Melting of a DNA hairpin without hyperchromism

UV absorbance spectroscopy is the most common method for detecting nucleic acid structural transitions and obtaining thermodynamic parameters. UV-detected melting has been used to determine stabilities of nucleic acid hairpins, duplexes, triplexes, and higher order structures and to determine thermodynamic effects of unusual or modified bases and mismatched base-pairs. We report that in some cases UV absorbance spectroscopy is an inadequate analytical technique for these purposes. Some critical transitions are invisible to UV absorbance spectroscopy. For example, the conversion of dodecamer d(CGCAAATTCGCG) from hairpin to random coil is not accompanied by hyperchromism. Circular dichroism (CD) spectroscopy (263 nm) clearly detects two transitions for this dodecamer, each giving a pronounced change in ellipiticity. The concentration dependence of the low-temperature transition and the concentration independence of the high-temperature transition indicate that the predominant state converts from duplex to hairpin to random coil as the temperature increases. These assignments are confirmed by comparison to oligonucleotides of similar sequence that undergo a hairpin to coil transition only. In contrast to CD spectroscopy, UV absorbance spectroscopy shows only a single transition. The transition detected by UV absorbance spectroscopy corresponds to the low-temperature transition detected by CD. UV absorbance spectroscopy does not detect the second transition at any wavelength (from 218 to 310 nm) (by changes) in either absorbance or its derivative with temperature.     

Apoptosis and its role in human disease

In a landmark paper published over two decades ago, Kerr et al. proposed the term apoptosis "for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations". In the ensuing years, this natural cell death process was studied at the basic science level, primarily with a view to understanding its roles in cancer and in the development and maintenance of the immune system. More recently, however, evidence has suggested a role for the failure of normal apoptosis control in many of the major diseases of the industrialized world. Though complex, apoptosis appears amenable to therapeutic intervention. The range of modern pharmaceutical strategies available to treat such disregulated gene-directed processes offers promise for advances in the control of cancer, immune system and neurodegenerative disorders, heart disease, and perhaps even the aging process itself.     

Natural ligands of the B cell adhesion molecule CD22 beta can be masked by 9-O-acetylation of sialic acids

CD22 beta is a B cell-restricted phosphoprotein expressed on the surface of mature resting B cells. It mediates interactions with other cells partly or exclusively via recognition of alpha 2-6-linked sialic acids on glycoconjugates. The sialylated N-linked oligosaccharides recognized best by CD22 beta are common to many glycoproteins, suggesting that additional regulatory mechanisms may exist. Since the exocyclic side chain of sialic acid is required for recognition, we explored the effects of a naturally occurring modification of the side chain, 9-O-acetylation. Semisynthetic N-linked oligosaccharides terminating with 9-O-acetylated, alpha 2-6-linked sialic acids showed markedly reduced binding to CD22 beta relative to their non-O-acetylated counterparts. Murine lymphoid cells were probed for natural CD22 beta ligands that might be O-acetylated using recombinant soluble forms of CD22 beta (CD22 beta Rg) and influenza C esterase (CHE-Fc, which specifically removes 9-O-acetyl esters from sialic acids). By flow cytometry analysis, CD22 beta Rg binding to splenic B cells and a subset of T cells was increased by pretreatment with CHE-Fc, indicating that some potential CD22 beta ligands are naturally "masked" by 9-O-acetylation. Unmasking of these CD22 beta ligands by removal of 9-O-acetyl esters from intact splenocytes substantially increases their CD22 beta-dependent adhesion in an in vitro adhesion assay. Probing of murine lymphoid tissue sections by CD22 beta Rg and CHE-Fc treatment demonstrates regionally restricted and differentially expressed patterns of distribution between masked and unmasked ligands. For example, lymph node-associated follicular B cells express high levels of CD22 beta ligands, none of which are masked by 9-O-acetylation. In contrast, the ligands on lymph node-associated dendritic cells are almost completely masked by 9-O-acetylation, suggesting that masking may regulate interactions between CD22 beta-positive B cells and dendritic cells. In the thymus, only medullary cells express CD22 beta ligands, and a significant portion of these are masked by 9-O-acetylation, particularly at the cortical-medullary junction. Thus, 9-O-acetylation of sialic acids on immune cells is in a position to negatively regulate CD22 beta adhesion events in a manner depending on both cell type and tissue localization.     

