Tanıl Kocagöz

Synthesis and antimycobacterial activity of some phthalimide derivatives

Structurally modified phthalimide derivatives were prepared through condensation of phthalic and tetrafluorophthalic anhydride with selected sulfonamides with variable yields. All compounds were screened for their antimycobacterium activity against Mycobacterium tuberculosis H37Ra (ATCC 25177) using a micro broth dilution technique. The fluorinated derivatives (compounds 2c, 2d, 2f and 2h) had antimycobacterium activity comparable with classical sulfonamide drugs. The minimum inhibitory concentration (MIC) of compounds 2c, 2d, 2f and 2h was greater than that of isoniazid (MIC<0.02 μg/mL) and in vitro activity was greater than that of pyrazinamide, another first line antimycobacterium drug (MIC 50-100 μg/mL). The new compounds could be considered new lead compounds in the treatment of multi-drug resistant tuberculosis.

Mycobacterial Strains That Stimulate the Immune System Most Efficiently as Candidates for the Treatment of Bladder Cancer

Background: Intravesical bacillus Calmette-Guérin (BCG) application is widely used in the treatment of superficial bladder carcinoma. Despite being an effective therapy, the pathogenicity and lethal side effects of BCG limits its usage. Intensive research has been carried out to find less toxic and more potent therapeutic agents for the treatment of bladder cancer. Researchers have focused on Mycobacterium phlei as an alternative. The cell wall extract of M. phlei is sufficient for antitumoral activity. Our preliminary experiments indicate that the fractions rich in cell wall proteins cause activation of tumor necrosis factor (TNF)-α and interleukin (IL)-12. This study aims to identify powerful and less harmful mycobacteria among 88 strains in terms of how they stimulate the immune system. Methods: Eighty-eight mycobacterial strains were grown in Middlebrook 7H9 medium. The bacterial cells were sonicated after heat treatment. The supernatants were incubated with the monocytic cell line THP-1, followed by measurement of TNF-α and IL-12 response. Results and Conclusion: In addition to M. phlei, the following 12 mycobacterial strains were selected as candidates for superficial bladder tumor treatment: M. agri, M. aichiense, M. aurum, M. brumae, M. chitae, M. chubuense, M. diernhoferi, M. gadium, M. murale, M. obuense, M. tokaiense and M. vaccae.

Leishmaniasis in Turkey: first clinical isolation of Leishmania major from 18 autochthonous cases of cutaneous leishmaniasis in four geographical regions.

To report isolation of Leishmania major strains obtained from 18 Turkish autochthonous cutaneous leishmaniasis (CL) patients infected with L. major between 2011 and 2014.

Efficiency of the TK Culture System in the diagnosis of tuberculosis.

We have evaluated the efficiency of the TK Rapid Mycobacterial Culture System in isolating mycobacteria from clinical samples and in susceptibility testing. The TK Medium indicates mycobacterial growth by changing its color from red to yellow. During a 1-year period, 16,303 clinical samples were inoculated to TK selective (TK SLC) and Löwenstein-Jensen media (LJ). Mycobacteria were isolated in 2150 (13.04%) samples in at least 1 type of medium. While LJ isolated mycobacteria from 1920 (11.69%) of all samples, TK SLC isolated 2070 (12.63%). Among all positives, the isolation rates for LJ and TK SLC were 89.30% and 96.27%, respectively. Contamination of cultures by other organisms was observed in 878 (5.33%) LJ tubes and in 90 (0.55%) TK SLC tubes. On average, time-to-growth detection was 15.57 days in TK SLC and 25.14 days in LJ. The modes of time-to-growth detection were 12 and 25 days for TK SLC and LJ, respectively. The reliability of antimycobacterial susceptibility testing was checked by 36 Mycobacterium tuberculosis strains with known susceptibility patterns which were obtained from the World Health Organization collection and by participating in an external quality control program. All susceptibility results, except for a few borderline-resistant strains, were consistent with the expected susceptibility patterns. The TK Rapid Mycobacterial Culture System is a practical and reliable automated system that shortens the time required for both culture and susceptibility results. All types of TK Media are ready to use, saving time and effort as well as drastically reducing contamination during testing.

