational research.

The integration and analysis of large datasets in translational research has become an increasingly challenging problem. We propose a collaborative approach to integrate established data management platforms with existing analytical systems to fill the hole in the value chain between data collection and data exploitation. Our proposal in particular ensures data security and provides support for widely distributed teams of researchers. As a successful example for such an approach, we describe the implementation of a unified single platform that combines capabilities of the knowledge management platform tranSMART and the data analysis system Genedata Analyst™. The combined end-to-end platform helps to quickly find, enter, integrate, analyze, extract, and share patient- and drug-related data in the context of translational R&D projects.

Approaches for drug delivery with intracortical probes.

Intracortical microprobes allow the precise monitoring of electrical and chemical signaling and are widely used in neuroscience. Microelectromechanical system (MEMS) technologies have greatly enhanced the integration of multifunctional probes by facilitating the combination of multiple recording electrodes and drug delivery channels in a single probe. Depending on the neuroscientific application, various assembly strategies are required in addition to the microprobe fabrication itself. This paper summarizes recent advances in the fabrication and assembly of micromachined silicon probes for drug delivery achieved within the EU-funded research project NeuroProbes. The described fabrication process combines a two-wafer silicon bonding process with deep reactive ion etching, wafer grinding, and thin film patterning and offers a maximum in design flexibility. By applying this process, three general comb-like microprobe designs featuring up to four 8-mm-long shafts, cross sections from 150×200 to 250×250 µm², and different electrode and fluidic channel configurations are realized. Furthermore, we discuss the development and application of different probe assemblies for acute, semichronic, and chronic applications, including comb and array assemblies, floating microprobe arrays, as well as the complete drug delivery system NeuroMedicator for small animal research.

Controlled delivery of naltrexone by an intraoral device: in vivo study on human subjects.

Naltrexone is widely used in the treatment of opiate addiction but its current peroral administration is characterized by low bioavailability with various side effects. The development of a long-acting transbuccal delivery device (IntelliDrug) for NLX may be useful to improve patient compliance and the therapy effectiveness. The aims of the study are (a) to test basic safety and effectiveness of controlled transbuccal drug delivery on human subjects; (b) to compare NLX bioavailability following transbuccal delivery vs per os conventional delivery; and (c) to test the hypothesis that transbuccal delivery is more efficient than the conventional route. In this randomized cross-over pilot study, 12 healthy subjects received in a different order 2 types of NLX administration, per os or transbuccal delivery, based on which group they were randomized to. For per os administration 50mg NLX tablets were used, while for transbuccal administration, a NLX-loaded prototype of the IntelliDrug device was fixed on patients' dental arch. Serial blood samples were drawn and analysed for the NLX concentration. The IntelliDrug prototype functioned properly and it did not exert any adverse side-effect. The transbuccal route resulted in administration efficiency 4-17 times higher than conventional per os route. Transbuccal delivery of NLX appears to be a more efficient drug administration route compared to peroral one. It allows to reach a given therapeutic blood level using a small drug dose.

Do epigenetic pathways initiate late onset Alzheimer disease (LOAD): towards a new paradigm.

Late onset Alzheimer's disease (LOAD) is a non-familial, progressive neurodegenerative disease and the most prominent form of dementia in the elderly. Accumulating evidence suggests that LOAD not only results from the combined effects of variation in a number of genes and environmental factors, but also from epigenetic abnormalities such as histone modifications or DNA methylation. In comparison to monogenic diseases, LOAD exhibits numerous anomalies that suggest an epigenetic component in disease etiology. Evidence against a monogenic course and for an epigenetic component include: 1) the dominance of sporadic cases over familial ones and the low estimated concordance rates for monozygotic twins; 2) gender specific susceptibility and course of disease; 3) parent-of-origin effects, and late age of onset; 4) brain chromatin abnormalities, non-Mendelian inheritance patterns, and atypical levels of folate and homocysteine; and 5) monoallelic expression patterns of susceptibility genes [1]. The epigenome is particularly susceptible to deregulation during early embryonic and neonatal periods and thus disturbances during these periods can have latent lasting effects. The Latent Early-life Associated Regulation (LEARn) model attempts to explain these consequences from a brain specific point of view. In the present review we present the evidence that support the role of epigenetics in the development of AD and explore the potential pathways and mechanisms that may be involved.

