Magdy A. Amin

A capacitive immunosensor for detection of cholera toxin.

Contamination of food with biological toxins as well as their potential use as weapons of mass destruction has created an urge for rapid and cost effective analytical techniques capable of detecting trace amounts of these toxins. This paper describes the development of a sensitive method for detection of cholera toxin (CT) using a flow-injection capacitive immunosensor based on self-assembled monolayers. The sensing surface consists of monoclonal antibodies against the B subunit of CT (anti-CT), immobilized on a gold transducer. Experimental results show that the immunosensor responded linearly to CT concentrations in the range from 1.0x10(-13) to 1.0x10(-10) M under optimized conditions. The limit of detection (LOD) was 1.0x10(-14) M. Two more analytical methods were employed for detection of CT using the same antibody namely, sandwich ELISA and surface plasmon resonance (SPR)-based immunosensor. The former had an LOD of 1.2x10(-12) M and a working range from 3.7x10(-11) to 2.9x10(-10) M whereas, the later had an LOD of 1.0x10(-11) M and a linearity ranging from 1.0x10(-9) to 1.0x10(-6) M. These results demonstrate that the developed capacitive immunosensor system has a higher sensitivity than the other two techniques. The binding affinity of CT to the immobilized anti-CT was determined using the SPR-based immunosensor and an association constant (K(A)) of 1.4x10(9) M(-1) was estimated.

Laccase production by Pleurotus ostreatus and its application in synthesis of gold nanoparticles

In this work, the production of fungal laccase was optimized from local isolate of Pleurotus ostreatus using solid state fermentation. Factorial design was used to study the effect of several nutrients on enzyme production. Purification and characterization of the enzyme and the effect of temperature, pH and gamma radiation on fungal growth and enzyme production was investigated. Optimization of production conditions yielded an enzyme with activity over 32,450 IU/g of fermented substrate. Factorial design was capable of establishing the conditions that multiplied the activity of the enzyme several folds, consequently, reducing the cost of production. The enzyme was capable of decolorizing several dyes with over 80% reduction in color confirming the aromatic degrading capability of laccase. The enzyme was also used in the synthesis of gold nanoparticles, proving that laccase from Pleurotus ostreatus has a strong potential in several industrial applications.

A novel competitive capacitive glucose biosensor based on concanavalin A-labeled nanogold colloids assembled on a polytyramine-modified gold electrode

A highly sensitive competitive capacitive glucose biosensor was constructed based on gold nanoparticles, which were employed as a platform to immobilize concanavalin A (Con A). Gold nanoparticles were fixed on a gold electrode, on which a layer of polytyramine was preformed via electrochemical polymerization. The sensing mechanism is based on the competitive dissociation of a glucose polymer or a glycoconjugate from the glycoligand binding sites of immobilized Con A by the added glucose. To further improve the sensor response, several glucose polymers as well as a synthesized glycoconjugate using the periodate method, were screened. Consequently, dextran (MW 39 kDa) was selected and the feasibility of the proposed biosensor was evaluated for a competitive assay of glucose. Experimental results show that the biosensor responded linearly to glucose in the range from 1.0 x 10(-6) to 1.0 x 10(-2) M, corresponding to 0.18 microg mL(-1) to 1.8 mg mL(-1) of glucose with a detection limit of 1.0 x 10(-6) M under optimized conditions. The studied biosensor exhibited a response time of about 15 min and a neglectable loss in sensitivity after 10 repeated analytical cycles.

Kinetics and metabolic versatility of highly tolerant phenol degrading Alcaligenes strain TW1.

A bacterium that could completely metabolize phenol in batch culture supplied with up to 1200 mg phenol l(-1) at room temperature (25 degrees C) was isolated from the activated sludge of the industrial wastewater treatment plant of a Coke company (Cairo, Egypt). Morphological and physiological characterization showed strain TW1 was a motile, strictly aerobic, gram negative and short-rod occurring singly or in clusters. Partial 16S rRNA gene sequence analysis revealed strain TW1 belonged to the beta group of Proteobacteria, showing 100% identity to Alcaligenes SCTI. Strain TW1 aerobically grew on a number of monocyclic aromatic compounds (hydroquinone, catechol and o-cresol) as well as polycyclic aromatic compounds (pyrene, phenanthrene and naphthalene). The growth of Alcaligenes TW1 on phenol as sole carbon and energy source (25 degrees C) was well described by the Haldane kinetics model with a maximal specific growth rate of 0.58 h(-1), a half-saturation constant of 10 mg l(-1), and a substrate inhibition constant of 152-550 mg l(-1). The biomass yield coefficient ranged from 0.55 to 0.64 mg dry cell mass/mg phenol. Due to its high tolerance to phenol and high metabolic versatility, Alcaligenes sp. TW1 is considered an excellent candidate for the biotreatment of high strength phenol-laden industrial wastewaters.

