COVID-19 Severity Shifts the Cytokine Milieu Toward a Proinflammatory State in Egyptian Patients: A Cross-Sectional Study.

Despite extensive research to decipher the immunological basis of coronavirus disease (COVID-19), limited evidence on immunological correlates of COVID-19 severity from MENA region and Egypt was reported. In a single-center cross-sectional study, we have analyzed 25 cytokines that are related to immunopathologic lung injury, cytokine storm, and coagulopathy in plasma samples from 78 hospitalized Egyptian COVID-19 patients in Tanta University Quarantine Hospital and 21 healthy control volunteers between April 2020 and September 2020. The enrolled patients were divided into 4 categories based on disease severity, namely mild, moderate, severe, and critically ill. Interestingly, interleukin (IL)-1-α, IL-2Rα, IL-6, IL-8, IL-18, tumor necrosis factor-alpha (TNF-α), FGF1, CCL2, and CXC10 levels were significantly altered in severe and/or critically ill patients. Moreover, principal component analysis (PCA) demonstrated that severe and critically ill COVID-19 patients cluster based on specific cytokine signatures that distinguish them from mild and moderate COVID-19 patients. Specifically, levels of IL-2Rα, IL-6, IL-10, IL-18, TNF-α, FGF1, and CXCL10 largely contribute to the observed differences between early and late stages of COVID-19 disease. Our PCA showed that the described immunological markers positively correlate with high D-dimer and C-reactive protein levels and inversely correlate with lymphocyte counts in severe and critically ill patients. These data suggest a disordered immune regulation, particularly in severe and critically ill Egyptian COVID-19 patients, manifested as overactivated innate immune and dysregulated T-helper1 responses. Additionally, our study emphasizes the importance of cytokine profiling to identify potentially predictive immunological signatures of COVID-19 disease severity.

A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases.

The quantitation of liberated reducing sugars by the copper-bicinchoninic acid (BCA) assay provides a highly sensitive method for the measurement of glycoside hydrolase (GH) activity, particularly on soluble polysaccharide substrates. Here we describe a straightforward method adapted to low-volume polymerase chain reaction (PCR) tubes that enables the rapid, parallel determination of GH kinetics in applications ranging from initial activity screening and assay optimization to precise Michaelis-Menten analysis.

Efficient adsorption of cesium cations and chromate anions by one-step process using surfactant-modified zeolite.

Natural zeolite is organically modified with the surfactant cetyltrimethylammonium bromide (CTAB) and employed as a dual-function material for simultaneous adsorption of Cs+ cations and HCrO4- anions from aqueous solutions. Unmodified and modified zeolites are characterized by Fourier transform infrared (FTIR), dynamic light scattering (DLS), nitrogen adsorption-desorption isotherms, and X-ray diffraction (XRD). The results showed that CTAB-zeolite had the efficiency to simultaneously adsorb the concerned species in the pH range 2.5-4.2. The kinetic data showed that 90 and 300 min for Cs(I) and Cr(VI), respectively, were sufficient to attain equilibrium and the data are well-fitted by the double-exponential kinetic model. Of the studied adsorption isotherm models, Redlich-Peterson was the best one for describing the equilibrium adsorption isotherms. Values of ∆H°, ∆S°, and ∆G° for the present adsorption processes are estimated. CTAB-zeolite exhibited adsorption capacities of 0.713 and 1.216 mmol/g for Cs(I) and Cr(VI), respectively, which are comparable with the data reported in the literature. The adsorption mechanism of the concerned (radio)toxicants is proposed.

Elastoplastic Indentation Response of Sigmoid/Power Functionally Graded Ceramics Structures.

Due to the applicability of new advanced functionally graded materials (FGMs) in numerous tribological systems, this manuscript aims to present computational and empirical indentation models to investigate the elastoplastic response of FG substrate under an indention process with spherical rigid punch. The spatial variation of the ceramic volume fraction through the specimen thickness is portrayed using the power law and sigmoid functions. The effective properties of two-constituent FGM are evaluated by employing a modified Tamura-Tomota-Ozawa (TTO) model. Bilinear hardening behavior is considered in the analysis. The finite element procedure is developed to predict the contact pressure, horizontal displacement, vertical deformation, and permanent deformation of FG structure under the rigid cylindrical indentation. The empirical forms for permanent deformation were evaluated and assigned. Model validation with experimental works was considered. The convergence of the mesh and solution procedure was checked. Numerical studies were performed to illustrate the influence of gradation function, gradation index, and indentation parameters on the contact pressure, von Mises stresses, horizontal/vertical displacements, and permanent plastic deformation. The present model can help engineers and designers in the selection of an optimum gradation function and gradation index based on their applications.

