Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition

BACKGROUND: Cationic lipid DNA complexes based on DOTAP (1,2-dioleoyl-3-(trimethyammonium) propane) and mixtures of DOTAP and cholesterol (DC) have been previously optimized for transfection efficiency in the absence of serum and used as a non-viral gene delivery system. To determine whether DOTAP and DC lipid DNA complexes could be obtained with increased transfection effciency in the presence of high serum concentrations, the composition of the complexes was varied systematically and a total of 162 different complexes were analyzed for transfection efficiency in the presence and absence of high serum concentrations. RESULTS: Increasing the ratio of DOTAP or DC to DNA led to a dose dependent enhancement of transfection efficiency in the presence of high serum concentrations up to a ratio of approximately 128 nmol lipid/μg DNA. Transfection efficiency could be further increased for all ratios of DOTAP and DC to DNA by addition of the DNA condensing agent protamine sulfate (PS). For DOTAP DNA complexes with ratios of ≤ 32 nmol/μg DNA, peak transfection efficiencies were obtained with 4 μg PS/μg DNA. In contrast, increasing the amount of PS of DC complexes above 0.5 μg PS /μg DNA did not lead to significant further increases in transfection efficiency in the presence of high serum concentrations. Four complexes, which had a similar high transfection efficiency in cell culture in the presence of low serum concentrations but which differed largely in the lipid to DNA ratio and the amount of PS were selected for further analysis. Intravenous injection of the selected complexes led to 22-fold differences in transduction efficiency, which correlated with transfection efficiency in the presence of high serum concentrations. The complex with the highest transfection efficiency in vivo consisted of 64 nmol DC/ 16 μg PS/ μg DNA. Physical analysis revealed a predicted size of 440 nm and the highest zeta potential of the complexes analyzed. CONCLUSIONS: Optimization of cationic lipid DNA complexes for transfection efficiency in the presence of high concentrations of serum led to the identification of a DC complex with high transduction efficiency in mice. This complex differs from previously described ones by higher lipid to DNA and PS to DNA ratios. The stability of this complex in the presence of high concentrations of serum and its high transduction efficiency in mice suggests that it is a promising candidate vehicle for in vivo gene delivery

A cloud-based Analytics-Platform for user-centric Internet of Things domains – Prototype and Performance Evaluation

Data analytics have the potential to increase the value of data emitted from smart devices in user-centric Internet of Things environments, such as smart home, drastically. In order to allow businesses and end-consumers alike to tap into this potential, appropriate analytics architectures must be present. Current solutions in this field do not tackle all of the diverse challenges and requirements, which were identified in previous research. Specifically, personalized, extensible analytics solutions, which still offer the means to address big data problems are scarce. In this paper, we therefore present an architectural solution, which was specifically designed to address the named challenges. Furthermore, we offer insights into the prototypical implementation of the proposed concept as well as an evaluation of its performance against traditional big data architectures

Streaming Analytics in User-centric Internet of Things Domains: A Fog-enabled System Architecture for Smart Home Applications

A smart home is an apartment or a house in which smart devices communicate with each other to improve key areas of daily life, such as comfort, security or energy consumption. Therefore, the smart home domain is user-centric and exhibits characteristics that distinguish it from other application domains of the Internet of Things. Specifically, this concerns the existence of different 'regular' and 'smart' devices, but also the basic arrangement of each household, which is highly individual. As a result, the realization of analytics scenarios in the smart home domain is influenced by household-specific requirements regarding the configuration, composition and execution of analytics tasks. Existing approaches in scientific literature cover the resulting architectural challenges only insufficiently. With the emergence of new computing paradigms, architectural concepts and technologies, new opportunities for analytics approaches, which enable individual household insights, become evident. For this reason, the objective of this work is the design of an Internet of Things analytics architecture for smart home applications, which supports the flexible deployment of analytics pipelines, therefore enabling the generation of individual household insights. In order to achieve this goal, challenges for Internet of Things analytics architectures are identified and analyzed by conducting a literature review. Based on the resulting challenges catalog, an architectural model is designed that facilitates the processing and analysis of streaming data from smart devices of different kinds. The developed architecture utilizes the fog computing paradigm, therefore allowing the deployment and execution of analytics pipelines in the cloud as well as at edge of the network. The architectural model is the foundation for a prototype, which is implemented to evaluate the proposed solution. The evaluation is performed by conducting several experiments, which are designed in order to validate the prototypes feasibility to address the found challenges. The main contributions of this work are a challenges catalog for Internet of Things analytics architectures, an architectural model for analytics in smart home applications as well as a prototype, which is based on it

