Professor David W. Denning an extraordinary high achiever in clinical and translational science

It is often wondered as to what part of all the research performed around the globe actually leaves the lab and reaches the real people and betters their lives. In case of Professor David W. Denning's research, a large part does. As a prominent international face in the field of medical mycology, he translates his research into clinical applications and takes medical problems back to his lab, in order to develop potential solutions. e is a professor of Infectious Diseases in Global Health and a consultant in infectious diseases and medical mycology, Wythenshawe Hospital and University of Manchester. He is also the founding president of the Global Action Fund for Fungal Infections [GAFFI] and the leader of Leading International Fungal Education [LIFE]. He supervises a strong research group funded by grants from the UK, USA, and Europe, as well as multiple pharmaceutical companies, with research grant income of more than 22 million pounds to Manchester University, since 1990

Expression optimization of anti-CD22 scFv-Apoptin fusion protein using experimental design methodology

Background: Design of experiments is a rapid and cost-effective approach for optimization of recombinant protein production process. In our previous study, we generated a potent dual-acting fusion protein, anti-CD22 scFv-apoptin, to target B-cell malignant cell lines. In the present investigation, we report the effect of different variables on the expression levels of this fusion protein

Methods: Four variables [cell optical density at induction, IPTG concentration, induction temperature, and induction time] were tested using experimental design

Results: Our findings demonstrated that among the examined variables, only the induction time had a significant positive effect on the protein expression yield

Conclusion: Experimental design was successfully applied in this study. The optimized condition obtained in the current study can be applied in future commercial production of this novel fusion protein

Identification and evaluation of novel drug targets against the human fungal pathogen Aspergillus fumigatus with elaboration on the possible role of RNA-binding protein

Bakground: Aspergillus fumigatus is an airborne opportunistic fungal pathogen that can cause fatal infections in immunocompromised patients. Although the current anti-fungal therapies are relatively efficient, some issues such as drug toxicity, drug interactions, and the emergence of drug-resistant fungi have promoted the intense research toward finding the novel drug targets

Methods: In search of new antifungal drug targets, we have used a bioinformatics approach to identify novel drug targets. We compared the whole proteome of this organism with yeast Saccharomyces cerevisiae to come up with 153 specific proteins. Further screening of these proteins revealed 50 potential molecular targets in A. fumigatus. Amongst them, RNA-binding protein [RBP] was selected for further examination. The aspergillus fumigatus RBP [AfuRBP], as a peptidylprolyl isomerase, was evaluated by homology modeling and bioinformatics tools. RBP-deficient mutant strains of A. fumigatus were generated and characterized. Furthermore, the susceptibility of these strains to known peptidylprolyl isomerase inhibitors was assessed

Results: AfuRBP-deficient mutants demonstrated a normal growth phenotype. MIC assay results using inhibitors of peptidylprolyl isomerase confirmed a higher sensitivity of these mutants compared to the wild type

Conclusion: Our bioinformatics approach revealed a number of fungal-specific proteins that may be considered as new targets for drug discovery purposes. Peptidylprolyl isomerase, as a possible drug target, was evaluated against two potential inhibitors and the promising results were investigated mechanistically. Future studies would confirm the impact of such target on the antifungal discovery investigations

Effect of cysteamine on cell growth and IgG[4] production in recombinant Sp2.0 cells

The manipulation of redox potential in secretory pathway by thiol reducing agents can be a strategy to improve the production levels of disulfide-bonded proteins including recombinant antibodies. Here we have studied the influence of cysteamine on viability and the production level of IgG[4] in Sp2.0 cells. For this purpose, the recombinant Sp2.0 cells producing an anti CD33 IgG[4], were subjected to different concentrations of cysteamine. At concentrations of 2, 4 and 5 mM cysteamine, the secreted levels of IgG[4] did not change significantly. However, in concentration of 7 mM cysteamine, a significant decrease was observed in IgG[4] levels which may indicate the cytotoxicity of this compound in higher concentrations. Our results show that the cysteamine treatment reduces the cell viability in a dose-dependent manner. Also it was observed that 2 mM cysteamine had no late effect on IgG4 production level and only at day 3, this concentration of cysteamine decreased the cell viability significantly. To test whether the addition of cysteamine can affect the expression level of protein disulfide isomerase, RT-PCR analysis was carried out. The results revealed that cysteamine does not affect the PDI transcription and expression level of IgG[4] in this type of recombinant cells

