A single-channel virtual receiving array using a time-reversal chaotic cavity.

Chaotic reverberation in a cavity, when coupled with time reversal acoustics, can be harnessed to build a perfect time-reversal mirror for transmitting and receiving highly focused sounds with a small number of transducers. In this article, a virtual receiving array, comprised of a single receiving transducer and a chaotic cavity, is developed based on time reversal processing of the reverberation inside the cavity. A prototype array, having 10 × 10 virtual receiving elements, is built and evaluated against a comparable physical array in terms of its localization and waveform reproduction capabilities. It turns out that the most crucial factor in the success of a virtual array is the ergodicity of its chaotic cavity, the exact mathematical expression for which is also derived. The virtual receiving array presented here may find some niche applications in reverberant environments, where a physical array turns out to be too costly or cumbersome to operate.

Human embryonic stem cells have enhanced repair of multiple forms of DNA damage.

Embryonic stem cells need to maintain genomic integrity so that they can retain the ability to differentiate into multiple cell types without propagating DNA errors. Previous studies have suggested that mechanisms of genome surveillance, including DNA repair, are superior in mouse embryonic stem cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary fibroblasts (WI-38, hs27) and, with the exception of UV-C damage, HeLa cells. Microarray gene expression analysis showed that mRNA levels of several DNA repair genes are elevated in human embryonic stem cells compared with their differentiated forms (embryoid bodies). These data suggest that genomic maintenance pathways are enhanced in human embryonic stem cells, relative to differentiated human cells.

Intrinsic ssDNA annealing activity in the C-terminal region of WRN.

Werner syndrome (WS) is a rare autosomal recessive disorder in humans characterized by premature aging and genetic instability. WS is caused by mutations in the WRN gene, which encodes a member of the RecQ family of DNA helicases. Cellular and biochemical studies suggest that WRN plays roles in DNA replication, DNA repair, telomere maintenance, and homologous recombination and that WRN has multiple enzymatic activities including 3' to 5' exonuclease, 3' to 5' helicase, and ssDNA annealing. The goal of this study was to map and further characterize the ssDNA annealing activity of WRN. Enzymatic studies using truncated forms of WRN identified a C-terminal 79 amino acid region between the RQC and the HRDC domains (aa1072-1150) that is required for ssDNA annealing activity. Deletion of the region reduced or eliminated ssDNA annealing activity of the WRN protein. Furthermore, the activity appears to correlate with DNA binding and oligomerization status of the protein.

Deficiency in 3'-phosphoglycolate processing in human cells with a hereditary mutation in tyrosyl-DNA phosphodiesterase (TDP1).

Tyrosyl-DNA phosphodiesterase (TDP1) is a DNA repair enzyme that removes peptide fragments linked through tyrosine to the 3' end of DNA, and can also remove 3'-phosphoglycolates (PGs) formed by free radical-mediated DNA cleavage. To assess whether TDP1 is primarily responsible for PG removal during in vitro end joining of DNA double-strand breaks (DSBs), whole-cell extracts were prepared from lymphoblastoid cells derived either from spinocerebellar ataxia with axonal neuropathy (SCAN1) patients, who have an inactivating mutation in the active site of TDP1, or from closely matched normal controls. Whereas extracts from normal cells catalyzed conversion of 3'-PG termini, both on single-strand oligomers and on 3' overhangs of DSBs, to 3'-phosphate termini, extracts of SCAN1 cells did not process either substrate. Addition of recombinant TDP1 to SCAN1 extracts restored 3'-PG removal, allowing subsequent gap filling on the aligned DSB ends. Two of three SCAN1 lines examined were slightly more radiosensitive than normal cells, but only for fractionated radiation in plateau phase. The results suggest that the TDP1 mutation in SCAN1 abolishes the 3'-PG processing activity of the enzyme, and that there are no other enzymes in cell extracts capable of processing protruding 3'-PG termini. However, the lack of severe radiosensitivity suggests that there must be alternative, TDP1-independent pathways for repair of 3'-PG DSBs.

Development of disposable membrane hydrophones for a frequency range from 1MHz to 10MHz.

A method for fabricating disposable membrane hydrophones is presented. The disposable hydrophones are intended for onetime use in such damaging environments as chemically contaminating fluids and high-amplitude (peak amplitude ∼100MPa) shock wave fields, where the use of commercial membrane hydrophones is not recommended. Fabrication of a hydrophone is done using only off-the-shelf components and hand tools, which translates into ease of fabrication and orders-of-magnitude reduction in unit cost. In particular, poling and sputtering, the two processes that are chiefly responsible for the cost and difficulty associated with the conventional fabrication method, are replaced with the use of pre-poled polyvinylidene fluoride (PVDF) films and polyethylene terephthalate (PET)-coated aluminum foils, respectively. Despite the seemingly crude construction, these disposable hydrophones can exhibit voltage sensitivity response that compares favorably with that of commercial hydrophones. For example, one prototype having a 2mm×2mm active element shows the end-of-cable voltage sensitivity of -270 (±1.9) dB re 1V/µPa over the frequency range of 1-10MHz.