Massenspektrometrische Untersuchungen an Spermien-Phospholipidmembranen

Fettleibigkeit und Adipositas haben in den letzten Jahren weltweit drastisch zugenommen. Die Fettleibigkeit geht nicht nur mit einer verringerten Lebensqualität einher, sondern ist auch mit verschiedenen Folgeerkrankungen, wie kardiovaskulären Erkrankungen (z.B. Arteriosklerose) und metabolische Erkrankungen (z.B. Diabetes) assoziiert. Vorliegende Studien belegen einen Zusammenhang zwischen Diabetes und männlicher Infertilität. In der vorliegenden Arbeit wurden daher die Spermienmembran wie auch das Seminalplasma (SP) mittels matrix-assisted laser desorption and ionisation time-of-flight Massenspektrometrie (MALDI-TOF MS) analysiert, um mögliche Lipid-Biomarker für die Fertilität bzw. Infertilität zu finden. Dafür wurde zunächst die MALDI-TOF MS Methode mit relevanten Standardlipiden optimiert. Anschließend konnte sowohl das Phospholipidmuster des SP mit dem Spermien verglichen werden als auch die Spermienlipide von normalgewichtigen und fettleibigen Probanden. Durch diese Analyse konnte das Phosphatidylcholin/Lysophosphatidylcholin (PC/LPC)-Verhältnis, aber auch ein stark erhöhter Sphingomyelin (SM)-Anteil bei den fettleibigen Probanden als Qualitätsmarker gefunden werden. Des Weiteren wurden im Rahmen dieser Arbeit murine Spermien aus dem Caput und dem Cauda der Epididymis mittels MALDI-TOF MS analysiert und das Phospholipidmuster miteinander verglichen. Es konnte damit gezeigt werden, dass die murinen Spermien einen wesentlich höheren Anteil an Stearinsäureresten aufweisen, die humanen Spermien hingegen vor allem durch Palmitinsäurereste charakterisiert sind. In den Spermienmembranen aus dem Cauda und dem Caput gab es wesentliche Unterschiede im Phospholipidmuster. Spermienmembranen aus dem Caput besitzen einen hohen Anteil an PC und Phosphatidylethanolamin (PE). Die Spermienmembranen aus dem Cauda hingegen enthalten mehr SM, und auch einen höheren Anteil an LPCs und Formyl-LPC. Diese Arbeit konnte somit zeigen, dass die Reifungsprozesse in der Epididymis auch die Phospholipid-Zusammensetzung betreffen

Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases

Elevated low-density lipoprotein (LDL) levels induce activation of the p38 mitogen-activated protein kinase (MAPK), a stress-activated protein kinase potentially participating in the development of atherosclerosis. The nature of the lipoprotein components inducing p38 MAPK activation has remained unclear however. We show here that both LDLs and high-density lipoproteins (HDLs) have the ability to stimulate the p38 MAPKs with potencies that correlate with their cholesterol content. Cholesterol solubilized in methyl-β-cyclodextrin was sufficient to activate the p38 MAPK pathway. Liposomes made of phosphatidylcholine (PC) or sphingomyelin, the two main phospholipids found in lipoproteins, were unable to stimulate the p38 MAPKs. In contrast, PC liposomes loaded with cholesterol potently activated this pathway. Reducing the cholesterol content of LDL particles lowered their ability to activate the p38 MAPKs. Cell lines representative of the three main cell types found in blood vessels (endothelial cells, smooth muscle cells and fibroblasts) all activated their p38 MAPK pathway in response to LDLs or cholesterol-loaded PC liposomes. These results indicate that elevated cholesterol content in lipoproteins, as seen in hypercholesterolemia, favors the activation of the stress-activated p38 MAPK pathway in cells from the vessel wall, an event that might contribute to the development of atherosclerosi