Isolation and characterization of a monoclonal anti-quadruplex DNA antibody from autoimmune "viable motheaten" mice

A cell line that produces an autoantibody specific for DNA quadruplex structures has been isolated and cloned from a hybridoma library derived from 3-month-old nonimmunized autoimmune, immunodeficient "viable motheaten" mice. This antibody has been tested extensively in vitro and found to bind specifically to DNA quadruplex structures formed by two biologically relevant sequence motifs. Scatchard and nonlinear regression analyses using both one- and two-site models were used to derive association constants for the antibody-DNA binding reactions. In both cases, quadruplexes had higher association constants than triplex and duplex molecules. The anti-quadruplex antibody binds to the quadruplex formed by the promoter-region-derived oligonucleotide d(CGCG4GCG) (Ka = 3.3 x 10(6) M-1), and has enhanced affinity for telomere-derived quadruplexes formed by the oligonucleotides d(TG4) and d(T2G4T2G4T2G4T2G4) (Ka = 5.38 x 10(6) and 1.66 x 10(7) M-1, respectively). The antibody binds both types of quadruplexes but has preferential affinity for the parallel four-stranded structure. In vitro radioimmunofilter binding experiments demonstrated that purified anti-DNA quadruplex antibodies from anti-quadruplex antibody-producing tissue culture supernatants have at least 10-fold higher affinity for quadruplexes than for triplex and duplex DNA structures of similar base composition and length. The antibody binds intramolecular DNA triplexes formed by d(G4T3G4T3C4) and d(C4T3G4T3G4), and the duplex d(CGCGCGCGCG)2 with an affinities of 6. 76 x 10(5), 5.59 x 10(5), and 8.26 x 10(5) M-1, respectively. Competition experiments showed that melted quadruplexes are not effective competitors for antibody binding when compared to native structures, confirming that the quadruplex is bound structure-specifically. To our knowledge, this is the first immunological reagent known to specifically recognize quadruplex structures. Subsequent sequence analysis demonstrates homologies between the antibody complementarity determining regions and sequences from Myb family telomere binding proteins, which are hypothesized to control cell aging via telomeric DNA interactions. The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.     

SENFIS: a Sensor Node File System for increasing the scalability and reliability of Wireless Sensor Networks applications

In the last years the Wireless Sensor Networks’ (WSN) technology has been increasingly employed in various application domains. The extensive use of WSN posed new challenges in terms of both scalability and reliability. This paper proposes Sensor Node File System (SENFIS), a novel file system for sensor nodes, which addresses both scalability and reliability concerns. SENFIS can be mainly used in two broad scenarios. First, it can transparently be employed as a permanent storage for distributed TinyDB queries, in order to increase the reliability and scalability. Second, it can be directly used by a WSN application for permanent storage of data on the WSN nodes. The experimental section shows that SENFIS implementation makes an efficient use of resources in terms of energy consumption, memory footprint, flash wear levelling, while achieving execution times similarly with existing WSN file systems.     

New Possibilities in Image Diffusion and Sharpening via High-Order Sobolev Gradient Flows

We study the use of high-order Sobolev gradients for PDE-based image smoothing and sharpening, extending our previous work on this problem. In particular, we study the gradient descent equation on the heat equation energy functional obtained by modifying the usual metric on the space of images, which is the L ² metric, to a weighted H ^k Sobolev metric. We present existence and uniqueness results which show that the Sobolev diffusion PDE are well-posed both in the forward and backward direction. Furthermore, we perform a Fourier analysis on the scale space generated by the Sobolev PDE and show that as the order of the Sobolev metric tends to infinity, the Sobolev gradients converge to a Gaussian smoothed L ² gradient. We then present experimental results which exploit the theoretical stability results by applying the various Sobolev gradient flows in the backward direction for image sharpening effects. Furthermore, we show that as the Sobolev order is increased, the sharpening effects become more global in nature and more immune to noise.     

From Autonomy to Cooperative Traded Control of Humanoid Manipulation Tasks with Unreliable Communication

In this paper, we present our system design, operational procedure, testing process, field results, and lessons learned for the valve-turning task of the DARPA Robotics Challenge (DRC). We present a software framework for cooperative traded control that enables a team of operators to control a remote humanoid robot over an unreliable communication link. Our system, composed of software modules running on-board the robot and on a remote workstation, allows the operators to specify the manipulation task in a straightforward manner. In addition, we have defined an operational procedure for the operators to manage the teleoperation task, designed to improve situation awareness and expedite task completion. Our testing process, consisting of hands-on intensive testing, remote testing, and remote practice runs , demonstrates that our framework is able to perform reliably and is resilient to unreliable network conditions. We analyze our approach, field tests, and experience at the DRC Trials and discuss lessons learned which may be useful for others when designing similar systems.