Predictive Computational Model for Damage Behavior of Metal-Matrix Composites Emphasizing the Effect of Particle Size and Volume Fraction.

In this paper, an integrated numerical model is proposed to investigate the effects of particulate size and volume fraction on the deformation, damage, and failure behaviors of particulate-reinforced metal matrix composites (PRMMCs). In the framework of a random microstructure-based finite element modelling, the plastic deformation and ductile cracking of the matrix are, respectively, modelled using Johnson-Cook constitutive relation and Johnson-Cook ductile fracture model. The matrix-particle interface decohesion is simulated by employing the surface-based-cohesive zone method, while the particulate fracture is manipulated by the elastic-brittle cracking model, in which the damage evolution criterion depends on the fracture energy cracking criterion. A 2D nonlinear finite element model was developed using ABAQUS/Explicit commercial program for modelling and analyzing damage mechanisms of silicon carbide reinforced aluminum matrix composites. The predicted results have shown a good agreement with the experimental data in the forms of true stress-strain curves and failure shape. Unlike the existing models, the influence of the volume fraction and size of SiC particles on the deformation, damage mechanism, failure consequences, and stress-strain curve of A359/SiC particulate composites is investigated accounting for the different possible modes of failure simultaneously.

The role of trans-thoracic echocardiography in the assessment of aortic annular diameter.

ABSTRACT: We aimed to compare two-dimension transthoracic echocardiogram (2D-TTE) and three-dimension transthoracic echocardiogram (3D-TTE) measurements of the aortic annular diameter using multi-detector CT (MDCT) as a gold standard.This prospective observational study included 50 consecutive patients who came to the cardiology department, Al-Azhar University Hospital, New Damietta, for MDCT coronary angiography. The study was carried out in the period from July 2016 until February 2017. All patients were subjected to informed consent, clinical history, physical examination, transthoracic echocardiography 2D and 3D, and MDCT.The aortic annular areas measured by MDCT and 3D-TTE were significantly larger than areas by 2D-TTE. A good correlation (r = 0.82) was observed between the areas obtained by 3D-TTE and MDCT; however, the correlation between the values by 2D-TTE and MDCT was rough (r = 0.30). Eccentricity Index (EI) values in 28% of the patients were greater than 0.1, that is, the aortic annulus was elliptical.Accuracy of aortic annular diameter measurement by 3D-TTE was superior to that by 2D-TTE. Three-D TTE and MDCT revealed that the shape of the aortic annulus was elliptical in 28% to 30% respectively of study subjects. There is a strong concordance between the minimum and the maximum diameter determine by 3D-TTE and MDCT.

New Family of Carbohydrate-Binding Modules Defined by a Galactosyl-Binding Protein Module from a Cellvibrio japonicus Endo-Xyloglucanase.

Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, for example, by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is a valuable source for CAZyme and CBM discovery and characterization due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (X181), and a glycoside hydrolase family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, CjGH5F, is an efficient and specific endo-xyloglucanase (M. A. Attia, C. E. Nelson, W. A. Offen, N. Jain, et al., Biotechnol Biofuels 11:45, 2018, https://doi.org/10.1186/s13068-018-1039-6). In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest products of CjGH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes. IMPORTANCE This study reveals carbohydrate-binding module family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the carbohydrate-active enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.

Heterogeneity Aware Two-Stage Group Testing.

