Interlaboratory evaluation of rat hepatic gene expression changes induced by methapyrilene.

Several studies using microarrays have shown that changes in gene expression provide information about the mechanism of toxicity induced by xenobiotic agents. Nevertheless, the issue of whether gene expression profiles are reproducible across different laboratories remains to be determined. To address this question, several members of the Hepatotoxicity Working Group of the International Life Sciences Institute Health and Environmental Sciences Institute evaluated the liver gene expression profiles of rats treated with methapyrilene (MP). Animals were treated at one facility, and RNA was distributed to five different sites for gene expression analysis. A preliminary evaluation of the number of modulated genes uncovered striking differences between the five different sites. However, additional data analysis demonstrated that these differences had an effect on the absolute gene expression results but not on the outcome of the study. For all users, unsupervised algorithms showed that gene expression allows the distinction of the high dose of MP from controls and low dose. In addition, the use of a supervised analysis method (support vector machines) made it possible to correctly classify samples. In conclusion, the results show that, despite some variability, robust gene expression changes were consistent between sites. In addition, key expression changes related to the mechanism of MP-induced hepatotoxicity were identified. These results provide critical information regarding the consistency of microarray results across different laboratories and shed light on the strengths and limitations of expression profiling in drug safety analysis

Polyglycerol-based amphiphilic dendrons as potential siRNA carriers for in vivo applications

The development of nonviral synthetic vectors for clinical application of gene therapy using siRNA transfection technology is of particular importance for treatment of human diseases, which is yet an unsolved challenge. By employing a rational design approach, we have synthesized a set of well-defined, low- molecular-weight dendritic polyglycerol-based amphiphiles, which are decorated peripherally with the DAPMA (N,N-di-(3-aminopropyl)-N-(methyl)amine) moiety. The main differences that were introduced in the structural motif relate to dendron generation and the type of linker between the hydrophilic and hydrophobic segment. The synthesized amphiphiles were then characterized for their aggregation behaviour and further evaluated with respect to their siRNA transfection potential by comparing their physico-chemical and biological features. Our findings demonstrated that all four synthesized amphiphiles yielded high gene binding affinities. Furthermore, the ester-linked compounds (G1-Ester-DAPMA, G2-Ester-DAPMA) revealed noticeable gene silencing in vitro without affecting the cell viability in the tumor cell line 786-O. Remarkably, neither G1-Ester-DAPMA nor G2-Ester-DAPMA induced inflammatory side effects after systemic administration in vivo, which is noteworthy because such highly positively charged compounds are typically associated with toxicity concerns which in turn supports their prospective application for in vivo purposes. Therefore, we believe that these structures may serve as new promising alternatives for nonviral siRNA delivery systems and have great potential for further synthetic modifications

Polyglycerol-based amphiphilic dendrons as potential siRNA carriers for in vivo applications

The development of nonviral synthetic vectors for clinical application of gene therapy using siRNA transfection technology is of particular importance for treatment of human diseases, which is yet an unsolved challenge. By employing a rational design approach, we have synthesized a set of well-defined, low- molecular-weight dendritic polyglycerol-based amphiphiles, which are decorated peripherally with the DAPMA (N,N-di-(3-aminopropyl)-N-(methyl)amine) moiety. The main differences that were introduced in the structural motif relate to dendron generation and the type of linker between the hydrophilic and hydrophobic segment. The synthesized amphiphiles were then characterized for their aggregation behaviour and further evaluated with respect to their siRNA transfection potential by comparing their physico-chemical and biological features. Our findings demonstrated that all four synthesized amphiphiles yielded high gene binding affinities. Furthermore, the ester-linked compounds (G1-Ester-DAPMA, G2-Ester-DAPMA) revealed noticeable gene silencing in vitro without affecting the cell viability in the tumor cell line 786-O. Remarkably, neither G1-Ester-DAPMA nor G2-Ester-DAPMA induced inflammatory side effects after systemic administration in vivo, which is noteworthy because such highly positively charged compounds are typically associated with toxicity concerns which in turn supports their prospective application for in vivo purposes. Therefore, we believe that these structures may serve as new promising alternatives for nonviral siRNA delivery systems and have great potential for further synthetic modifications

Integrated Epigenetics of Human Breast Cancer: Synoptic Investigation of Targeted Genes, MicroRNAs and Proteins upon Demethylation Treatment

The contribution of aberrant DNA methylation in silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations are reversible, it became of interest to determine the effects of the 5-aza-2'-deoxycytidine (DAC) demethylation therapy in breast cancer at different molecular levels

Stand und neuere Konzeptionen einer zwischenbetrieblichen Integration der EDV im Gueterverkehr

Bibliothek Weltwirtschaft Kiel A155,784 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Tetrode recording of local neuronal ensembles provides insight into coding mechanisms of short-term memory in macaque prefrontal cortex

As most cortical neurons are broadly tuned to various stimulus parameters, it is inevitable that individual neurons participate in the representation of more than one visual object. Vice versa, accurate representations of individual objects for example in short-term memory that can support reliable decisions require the participation of large neuronal populations. To provide evidence in favor of population codes, we have recently analyzed simultaneously recorded multi- and single-unit signals derived from arrays of single-ended microelectrodes (Waizel et al., SfN 2007). Multi-contact electrodes like tetrodes (tts) which have a real 3D-structure provide signals that allow for estimating the position of the recorded neurons by triangulation. Here we set out to study whether recording from 3D-tts would improve the quality of sorting and hence allow for the extraction of more information about the stimulus. Based on single trial firing rate values, we calculated one-way ANOVAs at 1 significance thresholds and performed subsequent posthoc comparison (Scheffé) in order to detect stimulus selectivity and determine stimulus specificity for the activity at each single site, respectively. In order to investigate the coding of distributed neuronal ensembles, we computed binary activity patterns for all active electrodes in the array and determined their stimulus selectivity and specificity. Compared to what we found previously in single microelectrode recordings, the number of object selective or even specific recording sites increased up to 3 times which provides highly specific sites in 3 out of 4 sessions (3000 trials, 13.5 million spikes). Given that our monkeys always performed the memory task with a set of twenty visual stimuli, we found highly specific sites coding for only one object which revealed up to 18 of 19 possible pairwise comparisons. According to the proposal that single neurons participate in more than one specific object representation we also found bi- or even tri-object-dependant sites (average 27 significant pairwise comparisons per session) and never non-systematic object specificity. As clusters of triangulation-reconstructed spikes tend to have inter-cluster regions with smooth transitions which could potentially reflect synchronous spikes, we wanted to know how much information could be carried by these signals. After removing spikes between clusters, we found object specificity highly decreased (in one session only 6 out of previous 38 significant pairwise comparisons remained). These results suggest that the use of tetrodes with a real 3D-structure provides more information about neuronal object representations

Splitting local ensembles by spike-sorting degrades the quality of neuronal object representations in macaque prefrontal cortex

As most cortical neurons are broadly tuned to various stimulus parameters, accurate representations of individual objects in short-term memory that can support the monkey’s decision need to be based on the activity of neuronal ensembles. We have in the past (Städtler et al., SfN 2006) analyzed simultaneously recorded multi-unit signals and found to our surprise that stimulus selectivity at individual sites was higher than what had been published for single units. We had expected that a signal that consists of the activity of several neurons should provide less selectivity because combining broadly tuned activity should result in even broader tuning by blurring. In order to test whether splitting up the multi-unit signal improves or degrades stimulus selectivity, we sorted multi-unit activity from 97 sites by means of semi-automatic PCA-based clustering (Chen et al., in prep.) into 413 sorted units. As for the multi-unit analysis, we calculated one-way ANOVAs (p<0.01) to detect stimulus selectivity and determined stimulus specificity by subsequent posthoc comparison (Scheffé). Only 11.6 of responsive sorted-units exhibited object-selective responses compared to 18.6 of responsive multi-units. Even more detrimental was the effect of sorting on stimulus specificity: only 2.9 of sorted units allowed for the discrimination of objects compared to 7.2 of the unsorted multi-unit signals. If this result is not due to a problem with detectability of weak signals, it suggests that the selectivity of local ensembles might be caused by coordination of their members. However, before we can directly address this possibility we need to improve our spike sorting methods such that temporally overlapping spike waveforms can unequivocally assigned to subsets of local neurons. For the time being we asked whether selectivity and specificity of distributed activity patterns based on sorted units combined across sites would profit from the contribution of a larger number of units, the prediction being that more independent signals could in principle provide more differentiating patterns. We therefore calculated binary activity patterns derived from epochs with a significant difference in firing rate compared to baseline. Distributed patterns of sorted activity provided a lower percentage of distinguishable patterns than the same analysis revealed for multi-unit signals. These results show that decomposition of local ensemble activity has direct impact on the quality of stimulus representations which in turn suggests that stimulus coding in the short-term memory of prefrontal cortex seems to depend on the coordinated activity of neuronal populations

Interlaboratory evaluation of rat hepatic gene expression changes induced by methapyrilene.

Several studies using microarrays have shown that changes in gene expression provide information about the mechanism of toxicity induced by xenobiotic agents. Nevertheless, the issue of whether gene expression profiles are reproducible across different laboratories remains to be determined. To address this question, several members of the Hepatotoxicity Working Group of the International Life Sciences Institute Health and Environmental Sciences Institute evaluated the liver gene expression profiles of rats treated with methapyrilene (MP). Animals were treated at one facility, and RNA was distributed to five different sites for gene expression analysis. A preliminary evaluation of the number of modulated genes uncovered striking differences between the five different sites. However, additional data analysis demonstrated that these differences had an effect on the absolute gene expression results but not on the outcome of the study. For all users, unsupervised algorithms showed that gene expression allows the distinction of the high dose of MP from controls and low dose. In addition, the use of a supervised analysis method (support vector machines) made it possible to correctly classify samples. In conclusion, the results show that, despite some variability, robust gene expression changes were consistent between sites. In addition, key expression changes related to the mechanism of MP-induced hepatotoxicity were identified. These results provide critical information regarding the consistency of microarray results across different laboratories and shed light on the strengths and limitations of expression profiling in drug safety analysis