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The complete enzymatic degradation of lignocellulosic biomass requires the cooperative action of cellulosic, hemicellulosic, and lignolytic enzymes such as cellulase, xylanase, laccase, galactosidase, and arabinofuranosidase. Arabinofuranosidases (E.C 3.2.1.55), which belong to the glycoside hydrolase family of enzymes, hydrolyze the 1,3- and 1,5-α-arabinosyl bonds in L-arabinose- containing molecules. L-arabinoses are present in hemicellulosic part of lignocellulosic biomass. Arabinofuranosidases also play an important role in the complete hydrolysis of arabinoxylans. Analysis of the genome project and CAZY database revealed two putative arabinofuranosidase genes in the A. acidocaldarius genome. The aim of the study was cloning, heterologous expression, purification and biochemical characterization of the arabinofuranosidase enzyme encoded in A. acidocaldarius genome. For this purpose, the AbfA gene of the arabinofuranosidase protein was cloned into the pQE-40 vector, heterologously expressed in E. coli BL21 GOLD (DE3) and successfully purified using His-Tag. Biochemical characterization of the purified enzyme revealed that A. acidocaldarius arabinofuranosidase exhibited activity over a wide pH and temperature range with optimum activity at 45 ºC and pH 6.5 in phosphate buffer towards 4-nitrophenyl-α-L-arabinofuranoside as the substrate. In addition, the enzyme is highly stable over wide range of temperature and maintaining 60% of its activity after 90 min of incubation at 80 ºC. Through the bioinformatics studies, the homology model of A. acidocaldarius arabinofuranosidase was generated and the substrate binding site and residues located in this site were identified. Further molecular docking analysis revealed that the substrate located in the catalytically active pose and, residues N174, E175, and E294 have direct interaction with 4-nitrophenyl-α-L-arabinofuranoside. Moreover, based on phylogenetic analysis, A. acidocaldarius arabinofuranosidase exists in the sub-group of intracellular arabinofuranosidases, and G. stearothermophilus and B.subtilis arabinofuranosidases are close relatives of A. acidocaldarius arabinofuranosidase. This is the first study to report the gene cloning, recombinant expression and biochemical and bioinformatic characterization of an auxiliary GH51 arabinofuranosidase from an acidothermophilic bacterium A. acidocaldarius. © 2023, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.

Enzymes are successfully used in many industrial process, such as food and paper industries. Enzymes, which are derived from microorganisms living under mild conditions, are not suitable for industrial processes performed at high temperature, acidic or basic reaction conditions. Thus, enzymes derived from extremophilic microorganisms are needed for these hard industrial conditions. Alicyclobacillus acidocaldarius DSM 446 with an optimum growth temperature of 60–65°C, is an acidothermophilic bacterium that grows in acidic hot springs. In addition to growth in both acidic and hot springs, having a complete genome project makes the organism an important source for enzymes that are used in harsh industrial conditions. Arabinofuranosidases (E.C 3.2.1.55; AFases) belong to glycoside hydrolase family enzymes. AFases hydrolyzes 1,3- and 1,5-α-arabinosyl bounds of L-arabinose containing molecules. L-arabinoses are present in hemicellulosic part of lignocellulosic biomass. AFases also play an important role in the complete hydrolysis of arabinoxylans. Analysis of the genome project and CAZY database revealed two putative arabinofuranosidase genes in the A. acidocaldarius genome. In this study, we aimed cloning, heterologous expression, purification and biochemical characterization of the α-N-Arabinofuranosidase enzyme encoded in A. acidocaldarius genome. For this purpose, the gene of α-N-Arabinofuranosidase protein was cloned into pQE-40 vector and expressed heterologously in E. coli BL21 GOLD (DE3). Biochemical characterization of the purified enzyme revealed that A. acidocaldarius α-N-Arabinofuranosidase enzyme showed optimum activity at 45 ºC and pH 6.5 in phosphate buffer. Furthermore, enzyme has 60% activity after 90 min. incubation at 80 ºC. A. acidocaldarius α-N-Arabinofuranosidase enzyme has high potential to be used as accessory enzyme for the complete degradation of arabinoxylans.

Yersinia outer protein M (YopM) is one of the effector proteins and essential for virulence. YopM is delivered by the Yersinia type III secretion system (T3SS) into the host cell, where it shows immunosuppressive effect through interaction with host proteins. Therefore, protein-protein interactions of YopM is significant to understand its molecular mechanism. In this study, we aimed to explore protein protein interactions of YopM with the two components of T3SS, namely LcrV and LcrG. We used bimolecular fluorescence complementation (BiFC) assay and monitored the reassembly of green fluorescence protein in Escherichia coli. As an indicator of the protein-protein interaction, we monitored the in vivo reconstitution of fluorescence by measuring fluorescence intensity and imaging the cells under fluorescence microscope. We showed, for the first time, that YopM interacts with LcrG, but not with LcrV. Here, we propose BiFC assay as a simple method to screen novel interaction partners of YopM. (c) 2021 Elsevier Inc. All rights reserved.

