Investigation of the functional roles of host cell proteins involved in Coronavirus infection using highly specific and scalable RNA interference (RNAi) approach

Since its identification in the 1990s, the RNA interference (RNAi) pathway has proven extremely useful in elucidating the function of proteins in the context of cells and even whole organisms. In particular, this sequence-specific and powerful loss-of-function approach has greatly simplified the study of the role of host cell factors implicated in the life cycle of viruses. Here, we detail the RNAi method we have developed and used to specifically knock down the expression of ezrin, an actin binding protein that was identified by yeast two-hybrid screening to interact with the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) spike (S) protein. This method was used to study the role of ezrin, specifically during the entry stage of SARS-CoV infection

Designing a videoconference system for active networks

Active networks are receiving increasing attention due to their promises of great flexibility in tailoring services to applications. This capability stems from the exploitation of network devices whose behavior can be changed dynamically by applications, possibly using technologies and architectures originally conceived for mobile code systems. Notwithstanding the promises of active networks, real-world applications that clearly benefit by them are still missing. In this work we describe the design of a videoconference system conceived expressly for operation over active networks. The goal of this activity is to pinpoint the benefits that mobile code and active networks bring in this application domain and to provide insights for the exploitation of these concepts in other application domain

Kinesin Is an Evolutionarily Fine-Tuned Molecular Ratchet-and-Pawl Device of Decisively Locked Direction

Conventional kinesin is a dimeric motor protein that transports membranous organelles toward the plus-end of microtubules (MTs). Individual kinesin dimers show steadfast directionality and hundreds of consecutive steps, yetthe detailed physical mechanism remains unclear. Here we compute free energies for the entire dimer-MT system for all possible interacting configurations by taking full account of molecular details. Employing merely first principles and several measured binding and barrier energies, the system-level analysis reveals insurmountable energy gaps between configurations, asymmetric ground state caused by mechanically lifted configurational degeneracy, and forbidden transitions ensuring coordination between both motor domains for alternating catalysis. This wealth of physical effects converts a kinesin dimer into a molecular ratchet-and-pawl device, which determinedly locks the dimer's movement into the MT plus-end and ensures consecutive steps in hand-over-hand gait.Under a certain range of extreme loads, however, the ratchet-and-pawl device becomes defective but not entirely abolished to allow consecutive back-steps. This study yielded quantitative evidence that kinesin's multiple molecular properties have been evolutionarily adapted to fine-tune the ratchet-and-pawl device so as to ensure the motor's distinguished performance.Comment: 10 printed page

Sensory neuron–derived NaV1.7 contributes to dorsal horn neuron excitability

Expression of the voltage-gated sodium channel NaV1.7 in sensory neurons is required for pain sensation. We examined the role of NaV1.7 in the dorsal horn of the spinal cord using an epitope-tagged NaV1.7 knock-in mouse. Immuno–electron microscopy showed the presence of NaV1.7 in dendrites of superficial dorsal horn neurons, despite the absence of mRNA. Rhizotomy of L5 afferent nerves lowered the levels of NaV1.7 in the dorsal horn. Peripheral nervous system–specific NaV1.7 null mutant mice showed central deficits, with lamina II dorsal horn tonic firing neurons more than halved and single spiking neurons more than doubled. NaV1.7 blocker PF05089771 diminished excitability in dorsal horn neurons but had no effect on NaV1.7 null mutant mice. These data demonstrate an unsuspected functional role of primary afferent neuron-generated NaV1.7 in dorsal horn neurons and an expression pattern that would not be predicted by transcriptomic analysis

Membrane electrode assemblies with low noble metal loadings for hydrogen production from solid polymer electrolyte water electrolysis

High performance membrane electrode assemblies (MEAs) with low noble metal loadings (NMLs) were developed for solid polymer electrolyte (SPE) water electrolysis. The electro- chemical and physical characterization of the MEAs was performed by IeV curves, elec- trochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). Even though the total NML was lowered to 0.38 mg cm-2, it still reached a high performance of 1.633 V at 2 A cm-2 and 80 o C, with IrO2 as anode catalyst. The influences of the ionomer content in the anode catalyst layer (CL) and the cell temperature were investigated with the purpose of optimizing the performance. SEM and EIS measurements revealed that the MEA with low NML has very thin porous cathode and anode CLs that get intimate contact with the electrolyte membrane, which makes a reduced mass transport limitation and lower ohmic resistance of the MEA. A short-term water electrolysis operation at 1 A cm-2 showed that the MEA has good stability: the cell voltage maintained at ~1.60 V without distinct degradation after 122 h operation at 80 o C and atmospheric pressure.Web of Scienc