Gene rearrangements induced by the DNA double-strand cleaving agent neocarzinostatin: conservative non-homologous reciprocal exchanges in an otherwise stable genome.

Among a collection of 74 aprt mutations induced by treatment of plateau phase Chinese hamster ovary CHO cells with the radiomimetic DNA double-strand cleaving agent neocarzinostatin, nine were large-scale rearrangements. Molecular analysis indicated that all nine were highly conservative, non-homologous reciprocal exchanges, most of which were intrachromosomal as determined by fluorescence in situ hybridization. All but one of the parental sequences contained potential double-strand cleavage sites positioned such that the observed rearrangements could be explained by drug-induced double-strand breakage followed by trimming, templated patching and ligation of the exchanged ends. Predicted non-complementary 3' overhangs were often preserved in the newly formed junctions, suggesting alignment-based fill-in of the overhangs. Banding of metaphase spreads of these mutants, and of a number of mutants induced by the functionally similar compound bleomycin, revealed that bleomycin-induced reciprocal exchange mutants had multiple additional chromosome alterations and considerable chromosomal heterogeneity within each mutant line. In contrast, neocarzinostatin-induced reciprocal exchange mutants, as well as bleomycin-induced base substitution and single base deletion mutants, retained stable pseudodiploid karyotypes similar to that of the parent line. Thus, some reciprocal exchanges arising from misjoining of double-strand breaks were associated with global chromosomal instability, while other ostensibly similar events were not.

Requirement for XRCC4 and DNA ligase IV in alignment-based gap filling for nonhomologous DNA end joining in vitro.

In the nonhomologous end joining pathway of DNA double-strand break repair, the ligation step is catalyzed by a complex of XRCC4 and DNA ligase IV. Extracts of CHO-K1 cells are able to accurately rejoin a site-specific free radical-mediated double-strand break with partially complementary overhangs, by a mechanism involving alignment-based gap filling followed by ligation. Extracts of XR-1 cells, which lack XRCC4 and DNA ligase IV, carried out neither gap filling nor ligation. Supplementation of the extracts with recombinant XRCC4/ligase IV, but not with XRCC4 alone, restored gap filling and accurate end joining. The results imply that XRCC4 and ligase IV are essential for alignment-based gap filling, as well as for final ligation of the breaks.

Effect of alumina composition and surface integrity in alumina/epoxy composites on the ultrasonic attenuation properties.

We report a method of fabricating backing blocks for ultrasonic imaging transducers, using alumina/epoxy composites. Backing blocks contain scatterers such as alumina particles interspersed in the epoxy matrix for the effective scattering and attenuation of ultrasound. Here, the surface integrity can be an issue, where the composite material may be damaged during machining because of differences in strength, hardness and brittleness of the hard alumina particles and the soft epoxy matrix. Poor surface integrity results in the formation of air cavities between the backing block and the piezoelectric element upon assembly, hence the increased reflection off the backing block and the eventual degradation in image quality. Furthermore, with an issue of poor surface integrity due to machining, it is difficult to increase alumina as scatterers more than a specific mass fraction ratio. In this study, we increased the portion of alumina within epoxy matrix by obtaining an enhanced surface integrity using a net shape fabrication method, and verified that this method could allow us to achieve higher ultrasonic attenuation. Backing blocks were net-shaped with various mass fractions of alumina to characterize the formability and the mechanical properties, including hardness, surface roughness and the internal micro-structure, which were compared with those of machined backing blocks. The ultrasonic attenuation property of the backing blocks was also measured.

Pathways and functions of the Werner syndrome protein.

Mutations in human WRN (also known as RECQ3) gene give rise to a rare autosomal recessive genetic disorder, Werner syndrome (WS). WS is a premature aging disease characterized by predisposition to cancer and early onset of symptoms related to normal aging including osteoporosis, ocular cataracts, graying and loss of hair, diabetes mellitus, arteriosclerosis, and atherosclerosis. This review focuses on the functional role of Werner protein (WRN) in guarding the genetic stability of cells, particularly by playing an integral role in the base excision repair, and at the telomere ends. Furthermore, in-depth biochemical investigations have significantly advanced our understanding of WRN protein regarding its binding partners and the site of protein-protein interaction. The mapping analysis of protein interaction sites in WRN for most of its binding partners have revealed a common site of protein-protein interaction in the RecQ conserved (RQC) region of WRN.