A multipurpose capacitive biosensor for assay and quality control of human immunoglobulin G.

We report a flow‐injection biosensor system with a capacitive transducer for assay and quality control of human immunoglobulin G (hIgG). The sensing platform is based on self‐assembled monolayers (SAMs) of carboxylic acid terminated alkyl‐thiols with covalently attached concanavalin A. The electrochemical characteristics of the sensor surface were assessed by cyclic voltammetry using a permeable redox couple (potassium ferricyanide). The developed biosensor proved capable of performing a sensitive label‐free assay of hIgG with a detection limit of 1.0 µg mL−1. The capacitance response depended linearly on hIgG concentration over the range from 5.0 to 100 µg mL−1, in a logarithmic plot. Typical measurements were performed in 15 min and up to 18 successive assays were achieved without significant loss of sensitivity using a single electrode. In addition, the biosensor can detect hIgG aggregates with concentrations as low as 0.01% of the total hIgG content (5.0 µg mL−1). Hence, it represents a potential post‐size‐exclusion chromatography–UV (post‐SEC–UV) binding assay for in‐process quality control of hIgG, which cannot be detected by SEC–UV singly at concentrations below 0.3% of the total hIgG content. Biotechnol. Bioeng. 2009; 104: 312–320

Antimicrobial Resistance Pattern and Their Beta-Lactamase Encoding Genes among Pseudomonas aeruginosa Strains Isolated from Cancer Patients

This study was designed to investigate the prevalence of metallo-β-lactamases (MBL) and extended-spectrum β-lactamases (ESBL) in P. aeruginosa isolates collected from two different hospitals in Cairo, Egypt. Antibiotic susceptibility testing and phenotypic screening for ESBLs and MBLs were performed on 122 P. aeruginosa isolates collected in the period from January 2011 to March 2012. MICs were determined. ESBLs and MBLs genes were sought by PCR. The resistant rate to imipenem was 39.34%. The resistance rates for P. aeruginosa to cefuroxime, cefoperazone, ceftazidime, aztreonam, and piperacillin/tazobactam were 87.7%, 80.3%, 60.6%, 45.1%, and 25.4%, respectively. Out of 122 P. aeruginosa, 27% and 7.4% were MBL and ESBL, respectively. The prevalence of bla VIM-2, bla OXA-10-, bla VEB-1, bla NDM-, and bla IMP-1-like genes were found in 58.3%, 41.7%, 10.4%, 4.2%, and 2.1%, respectively. GIM-, SPM-, SIM-, and OXA-2-like genes were not detected in this study. OXA-10-like gene was concomitant with VIM-2 and/or VEB. Twelve isolates harbored both OXA-10 and VIM-2; two isolates carried both OXA-10 and VEB. Only one strain contained OXA-10, VIM-2, and VEB. In conclusion, bla VIM-2- and bla OXA-10-like genes were the most prevalent genes in P. aeruginosa in Egypt. To our knowledge, this is the first report of bla VIM-2, bla IMP-1, bla NDM, and bla OXA-10 in P. aeruginosa in Egypt.

Microevolution of highly pathogenic avian influenza A(H5N1) viruses isolated from humans, Egypt, 2007-2011.

We analyzed highly pathogenic avian influenza A(H5N1) viruses isolated from humans infected in Egypt during 2007–2011. All analyzed viruses evolved from the lineage of subtype H5N1 viruses introduced into Egypt in 2006; we found minimal evidence of reassortment and no exotic introductions. The hemagglutinin genes of the viruses from 2011 formed a monophyletic group within clade 2.2.1 that also included human viruses from 2009 and 2010 and contemporary viruses from poultry; this finding is consistent with zoonotic transmission. Although molecular markers suggestive of decreased susceptibility to antiviral drugs were detected sporadically in the neuraminidase and matrix 2 proteins, functional neuraminidase inhibition assays did not identify resistant viruses. No other mutations suggesting a change in the threat to public health were detected in the viral proteomes. However, a comparison of representative subtype H5N1 viruses from 2011 with older subtype H5N1 viruses from Egypt revealed substantial antigenic drift.

Acinetobacter baumannii universal stress protein A plays a pivotal role in stress response and is essential for pneumonia and sepsis pathogenesis.

