Inducción de Apoptosis in vitro en hepatocitos de rata por ciclosporina A

In rat hepatocytes and isolated liver mitochondrial fractions, CsA is often used as a specific inhibitor of mitochondrial Ca2+ release and as a specific blocker of mitochondrial membrane potential and permeability transition (MPT), which are all processes involved in the inhibition of apoptosis. However, neither inhibition nor induction of apoptosis by CsA has yet been described in the rat hepatocyte primary culture during incubation for 4 and 20 hours. It was the purpose of the present study to examine by means of morphological and biochemical criteria the effects of CsA on apoptosis, and to characterize the underlying mechanisms. Rat hepatocytes were cultured for 4 or 20 hours with CsA at concentrations of 0, 10, 25 and 50 μM. Chromatin condensation and fragmentation, DNA fragmentation (TUNEL), membrane phosphatidylserine distribution (Annexin V), caspase-1, -3 and -6 activity, mitochondrial membrane potential (Rhodamine 123), and cytochrome c release into the cytosol were investigated. Four hours after CsA treatment, chromatin condensation and fragmentation, and the number of TUNEL- and Annexin V-positive cells increased dose dependentlywithout any observable enzyme leakage, which indicated the integrity of the outer cell membrane. After 20 hours of CsA incubation apoptosis parameters were further increased and were accompanied by the increased activity of the cysteine protease, caspase-3 (CPP 32), and slightly increased caspase-6 (Mch 2), but not caspase-1 (ICE). The specific caspase-3 inhibitor, Ac-DEVD-CHO, inhibited caspase-3 activation and attenuated CsA-induced apoptosis and LDH leakage. The caspase-6 inhibitor, Ac-VEIDCHO, only marginally inhibited CsA-induced apoptosis. Decreased mitochondrial membrane potential and cytochrome c release went in parallel with ultrastructural mitochondrial changes and might be regarded as early events which trigger the apoptosis cascade. Transmission electron microscopy (TEM) confirmed an increase in the number of necrotic cells after 20 hours, but not after 4 hours, compared with controls.La cefalosporina A (CsA) se utiliza frecuentemente en hepatocitos de rata y fracciones mitocondriales hepáticas aisladas como inhibidor específico de la liberación de Ca2+ y como bloqueador selectivo de la permeabilidad y del potencial de membrana mitocondriales, procesos implicados en la inhibición de la apoptosis. Sin embargo, hasta ahora no se ha descrito ni la inhibición ni la inducción de apoptosis por CsA en cultivos primarios de hepatocitos de rata tras su incubación por un periodo de 4 a 20 horas. El propósito de este estudio ha sido examinar con criterios morfológicos y bioquímicos los efectos de la CsA sobre la apoptosis y esclarecer las características de los mecanismos subyacentes. Los hepatocitos de rata se cultivaron durante 4-20 h. con CsA a concentraciones de 0, 10, 25 y 50 μM. Se investigaron los fenómenos de condensación y fragmentación de la cromatina, fragmentación de ADN (TUNEL), distribución de fostatidilcolina en la membrana (Anexina V), así como la actividades enzimática de caspasas 1, -3 y –6, el potencial de membrana mitocondrial (Rhodamina 123) y la liberación de citocromo C en el citosol. Tras cuatro horas de incubación con CsA, la condensación y fragmentación de la cromatina y el número de células TUNEL y Anexina V positivas aumentaron en función de la dosis sin que se observara pérdida enzimática, lo que indica la integridad de la membrana celular externa. Después de 20 horas de incubación con CsA, experimentaron un mayor incremento acompañado del aumento de las actividad de cistein-proteasa, caspasa-3 (CPP32) y de un ligero incremento de caspasa-6 (Mch 2), pero no de caspasa-1 (ICE). El inhibidor específico de caspasa-3, Ac-DEVD-CHO,inhibió la activación de caspasa-3 y atenuó la apoptosis inducida por CsA y la pérdida de LDH. El inhibidor de caspasa-6, Ac-VEID-CHO, únicamente inhibió la apoptosis inducida por CsA. El descenso de potencial de membrana mitocondrial y la liberación de citocromo C fueron paralelos a los cambios de ultraestructuras mitocondriales y pudieran considerarse reacciones tempranas que desencadenan la cascada de fenómenos apoptóticos. La microscopia de transmisión electrónica (TEM) confirmó el incremento del número de células necróticas al cabo de 20 horas, pero no tras 4 horas de incubación, en comparación con los controles

