Enhanced gene trapping in mouse embryonic stem cells

Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes

Leukotriene and purinergic receptors are involved in the hyperpolarizing effect of glucagon in liver cells

AbstractThe pancreatic hormone glucagon hyperpolarizes the liver cell membrane. In the present study, we investigated the cellular signalling pathway of glucagon-induced hyperpolarization of liver cells by using the conventional microelectrode method. The membrane potential was recorded in superficial liver cells of superfused mouse liver slices. In the presence of the K+ channel blockers tetraethylammonium (TEA, 1 mmol/l) and Ba2+ (BaCl2, 5 mmol/l) and the blocker of the Na+/K+ ATPase, ouabain (1 mmol/l), no glucagon-induced hyperpolarization was observed confirming previous findings. The hyperpolarizing effect of glucagon was abolished by the leukotriene B4 receptor antagonist CP 195543 (0.1 mmol/l) and the purinergic receptor antagonist PPADS (5 μmol/l). ATPγS (10 μmol/l), a non-hydrolyzable ATP analogue, induced a hyperpolarization of the liver cell membrane similar to glucagon. U 73122 (1 μmol/l), a blocker of phospholipase C, prevented both the glucagon- and ATPγS-induced hyperpolarization. These findings suggest that glucagon affects the hepatic membrane potential partly by inducing the formation and release of leukotrienes and release of ATP acting on purinergic receptors of the liver cell membrane

Functional connectivity changes in the delta frequency band following trauma treatment in complex trauma and dissociative disorder patients

ObjectivePhase-oriented trauma treatment is efficacious in the treatment of complex trauma and dissociative disorder patients. However, the neural correlates of this therapeutic effect are not yet well-understood. In the current study we investigated whether patients show a strengthening in functional network connectivity in the delta frequency band (1–3.5 Hz) over the course of phase-oriented inpatient trauma treatment while they performed an emotion regulation task. Further, we examined whether neural changes were associated with symptom reduction and improvement in emotion regulation skills.MethodsBefore and after 8 weeks of treatment, electroencephalography (EEG) was acquired in patients (n = 28) with a complex posttraumatic stress disorder (cPTSD) or complex dissociative disorder (CDD). They also completed clinical and emotion regulation questionnaires. To delimit data variability, patients participated as one dissociative part that is referred to as Apparently Normal Part (ANP). Patients' data were compared to a matched healthy control croup (n = 38), also measured twice.ResultsPrior to treatment, functional connectivity was significantly lower in patients compared to controls during cognitive reappraisal of unpleasant pictures and passive viewing of unpleasant and neutral pictures. These hypoconnected networks largely overlapped with networks typically activated during the recall of (emotional) autobiographical memories. Functional connectivity strength within these networks significantly increased following treatment and was comparable to controls. Patients showed symptom reduction across various clinical domains and improvement in the use of cognitive reappraisal as emotion regulation strategy. Treatment-related network normalizations were not related to changes in questionnaire data.ConclusionPhase-oriented treatment may strengthen connections between regions that are activated during autobiographical recall. These findings encourage further investigation of this circuitry as a therapeutic target in cPTSD and CDD patients.Clinial trial registrationwww.ClinicalTrials.gov, identifier: NCT02459340, https://www.kofam.ch/de/studienportal/suche/149284/studie/26681

Impact of aging : sporadic, and genetic risk factors on vulnerability to apoptosis in Alzheimer's disease

The identification of specific genetic (presenilin-1 [PS1] and amyloid precursor protein [APP] mutations) and environmental factors responsible for Alzheimer's disease (AD) has revealed evidence for a shared pathway of neuronal death. Moreover, AD-specific cell defects may be observed in many other nonneuronal cells (e.g., lymphocytes). Thus, lymphocytes may serve as a cellular system in which to study risk factors of sporadic, as well as genetic AD in vivo. The aim of our present study was to clarify whether lymphocytes bearing genetic or sporadic risk factors of AD share an increased susceptibility to cell death. Additionally we examined whether a cell typespecific vulnerability pattern was present and how normal aging, the main risk factor of sporadic AD, contributes to changes in susceptibility to cell death. Here, we report that lymphocytes affected by sporadic or genetic APP and PS1 AD risk factors share an increased vulnerability to cell death and exhibit a similar cell type-specific pattern, given that enhanced vulnerability was most strongly developed in the CD4+ T-cell subtype. In this paradigm, sporadic risk factors revealed the highest impact on cell type-specific sensitivity of CD4+ T cells to apoptosis. In contrast, normal aging results in an increased susceptibility to apoptosis of both, CD4+ and CD8+ T cells

Functional connectivity changes in the delta frequency band following trauma treatment in complex trauma and dissociative disorder patients

Objective: Phase-oriented trauma treatment is efficacious in the treatment of complex trauma and dissociative disorder patients. However, the neural correlates of this therapeutic effect are not yet well-understood. In the current study we investigated whether patients show a strengthening in functional network connectivity in the delta frequency band (1–3.5 Hz) over the course of phase-oriented inpatient trauma treatment while they performed an emotion regulation task. Further, we examined whether neural changes were associated with symptom reduction and improvement in emotion regulation skills. Methods: Before and after 8 weeks of treatment, electroencephalography (EEG) was acquired in patients (n = 28) with a complex posttraumatic stress disorder (cPTSD) or complex dissociative disorder (CDD). They also completed clinical and emotion regulation questionnaires. To delimit data variability, patients participated as one dissociative part that is referred to as Apparently Normal Part (ANP). Patients' data were compared to a matched healthy control croup (n = 38), also measured twice. Results: Prior to treatment, functional connectivity was significantly lower in patients compared to controls during cognitive reappraisal of unpleasant pictures and passive viewing of unpleasant and neutral pictures. These hypoconnected networks largely overlapped with networks typically activated during the recall of (emotional) autobiographical memories. Functional connectivity strength within these networks significantly increased following treatment and was comparable to controls. Patients showed symptom reduction across various clinical domains and improvement in the use of cognitive reappraisal as emotion regulation strategy. Treatment-related network normalizations were not related to changes in questionnaire data. Conclusion: Phase-oriented treatment may strengthen connections between regions that are activated during autobiographical recall. These findings encourage further investigation of this circuitry as a therapeutic target in cPTSD and CDD patients

Resting-state functional connectivity in patients with a complex PTSD or complex dissociative disorder before and after inpatient trauma treatment

INTRODUCTION: Recent research suggests that traumatized patients are characterized by disrupted resting-state functional connectivity. We examined whether neural networks involved in resting-state change over the course of a phase-oriented inpatient treatment for complex traumatized and dissociative disorder patients. We also investigated associations between these network alterations and clinical symptoms and emotion regulation skills. METHODS: Pre- and post-treatment, electroencephalography (EEG) was recorded during resting-state in patients (n = 23) with a complex dissociative disorder (CDD) or complex posttraumatic stress disorder (cPTSD). Patients also completed clinical and emotion regulation questionnaires. To reduce variance in the collected data, patients were exclusively tested as one prototypical dissociative part referred to as Apparently Normal Part (ANP). Functional network connectivity was examined and compared with a matched healthy control group (n = 37), also measured twice. RESULTS: Prior to treatment and compared with controls, patients had a significantly lower functional connectivity strength within eyes-open and eyes-closed resting-state networks in the theta and alpha frequency band. Following treatment, functional connectivity strength within these networks was comparable to the control group and comprised areas belonging to the default mode network (DMN) and prefrontal as well as anterior cingulate control regions. Treatment-related network normalizations in the theta frequency band were associated with a self-reported increase in the use of cognitive reappraisal strategies and reduction in emotion regulation difficulties. CONCLUSION: Phase-oriented trauma treatment can strengthen resting-state network connectivity and can increase the capacity of complex traumatized and dissociative patients as ANP to handle emotional challenges effectively

Nicht-harmonische Schwingungen am Huygens-Raebiger Pendel

In der Lehramtsausbildung haben im physikalischen Praktikum zunehmend computergestützte Versuche ihren Platz gefunden. Hierbei ist nicht nur die computergestützte Messwertaufnahme von Vorteil. Die Studierenden haben auch bei der Auswertung die Möglichkeit sich mit vergleichsweise geringem Aufwand mit mathematischen Modellierungen von physikalischen Sachverhalten zu beschäftigen. Aus einer Reihe entwickelter Versuche für das physikalische Praktikum an der Pädagogischen Hochschule Freiburg beschäftigt sich ein Versuch mit den Unterschieden zwischen einer harmonischen Schwingung und einer nicht-harmonischen Schwingung. Es soll mit diesem Versuch gezeigt werden, dass für große Winkel ein physikalisches Pendel keine harmonische Schwingung vollzieht und dass dieser Effekt bei längeren Pendeln größer ist als bei kürzeren. In diesem Zusammenhang werden die Grenzen einer mathematischen Modellierung mit Hilfe eines CAS veranschaulicht. Als physikalisches Pendel wird das Huygens-Raebiger Pendel verwendet – ein Pendel das sowohl gekoppelt als ein Pendel schwingen kann, als auch entkoppelt als sieben „kleine“, sich durch die Länge der Stange unterscheidende, Pendel. Im Folgenden werden der Versuch und die Modellierung der Messergebnisse vorgestellt