Membrane electrode assemblies with low noble metal loadings for hydrogen production from solid polymer electrolyte water electrolysis

High performance membrane electrode assemblies (MEAs) with low noble metal loadings (NMLs) were developed for solid polymer electrolyte (SPE) water electrolysis. The electro- chemical and physical characterization of the MEAs was performed by IeV curves, elec- trochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). Even though the total NML was lowered to 0.38 mg cm-2, it still reached a high performance of 1.633 V at 2 A cm-2 and 80 o C, with IrO2 as anode catalyst. The influences of the ionomer content in the anode catalyst layer (CL) and the cell temperature were investigated with the purpose of optimizing the performance. SEM and EIS measurements revealed that the MEA with low NML has very thin porous cathode and anode CLs that get intimate contact with the electrolyte membrane, which makes a reduced mass transport limitation and lower ohmic resistance of the MEA. A short-term water electrolysis operation at 1 A cm-2 showed that the MEA has good stability: the cell voltage maintained at ~1.60 V without distinct degradation after 122 h operation at 80 o C and atmospheric pressure.Web of Scienc

Disseny d'un sistema d'indicadors ambientals en camins i diagnosi socioambiental del Camí de Cavalls : sector sud-oest

Menorca va ser declarada Reserva de la Biosfera per la UNESCO l'any 1993. Entre d'altres, un dels aspectes més emblemàtics de l'Illa és el Camí de Cavalls. Preservar la diversitat que aquest ofereix comporta una gestió complexa. Per això, és necessària per una millor gestió ambiental la realització prèvia d'una diagnosi per determinar en quina situació es troba actualment el Camí, i així poder optimitzar els mecanismes de gestió de l'espai. Aquesta diagnosi té com a objectius desenvolupar un sistema d'indicadors ambientals i socioeconòmics i validar-los per avaluar els canvis que es poden donar en el litoral de Menorca en els propers anys en relació a aquest espai. Dins d'aquest marc de treball, el sistema d'indicadors dissenyat a nivell teòric ha estat validat a través d'un exhaustiu treball de camp. Amb aquest objectiu s'ha volgut crear un disseny eficient, de forma que qualsevol persona que vulgui aplicar-lo al Camí de Cavalls o en altres tipologies de camí pugui fer-ho d'una manera correcte.Nota: Aquest document conté originàriament altre material i/o programari només consultable a la Biblioteca de Ciència i Tecnologia.Aquest projecte està relacionat amb el titulat "Disseny d'un sistema d'indicadors en camins i diagnosi socioambiental del Camí de Cavalls de Menorca 2008: sector nord-est", redactat per Dídac Masana, Marta Morros i Esther Sanyé

Ezrin interacts with the SARS coronavirus spike protein and restrains infection at the entry stage

© 2012 Millet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process. Methodology/Principal Findings: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S pseudotyped particles and potentiated S-dependent membrane fusion. Conclusions/Significance: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.This work was supported by the Research Grant Council of Hong Kong (RGC#760208)and the RESPARI project of the International Network of Pasteur Institutes

Community health workers improve contact tracing among immigrants with tuberculosis in Barcelona

Background: The important increase in immigration during recent years has changed the epidemiology and control strategies for tuberculosis (TB) in many places. This study evaluates the effectiveness of intervention with community health workers (CHW) to improve contact tracing among immigrants. Methods: The study included all TB cases detected by the Barcelona TB Program from 2000 to 2005 and compared a period without CHW intervention (2000-2002) to a period with CHW intervention (2003-2005). The influence on contact tracing of sex, age, hospital of diagnosis, district of residence, birthplace, HIV, homeless and CHW intervention was analysed by logistic regression. Odds ratio (OR) and 95 % confidence intervals (CI) were calculated. Results: 960 foreign born TB cases were detected, 388 in the intervention period. Contact tracing was performed on 65,7 % of 201 smear-positive cases during the pre-intervention period compared to 81.6 % of 152 smear-positive TB cases during the intervention period (p < 0.001). Risk factors associated with incomplete contact tracing of smear-positive index cases included being diagnosed in two hospitals without contact tracing TB unit (OR = 3.5; CI:1.4-8.9) and (OR = 4.6; CI:1.6-13.5) respectively, birth place in India-Pakistan (OR = 4.4; CI:1.9-10.3) or North Africa (OR = 4.3; CI:1.8-10.5), having an unknown residence (OR = 5.4; CI:1.6-18.0), being HIV-infected (OR = 6.1; CI:2.5-14.8