Publications by authors named "Mohamed A Attia"

23 Publications

  • Page 1 of 1

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

Medicine (Baltimore) 2021 Mar;100(9):e24682

Faculty of Medicine, Al-Azhar University, Damietta.

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.
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http://dx.doi.org/10.1097/MD.0000000000024682DOI Listing
March 2021

Characterization of a galactosyl-binding protein module from a endo-xyloglucanase defines a new family of Carbohydrate Binding Modules.

Appl Environ Microbiol 2020 Dec 18. Epub 2020 Dec 18.

Michael Smith Laboratories, Department of Chemistry, Department of Botany, and Department of Biochemistry and Molecular Biology, University of British Columbia, Canada

Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, by increasing substrate affinity. The Gram-negative soil saprophyte is 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 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, GH5F, is an efficient and specific endo-xyloglucanase [Attia 2018. , : 45]. 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 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 product of GH5F. 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 , this study will guide future bioinformatic and functional analyses across microbial (meta)genomes. 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.
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http://dx.doi.org/10.1128/AEM.02634-20DOI Listing
December 2020

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

AAPS PharmSciTech 2020 Feb 3;21(3):88. Epub 2020 Feb 3.

James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, Ohio, USA.

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.
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http://dx.doi.org/10.1208/s12249-020-1618-2DOI Listing
February 2020

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

J Biol Chem 2019 09 19;294(36):13233-13247. Epub 2019 Jul 19.

Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, British Columbia V6T 1Z4, Canada

Glycoside hydrolase family 74 (GH74) is a historically important family of -β-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: , -dissociative, and -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 β-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.
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http://dx.doi.org/10.1074/jbc.RA119.009861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737227PMC
September 2019

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

Appl Radiat Isot 2019 Mar 5;145:85-94. Epub 2018 Dec 5.

Chemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt.

A magnetic hydroxyapatite composite (CaHAP/NF) derived from calcium hydroxyapatite [Ca(PO)(OH)] and nickel ferrite [NiFeO] 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 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 FeCl and EDTA solutions. More importantly, both FeCl 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.
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http://dx.doi.org/10.1016/j.apradiso.2018.12.003DOI Listing
March 2019

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

Org Biomol Chem 2019 01;17(2):398

Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, British Columbia V6T 1Z4, Canada.

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.
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http://dx.doi.org/10.1039/c8ob90185fDOI Listing
January 2019

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

Org Biomol Chem 2018 11;16(45):8732-8741

Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, British Columbia V6T 1Z4, Canada. and Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada and Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.

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-β-glycoside mechanism-based inhibitor based on the highly branched XyG repeating motif XXXG (Xyl3Glc4: ([α-d-Xylp-(1→6)]-β-d-Glcp-(1→4)-[α-d-Xylp-(1→6)]-β-d-Glcp-(1→4)-[α-d-Xylp-(1→6)]-β-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)-β-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.
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http://dx.doi.org/10.1039/c8ob02250jDOI Listing
November 2018

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.

Mol Pharm 2018 06 7;15(6):2194-2205. Epub 2018 May 7.

Department of Pharmaceutics, Faculty of Pharmacy , Assiut University , Assiut 71526 , Egypt.

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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00047DOI Listing
June 2018

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

Biotechnol Biofuels 2018 17;11:45. Epub 2018 Feb 17.

1Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4 Canada.

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 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 have been functionally characterized, the requisite backbone-cleaving -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 -xyloglucanases in . 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 -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 . Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the -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 . The detailed structural-functional characterization of three distinct GH5_4 -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.
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http://dx.doi.org/10.1186/s13068-018-1039-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816542PMC
February 2018

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

J Laparoendosc Adv Surg Tech A 2018 Jun 9;28(6):755-759. Epub 2018 Feb 9.

Pediatric Surgery, Department of Surgery, Faculty of Medicine, Tanta University , Tanta, Egypt .

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.
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http://dx.doi.org/10.1089/lap.2017.0273DOI Listing
June 2018

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

Int J Pharm 2018 Jan 8;535(1-2):148-156. Epub 2017 Nov 8.