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response

Streaming Analytics in User-centric Internet of Things Domains: A Fog-enabled System Architecture for Smart Home Applications

A smart home is an apartment or a house in which smart devices communicate with each other to improve key areas of daily life, such as comfort, security or energy consumption. Therefore, the smart home domain is user-centric and exhibits characteristics that distinguish it from other application domains of the Internet of Things. Specifically, this concerns the existence of different 'regular' and 'smart' devices, but also the basic arrangement of each household, which is highly individual. As a result, the realization of analytics scenarios in the smart home domain is influenced by household-specific requirements regarding the configuration, composition and execution of analytics tasks. Existing approaches in scientific literature cover the resulting architectural challenges only insufficiently. With the emergence of new computing paradigms, architectural concepts and technologies, new opportunities for analytics approaches, which enable individual household insights, become evident. For this reason, the objective of this work is the design of an Internet of Things analytics architecture for smart home applications, which supports the flexible deployment of analytics pipelines, therefore enabling the generation of individual household insights. In order to achieve this goal, challenges for Internet of Things analytics architectures are identified and analyzed by conducting a literature review. Based on the resulting challenges catalog, an architectural model is designed that facilitates the processing and analysis of streaming data from smart devices of different kinds. The developed architecture utilizes the fog computing paradigm, therefore allowing the deployment and execution of analytics pipelines in the cloud as well as at edge of the network. The architectural model is the foundation for a prototype, which is implemented to evaluate the proposed solution. The evaluation is performed by conducting several experiments, which are designed in order to validate the prototypes feasibility to address the found challenges. The main contributions of this work are a challenges catalog for Internet of Things analytics architectures, an architectural model for analytics in smart home applications as well as a prototype, which is based on it

Massenspektrometrische Untersuchungen an Spermien-Phospholipidmembranen

Fettleibigkeit und Adipositas haben in den letzten Jahren weltweit drastisch zugenommen. Die Fettleibigkeit geht nicht nur mit einer verringerten Lebensqualität einher, sondern ist auch mit verschiedenen Folgeerkrankungen, wie kardiovaskulären Erkrankungen (z.B. Arteriosklerose) und metabolische Erkrankungen (z.B. Diabetes) assoziiert. Vorliegende Studien belegen einen Zusammenhang zwischen Diabetes und männlicher Infertilität. In der vorliegenden Arbeit wurden daher die Spermienmembran wie auch das Seminalplasma (SP) mittels matrix-assisted laser desorption and ionisation time-of-flight Massenspektrometrie (MALDI-TOF MS) analysiert, um mögliche Lipid-Biomarker für die Fertilität bzw. Infertilität zu finden. Dafür wurde zunächst die MALDI-TOF MS Methode mit relevanten Standardlipiden optimiert. Anschließend konnte sowohl das Phospholipidmuster des SP mit dem Spermien verglichen werden als auch die Spermienlipide von normalgewichtigen und fettleibigen Probanden. Durch diese Analyse konnte das Phosphatidylcholin/Lysophosphatidylcholin (PC/LPC)-Verhältnis, aber auch ein stark erhöhter Sphingomyelin (SM)-Anteil bei den fettleibigen Probanden als Qualitätsmarker gefunden werden. Des Weiteren wurden im Rahmen dieser Arbeit murine Spermien aus dem Caput und dem Cauda der Epididymis mittels MALDI-TOF MS analysiert und das Phospholipidmuster miteinander verglichen. Es konnte damit gezeigt werden, dass die murinen Spermien einen wesentlich höheren Anteil an Stearinsäureresten aufweisen, die humanen Spermien hingegen vor allem durch Palmitinsäurereste charakterisiert sind. In den Spermienmembranen aus dem Cauda und dem Caput gab es wesentliche Unterschiede im Phospholipidmuster. Spermienmembranen aus dem Caput besitzen einen hohen Anteil an PC und Phosphatidylethanolamin (PE). Die Spermienmembranen aus dem Cauda hingegen enthalten mehr SM, und auch einen höheren Anteil an LPCs und Formyl-LPC. Diese Arbeit konnte somit zeigen, dass die Reifungsprozesse in der Epididymis auch die Phospholipid-Zusammensetzung betreffen

