Incidence of Common Herpesviruses in Colonic Mucosal Biopsies Following Hematopoietic Stem Cell Transplantation.

Intestinal complications are common after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, only scarce data concern herpesvirus incidence in the colonic mucosa post-HSCT. Our purpose was to assess the frequency and clinical significance of cytomegalovirus (CMV), Epstein−Barr virus (EBV), human herpesvirus type 6 (HHV6), and herpes simplex virus (HSV) in the colonic mucosa post-HSCT. The study group included 119 patients of different ages, mostly with leukemias and lymphomas, subjected to allo-HSCT from haploidentical related (48%) or HLA-compatible donors (52%). In total, 155 forceps biopsies of the colonic mucosa were taken in cases of severe therapy-resistant intestinal syndrome post-HSCT. Most samples were taken from the descending, sigmoid, and transverse colon. Intestinal GVHD or local infections were assessed clinically and by histology. EBV, CMV, HSV, and HHV6 were tested in colonic mucosal lysates with commercial PCR assays. HSV was found in <8% of colonic samples, along with high HHV6 and CMV positivity (up to 62% and 35%, respectively) and a higher EBV incidence at 5−6 months post-HSCT (35%). For CMV and EBV, significant correlations were revealed between their rates of detection in blood and colonic mucosa (r = 0.489 and r = 0.583; p < 0.05). No significant relationships were found between the presence of herpesviruses and most patients' characteristics. EBV positivity in colonic samples was correlated with delayed leukocyte and platelet recovery post-HSCT. Higher EBV frequency in the colonic mucosa was found in deceased patients (56% versus 21%, p = 0.02). The correlations among EBV positivity in the colon, lethality rates and delayed hematopoietic reconstitution suggest some relationship with systemic and local EBV reactivation post-transplant.

SAW RFID Devices Using Connected IDTs as an Alternative to Conventional Reflectors for Harsh Environments.

Remote interrogation of surface acoustic wave identification tag (ID-tags) imposes a high signal amplitude which is related to a high coupling coefficient value ( K2 ) and low propagation losses ( α ). In this article, we propose and discuss an alternative configuration to the standard one. Here, we replaced the conventional configuration, i.e., one interdigital transducer (IDT) and several reflectors, by a series of electrically connected IDTs. The goal is to increase the amplitude of the detected signal using direct transmission between IDTs instead of the reflection from passive reflectors. This concept can, therefore, increase the interrogation scope of ID-tags made on a conventional substrate with high K2 value. Moreover, it can also be extended to suitable substrates for harsh environments, such as high-temperature environments: the materials used exhibit limited performances (low K2 value and relatively high propagation losses) and are, therefore, rarely used for identification applications. The concept was first tested and validated using the lithium niobate 128° Y-X cut substrate, which is commonly used in ID-tags. A good agreement between experimental and numerical results was obtained for the promising concept of connected IDTs. The interesting features of the structure were also validated using a langasite substrate, which is well-known to operate at very high temperatures. Performances of both substrates (lithium niobate and langasite) were tested with an in situ RF characterization up to 600 °C. Unexpected results regarding the resilience of devices based on congruent lithium niobate were obtained.

High-Temperature SAW Resonator Sensors: Electrode Design Specifics.

Surface acoustic wave (SAW) sensors are steadily paving the way to wider application areas. Their main benefit consisting in the possibility of wireless interrogation with the radio frequency interrogation signal being the only energy source for the reradiated signal. This feature is getting more and more attractive with the growing demand in monitoring multiple industrial objects difficult to access by wired sensors in harsh environments. Among such wider applications, the possibility of making measurements of temperature, deformation, vibrations, and some other parameters at temperatures in the range of 300 °C-1000 °C look quite promising. This paper concentrates on specific features of the SAW resonator-based sensors operation at this temperature range. High-temperature influences the material choice and thus the properties of SAW resonators design peculiarities intended for use at high temperature. It is suggested that preferable designs should use synchronous resonators with relatively thick electrodes (10% of wavelength) based on Ir or Pt alloys while benefiting from the possibilities of specific designs that could reduce the negative impact of thick electrodes on the manufacturing in quantity. This solution benefits from lower resonance frequency scatter because of the automatic compensation of SAW velocity decrease due to electrode metallization ratio increase. This compensation originates from the resonance frequency increase that is related to the decrease of the Bragg bandwidth defined by the reflection. It is shown in modeling examples that the value of metallization ratio at which this compensation occurs is close to 65%-70%.

SAW Temperature Sensor on Quartz.

