A novel thermophilic recombinant obligate xylobiohydrolase (AcGH30A) from Acetivibrio clariflavus orchestrates the deconstruction of xylan polysaccharides.

GH30 xylobiohydrolases, an expanding enzyme category, need deeper insights for optimal use. The primary aim of this study was to characterize a new xylobiohydrolase, AcGH30A of GH30 family from Acetivibrio clariflavus. The gene encoding AcGH30A was cloned using pET28a(+) vector and expressed in E. coli BL21(DE3) cells. AcGH30A was purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of AcGH30A showed molecular mass of ~58 kDa. AcGH30A showed optimum temperature 80 °C and optimum pH 7.0. AcGH30A was stable (maintaining >80 % of control activity) in pH range, 4-7 and temperature range, 30 °C -70 °C when incubated for 90 min. AcGH30A displayed melting temperature, 72 °C and half-life, 21 days at 4 °C. The enzyme activity of AcGH30A was enhanced by 10 mM Ca2+ and Mg2+ ions by 25 % and 21 %, respectively, whereas 10 mM Co2+, Zn2+, Fe2+, and Cu2+ ions significantly reduced it. AcGH30A showed activity against various xylan polysaccharides displaying highest Vmax, 139 U.mg-1 and KM, 0.71 mg.ml-1 against 4-O-methyl glucuronoxylan under optimum conditions. TLC, HPLC and LC-MS analyses of AcGH30A hydrolyzed products from xylan substrates revealed the release of sole product, xylobiose, confirming it as an obligate xylobiohydrolase. AcGH30A being a highly thermostable enzyme can be potentially utlilized in various biotechnological applications.

Open or closed abdomen post laparotomy to control severe abdominal sepsis: a survival analysis.

INTRODUCTION: severe abdominal sepsis, accompained by diffuse peritonitis, poses a significant challenge for most surgeons. It often requires repetitive surgical interventions, leading to complications and resulting in high morbidity and mortality rates. The open abdomen technique, facilitated by applying a negative-pressure wound therapy (NPWT), reduces the duration of the initial surgical procedure, minimizes the accumulation of secretions and inflammatory mediators in the abdominal cavity and lowers the risk of abdominal compartment syndrome and its associated complications. Another approach is primary closure of the abdominal aponeurosis, which involves suturing the layers of the abdominal wall. METHODS: the objective of this study is to conduct a survival analysis comparing the treatment of severe abdominal sepsis using open abdomen technique versus primary closure after laparotomy in a public hospital in the South of Brazil. We utilized data extracted from electronic medical records to perform both descriptive and survival analysis, employing the Kaplan-Meier curve and a log-rank test. RESULTS: the study sample encompassed 75 laparotomies conducted over a span of 5 years, with 40 cases employing NPWT and 35 cases utilizing primary closure. The overall mortality rate observed was 55%. Notably, survival rates did not exhibit statistical significance when comparing the two methods, even after stratifying the data into separate analysis groups for each technique. CONCLUSION: recent publications on this subject have reported some favorable outcomes associated with the open abdomen technique underscoring the pressing need for a standardized approach to managing patients with severe, complicated abdominal sepsis.

Spatial distribution of mortality from colorectal cancer in the southern region of Brazil.

Colorectal cancer (CRC) is the leading cause of death due to cancer worldwide. In Brazil, it is the second most frequent cancer in men and women, with a mortality reaching 9.4% of those diagnosed. The aim of this study was to analyze the spatial heterogeneity of CRC deaths among municipalities in south Brazil, from 2015 to 2019, in different age groups (50-59 years, 60-69 years, 70-79 years, and 80 years old or more) and identify the associated variables. Global Spatial Autocorrelation (Moran's I) and Local Spatial Autocorrelation (LISA) analyses were used to evaluate the spatial correlation between municipalities and CRC mortality. Ordinary Least Squares (OLS) and Geographically Weighted Regression (GWR) were applied to evaluate global and local correlations between CRC deaths, sociodemographic, and coverage of health care services. For all age groups, our results found areas with high CRC rates surrounded by areas with similarly high rates mainly in the Rio Grande do Sul state. Even as factors associated with CRC mortality varied according to age group, our results suggested that improved access to specialized health centers, the presence of family health strategy teams, and higher rates of colonoscopies are protective factors against colorectal cancer mortality in southern Brazil.

Carbohydrate Depolymerization by Intricate Cellulosomal Systems.

