Enzibióticos bactericidas mejorados frente a neumococo y otras bacterias

La presente invención se encuadra dentro del campo de la biotecnología. En la presente invención se presenta una secuencia polipeptídica derivada del módulo de unión a pared del enzima lítica del fago Cp7 (Cpl-7), que permite la construcción de nuevas enzimas líticas con actividad bactericida mejorada y amplio espectro. Igualmente, en esta invención se incluyen enzimas quiméricas que contienen dicho módulo de unión a pared mejorado y se dan ejemplos de su actividad frente a especies Gram-positivas y Gram-negativas.Peer reviewedConsejo Superior de Investigaciones Científicas, CIBER Enfermedades Respiratorias (CIBERES)A2 Solicitud de patente sin informe sobre el estado de la técnic

Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB

Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening?closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA

Real-time Relaxation and Kinetics in Hot Scalar QED: Landau Damping

The real time evolution of field condensates with soft length scales k^{-1}>(eT)^{-1} is solved in hot scalar electrodynamics, with a view towards understanding relaxational phenomena in the QGP and the electroweak plasma. We find that transverse gauge invariant non-equilibrium expectation values of fields relax via {\em power laws} to asymptotic amplitudes that are determined by the quasiparticle poles. The long time relaxational dynamics and relevant time scales are determined by the behaviour of the retarded self-energy not at the small frequencies, but at the Landau damping thresholds. This explains the presence of power laws and not of exponential decay. Furthermore, we derive the influence functional, the Langevin equation and the fluctuation-dissipation theorem for the soft modes, identifying the correlation functions that emerge in the classical limit. We show that a Markovian approximation fails to describe the dynamics {\em both} at short and long times. We also introduce a novel kinetic approach that goes beyond the standard Boltzmann equation and incorporates off-shell processes and find that the distribution function for soft quasiparticles relaxes with a power law through Landau damping. We also find an unusual dressing dynamics of bare particles and anomalous (logarithmic) relaxation of hard quasiparticles.Comment: 41 pages, 5 figures, uses revtex, replaced with version to appear in Phys. Rev.

Chapter 1

Experimenting is fundamental to the training process of all scientists and engineers. While experiments have been traditionally done inside laboratories, the emergence of Information and Communication Technologies added two alter-natives accessible anytime, anywhere. These two alternatives are known as virtual and remote labs, and are sometimes indistinguishably referred as online labs. Sim-ilarly to other instructional technologies, virtual and remote labs require some ef-fort from teachers in integrating them into curricula, taking into consideration sev-eral factors that affect their adoption (i.e. cost) and their educational effectiveness (i.e. benefit). This chapter analyses these two dimensions and sustains the case where only through international cooperation it is possible to serve the large num-ber of teachers and students involved in engineering education. It presents an ex-ample in the area of Electrical and Electronics Engineering, based on a remote lab named Virtual Instruments System in Reality, and it then describes how a number of European and Latin-American institutions have been cooperating under the scope of an Erasmus+ project2, for spreading its use in Brazil and Argentina.info:eu-repo/semantics/publishedVersio

Erratum: Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017

Interpretation: By quantifying levels and trends in exposures to risk factors and the resulting disease burden, this assessment offers insight into where past policy and programme efforts might have been successful and highlights current priorities for public health action. Decreases in behavioural, environmental, and occupational risks have largely offset the effects of population growth and ageing, in relation to trends in absolute burden. Conversely, the combination of increasing metabolic risks and population ageing will probably continue to drive the increasing trends in non-communicable diseases at the global level, which presents both a public health challenge and opportunity. We see considerable spatiotemporal heterogeneity in levels of risk exposure and risk-attributable burden. Although levels of development underlie some of this heterogeneity, O/E ratios show risks for which countries are overperforming or underperforming relative to their level of development. As such, these ratios provide a benchmarking tool to help to focus local decision making. Our findings reinforce the importance of both risk exposure monitoring and epidemiological research to assess causal connections between risks and health outcomes, and they highlight the usefulness of the GBD study in synthesising data to draw comprehensive and robust conclusions that help to inform good policy and strategic health planning

Ten millennia of hepatitis B virus evolution

Hepatitis B virus (HBV) has been infecting humans for millennia and remains a global health problem, but its past diversity and dispersal routes are largely unknown. We generated HBV genomic data from 137 Eurasians and Native Americans dated between ~10,500 and ~400 years ago. We date the most recent common ancestor of all HBV lineages to between ~20,000 and 12,000 years ago, with the virus present in European and South American hunter-gatherers during the early Holocene. After the European Neolithic transition, Mesolithic HBV strains were replaced by a lineage likely disseminated by early farmers that prevailed throughout western Eurasia for ~4000 years, declining around the end of the 2nd millennium BCE. The only remnant of this prehistoric HBV diversity is the rare genotype G, which appears to have reemerged during the HIV pandemic