James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH 45267 USA. Electronic address:

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 (P = 8.43 ± 0.14 × 10 cm/s and vs. 2.76 ± 0.42 × 10 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.
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http://dx.doi.org/10.1016/j.ijpharm.2017.10.064DOI Listing
January 2018

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

Environ Microbiol 2017 Dec 7;19(12):5025-5039. Epub 2017 Dec 7.

Department of Biological Sciences, University of Maryland, Baltimore County, MD, USA.

Lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), β(1→3)/β(1→4) mixed-linkage glucan (MLG) and β(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 β-glucans. Despite high protein sequence similarity and partially overlapping activity profiles on disaccharides, these β-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports β(1→3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives β(1→3) glucan utilization. Finally, Bgl3D is the crucial β-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.
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http://dx.doi.org/10.1111/1462-2920.13959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858740PMC
December 2017

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

Drug Dev Ind Pharm 2017 Nov 21;43(11):1780-1791. Epub 2017 Jun 21.

a Department of Pharmaceutics, Faculty of Pharmacy , Assiut University , Assiut , Egypt.

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.
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http://dx.doi.org/10.1080/03639045.2017.1339082DOI Listing
November 2017

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

Methods Mol Biol 2017 ;1588:3-14

Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4, Canada.

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.
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http://dx.doi.org/10.1007/978-1-4939-6899-2_1DOI Listing
April 2017

Gold nanoparticles enhance 5-fluorouracil anticancer efficacy against colorectal cancer cells.

Int J Pharm 2016 Nov 29;513(1-2):648-658. Epub 2016 Sep 29.

Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt.

5-Fluorouracil (5-FU), an antimetabolite drug, is extensively used in the treatment solid tumors. However, its severe side effects limit its clinical benefits. To enhance 5-FU anticancer efficacy and reduce its side effects it was loaded onto gold nanoparticles (GNPs) using two thiol containing ligands, thioglycolic acid (TGA) and glutathione (GSH). The GNPs were prepared at different 5-FU/ligand molar ratios and evaluated using different techniques. Anticancer efficacy of 5-FU/GSH-GNPs was studied using flow cytometry in cancerous tissue obtained from patients having colorectal cancer. The GNPs were spherical in shape and had a size of ∼9-17nm. Stability of the GNPs and drug release were studied as a function of salt concentration and solution pH. Maximum 5-FU loading was achieved at 5-FU/ligand molar ratio of 1:1 and 2:1 for TGA-GNPs and GSH-GNPs, respectively. GNPs coating with pluronic F127 improved their stability against salinity. 5-FU release from GNPs was slow and pH-dependent. 5-FU/GSH-GNPs induced apoptosis and stopped the cell cycle progression in colorectal cancer cells. They also had a 2-fold higher anticancer effect compared with free 5-FU. These results confirm the potential of GNPs to enhance 5-FU anticancer efficacy.
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http://dx.doi.org/10.1016/j.ijpharm.2016.09.076DOI Listing
November 2016

Recent structural insights into the enzymology of the ubiquitous plant cell wall glycan xyloglucan.

Curr Opin Struct Biol 2016 10 28;40:43-53. Epub 2016 Jul 28.

Michael Smith Laboratories and Department of Chemistry, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4, Canada. Electronic address:

The xyloglucans (XyGs) constitute a family of highly decorated β(1→4)-glucans whose members are widespread and abundant across the plant kingdom. As such, XyGs constitute a significant reserve of metabolically accessible monosaccharides for diverse phytopathogenic, saprophytic, and gut symbiotic micro-organisms. To overcome the intrinsic stability of the diverse glycosidic bonds in XyGs, bacteria and fungi have evolved extensive repertoires of xyloglucan-active enzymes from manifold families, whose exquisitely adapted tertiary structures are recently coming to light.
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http://dx.doi.org/10.1016/j.sbi.2016.07.005DOI Listing
October 2016

Direct Detection of Burkholderia cepacia in Susceptible Pharmaceutical Products Using Semi-Nested PCR.

PDA J Pharm Sci Technol 2016 Mar-Apr;70(2):99-108. Epub 2016 Jan 21.

Microbiology and Immunology Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt; and.