Cellulosomes are multi-enzymatic nanomachines that have been fine-tuned through evolution to efficiently deconstruct plant biomass. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between the various enzyme-borne dockerin modules and the multiple copies of the cohesin modules located on the scaffoldin subunit. Recently, designer cellulosome technology was established to provide insights into the architectural role of catalytic (enzymatic) and structural (scaffoldin) cellulosomal constituents for the efficient degradation of plant cell wall polysaccharides. Owing to advances in genomics and proteomics, highly structured cellulosome complexes have recently been unraveled, and the information gained has inspired the development of designer-cellulosome technology to new levels of complex organization. These higher-order designer cellulosomes have in turn fostered our capacity to enhance the catalytic potential of artificial cellulolytic complexes. In this chapter, methods to produce and employ such intricate cellulosomal complexes are reported.

Structure-function studies can improve binding affinity of cohesin-dockerin interactions for multi-protein assemblies.

The cellulosome is an elaborate multi-enzyme structure secreted by many anaerobic microorganisms for the efficient degradation of lignocellulosic substrates. It is composed of multiple catalytic and non-catalytic components that are assembled through high-affinity protein-protein interactions between the enzyme-borne dockerin (Doc) modules and the repeated cohesin (Coh) modules present in primary scaffoldins. In some cellulosomes, primary scaffoldins can interact with adaptor and cell-anchoring scaffoldins to create structures of increasing complexity. The cellulosomal system of the ruminal bacterium, Ruminococcus flavefaciens, is one of the most intricate described to date. An unprecedent number of different Doc specificities results in an elaborate architecture, assembled exclusively through single-binding-mode type-III Coh-Doc interactions. However, a set of type-III Docs exhibits certain features associated with the classic dual-binding mode Coh-Doc interaction. Here, the structure of the adaptor scaffoldin-borne ScaH Doc in complex with the Coh from anchoring scaffoldin ScaE is described. This complex, unlike previously described type-III interactions in R. flavefaciens, was found to interact in a dual-binding mode. The key residues determining Coh recognition were also identified. This information was used to perform structure-informed protein engineering to change the electrostatic profile of the binding surface and to improve the affinity between the two modules. The results show that the nature of the residues in the ligand-binding surface plays a major role in Coh recognition and that Coh-Doc affinity can be manipulated through rational design, a key feature for the creation of designer cellulosomes or other affinity-based technologies using tailored Coh-Doc interactions.

Dispositivo com câmera para treinamento de intubação orotraqueal: possibilidade de ensino médico em período pandêmico / Device with camera in orotracheal intubation training: possibility of medical education a pandemic period

Resumo: Introdução: A intubação orotraqueal é um procedimento importante no manejo da via aérea, principalmente quando realizada em situações de emergência. A videolaringoscopia é um artificio que facilita a visualização da glote ao auxiliar a intubação. Objetivo: Este estudo teve como objeto acoplar uma câmera de vídeo a um laringoscópio convencional do tipo Macintosh para possibilitar e orientar o treinamento da intubação orotraqueal. Método: O uso de uma câmera acoplada a um laringoscópio convencional permite a visibilização direta e indireta da glote. As imagens da câmera podem ser transmitidas por wi-fi e compartilhadas para dispositivos e plataformas eletrônicos, visando ao ensino presencial ou remoto da intubação orotraqueal. Resultado: A utilização do dispositivo artesanal como método de ensino de intubação orotraqueal permite ao docente ensinar a teoria do procedimento e orientar e corrigir a execução realizada pelo acadêmico. Esse feedback no treinamento prático pode ser realizado presencialmente ou por via remota. Conclusão: O uso do dispositivo artesanal de videolaringoscopia no ensino médico é uma ferramenta de baixo custo para aperfeiçoar o treinamento de intubação orotraqueal convencional.

Abstract: Introduction: The orotracheal intubation is an important procedure in airway management, especially when performed in emergency situations. Video-laryngoscopy is an artifice that facilitates visualization of the glottis, aiding intubation. Objective: This study aimed to attach a video camera to a conventional Macintosh-type laryngoscope to enable and train intubation or otracheal training. Method: The use of a camera coupled to a conventional laryngoscope allows direct and indirect visualization of the glottis. Camera images can be transmitted over wi-fi and shared to electronic devices and platforms, aiming at teaching in person or remotely about orotracheal intubation. Result: The use of the device as a method of teaching intubation or otracheal, allows teaching to teach the theory of procedure and training the execution performed by the artisanal method. This feedback in the practical training of orotracheal intubation in mannequins can be performed in person or remotely. Conclusion: The use of handcrafted video laryngoscopy device in medical education is a low-cost tool to improve conventional orotracheal intubation training.

The Role of Pyrazolopyridine Derivatives on Different Steps of Herpes Simplex Virus Type-1 In Vitro Replicative Cycle.

Herpes simplex virus type-1 (HSV-1) infection causes several disorders, and acyclovir is used as a reference compound. However, resistant strains are commonly observed. Herein, we investigate the effects of N-heterocyclic compounds (pyrazolopyridine derivatives), named ARA-04, ARA-05, and AM-57, on HSV-1 in vitro replication. We show that the 50% effective concentration (EC50) values of the compounds ARA-04, ARA-05, and AM-57 were 1.00 ± 0.10, 1.00 ± 0.05, and 0.70 ± 0.10 µM, respectively. These compounds presented high 50% cytotoxic concentration (CC50) values, which resulted in a selective index (SI) of 1000, 1000, and 857.1 for ARA-04, ARA-05, and AM-57, respectively. To gain insight into which step of the HSV-1 replication cycle these molecules would impair, we performed adsorption and penetration inhibition assays and time-of-addition experiments. Our results indicated that ARA-04 and ARA-05 affected viral adsorption, while AM-57 interfered with the virus replication during its α- and γ-phases and decreased ICP27 content during initial and late events of HSV-1 replication. In addition, we also observed that AM-57 caused a strong decrease in viral gD content, which was reinforced by in silico calculations that suggested AM-57 interacts preferentially with the viral complex between a general transcription factor and virion protein (TFIIBc-VP16). In contrast, ARA-04 and ARA-05 interact preferentially in the proteins responsible for the viral adsorption process (nectin-1 and glycoprotein). Thus, our results suggest that the 1H-pyrazolo[3,4-b]pyridine derivatives inhibit the HSV-1 replicative cycle with a novel mechanism of action, and its scaffold can be used as a template for the synthesis of promising new molecules with antiviral effects, including to reinforce the presented data herein for a limited number of molecules.

Salinity-dependent modulation by protein kinases and the FXYD2 peptide of gill (Na+, K+)-ATPase activity in the freshwater shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

The geographical distribution of aquatic crustaceans is determined by ambient factors like salinity that modulate their biochemistry, physiology, behavior, reproduction, development and growth. We investigated the effects of exogenous pig FXYD2 peptide and endogenous protein kinases A and C on gill (Na+, K+)-ATPase activity, and characterized enzyme kinetic properties in a freshwater population of Macrobrachium amazonicum in fresh water (<0.5 ‰ salinity) or acclimated to 21 ‰S. Stimulation by FXYD2 peptide and inhibition by endogenous kinase phosphorylation are salinity-dependent. While without effect in shrimps in fresh water, the FXYD2 peptide stimulated activity in salinity-acclimated shrimps by ≈50 %. PKA-mediated phosphorylation inhibited gill (Na+, K+)-ATPase activity by 85 % in acclimated shrimps while PKC phosphorylation markedly inhibited enzyme activity in freshwater- and salinity-acclimated shrimps. The (Na+, K+)-ATPase in salinity-acclimated shrimp gills hydrolyzed ATP at a Vmax of 54.9 ± 1.8 nmol min-1 mg-1 protein, corresponding to ≈60 % that of freshwater shrimps. Mg2+ affinity increased with salinity acclimation while K+ affinity decreased. (Ca2+, Mg2+)-ATPase activity increased while V(H+)- and Na+- or K+-stimulated activities decreased on salinity acclimation. The 120-kDa immunoreactive band expressed in salinity-acclimated shrimps suggests nonspecific α-subunit phosphorylation by PKA and/or PKC. These alterations in (Na+, K+)-ATPase kinetics in salinity-acclimated M. amazonicum may result from regulatory mechanisms mediated by phosphorylation via protein kinases A and C and the FXYD2 peptide rather than through the expression of a different α-subunit isoform. This is the first demonstration of gill (Na+, K+)-ATPase regulation by protein kinases in freshwater shrimps during salinity challenge.

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.

Effect of Dietary Laminaria digitata with Carbohydrases on Broiler Production Performance and Meat Quality, Lipid Profile, and Mineral Composition.