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy., TUBITAK [120R021]; Republic of Turkey Ministry of Development [2010K120810/2020K12150700, 2016K121190], The study was supported by TUBITAK (Project number: 120R021). The Republic of Turkey Ministry of Development provided the support for the infrastructure of EGEMATAL (Ege University/Izmir) via 2010K120810/2020K12150700 and 2016K121190 grants.

Pesticide use in agriculture is one of the threats to water safety. Therefore, detection of pesticide residues is crucial for human health. Compared to conventional chromatographic methods, aptasensors are promising tools for fast, cheap and sensitive detection of environmental contaminants. To the best of our knowledge, such an aptasensor has not been reported for imidacloprid (Imi) which is one of the most widely used pesticides. in order to meet this demand, we initially selected two novel aptamers designated as 'Apta-1' and 'Apta-2' by graphene oxide-SELEX (GO-SELEX) method. Then, these aptamers were used to fabricate the gold electrode-based aptasensor platforms and characterized by using electrochemical methods such as cyclic voltammetry, and electrochemical impedance spectroscopy as well as X-Ray photoelectron spectroscopy. It was found that the limit of detection value of Apta-1 based sensor for the Imi was found better than Apta-2 based system, although linear ranges were similar. Based on that finding, Apta-1 based system was further tested against possible interference molecules. the proposed platform was successfully used for detection of very low concentrations of Imi in the range of ng/mL. Thus, it eliminates the need for sample pre-treatment and enables a practical analysis in real wastewater samples., EUEuropean Union (EU); FCT under the ERA-NET Cofund WaterWorks 2015 Call [Water-Works2015-AGRINUPES]; Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [117Y195]; Republic of Turkey, Ministry of DevelopmentTurkiye Cumhuriyeti Kalkinma Bakanligi [2016K121190], The authors would like to thank the EU and FCT for funding, in the frame of the collaborative international consortium AGRINUPES financed under the ERA-NET Cofund WaterWorks 2015 Call (Project code: Water-Works2015-AGRINUPES, Project name: Integrated monitoring and control of water, nutrients and plant protection products towards a sustainable agricultural sector). This ERA-NET is an integral part of the 2016 Joint Activities developed by the Water Challenges for a Changing World Joint Programme Initiative (Water JPI). This research was supported by the Scientific and Technological Research Council of Turkey (TUBITAK, Project Grant No: 117Y195). This work was also partially supported by Republic of Turkey, Ministry of Development (Project Grant No: 2016K121190). Ege University Central Research Testing and Analysis Laboratory Research and Application Center (EGE-MATAL) chromatography laboratories were used in this study. Also, we thank to Turkish Water Institute (SUEN) due to their contributions as one of the partner Institute in Turkey.

Synthetic cannabinoids (SCs) are the large group of abused drugs and detection of them is still a challenge. Hence, new methods for analysis of SCs are being investigated. We aimed to develop a novel system for selective analysis of SCs. First, various custom-tailored aptamers against the target SCs were selected through GO-SELEX process. Toggling between different SC analytes during successive rounds of selection was performed to generate cross-reactive aptamers. Then, the amino-capped aptamers were synthesized and easily attached to the cysteamine-covered gold electrodes. Analytical parameters and selectivity of the aptasensors were compared by using electrochemical techniques. After comparison of the analytical features and selectivity towards target analytes, one of the aptamers designated as Apta-1 was chosen for further measurements. the aptasensor was tested by using differential pulse voltammetry technique against JWH-018 (5-pentanoic acid), selected as a model for SCs. the linearity and limit of detection were determined as 0.01-1.0 ng/mL and 0.036 ng/mL. Finally, sample application in synthetic urine samples was successfully performed with standard addition method, as confirmed by LC-QTOF/MS. JWH-018 (4-hydroxypentyl), JWH-073 (3-hydroxybutyl), JWH-250 (5-hidroxypentyl) and HU-210 were used to test the selectivity of the aptasensor and the system was shown to recognize all these SCs. Also other illegal drugs did not significantly interfere with the signal responses., Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [117Z152]; Republic of Turkey, Ministry of DevelopmentTurkiye Cumhuriyeti Kalkinma Bakanligi [2016K121190]; Ege University Central Research Testing and Analysis Laboratory Research and Application Center (EGE-MATAL), This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK, Project Grant No: 117Z152). This work was also supported by Republic of Turkey, Ministry of Development (Project Grant No: 2016K121190) and Ege University Central Research Testing and Analysis Laboratory Research and Application Center (EGE-MATAL) chromatography laboratories were used in this study.