Sequences type analysis of Candida albicans isolates from Iranian human immunodeficiency virus infected patients with oral candidiasis

The growing number of immunocompromised individuals has increased the incidence of infections caused by Candida species during the recent decades. Typing of C. albicans on the basis of DNA sequences at multiple loci has greatly advanced our knowledge about the epidemiology and phylogeny of candidiasis. The aim of this study was to evaluate the diversity, and genetic relationships among C. albicans isolates obtained from HIV patients in Iran using multilocus sequence typing [MLST] method. We analyzed 25 C. albicans isolates obtained from HIV positive patients referred to Iranian Research Center for HIV/AIDS. After diagnostic test and DNA extraction C. albicans isolates were typed using the original MLST scheme explained previously include of six loci: ACC1, VPS13, GLN4, ADP1, RPN2, and SYA1. Fifty one [2.17%] nucleotide sites were found to be polymorphic; all were found to be heterozygous in at least one isolate. For the 25 clinical isolates, 22 diploid sequence types were defined by the genotypes identified from the six loci. The MLST data suggest a relatively high level of divergence in the population structure of C. albicans isolated from HIV infected patients. These findings indicate that in these patients there is a favorable context for the growth of potential pathogenic C. albicans. We found no association between fluconazole resistance, highly active antiretroviral therapy [HAART] receiving and either sequence type or group

[ effects of recombinant IgG4 expression on the growth of Sp2.0 cell lines]

The study of physiological changes in recombinant cell lines provides useful information to improve production performance. In this study, we investigate the effects of an anti-CD33 chimeric IgG4 expression on Sp2.0 cell growth. Variable region genes of light and heavy chains of monoclonal antibody produced by M195 were cloned in pFUSE-CLIg-hk and pFUSE-CHIg-hG4 expression vectors, respectively. Transfection of recombinant plasmids into Sp2.0 cell lines was performed using lipofectamine in two steps. Positive transformant cells were isolated and subjected to PCR, RT-PCR and Western blot analysis to confirm the integration of gene cassettes and the expression of recombinant IgG4. To assess the growth parameters, recombinant and parent Sp2.0 cell lines were seeded at a density of 1×10[5] cells/ml in duplicate into 12-well plates. For nine days, culture plates were sampled daily and viable cell count and viability determined. The results of PCR, RT-PCR and Western blot analyses confirmed the generation of stable producer cell lines. In recombinant cells, the maximum cell density decreased by 46%. However, it was observed that IgG4 expression had no effect on cell viability of these transfectants. Our results showed that the expression of recombinant IgG4 can change growth parameters in Sp2.0 cell lines that express the pFUSE-CHIg-hG4-pFUSE-CLIg-hk construct

Generation of a uracil auxotroph strain of the probiotic yeast Saccharomyces boulardii as a host for the recombinant protein production

