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"Nava, A"
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The PDZ-Binding Motif of Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Is a Determinant of Viral Pathogenesis
A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation.
Journal Article
Avatars and virtual agents – relationship interfaces for the elderly
In the Digital Era, the authors witness a change in the relationship between the patient and the care-giver or Health Maintenance Organization's providing the health services. Another fact is the use of various technologies to increase the effectiveness and quality of health services across all primary and secondary users. These technologies range from telemedicine systems, decision making tools, online and self-services applications and virtual agents; all providing information and assistance. The common thread between all these digital implementations, is they all require human machine interfaces. These interfaces must be interactive, user friendly and inviting, to create user involvement and cooperation incentives. The challenge is to design interfaces which will best fit the target users and enable smooth interaction especially, for the elderly users. Avatars and Virtual Agents are one of the interfaces used for both home care monitoring and companionship. They are also inherently multimodal in nature and allow an intimate relation between the elderly users and the Avatar. This study discusses the need and nature of these relationship models, the challenges of designing for the elderly. The study proposes key features for the design and evaluation in the area of assistive applications using Avatar and Virtual agents for the elderly users.
Journal Article
Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression
The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels.
Journal Article
Sixty years of global progress in managed aquifer recharge
The last 60 years has seen unprecedented groundwater extraction and overdraft as well as development of new technologies for water treatment that together drive the advance in intentional groundwater replenishment known as managed aquifer recharge (MAR). This paper is the first known attempt to quantify the volume of MAR at global scale, and to illustrate the advancement of all the major types of MAR and relate these to research and regulatory advancements. Faced with changing climate and rising intensity of climate extremes, MAR is an increasingly important water management strategy, alongside demand management, to maintain, enhance and secure stressed groundwater systems and to protect and improve water quality. During this time, scientific research—on hydraulic design of facilities, tracer studies, managing clogging, recovery efficiency and water quality changes in aquifers—has underpinned practical improvements in MAR and has had broader benefits in hydrogeology. Recharge wells have greatly accelerated recharge, particularly in urban areas and for mine water management. In recent years, research into governance, operating practices, reliability, economics, risk assessment and public acceptance of MAR has been undertaken. Since the 1960s, implementation of MAR has accelerated at a rate of 5%/year, but is not keeping pace with increasing groundwater extraction. Currently, MAR has reached an estimated 10 km3/year, ~2.4% of groundwater extraction in countries reporting MAR (or ~1.0% of global groundwater extraction). MAR is likely to exceed 10% of global extraction, based on experience where MAR is more advanced, to sustain quantity, reliability and quality of water supplies.
Journal Article
Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis
Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence.
Journal Article
Length-scale dependency of biomimetic hard-soft composites
Biomimetic composites are usually made by combining hard and soft phases using, for example, multi-material additive manufacturing (AM). Like other fabrication methods, AM techniques are limited by the resolution of the device, hence, setting a minimum length scale. The effects of this length scale on the performance of hard-soft composites are not well understood. Here, we studied how this length scale affects the fracture toughness behavior of single-edge notched specimens made using random, semi-random, and ordered arrangements of the hard and soft phases with five different ratios of hard to soft phases. Increase in the length scale (40 to 960 μm) was found to cause a four-fold drop in the fracture toughness. The effects of the length scale were also modulated by the arrangement and volumetric ratio of both phases. A decreased size of the crack tip plastic zone, a crack path going through the soft phase, and highly strained areas far from the crack tip were the main mechanisms explaining the drop of the fracture toughness with the length scale.
Journal Article
Cervical Cancer Genetic Susceptibility: A Systematic Review and Meta-Analyses of Recent Evidence
Cervical cancer (CC) has one of the highest mortality rates among women worldwide. Several efforts have been made to identify the genetic susceptibility factors underlying CC development. However, only a few polymorphisms have shown consistency among studies.
We conducted a systematic review of all recent case-control studies focused on the evaluation of single nucleotide polymorphisms (SNPs) and CC risk, stringently following the \"PRISMA\" statement recommendations. The MEDLINE data base was used for the search. A total of 100 case-control studies were included in the meta-analysis. Polymorphisms that had more than two reports were meta-analyzed by fixed or random models according to the heterogeneity presented among studies.
We found significant negative association between the dominant inheritance model of p21 rs1801270 polymorphism (C/A+A/A) and CC (pooled OR = 0.76; 95%CI: 0.63-0.91; p<0.01). We also found a negative association with the rs2048718 BRIP1 polymorphism dominant inheritance model (T/C+C/C) and CC (pooled OR = 0.83; 95%CI: 0.70-0.98; p = 0.03), as well as with the rs11079454 BRIP1 polymorphism recessive inheritance model and CC (pooled OR = 0.79; 95%CI: 0.63-0.99; p = 0.04). Interestingly, we observed a strong tendency of the meta-analyzed studies to be of Asiatic origin (67%). We also found a significant low representation of African populations (4%).
Our results provide evidence of the negative association of p21 rs1801270 polymorphism, as well as BRIP1 rs2048718 and rs11079454 polymorphisms, with CC risk. This study suggests the urgent need for more replication studies focused on GWAS identified CC susceptibility variants, in order to reveal the most informative genetic susceptibility markers for CC across different populations.
Journal Article
Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine
A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.
Journal Article
Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis
Viroporins are viral proteins with ion channel (IC) activity that play an important role in several processes, including virus replication and pathogenesis. While many coronaviruses (CoVs) encode two viroporins, severe acute respiratory syndrome CoV (SARS-CoV) encodes three: proteins 3a, E, and 8a. Additionally, proteins 3a and E have a PDZ-binding motif (PBM), which can potentially bind over 400 cellular proteins which contain a PDZ domain, making them potentially important for the control of cell function. In the present work, a comparative study of the functional motifs included within the SARS-CoV viroporins was performed, mostly focusing on the roles of the IC and PBM of E and 3a proteins. Our results showed that the full-length E and 3a proteins were required for maximal SARS-CoV replication and virulence, whereas viroporin 8a had only a minor impact on these activities. A virus missing both the E and 3a proteins was not viable, whereas the presence of either protein with a functional PBM restored virus viability. E protein IC activity and the presence of its PBM were necessary for virulence in mice. In contrast, the presence or absence of the homologous motifs in protein 3a did not influence virus pathogenicity. Therefore, dominance of the IC and PBM of protein E over those of protein 3a was demonstrated in the induction of pathogenesis in mice. IMPORTANCE Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines. Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines.
Journal Article