An aptamer-based electrochemical biosensor was designed and fabricated for methamphetamine (METH) anal-ysis. Several aptamer sequences developed by the GO-SELEX method were immobilized onto gold electrodes (GE) via gold-thiol affinity. Surface characterizations were performed by electrochemical methods including cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectrometry (EIS). GE/Apta-4/METH aptasensor was selected for further studies since the best results were obtained with the Apta-4 sequence in terms of analytical parameters. Accordingly, it exhibited good selectivity for METH analysis with a wide linear range of 0.1-50 ng/mL and a low limit of detection (LOD) which was found to be 0.467 ng/mL. The proposed aptasensor platform displayed good selectivity for the detection of METH in synthetic urine samples spiked with METH., Republic of Turkey, Ministry of Development [2016 K121190]; Ege University Scienti fi c Research Projects Coordination Unit [18 -FEN -006], This work was supported by the Republic of Turkey, Ministry of Development (Project Grant No: 2016 K121190) and partially by Ege University Scienti fi c Research Projects Coordination Unit (Project Grant No: 18 -FEN -006) . We thank Ege University Central Research Testing and Analysis Laboratory Research and Application Center (EGE-MATAL) for ITC measurements.

The increasing mutation frequency of the SARS-CoV-2 virus and the emergence of successive variants have made correct diagnosis hard to perform. Developing efficient and accurate methods to diagnose infected patients is crucial to effectively mitigate the pandemic. Here, we developed an electrochemical immunosensor based on SARS-CoV-2 antibody cocktail-conjugated magnetic nanoparticles for the sensitive and accurate detection of the SARS-CoV-2 virus and its variants in nasopharyngeal swabs. The application of the antibody cocktail was compared with commercially available anti-SARS-CoV-2 S1 (anti-S1) and anti-S2 monoclonal antibodies. After optimization and calibration, the limit of detection (LOD) determination demonstrated a LOD = 0.53-0.75 ng/ mL for the antibody cocktail-based sensor compared with 0.93 ng/mL and 0.99 ng/mL for the platforms using anti-S1 and anti-S2, respectively. The platforms were tested with human nasopharyngeal swab samples pre diagnosed with RT-PCR (10 negatives and 40 positive samples). The positive samples include the original, alpha, beta, and delta variants (n = 10, for each). The polyclonal antibody cocktail performed better than commercial anti-S1 and anti-S2 antibodies for all samples reaching 100% overall sensitivity, specificity, and accuracy. It also showed a wide range of variants detection compared to monoclonal antibody-based platforms. The present work proposes a versatile electrochemical biosensor for the indiscriminate detection of the different variants of SARS-CoV-2 using a polyclonal antibody cocktail. Such diagnostic tools allowing the detection of variants can be of great efficiency and economic value in the fight against the ever-changing SARS-CoV-2 virus., Scientific and Technological Research Council of Turkey (TUBITAK) [120R021]; Republic of Turkey, Ministry of Development [2016K121190], This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK, Project Grant No: 120R021) and Republic of Turkey, Ministry of Development (Project Grant No: 2016K121190) . The authors are thankful to the EGE-MATAL for providing its facilities for all experimental stages.

The global pandemic of COVID-19 continues to be an important threat, especially with the fast transmission rate observed after the discovery of novel mutations. In this perspective, prompt diagnosis requires massive economical and human resources to mitigate the disease. The current study proposes a rational design of a colorimetric lateral flow immunoassay (LFA) based on the repurposing of human samples to produce COVID-19-specific antigens and antibodies in combination with a novel dye-loaded polymersome for naked-eye detection. A group of 121 human samples (61 serums and 60 nasal swabs) were obtained and analyzed by RT-PCR and ELISA. Pooled samples were used to purify antibodies using affinity chromatography, while antigens were purified via magnetic nanoparticles-based affinity. The purified proteins were confirmed for their specificity to COVID-19 via commercial LFA, ELISA, and electrochemical tests in addition to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Polymersomes were prepared using methoxy polyethylene glycol-b-polycaprolactone (mPEG-b-PCL) diblock copolymers and loaded with a Coomassie Blue dye. The polymersomes were then functionalized with the purified antibodies and applied for the preparation of two types of LFA (antigen test and antibody test). Overall, the proposed diagnostic tests demonstrated 93 and 92.2% sensitivity for antigen and antibody tests, respectively. The repeatability (92-94%) and reproducibility (96-98%) of the tests highlight the potential of the proposed LFA. The LFA test was also analyzed for stability, and after 4 weeks, 91-97% correct diagnosis was observed. The current LFA platform is a valuable assay that has great economical and analytical potential for widespread applications., EGE University, Research Foundation [TOA2020-21862]; Republic of Turkey, Ministry of Development [2010K120810/2020K12150700, 2016K121190], We send our acknowledgment to the staff of EGE University Hospital who are on the front line fighting against the COVID19 pandemic. FTIR and SEM analyses were performed at EGE MATAL (Ege University/Izmir). EGE University, Research Foundation was acknowledged for the financial support (TOA2020-21862). Republic of Turkey, Ministry of Development provided the support for the infrastructure of EGE MATAL (Ege University/Izmir) via 2010K120810/2020K12150700 and 2016K121190 grants.

On-site detection of substance abuse is an important approach in the preventive and intervention protocols implementations. It is known that the traditional methods are heavy, time-consuming, and need a high level of logistical requirements. As such, biosensors represent great potential to simplify and improve substance abuse detection. in this study, we have designed a functionalized screen-printed electrode (SPE) electrochemical biosensor with cobalt oxide nanoparticles and single-chain antibody fragments (scFvs) for cocaine detection. Different electrochemical techniques such as differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectrometry were used to examine the functionality of the designed biosensor. Furthermore, SEM observations were performed to observe the surface changes after functionalization. the results showed that the linearity ranged between 5.0 and 250 ng/mL and a detection limit of 3.6 ng/mL (n = 6). These results were compared to results obtained from Q-TOF/MS where four different matrices (serum, sweat, urine, and saliva) were spiked with 100 ng/mL cocaine and were analyzed by both methods (Biosensor and Q-TOF/MS). Results showed a higher performance of the biosensor compared to traditional methods. in addition, the selectivity of the biosensor was shown in the presence of different interferents where the designed platform showed a specific response to only cocaine. in conclusion, the designed biosensor proposes great potential for portable and on-site substance abuse detection in addition to boasting the capability of reuse of the SPE and thus, reducing the costs related to such applications., Republic of Turkey, Ministry of DevelopmentTurkiye Cumhuriyeti Kalkinma Bakanligi [2016K121190]; Aliye Uster Foundation, This work was supported by the Republic of Turkey, Ministry of Development (Project Grant No: 2016K121190). Part of the work was funded by Aliye Uster Foundation.

Non-viral gene delivery systems have great potential for safe and efficient gene therapy, while inefficient cellular and nuclear uptake remain as the major hurdles. Novel approaches are needed to enhance the transfection efficiency of non-viral vectors. in accordance with this need, the objective of this study was to construct a nonviral vector that could achieve gene delivery without using additional lipid-based transfection agent. We aimed to impart self-delivery property to a non-viral vector by using the cell and nucleus penetrating properties of YopM proteins from the three Yersinia spp. (Y. pestis, Y. enterocolotica and Y. pseudotuberculosis). Plasmid DNA (pDNA) encoding green fluorescent protein (GFP) was labeled with quantum dots (QDs) via peptide-nucleic acid (PNA) recognition site. Recombinant YopM protein was then attached to the conjugate via a second PNA recognition site. the YopM- QDs- pDNA conjugate was transfected into HeLa cells without using additional transfection reagent. All three conjugates produced GFP fluorescence, indicating that the plasmid was successfully delivered to the nucleus. As control, naked pDNA was transfected into the cells by using a commercial transfection reagent. the Y. pseudotuberculosis YopM-functionalized conjugate achieved the highest GFP expression, compared to other two YopM proteins and the transfection reagent. To the best of our knowledge, YopM protein was used for the first time in a non-viral gene delivery vector., Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [113Z379]; Republic of Turkey, Ministry of Development, Turkey [2016K121190], This study was financially supported by the Scientific and Technological Research Council of Turkey (TUBITAK, Grant number; 113Z379). We also acknowledge the fellowships to O.U and F.B.B. from the Republic of Turkey, Ministry of Development, Turkey (Grant number 2016K121190).

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays are the gold standard for virus diagnosis. Point-of-care (POC) technologies have shown great progress during this period. Herein, we propose a novel fuchsine dye-loaded polymersome for a colorimetric paper-based dot blot spike protein diagnostic assay for COVID-19 via smartphone-assisted sensing. The prepared platform aimed to create an adaptable tool that competes with traditional nanoparticle-based assays employing gold and silver. Analytical characterization and application of the testing platform showed high sensitivity (10 times better than gold nanoparticles), stability, fast turnaround, and reproducibility. The potential and possibilities demonstrated by the current platform could be observed in its adaptability for different markers and pathologies. In addition, smartphone-assisted sensing emphasizes the ability to use the tool at home by common peoples which can lower the burden on the healthcare facilities and reach more underdeveloped regions., Research Foundation of Ege University, Turkey [TOA-2020-21862]; Republic of Turkey, Ministry of Development [2010K120810/2020K12150700, 2016K121190], The research project was supported by the Research Foundation of Ege University, Turkey (Project number: TOA-2020-21862) . Republic of Turkey, Ministry of Development provided the support for the infra-structure of EGE MATAL (Ege University/Izmir) via 2010K120810/2020K12150700 and 2016K121190 grants.

Salmonella Enteritidis is an important pathogen that can invade the intestinal cells of its host causing salmonellosis. SipA protein, an effector protein secreted by T3SS, maintains invasion of host cells more efficient. Thus, inhibitory aptamers against SipA protein were developed using magnetic bead-based Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. the enriched sequences were obtained after 9 SELEX rounds. Among which, an aptamer namely Apt17 displayed Kd values equivalent to 114.9 and 63.4 nM at 27 degrees C and 37 degrees C, respectively. the effect of Apt17 on adhesion and invasion of Caco-2 cells by the tested strains was determined. While the adhesion and invasion of Salmonella Enteritidis TM 6 were inhibited by 70% and 37.7%, those of Salmonella Enteritidis TM 68 were inhibited by 45.71% and 39.5% respectively. These results represent a corner stone for future studies that could aim to develop putative inhibitors against Salmonellosis. (C) 2020 Elsevier B.V. All rights reserved., Scientific Research Unit of Ege UniversityEge University [2017-FEN-038], This work was financially supported by the Scientific Research Unit of Ege University (Project number: 2017-FEN-038).

Aptamer-based point-of-care (POC) diagnostics platforms may be of substantial benefit in forensic analysis as they provide rapid, sensitive, user-friendly, and selective analysis tools for detection. Aptasensors have not yet been adapted commercially. However, the significance of the applications of aptasensors in the literature exceeded their potential. Herein, in this review, a bottom-up approach is followed to describe the aptasensor development and application procedure, starting from the synthesis of the corresponding aptamer sequence for the selected analyte to creating a smart surface for the sensitive detection of the molecule of interest. Optical and electrochemical biosensing platforms, which are designed with aptamers as recognition molecules, detecting abused drugs are critically reviewed, and existing and possible applications of different designs are discussed. Several potential disciplines in which aptamer-based biosensing technology can be of greatest value, including forensic drug analysis and biological evidence, are then highlighted to encourage researchers to focus on developing aptasensors in these specific areas.

Mercury ion (Hg

Sarcosine is an amino acid derivative, which is considered as a key metabolite in various metabolic processes. Therefore, simple and sensitive detection methods are needed for further understanding its metabolic role and diagnostic value. In this study, we developed a novel method that meets the need for practical and sensitive detection in a complex medium mimicking urine conditions. For this aim, we selected sarcosine-specific DNA aptamers using graphene oxide-assisted systemic evolution of ligands by exponential enrichment (GO-SELEX). The candidate aptamers were labeled with 6-carboxyfluorescein (6-FAM) at their 5' ends. Two aptamers, namely 9S and 13S produced a significant fluorescence signal upon sarcosine binding. Both aptamers enabled a sensitive analysis with a detection limit of 0.5 pM. The linear detection ranged between 5 pM and 50 mu M for 9S aptamer, while 13S aptamer enabled a wider linear detection range between 5 pM and 500 mu M. The aptamer-based assay allowed rapid detection with no need for chemical derivatization of sarcosine and sophisticated instruments. Moreover, the aptamer-based assay was free of interference from urea and human serum albumin. (C) 2019 Elsevier B.V. All rights reserved.

Evran, Serap/0000-0001-6676-4888, WOS: 000491221600003, PubMed: 31411759, Mercury ion (Hg2+) is a universal pollutant and its detection is crucial for public healthcare. in this study, we developed a novel fluorescent biosensor by construction of a protein fusion between the mercury-sensing transcription factor MerR and enhanced yellow fluorescent protein (EYFP). Hg2+-induced conformational change of MerR was transduced into fluorescence signal. Fluorescence intensity of the biosensor protein decreased with increasing concentrations of Hg2+ and a linear response was obtained in the range of 0.5-40 nM. the limit of detection was 0.5 nM, which was much lower than the maximum allowed level in water. the biosensor specificity was highly dependent on type and concentration of metal ion. the biosensor exhibited high specificity in a mixture of metal ions at 0.5 nM concentration. However, the interference effect was more pronounced at 40 nM concentration of metal ions. the measurement was completed in less than 1 Min with no need for sample preparation or preincubation steps. the biosensor achieved accurate and reliable detection in the spiked drinking water sample, as validated by the inductively coupled plasma optical emission spectrometry., Ege University Scientific Research Projects Coordination UnitEge University [2011 FEN 035] Funding Source: Medline

