DNA Sequence Determinants Controlling Affinity, Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis.

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions

Yeast Irc6p is a novel type of conserved clathrin coat accessory factor related to small G proteins.

Clathrin coat accessory proteins play key roles in transport mediated by clathrin-coated vesicles. Yeast Irc6p and the related mammalian p34 are putative clathrin accessory proteins that interact with clathrin adaptor complexes. We present evidence that Irc6p functions in clathrin-mediated traffic between the trans-Golgi network and endosomes, linking clathrin adaptor complex AP-1 and the Rab GTPase Ypt31p. The crystal structure of the Irc6p N-terminal domain revealed a G-protein fold most related to small G proteins of the Rab and Arf families. However, Irc6p lacks G-protein signature motifs and high-affinity GTP binding. Also, mutant Irc6p lacking candidate GTP-binding residues retained function. Mammalian p34 rescued growth defects in irc6 cells, indicating functional conservation, and modeling predicted a similar N-terminal fold in p34. Irc6p and p34 also contain functionally conserved C-terminal regions. Irc6p/p34-related proteins with the same two-part architecture are encoded in genomes of species as diverse as plants and humans. Together these results define Irc6p/p34 as a novel type of conserved clathrin accessory protein and founding members of a new G protein-like family

Control of DNA minor groove width and Fis protein binding by the purine 2-amino group.

The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis-DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not contacted by the Fis protein, reduce binding affinities up to 2000-fold over A/T-rich sequences. We show here through multiple X-ray structures and binding properties of Fis-DNA complexes containing base analogs that the 2-amino group on guanine is the primary molecular determinant controlling minor groove widths. Molecular dynamics simulations of free-DNA targets with canonical and modified bases further demonstrate that sequence-dependent narrowing of minor groove widths is modulated almost entirely by the presence of purine 2-amino groups. We also provide evidence that protein-mediated phosphate neutralization facilitates minor groove compression and is particularly important for binding to non-optimally shaped DNA duplexes

Influencia de la respiración diafragmática en la motricidad fina

El objetivo de esta investigación fue determinar si la respiración diafragmática influye en la ejecución de una tarea de motricidad fina. Se utilizó un diseño experimental con postprueba únicamente. Los participantes fueron 16 mujeres con edades entre los 18 y 25 años; al grupo experimental se le entrenó en respiración diafragmática y posteriormente se evaluó su motricidad fina con la prueba Purdue Pegboard. Durante la ejecución de la prueba se midió la frecuencia respiratoria por minuto, se registraron 6 clases de errores y los ensayos correctos. Los resultados sugieren que el patrón respiratorio diafragmático influye positivamente sobre la ejecución motriz, lo cual soporta nuevas preguntas de investigación. Se discute el papel del aprendizaje en la modificación del patrón respiratorio y la influencia que pueden tener variables cognoscitivas y ambientales en este tipo de experimentos orientados a fundamentar la práctica clínica.The objective of this research was to establish whether the diaphragmatic breathing affects the performance o fan fine motricity work. An experimental posttest was used. The participants were 16 women between 18 an 25 years. The experimental group was trained in diaphragmatic breathing and further evaluated in fine motricity on the purdue pegmoard. During the execution of the test the breathing frequency per minute was measured and were registered 6 kinds of mistake altogether the correct trials. The results suggest that the diaphragmatic breathing pattern influences on the motricity executions, which supports new investigation questions. The roll of learning on the breathing pattern modification is discussed in this sort of experiments oriented to base the clinical practice

Antimicrobial and Antibiofilm Peptides

The increasing onset of multidrug-resistant bacteria has propelled microbiology research towards antimicrobial peptides as new possible antibiotics from natural sources. Antimicrobial peptides are short peptides endowed with a broad range of activity against both Gram-positive and Gram-negative bacteria and are less prone to trigger resistance. Besides their activity against planktonic bacteria, many antimicrobial peptides also show antibiofilm activity. Biofilms are ubiquitous in nature, having the ability to adhere to virtually any surface, either biotic or abiotic, including medical devices, causing chronic infections that are difficult to eradicate. The biofilm matrix protects bacteria from hostile environments, thus contributing to the bacterial resistance to antimicrobial agents. Biofilms are very difficult to treat, with options restricted to the use of large doses of antibiotics or the removal of the infected device. Antimicrobial peptides could represent good candidates to develop new antibiofilm drugs as they can act at different stages of biofilm formation, on disparate molecular targets and with various mechanisms of action. These include inhibition of biofilm formation and adhesion, downregulation of quorum sensing factors, and disruption of the pre-formed biofilm. This review focuses on the proprieties of antimicrobial and antibiofilm peptides, with a particular emphasis on their mechanism of action, reporting several examples of peptides that over time have been shown to have activity against biofilm

Structural and functional characterization of a novel recombinant antimicrobial peptide from hermetia illucens