We hypothesized that dietary inclusion of 15% Laminaria digitata, supplemented or not with carbohydrases, could improve the nutritional value of poultry meat without impairing animal growth performance. A total of 120 22-day old broilers were fed the following dietary treatments (n = 10) for 14 days: cereal-based diet (control); control diet with 15% L. digitata (LA); LA diet with 0.005% Rovabio® Excel AP (LAR); LA diet with 0.01% alginate lyase (LAE). Final body weight was lower and feed conversion ratio higher with LA diet than with the control. The ileal viscosity increased with LA and LAR diets relative to control but without differences between LAE and control. The pH of thigh meat was higher, and the redness value of breast was lower with LA diet than with control. Meat overall acceptability was positively scored for all treatments. The γ-tocopherol decreased, whereas total chlorophylls and carotenoids increased in meat with alga diets relative to control. The percentage of n-3 polyunsaturated fatty acids (PUFA) and accumulation of bromine and iodine in meat increased with alga diets compared with control. Feeding 15% of L. digitata to broilers impaired growth performance but enhanced meat quality by increasing antioxidant pigments, with beneficial effects on n-3 PUFA and iodine.

Highly efficient, processive and multifunctional recombinant endoglucanase RfGH5_4 from Ruminococcus flavefaciens FD-1 v3 for recycling lignocellulosic plant biomasses.

Gene encoding endoglucanase, RfGH5_4 from R. flavefaciens FD-1 v3 was cloned, expressed in Escherichia coli BL21(DE3) cells and purified. RfGH5_4 showed molecular size 41 kDa and maximum activity at pH 5.5 and 55 °C. It was stable between pH 5.0-8.0, retaining 85% activity and between 5 °C-45 °C, retaining 75% activity, after 60 min. RfGH5_4 displayed maximum activity (U/mg) against barley ß-D-glucan (665) followed by carboxymethyl cellulose (450), xyloglucan (343), konjac glucomannan (285), phosphoric acid swollen cellulose (86), beechwood xylan (21.7) and carob galactomannan (16), thereby displaying the multi-functionality. Catalytic efficiency (mL.mg-1 s-1) of RfGH5_4 against carboxymethyl cellulose (146) and konjac glucomannan (529) was significantly high. TLC and MALDI-TOF-MS analyses of RfGH5_4 treated hydrolysates of cellulosic and hemicellulosic polysaccharides displayed oligosaccharides of degree of polymerization (DP) between DP2-DP11. TLC, HPLC and Processivity-Index analyses revealed RfGH5_4 to be a processive endoglucanase as initially, for 30 min it hydrolysed cellulose to cellotetraose followed by persistent release of cellotriose and cellobiose. RfGH5_4 yielded sufficiently high Total Reducing Sugar (TRS, mg/g) from saccharification of alkali pre-treated sorghum (72), finger millet (62), sugarcane bagasse (38) and cotton (27) in a 48 h saccharification reaction. Thus, RfGH5_4 can be considered as a potential endoglucanase for renewable energy applications.

Influence of Chlorella vulgaris on growth, digestibility and gut morphology and microbiota of weaned piglet.

The purpose of this study was to evaluate the impact of Chlorella vulgaris (5% in the diet), supplemented or not with two exogenous carbohydrase mixtures on piglets' performance, nutrient digestibility and gut morphology, fermentation products and microbiota. Forty-four male piglets weaned at 28 days of age, with 11.2 ± 0.46 kg of live weight, were used and assigned to 1 of 4 dietary treatments: cereal and soybean meal based-diet (control, n = 11), control diet with 5% of C. vulgaris (CH, n = 10), CH diet supplemented with 0.005% of Rovabio® Excel AP (CH + R, n = 10) and CH diet supplemented with 0.01% of a recombinant 4-carbohydrase mixture (CH + M, n = 11). Growth performance was not changed by the of C. vulgaris inclusion during 21 days of trial. However, total tract apparent digestibility of nutritional fractions was negatively impacted by the inclusion. In addition, the viscosity of duodenum plus jejunum contents slightly increased in all groups fed with the microalga. In contrast, dietary microalga increased duodenum villus height and promoted a healthier gut microbiota, with higher abundance of some specific bacterial taxa (Colidextribacter, Oscillospira and Lactobacillus). This study indicates that the dietary inclusion of 5% C. vulgaris improves piglets' gut health without impairing performance. Data also indicate that C. vulgaris reduces nutrient digestibility but promotes compensatory developments of gut mucosa and prebiotic effects. Dietary supplementation with exogenous carbohydrases does not seem to be necessary for this inclusion level. Therefore, the incorporation of CH as a sustainable feed ingredient in piglets' nutrition is a viable alternative approach.

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.

From Cancer Therapy to Winemaking: The Molecular Structure and Applications of ß-Glucans and ß-1, 3-Glucanases.

ß-glucans are a diverse group of polysaccharides composed of ß-1,3 or ß-(1,3-1,4) linked glucose monomers. They are mainly synthesized by fungi, plants, seaweed and bacteria, where they carry out structural, protective and energy storage roles. Because of their unique physicochemical properties, they have important applications in several industrial, biomedical and biotechnological processes. ß-glucans are also major bioactive molecules with marked immunomodulatory and metabolic properties. As such, they have been the focus of many studies attesting to their ability to, among other roles, fight cancer, reduce the risk of cardiovascular diseases and control diabetes. The physicochemical and functional profiles of ß-glucans are deeply influenced by their molecular structure. This structure governs ß-glucan interaction with multiple ß-glucan binding proteins, triggering myriad biological responses. It is then imperative to understand the structural properties of ß-glucans to fully reveal their biological roles and potential applications. The deconstruction of ß-glucans is a result of ß-glucanase activity. In addition to being invaluable tools for the study of ß-glucans, these enzymes have applications in numerous biotechnological and industrial processes, both alone and in conjunction with their natural substrates. Here, we review potential applications for ß-glucans and ß-glucanases, and explore how their functionalities are dictated by their structure.

Effect of Fe3+ on Na,K-ATPase: Unexpected activation of ATP hydrolysis.

Iron is a key element in cell function; however, its excess in iron overload conditions can be harmful through the generation of reactive oxygen species (ROS) and cell oxidative stress. Activity of Na,K-ATPase has been shown to be implicated in cellular iron uptake and iron modulates the Na,K-ATPase function from different tissues. In this study, we determined the effect of iron overload on Na,K-ATPase activity and established the role that isoforms and conformational states of this enzyme has on this effect. Total blood and membrane preparations from erythrocytes (ghost cells), as well as pig kidney and rat brain cortex, and enterocytes cells (Caco-2) were used. In E1-related subconformations, an enzyme activation effect by iron was observed, and in the E2-related subconformations enzyme inhibition was observed. The enzyme's kinetic parameters were significantly changed only in the Na+ curve in ghost cells. In contrast to Na,K-ATPase α2 and α3 isoforms, activation was not observed for the α1 isoform. In Caco-2 cells, which only contain Na,K-ATPase α1 isoform, the FeCl3 increased the intracellular storage of iron, catalase activity, the production of H2O2 and the expression levels of the α1 isoform. In contrast, iron did not affect lipid peroxidation, GSH content, superoxide dismutase and Na,K-ATPase activities. These results suggest that iron itself modulates Na,K-ATPase and that one or more E1-related subconformations seems to be determinant for the sensitivity of iron modulation through a mechanism in which the involvement of the Na, K-ATPase α3 isoform needs to be further investigated.

Impact of Chlorella vulgaris as feed ingredient and carbohydrases on the health status and hepatic lipid metabolism of finishing pigs.

The implication of high dietary level of Chlorella vulgaris, individually and supplemented with two carbohydrase mixtures, on pigs' health and liver metabolism was assessed in this study. Forty crossbred (Large White × Landrace sows crossed with Pietrain boars) entire male pigs were randomly allocated to the following feeding treatments (n = 10): cereal-soybean meal basal diet (control); basal diet with 5% C. vulgaris; basal diet with 5% C. vulgaris supplemented with 0.005% Rovabio® Excel AP; and basal diet with 5% C. vulgaris supplemented with 0.01% of a preselected four-CAZyme mixture. The trial lasted from 59.1 ± 5.69 kg of initial live weight to 101 ± 1.9 kg of slaughter weight. Data indicate that this high dietary level of C. vulgaris has impact on several blood parameters of finishing pigs. However, the most relevant health outcome observed was a strong immunosuppressive effect promoted by the microalga, which increases pigs' susceptibility to infection diseases. In addition, the dietary incorporation of C. vulgaris reduced the systemic antioxidant capacity of pigs. In turn, the dietary supplementation with the four-CAZyme mixture promoted a clear decrease on some blood parameters compared with the control group. Regarding hepatic lipids, pigs fed C. vulgaris diets, had an increased hepatic content of n-3 PUFA, with a consequent decrease on the n-6/n-3 ratio. In conclusion, the use of C. vulgaris as feed ingredient appears to be safe under controlled experimental conditions. However, it is imperative test it in industrial production systems, with more stressful and less hygienic environments.