The hydrophobic nature of cell membranes is one of the major obstacles in the therapeutic delivery of nucleic acids and drug-loaded nanoparticles. Cell-penetrating peptides (CPPs) have the ability to pass biological membranes and enter cells. Due to this intrinsic property, CPPs are employed as vectors for intracellular delivery of nucleic acids and nanoparticles. In this chapter, we first briefly describe the classification and uptake mechanisms of CPPs. Then, we describe the recent therapeutic applications of CPP-modified nanoparticles as drug carriers. In this context, we give an overview of covalent and noncovalent conjugation of CPPs. The second part involves the use of CPPs in nonviral delivery of nucleic acids. Although viral vectors are highly efficient systems for introducing genes, the safety issues with viral systems need to be considered. Nanoparticle-based nonviral vectors provide an attractive alternative, but their gene transfection efficiency is very low. Therefore, novel design strategies are needed to enhance the efficiency. We summarize the use of CPPs in enhancing gene transfer efficiency of nonviral vectors. Besides the clinical potential of currently known CPPs, we also discuss the limitations and the need for designing novel CPPs.

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Inefficient delivery of antibiotics to the infection microenvironment, side effects of high-dose antibiotic use, as well as antimicrobial drug resistance are major global issues that remain to be solved. Efficient drug delivery systems and novel antimicrobial treatment strategies are urgently needed. Cell-specific aptamers are excellent molecules for specific targeting of microbial cells. Aptamers are single-stranded DNA or RNA molecules, which are able to selectively bind to their targets with high affinity. In this chapter, we first describe the methods for in vitro selection of aptamers against the whole pathogenic cell. Then we present an overview of current cell-specific aptamer-conjugated nanoparticles. In addition, we address two issues: nanoparticle-based antimicrobial gene delivery and modification of nanocarriers with aptamers.

A simple approach for the preparation of a clickable poly(ethylene glycol)-based hydrogel as a polymeric support for protein immobilization via photoinitiated free radical polymerization of poly(ethylene glycol) diacrylate and propargyl acrylate is established. Bioconjugation to the obtained gels was achieved by azide–alkyne click reaction with azido-functionalized anti-immunoglobulin G (anti-IgG) and anti-His tag antibodies. Evaluation of the affinities of the PEG hydrogels to the corresponding substrates (IgG and His-tagged YFP, respectively) indicates their specific binding capability.

We developed a fluorescent protein construct by genetically fusing green fluorescent protein (GFP) to aspartate dehydrogenase from Thermotoga maritima. The fusion protein was cloned, heterologously expressed in Escherichia coli cells, and purified by Ni-chelate affinity chromatography. It was then introduced into a measurement cuvette to monitor its fluorescence signal. Aspartate dehydrogenase functioned as the biorecognition element, and aspartate-induced conformational change was converted to a fluorescence signal by GFP. The recombinant protein responded to l-aspartate (l-Asp) linearly within the concentration range of 1-50 mM, and it was capable of giving a fluorescence signal in 1 Min. Although a linear response was also observed for l-Glu, the fluorescence signal was 2.7 times lower than that observed for l-Asp. In the present study, we describe two novelties: development of a genetically encoded fluorescent protein construct for monitoring of l-Asp in vitro, and employment of aspartate dehydrogenase scaffold as a biorecognition element. A few genetically encoded amino-acid biosensors have been described in the literature, but to our knowledge, a protein has not been constructed solely for determination of l-Asp. Periplasmic ligand binding proteins offer high binding affinity in the micromolar range, and they are frequently used as biorecognition elements. Instead of choosing a periplasmic l-Asp binding protein, we attempted to use the substrate specificity of aspartate dehydrogenase enzyme.

Lysozyme from egg white was modified by covalent attachment of an oleyl group to the free amino groups of lysozyme. The aim of the chemical modification was to develop an effective antimicrobial lysozyme derivative against both gram-negative and gram-positive bacteria. Lysozyme with various degrees of modification was obtained by changing oleoyl chloride/lysozyme mass ratio. Lysozyme derivatives evidently exhibited an antimicrobial effect against Escherichia coli (ATCC 29998). The modification slightly changed the antimicrobial effect of lysozyme derivative against Staphylococcus aureus (ATCC 121002). Since there was a positive correlation between the modification degree and the antimicrobial effect against E. coli, it was concluded that the change in antimicrobial behavior was due to an increase in hydrophobicity of the enzyme molecule enabling it to penetrate through the bacterial membrane of E. coli. It was also shown that oleoyl chloride with an MIC value of 10 mg/mL was effective against both E. coli and S. aureus.