Antibiotics are commonly used to treat pathogenic bacteria, but their prolonged use con-tributes to the development and spread of drug-resistant microorganisms raising the challenge to find new alternative drugs. Antimicrobial peptides (AMPs) are small/medium molecules ranging 10–100 residues synthesized by all living organisms and playing important roles in the defense sys-tems. These features, together with the inability of microorganisms to develop resistance against the majority of AMPs, suggest that these molecules might represent effective alternatives to clas-sical antibiotics. Because of their high biodiversity, with over one million described species, and their ability to live in hostile environments, insects represent the largest source of these molecules. However, production of insect AMPs in native forms is challenging. In this work we investigate a defensin-like antimicrobial peptide identified in the Hermetia illucens insect through a combination of transcriptomics and bioinformatics approaches. The C-15867 AMP was produced by recombi-nant DNA technology as a glutathione S-transferase (GST) fusion peptide and purified by affinity chromatography. The free peptide was then obtained by thrombin proteolysis and structurally characterized by mass spectrometry and circular dichroism analyses. The antibacterial activity of the C-15867 peptide was evaluated in vivo by determination of the minimum inhibitory concentration (MIC). Finally, crystal violet assays and SEM analyses suggested disruption of the cell membrane architecture and pore formation with leaking of cytosolic material

Characterization of the proteins involved in the DNA repair mechanism in M. smegmatis.

: Several alkylating agents that either occur in the environment or are self-produced can cause DNA-damaging injuries in bacterial cells. Therefore, all microorganisms have developed repair systems that are able to counteract DNA alkylation damage. The adaptive response to alkylation stress in Escherichia coli consists of the Ada operon, which has been widely described; however, the homologous system in Mycobacterium tuberculosis (MTB) has been shown to have a different genetic organization but it is still largely unknown. In order to describe the defense system of MTB, we first investigated the proteins involved in the repair mechanism in the homologous non-pathogenic mycobacterium M. smegmatis. Ogt, Ada-AlkA and FadE8 proteins were recombinantly produced, purified and characterized. The biological role of Ogt was examined using proteomic experiments to identify its protein partners in vivo under stress conditions. Our results suggested the formation of a functional complex between Ogt and Ada-AlkA, which was confirmed both in silico by docking calculations and by gel filtration chromatography. We propose that this stable association allows the complex to fulfill the biological roles exerted by Ada in the homologous E. coli system. Finally, FadE8 was demonstrated to be structurally and functionally related to its E. coli homologous, Aid

Redes corporativas de voz VS telefonía IP: modelo de análisis y diseño

La integración de la voz y los datos en una isma red no es una idea novedosa pues en las últimas décadas han surgido soluciones de distintos fabricantes y operadores, permitiendo la utilización de las redes WAN de datos de las empresas (típicamente conexiones punto apunto y frame relay) para la transmisión del tráfico de voz. Sin embargo, la falta de estándares, así como el largo plazo de amortización de este tipo de soluciones no han permitido una amplia implantación de éstas. En este artículo, se realiza un análisis de la red de datos y de telefonía de na empresa nacional de Telecomunicaciones para determinar los requerimientos en la convergencia de telefonía IP como alternativa frente a la red d voz tradicional. Para ello se desarrolla un modelo evaluativo de tres etapas: Dimensionamiento y diseño de la red de Telefonía en forma parametrizada, Análisis de Telefonía IP considerando diferenes plataforma, con la evaluación económica de las alternativas, y por último: Determinación de la viabilidad operativa del área de Telefonía privada.as well as VoIP service

The antimicrobial peptide Temporin L impairs E. coli cell division by interacting with FtsZ and the divisome complex

Background: The comprehension of the mechanism of action of antimicrobial peptides is fundamental for the design of new antibiotics. Studies performed looking at the interaction of peptides with bacterial cells offer a faithful picture of what really happens in nature. Methods: In this work we focused on the interaction of the peptide Temporin L with E. coli cells, using a variety of biochemical and biophysical techniques that include: functional proteomics, docking, optical microscopy, TEM, DLS, SANS, fluorescence. Results: We identified bacterial proteins specifically interacting with the peptides that belong to the divisome machinery; our data suggest that the GTPase FtsZ is the specific peptide target. Docking experiments supported the FtsZ-TL interaction; binding and enzymatic assays using recombinant FtsZ confirmed this hypothesis and revealed a competitive inhibition mechanism. Optical microscopy and TEM measurements demonstrated that, upon incubation with the peptide, bacterial cells are unable to divide forming long necklace-like cell filaments. Dynamic light scattering studies and Small Angle Neutron Scattering experiments performed on treated and untreated bacterial cells, indicated a change at the nanoscale level of the bacterial membrane. Conclusions: The peptide temporin L acts by a non-membrane-lytic mechanism of action, inhibiting the divisome machinery. General significance: Identification of targets of antimicrobial peptides is pivotal to the tailored design of new antimicrobials