Design, Synthesis, and Molecular Docking Studies of a Conjugated Thiadiazole-Thiourea Scaffold as Antituberculosis Agents.

In view of the emergence and frequency of multidrug-resistant and extensively drug-resistant tuberculosis and consequences of acquired resistance to clinically used drugs, we undertook the design and synthesis of novel prototypes that possess the advantage of the two pharmacophores of thiourea and 1,3,4-thiadiazole in a single molecular backbone. Three compounds from our series were distinguished from the others by their promising activity profiles against Mycobacterium tuberculosis strain H37Rv. Compounds 11 and 19 were the most active representatives with minimum inhibitory concentration (MIC) values of 10.96 and 11.48 µM, respectively. Compound 15 was shown to inhibit M. tuberculosis strain H37Rv with an MIC value of 17.81 µM. Cytotoxicity results in the Vero cell line showed that these three derivatives had selectivity indices between 1.8 and 8.7. In order to rationalize the biological results of our compounds, molecular docking studies with the enoyl acyl carrier protein reductase (InhA) of M. tuberculosis were performed and compounds 11, 15, and 19 were found to have good docking scores in the range of -7.12 to -7.83 kcal/mol.

Lateral flow assay for rapid differentiation of Mycobacterium tuberculosis complex and 97 species of mycobacteria other than tuberculosis grown in Löwenstein-Jensen and TK-SLC medium

BACKGROUND Mycobacterial antigen MPB64 is a secretory protein specific for Mycobacterium tuberculosis complex. A lateral flow immunochromatographic assay (ICA) is a method used for the rapid differentiation of M. tuberculosis complex. AIM We aimed to evaluate the performance of ICA in rapid differentiation of M. tuberculosis complex from 97 Mycobacterium species other than tuberculosis (MOTT), which are grown in Lφwenstein-Jensen and TK-selective (SLC) medium. MATERIALS AND METHODS The study was performed in our laboratory between January 2009 and January 2010. A total of 394 isolates consisting of reference strains of 34 M. tuberculosis from World Health Organization (WHO) collection, 97 different MOTT bacilli, 7 Mycobacterium bovis BCG substrains and total 256 clinical Mycobacterium isolates were tested by ICA, which is based on anti-MPB64 monoclonal antibodies. All the strains were inoculated onto a TK-SLC (selective) medium and Lowenstein-Jensen medium. TK-SLC is a new rapid mycobacterial culture medium that indicates mycobacterial growth by colour change. RESULTS The growth of mycobacterial strains was observed in 10-12 days on TK-SLC medium. ICA test was performed in 15 minutes. All strains belonging to M. tuberculosis complex group were found positive and all MOTT species were found negative on ICA slides. The results were confirmed with nucleic acid amplification by polymerase chain reaction (PCR) using primers specific for M. tuberculosis complex. CONCLUSION With the additive effect of growth on TK-SLC medium in 10-12 days, the mycobacterial antigen MPB64 is a very useful and specific tool in rapid differentiation of M. tuberculosis and MOTT grown in culture.

Antimicrobial activities of phosphonium containing polynorbornenes

In this study, amphiphilic polyoxanorbornene with different alkyl and aromatic phosphonium side chains was synthesized. The biological activities of these polymers were determined by the minimal inhibitory concentration (MIC) against E. coli, S. aureus, M. tuberculosis and the yeast C. albicans, and cytotoxicity studies on red blood cells were performed. A series of polymers with different alkyl and aromatic substituents (methyl, ethyl, tripropyl, tert-butyl, phenyl, and tris 4-methoxyphenyl) and two types different molecular weight, 3000 g mol−1 and 10000 g mol−1, were prepared. It was observed that the biological activity of the polymers with aromatic group substituents had an MIC of 16, 8, 64 and 128 μg mL−1 against E. coli, S. aureus, M. tuberculosis and C. albicans, respectively, while those with non-aromatic carbons had a higher MIC compared to those with aromatic carbons. The aromaticity of the repeat unit had impressive effects on hemolytic activities as well. Zeta potential measurements of E. coli incubated with active and inactive polymer concentration revealed a relationship between the MIC and membrane surface charge density. Polymers bearing aromatic groups killed the bacteria with widespread damage after the polymers, holding the threshold concentration, were added to the bacteria.