Burkholderia cepaciahas recently received a considerable attention as one of the major risks in susceptible pharmaceutical products. This microorganism can easily propagate and cause vast and severe contamination, especially to the water supplies for pharmaceutical companies. Moreover, it proliferates within the products and can cause severe infections for humans. Therefore, fast and sensitive detection of these bacteria is of a great demand. The present study introduces improved application of a polymerase chain reaction assay with relatively high sensitivity and specificity for the direct detection ofB. cepaciafrom the aqueous pharmaceutical products. A semi-nested polymerase chain reaction approach using the primer set BCR1/BCR2 followed by BCR1/Mr yielding a 465 bp fragment of the recA gene was applied and tested using both crude lysate from isolated colonies and DNA directly extracted from artificially prepared and spiked reference syrup. The polymerase chain reaction assay showed no interference with other bacterial reference and environmental strains tested, includingStaphylococcus aureusATCC® 6538,Pseudomonas aeruginosaATCC® 9027,Escherichia coliATCC® 8739,Salmonella abonyNCTC® 6017,Bacillus subtilisATCC® 6633,Micrococcus luteus, Staphylococcus warneri, Pseudomonas fluorescens, Pseudomonas putida, andRalstonia pickettii Moreover, this semi-nested assay showed a detection limit of around 10 colony-forming units per sample and could detectB. cepaciastrains isolated from a municipal pre-treated potable water tank. Comparing the results for detection ofB. cepaciain 100 randomly collected commercial syrup preparations using both conventional standard method and polymerase chain reaction assay revealed thatB. cepaciawas detected in two samples using polymerase chain reaction assay while all samples showed negative results by conventional culturing and biochemical methods. These results highlight the advantage of using this polymerase chain reaction assay to detectB. cepaciain contaminated pharmaceutical products and even water for pharmaceutical purposes, without the need of culturing or pre-enrichment, where it may give false-negative results and may be misidentified when biochemically tested.
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http://dx.doi.org/10.5731/pdajpst.2015.006049DOI Listing
January 2018

Laparoscopic Interrupted Muscular Arch Repair in Recurrent Unilateral Inguinal Hernia Among Children.

J Laparoendosc Adv Surg Tech A 2015 Aug 6;25(8):675-80. Epub 2015 May 6.

Section of Pediatric Surgery, Department of Surgery, Faculty of Medicine, Tanta University , Tanta, Egypt .

Purpose: We present a procedure of suturing the transversus abdominis muscular arch to the ileopubic tract laparoscopically in order to repair recurrent unilateral pediatric inguinal hernia (PIH).

Patients And Methods: Twenty-five children with recurrent unilateral PIH were treated during a 5-year period in a tertiary academic center. All cases were subjected to laparoscopic hernia repair and discharged the next morning. Sutures were placed from the muscular arch to the ileopubic tract, avoiding the spermatic vessels and duct, in an interrupted manner using 2/0-3/0 polypropylene (Prolene®; Ethicon, Somerville, NJ) or polyglactin 910 (Vicryl®; Ethicon) sutures. In 4 cases, a rectangular purse-string-like suture was added to narrow the internal ring defect. Operative findings and postoperative results and complications were assessed. The patients were followed up for a period that ranged between 6 and 60 months.

Results: There were 23 boys and 2 girls. Operative age ranged between 18 months and 15 years. Three or four sutures were placed in each case. In 4 cases, an additional rectangular purse-string-like suture was added. Operative time ranged between 35 and 70 minutes, and there was no conversion. Mild scrotal edema was reported in 4 cases and port-site infection in 2 cases; all cases were treated conservatively. One case of recurrence among boys was reported, but there was no case of testicular atrophy. Cosmetic outcomes were excellent.

Conclusions: Laparoscopic interrupted muscular arch repair is a feasible and safe technique in the reconstruction of the inguinal canal in recurrent unilateral PIH. Larger studies and long-term follow-up are needed to support our encouraging results.
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http://dx.doi.org/10.1089/lap.2014.0305DOI Listing
August 2015

Poly(ethylene glycol)-block-poly(ε-caprolactone) nanomicelles for the solubilization and enhancement of antifungal activity of sertaconazole.

Curr Drug Deliv 2014 ;11(6):753-62

Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut, 71526, Egypt.