Autocorrected off-axis holography of two-dimensional materials

The reduced dimensionality in two-dimensional materials leads to a wealth of unusual properties, which are currently explored for both fundamental and applied sciences. In order to study the crystal structure, edge states, the formation of defects and grain boundaries, or the impact of adsorbates, high-resolution microscopy techniques are indispensable. Here we report on the development of an electron holography (EH) transmission electron microscopy (TEM) technique, which facilitates high spatial resolution by an automatic correction of geometric aberrations. Distinguished features of EH beyond conventional TEM imaging are gap-free spatial information signal transfer and higher dose efficiency for certain spatial frequency bands as well as direct access to the projected electrostatic potential of the two-dimensional material. We demonstrate these features with the example of h-BN, for which we measure the electrostatic potential as a function of layer number down to the monolayer limit and obtain evidence for a systematic increase of the potential at the zig-zag edges

Ginkgo biloba Extract EGb 761 Improves Vestibular Compensation and Modulates Cerebral Vestibular Networks in the Rat

Unilateral inner ear damage is followed by behavioral recovery due to central vestibular compensation. The dose-dependent therapeutic effect of Ginkgo biloba extract EGb 761 on vestibular compensation was investigated by behavioral testing and serial cerebral [18F]-Fluoro-desoxyglucose ([18F]-FDG)-μPET in a rat model of unilateral labyrinthectomy (UL). Five groups of 8 animals each were treated with EGb 761-supplemented food at doses of 75, 37.5 or 18.75 mg/kg body weight 6 weeks prior and 15 days post UL (groups A,B,C), control food prior and EGb 761-supplemented food (75 mg/kg) for 15 days post UL (group D), or control food throughout (group E). Plasma levels of EGb 761 components bilobalide, ginkgolide A and B were analyzed prior and 15 days post UL. Behavioral testing included clinical scoring of nystagmus, postural asymmetry, head roll tilt, body rotation during sensory perturbation and instrumental registration of mobility in an open field before and 1, 2, 3, 5, 7, 15 days after UL. Whole-brain [18F]-FDG-μPET was recorded before and 1, 3, 7, 15 days after UL. The EGb 761 group A (75 mg/kg prior/post UL) showed a significant reduction of nystagmus scores (day 3 post UL), of postural asymmetry (1, 3, 7 days post UL), and an increased mobility in the open field (day 7 post UL) as compared to controls (group E). Application of EGb 761 at doses of 37.5 and 18.75 mg/kg prior/post UL (groups B,C) resulted in faster recovery of postural asymmetry, but did not influence mobility relative to controls. Locomotor velocity increased with higher plasma levels of ginkgolide A and B. [18F]-FDG-μPET revealed a significant decrease of the regional cerebral glucose metabolism (rCGM) in the vestibular nuclei and cerebellum and an increase in the hippocampal formation with higher plasma levels of ginkgolides and bilobalide 1 and 3 days post UL. Decrease of rCGM in the vestibular nucleus area and increase in the hippocampal formation with higher plasma levels persisted until day 15 post UL. In conclusion, Ginkgo biloba extract EGb 761 improves vestibulo-ocular motor, vestibulo-spinal compensation, and mobility after UL. This rat study supports the translational approach to investigate EGb 761 at higher dosages for acceleration of vestibular compensation in acute vestibular loss

Ginkgo biloba Extract EGb 761 Improves Vestibular Compensation and Modulates Cerebral Vestibular Networks in the Rat