Zebrafish embryos as an alternative to animal experiments--a commentary on the definition of the onset of protected life stages in animal welfare regulations.

Worldwide, the zebrafish has become a popular model for biomedical research and (eco)toxicology. Particularly the use of embryos is receiving increasing attention, since they are considered as replacement method for animal experiments. Zebrafish embryos allow the analysis of multiple endpoints ranging from acute and developmental toxicity determination to complex functional genetic and physiological analysis. Particularly the more complex endpoints require the use of post-hatched eleutheroembryo stages. According to the new EU Directive 2010/63/EU on the protection of animals used for scientific purposes, the earliest life-stages of animals are not defined as protected and, therefore, do not fall into the regulatory frameworks dealing with animal experimentation. Independent feeding is considered as the stage from which free-living larvae are subject to regulations for animal experimentation. However, despite this seemingly clear definition, large variations exist in the interpretation of this criterion by national and regional authorities. Since some assays require the use of post-hatched stages up to 120 h post fertilization, the literature and available data are reviewed in order to evaluate if this stage could still be considered as non-protected according to the regulatory criterion of independent feeding. Based on our analysis and by including criteria such as yolk consumption, feeding and swimming behavior, we conclude that zebrafish larvae can indeed be regarded as independently feeding from 120 h after fertilization. Experiments with zebrafish should thus be subject to regulations for animal experiments from 120 h after fertilization onwards.

Bioavailability in vivo of naltrexone following transbuccal administration by an electronically-controlled intraoral device: a trial on pigs.

Naltrexone (NLX), an opioid antagonist, is widely used in the treatment of opiate addiction, alcoholism and smoking cessation. Its current peroral administration induces various adverse side effects and has limited efficacy since bioavailability and patient compliance are poor. The development of a long-acting drug delivery system of NLX may overcome the current drawbacks and help in the improvement of treatment of addiction. The primary endpoints of this study were: a) to compare the NLX bioavailability and pharmacokinetics after delivering a single transbuccal dose, released by a prototype of intraoral device, versus an intravenous (I.V.) bolus of the same drug dose; b) to verify the functioning of a prototype of a new intraoral device in vivo; c) to evaluate the permeation enhancement effect of iontophoresis; d) to assess any histomorphological changes in the buccal mucosa after transbuccal delivery. The system was tested on 6 pigs in a cross-over trial. Venous blood samples were drawn at a fixed timetable from the beginning of drug administration and analyzed for the presence of NLX, using an LC/MS/MS method. A punch biopsy was performed for histological analysis after the final experiment. The administration of I.V. NLX induced a sharp increase in blood levels after 5 min and then a steep decrease. In contrast, transmucosal delivery resulted in a gradual increase in blood NLX levels, reaching its peak after 90 min, followed by a slow decrease. After 6h the blood levels of NLX delivered through the buccal mucosa were higher as compared to I.V. administration. No signs of flogosis or tissue damage were histologically highlighted. These results suggest that buccal delivery by an intraoral electronic device could potentially induce long-lasting, continuous and controlled blood levels of NLX, avoiding at the same time spikes of drug plasma levels typical of the I.V. administration route.

No association of TDP-43 with sporadic frontotemporal dementia.

A hyperphosphorylated, ubiquitinated form of TDP-43, known as pathologic TDP-43, was shown to be a central component of ubiquitin-positive, tau-negative and alpha-synuclein-negative inclusions in frontotemporal lobar degeneration (FTLD-U) and amytrophic lateral sclerosis (ALS). To investigate the role of the TDP-43 gene in sporadic forms of frontotemporal dementia (FTD), we genotyped 10 single nucleotide polymorphisms covering the entire TDP-43 genomic region, including the MASP2 gene in 173 patients with sporadic FTD (including 7 patients that were diagnosed with FTD and ALS) and 184 matched controls from Germany. Although we could observe a weak trend towards a potential disease association in a few FTD/ALS patients, no significant association with sporadic FTD could be demonstrated. There is no evidence, that common variants in TDP-43 confer a strong risk to the development of sporadic FTD.