ALEPH: a network-oriented approach for the generation of fragment-based libraries and for structure interpretation.

The analysis of large structural databases reveals general features and relationships among proteins, providing useful insight. A different approach is required to characterize ubiquitous secondary-structure elements, where flexibility is essential in order to capture small local differences. The ALEPH software is optimized for the analysis and the extraction of small protein folds by relying on their geometry rather than on their sequence. The annotation of the structural variability of a given fold provides valuable information for fragment-based molecular-replacement methods, in which testing alternative model hypotheses can succeed in solving difficult structures when no homology models are available or are successful. ARCIMBOLDO_BORGES combines the use of composite secondary-structure elements as a search model with density modification and tracing to reveal the rest of the structure when both steps are successful. This phasing method relies on general fold libraries describing variations around a given pattern of β-sheets and helices extracted using ALEPH. The program introduces characteristic vectors defined from the main-chain atoms as a way to describe the geometrical properties of the structure. ALEPH encodes structural properties in a graph network, the exploration of which allows secondary-structure annotation, decomposition of a structure into small compact folds, generation of libraries of models representing a variation of a given fold and finally superposition of these folds onto a target structure. These functions are available through a graphical interface designed to interactively show the results of structure manipulation, annotation, fold decomposition, clustering and library generation. ALEPH can produce pictures of the graphs, structures and folds for publication purposes

March1-dependent modulation of donor MHC II on CD103+ dendritic cells mitigates alloimmunity.

In transplantation, donor dendritic cells (do-DCs) initiate the alloimmune response either by direct interaction with host T cells or by transferring intact donor MHC to host DCs. However, how do-DCs can be targeted for improving allograft survival is still unclear. Here we show CD103+ DCs are the major do-DC subset involved in the acute rejection of murine skin transplants. In the absence of CD103+ do-DCs, less donor MHC-II is carried to host lymph nodes, fewer allogenic T cells are primed and allograft survival is prolonged. Incubation of skin grafts with the anti-inflammatory mycobacterial protein DnaK reduces donor MHC-II on CD103+DCs and prolongs graft survival. This effect is mediated through IL-10-induced March1, which ubiquitinates and decreases MHC-II levels. Importantly, in vitro pre-treatment of human DCs with DnaK reduces their ability to prime alloreactive T cells. Our findings demonstrate a novel therapeutic approach to dampen alloimmunity by targeting donor MHC-II on CD103+DCs

Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER.

Structure determination of novel biological macromolecules by X-ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre-existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main-chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose

Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER.

Structure determination of novel biological macromolecules by X-ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre-existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main-chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose

Insect pest management with sex pheromone precursors from engineered oilseed plants

Pheromones have become an environmentally friendly alternative to conventional insecticides for pest control. Most current pheromone-based pest control products target lepidopteran pests of high-value crops, as today’s manufacturing processes cannot yet produce pheromones at low enough costs to enable their use for lower-value crops, especially commodity crops. Camelina sativa seeds genetically modified to express (Z)-11-hexadecenoic acid, a sex pheromone precursor of several moth species, provided the oil from which the precursor was isolated, purified and transformed into the final pheromone. Trap lures containing this pheromone were then assessed for their capacity to manage moth pests in the field. Plant-derived pheromone lures proved equally effective as synthetic pheromone lures in monitoring the diamondback moth, Plutella xylostella, in cabbage and disrupting mating of cotton bollworm, Helicoverpa armigera, in common bean fields. Our study demonstrates the biological efficacy and economic feasibility of pheromone production in plant factories by metabolic engineering of an oilseed crop

Evidence for lack of direct causality between pain and affective disturbances in a rat peripheral neuropathy model

[Epub ahead of print]Chronic pain is frequently accompanied by the manifestation of emotional disturbances and cognitive deficits. While a causality relation between pain and emotional/cognitive disturbances is generally assumed, several observations suggest a temporal dissociation and independent mechanisms. We therefore studied Sprague-Dawley rats that presented a natural resistance to pain manifestation in a neuropathy model (spared nerve injury [SNI]) and compared their performance in a battery of behavioral paradigms-anxiety, depression and fear memory-with animals that presented a pain phenotype. Afterward, we performed an extensive volumetric analysis across prefrontal, orbitofrontal and insular cortical areas. The majority of SNI animals manifested mechanical allodynia (low threshold [LT]), but 13% were similar to Sham controls (high threshold [HT]). Readouts of spontaneous hypersensivity (paw flinches) were also significantly reduced in HT and correlated with allodynia. To increase the specificity of our findings, we segregated the SNI animals in those with left (SNI-L) and right (SNI-R) lesions and the lack of association between pain and behavior still remains. Left-lesioned animals, independent of the LT or HT phenotype, presented increased anxiety-like behaviors and decreased well-being. In contrast, we found that the insular cortex (agranular division) was significantly smaller in HT than in LT. To conclude, pain and emotional disturbances observed following nerve injury are to some extent segregated phenomena. Also, HT and LT SNI presented differences in insular volumes, an area vastly implicated in pain perception, suggesting a supraspinal involvement in the manifestation of these phenotypes.This work has been funded by the European Regional Development Fund (FEDER), through the Competitiveness Factors Operational Programme (COMPETE) and the Northern Portugal Regional Operational Programme (NORTE 2020) under the Portugal 2020 Partnership Agreement (project NORTE-01-0145-FEDER-000023). It was also funded by National and International funds, through the Foundation for Science and Technology (FCT), under the scope of the projects POCI-01-0145- FEDER-007038 and PTDC/NEU-SCC/5301/2014 and by the IASP Early Career Research Grant 2015. Researchers were supported by FCT grant numbers PD/BD/114117/2015 (MRG via Inter-University Doctoral Programme in Ageing and Chronic Disease, PhDOC), SFRH/BD/109111/2015 (AMC via PhD Program in Health Sciences), SFRH/BD/52291/2013 (ME via PhDOC), SFRH/BD/89936/2012 (SB via PhD Program in Health Sciences (Applied)), PDE/BDE/113604/2015 (RM via PhD Program in Health Sciences (Applied)), PDE/BDE/113601/2015 (PSM via PhD Program in Health Sciences (Applied)) and SFRH/BPD/80118/2011 (HA). ARS integrated the Master Programme in Health Sciences of the School of Medicine, University of Minho.info:eu-repo/semantics/acceptedVersio

Statistical Analysis of Factors Influencing Corrosion in Concrete Structures

The use of mathematical and statistical models has allowed the description of the behavior of many natural phenomena. However, their application in civil engineering particularly, for the analysis of the corrosion behavior, has been overlooked in recent studies. In the present work, a factorial analysis with subdivided parcels design were conducted to evaluate the corrosion rate behavior of arrangements of protected and not protected steel bars found in reinforced concrete specimens subject to two different aggressive media. The results showed that, in comparison to sulphate ions, the effect of chloride ions on the corrosion rate was not statistically significant. In the same way, protected and not protected segments on the steel bars (i.e., steel bar condition) did not have a significant effect on the corrosion rate. The only factor found to significantly affect the corrosion rate was the variability of the beam fabrication process (i.e., beam factor)