Computational and SAXS-based structure insights of pectin acetyl esterase (CtPae12B) of family 12 carbohydrate esterase from Clostridium thermocellum ATCC 27405.

Pectin is a complex form of polysaccharide and is composed of several structural components that require the concerted action of several pectinases for its complete degradation. In this study, in silico and solution structure of a pectin acetyl esterase (CtPae12B) of family 12 carbohydrate esterase (CE12) from Clostridium thermocellum was determined. The CtPae12B modelled structure, showed a new α/ß hydrolase fold, similar to the fold found in the crystal structures of its nearest homologues from CE12 family, which differed from α/ß hydrolase fold found in glycoside hydrolases. In the active site of CtPae12B, two loops (loop1 and loop6) play an important role in the formation of a catalytic triad Ser15-Asp187-His190, where Ser15 acts as a nucleophile. The structural stability of CtPae12B and its catalytic site was detected by performing molecular dynamic (MD) simulation which showed stable and compact conformation of the structure. Molecular docking method was employed to analyse the conformations of various suitable ligands docked at the active site of CtPae12B. The stability and structural specificity of the catalytic residues with the ligand, 4-nitrophenyl acetate (4-NPA) was confirmed by MD simulation of CtPae12B-4NPA docked complex. Moreover, it was found that the nucleophile Ser15, forms hydrophobic interaction with 4-NPA in the active site to complete covalent catalysis. Small angle X-ray scattering analysis of CtPae12B at 3 mg/mL displayed elongated, compact and monodispersed nature in solution. The ab initio derived dummy model showed that CtPae12B exists as a homotrimer at 3 mg/mL which was also confirmed by dynamic light scattering.Communicated by Ramaswamy H. Sarma.

Mapping Molecular Recognition of ß1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus.

A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage ß1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBPMLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBPMLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBPMLG-A with a ß1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward ß1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of ß1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial ß1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the ß-glucan backbone imposed by the ß1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBPMLG-A to import long ß1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of ß1,3-1,4-glucans. IMPORTANCE With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBPMLG-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage ß1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBPMLG-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of Bacteroidetes, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.

An experimental pleural drainage device in hypertensive pneumothorax.

PURPOSE: To develop a specific device for pleural drainage in hypertensive pneumothorax. METHODS: The prototype was modeled from the free version of a 3D modeling application, printed on a 3D printer using ABS® plastic material, and tested in a pleural drainage simulator. RESULTS: Pleural drainage in the simulator using the prototype was feasible and reproducible. CONCLUSIONS: While the prototype is functional in the simulator, it requires improvement and refinement for use in humans.

A trimodular family 16 glycoside hydrolase from the cellulosome of Ruminococcus flavefaciens displays highly specific licheninase (EC 3.2.1.73) activity.

Cellulosomes are highly complex cell-bound multi-enzymatic nanomachines used by anaerobes to break down plant carbohydrates. The genome sequence of Ruminococcus flavefaciens revealed a remarkably diverse cellulosome composed of more than 200 cellulosomal enzymes. Here we provide a detailed biochemical characterization of a highly elaborate R. flavefaciens cellulosomal enzyme containing an N-terminal dockerin module, which anchors the enzyme into the multi-enzyme complex through binding of cohesins located in non-catalytic cell-bound scaffoldins, and three tandemly repeated family 16 glycoside hydrolase (GH16) catalytic domains. The DNA sequence encoding the three homologous catalytic domains was cloned and hyper-expressed in Escherichia coli BL21 (DE3) cells. SDS-PAGE analysis of purified His6 tag containing RfGH16_21 showed a single soluble protein of molecular size ~89 kDa, which was in agreement with the theoretical size, 89.3 kDa. The enzyme RfGH16_21 exhibited activity over a wide pH range (pH 5.0-8.0) and a broad temperature range (50-70 °C), displaying maximum activity at an optimum pH of 7.0 and optimum temperature of 55 °C. Substrate specificity analysis of RfGH16_21 revealed maximum activity against barley ß-d-glucan (257 U mg-1) followed by lichenan (247 U mg-1), but did not show significant activity towards other tested polysaccharides, suggesting that it is specifically a ß-1,3-1,4-endoglucanase. TLC analysis revealed that RfGH16_21 hydrolyses barley ß-d-glucan to cellotriose, cellotetraose and a higher degree of polymerization of gluco-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a fairly high, active and thermostable bacterial endo-glucanase which may find considerable biotechnological potentials.