Saccharomyces boulardii [S. boulardii] is the best known probiotic yeast. The genetic engineering of this probiotic strain requires the availability of appropriate mutants to accept various gene constructs carrying different selection markers. As the auxotrophy selection markers are under focus, we have generated a ura3 auxotroph mutant of S. boulardii for use in further genetic manipulations. Classical UV mutagenesis was used for the generation of auxotroph mutants. The mutants were selected in the presence of 5-FOA [5-Fluoroorotic acid], uracil and uridine. Uracil auxotrophy phenotype was confirmed by the ability of mutants to grow in the presence of uracil and the lack of growth in the absence of this compound. To test whether the uracil auxotrophy phenotype is due to the inactivation of URA3, the mutants were transformed with a plasmid carrying the gene. An in vitro assay was used for the analysis of acid and bile resistance capacity of these mutants. Three mutants were found to be ura3 auxotroph as they were able to grow only in the presence of uracil. When the URA3 gene was added, these mutants were able to grow normally in the absence of uracil. Further in vitro analysis showed that the acid and bile resistance capacity of one of these mutants is intact and similar to the wild type. A uracil auxotroph mutant of the probiotic yeast, S. boulardii, was generated and characterized. This auxotroph strain may have potential applications in the production and delivery of the recombinant pharmaceutics into the intestinal lumen

Cloning and expression of gumboro VP2 antigen in Aspergillus niger

Infectious Bursal Disease Virus [IBDV] causes a highly immunosuppressive disease in chickens and is a pathogen of major economic importance to the poultry industry worldwide. The VP2 protein is the major host-protective immunogen of IBDV and has been considered as a potential subunit vaccine against the disease. VP2 coding sequence was cloned in an inducible fungal vector and the protein was expressed in Aspergillus niger [A. niger]. Aiming at a high level of expression, a multicopy AMA1-pyrG-based episomal construct driven by a strong inducible promoter, glaA, was prepared and used in transformation of A. niger pyrG[-] protoplasts. SDS-PAGE and western blot analysis was carried out to confirm the expression of the protein. A number of pyrG[+] positive transformants were isolated and the presence of expression cassette was confirmed. Western blot analysis of one of these recombinant strains using monospecific anti-VP2 antibodies demonstrated the successful expression of the protein. The recombinant protein was also detected by serum obtained from immunized chicken. In the present study, we have generated a recombinant A. niger strain expressing VP2 protein intracellulary. This recombinant strain of A. niger may have potential applications in oral vaccination against IBDV in poultry industry

Antifungal indole and pyrrolidine-2,4-dione derivative peptidomimetic lead design based on in silico study of bioactive peptide families

The rise of opportunistic fungal infections highlights the need for development of new antimicrobial agents. Antimicrobial Peptides [AMPs] and Antifungal Peptides [AFPs] are among the agents with minimal resistance being developed against them, therefore they can be used as structural templates for design of new antimicrobial agents. In the present study four antifungal peptidomimetic structures named C[1] to C[4] were designed based on plant defensin of Pisum sativum. Minimum inhibitory concentrations [MICs] for these structures were determined against Aspergillus niger N402, Candida albicans ATCC 10231, and Saccharomyces cerevisiae PTCC 5052. C[1] and C[2] showed more potent antifungal activity against these fungal strains compared to C[3] and C[4]. The structure C[2] demonstrated a potent antifungal activity among them and could be used as a template for future study on antifungal peptidomemetics design. Sequences alignments led to identifying antifungal decapeptide [KTCENLADTY] named KTC-Y, which its MIC was determined on fungal protoplast showing 25 [micro g/ml] against Aspergillus fumigatus Af293. The present approach to reach the antifungal molecules seems to be a powerful approach in design of bioactive agents based on AMP mimetic identification

[ comparison of bone morphogenetic protein-7 mutant expression in prokaryotic and eukaryotic hosts]