Sertaconazole nitrate is a broad spectrum imidazole antifungal agent with antibacterial and anti-inflammatory properties. However, its lipophilic nature and very poor aqueous solubility limit its use in the clinic. The aim of this study was to develop and characterize poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) polymeric nanomicelles for the solubilization and enhancement of sertaconazole antifungal activity. Sertaconazole was incorporated into PEG-b- PCL polymeric nanomicelles by a co-solvent evaporation method and micelle size, drug loading capacity and drug release properties were determined. The antifungal properties of nanomicelle-loaded drug were evaluated in Fusarium miscanthi, Microsporum canis, and Trichophyton mentagrophytes isolated, respectively from fungal keratitis, ringworm, and tinea corporis. PEG-b-PCL formed nanomicelles in aqueous solution with a diameter ranging from 40-80 nm, depending on the polymer composition and level of drug loading. Drug loading properties of the nanomicelles were dependent on the PCL block molecular weight and drug/polymer weight feed ratio. Drug encapsulation efficiency of up to 85% was achieved and this resulted in more than 80-fold enhancement in sertaconazole aqueous solubility at polymer concentration of 0.2%. Drug release studies showed an initial burst release followed by sustained drug release for 72 hours. In vitro antimycotic studies showed that nanomicelle-incorporated sertaconazole inhibited fungal growth in a concentration dependent manner. Further, it was more effective than the free drug in inhibiting the growth of Fusarium miscanthi and Microsporum canis. These results confirm the utility of PEG-b-PCL nanomicelles in enhancing the aqueous solubility and antifungal activity of sertaconazole or other similar antifungal drugs.
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http://dx.doi.org/10.2174/1567201811666140605151923DOI Listing
September 2015

HLA-G expression as a prognostic indicator in B-cell chronic lymphocytic leukemia.

Acta Haematol 2014 15;132(1):53-8. Epub 2014 Feb 15.

Department of Clinical Pathology, Tanta University, Faculty of Medicine, Tanta, Egypt.

Background: The expression of human leukocyte antigen (HLA)-G was studied in certain malignancies and its role in escaping from immunosurveillance in cancers was proposed since HLA-G is a non-conventional HLA class I molecule that protects the fetus from immunorecognition during pregnancy. Some particles involved in the regulation of an immune system might represent prognostic value for B-cell chronic lymphocytic leukemia (B-CLL). The identification of novel prognostic factors in B-CLL may help define patient subgroups that may benefit from early therapeutic intervention.

Objective: To evaluate the prognostic significance of HLA-G expression in B-CLL patients and its relationship with other well-established prognostic markers.

Methodology: Thirty B-CLL patients diagnosed by clinical, morphological and immunophenotyping criteria were studied for HLA-G expression by flow cytometry. The relationship between HLA-G expression and some known prognostic markers was evaluated.

Results: HLA-G was expressed in 36.7% of CLL patients at diagnosis, with a mean expression level of 35.31 ± 12.35%. A significant association between HLA-G expression and common prognostic markers of progressive disease was detected. The group of patients with positive HLA-G expression showed significantly higher absolute lymphocyte counts and serum levels of LDH and β2-microglobulin, lower platelet counts, positive CD38 expression and advanced stages of Binet clinical staging.

Conclusion: The present study demonstrated that HLA-G expression correlates with prognostic markers of a poor B-CLL outcome, mainly Binet clinical staging and CD38 expression by B-CLL cells, which indicates that this parameter may play a role as an important prognosticator of disease progression and consequently targeted therapy in B-CLL.
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http://dx.doi.org/10.1159/000353757DOI Listing
September 2014

Formulation and evaluation of metoclopramide solid lipid nanoparticles for rectal suppository.

J Pharm Pharmacol 2013 Nov 15;65(11):1607-21. Epub 2013 Sep 15.

Department of Pharmaceutical Technology, Faculty of Pharmacy and Biotechnology, German University in Cairo, GUC, Cairo, Egypt.

Objectives: The purpose of this study was to formulate and characterize metoclopramide solid lipid nanoparticles (MCP-SLNs) and incorporating it into suppository bases for treatment of nausea and vomiting, produced with chemotherapeutic agents, using one dose per day.

Methods: MCP-SLNs was prepared using high shear homogenization (hot homogenization) technique using different surfactants (tween 80, poloxamer 407, poloxamer 188 and cremophore) in two different concentrations (2.5% and 5%) then solid lipid nanoparticle (SLN), whose release percentage above 50%, was incorporated into suppository for treatment of nausea and vomiting. The prepared SLN and suppositories were then evaluated and characterized.