Nicht-harmonische Schwingungen am Huygens-Raebiger Pendel

In der Lehramtsausbildung haben im physikalischen Praktikum zunehmend computergestützte Versuche ihren Platz gefunden. Hierbei ist nicht nur die computergestützte Messwertaufnahme von Vorteil. Die Studierenden haben auch bei der Auswertung die Möglichkeit sich mit vergleichsweise geringem Aufwand mit mathematischen Modellierungen von physikalischen Sachverhalten zu beschäftigen. Aus einer Reihe entwickelter Versuche für das physikalische Praktikum an der Pädagogischen Hochschule Freiburg beschäftigt sich ein Versuch mit den Unterschieden zwischen einer harmonischen Schwingung und einer nicht-harmonischen Schwingung. Es soll mit diesem Versuch gezeigt werden, dass für große Winkel ein physikalisches Pendel keine harmonische Schwingung vollzieht und dass dieser Effekt bei längeren Pendeln größer ist als bei kürzeren. In diesem Zusammenhang werden die Grenzen einer mathematischen Modellierung mit Hilfe eines CAS veranschaulicht. Als physikalisches Pendel wird das Huygens-Raebiger Pendel verwendet – ein Pendel das sowohl gekoppelt als ein Pendel schwingen kann, als auch entkoppelt als sieben „kleine“, sich durch die Länge der Stange unterscheidende, Pendel. Im Folgenden werden der Versuch und die Modellierung der Messergebnisse vorgestellt

Enhanced ROS-generation in lymphocytes from Alzheimer’s patients

Introduction: Reactive oxygen species (ROS) have been implicated in neurodegeneration and seem to be involved in the physiology and pathophysiology of several diseases, including normal aging and Alzheimer’s disease (AD). Enhanced ROS production in aging or AD is not restricted to the brain, but can also been seen in several peripheral tissues. The objective of the present study was to evaluate whether the mechanisms involved in the generation of oxidative stress in normal senescence and Alzheimer’s disease are identical or not. Methods: We analysed intracellular basal levels of ROS in lymphocytes from AD patients and healthy young and aged not-demented subjects as well as ROS levels following stimulation with d-ribose and staurosporine in all three groups. ROS levels were measured by flow cytometry using the intracellular fluorescence dye dihydrorhodamine123 (DHR123). Results: Our study shows that AD lymphocytes have increased basal levels of ROS, low susceptibility to ROS stimulation by 2-deoxy-D-ribose (dRib) and an increased response to staurosporine when compared with age-matched controls. Discussion: The data suggest that the defect(s) responsible for enhanced ROS production in AD may involve different or additional biological pathways than those involved in enhanced ROS generation during aging

Characterization of a Novel Human Type II Epithelial Keratin K1b, Specifically Expressed in Eccrine Sweat Glands

In this study, we show that a novel human type II epithelial keratin, K1b, is exclusively expressed in luminal duct cells of eccrine sweat glands. Taking this luminal K1b expression as a reference, we have used antibodies against a plethora of epithelial keratins to systematically investigate their expression in the secretory globule and the two-layered sweat duct, which was divided into the intraglandular, intradermal, and intraepidermal (acrosyringium) segments, the latter being further subdivided into the sweat duct ridge and upper intraepidermal duct. We show that (i) each of the eccrine sweat gland tissue compartments expresses their own keratin patterns, (ii) the peripheral and luminal duct layers exhibit a sequential keratin expression, with both representing self-renewing cell layers, (iii) the intradermal duct and the sweat duct ridge display hitherto unknown length variations, and (iv) out of all cell layers, the luminal cell layer is the most robust layer and expresses the highest number of keratins, these being concentrated at the apical side of the cells to form the cuticle. We provide evidence that the cellular and intercellular properties of the peripheral and the luminal layers reflect adaptations to different functions