Force Spectroscopy with Dual-Trap Optical Tweezers: Molecular Stiffness Measurements and Coupled Fluctuations Analysis

ABSTRACT Dual-trap optical tweezers are often used in high-resolution measurements in single-molecule biophysics. Such measurements can be hindered by the presence of extraneous noise sources, the most prominent of which is the coupling of fluctuations along different spatial directions, which may affect any optical tweezers setup. In this article, we analyze, both from the theoretical and the experimental points of view, the most common source for these couplings in dual-trap optical-tweezers setups: the misalignment of traps and tether. We give criteria to distinguish different kinds of misalignment, to estimate their quantitative relevance and to include them in the data analysis. The experimental data is obtained in a, to our knowledge, novel dual-trap optical-tweezers setup that directly measures forces. In the case in which misalignment is negligible, we provide a method to measure the stiffness of traps and tether based on variance analysis. This method can be seen as a calibration technique valid beyond the linear trap region. Our analysis is then employed to measure the persistence length of dsDNA tethers of three different lengths spanning two orders of magnitude. The effective persistence length of such tethers is shown to decrease with the contour length, in accordance with previous studies.

Cluster glasses of ultrasoft particles

We present molecular dynamics (MD) simulations results for dense fluids of ultrasoft, fully penetrable particles. These are a binary mixture and a polydisperse system of particles interacting via the generalized exponential model, which is known to yield cluster crystal phases for the corresponding monodisperse systems. Because of the dispersity in the particle size, the systems investigated in this work do not crystallize and form disordered cluster phases. The clusteringtransition appears as a smooth crossover to a regime in which particles are mostly located in clusters, isolated particles being infrequent. The analysis of the internal cluster structure reveals microsegregation of the big and small particles, with a strong homo-coordination in the binary mixture. Upon further lowering the temperature below the clusteringtransition, the motion of the clusters" centers-of-mass slows down dramatically, giving way to a cluster glass transition. In the cluster glass, the diffusivities remain finite and display an activated temperature dependence, indicating that relaxation in the cluster glass occurs via particle hopping in a nearly arrested matrix of clusters. Finally we discuss the influence of the microscopic dynamics on the transport properties by comparing the MD results with Monte Carlo simulations.

On the role of G protein-coupled receptors oligomerization

The existence of a supramolecular organization of the G protein-coupled receptor (GPCR) is now being widely accepted by the scientific community. Indeed, GPCR oligomers may enhance the diversity and performance by which extracellular signals are transferred to the G proteins in the process of receptor transduction, although the mechanism that underlies this phenomenon still remains unsolved. Recently, it has been proposed that a trans-conformational switching model could be the mechanism allowing direct inhibition/activation of receptor activation/inhibition, respectively. Thus, heterotropic receptor-receptor allosteric regulations are behind the GPCR oligomeric function. In this paper we want to revise how GPCR oligomerization impinges on several important receptor functions like biosynthesis, plasma membrane diffusion or velocity, pharmacology and signaling. In particular, the rationale of receptor oligomerization might lie in the need of sensing complex whole cell extracellular signals and translating them into a simple computational model.

Broken colinearity of the amphioxus Hox cluster.

Background In most eumetazoans studied so far, Hox genes determine the identity of structures along the main body axis. They are usually linked in genomic clusters and, in the case of the vertebrate embryo, are expressed with spatial and temporal colinearity. Outside vertebrates, temporal colinearity has been reported in the cephalochordate amphioxus (the least derived living relative of the chordate ancestor) but only for anterior and central genes, namely Hox1 to Hox4 and Hox6. However, most of the Hox gene expression patterns in amphioxus have not been reported. To gain global insights into the evolution of Hox clusters in chordates, we investigated a more extended expression profile of amphioxus Hox genes. Results Here we report an extended expression profile of the European amphioxus Branchiostoma lanceolatum Hox genes and describe that all Hox genes, except Hox13, are expressed during development. Interestingly, we report the breaking of both spatial and temporal colinearity for at least Hox6 and Hox14, which thus have escaped from the classical Hox code concept. We show a previously unidentified Hox6 expression pattern and a faint expression for posterior Hox genes in structures such as the posterior mesoderm, notochord, and hindgut. Unexpectedly, we found that amphioxus Hox14 had the most divergent expression pattern. This gene is expressed in the anterior cerebral vesicle and pharyngeal endoderm. Amphioxus Hox14 expression represents the first report of Hox gene expression in the most anterior part of the central nervous system. Nevertheless, despite these divergent expression patterns, amphioxus Hox6 and Hox14 seem to be still regulated by retinoic acid. Conclusions Escape from colinearity by Hox genes is not unusual in either vertebrates or amphioxus and we suggest that those genes escaping from it are probably associated with the patterning of lineage-specific morphological traits, requiring the loss of those developmental constraints that kept them colinear.