For biomedical applications, narrow temperature range and high sensor accuracy requirements define the need for high temperature sensitivity. Wireless SAW sensors connected to antennas need a reference element to account for changes in electromagnetic coupling between the transmitter and receiver antennas. A pair of sensors with different temperature sensitivities may serve as a self-referenced sensor assembly. This justifies the need for materials with useful SAW resonator properties and with the largest difference between temperature coefficients of frequency (TCF) for a resonator pair on a single substrate. We have identified several cuts of quartz having useful properties with a TCF difference up to 140 ppm/°C for a pair of resonators on a single substrate. As a rule, placing such resonators on a single substrate requires their rotation by up to 90° relative to each other. The limited range of cuts presents a unique opportunity to place both resonators along the X+90° direction with one resonator using Bleustein-Gulyaev-Shimizu (BGS) waves (with electrodes placed along the x-axis) and the other one (with electrodes inclined by about ±10° to the x-axis) using quasi-Rayleigh waves. These cuts are close to the 70°Y cut where a high TCF difference is reached together with acceptable characteristics of the resonators. Resonators were designed for all useful cuts (including the 70°Y cut) and tested. The use of different periods in reflectors and interdigital transducer (IDT) together with individual choice of gaps between reflectors and IDT meant achieving low spurious content in resonator responses. The quality factors reached values up to 3500 at central frequencies around 915 MHz for both BGS and quasi-Rayleigh types of waves. The measured difference of the TCF is about 138 ppm/°C on 70°Y cut that is close to the calculated value.

The actin-binding domain of cortactin is dynamic and unstructured and affects lateral and longitudinal contacts in F-actin.

Cortactin is an F-actin- and Arp2/3 complex-binding protein, implicated in the regulation of cytoskeleton dynamics and cortical actin-assembly. The actin-binding domain of cortactin consists of a 6.5 tandem repeat of a 37-amino acid sequence known as the cortactin repeat (residues 80-325). Using a combination of structure prediction, circular dichroism, and cysteine crosslinking, we tested a recently published three-dimensional model of the cortactin molecule in which the cortactin repeat is folded as a globular helical domain [Zhang et al., 2007, Mol Cell 27:197-213]. We show that the cortactin repeat is unstructured in solution. Thus, wild type and mutant constructs of the cortactin repeat, containing pairs of cysteines at positions 112 and 246, 83 and 112, 83 and 246, and 83 and 306, could be readily crosslinked with reagents of varying lengths (0-9.6 A). Using yeast actin cysteine mutants, we also show that cortactin inhibits disulfide and dibromobimane crosslinking across the lateral and longitudinal interfaces of actin subunits in the filament, suggesting a weakening of intersubunits contacts. Our results are in disagreement with the proposed model of the cortactin molecule and have important implications for our understanding of cortactin regulation of cytoskeleton dynamics.

Effects of binding factors on structural elements in F-actin.

Understanding the dynamics of the actin filament is essential to a detailed description of their interactions and role in the cell. Previous studies have linked the dynamic properties of actin filaments (F-actin) to three structural elements contributing to a hydrophobic pocket, namely, the hydrophobic loop, the DNase I binding loop, and the C-terminus. Here, we examine how these structural elements are influenced by factors that stabilize or destabilize F-actin, using site-directed spin-labeled (SDSL) electron paramagnetic resonance (EPR), fluorescence, and cross-linking techniques. Specifically, we employ cofilin, an actin destabilizing protein that binds and severs filaments, and phalloidin, a fungal toxin that binds and stabilizes F-actin. We find that cofilin shifts both the DNase I binding loop and the hydrophobic loop away from the C-terminus in F-actin, as demonstrated by weakened spin-spin interactions, and alters the environment of spin probes on residues of these two loops. In contrast, although phalloidin strongly stabilizes F-actin, it causes little or no local change in the environment of the loop residues. This indicates that the stabilizing effect of phalloidin is achieved mainly through constraining structural fluctuations in F-actin and suggests that factors and interactions that control these fluctuations have an important role in the cytoskeleton dynamics.

Actin hydrophobic loop 262-274 and filament nucleation and elongation.

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a "parked" conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by approximately 5 degrees per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by approximately 1 degrees per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.

One-port SAW resonators using natural SPUDT substrates.

We discuss the design of one-port surface acoustic wave (SAW) resonators using substrates with a partial or total degree of directivity, that is, the natural single-phase unidirectional transducer (N-SPUDT) effect. A general design method gives a resonance when all three frequencies (the required resonance frequency and the Bragg frequencies) are different. A second method has been derived from the resonance condition for a symmetrical substrate. Two further methods incorporate lamda/4 gaps. The capacitance ratio is presented as a function of the phase of the electrode reflection coefficient. The simulations use data for the N-SPUDT orientation of langasite. The reflection coefficient for Al electrodes has been calculated from finite element modeling (FEM) analysis. The approximate perturbation theory is found to agree well for small film thickness (h/lamda < 2%). The phase of the reflection coefficient is typically 150 degrees, not quite the ideal value of 180 degrees. Measurements on resonators using Al and Cu films are presented.

Hydrophobic loop dynamics and actin filament stability.