Key Findings: Formulation of poloxamer 407 with compritol and drug (F9) produced highest in-vitro % release (80%). Transmission electron microscopy showed that SLN had round and spherical shape in form of solid dispersion or drug-enriched core. Particle size analysis of SLN showed a size range of 24.99-396.8 nm. Negative zeta potential proves complete drug entrapment. In-vivo study of MCP-SLN suppositories produced the same %GE as the market metoclopramide (MCP) suppository (Primperan) with sustained release effect.

Conclusion: MCP-SLN suppositories (formula F) can reverse decrease in %GE because of emesis with sustained release effect. So it succeeded to be an alternative to MCP suppositories with no multiple dosing.
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http://dx.doi.org/10.1111/jphp.12136DOI Listing
November 2013

Prognostic value of soluble angiopoietin-2 and soluble Tie-2 in Egyptian patients with acute myeloid leukemia.

Turk J Haematol 2010 Dec;27(4):282-8

Clinical Pathology Department, Faculty of Medicine, Tanta University, 31511 Tanta, Egypt Phone: 0020403348954 E-mail:

Objective: Angiogenesis plays a critical role in the development and growth of solid tumors and hematologic malignancies. The system involving angiopoietin-2 [Ang-2] and its receptor Tie-2 appears to play an important role in tumor angiogenesis and in the biology of hematological and non-hematological malignancies. We evaluated the levels of soluble (s)Ang-2 and sTie-2 in acute myeloid leukemia (AML) patients and investigated the impact of their circulating levels on the overall survival in those patients.

Methods: Ang-2 and Tie-2 were measured in plasma samples from AML patients and controls using enzyme-linked immunosorbent assay (ELISA).

Results: The levels of sAng-2 and sTie-2 were significantly higher in AML patients (2382.1±1586.1 pg/ml and 6.74±3.47 ng/ml, respectively) than in controls (649.5±402.6 pg/ml and 2.63±0.57 ng/ml, respectively; p<0.01). AML patients with high levels of sAng-2 and sTie-2 (≥2500 pg/ml and ≥8 ng/ml, respectively) had significantly shorter overall survival than those patients with low levels (<2500 pg/ml and <8 ng/ml, respectively).

Conclusion: The results of our study demonstrated the prognostic significance of circulating sAng-2 and sTie-2 in AML patients. Modulation of the angiopoietin / Tie-2 axis may be a promising approach to improve the outcome in those patients.
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http://dx.doi.org/10.5152/tjh.2010.50DOI Listing
December 2010

Fidelity and reproducibility of antisense RNA amplification for the study of gene expression in human CD34+ haemopoietic stem and progenitor cells.

Br J Haematol 2003 Aug;122(3):498-505

Department of Cellular and Molecular Medicine, St. George's Hospital Medical School, London, UK.

Microarrays provide a powerful tool for the study of haemopoietic stem and progenitor cells (HSC). Because of the low frequency of HSC, it is rarely feasible to obtain enough mRNA for microarray hybridizations, and amplification will be necessary. Antisense RNA (aRNA) amplification is reported to give high-fidelity amplification, but most studies have used only qualitative validation. Before applying aRNA amplification to the study of HSC, we wished to determine its fidelity and reproducibility, and whether statistically significant results can be obtained. We found that aRNA amplification introduced biases into relative RNA abundance. However, these biases were extremely consistent, and valid comparisons could be made, if amplified RNA was compared with amplified RNA. By applying this method to the effect of interferon-gamma and tumour necrosis factor-alpha on normal primary CD34+ HSC, biologically significant differences could be detected, including potential mechanisms for resistance of CD34+ cells to CD95-mediated apoptosis and evidence of the differentiating effects of the cytokines. Differences of twofold or less were detected, and most of these differences attained statistical significance after triplicate experiments. These data demonstrate that aRNA amplification can be used with microarray hybridization to study the transcriptional profiles of small numbers of primary CD34+ HSC.
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http://dx.doi.org/10.1046/j.1365-2141.2003.04440.xDOI Listing
August 2003