The Plesiomonas shigelloides wbO1 gene cluster and the role of O1-antigen LPS in pathogenicity

The Plesiomonas shigelloides 302-73 strain (serotype O1) wb gene cluster encodes 15 proteins which are consistent with the chemical structure of the O1-antigen lypopolysaccharide (LPS) previously described for this strain. The P. shigelloides O1-antigen LPS export uses the Wzy-dependent pathway as correspond to heteropolysaccharides structures. By the isolation of two mutants lacking this O1-antigen LPS, we could establish that the presence of the O1-antigen LPS is crucial for to survive in serum mainly to become resistant to complement. Also, it is an important factor in the bacterial adhesion and invasion to some eukaryotic cells, and in the ability to form biofilms. This is the first report on the genetics from a P. shigelloides O-antigen LPS cluster (wb) not shared by Shigella like P. shigelloides O17, the only one reported until now.

Rapid analysis of multiclass antibiotic residues and some of their metabolites in hospital, urban wastewater and river water by ultra-highperformance liquid chromatography coupled to quadrupole-linear ion trap tandem mass spectrometry

The present work describes the development of a fast and robust analytical method for the determination of 53 antibiotic residues, covering various chemical groups and some of their metabolites, in environmental matrices that are considered important sources of antibiotic pollution, namely hospital and urban wastewaters, as well as in river waters. The method is based on automated off-line solid phase extraction (SPE) followed by ultra-high-performance liquid chromatography coupled to quadrupole linear ion trap tandem mass spectrometry (UHPLC–QqLIT). For unequivocal identification and confirmation, and in order to fulfill EU guidelines, two selected reaction monitoring (SRM) transitions per compound are monitored (the most intense one is used for quantification and the second one for confirmation). Quantification of target antibiotics is performed by the internal standard approach, using one isotopically labeled compound for each chemical group, in order to correct matrix effects. The main advantages of the method are automation and speed-up of sample preparation, by the reduction of extraction volumes for all matrices, the fast separation of a wide spectrum of antibiotics by using ultra-high-performance liquid chromatography, its sensitivity (limits of detection in the low ng/L range) and selectivity (due to the use of tandem mass spectrometry) The inclusion of β-lactam antibiotics (penicillins and cephalosporins), which are compounds difficult to analyze in multi-residue methods due to their instability in water matrices, and some antibiotics metabolites are other important benefits of the method developed. As part of the validation procedure, the method developed was applied to the analysis of antibiotics residues in hospital, urban influent and effluent wastewaters as well as in river water samples

Subtypes of adolescents with substance use disorders and psychiatric comorbidity using cluster and discriminant analysis of MMPI-A profiles

The main aim of this study was to replicate and extend previous results on subtypes of adolescents with substance use disorders (SUD), according to their Minnesota Multiphasic Personality Inventory for adolescents (MMPI-A) profiles. Sixty patients with SUD and psychiatric comorbidity (41.7% male, mean age = 15.9 years old) completed the MMPI-A, the Teen Addiction Severity Index (T-ASI), the Child Behaviour Checklist (CBCL), and were interviewed in order to determine DSMIV diagnoses and level of substance use. Mean MMPI-A personality profile showed moderate peaks in Psychopathic Deviate, Depression and Hysteria scales. Hierarchical cluster analysis revealed four profiles (acting-out, 35% of the sample; disorganized-conflictive, 15%; normative-impulsive, 15%; and deceptive-concealed, 35%). External correlates were found between cluster 1, CBCL externalizing symptoms at a clinical level and conduct disorders, and between cluster 2 and mixed CBCL internalized/externalized symptoms at a clinical level. Discriminant analysis showed that Depression, Psychopathic Deviate and Psychasthenia MMPI-A scales correctly classified 90% of the patients into the clusters obtained.

Identification of new transformation products during enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass spectrometer LTQ-Orbitrap

This work describes the formation of transformation products (TPs) by the enzymatic degradation at laboratory scale of two highly consumed antibiotics: tetracycline (Tc) and erythromycin (ERY). The analysis of the samples was carried out by a fast and simple method based on the novel configuration of the on-line turbulent flow system coupled to a hybrid linear ion trap – high resolution mass spectrometer. The method was optimized and validated for the complete analysis of ERY, Tc and their transformation products within 10 min without any other sample manipulation. Furthermore, the applicability of the on-line procedure was evaluated for 25 additional antibiotics, covering a wide range of chemical classes in different environmental waters with satisfactory quality parameters. Degradation rates obtained for Tc by laccase enzyme and ERY by EreB esterase enzyme without the presence of mediators were ∼78% and ∼50%, respectively. Concerning the identification of TPs, three suspected compounds for Tc and five of ERY have been proposed. In the case of Tc, the tentative molecular formulas with errors mass within 2 ppm have been based on the hypothesis of dehydroxylation, (bi)demethylation and oxidation of the rings A and C as major reactions. In contrast, the major TP detected for ERY has been identified as the “dehydration ERY-A”, with the same molecular formula of its parent compound. In addition, the evaluation of the antibiotic activity of the samples along the enzymatic treatments showed a decrease around 100% in both cases

Identification of metabolites and thermal transformation products of quinolones in raw cow milk by liquid chromatography coupled to high resolution mass spectrometry

The presence of residues of antibiotics, metabolites, and thermal transformation products (TPs), produced during thermal treatment to eliminate pathogenic microorganisms in milk, could represent a risk for people. Cow"s milk samples spiked with enrofloxacin (ENR), ciprofloxacin (CIP), difloxacin (DIF), and sarafloxacin (SAR) and milk samples from cows medicated with ENR were submitted to several thermal treatments. The milk samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) to find and identify TPs and metabolites. In this work, 27 TPs of 4 quinolones and 24 metabolites of ENR were found. Some of these compounds had been reported previously, but others were characterized for the first time, including lactose-conjugated CIP, the formamidation reaction for CIP and SAR, and hydroxylation or ketone formation to produce three different isomers for all quinolones studied.

A Forwarding Cooperation Protocol for Plain and Cluster-based Ad Hoc Networks

Peer-reviewed

Emergence and development of a financial cluster: the evolution of Andorra’s banking deposits in the long-term, 1931-2007

This paper explores the origins of Andorra’s financial cluster. It shows that the free movement of currency, the protection of infant industry, and geographical concentration lie at the foundation of the cluster’s competitive advantage. Drawing on a new set of data, the paper also provides for the first time an estimate of the total deposits held by Andorra’s banks between 1931 and 2007. Based on this new information, the paper reaches the conclusion that the development of the cluster went through four distinct phases in which large companies, acting as leaders, played an important role in enhancing the cluster’s business capabilities.

Identification of metabolites and thermal transformation products of quinolones in raw cow milk by liquid chromatography coupled to high resolution mass spectrometry.

The presence of residues of antibiotics, metabolites, and thermal transformation products (TPs), produced during thermal treatment to eliminate pathogenic microorganisms in milk, could represent a risk for people. Cow"s milk samples spiked with enrofloxacin (ENR), ciprofloxacin (CIP), difloxacin (DIF), and sarafloxacin (SAR) and milk samples from cows medicated with ENR were submitted to several thermal treatments. The milk samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) to find and identify TPs and metabolites. In this work, 27 TPs of 4 quinolones and 24 metabolites of ENR were found. Some of these compounds had been reported previously, but others were characterized for the first time, including lactose-conjugated CIP, the formamidation reaction for CIP and SAR, and hydroxylation or ketone formation to produce three different isomers for all quinolones studied.

Cómo aplicar un cluster jerárquico en SPSS

L'anàlisi de conglomerats o cluster és una tècnica multivariant que busca agrupar elements o variables tractant d'aconseguir la màxima homogeneïtat en cada grup i la major diferència entre ells, mitjançant una estructura jerarquitzada per poder decidir quin nivell jeràrquic és el més apropiat per establir la classificació. El programa SPSS disposa de tres tipus d'anàlisi de conglomerats: l'anàlisi de conglomerats jeràrquic, bietàpic i de K mitjanes. Aplicarem el mètode jeràrquic com el més idoni per determinar el nombre òptim de conglomerats existent en les dades i el contingut dels mateixos per al nostre cas pràctic.

A Fair and Secure Cluster Formation Process for Ad Hoc Networks

An efficient approach for organizing large ad hoc networks is to divide the nodesinto multiple clusters and designate, for each cluster, a clusterhead which is responsible forholding intercluster control information. The role of a clusterhead entails rights and duties.On the one hand, it has a dominant position in front of the others because it manages theconnectivity and has access to other node¿s sensitive information. But on the other hand, theclusterhead role also has some associated costs. Hence, in order to prevent malicious nodesfrom taking control of the group in a fraudulent way and avoid selfish attacks from suitablenodes, the clusterhead needs to be elected in a secure way. In this paper we present a novelsolution that guarantees the clusterhead is elected in a cheat-proof manner.