DNA end sequestration by DNA-dependent protein kinase and end joining of sterically constrained substrates in whole-cell extracts.

Extracts of Xenopus eggs and of cultured human and hamster cells have the capacity to join nonhomologous DNA ends, and all do so with similar specificity. To examine the formation of repair complexes on DNA under conditions of end joining, end-labeled fragments were incubated with the various extracts and then subjected to DNase-I footprinting. Human and Xenopus extracts produced footprints virtually identical to that of purified DNA-dependent protein kinase holoenzyme (Ku plus DNA-PKcs), with protection of the terminal 28 bp. Extracts of hamster cells were more variable, but usually produced a 16-bp footprint, similar to that of Ku alone. In all cases a 28-bp holoenzyme-like footprint was associated with wortmannin-sensitive end joining, minimal 3'-5' exonucleolytic resection, and a predominance of accurate end-joining products. To determine whether the short segments of DNA occupied by Ku and DNA-PK were sufficient to support end joining, Y-shaped substrates were constructed in which only one arm was available for end joining. A Y substrate with a 31-bp arm bearing a partially cohesive 3' overhang was accurately joined by a Xenopus egg extract, whereas a substrate with a 21-bp arm was not. Surprisingly, a human cell extract did not join the Y substrates at all. The results suggest that differences in wortmannin sensitivity and in the distribution of in vitro end-joining products may be attributable to the variations in the levels of DNA-PKcs in the extracts. In addition, end joining in human extracts appears to involve interactions with significantly longer segments of DNA than the approximately 28 bp occupied by DNA-PK.

Mechanism of Werner DNA helicase: POT1 and RPA stimulates WRN to unwind beyond gaps in the translocating strand.

WRN belongs to the RecQ family of DNA helicases and it plays a role in recombination, replication, telomere maintenance and long-patch base excision repair. Here, we demonstrate that WRN efficiently unwinds DNA substrates containing a 1-nucleotide gap in the translocating DNA strand, but when the gap size is increased to 3-nucleotides unwinding activity significantly declines. In contrast, E. coli UvrD (3'-->5' helicase), which recognizes nicks in DNA to initiate unwinding, does not unwind past a 1-nucleotide gap. This unique ability of WRN to bypass gaps supports its involvement in DNA replication and LP-BER where such gaps can be produced by glycosylases and the apurinic/apyrimidinic endonuclease 1 (APE1). Furthermore, we tested telomere repeat binding factor 2 (TRF2), both variants 1 and 2 of protector of telomeres 1 (POT1v1 and POT1v2) and RPA on telomeric DNA substrates containing much bigger gaps than 3-nucleotides in order to determine whether unwinding could be facilitated through WRN-protein interaction. Interestingly, POT1v1 and RPA are capable of stimulating WRN helicase on gapped DNA and 5'-overhang substrates, respectively.

Inhibitory effect of tramadol on vasorelaxation mediated by ATP-sensitive K+ channels in rat aorta.

PURPOSE: Tramadol produces a conduction block similar to lidocaine by exerting a local anesthetic-like effect. The aims of this in vitro study were to determine the effects of tramadol on the vasorelaxant response induced by the adenosine triphosphate-sensitive K(+) (K(ATP)) channel opener, levcromakalim, in an endothelium-denuded rat aorta, and to determine whether this effect of tramadol is stereoselective. METHODS: The effects of tramadol (racemic, R(-) and S(+): 10(-6), 10(-5), 5 x 10(-5) M), and glibenclamide on the levcromakalim dose-response curve were assessed in aortic rings that had been pre-contracted with phenylephrine. In the rings pretreated independently with naloxone, and glibenclamide, the levcromakalim dose-response curves were generated in the presence or absence of tramadol. The effect of tramadol on the dose-response curve of diltiazem was assessed. RESULTS: Racemic, R(-) and S(+) tramadol (10(-5), 5 x 10(-5) M) attenuated (P < 0.0001) levcromakalim-induced relaxation in the ring with or without naloxone in a dose-dependent manner. The magnitude of the R(-)-tramadol-induced attenuation of vasorelaxant response induced by levcromakalim was greater (P < 0.05) than that induced by S(+)-tramadol. Glibenclamide almost abolished the levcromakalim-induced relaxation. Tramadol, 5 x 10(-5) M, did not significantly alter the diltiazem-induced relaxation. CONCLUSION: These results suggest that a supraclinical dose (10(-5) M) of tramadol [racemic, R(-) and S(+)] attenuates the vasorelaxation mediated by the K(ATP) channels in the rat aorta. The R(-) tramadol-induced attenuation of vasorelaxation induced by levcromaklim was more potent than that induced by S(+) tramadol. This attenuation is independent of opioid receptor activation.

