Algorithm of a Single Chip Acoustic Echo Canceller Using Cascaded Cross Spectral Estimation

This paper details the algorithm used by a low cost, single chip acoustic echo canceller. The algorithm is based on classical cross spectral estimation. It is employed in a cascaded filter structure where a short, fast filter operates on the output of a longer but slower filter to optimize the tracking performance of changes in the echo path without affecting the steady state performance. This combination allows the use of a fixed configuration for a wide range of acoustic environments

Carnuntum. Roman city on Danube (1st - 4th Century AD)

This thesis deals with questions of urban development in the Roman city of Camuntum. The main foeus is foremost to specify and review the global strueture of settlement, urbanism and civilian architecture during the first four hunderts AD. In the first chapters provide an overview of loeation and historical situation of Camuntum. In the following chapters the description of the urban settlement is divided jnto "civilian" and , "military" territory. including description of several archaeologieal findings and monuments, maily publie buildings and religious areas. Furthermore, questions related to typology of dwelling-houses and their interior have not been omitted. The thesis also tries to summarize other topics associated with the area of Roman provinees, for example trade and burialground. The last part of the thesis is devoted to comparison and parallels with other important sites in the area of Rhine and Danube

Non-Muscle Myosin II Regulates Neuronal Actin Dynamics by Interacting with Guanine Nucleotide Exchange Factors

<div><p>Background</p><p>Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family.</p><p>Principal Findings</p><p>NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II–GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II–GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation.</p><p>Conclusion</p><p>Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF–Rho GTPase signaling pathway.</p></div

Blebbistatin alters growth cone actin dynamics through Cdc42 activation in cultured hippocampal neurons.

<p>A. Cultured hippocampal (HP) neurons at 2 days <i>in vitro</i> (DIV) were incubated with or without 50 µM blebbistatin (BBS) for 30 min, and then BBS was removed. Growth cones were stained for actin with TRITC-labeled phalloidin. Arrowheads indicate actin arcs. Scale bar, 10 µm. B. HP neurons were transfected with the Raichu-Cdc42 probe for 1 day and then incubated with or without 50 µM BBS for 10 min. Representative ratio images of FRET/CFP after BBS treatment are shown in the intensity-modulated display (IMD) mode (left). Bar graphs represent the relative emission ratio (FRET/CFP) of the whole cell area (right). The number of cells examined for each sample was 28 (with BBS) or 49 (without BBS). Error bars are ± SD. *, <i>P</i><0.05. C. GST-PBD pulldown assay for activated Cdc42. HP neurons were incubated with or without 50 µM BBS for the indicated times and then lysed. Equal amounts of protein from each lysate were incubated with GST-PBD immobilized on glutathione-Sepharose. Total and GST-PBD–bound Cdc42 was probed by immunoblotting with anti-Cdc42 antibody. The data are representative of three independent experiments. D. Quantitative analysis of immunoblots from three independent experiments shown in C. The data were quantified by densitometric analysis using Image J software.</p

The effect of overexpression of the βPIX DH domain on growth cone formation and neurite branching.

<p>A. PC12 cells were transfected with plasmids encoding GFP (control), GFP-βPIX DH^wt, or GFP-βPIX DH^mt. Lysates were immunoprecipitated with an anti-βPIX monoclonal antibody, and immunoprecipitates were probed for NM IIB or βPIX (top). To monitor the expression of transfected genes, immunoblotting for GFP was performed (bottom). The blot is representative of three independent experiments. B. Quantitative analysis of immunoblots from three experiments shown in A. C. GST-PBD pulldown assay for Cdc42 activation. Total (input) and GST-PBD–bound Cdc42 was immunoblotted with an anti-Cdc42 antibody (top). The expression of transfected genes was monitored by immunoblotting for GFP (bottom). The blot is representative of three independent experiments. D. Quantitative analysis of immunoblots from three experiments shown in C. E. Cultured HP neurons were transfected with plasmids encoding GFP (control), GFP-DH^wt or GFP-DH^mt. Cells were stained for actin (red), and expression of transfected GFP constructs (green) was examined by fluorescence microscopy. Scale bar, 10 µm.</p

Blebbistatin alters localization of GEFs.

<p>A. Schematic diagram of growth cone zones used for quantification of GEF localization. Peripheral actin-stained areas, pA and pA', are shaded in red. B. Time course of βPIX localization in the actin-positive area after BBS treatment. Cultured HP neurons were incubated with 50 µM BBS for the indicated times and then double-stained with TRITC-labeled phalloidin for actin (red) and anti-βPIX antibody (green). The dotted lines indicate the peripheral actin–positive area. Scale bar, 10 µm. C. Quantification of βPIX localization in the peripheral actin–positive area. Mean fluorescence intensity of βPIX after BBS treatment was expressed as a relative ratio of pA'/pA. The mean intensity for pA in the BBS-untreated growth cones were set to 1. D. Quantification of the peripheral actin–positive area. The size of the peripheral actin–positive area after BBS treatment was expressed as a relative ratio of pA'/pA. The size for pA in the BBS-untreated growth cones were set to 1. The number of cells examined was as follows: for time 0, <i>n</i> = 12; for 15 min, <i>n</i> = 10; for 30 min, <i>n</i> = 10. Error bars are ± SD. *, <i>P</i><0.05. E. BBS-induced alterations in localization of GEFs in a distal axon. Cultured HP neurons were incubated without or with 50 µM BBS for 30 min and co-stained for actin (red) and the indicated GEF (green). White arrowheads indicate the ends of branched neurites on the distal axon. Note the absence of branching on the distal axon in BBS-untreated cells. Scale bar, 10 µm.</p

Blebbistatin dissociates βPIX and NM IIB in growth cones.

<p>A. BBS-induced dissociation of NM IIB–βPIX complex. Cultured HP neurons were incubated with or without 50 µM BBS for 30 min. Lysates were immunoprecipitated with anti-NM IIB antibody, and immunoblotted for βPIX and NM IIB. To ensure equal loading, lysates were immunoblotted with anti-βPIX antibody. B. Time-dependent dissociation of the NM IIB–βPIX complex. PC12 cells were incubated with 50 µM BBS for the indicated times. Lysates were processed as described above. C. Schematic diagram of the growth cone zones used for quantification of the NM IIB–βPIX interaction. Total actin–stained areas, A and A′, are shaded in red. D. <i>In situ</i> proximity ligation assay. HP neurons were processed using the Duolink <i>In Situ</i> Detection Reagents. Anti-βPIX and NM IIB antibodies were used as primary antibodies. Red spots represent the interaction of NM IIB and βPIX. To visualize actin structures in growth cones, cells were stained with Alexa Fluor 488–conjugated phalloidin (green). Scale bar, 10 µm. E. Quantification of association of βPIX with NM IIB. Fluorescence intensity of the NM IIB–βPIX complex before and after BBS treatment was expressed as a relative ratio of A′/A. The relative ratio in the BBS-untreated growth cones was set to 1. The number of cells examined was as follows: for time 0, <i>n</i> = 20; for 15 min, <i>n</i> = 25; for 30 min, <i>n</i> = 25. Error bars are ± SD. *, <i>P</i><0.05.</p

Cdc42–PAK1 pathway mediates dissociation of the NMII-GEF complex in the NGF signaling pathway.

<p>A. PC12 cells were transfected with GFP-tagged constructs for active Cdc42 (Cdc42V12), RhoA (RhoAV14) or GFP alone for 24 h. Lysates were immunoprecipitated with anti-NM IIB or anti-MLC antibodies, and immunoprecipitates were immunoblotted for phosphorylated and total NM IIB (top), phosphorylated (Ser18/Thr19) and total MLC (2nd panel), or the indicated GEFs (3rd panel). The expression of transfected genes was assessed by immunoblotting for GFP (bottom). B. Cells were transfected with GFP-tagged wild-type Cdc42 and stimulated with 100 ng/ml NGF for the indicated times. A GST-PBD pulldown assay was performed to measure Cdc42 activation (top). NGF-stimulated lysates were immunoprecipitated with anti-NM IIB (middle) or βPIX (bottom) antibodies, and immunoprecipitates were immunoblotted for the indicated proteins. Phosphorylated NM IIB (pNM IIB) was detected by anti-phospho-threonine antibody. C. Non-transfected cells were stimulated with 100 ng/ml NGF for the indicated times. A GST-PBD pulldown assay was performed to measure Cdc42 activation (top). Lysates were immunoprecipitated with anti-βPIX antibody, and immunoprecipitates were immunoblotted for the indicated proteins (bottom). D and E. Cells were transfected with GFP-tagged constructs for WT or DN-Cdc42 (Cdc42N17) (D) or Myc-tagged constructs for WT or DN-PAK1 (H83/86L, K299R) (E), and then treated with or without 100 ng/ml NGF for 10 min. Lysates were immunoprecipitated with anti-NM IIB antibody, and immunoprecipitates were immunoblotted for the indicated proteins (top). The expression of transfected Cdc42 and PAK1 constructs was confirmed by immunoblotting with anti-GFP and anti-Myc antibody, respectively (bottom). The data are representative of three independent experiments.</p