Peptide Antibiotics Developed by Mimicking Natural Antimicrobial Peptides

Antimicrobial peptides are widely preferred drugs for infectious disease treatment. Inspired from natural antimicrobial peptides, short peptides showing good antibacterial activity are designed in this study. The peptides consisted of repeating hydrophobic and positively charged amino acids, positioned on one side of the alpha helix. Arginine in peptides resulted in better activity compared to lysine. Having positively charged amino acids at both ends, created better activity for Escherichia coli compared to Staphylococcus aureus, and only at one end, created comparable activities for both organisms. Positioning of arginines on one side in zigzag form prominently increased the activity compared to positioning on linear axis. Elongating hydrophobic tail resulted in self-binding and eliminated the antibacterial activity. Molecular dynamic simulations suggested that a single molecule is capable of creating hydrophilic channel in membrane. Electron microscopic examination of staphylococci treated with these peptides revealed that the bacteria split into halves. Docking studies revealed that the peptides strongly bind to the major peptidoglycan synthesizing membrane protein, glycosyltransferase. The unique composition and design of these peptides revealed a promising antibacterial activity that may further lead to the development of new antimicrobial compounds effective to multi-drug resistant organisms.

Detection of nuclease activity using a simple fluorescence based biosensor

Nucleases are associated with a variety of diseases as they catalyze the degradation of nucleic acids. Their presence as a contaminant could also be a major concern in several molecular biology-related analyses where DNAse and RNAse free environments are required. Here, we report a simple, fluorescence-based biosensor for nuclease detection. The sensor utilizes only one fluorescent molecule attached to a ssDNA molecule building upon a fluorescence-quenching phenomenon taking place between a fluorescent dye and a nucleotide base. The intensity of the fluorescent molecule is quenched by covalently attaching to ssDNA and upon digestion of the ssDNA the fluorescence signal increases dramatically. Consequently, the fluorescence signal can be used to determine the activity of nuclease enzymes for a variety of applications ranging from molecular biology to clinical diagnostics. Compared to available methods, our sensor does not use any additional substances such as cationic polymers, carbon nanotubes or nanoparticles, graphene oxide or silver nanoclusters or a quencher like DABCYL to quench a fluorophore. The simplicity of the detection method makes this an attractive sensor as a low cost, fast and convenient system that can be incorporated into multiple readers for widespread use in virtually all molecular biology-related analyses.

Multiplex-PCR-Based Screening and Computational Modeling of Virulence Factors and T-Cell Mediated Immunity in Helicobacter pylori Infections for Accurate Clinical Diagnosis

The outcome of H. pylori infection is closely related with bacterias virulence factors and host immune response. The association between T cells and H. pylori infection has been identified, but the effects of the nine major H. pylori specific virulence factors; cagA, vacA, oipA, babA, hpaA, napA, dupA, ureA, ureB on T cell response in H. pylori infected patients have not been fully elucidated. We developed a multiplex- PCR assay to detect nine H. pylori virulence genes with in a three PCR reactions. Also, the expression levels of Th1, Th17 and Treg cell specific cytokines and transcription factors were detected by using qRT-PCR assays. Furthermore, a novel expert derived model is developed to identify set of factors and rules that can distinguish the ulcer patients from gastritis patients. Within all virulence factors that we tested, we identified a correlation between the presence of napA virulence gene and ulcer disease as a first data. Additionally, a positive correlation between the H. pylori dupA virulence factor and IFN-γ, and H. pylori babA virulence factor and IL-17 was detected in gastritis and ulcer patients respectively. By using computer-based models, clinical outcomes of a patients infected with H. pylori can be predicted by screening the patients H. pylori vacA m1/m2, ureA and cagA status and IFN-γ (Th1), IL-17 (Th17), and FOXP3 (Treg) expression levels. Herein, we report, for the first time, the relationship between H. pylori virulence factors and host immune responses for diagnostic prediction of gastric diseases using computer—based models.