The human Werner syndrome protein stimulates repair of oxidative DNA base damage by the DNA glycosylase NEIL1.

The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (K(D) = 60 nM) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.

Modulation of Werner syndrome protein function by a single mutation in the conserved RecQ domain.

Naturally occurring mutations in the human RECQ3 gene result in truncated Werner protein (WRN) and manifest as a rare premature aging disorder, Werner syndrome. Cellular and biochemical studies suggest a multifaceted role of WRN in DNA replication, DNA repair, recombination, and telomere maintenance. The RecQ C-terminal (RQC) domain of WRN was determined previously to be the major site of interaction for DNA and proteins. By using site-directed mutagenesis in the WRN RQC domain, we determined which amino acids might be playing a critical role in WRN function. A site-directed mutation at Lys-1016 significantly decreased WRN binding to fork or bubble DNA substrates. Moreover, the Lys-1016 mutation markedly reduced WRN helicase activity on fork, D-loop, and Holliday junction substrates in addition to reducing significantly the ability of WRN to stimulate FEN-1 incision activities. Thus, DNA binding mediated by the RQC domain is crucial for WRN helicase and its coordinated functions. Our nuclear magnetic resonance data on the three-dimensional structure of the wild-type RQC and Lys-1016 mutant proteins display a remarkable similarity in their structures.

Physical and functional mapping of the replication protein a interaction domain of the werner and bloom syndrome helicases.

The single-stranded DNA-binding protein replication protein A (RPA) interacts with several human RecQ DNA helicases that have important roles in maintaining genomic stability; however, the mechanism for RPA stimulation of DNA unwinding is not well understood. To map regions of Werner syndrome helicase (WRN) that interact with RPA, yeast two-hybrid studies, WRN affinity pull-down experiments and enzyme-linked immunosorbent assays with purified recombinant WRN protein fragments were performed. The results indicated that WRN has two RPA binding sites, a high affinity N-terminal site, and a lower affinity C-terminal site. Based on results from mapping studies, we sought to determine if the WRN N-terminal region harboring the high affinity RPA interaction site was important for RPA stimulation of WRN helicase activity. To accomplish this, we tested a catalytically active WRN helicase domain fragment (WRN(H-R)) that lacked the N-terminal RPA interaction site for its ability to unwind long DNA duplex substrates, which the wild-type enzyme can efficiently unwind only in the presence of RPA. WRN(H-R) helicase activity was significantly reduced on RPA-dependent partial duplex substrates compared with full-length WRN despite the presence of RPA. These results clearly demonstrate that, although WRN(H-R) had comparable helicase activity to full-length WRN on short duplex substrates, its ability to unwind RPA-dependent WRN helicase substrates was significantly impaired. Similarly, a Bloom syndrome helicase (BLM) domain fragment, BLM(642-1290), that lacked its N-terminal RPA interaction site also unwound short DNA duplex substrates similar to wild-type BLM, but was severely compromised in its ability to unwind long DNA substrates that full-length BLM helicase could unwind in the presence of RPA. These results suggest that the physical interaction between RPA and WRN or BLM helicases plays an important role in the mechanism for RPA stimulation of helicase-catalyzed DNA unwinding.

Implication of DNA polymerase lambda in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts.

Accurate repair of free radical-mediated DNA double-strand breaks by the nonhomologous end joining pathway requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nuclear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/ligase IV), were capable of accurately rejoining model double-strand break substrates with a 1- or 2-base gap, and the gap-filling step was dependent on XRCC4/ligase IV. To determine what polymerase was responsible for gap filling, end joining was examined in the presence of polyclonal antibodies against each of two prime candidate enzymes, DNA polymerases mu and lambda, both of which were present in the extracts. For a DNA substrate with partially complementary 3' overhangs and a 2-base gap, antibodies to polymerase lambda completely eliminated both gap filling and accurate end joining, whereas antibodies to polymerase mu had little effect. Immunodepletion of polymerase lambda, but not polymerase mu, likewise blocked both gap filling and end joining, and both functions could be restored by addition of recombinant polymerase lambda. Recombinant polymerase mu, and a truncated polymerase lambda lacking the Brca1 C-terminal domain, were at least 10-fold less active in restoring gap filling to the immunodepleted extracts, and polymerase beta was completely inactive. The results suggest that polymerase lambda is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity.