Recalcitrant cell wall of Ulva lactuca seaweed is degraded by a single ulvan lyase from family 25 of polysaccharide lyases.

Green macroalgae, e.g., Ulva lactuca, are valuable bioactive sources of nutrients; but algae recalcitrant cell walls, composed of a complex cross-linked matrix of polysaccharides, can compromise their utilization as feedstuffs for monogastric animals. This study aimed to evaluate the ability of pre-selected Carbohydrate-Active enZymes (CAZymes) and sulfatases to degrade U. lactuca cell walls and release nutritive compounds. A databank of 199 recombinant CAZymes and sulfatases was tested in vitro for their action towards U. lactuca cell wall polysaccharides. The enzymes were incubated with the macroalga, either alone or in combination, to release reducing sugars and decrease fluorescence intensity of Calcofluor White stained cell walls. The individual action of a polysaccharide lyase family 25 (PL25), an ulvan lyase, was shown to be the most efficient in cell wall disruption. The ulvan lyase treatment, in triplicate measures, promoted the release of 4.54 g/L (P < 0.001) reducing sugars, a mono- and oligosaccharides release of 11.4 and 11.2 mmol/100 g of dried alga (P < 0.01), respectively, and a decrease of 41.7% (P < 0.001) in cell wall fluorescence, in comparison to control. The ability of ulvan lyase treatment to promote the release of nutritional compounds from alga biomass was also evaluated. A release of some monounsaturated fatty acids was observed, particularly the health beneficial 18:1c9 (P < 0.001). However, no significant release of total fatty acids (P > 0.05), proteins (P = 0.861) or pigments (P > 0.05) was found. These results highlight the capacity of a single recombinant ulvan lyase (PL25 family) to incompletely disrupt U. lactuca cell walls. This enzyme could enhance the bioaccessibility of U. lactuca bioactive products with promising utilization in the feed industry.

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction.

In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.

An individual alginate lyase is effective in the disruption of Laminaria digitata recalcitrant cell wall.

In the present study, 199 pre-selected Carbohydrate-Active enZymes (CAZymes) and sulfatases were assessed, either alone or in combination, to evaluate their capacity to disrupt Laminaria digitata cell wall, with the consequent release of interesting nutritional compounds. A previously characterized individual alginate lyase, belonging to the family 7 of polysaccharide lyases (PL7) and produced by Saccharophagus degradans, was shown to be the most efficient in the in vitro degradation of L. digitata cell wall. The alginate lyase treatment, compared to the control, released up to 7.11 g/L of reducing sugars (p < 0.001) and 8.59 mmol/100 g dried alga of monosaccharides (p < 0.001), and reduced cell wall fluorescence intensity by 39.1% after staining with Calcofluor White (p = 0.001). The hydrolysis of gel-forming polymer alginate by the alginate lyase treatment could prevent the trapping of fatty acids and release beneficial monounsaturated fatty acids, particularly 18:1c9 (p < 0.001), to the extracellular medium. However, no liberation of proteins (p > 0.170) or pigments (p > 0.070) was observed. Overall, these results show the ability of an individual alginate lyase, from PL7 family, to partially degrade L. digitata cell wall under physiological conditions. Therefore, this CAZyme can potentially improve the bioavailability of L. digitata bioactive compounds for monogastric diets, with further application in feed industry.

A two-enzyme constituted mixture to improve the degradation of Arthrospira platensis microalga cell wall for monogastric diets.

The main goal of this study was to test a rational combination of pre-selected carbohydrate-active enzymes (CAZymes) and sulphatases, individually or in combination, in order to evaluate its capacity to disrupt Arthrospira platensis cell wall, allowing the release of its valuable nutritional bioactive compounds. By the end, a two-enzyme constituted mixture (Mix), composed by a lysozyme and a α-amylase, was incubated with A. platensis suspension. The microalga cell wall disruption was evaluated through the amount of reducing sugars released from the cell wall complemented with the oligosaccharide profile by HPLC. An increase of the amount of reducing sugars up to 2.42 g/L in microalgae treated with the Mix relative to no treatment (p < .05), as well as a 7-fold increase of oligosaccharides amount (p < .001), were obtained. With resort of fluorescence microscopy, a 36% reduction of fluorescence intensity (p < .001) was observed using Calcofluor White staining. In the supernatant, the Mix caused a 1.34-fold increase in protein content (p = .018) relative to the control. Similarly, n-6 polyunsaturated fatty acids (PUFA) (p = .007), in particular 18:2n-6 (p = .016), monounsaturated fatty acids (MUFA) (p = .049) and chlorophyll a (p = .025) contents were higher in the supernatant of microalgae treated with the enzyme mixture in relation to the control. Taken together, these results point towards the disclosure of a novel two-enzyme mixture able to partial degrade A. platensis cell wall, improving its nutrients bioavailability for monogastric diets with the cost-effective advantage use of microalgae in animal feed industry.

Immobilization of bacterial feruloyl esterase on mesoporous silica particles and enhancement of synthetic activity by hydrophobic-modified surface.

Here, we demonstrated the immobilization of bacterial feruloyl esterase (FAE) from Butyrivibrio sp. XPD2006, Lactobacillus crispatus, Butyrivibrio sp. AE2015, Ruminococcus albus, Cellulosilyticum ruminicola and Clostridium cellulovorans on SBA-15 and their ability to synthesize butyl ferulate (BFA). The BFae2 from Butyrivibrio sp. XPD2006 showed the best catalytic efficiency. High BFA yield was produced when the immobilization of BFae2 took place with a high protein loading and narrow pore sized SBA-15, suggesting alteration of enzyme behavior due to the crowding environment in SBA-15. Grafting of SBA-15 with octyl moieties led to shrinking pore size and resulted in 2.5-fold increment of BFA activity compared to the free enzyme and 70%mol BFA was achieved. The BFae2 encapsulated in hydrophobic-modified SBA-15 endured up to seven reaction cycles while the BFA activity remained above 60%. This is the first report showing the superior performance of hydrophobic-modified surface to entrap FAE to produce fatty phenolic esters.

High-Throughput Production of a New Library of Human Single and Tandem PDZ Domains Allows Quantitative PDZ-Peptide Interaction Screening Through High-Throughput Holdup Assay.

PDZ domains recognize PDZ Binding Motifs (PBMs) at the extreme C-terminus of their partner proteins. The human proteome contains 266 identified PDZ domains, the PDZome, spread over 152 proteins. We previously developed the "holdup" chromatographic assay for high-throughput determination of PDZ-PBM affinities. In that work, we had used an expression library of 241 PDZ constructs (the "PDZome V.1"). Here, we cloned, produced, and characterized a new bacterial expression library ("PDZome V.2"), which comprises all the 266 known human PDZ domains as well as 37 PDZ tandem constructs. To ensure the best expression level, folding, and solubility, all construct boundaries were redesigned using available structural data and all DNA sequences were optimized for Escherichia coli expression. Consequently, all the PDZ constructs are produced in a soluble form. Precise quantification and quality control were carried out. The binding profiles previously published using "PDZome V.1" were reproduced and completed using the novel "PDZome V.2" library. We provide here the detailed description of the high-throughput protocols followed through the PDZ gene synthesis and cloning, PDZ production, holdup assay and data treatment.

Novel combination of feed enzymes to improve the degradation of Chlorella vulgaris recalcitrant cell wall.

In this study, a rational combination of 200 pre-selected Carbohydrate-Active enzymes (CAZymes) and sulfatases were tested, individually or combined, according to their ability to degrade Chlorella vulgaris cell wall to access its valuable nutritional compounds. The disruption of microalgae cell walls by a four-enzyme mixture (Mix) in comparison with the control, enabled to release up to 1.21 g/L of reducing sugars (p < 0.001), led to an eight-fold increase in oligosaccharides release (p < 0.001), and reduced the fluorescence intensity by 47% after staining with Calcofluor White (p < 0.001). The Mix treatment was successful in releasing proteins (p < 0.001), some MUFA (p < 0.05), and the beneficial 18:3n-3 fatty acid (p < 0.05). Even if no variation was detected for chlorophylls (p > 0.05), total carotenoids were increased in the supernatant (p < 0.05) from the Mix treatment, relative to the control. Taken together, these results indicate that this four-enzyme Mix displays an effective capacity to degrade C. vulgaris cell wall. Thus, these enzymes may constitute a good approach to improve the bioavailability of C. vulgaris nutrients for monogastric diets, in particular, and to facilitate the cost-effective use of microalgae by the feed industry, in general.

Neuro-immune regulation of mucosal physiology.

Mucosal barriers constitute major body surfaces that are in constant contact with the external environment. Mucosal sites are densely populated by a myriad of distinct neurons and immune cell types that sense, integrate and respond to multiple environmental cues. In the recent past, neuro-immune interactions have been reported to play central roles in mucosal health and disease, including chronic inflammatory conditions, allergy and infectious diseases. Discrete neuro-immune cell units act as building blocks of this bidirectional multi-tissue cross-talk, ensuring mucosal tissue health and integrity. Herein, we will focus on reciprocal neuro-immune interactions in the airways and intestine. Such neuro-immune cross-talk maximizes sensing and integration of environmental aggressions, which can be considered an important paradigm shift in our current views of mucosal physiology and immune regulation.

Genome-wide association study and functional analysis of feet and leg conformation traits in Nellore cattle.

Feet and leg conformation is evaluated as a subset of conformational structure traits in dairy and beef cattle and is related to the feet and leg quality that can compromise the animals' productive performance and longevity. The aim of this study was to perform a genome-wide association study (GWAS) of two traits related to feet and leg conformation in Nellore cattle to identify chromosomal regions related to the expression of these traits. Phenotypic and pedigree data from 104,725 animals and genotypes from 1,435 animals and 407,730 SNPs were used. Feet and leg structure was evaluated as a binary trait (FL1) to identify yearling animals with feet and leg problems or as categorical score (FL2) to assess the overall quality of their feet and leg. The top ten 1-Mb windows that explained the largest proportion of the total genetic variance were identified and functional enrichment analyses were performed. The 10 windows with large effects obtained for FL1 are located on chromosomes 1, 2, 6, 7, 8, 10, and 14, and together explained 8.96% of the additive genetic variance. For FL2, these windows are located on chromosomes 1, 7, 10, 11, 18, 20, 22, 28, and 29, explaining 8.98% of the additive genetic variance. Several candidate genes were identified, including DLX2 which is associated with osteogenic differentiation, IL-1ß and IL-1A associated with some properties of articular cartilage, PiT1 which plays an important role in bone physiology, and CTSL associated with rheumatoid arthritis. The results presented here should contribute to a better understanding of the genetic and physiologic mechanisms regulating both traits, and identifies candidate genes for future investigation of causal mutations.

Neuronal regulation of type 2 innate lymphoid cells via neuromedin U.

Group 2 innate lymphoid cells (ILC2s) regulate inflammation, tissue repair and metabolic homeostasis, and are activated by host-derived cytokines and alarmins. Discrete subsets of immune cells integrate nervous system cues, but it remains unclear whether neuron-derived signals control ILC2s. Here we show that neuromedin U (NMU) in mice is a fast and potent regulator of type 2 innate immunity in the context of a functional neuron-ILC2 unit. We found that ILC2s selectively express neuromedin U receptor 1 (Nmur1), and mucosal neurons express NMU. Cell-autonomous activation of ILC2s with NMU resulted in immediate and strong NMUR1-dependent production of innate inflammatory and tissue repair cytokines. NMU controls ILC2s downstream of extracellular signal-regulated kinase and calcium-influx-dependent activation of both calcineurin and nuclear factor of activated T cells (NFAT). NMU treatment in vivo resulted in immediate protective type 2 responses. Accordingly, ILC2-autonomous ablation of Nmur1 led to impaired type 2 responses and poor control of worm infection. Notably, mucosal neurons were found adjacent to ILC2s, and these neurons directly sensed worm products and alarmins to induce NMU and to control innate type 2 cytokines. Our work reveals that neuron-ILC2 cell units confer immediate tissue protection through coordinated neuroimmune sensory responses.