WOS: 000358017700001, PubMed ID: 25680359, Neopullulanase, a glycosyl hydrolase from Bacillus stearothermophilus (bsNpl), is a potentially valuable enzyme for starch and detergent industries. However, as the protein is not active at elevated temperatures and high surfactant concentrations, we aimed to increase its stability by rational enzyme design. Nine potentially destabilizing cavities were identified in the crystal structure of the enzyme. Based on computational predictions, these cavities were filled by residues with bulkier side chains. The five Asp46Glu, Val239Leu, Val404Leu, Ser407Thr and Ala566Leu exchanges resulted in a drastic stabilization of bsNpl against inactivation by heat and detergents. The catalytic activity of the variants was identical to the wild-type enzyme., TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [210T074]; BMBFFederal Ministry of Education & Research (BMBF) [01DL12022], This work was supported by a joint Turkish-German collaboration project of TUBITAK (IntenC project number 210T074) and BMBF (project number 01DL12022).

His-tagged Alcohol dehydrogenase was produced as a recombinant protein in E. coli. Afterwards, isolation and immobilization of the enzyme was carried in one-step via copper modified magnetic nanoparticles (MNPs) by the effect of interactions between Cu and histidine. The resulting enzyme bound MNPs was then attached to the surface of carbon paste electrode by the magnetic force and used as an electrochemical biosensor for the alcohol sensing applications.

Circularly permuted green fluorescent protein (cGFP) was inserted into the hyperthermophilic maltose binding protein at two different locations. cGFP was inserted between amino acid residues 206 and 207, or fused to the N-terminal of maltose binding protein from Thermotoga maritima. The cloned DNA constructs were expressed in Escherichia coli cells, and purified by metal chelate affinity chromatography. Conformational change upon ligand binding was monitored by the increase in fluorescence intensity. Both of the fusion proteins developed significant fluorescence change at 0.5 mM maltose concentration, whereas their maltose binding affinities and optimum incubation times were different. Fluorescent biosensors based on mesophilic maltose binding proteins have been described in the literature, but there is a growing interest in biosensors based on thermostable proteins. Therefore, the developed protein constructs could be models for thermophilic protein-based fluorescent biosensors.

The oxidative stability of α-amylase (AmyC) from Thermotoga maritima was improved by mutating the methionine residues at positions 43 and 44, 55, and 62 to oxidative-resistant alanine residues. The most resistant M55A variant showed 50% residual activity in the presence of 100 mM H2O2, whereas the wild-type enzyme was inactive.

We developed a fluorescent protein construct by genetically fusing green fluorescent protein (GFP) to aspartate dehydrogenase from Thermotoga maritima. The fusion protein was cloned, heterologously expressed in Escherichia coli cells, and purified by Ni-chelate affinity chromatography. It was then introduced into a measurement cuvette to monitor its fluorescence signal. Aspartate dehydrogenase functioned as the biorecognition element, and aspartate-induced conformational change was converted to a fluorescence signal by GFP. The recombinant protein responded to l-aspartate (l-Asp) linearly within the concentration range of 1-50 mM, and it was capable of giving a fluorescence signal in 1 Min. Although a linear response was also observed for l-Glu, the fluorescence signal was 2.7 times lower than that observed for l-Asp. In the present study, we describe two novelties: development of a genetically encoded fluorescent protein construct for monitoring of l-Asp in vitro, and employment of aspartate dehydrogenase scaffold as a biorecognition element. A few genetically encoded amino-acid biosensors have been described in the literature, but to our knowledge, a protein has not been constructed solely for determination of l-Asp. Periplasmic ligand binding proteins offer high binding affinity in the micromolar range, and they are frequently used as biorecognition elements. Instead of choosing a periplasmic l-Asp binding protein, we attempted to use the substrate specificity of aspartate dehydrogenase enzyme.

PubMed ID: 22490958, Phosphoribosylanthranilate (PRA) isomerase (TrpF) and tryptophan synthase ?-subunit (TrpA) are (ß?)8-barrel enzymes that are involved in the biosynthesis of tryptophan. They contain a conserved phosphate binding site, which indicates a common evolutionary origin. In order to experimentally back this hypothesis, we have established TrpF activity on the scaffold of TrpA from Salmonella typhimurium using protein engineering. Based on the superposition of crystal structures with bound ligands, two residues in the active site of TrpA were replaced with catalytic residues from TrpF using site-directed mutagenesis. This TrpA variant as well as wild-type TrpA were each subjected to random mutagenesis using error-prone PCR. The two resulting trpA gene libraries were used to transform an auxotrophic Escherichia coli trpF deletion strain, and TrpA variants with PRA isomerisation activity were isolated by in vivo complementation. The amino acid substitutions of the selected TrpA variants were recombined by DNA shuffling, again followed by complementation in vivo. Several TrpA variants were produced in E. coli and purified, and their catalytic TrpF activities were determined in vitro by steady-state enzyme kinetics. Our results support that TrpA and TrpF have evolved by gene duplication and diversification from each other or a common predecessor, and provide insights into the minimum requirements for the catalysis of PRA isomerisation. © The Author 2012. Published by Oxford University Press., German Academic Exchange Service London 2006 FEN 001, 2007 BIL 011 European Molecular Biology Organization: 106M111 Deutscher Akademischer Austauschdienst National Council for Scientific Research, We acknowledge fellowships to S.E. from the Scientific and Technological Research Council of Turkey (TUBITAK), the German Academic Exchange Service (DAAD) and the European Molecular Biology Organization (EMBO). This work was supported by research grants from TUBITAK (106M111) and Ege University Research Foundation (2006 FEN 001 and 2007 BIL 011).

