Aymen S. Yassin

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.

Monolithic Microfluidic Mixing-Spraying Devices for Time-Resolved Cryo-Electron Microscopy

The goal of time-resolved cryo-electron microscopy is to determine structural models for transient functional states of large macromolecular complexes such as ribosomes and viruses. The challenge of time-resolved cryo-electron microscopy is to rapidly mix reactants, and then, following a defined time interval, to rapidly deposit them as a thin film and freeze the sample to the vitreous state. Here we describe a methodology in which reaction components are mixed and allowed to react, and are then sprayed onto an EM grid as it is being plunged into cryogen. All steps are accomplished by a monolithic, microfabricated silicon device that incorporates a mixer, reaction channel, and pneumatic sprayer in a single chip. We have found that microdroplets produced by air atomization spread to sufficiently thin films on a millisecond time scale provided that the carbon supporting film is made suitably hydrophilic. The device incorporates two T-mixers flowing into a single channel of four butterfly-shaped mixing elements that ensure effective mixing, followed by a microfluidic reaction channel whose length can be varied to achieve the desired reaction time. The reaction channel is flanked by two ports connected to compressed humidified nitrogen gas (at 50 psi) to generate the spray. The monolithic mixer-sprayer is incorporated into a computer-controlled plunging apparatus. To test the mixing performance and the suitability of the device for preparation of biological macromolecules for cryo-EM, ribosomes and ferritin were mixed in the device and sprayed onto grids. Three-dimensional reconstructions of the ribosomes demonstrated retention of native structure, and 30S and 50S subunits were shown to be capable of reassociation into ribosomes after passage through the device.

Viral etiologies of lower respiratory tract infections among Egyptian children under five years of age

BackgroundLower respiratory tract infections (LRTI) are responsible for a considerable number of deaths among children, particularly in developing countries. In Egypt and the Middle East region, there is a lack of data regarding the viral causes of LRTI. In this study, we aimed to identify the relative prevalence of various respiratory viruses that contribute to LRTIs in young children. Although, nucleic acid-based methods have gained importance as a sensitive tool to determine the viral infections, their use is limited because of their prohibitive cost in low-income countries. Therefore, we applied three different laboratory methods, and presented the different virus prevalence patterns detected by each method.MethodsWe collected nasopharyngeal aspirate samples, demographic data and, clinical data from 450 children under five years of age who presented with LRTI at Abou El Reesh hospital in Cairo during a one-year period. To identify the viral causes of the LRTI we used direct fluorescence assay, real-time reverse-transcriptase polymerase chain reaction (rt-RT-PCR), and shell vial culture. We tested for eight major respiratory viruses.ResultsTwo hundred sixty-nine patients (59.9%) had a viral infection, among which 10.8% had a co-infection with two or more viruses. By all three methods, respiratory syncytial virus (RSV) was the most predominant, and parainfluenza virus type 2 (HPIV-2), influenza B virus (FLUBV) were the least predominant. Other viral prevalence patterns differed according to the detection method used. The distribution of various viruses among different age groups and seasonal distribution of the viruses were also determined.ConclusionsRSV and human adenovirus were the most common respiratory viruses detected by rt-RT-PCR. Co-infections were found to be frequent among children and the vast majority of co-infections were detected by nucleic acid-based detection assays.

Insertion domain within mammalian mitochondrial translation initiation factor 2 serves the role of eubacterial initiation factor 1

Mitochondria have their own translational machineries for the synthesis of thirteen polypeptide chains that are components of the complexes that participate in the process of oxidative phosphorylation (or ATP generation). Translation initiation in mammalian mitochondria requires two initiation factors, IF2mt and IF3mt, instead of the three that are present in eubacteria. The mammalian IF2mt possesses a unique 37 amino acid insertion domain, which is known to be important for the formation of the translation initiation complex. We have obtained a three-dimensional cryoelectron microscopic map of the mammalian IF2mt in complex with initiator and the eubacterial ribosome. We find that the 37 amino acid insertion domain interacts with the same binding site on the ribosome that would be occupied by the eubacterial initiation factor IF1, which is absent in mitochondria. Our finding suggests that the insertion domain of IF2mt mimics the function of eubacterial IF1, by blocking the ribosomal aminoacyl-tRNA binding site (A site) at the initiation step.

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.

Optimization of rhamnolipid production by biodegrading bacterial isolates using Plackett-Burman design.

Biosurfactants are biological surfactants produced by microorganisms. Pseudomonas species are well known for the production of the rhamnolipid biosurfactant. In this work, the production of rhamnolipid biosurfactant by Pseudomonas spp. was investigated and further optimized. Two Plackett-Burman designs to study the effect of carbon source, nitrogen source, C/N ratio, iron concentration, magnesium concentration, phenol toxicity, pH, temperature, agitation and sampling time were tested. The first design revealed an optimization that increased biosurfactant productivity by almost two to fivefolds for the tested isolates. However, using the second design showed no remarkable increase in biosurfactant productivity. An additional validation run was adopted using the predicted optimal medium with predicted optimal conditions. The validation run showed remarkable increase in the productivity of the tested isolates. The use of microorganisms with biodegradation ability coupled with optimization of the parameters affecting productivity provides an efficient strategy for biosurfactant production.