Stability and Ligand Promiscuity of Type A Carbohydrate-binding Modules Are Illustrated by the Structure of Spirochaeta thermophila StCBM64C.

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A carbohydrate-binding modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a co-planar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

Complexity of the Ruminococcus flavefaciens FD-1 cellulosome reflects an expansion of family-related protein-protein interactions.

Protein-protein interactions play a vital role in cellular processes as exemplified by assembly of the intricate multi-enzyme cellulosome complex. Cellulosomes are assembled by selective high-affinity binding of enzyme-borne dockerin modules to repeated cohesin modules of structural proteins termed scaffoldins. Recent sequencing of the fiber-degrading Ruminococcus flavefaciens FD-1 genome revealed a particularly elaborate cellulosome system. In total, 223 dockerin-bearing ORFs potentially involved in cellulosome assembly and a variety of multi-modular scaffoldins were identified, and the dockerins were classified into six major groups. Here, extensive screening employing three complementary medium- to high-throughput platforms was used to characterize the different cohesin-dockerin specificities. The platforms included (i) cellulose-coated microarray assay, (ii) enzyme-linked immunosorbent assay (ELISA) and (iii) in-vivo co-expression and screening in Escherichia coli. The data revealed a collection of unique cohesin-dockerin interactions and support the functional relevance of dockerin classification into groups. In contrast to observations reported previously, a dual-binding mode is involved in cellulosome cell-surface attachment, whereas single-binding interactions operate for cellulosome integration of enzymes. This sui generis cellulosome model enhances our understanding of the mechanisms governing the remarkable ability of R. flavefaciens to degrade carbohydrates in the bovine rumen and provides a basis for constructing efficient nano-machines applied to biological processes.

Type I IFN Inhibits Alternative Macrophage Activation during Mycobacterium tuberculosis Infection and Leads to Enhanced Protection in the Absence of IFN-γ Signaling.

Tuberculosis causes ∼1.5 million deaths every year, thus remaining a leading cause of death from infectious diseases in the world. A growing body of evidence demonstrates that type I IFN plays a detrimental role in tuberculosis pathogenesis, likely by interfering with IFN-γ-dependent immunity. In this article, we reveal a novel mechanism by which type I IFN may confer protection against Mycobacterium tuberculosis infection in the absence of IFN-γ signaling. We show that production of type I IFN by M. tuberculosis-infected macrophages induced NO synthase 2 and inhibited arginase 1 gene expression. In vivo, absence of both type I and type II IFN receptors led to strikingly increased levels of arginase 1 gene expression and protein activity in infected lungs, characteristic of alternatively activated macrophages. This correlated with increased lung bacterial burden and pathology and decreased survival compared with mice deficient in either receptor. Increased expression of other genes associated with alternatively activated macrophages, as well as increased expression of Th2-associated cytokines and decreased TNF expression, were also observed. Thus, in the absence of IFN-γ signaling, type I IFN suppressed the switching of macrophages from a more protective classically activated phenotype to a more permissive alternatively activated phenotype. Together, our data support a model in which suppression of alternative macrophage activation by type I IFN during M. tuberculosis infection, in the absence of IFN-γ signaling, contributes to host protection.

Shared and divergent pathways for flower abscission are triggered by gibberellic acid and carbon starvation in seedless Vitis vinifera L.

BACKGROUND: Abscission is a highly coordinated developmental process by which plants control vegetative and reproductive organs load. Aiming at get new insights on flower abscission regulation, changes in the global transcriptome, metabolome and physiology were analyzed in 'Thompson Seedless' grapevine (Vitis vinifera L.) inflorescences, using gibberellic acid (GAc) spraying and shading as abscission stimuli, applied at bloom. RESULTS: Natural flower drop rates increased from 63.1% in non-treated vines to 83% and 99% in response to GAc and shade treatments, respectively. Both treatments had a broad effect on inflorescences metabolism. Specific impacts from shade included photosynthesis inhibition, associated nutritional stress, carbon/nitrogen imbalance and cell division repression, whereas GAc spraying induced energetic metabolism simultaneously with induction of nucleotide biosynthesis and carbon metabolism, therefore, disclosing alternative mechanisms to regulate abscission. Regarding secondary metabolism, changes in flavonoid metabolism were the most represented metabolic pathways in the samples collected following GAc treatment while phenylpropanoid and stilbenoid related pathways were predominantly affected in the inflorescences by the shade treatment. However, both GAc and shade treated inflorescences revealed also shared pathways, that involved the regulation of putrescine catabolism, the repression of gibberellin biosynthesis, the induction of auxin biosynthesis and the activation of ethylene signaling pathways and antioxidant mechanisms, although often the quantitative changes occurred on specific transcripts and metabolites of the pathways. CONCLUSIONS: Globally, the results suggest that chemical and environmental cues induced contrasting effects on inflorescence metabolism, triggering flower abscission by different mechanisms and pinpointing the participation of novel abscission regulators. Grapevine showed to be considered a valid model to study molecular pathways of flower abscission competence acquisition, noticeably responding to independent stimuli.

Flower abscission in Vitis vinifera L. triggered by gibberellic acid and shade discloses differences in the underlying metabolic pathways.

Understanding abscission is both a biological and an agronomic challenge. Flower abscission induced independently by shade and gibberellic acid (GAc) sprays was monitored in grapevine (Vitis vinifera L.) growing under a soilless greenhouse system during two seasonal growing conditions, in an early and late production cycle. Physiological and metabolic changes triggered by each of the two distinct stimuli were determined. Environmental conditions exerted a significant effect on fruit set as showed by the higher natural drop rate recorded in the late production cycle with respect to the early cycle. Shade and GAc treatments increased the percentage of flower drop compared to the control, and at a similar degree, during the late production cycle. The reduction of leaf gas exchanges under shade conditions was not observed in GAc treated vines. The metabolic profile assessed in samples collected during the late cycle differently affected primary and secondary metabolisms and showed that most of the treatment-resulting variations occurred in opposite trends in inflorescences unbalanced in either hormonal or energy deficit abscission-inducing signals. Particularly concerning carbohydrates metabolism, sucrose, glucose, tricarboxylic acid metabolites and intermediates of the raffinose family oligosaccharides pathway were lower in shaded and higher in GAc samples. Altered oxidative stress remediation mechanisms and indolacetic acid (IAA) concentration were identified as abscission signatures common to both stimuli. According to the global analysis performed, we report that grape flower abscission mechanisms triggered by GAc application and C-starvation are not based on the same metabolic pathways.

Overexpression, crystallization and preliminary X-ray characterization of Ruminococcus flavefaciens scaffoldin C cohesin in complex with a dockerin from an uncharacterized CBM-containing protein.

Cellulosomes are massive cell-bound multienzyme complexes tethered by macromolecular scaffolds that coordinate the efforts of many anaerobic bacteria to hydrolyze plant cell-wall polysaccharides, which are a major untapped source of carbon and energy. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between cohesin modules, located in the scaffold, and dockerin modules, found in the enzymes and other cellulosomal proteins. The proposed cellulosomal architecture for Ruminococcus flavefaciens strain FD-1 consists of a major scaffoldin (ScaB) that acts as the backbone to which other components attach. It has nine cohesins and a dockerin with a fused X-module that binds to the cohesin on ScaE, which in turn is covalently attached to the cell wall. The ScaA dockerin binds to ScaB cohesins allowing more carbohydrate-active modules to be assembled. ScaC acts as an adaptor that binds to both ScaA and selected ScaB cohesins, thereby increasing the repertoire of dockerin-bearing proteins that integrate into the complex. In previous studies, a screen for novel cohesin-dockerin complexes was performed which led to the identification of a total of 58 probable cohesin-dockerin pairs. Four were selected for subsequent structural and biochemical characterization based on the quality of their expression and the diversity in their specificities. One of these is C12D22, which comprises the cohesin from the adaptor ScaC protein bound to the dockerin of a CBM-containing protein. This complex has been purified and crystallized, and data were collected to resolutions of 2.5 Š(hexagonal, P65), 2.16 Š(orthorhombic, P212121) and 2.4 Š(orthorhombic, P21212) from three different crystalline forms.