It has been postulated that the hydrophobic loop of actin (residues 262-274) swings out and inserts into the opposite strand in the filament, stabilizing the filament structure. Here, we analyzed the hydrophobic loop dynamics utilizing four mutants that have cysteine residues introduced at a single location along the yeast actin loop. Lateral, copper-catalyzed disulfide cross-linking of the mutant cysteine residues to the native C374 in the neighboring strand within the filament was fastest for S265C, followed by V266C, L267C, and then L269C. Site-directed spin labeling (SDSL) studies revealed that C265 lies closest to C374 within the filament, followed by C266, C267, and then C269. These results are not predicted by the Holmes extended loop model of F-actin. Furthermore, we find that disulfide cross-linking destroys L267C and L269C filaments; only small filaments are observed via electron microscopy. Conversely, phalloidin protects the L267C and L269C filaments and inhibits their disulfide cross-linking. Combined, our data indicate that, in solution, the loop resides predominantly in a "parked" position within the filament but is able to dynamically populate other conformational states which stabilize or destabilize the filament. Such states may be exploited within a cell by filament-stabilizing and -destabilizing factors.

Conformational dynamics of loop 262-274 in G- and F-actin.

According to the original Holmes model of F-actin structure, the hydrophobic loop 262-274 stabilizes the actin filament by inserting into a pocket formed at the interface between two protomers on the opposing strand. Using a yeast actin triple mutant, L180C/L269C/C374A [(LC)(2)CA], we showed previously that locking the hydrophobic loop to the G-actin surface by a disulfide bridge prevents filament formation. We report here that the hydrophobic loop is mobile in F- as well as in G-actin, fluctuating between the extended and parked conformations. Copper-catalyzed, brief air oxidation of (LC)(2)CA F-actin on electron microscopy grids resulted in the severing of thin filaments and their conversion to amorphous aggregates. Disulfide, bis(methanethiosulfonate) (MTS), and dibromobimane (DBB) cross-linking reactions proceeded in solution at a faster rate with G- than with F-actin. Cross-linking of C180 to C269 by DBB (4.4 A) in either G- or F-actin resulted in shorter and less stable filaments. The cross-linking with a longer MTS-6 reagent (9.6 A) did not impair actin polymerization or filament structure. Myosin subfragment 1 (S1) and tropomyosin inhibited the disulfide cross-linking of phalloidin-stabilized F-actin. Electron paramagnetic resonance measurements with nitroxide spin-labeled actin revealed strong spin-spin coupling and a similar mean interspin distance ( approximately 10 A) in G- and in F-actin, with a broader distance distribution in G-actin. These results show loop 262-274 fluctuations in G- and F-actin and correlate loop dynamics with actin filament formation and stability.

Synthesis and biological evaluation of 6/7-exo-methyl-3beta-(4-iodo)phenyltropane-2beta-carboxylic acid methyl esters.

6beta/7beta-Methyl-2-methoxycarbonyltropinones (3a, 3b) were synthesized and used as starting materials in the synthesis of 6beta/7beta-methyl-2beta-methoxycarbonyl-3beta-phenyltropanes (6a, 6b), 6beta/7beta-methyl-2beta-methoxycarbonyl-3beta-(4-iodo)phenyltropanes (7a, 7b) and 6beta-methyl-2beta-methoxycarbonyl-3beta-(4-iodo)phenylnortropane (8). The effect of 6/7-groups was evaluated by in vitro receptor binding to dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters. Introduction of a methyl group at the 6- or 7-position diminished the overall affinity for the transporters, though mostly to NET. In vivo locomotor tests were performed in mice for compounds 7a and 8. Compound 8 had no apparent effect on locomotor activity. Compound 7a increased locomotion in a wide dose range, but was much less potent than a reference compound, 2beta-carbomethoxy-3beta-(4-iodo)phenyl-tropane (beta-CIT).

Actin-destabilizing factors disrupt filaments by means of a time reversal of polymerization.

Actin, one of the most highly conserved and abundant eukaryotic proteins, is constantly being polymerized and depolymerized within cells as part of cellular motility, tissue formation and repair, and embryonic development. Many proteins exist that bind to monomeric or filamentous (F) forms of actin to regulate the polymerization state. It has become increasingly apparent that the ability of different proteins to bind to and regulate actin filament dynamics depends on the ability of the filament to exist in altered conformations. Yet, little is known about how these conformational changes occur at the molecular level. We have destabilized F-actin filaments by forming a disulfide that locks the "hydrophobic plug" to the body of the actin subunit or by altering the C terminus of actin with a tetramethylrhodamine label. We also examined F-actin filaments at short times after the initiation of polymerization. In all three cases, a substantial fraction of protomers can be found in a "tilted" state that also is induced by actin depolymerizing factor/cofilin proteins. These observations suggest that F-actin filaments are annealed over time into a stable filament and that actin-depolymerizing proteins can effect a time reversal of polymerization.