A molecular investigative approach to an outbreak of acute hemorrhagic conjunctivitis in Egypt, October 2010

BackgroundDuring October 2010, Egypt reported an outbreak of acute hemorrhagic conjunctivitis (AHC). A total of 1831 cases were reported from three governorates; 1703 cases in El Daqahliya, 92 cases in Port Said, and 36 in Damietta. The purpose of this study was to identify and characterize the causative agent associated with this outbreak.MethodsThe U.S. Naval Medical Research Unit No.3 (NAMRU-3) was contacted by the Egyptian Ministry of Health and Population to perform diagnostic laboratory testing on eighteen conjunctival swabs from patients with conjunctivitis from El Daqahliya Governorate. Conjunctival swabs were tested by molecular methods for human adenovirus (HAdV) and enteroviruses (EV). Virus isolation was performed; the isolated virus was further characterized by molecular typing and phylogenetic analysis.ResultsThe majority of the samples (17/18) were positive for enterovirus and all were negative for HAdV. Molecular typing and sequencing of the isolated virus revealed the presence of coxsackievirus A24 variant. Phylogenetic analysis based on the VP1 and 3C regions demonstrated that the Egyptian viruses belonged to Genotype IV and are closely related to coxsackievirus A24 variant, reported in a similar outbreak in China in August 2010.ConclusionsThis study strongly suggests that coxsackievirus A24 variant was associated with the acute hemorrhagic conjunctivitis outbreak reported in Egypt in October 2010. There is a possibility that the same strain of CV-A24v was implicated in the AHC outbreaks in both China and Egypt in 2010.

Fluoroquinolone resistant mechanisms in methicillin-resistant Staphylococcus aureus clinical isolates in Cairo, Egypt

INTRODUCTION Methicillin-resistant Staphylococcus aureus (MRSA) is a persistent problem in community and health care settings. Fluoroquinolones are among the drugs of choice used to treat MRSA infections. This study aims to identify different mechanisms of fluoroquinolne resistance in local MRSA random sampling isolates in Cairo, Egypt. METHODOLOGY A total of 94 clinical isolates of S. aureus were collected from two major University hospitals in Cairo. Identification was confirmed by appropriate morphological, cultural, and biochemical tests. The antibiotic susceptibility pattern was determined for all isolates. The possible involvement of efflux pumps in mediating fluoroquinolone resistance as well as changes in the quinolone resistance determining region (QRDR) of gyrA and gyrB genes were investigated RESULTS A total of 45 isolates were found to be MRSA, among which 26 isolates were found to be fluoroquinolone-resistant. The MIC values of the tested fluoroquinolones in the presence of the efflux pump inhibitors omeprazole and piperine were reduced. Measuring the uptake of ciprofloxacin upon the addition of the efflux pump inhibitor omeprazole, an increased level of accumulation was observed. Non-synonymous and silent mutations were detected in the QRDR of gyrA and gyrB genes. CONCLUSIONS These results shed light on some of the resistance patterns of MRSA strains isolated from local health care settings in Cairo, Egypt. The resistance of these MRSA towards fluoroquinolones does not depend only on mutation in target genes; other mechanisms of resistance such as the permeability effect, efflux pumps and decreased availability of quinolones at the target site can also be involved.

Utilization of Crude Glycerol as a Substrate for the Production of Rhamnolipid by Pseudomonas aeruginosa

Biosurfactants are produced by bacteria or yeast utilizing different substrates as sugars, glycerol, or oils. They have important applications in the detergent, oil, and pharmaceutical industries. Glycerol is the product of biodiesel industry and the existing glycerol market cannot accommodate the excess amounts generated; consequently, new markets for refined glycerol need to be developed. The aim of present work is to optimize the production of microbial rhamnolipid using waste glycerol. We have developed a process for the production of rhamnolipid biosurfactants using glycerol as the sole carbon source by a local Pseudomonas aeruginosa isolate that was obtained from an extensive screening program. A factorial design was applied with the goal of optimizing the rhamnolipid production. The highest production yield was obtained after 2 days when cells were grown in minimal salt media at pH 6, containing 1% (v/v) glycerol and 2% (w/v) sodium nitrate as nitrogen source, at 37°C and at 180 rpm, and reached 2.164 g/L after 54 hours (0.04 g/L h). Analysis of the produced rhamnolipids by TLC, HPLC, and FTIR confirmed the nature of the biosurfactant as monorhamnolipid. Glycerol can serve as a source for the production of rhamnolipid from microbial isolates providing a cheap and reliable substrate.

Gas-Assisted Annular Microsprayer for Sample Preparation for Time-Resolved Cryo-Electron Microscopy

Time-resolved cryo electron microscopy (TRCEM) has emerged as a powerful technique for transient structural characterization of isolated biomacromolecular complexes in their native state within the time scale of seconds to milliseconds. For TRCEM sample preparation, microfluidic device [9] has been demonstrated to be a promising approach to facilitate TRCEM biological sample preparation. It is capable of achieving rapidly aqueous sample mixing, controlled reaction incubation, and sample deposition on electron microscopy (EM) grids for rapid freezing. One of the critical challenges is to transfer samples to cryo-EM grids from the microfluidic device. By using microspraying method, the generated droplet size needs to be controlled to facilitate the thin ice film formation on the grid surface for efficient data collection, while not too thin to be dried out before freezing, i.e., optimized mean droplet size needs to be achieved. In this work, we developed a novel monolithic three dimensional (3D) annular gas-assisted microfluidic sprayer using 3D MEMS (MicroElectroMechanical System) fabrication techniques. The microsprayer demonstrated dense and consistent microsprays with average droplet size between 6-9 μm, which fulfilled the above droplet size requirement for TRCEM sample preparation. With droplet density of around 12-18 per grid window (window size is 58×58 μm), and the data collectible thin ice region of >50% total wetted area, we collected ~800-1000 high quality CCD micrographs in a 6-8 hour period of continuous effort. This level of output is comparable to what were routinely achieved using cryo-grids prepared by conventional blotting and manual data collection. In this case, weeks of data collection process with the previous device [9] has shortened to a day or two. And hundreds of microliter of valuable sample consumption can be reduced to only a small fraction.