Functional dissection of the conjugative coupling protein TrwB

Tesis Doctoral presentada en el Departamento de Biología Molecular de la Facultad de Medicina de la Universidad de Cantabria para la obtención del grado de Doctor en Ciencias biológicas.[ES]: Los sistemas de secreción tipo IV (T4SS) son una gran familia de sistemas de secreción que se encuentra ampliamente distribuidos entre las bacterias, y que presentan roles biológicos muy diferentes a pesar de la gran homología que que comparten entre sí, siendo imprescindibles para la conjugación o para la virulencia de aquellas cepas que los codifican. En ete trabajo, el estudio de dos T4SS implicados en transferencia de DNA y virulencia, nos ha permitido manipular sus componentes, consiguiendo movilizar DNA desde Bartonella hacia células humanas. Esto podría sentar las bases para una herramienta de transferencia e integración sitio-específica en el genoma humano de DNA de cualquier origen y longitud. Esta sería una herramienta de gran valor en el campo de la terapia génica. Para ello, primero realizamos un estudio comparativo de los T4SS Trw de R388 y Bartonella, estableciendo los componentes que pueden ser intercambiados, estructural y funcionalmente. Además, hemos desarrollado un análisis mutacional de la proteína acopladora de R388 (TrwB). Hemos creado mutantes que interaccionan más fuertemente con el T4SS y analizado el efecto de estas mutaciones junto con otras presentes en otras regiones de la proteína con distintas funciones. Para ver el efecto de las mutaciones en la funcionalidad de TrwB, además hemos desarrollado un sistema donde las condiciones limitantes de TrwB descubren los efectos de estas mutaciones, ya que en el sistema stándar, la elevada cantidad de TrwB a menudo impide ver los efectos de dichas mutaciones. Por último, hemos conseguido movilizar DNA de Bartonella a células humanas, estudiando las implicaciones en este procesos de los distintos T4SS presentes en la bacteria.[EN]: The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in TrwB cytoplasmic domain on the basis of the crystal structure of TrwBΔN70, targeting the NTP-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, being also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel are important for TrwB function, and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained in the transmembranal helices; specifically, one Proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.El presente trabajo ha sido realizado gracias a las becas predoctorales concedidas por la Fundación Marqués de Valdecilla y la Universidad de Cantabria. Durante este periodo se ha realizado una estancia de 3 meses en el laboratorio del Dr. Christoph Dehio (Biozentrum, Basel, Suiza) gracias a una beca EMBO.This work was supported by grants BIO2008-00133 from the Spanish Ministry of Science and Innovation and API 07/01 from the Fundación Marqués de Valdecilla to ML; and grants LSHM-CT-2005_019023 from the European Commission, BFU2008-00995/BMC from the Spanish Ministry of Science and Innovation, and RETICS research network RD06/0008/1012, Instituto de Salud Carlos III, Spanish Ministry of Health, to FC. HP and DL were the recipients of predoctoral fellowships from the University of Cantabria and JAE-predoc (CSIC), respectively.Peer Reviewe

Structural independence of conjugative coupling protein TrwB from its Type IV secretion machinery

The stability of components of multiprotein complexes often relies on the presence of the functional complex. To assess structural dependence among the components of the R388 Type IV secretion system (T4SS), the steady-state level of several Trw proteins was determined in the absence of other Trw components. While several Trw proteins were affected by the lack of others, we found that the coupling protein TrwB is not affected by the absence of other T4SS components, nor did its absence alter significantly the levels of integral components of the complex, underscoring the independent role of the coupling protein on the T4SS architecture. The cytoplasmic ATPases TrwK (VirB4) and TrwD (VirB11) were affected by the absence of several core complex components, while the pilus component TrwJ (VirB5) required the presence of all other Trw proteins (except for TrwB) to be detectable. Overall, the results delineate a possible assembly pathway for the T4SS of R388. We have also tested structural complementation of TrwD (VirB11) and TrwJ (VirB5) by their homologues in the highly related Trw system of Bartonella tribocorum (Bt). The results reveal a correlation with the functional complementation data previously reported. © 2013 Elsevier Inc.This work was supported by Grant BIO2010-11623-E to ML. Work in FC lab was supported by Spanish Ministry of Education (BFU2011-26608), and European VII Framework Program Grants no. 248919/FP7-ICT-2009-4 and 282004/FP7-HEALTH.2011.2.3.1-2. DL was a recipient of a JAE-PRE predoctoral fellowship from the CSIC (Spain).Peer Reviewe

Improved lethal effect of a phage pneumococcal lysozyme by changing the net charge

3 p.-1 tab.Bacteriophage lytic murein-hydrolases have been proposed as an efficient way to fight bacterial infections. However, the use of these enzymes is normally restricted to Gram-positive bacteria since the outer membrane of the Gram-negative bacteria hampers the access of the protein to its peptidoglycan substrate. All the murein hydrolases reported in the pneumococcal system, both from host or phage origin, depend on the aminoalcohol choline to be fully active. There is only a unique exception to this rule, the Cpl-7 lysozyme, encoded by the lytic pneumococcal phage Cp-7, which instead of the common cell wall binding module recognizing choline, harbors a completely different cell wall module. Studies developed in our laboratories have allowed the improvement of Cpl-7 antibacterial activity by reducing the net charge from -28.8 to -10.85 introducing 15 amino acid substitutions in the cell wall binding module. This modified enzyme, Cpl-7S, was capable to lyse not only the pneumococcal strains tested, including the antibiotic-multiresistant D48 strain, but also a variety of Gram-positive bacteria. We have also designed a standard protocol to destabilize the outer membrane of Gram-negative bacteria and turning them susceptible to the action of Cpl-7S. In this communication, we will present the results of Cpl-7S, which constitutes a promising alternative to kill not only pneumococcal cells but also other important pathogens like Streptococcus pyogenes, and even Gram-negative bacteria after sensitization of the outer membrane. This improved lysozyme, Cpl-7S, has also been tested in zebra fish embryos and conclusions drawn from this new animal model will be discussed.Peer reviewe

Transfer of R388 derivatives by a pathogenesis-associated type IV secretion system into both bacteria and human cells

Trabajo presentado al: "Workshops Current Trends in Biomedicine" organizado por la Universidad Internacional de Andalucía y celebrado en Baeza (España) del 24 al 26 de octubre de 2011.Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A ΔvirB mutant strain was transfer deficient, while a ΔtrwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs.This work was supported by grants BIO2008-00133 and BIO2007-63656 from the Spanish Ministry of Science and Innovation to M.L. and F.J.S., respectively, and by grant 31003A-109925 from the Swiss National Science Foundation to C.D.Peer reviewe