Unilateral inner ear damage is followed by behavioral recovery due to central vestibular compensation. The dose-dependent therapeutic effect of Ginkgo biloba extract EGb 761 on vestibular compensation was investigated by behavioral testing and serial cerebral [18F]-Fluoro-desoxyglucose ([18F]-FDG)-μPET in a rat model of unilateral labyrinthectomy (UL). Five groups of 8 animals each were treated with EGb 761-supplemented food at doses of 75, 37.5 or 18.75 mg/kg body weight 6 weeks prior and 15 days post UL (groups A,B,C), control food prior and EGb 761-supplemented food (75 mg/kg) for 15 days post UL (group D), or control food throughout (group E). Plasma levels of EGb 761 components bilobalide, ginkgolide A and B were analyzed prior and 15 days post UL. Behavioral testing included clinical scoring of nystagmus, postural asymmetry, head roll tilt, body rotation during sensory perturbation and instrumental registration of mobility in an open field before and 1, 2, 3, 5, 7, 15 days after UL. Whole-brain [18F]-FDG-μPET was recorded before and 1, 3, 7, 15 days after UL. The EGb 761 group A (75 mg/kg prior/post UL) showed a significant reduction of nystagmus scores (day 3 post UL), of postural asymmetry (1, 3, 7 days post UL), and an increased mobility in the open field (day 7 post UL) as compared to controls (group E). Application of EGb 761 at doses of 37.5 and 18.75 mg/kg prior/post UL (groups B,C) resulted in faster recovery of postural asymmetry, but did not influence mobility relative to controls. Locomotor velocity increased with higher plasma levels of ginkgolide A and B. [18F]-FDG-μPET revealed a significant decrease of the regional cerebral glucose metabolism (rCGM) in the vestibular nuclei and cerebellum and an increase in the hippocampal formation with higher plasma levels of ginkgolides and bilobalide 1 and 3 days post UL. Decrease of rCGM in the vestibular nucleus area and increase in the hippocampal formation with higher plasma levels persisted until day 15 post UL. In conclusion, Ginkgo biloba extract EGb 761 improves vestibulo-ocular motor, vestibulo-spinal compensation, and mobility after UL. This rat study supports the translational approach to investigate EGb 761 at higher dosages for acceleration of vestibular compensation in acute vestibular loss

Standardized, systemic phenotypic analysis reveals kidney dysfunction as main alteration of Kctd1 I27N mutant mice

Background: Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically dominant mutant line HST014 was established and further analyzed. Methods: Analysis of the causative mutation as well as the standardized, systemic phenotypic analysis of the mutant line was carried out. Results: The causative mutation was detected in the potassium channel tetramerization domain containing 1 (Kctd1) gene which leads to the amino acid exchange Kctd1 I27N thereby affecting the functional BTB domain of the protein. This line is the first mouse model harboring a Kctd1 mutation. Kctd1 I27N homozygous mutant mice die perinatally. Standardized, systemic phenotypic analysis of Kctd1 I27N heterozygous mutants was carried out in the German Mouse Clinic (GMC). Systematic morphological investigation of the external physical appearance did not detect the specific alterations that are described in KCTD1 mutant human patients affected by the scalp-ear-nipple (SEN) syndrome. The main pathological phenotype of the Kctd1 I27N heterozygous mutant mice consists of kidney dysfunction and secondary effects thereof, without gross additional primary alterations in the other phenotypic parameters analyzed. Genome-wide transcriptome profiling analysis at the age of 4 months revealed about 100 differentially expressed genes (DEGs) in kidneys of Kctd1 I27N heterozygous mutants as compared to wild-type controls. Conclusions: In summary, the main alteration of the Kctd1 I27N heterozygous mutants consists in kidney dysfunction. Additional analyses in 9–21 week-old heterozygous mutants revealed only few minor effects

Data-driven Soft Sensors in the Process Industry

In the last two decades Soft Sensors established themselves as a valuable alternative to the traditional means for the acquisition of critical process variables, process monitoring and other tasks which are related to process control. This paper discusses characteristics of the process industry data which are critical for the development of data-driven Soft Sensors. These characteristics are common to a large number of process industry fields, like the chemical industry, bioprocess industry, steel industry, etc. The focus of this work is put on the data-driven Soft Sensors because of their growing popularity, already demonstrated usefulness and huge, though yet not completely realised, potential. A comprehensive selection of case studies covering the three most important Soft Sensor application fields, a general introduction to the most popular Soft Sensor modelling techniques as well as a discussion of some open issues in the Soft Sensor development and maintenance and their possible solutions are the main contributions of this work