Massenspektrometrische Untersuchungen an Spermien-Phospholipidmembranen

Fettleibigkeit und Adipositas haben in den letzten Jahren weltweit drastisch zugenommen. Die Fettleibigkeit geht nicht nur mit einer verringerten Lebensqualität einher, sondern ist auch mit verschiedenen Folgeerkrankungen, wie kardiovaskulären Erkrankungen (z.B. Arteriosklerose) und metabolische Erkrankungen (z.B. Diabetes) assoziiert. Vorliegende Studien belegen einen Zusammenhang zwischen Diabetes und männlicher Infertilität. In der vorliegenden Arbeit wurden daher die Spermienmembran wie auch das Seminalplasma (SP) mittels matrix-assisted laser desorption and ionisation time-of-flight Massenspektrometrie (MALDI-TOF MS) analysiert, um mögliche Lipid-Biomarker für die Fertilität bzw. Infertilität zu finden. Dafür wurde zunächst die MALDI-TOF MS Methode mit relevanten Standardlipiden optimiert. Anschließend konnte sowohl das Phospholipidmuster des SP mit dem Spermien verglichen werden als auch die Spermienlipide von normalgewichtigen und fettleibigen Probanden. Durch diese Analyse konnte das Phosphatidylcholin/Lysophosphatidylcholin (PC/LPC)-Verhältnis, aber auch ein stark erhöhter Sphingomyelin (SM)-Anteil bei den fettleibigen Probanden als Qualitätsmarker gefunden werden. Des Weiteren wurden im Rahmen dieser Arbeit murine Spermien aus dem Caput und dem Cauda der Epididymis mittels MALDI-TOF MS analysiert und das Phospholipidmuster miteinander verglichen. Es konnte damit gezeigt werden, dass die murinen Spermien einen wesentlich höheren Anteil an Stearinsäureresten aufweisen, die humanen Spermien hingegen vor allem durch Palmitinsäurereste charakterisiert sind. In den Spermienmembranen aus dem Cauda und dem Caput gab es wesentliche Unterschiede im Phospholipidmuster. Spermienmembranen aus dem Caput besitzen einen hohen Anteil an PC und Phosphatidylethanolamin (PE). Die Spermienmembranen aus dem Cauda hingegen enthalten mehr SM, und auch einen höheren Anteil an LPCs und Formyl-LPC. Diese Arbeit konnte somit zeigen, dass die Reifungsprozesse in der Epididymis auch die Phospholipid-Zusammensetzung betreffen

Streaming Analytics in User-centric Internet of Things Domains: A Fog-enabled System Architecture for Smart Home Applications

A smart home is an apartment or a house in which smart devices communicate with each other to improve key areas of daily life, such as comfort, security or energy consumption. Therefore, the smart home domain is user-centric and exhibits characteristics that distinguish it from other application domains of the Internet of Things. Specifically, this concerns the existence of different 'regular' and 'smart' devices, but also the basic arrangement of each household, which is highly individual. As a result, the realization of analytics scenarios in the smart home domain is influenced by household-specific requirements regarding the configuration, composition and execution of analytics tasks. Existing approaches in scientific literature cover the resulting architectural challenges only insufficiently. With the emergence of new computing paradigms, architectural concepts and technologies, new opportunities for analytics approaches, which enable individual household insights, become evident. For this reason, the objective of this work is the design of an Internet of Things analytics architecture for smart home applications, which supports the flexible deployment of analytics pipelines, therefore enabling the generation of individual household insights. In order to achieve this goal, challenges for Internet of Things analytics architectures are identified and analyzed by conducting a literature review. Based on the resulting challenges catalog, an architectural model is designed that facilitates the processing and analysis of streaming data from smart devices of different kinds. The developed architecture utilizes the fog computing paradigm, therefore allowing the deployment and execution of analytics pipelines in the cloud as well as at edge of the network. The architectural model is the foundation for a prototype, which is implemented to evaluate the proposed solution. The evaluation is performed by conducting several experiments, which are designed in order to validate the prototypes feasibility to address the found challenges. The main contributions of this work are a challenges catalog for Internet of Things analytics architectures, an architectural model for analytics in smart home applications as well as a prototype, which is based on it