Porcine pancreatic lipase was modified with Z‐proline via the constitution of amide bonds between the free amino groups of lipase and the carboxyl groups of Z‐proline, which were activated by 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide (EDC). Different amounts of Z‐proline were bound to lipase. Modification degree was determined by 2,4,6‐trinitrobenzene sulphonic acid (TNBS), by means of the decrease in free amino groups on lipase. The reason for choosing Z‐proline was its unique structural characteristics, protected amino groups, and its effect on protein conformation by reducing the flexibility of the lipase molecule, thus achieving stabilization against changes in pH and temperature. After the modification, porcine pancreatic lipase was found to have new physicochemical characteristics, such as optimum alkaline pH stability and thermal stability at elevated temperatures.

Helicobacter pylori gastrit ve duodenum ülserinin en önemli sebebidir. Bunun yanında Helicobacter pylori ve diğer bazı Helicobacter türlerine mide dışı dokularda da rastlanmaktadır. Karaciğer kanseri ve primer biliyer siroz gibi çeşitli karaciğer hastalıklarında da Helicobacter türlerinin rolü olabileceğine dair çalışmalar yayınlanmıştır. Amaç: Bu çalışmada çeşitli karaciğer hastalıklarında karaciğer dokusunda Helicobacter pylori ve diğer Helicobacter türlerinin varlığının araştırılması planlanmıştır. Materyal ve Metod: Farklı etyolojilere sahip kronik karaciğer hastalarında iğne biyopsisi ile elde edilen karaciğer biyopsilerinde PCR yöntemi ile Helicobacter pylori'nm üreaz genini ve Helicobacter türlerinin 16S ribozomal DNA'sını hedefleyen amplifikasyonlar yapılmıştır. Bulgular: Kırkbeşi kriptojenik karaciğer hastalığı, 30'u kronik HBV infeksiyonu, 21'i kronik HCV infeksiyonu, 17'si alkole bağlı olmayan karaciğer yağlanması, 15'i primer biliyer siroz, 12'si alkol dışı steatohepatit tanısı alan, 45'i diğer karaciğer hastalıklarına sahip 185 hastanın sadece "otoimmün hepatit+primer biliyer siroz overlap sendromu" tanısı alan birinde karaciğerde Helicobacter 16S ribozomal DNA'stna rastlanmıştır. Üreaz gen amplifikasyonu tüm hastalarda negatif bulunmuştur. Sonuç: Kronik karaciğer hastalıklarında karaciğer dokusunda Helicobacter türlerine ait genetik elemanlara rastlanmamış, bu nedenle kronik karaciğer hastalıklarının etyolojisinde Helicobacter türleriyle oluşan infeksiyonunun bir rolü olmadığı düşünülmüştür., Helicobacter pylori is the major etiologic agent in gastritis and duodenal ulcer. Helicobacter pylori and some other Helicobacter species have been found in extragastric tissues as well. Some reports suggested a relation between Helicobater infection and various liver diseases, such as liver cancer, and primary biliary cirrhosis. Aim: to investigate the genetic elements of Helicobacter pylori and other Helicobacter species in liver tissues of the patients with various liver diseases by PCR. Materials and Methods: a part of the urease gene of Helicobacter pylori, and 16S ribosomal DNA of Helicobacter species were determined from the liver tissues of patients with chronic liver disease by PCR. Results: 185 patients with various liver disease (45 had crip-togenic liver disease, 30 had chronic HBV infection, 21 HCV infection, 17 steatosis hepatis, 15 primary biliary cirrhosis and 12 had nonalcoholic steatohepatitis, and 45 other liver diseases) were included in the study. Only 1 patient with PBC and autoimmune hepatitis overlap syndrome had Helicobacter 16S ribosomal DNA in her liver tissue. None of the patients was found to be positive for the PCR of H. pylori urease gene. Conclusions: In this study, genetic elements of Helicobacter species were not detected in the liver tissues of the patients with chronic liver diseases. These results suggested that Helicobacter infections in the liver have almost no role in the pathogenesis of chronic liver diseases in our population.