Group testing refers to the process of testing pooled samples to reduce the total number of tests. Given the current pandemic, and the shortage of test supplies for COVID-19, group testing can play a critical role in time and cost efficient diagnostics. In many scenarios, samples collected from users are also accompanied with auxiliary information (such as demographics, history of exposure, onset of symptoms). Such auxiliary information may differ across patients, and is typically not considered while designing group testing algorithms. In this paper, we abstract such heterogeneity using a model where the population can be categorized into clusters with different prevalence rates. The main result of this work is to show that exploiting knowledge heterogeneity can further improve the efficiency of group testing. Motivated by the practical constraints and diagnostic considerations, we focus on two-stage group testing algorithms, where in the first stage, the goal is to detect as many negative samples by pooling, whereas the second stage involves individual testing to detect any remaining samples. For this class of algorithms, we prove that the gain in efficiency is related to the concavity of the number of tests as a function of the prevalence. We also show how one can choose the optimal pooling parameters for one of the algorithms in this class, namely, doubly constant pooling. We present lower bounds on the average number of tests as a function of the population heterogeneity profile, and also provide numerical results and comparisons.

Permeation-Enhancing Nanoparticle Formulation to Enable Oral Absorption of Enoxaparin.

This study tests the hypothesis that association complexes formed between enoxaparin and cetyltrimethylammonium bromide (CTAB) augment permeation across the gastrointestinal mucosa due to improved encapsulation of this hydrophilic macromolecule within biocompatible poly (lactide-co-glycolide, PLGA RG 503) nanoparticles. When compared with free enoxaparin, association with CTAB increased drug encapsulation efficiency within PLGA nanoparticles from 40.3 ± 3.4 to 99.1 ± 1.0%. Drug release from enoxaparin/CTAB PLGA nanoparticles was assessed in HBSS, pH 7.4 and FASSIFV2, pH 6.5, suggesting effective protection of PLGA-encapsulated enoxaparin from unfavorable intestinal conditions. The stability of the enoxaparin/CTAB ion pair complex was pH-dependent, resulting in more rapid dissociation under simulated plasma conditions (i.e., pH 7.4) than in the presence of a mild acidic gastrointestinal environment (i.e., pH 6.5). The intestinal flux of enoxaparin complexes across in vitro Caco-2 cell monolayers was greater when encapsulated within PLGA nanoparticles. Limited changes in transepithelial transport of PLGA-encapsulated enoxaparin complexes in the presence of increasing CTAB concentrations suggest a significant contribution of size-dependent passive diffusion as the predominant transport mechanism facilitating intestinal absorption. Graphical abstract.

Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74.

Glycoside hydrolase family 74 (GH74) is a historically important family of endo-ß-glucanases. On the basis of early reports of detectable activity on cellulose and soluble cellulose derivatives, GH74 was originally considered to be a "cellulase" family, although more recent studies have generally indicated a high specificity toward the ubiquitous plant cell wall matrix glycan xyloglucan. Previous studies have indicated that GH74 xyloglucanases differ in backbone cleavage regiospecificities and can adopt three distinct hydrolytic modes of action: exo, endo-dissociative, and endo-processive. To improve functional predictions within GH74, here we coupled in-depth biochemical characterization of 17 recombinant proteins with structural biology-based investigations in the context of a comprehensive molecular phylogeny, including all previously characterized family members. Elucidation of four new GH74 tertiary structures, as well as one distantly related dual seven-bladed ß-propeller protein from a marine bacterium, highlighted key structure-function relationships along protein evolutionary trajectories. We could define five phylogenetic groups, which delineated the mode of action and the regiospecificity of GH74 members. At the extremes, a major group of enzymes diverged to hydrolyze the backbone of xyloglucan nonspecifically with a dissociative mode of action and relaxed backbone regiospecificity. In contrast, a sister group of GH74 enzymes has evolved a large hydrophobic platform comprising 10 subsites, which facilitates processivity. Overall, the findings of our study refine our understanding of catalysis in GH74, providing a framework for future experimentation as well as for bioinformatics predictions of sequences emerging from (meta)genomic studies.

Correction: Synthesis and application of a highly branched, mechanism-based 2-deoxy-2-fluoro-oligosaccharide inhibitor of endo-xyloglucanases.

Correction for 'Synthesis and application of a highly branched, mechanism-based 2-deoxy-2-fluoro-oligosaccharide inhibitor of endo-xyloglucanases' by Namrata Jain et al., Org. Biomol. Chem., 2018, 16, 8732-8741.

Hydroxyapatite/NiFe2O4 superparamagnetic composite: Facile synthesis and adsorption of rare elements.