Acinetobacter baumannii is one of the most significant threats to global public health. This threat is compounded by the fact that A. baumannii is rapidly becoming resistant to all relevant antimicrobials. Identifying key microbial factors through which A. baumannii resists hostile host environment is paramount to the development of novel antimicrobials targeting infections caused by this emerging pathogen. An attractive target could be a molecule that plays a role in the pathogenesis and stress response of A. baumannii. Accordingly, the universal stress protein A (UspA) was chosen to be fully investigated in this study. A platform of A. baumannii constructs, expressing various levels of the uspA gene ranging from zero to thirteen folds of wild-type level, and a recombinant E. coli strain, were employed to investigate the role of UspA in vitro stress and in vivo pathogenesis. The UspA protein plays a significant role in protecting A. baumannii from H(2)O(2), low pH, and the respiratory toxin 2,4-DNP. A. baumannii UspA protein plays an essential role in two of the deadliest types of infection caused by A. baumannii; pneumonia and sepsis. This distinguishes A. baumannii UspA from its closely related homolog, the Staphylococcus aureus Usp2, as well as from the less similar Burkholderia glumae Usps. Heterologous and overexpression experiments suggest that UspA mediates its role via an indirect mechanism. Our study highlights the role of UspA as an important contributor to the A. baumannii stress and virulence machineries, and polishes it as a plausible target for new therapeutics.

Breast cancer associated a2 isoform vacuolar ATPase immunomodulates neutrophils: potential role in tumor progression.

In invasive breast cancer, tumor associated neutrophils (TAN) represent a significant portion of the tumor mass and are associated with increased angiogenesis and metastasis. Identifying the regulatory factors that control TAN behavior will help in developing ideal immunotherapies. Vacuolar ATPases (V-ATPases), multi-subunit proton pumps, are highly expressed in metastatic breast cancer cells. A cleaved peptide from a2 isoform V-ATPase (a2NTD) has immunomodulatory role in tumor microenvironment. Here, we report for the first time the role of V-ATPase in neutrophils modulation. In invasive breast cancer cells, a2NTD was detected and a2V was highly expressed on the surface. Immunohistochemical analysis of invasive breast cancer tissues revealed that increased neutrophil recruitment and blood vessel density correlated with increased a2NTD levels. In order to determine the direct regulatory role of a2NTD on neutrophils, recombinant a2NTD was used for the treatment of neutrophils isolated from the peripheral blood of healthy volunteers. Neutrophils treated with a2NTD (a2Neuɸ) showed increased secretion of IL-1RA, IL-10, CCL-2 and IL-6 that are important mediators in cancer related inflammation. Moreover, a2Neuɸ exhibited an increased production of protumorigenic factors including IL-8, matrix metaloprotinase-9 and vascular endothelial growth factor. Further, functional characterization of a2Neuɸ revealed that a2Neuɸ derived products induce in vitro angiogenesis as well as increase the invasiveness of breast cancer cells. This study establishes the modulatory effect of breast cancer associated a2V on neutrophils, by the action of a2NTD, which has a positive impact on tumor progression, supporting that a2V can be a potential selective target for breast cancer therapy.

Dissemination of VIM-2 producing Pseudomonas aeruginosa ST233 at tertiary care hospitals in Egypt

BackgroundPseudomonas aeruginosa is an important nosocomial pathogen, commonly causing infections in immunocompromised patients. The aim of this study was to examine the genetic relatedness of metallo-beta-lactamase (MBL) producing carbapenem resistant Pseudomonas aeruginosa clinical isolates collected from 2 tertiary hospitals in Cairo, Egypt using Multi Locus sequence typing (MLST).MethodsPhenotypic and genotypic detection of metallo-beta-lactamase for forty eight non-duplicate carbapenem resistant P. aeruginosa isolates were carried out. DNA sequencing and MLST were done.ResultsThe blaVIM-2 gene was highly prevalent (28/33 strains, 85%) among 33 MBL-positive P.aeruginosa isolates. MLST revealed eleven distinct Sequence Types (STs). A unique ST233 clone producing VIM-2 was documented by MLST in P.aeruginosa strains isolated from Cairo university hospitals. The high prevalence of VIM-2 producers was not due to the spread of a single clone.ConclusionsThe findings of the present study clearly demonstrate that clones of VIM-2 positive in our hospitals are different from those reported from European studies. Prevalence of VIM-2 producers of the same clone was detected from surgical specimens whereas oncology related specimens were showing diverse clones.