Cofilin (ADF) affects lateral contacts in F-actin.

The effect of yeast cofilin on lateral contacts between protomers of yeast and skeletal muscle actin filaments was examined in solution. These contacts are presumably stabilized by the interactions of loop 262-274 of one protomer with two other protomers on the opposite strand in F-actin. Cofilin inhibited several-fold the rate of interstrand disulfide cross-linking between Cys265 and Cys374 in yeast S265C mutant F-actin, but enhanced excimer formation between pyrene probes attached to these cysteine residues. The possibility that these effects are due to a translocation of the C terminus of actin by cofilin was ruled out by measurements of fluorescence resonance energy transfer (FRET) from tryptophan residues and ATP to acceptor probes at Cys374. Such measurements did not reveal cofilin-induced changes in FRET efficiency, suggesting that changes in Cys265-Cys374 cross-linking and excimer formation stem from the perturbation of loop 262-274 by cofilin. Changes in lateral interactions in F-actin were indicated also by the cofilin-induced partial release of rhodamine phalloidin. Disulfide cross-linking of S265C yeast F-actin inhibited strongly and reversibly the release of rhodamine phalloidin by cofilin. Overall, this study provides solution evidence for the weakening of lateral interactions in F-actin by cofilin.

ADF/cofilin use an intrinsic mode of F-actin instability to disrupt actin filaments.

Proteins in the ADF/cofilin (AC) family are essential for rapid rearrangements of cellular actin structures. They have been shown to be active in both the severing and depolymerization of actin filaments in vitro, but the detailed mechanism of action is not known. Under in vitro conditions, subunits in the actin filament can treadmill; with the hydrolysis of ATP driving the addition of subunits at one end of the filament and loss of subunits from the opposite end. We have used electron microscopy and image analysis to show that AC molecules effectively disrupt one of the longitudinal contacts between protomers within one helical strand of F-actin. We show that in the absence of any AC proteins, this same longitudinal contact between actin protomers is disrupted at the depolymerizing (pointed) end of actin filaments but is prominent at the polymerizing (barbed) end. We suggest that AC proteins use an intrinsic mechanism of F-actin's internal instability to depolymerize/sever actin filaments in the cell.

Locking the hydrophobic loop 262-274 to G-actin surface by a disulfide bridge prevents filament formation.

Models of F-actin structure predict the importance of hydrophobic loop 262-274 at the interface of subdomains 3 and 4 to interstrand interactions in filaments. If this premise is correct, prevention of the loop conformational change--its swinging motion--should abort filament formation. To test this hypothesis, we used site-directed mutagenesis to create yeast actin triple mutant (LC)2CA (L180C/L269C/C374A). This mutation places two cysteine residues in positions potentially enabling the locking of loop 262-274 to the monomer surface via disulfide formation. Exposure of the purified mutant to oxidation catalysts resulted in an increased electrophoretic mobility of actin on SDS PAGE and a loss of two cysteines by DTNB titrations, consistent with disulfide formation. The polymerization of un-cross-linked mutant actin by MgCl2 was inhibited strongly but could be restored to wild type actin levels by phalloidin and improved greatly through copolymerization with the wild-type actin. Light scattering measurements revealed nonspecific aggregation of the cross-linked actin under the same conditions. Electron microscopy confirmed the absence of filaments and the presence of amorphous aggregates in the cross-linked actin samples. Reduction of the disulfide bond by DTT restored normal actin polymerization in the presence of MgCl2 and phalloidin. These observations provide strong experimental support for a critical role of the hydrophobic loop 262-274 in the polymerization of actin into filaments.

Insufficiently charged isosteric analogue of spermine: interaction with polyamine uptake, and effect on Caco-2 cell growth.

We characterised a novel, charge-insufficient isosteric analogue of spermine, 11-[(amino)oxy]-4,9-diaza-1-aminoundecane (AOSPM). This analogue was synthesised by displacing aminopropyl group by aminooxyethyl group, the latter having pK(a) of about 5. Charge deficiency of the AOSPM molecule was fixed at a definite atom, while pK(a) of the rest nitrogen was similar to the parent polyamine. AOSPM competed with putrescine, spermidine and spermine for the uptake into the cell, and was accumulated in the cells in high amounts when exogenous polyamine synthesis was impaired. It was not recognised by the cells as growth-promoting polyamine, since it was unable to restore growth arrest due to polyamine deprivation. Like natural spermine, this polyamine analogue prevented oxidative DNA damage. AOSPM could be used not only as a tool to study polyamine homeostasis in the cell, but may have distinct applications either as radiation protector, a stable and non-toxic inhibitor of polyamine uptake or, as an appropriate vector, to enhance the uptake of impermeable compounds into the cell.