A magnetic hydroxyapatite composite (CaHAP/NF) derived from calcium hydroxyapatite [Ca10(PO4)6(OH)2] and nickel ferrite [NiFe2O4] was successfully synthesized by a coprecipitation method. The synthesized composite was characterized using Fourier transform infrared spectroscopy (FT-IR), X-Ray diffractometer (XRD), thermogravimetric differential thermal analysis (TG-DTA), scanning electron microscopy (SEM) and vibrating sample magnetometer (VSM). Results clarify that the composite is a crystalline in nature, thermally stable up to 800 °C and possesses a high porous structure. The synthesized CaHAP/NF composite is a superparamagnetic material easily separated from aqueous solutions and would dissociate to some extent in strongly acidic conditions. The synthesized material was successfully applied as a solid phase for separation of Eu(III) and Tb(III) ions from aqueous solutions. The effect of various parameters (e.g. solution pH, equilibrium time and ionic strength) on sorption process was studied in static conditions. The synthesized sorbent could be considered as an efficient candidate for separation and recovery of Eu(III) and Tb(III). The sorption process was very fast initially, reached equilibrium within 6 h of contact and independent of ionic strength. The maximum sorption capacity values were 137.35 and 130.43 mg g-1 for Eu(III) and Tb(III), respectively. Desorption of Eu(III) and Tb(III) from loaded sample was studied using various eluents and maximum recovery was obtained using FeCl3 and EDTA solutions. More importantly, both FeCl3 and EDTA were individually applied as eluents in chromatographic separation of Eu(III) and Tb(III) in CaHAP/NF packed column and the best separation results were obtained by EDTA.

Synthesis and application of a highly branched, mechanism-based 2-deoxy-2-fluoro-oligosaccharide inhibitor of endo-xyloglucanases.

Xyloglucan (XyG) is a complex polysaccharide that is ubiquitous and often abundant in the cell walls of terrestrial plants. XyG metabolism is therefore a key component of the global carbon cycle, and hence XyG enzymology is of significant fundamental and applied importance in biomass conversion. To facilitate structure-function analyses of XyG-specific endo-glucanases, we have synthesized a 2',4'-dinitrophenyl 2-deoxy-2-fluoro-ß-glycoside mechanism-based inhibitor based on the highly branched XyG repeating motif XXXG (Xyl3Glc4: ([α-d-Xylp-(1→6)]-ß-d-Glcp-(1→4)-[α-d-Xylp-(1→6)]-ß-d-Glcp-(1→4)-[α-d-Xylp-(1→6)]-ß-d-Glcp-(1→4)-d-Glcp. Key steps in the chemo-enzymatic synthesis included selective enzyme hydrolysis of XyG polysaccharide to produce the core heptasaccharide, per-O-acetylation, α-bromination, reductive glycal formation, electrophilic fluorination, SNAr glycosylation, and Zemplen deprotection. The resulting compound, XXXG(2F)-ß-DNP, specifically labelled the active sites of several endo-(xylo)glucanases by accumulation of a covalent glycosyl-enzyme intermediate, as revealed by intact protein mass spectrometry. Crystallography of a complex with a Cellvibrio japonicus Glycoside Hydrolase Family 5 (GH5) endo-xyloglucanase corroborated the covalent nature of the intermediate, and further revealed the anticipated specificity for the catalytic nucleophile of this anomeric-configuration-retaining glycosidase. This specificity complements that of an analogous XXXG N-bromoacetylglycosylamine inhibitor, which labelled the catalytic acid-base sidechain in the same enzyme [Attia, et al., Biotechnol. Biofuels, 2018, 11, 45]. We anticipate that these inhibitors may find continued use in mechanistic analyses of endo-(xylo)glucanases from diverse GH families.

Fluorouracil-Loaded Gold Nanoparticles for the Treatment of Skin Cancer: Development, in Vitro Characterization, and in Vivo Evaluation in a Mouse Skin Cancer Xenograft Model.

Fluorouracil (5-FU) is an antimetabolite drug used in the treatment of various malignancies, such as colon and skin cancers. However, its systemic administration results in severe side effects. Topical 5-FU delivery for the treatment of skin cancer could circumvent these shortcomings, but it is limited by the drug poor permeability through the skin. To enhance 5-FU efficacy against skin cancer and reduce its systemic side effects, it was loaded into a gold nanoparticle (GNP)-based topical delivery system. 5-FU was loaded onto GNPs capped with CTAB through ionic interactions between 5-FU and CTAB. GNPs were prepared at different 5-FU/CTAB molar ratios and evaluated using different techniques. GNP stability and drug release were studied as a function of salt concentration and solution pH. Optimum 5-FU/CTAB-GNPs were incorporated into gel and cream bases, and their ex vivo permeability was evaluated in mice dorsal skin. The in vivo anticancer efficacy of the same preparations was evaluated in A431 tumor-bearing mice. The GNPs had spherical shape and a size of ∼16-150 nm. Maximum 5-FU entrapment was achieved at 5-FU/CTAB molar ratio of 1:1 and pH 11.5. Drug release from GNPs was sustained and pH-dependent. 5-FU GNP gel and cream had around 2-fold higher permeability through mice skin compared with free 5-FU gel and cream formulations. Further, in vivo studies in a mouse model having A431 skin cancer cells implanted in the subcutaneous space showed that the GNP gel and cream achieved 6.8- and 18.4-fold lower tumor volume compared with the untreated control, respectively. These results confirm the potential of topical 5-FU/CTAB-GNPs to enhance drug efficacy against skin cancer.

In vitro and in vivo characterization of three Cellvibrio japonicus glycoside hydrolase family 5 members reveals potent xyloglucan backbone-cleaving functions.

BACKGROUND: Xyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved. RESULTS: Combined bioinformatic and transcriptomic analyses implicated three glycoside hydrolase family 5 subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here. CONCLUSIONS: Through a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural-functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatic predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications.

Algorithm of Laparoscopic Technique in Pediatric Inguinal Hernia: Results from Experience of 10 Years.

BACKGROUND: Many laparoscopic techniques have been evolved along years for pediatric inguinal hernia (PIH) with no standardization of technique. No single technique suits all varieties of hernia. PURPOSE: To propose an algorithm for allocation of PIH to laparoscopic technique based on internal ring (IR) diameter to improve outcomes. PATIENTS AND METHODS: Along 10 years, 459 cases with unilateral PIH were treated in Tanta University Hospital. In the first 5 years (phase I), 214 cases included then an algorithm for stratification was designed and applied in the second 5-year period (phase II), where 245 cases managed. This algorithm included evaluation of the hernia based on IR diameter as measured by the laparoscope from inside by a piece of suture. When the IR diameter is from 4 to 15 mm, complete sac disconnection is used. When IR diameter lies from 15 to 25 mm a purse string is added. When IR diameter is >2.5 cm or recurrent cases, the interrupted muscular arch repair after sac disconnection is used. RESULTS: In phase I, 170 boys and 44 girls from 6 to 180 months of age were treated. All cases managed by laparoscopy were 84 herniotomy, 82 by purse string, and 44 by interrupted muscular arch. In phase II, 180 boys and 65 girls from 3 to 180 months of age were included. Eighty were managed by herniotomy, 137 by purse string, and 25 by interrupted muscular arch. Recurrence rate decreased significantly in phase II. CONCLUSIONS: Application of Tanta algorithm reduces the recurrence rate significantly. The laparoscopic technique should be tailored according to criteria of each group of PIH to get the best outcome and reduced recurrence rate.

High payload nanostructured lipid carriers fabricated with alendronate/polyethyleneimine ion complexes.

Oral bioavailability of the anti-osteoporotic drug alendronate (AL) is limited to ≤ 1% due to unfavorable physicochemical properties. To augment absorption across the gastrointestinal mucosa, an ion pair complex between AL and polyethyleneimine (PEI) was formed and incorporated into nanostructured lipid carriers (NLCs) using a modified solvent injection method. When compared to free AL, ion pairing with PEI increased drug encapsulation efficiency in NLCs from 10% to 87%. Drug release from NLCs measured in vitro using fasted state simulated intestinal fluid, pH 6.5 (FaSSIF-V2) was significantly delayed after PEI complexation. Stability of AL/PEI was pH-dependent resulting in 10-fold faster dissociation of AL in FaSSIF-V2 than measured at pH 7.4. Intestinal permeation properties estimated in vitro across Caco-2 cell monolayers revealed a 3-fold greater flux of AL encapsulated as hydrophobic ion complex in NLCs when compared to AL solution (Papp = 8.43 ± 0.14 × 10-6 cm/s and vs. 2.76 ± 0.42 × 10-6 cm/s). Cellular safety of AL/PEI-containing NLCs was demonstrated up to an equivalent AL concentration of 2.5 mM. These results suggest that encapsulation of AL/PEI in NLCs appears a viable drug delivery strategy for augmenting oral bioavailability of this clinically relevant bisphosphonate drug and, simultaneously, increase gastrointestinal safety.

