Mohamed Morsi Ahmed

Co-circulation of three camel coronavirus species and recombination of MERS-CoVs in Saudi Arabia

Coronaviruses in the Middle East Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness and kills about a third of people infected. The virus is common in dromedary camels, which can be a source of human infections. In a survey for MERSCoV in over 1300 Saudi Arabian camels, Sabir et al. found that dromedaries share three coronavirus species with humans. Diverse MERS lineages in camels have caused human infections, which suggests that transfer among host species occurs quite easily. Haagmans et al. made a MERS-CoV vaccine for use in camels, using poxvirus as a vehicle. The vaccine significantly reduced virus excretion, which should help reduce the potential for transmission to humans, and conferred cross-immunity to camelpox infections. Science, this issue p. 81, p. 77 Several camel coronaviruses occur in the Middle East, and a recombinant lineage is linked to the recent human outbreaks of MERS. Outbreaks of Middle East respiratory syndrome (MERS) raise questions about the prevalence and evolution of the MERS coronavirus (CoV) in its animal reservoir. Our surveillance in Saudi Arabia in 2014 and 2015 showed that viruses of the MERS-CoV species and a human CoV 229E–related lineage co-circulated at high prevalence, with frequent co-infections in the upper respiratory tract of dromedary camels. viruses of the betacoronavirus 1 species, we found that dromedary camels share three CoV species with humans. Several MERS-CoV lineages were present in camels, including a recombinant lineage that has been dominant since December 2014 and that subsequently led to the human outbreaks in 2015. Camels therefore serve as an important reservoir for the maintenance and diversification of the MERS-CoVs and are the source of human infections with this virus.

Cell factories for insulin production

The rapid increase in the number of diabetic patients globally and exploration of alternate insulin delivery methods such as inhalation or oral route that rely on higher doses, is bound to escalate the demand for recombinant insulin in near future. Current manufacturing technologies would be unable to meet the growing demand of affordable insulin due to limitation in production capacity and high production cost. Manufacturing of therapeutic recombinant proteins require an appropriate host organism with efficient machinery for posttranslational modifications and protein refolding. Recombinant human insulin has been produced predominantly using E. coli and Saccharomyces cerevisiae for therapeutic use in human. We would focus in this review, on various approaches that can be exploited to increase the production of a biologically active insulin and its analogues in E. coli and yeast. Transgenic plants are also very attractive expression system, which can be exploited to produce insulin in large quantities for therapeutic use in human. Plant-based expression system hold tremendous potential for high-capacity production of insulin in very cost-effective manner. Very high level of expression of biologically active proinsulin in seeds or leaves with long-term stability, offers a low-cost technology for both injectable as well as oral delivery of proinsulin.

Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives

Escherichia coli is the most preferred microorganism to express heterologous proteins for therapeutic use, as around 30% of the approved therapeutic proteins are currently being produced using it as a host. Owing to its rapid growth, high yield of the product, cost-effectiveness, and easy scale-up process, E. coli is an expression host of choice in the biotechnology industry for large-scale production of proteins, particularly non-glycosylated proteins, for therapeutic use. The availability of various E. coli expression vectors and strains, relatively easy protein folding mechanisms, and bioprocess technologies, makes it very attractive for industrial applications. However, the codon usage in E. coli and the absence of post-translational modifications, such as glycosylation, phosphorylation, and proteolytic processing, limit its use for the production of slightly complex recombinant biopharmaceuticals. Several new technological advancements in the E. coli expression system to meet the biotechnology industry requirements have been made, such as novel engineered strains, genetically modifying E. coli to possess capability to glycosylate heterologous proteins and express complex proteins, including full-length glycosylated antibodies. This review summarizes the recent advancements that may further expand the use of the E. coli expression system to produce more complex and also glycosylated proteins for therapeutic use in the future.

Genetics similarity among four breeds of goat in Saudi Arabia detected by random amplified polymorphic DNA marker

Phylogeny analysis using random amplified polymorphic DNA (RAPD) markers was performed for studying genetic variation in four Saudi Arabia goat breeds, namely: Harri, Ardi, Habsi and Masri. Six goats from Harri breed, four each from both Ardi and Habsi breeds and five from Masri breed were used for the experiment. Four different 10 bp RAPD primers were employed for the polymerase chain reaction (PCR). The results obtained showed that Harri and Ardi goat breeds lay in the same group and share about 73.5% genetic similarity, while Habsi and Masri goat breeds were closer to each other more than the previous two breeds, where they share about 82.5% of genetic similarity. Key words : Goats, breeds, RAPD, genetic similarity.

Characterization of native fungi responsible for degrading crude oil from the coastal area of Yanbu, Saudi Arabia

ABSTRACT A total of 15 fungal isolates were obtained from oil-contaminated sites near the Red Sea in the Yanbu region. Based on the preliminary DCPIP (2,6-dichlorophenolindophenol) assay, three isolates showed promising oil degrading ability. The next-generation sequencing of the ITS-I and ITS-II internal transcribed spacer regions assigned the isolates to Aspergillus and Penicillium. Among these three strains, Y2 (Aspergillus oryzae) was the most efficient, degrading about 99% of the crude oil. The degradation rates were corroborated using spectrophotometric and gas chromatography–mass spectrometry analyses after two weeks of cultivation in Bushnell–Haas medium. All the three strains proved to be potent oil-degrading strains and, hence, can be utilized to degrade oil contaminants.

Isolation and identification of bacterial consortia responsible for degrading oil spills from the coastal area of Yanbu, Saudi Arabia

ABSTRACT Twenty-three crude-oil-degrading bacteria were isolated from oil-contaminated sites near the Red Sea. Based on a high growth rate on crude oil and on hydrocarbon degradation ability, four strains were selected from the 23 isolated strains for further study. These four strains were selected on the basis of dichlorophenolindophenol assay. The nucleotide sequences of the 16S rRNA gene showed that these isolated strains belonged to genus Pseudomonas and Nitratireductor. Among the four isolates, strains S5 (Pseudomonas sp., 95%) and 4b (Nitratireductor sp., 70%) were the most effective ones in degrading crude oil. Using a spectrophotometer and gas chromatography–mass spectrometry, degradation of more than 90% of the crude oil was observed after two weeks of cultivation in Bushnell–Haas medium. The results showed that these strains have the ability to degrade crude oil and may be used for environmental remediation.

Role of Toxicogenomics in the Development of Safe, Efficacious and Novel Anti-microbial Therapies

Over the last two decades, occurrence of bacterial resistance to commonly used antibiotics has necessitated the development of safer and more potent anti-microbial drugs. However, the development of novel antibiotics is severely hampered by adverse side effects, such as drug-induced liver toxicity. Several antibacterial drugs are known to have the potential to cause severe liver damage. The major challenge in developing novel anti-microbial drugs is to predict, with certain amount of probability, the drug-induced toxicity during the pre-clinical stages, thus optimizing and reducing the time and cost of drug development. Toxicogenomics approach is generally used to harness the potential of genomic tools and to understand the physiological basis of drug-induced toxicity based on the in-depth analysis of Metagenomic data sets, i.e., transcriptional, translational or metabolomic profiles. Toxicogenomics, therefore, represents a new paradigm in the drug development process, and is anticipated to play an invaluable role in future to develop safe and efficacious medicines, by predicting the toxic potential of a new chemical entity (NCE) in early stages of drug discovery. This review examines the toxicogenomic approach in predicting the safety/toxicity of novel anti-microbial drugs, and analyses the promises, pitfalls and challenges of applying this powerful technology to the drug development process.

The effect of Rhazya stricta aqueous leaves extract on MRSA genotypes in Jeddah province

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that is resistant to a large group of beta-lactam antibiotics. Rhazya stricta is a local shrub that grows naturally as a normal flora and is used as a medicinal plant by several nations for a lot of infectious diseases, caused by microorganisms. Therefore, the effect of the plant against different genotypes of methicillin-resistant S. aureus was tested in the present study. Molecular identification was done for the medical sampling of 44 MRSA and biodiversity approaches were applied to detect the mecA gene. The 16S rRNA genes analysis was performed for the construction of a phylogenetic tree. Later on, the antimicrobial effect of the plant leaves’ water extract was tested on different genotypes. MecA gene appeared in all isolates, except in methicillin-susceptible Staphylococcus aureus. The selected MRSA 16S rRNA sequences were sent to GenBank and six accession numbers (KM893010, KM893011, KP091274, KP091275, KP137513 and KP137514) were acquired. Also, an evolutionary analysis of these strains was done and a phylogenetic tree was constructed. Plant extracts showed that the interaction between pathogens and drugs is more efficient in a liquid environment than in a solid one.

Allele mining in the caprine alpha-lactalbumin (LALBA) gene of native Saudi origin

ABSTRACT Alpha-lactalbumin (α-LA) is a distinctive whey protein found in bovine milk and the milk of other mammalian species. Its main function in the lactating mammary glands is involved in lactose biosynthesis, which makes it a potential quantitative trait locus. The aim of this study is to determine the incidence of single nucleotide polymorphisms (SNPs) within the exonic and intronic portions of the caprine α-LA gene (LALBA) in native Saudi breeds (Ardi, Habsi and Harri) in relation to utility traits. Blood samples were collected from 300 animals (100 for each breed). Genomic DNA was extracted and a 268 bp fragment of the LALBA gene comprising exon 3 and its flanking area was amplified. Subsequent digestion with MvaI restriction endonuclease resulted in two different banding patterns: αLA A1/αLA A1 and αLA A1/αLA A2, and two allelic forms, i.e. αLA A1 and αLA A2. Nucleotide sequencing of the designated LALBA amplicons was done and, following successful BLAST analysis, the sequences were submitted to GenBank (NCBI Accession No. KM267632, KM267633, KM267634 and KP940442). SNPs were searched within breeds of Saudi origin and homology was sought among caprine, ovine and bovine species. A C > T transition was identified within the fifth nucleotide of the silent α-LA A2 allele. Phylogenetic analysis on the basis of LALBA nucleotide sequence of Saudi goats indicated similarity with evolutionarily related sheep, while the origin of bovine species and deer was located some distance away.

Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System

Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system.