Overexpression, purification, crystallization and preliminary X-ray characterization of the fourth scaffoldin A cohesin from Acetivibrio cellulolyticus in complex with a dockerin from a family 5 glycoside hydrolase.

Cellulosomes are cell-bound multienzyme complexes secreted by anaerobic bacteria that play a crucial role in carbon turnover by degrading plant cell walls to simple sugars. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between cohesin modules located in a molecular scaffold and dockerin modules found in cellulosomal enzymes. Acetivibrio cellulolyticus possesses a complex cellulosome arrangement which is organized by a primary enzyme-binding scaffoldin (ScaA), two anchoring scaffoldins (ScaC and ScaD) and an unusual adaptor scaffoldin (ScaB). A dockerin from a family 5 glycoside hydrolase (GH5), which was engineered to inactivate one of the two putative cohesin-binding interfaces, complexed with one of the ScaA cohesins from A. cellulolyticus has been purified and crystallized, and data were processed to a resolution of 1.57 Šin the orthorhombic space group P212121.

Construction of GH16 ß-glucanase mini-cellulosomes to improve the nutritive value of barley-based diets for broilers.

Anaerobic cellulolytic bacteria organize a comprehensive range of cellulases and hemicellulases in high molecular weight multienzyme complexes termed cellulosomes. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between cohesins and dockerins. This paper reports the production of mini-cellulosomes containing one (GH16-1C) or three (GH16-3C) copies of Clostridium thermocellum glucanase 16A (CtGlc16A). Barley ß-1,3-1,4-glucans are known to be antinutritive for monogastric animals, particularly for poultry. GH16-1C and GH16-3C were used to supplement barley-based diets for broilers. The data revealed that the two mini-cellulosomes effectively improved the nutritive value of barley-based diets for broilers. Analysis of mini-cellulosome molecular integrity revealed that linker sequences separating protein domains in scaffoldins and cellulosomal catalytic units are highly susceptible to proteolytic attack in vivo. The data suggest that linker protection could result in further improvements in enzyme efficacy to improve the nutritive value of barley-based diets for monogastric animals.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of glucuronoxylan xylanohydrolase (Xyn30A) from Clostridium thermocellum.

The modular carbohydrate-active enzyme belonging to glycoside hydrolase family 30 (GH30) from Clostridium thermocellum (CtXynGH30) is a cellulosomal protein which plays an important role in plant cell-wall degradation. The full-length CtXynGH30 contains an N-terminal catalytic module (Xyn30A) followed by a family 6 carbohydrate-binding module (CBM6) and a dockerin at the C-terminus. The recombinant protein has a molecular mass of 45 kDa. Preliminary structural characterization was carried out on Xyn30A crystallized in different conditions. All tested crystals belonged to space group P1 with one molecule in the asymmetric unit. Molecular replacement has been used to solve the Xyn30A structure.

Informational ergonomics and design: signage design for Monte Sião Camp.

This paper refers to the development of a signage system, driven by the vector of Signage Design and Informational Ergonomics associated with Regulatory Standards. The methodology of the Ergonomic Intervention of Moraes and Mont'Alvão (2003), in its early stages, and the Method of the Signage Pyramid of Calori (2007) were used to develop the research, data collection and analysis and to guide the design by a signaling system. The system contemplated by this job is called Mount Zion, a site of 19 hectares, which has signs of disturbance. As a result, we obtained a signaling system, with graphic features that refer to formal-site, capable of meeting the needs of orientation and displacement inherent to the site.