Comprehensive functional characterization of the glycoside hydrolase family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification.

Lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), ß(1→3)/ß(1→4) mixed-linkage glucan (MLG) and ß(1→3) glucan components of lignocellulose represent significant carbohydrate energy sources for saprophytic microorganisms. The bacterium Cellvibrio japonicus has a robust capacity for plant polysaccharide degradation, due to a genome encoding a large contingent of Carbohydrate-Active enZymes (CAZymes), many of whose specific functions remain unknown. Using a comprehensive genetic and biochemical approach, we have delineated the physiological roles of the four C. japonicus glycoside hydrolase family 3 (GH3) members on diverse ß-glucans. Despite high protein sequence similarity and partially overlapping activity profiles on disaccharides, these ß-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports ß(1→3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives ß(1→3) glucan utilization. Finally, Bgl3D is the crucial ß-glucosidase for XyG utilization. This study not only sheds the light on the metabolic machinery of C. japonicus, but also expands the repertoire of characterized CAZymes for future deployment in biotechnological applications. In particular, the precise functional analysis provided here serves as a reference for informed bioinformatics on the genomes of other Cellvibrio and related species.

Gold nanoparticles capped with benzalkonium chloride and poly (ethylene imine) for enhanced loading and skin permeability of 5-fluorouracil.

OBJECTIVE: To enhance 5-fluorouracil (5-FU) permeability through the skin by loading onto gold nanoparticles (GNPs) capped with two cationic ligands, benzalkonium chloride (BC) or poly (ethylene imine) (PEI). Whereas 5-FU has excellent efficacy against many cancers, its poor permeability through biological membranes and several adverse effects limit its clinical benefits. BC and PEI were selected to stabilize GNPs and to load 5-FU through ionic interactions. METHODS: 5-FU/BC-GNPs and 5-FU/PEI-GNPs were prepared at different 5-FU/ligand molar ratios and different pH values and were evaluated using different techniques. GNPs stability was tested as a function of salt concentration and storage time. 5-FU release from BC- and PEI-GNPs was evaluated as a function of solution pH. Ex vivo permeability studies of different 5-FU preparations were carried out using mice skin. RESULTS: 5-FU-loaded GNPs size and surface charge were dependent on the 5-FU/ligand molar ratios. 5-FU entrapment efficiency and loading capacity were dependent on the used ligand, 5-FU/ligand molar ratio and solution pH. Maximum drug entrapment efficiency of 59.0 ± 1.7% and 46.0 ± 1.1% were obtained for 5-FU/BC-GNPs and 5-FU/PEI-GNPs, respectively. 5-FU-loaded GNPs had good stability against salinity and after storage for 4 months at room temperature and at 4 °C. In vitro 5-FU release was pH- and ligand-dependent where slower release was observed at higher pH and for 5-FU/BC-GNPs. 5-FU permeability through mice skin was significantly higher for drug-loaded GNPs compared with drug-ligand complex or drug aqueous solution. CONCLUSION: Based on these results, BC- and PEI-GNPs might find applications as effective topical delivery systems of 5-FU.

A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases.

The quantitation of liberated reducing sugars by the copper-bicinchoninic acid (BCA) assay provides a highly sensitive method for the measurement of glycoside hydrolase (GH) activity, particularly on soluble polysaccharide substrates. Here, we describe a straightforward method adapted to low-volume polymerase chain reaction (PCR) tubes which enables the rapid, parallel determination of GH kinetics in applications ranging from initial activity screening and assay optimization, to precise Michaelis-Menten analysis.