Dll1 Haploinsufficiency in Adult Mice Leads to a Complex Phenotype Affecting Metabolic and Immunological Processes

BACKGROUND: The Notch signaling pathway is an evolutionary conserved signal transduction pathway involved in embryonic patterning and regulation of cell fates during development and self-renewal. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, involving as well other signal transduction pathways, and implicated in distinct human diseases. Delta-like 1 (Dll1) is one of the known ligands of the Notch receptors. The role of the Notch ligands is less well understood. Loss-of-function of Dll1 leads to embryonic lethality, but reduction of Delta-like 1 protein levels has not been studied in adult stage. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the haploinsufficient phenotype of Dll1 and a missense mutant Dll1 allele (Dll1(C413Y)). Haploinsufficiency leads to a complex phenotype with several biological processes altered. These alterations reveal the importance of Dll1 mainly in metabolism, energy balance and in immunology. The animals are smaller, lighter, with altered fat to lean ratio and have increased blood pressure and a slight bradycardia. The animals have reduced cholesterol and triglyceride levels in blood. At the immunological level a subtle phenotype is observed due to the effect and fine-tuning of the signaling network at the different levels of differentiation, proliferation and function of lymphocytes. Moreover, the importance of the proteolytic regulation of the Notch signaling network emphasized. CONCLUSIONS/SIGNIFICANCE: In conclusion, slight alterations in one player of Notch signaling alter the entire organism, emphasizing the fine-tuning character of this pathway in a high number of processes

Mutation in the mouse histone gene Hist2h3c1 leads to degeneration of the lens vesicle and severe microphthalmia

During an ENU (N-ethyl-N-nitrosourea) mutagenesis screen, we observed a dominant small-eye mutant mouse with viable homozygotes. A corresponding mutant line was established and referred to as Aey69 (abnormality of the eye #69). Comprehensive phenotyping of the homozygous Aey69 mutants in the German Mouse Clinic revealed only a subset of statistically significant alterations between wild types and homozygous mutants. The mutation causes microphthalmia without a lens but with retinal hyperproliferation. Linkage was demonstrated to mouse chromosome 3 between the markers D3Mit188 and D3Mit11. Sequencing revealed a 358 A- > C mutation (I1e120Leu) in the Hist2h3c1 gene and a 71 T- > C (Val24Ala) mutation in the Gja8 gene. Detailed analysis of eye development in the homozygous mutant mice documented a perturbed lens development starting -from the lens vesicle stage including decreasing expression of crystallins as well as of lens-specific transcription - factors like PITX3 and FOXE3. In contrast, we observed an early expression of retinal progenitor cells characterized by several markers including BRN3 (retinal ganglion cells) and OTX2 (cone photoreceptors). The changes in the retina at the early embryonic stages of E11.5-E15.5 happen in parallel with apoptotic processes in the lens at the respective stages. The excessive retinal hyperproliferation is characterized by an increased level of Ki67. The hyperproliferation, however, does not disrupt the differentiation and appearance of the principal retinal cell types at postnatal stages, even if the overgrowing retina covers finally the entire bulbus of the eye. Morpholino-mediated knock-down of the hist2h3ca1 gene in zebrafish leads to a specific perturbation of lens development. When injected into zebrafish zygotes, only the mutant mouse mRNA leads to severe malformations, ranging from cyclopia to severe microphthalmia. The wild-type Hist2h3c1 mRNA can rescue the morpholino-induced defects corroborating its specific function in lens development. Based upon these data, it is concluded that the ocular function of the Hist2h3c1 gene (encoding a canonical H3.2 variant) is conserved throughout evolution. Moreover, the data highlight also the importance of Hist2h3c1 in the coordinated formation of lens and retina during eye development