Bone morphogenetic protein-7 [BMP-7] is a multifunctional growth factor predominantly recognized for its osteoinductive properties. Due to the high cost of this protein, the availability of BMP-7 for treatment is limited. The heterologous production of recombinant hBMP-7 has been performed in a number of expression systems. In this study a novel form of BMP-7 was expressed in eukaryotic and prokaryotic hosts. For expression in the prokaryotic system, the novel protein was secreted to the periplasmic space of Escherichia coli using a pelB signal sequence followed by singlestep purification by Ni2+-charged column chromatography. In the mammalian cell expression system, we transferred a full-length cDNA encoding precursor of the novel protein to CHO cells then selected stable clones by using the appropriate antibiotic concentration. Expressions in both systems were confirmed by Western blot analysis. The novel recombinant protein was produced as a 36-38 kDa dimer in the CHO cell line and a 16 kDa monomer in the Escherichia coli system. Quantitative analysis according to ELISA showed that the expression levels of the mutant protein in the eukaryotic and prokaryotic expression systems were 40 ng/ml and 135 ng/ml of the culture media, respectively. In this study, the expression level in Escherichia coli was at least three times more than observed in the CHO cells. However, further optimization is required to obtain a dimer protein in Escherichia coli. The results show that periplasmic expression may be suitable for the production of complex proteins such as BMPs

Periplasmic expression of a novel human bone morphogenetic protein-7 mutant in escherichia coli

Bone Morphogenetic Proteins [BMPs] belong to the transforming growth factor-beta [TGF-beta] superfamily, and play an important role in bone metabolism. Recombinant forms of BMP-2 and BMP-7 are the only BMPs used clinically. In this study the mature part of human bone morphogenetic protein-7 [BMP-7] was engineered through substitution of the BMP-7 Nterminal sequence by heparin-binding site of BMP-2. This targeted substitution was made to enhance the binding affinity of the novel protein to the extracellular matrix components such as heparin and heparan sulfate proteoglycans [HSPGs]. The engineered protein was expressed in Escherichia coli [E.coli]. The PelB signal sequence was used to translocate soluble proteins into the periplasmic space of E.coli. The protein was purified from periplasmic extract using Ni-NTA chromatography and the SDS-PAGE and western blot analysis confirmed the successful expression of the novel protein. The novel hBMP-7 mutant was produced as approximately 16 kDa monomer. It was found that the heparin binding of this protein was approximately 50% more than that of the wild-type at a protein concentration of 500 ng/ml. The findings showed that the periplasmic expression may be suitable to produce complex proteins like BMPs

[In vitro antifungal susceptibility of oral candida species from Iranian HIV infected patients]

Oropharyngeal candidiasis and antifungal drug resistance are major problems in HIV positive patients. The increased reports of antifungal resistance and expanding therapeutic options prompted the determination of antifungal susceptibility profile of Candida species isolates in Iranian patients living with HIV/AIDS [PLWHA] in the present study. One hundred fifty oral samples from Iranian HIV positive patients were obtained and cultured on CHROMagar and Sabouraud's dextrose agar. All isolates were identified according to assimilation profile, germ tube, colony color and other conventional methods. Disk diffusion testing and Broth Microdilution of six antifungal agents were performed according to the methods described in CLSI. Candida albicans [50.2%] was the most frequent isolated yeast, followed by C. glabrata [22%]. Non-Candida albicans species were isolated from 71 [61%] positive cultures. 25.7% of Candida albicans isolates were resistant to fluconazole [MIC >/= 64 micro g/ml] as were 21.9% and 16.4% to ketoconazole and clotrimazole [MIC>0.125 micro g/ml], respectively. Resistance to polyene antifungals including amphotericin B and nystatin, and caspofungin were scarce. 57.7% of candida glabrata isolates were resistant to fluconazole, 31% to ketoconazole and 35% to clotrimazole. Screening for antifungal resistant candida isolates by disk diffusion or broth dilution methods in clinical laboratories is an ideal surveillance measure in the management of oral thrush in patients with HIV/AIDS. Although nystatin is widely used in clinical practice for HIV positive patients, there was no evidence of enhanced resistance to it. Regarding no resistance to caspofungin, its administration is suggested

Human tissue plasminogen activator expression in Escherichia coli using cytoplasmic and periplasmic cumulative power

Tissue plasminogen activator [tPA] is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli [E. coli] is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm