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Molecular Biotechnology

Arezoo Golestanipour, Maryam Nikkhah, Ali Aalami, Saman Hosseinkhani
Development of efficient, easy, and safe gene delivery methods is of great interest in the field of plant biotechnology. Considering the limitations of the usual transfection methods (such as transgene size and plant type), several new techniques have been tested for replacement. The success of some biological and synthetic nanostructures such as cell-penetrating peptides and carbon nanotubes in transferring macromolecules (proteins and nucleic acids) into mammalian cells provoked us to assess the ability of an engineered chimeric peptide and also arginine functionalized single-walled carbon nanotube in gene delivery to intact tobacco (Nicotiana tabacum var...
September 10, 2018: Molecular Biotechnology
Sébastien Sart, Spiros N Agathos
Mesenchymal stem cells and pluripotent stem cells are recognized as promising tools for tissue engineering, cell therapy, and drug screening. Their use in therapy requires the production of a sufficient number of cells committed to functional regenerative phenotypes. Time- and magnitude-controlled application of mechanical and biochemical cues is required to appropriately control the evolution of stem cell phenotype in 3D. The temporal monitoring of the impact of these cues on the diverse fates of individual stem cells is also needed to ensure the reliability of the differentiation processes...
September 8, 2018: Molecular Biotechnology
Saeed Khalili, Mohamad Javad Rasaee, Taravat Bamdad, Maysam Mard-Soltani, Majid Asadi Ghalehni, Abolfazl Jahangiri, Mohammad Hassan Pouriayevali, Mohammad Reza Aghasadeghi, Fatemeh Malaei
Nucleic acid immunization has recently exhibited a great promise for immunotherapy of various diseases. However, it is now clear that powerful strategies are imminently needed to improve their efficiency. In this regard, whole bacteriophage particles have been described as efficient DNA vaccine delivery vehicles, capable of circumventing the limitations of naked DNA immunization. Moreover, phage particles could be engineered to display specific peptides on their surfaces. Given these inherent characteristics of phages, we have designed a novel hybrid phage-DNA immunization vector using both M13 and pAAV plasmid elements...
September 4, 2018: Molecular Biotechnology
Monika M Golas, Sakthidasan Jayaprakash, Le T M Le, Zongpei Zhao, Violeta Heras Huertas, Ida S Jensen, Juan Yuan, Bjoern Sander
The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins...
September 3, 2018: Molecular Biotechnology
Wojciech Potocki, Alessandro Negri, Grażyna Peszyńska-Sularz, Krzysztof Hinc, Michał Obuchowski, Adam Iwanicki
Mucosal immunizations are convenient ways of vaccination, which do not require any trained personnel for administration. One of the major challenges for developing an effective mucosal vaccine is finding appropriate adjuvant. Bacillus subtilis endospores have been shown to help solving these obstacles while serving as a platform for presentation of both, antigens and adjuvants. In this study, we have successfully designed and constructed recombinant spores displaying an antigen/adjuvant chimeric protein. We have used a fragment of Clostridium difficile flagellar cap FliD protein as antigen and VQGEESNDK peptide, a fragment of human IL-1β, as adjuvant...
September 3, 2018: Molecular Biotechnology
Rahul Vasudeo Ramekar, Kyong-Cheul Park, Kyu Jin Sa, Ju Kyong Lee
Transposable elements account for up to 85% of the maize genome and have significant implications in crop-improvement and evolutionary analyses. The Mutator (Mu) transposon superfamily, a class of DNA transposons, comprises the most complex and active elements in the maize genome, suggesting a special role in plant evolution. Here, we designed a set of Mu-specific primers based on terminal invert repeats and used a transposon display (TD) method for genotyping. We analyzed the distribution pattern of Mu insertions in teosinte (wild relative), sorghum (distant relative), and domesticated maize accessions (dent, sweet, and waxy)...
September 3, 2018: Molecular Biotechnology
Yan Wang, Tingjun Lei, Qian Dai, Ping Ding, Tong Qiu, Yin Fang
Embryonic stem cells (ESCs) are self-renewing pluripotent cells, capable of differentiating into all somatic cell types. The molecular control of self-renewal is relatively well-characterized, whereas how ESCs exit pluripotent state to differentiate is poorly understood. Here we identify two genes are required for differentiation and dozens of intergenic regions that potentially regulate ESC differentiation. We used PiggyBac (PB) transposon-based approach to randomly mutate the genome of ESCs, and generated hundreds of clones that resisted differentiation signals...
August 31, 2018: Molecular Biotechnology
Yeliz Demir, Neslihan Dikbaş, Şükrü Beydemir
Phytase (myo-inositol hexaphosphate phosphohydrolase) belongs to phosphatases. It catalyzes the hydrolysis of phytate to less-phosphorylated inorganic phosphates and phytate. Phytase is used primarily for the feeding of simple hermit animals in order to increase the usability of amino acids, minerals, phosphorus and energy. In the present study, phytase isolation from the Lactobacillus coryniformis strain, isolated from Lor cheese sources, phytase purification and characterization were studied. The phytase was purified in simple three steps...
August 31, 2018: Molecular Biotechnology
Flavia Ferrantelli, Francesco Manfredi, Chiara Chiozzini, Simona Anticoli, Eleonora Olivetta, Claudia Arenaccio, Maurizio Federico
Eukaryotic cells constitutively produce nanovesicles of 50-150 nm of diameter, referred to as exosomes, upon release of the contents of multivesicular bodies (MVBs). We recently characterized a novel, exosome-based way to induce cytotoxic T lymphocyte (CTL) immunization against full-length antigens. It is based on DNA vectors expressing products of fusion between the exosome-anchoring protein Nef mutant (Nefmut ) with the antigen of interest. The strong efficiency of Nefmut to accumulate in MVBs results in the production of exosomes incorporating huge amounts of the desired antigen...
August 30, 2018: Molecular Biotechnology
Liliana López-Pliego, Liliana García-Ramírez, Emma Aurora Cruz-Gómez, Patricia Domínguez-Ojeda, Alejandra López-Pastrana, Luis Ernesto Fuentes-Ramírez, Cinthia Núñez, Miguel Castañeda
The GacS/A system in Azotobacter vinelandii regulates alginate and alkylresorcinols production through RsmZ1, a small regulatory RNA (sRNA) that releases the translational repression of the algD and arpR mRNAs caused by the RsmA protein. In the Pseudomonadaceae family, the Rsm-sRNAs are grouped into three families: RsmX, RmsY and RsmZ. Besides RsmZ1, A. vinelandii has six other isoforms belonging to the RsmZ family and another one to the RsmY. Environmental signals controlling rsmsRNAs genes in A. vinelandii are unknown...
July 10, 2018: Molecular Biotechnology
Rodrigo Arreola, José Luis Villalpando, Jonathan Puente-Rivera, Jorge Morales-Montor, Enrique Rudiño-Piñera, María Elizbeth Alvarez-Sánchez
Previously, metalloproteinase was isolated and identified from Trichomonas vaginalis, belonging to the aminopeptidase P-like metalloproteinase subfamily A/B, family M24 of clan MG, named TvMP50. The native and recombinant TvMP50 showed proteolytic activity, determined by gelatin zymogram, and a 50 kDa band, suggesting that TvMP50 is a monomeric active enzyme. This was an unexpected finding since other Xaa-Pro aminopeptidases/prolidases are active as a biological unit formed by dimers/tetramers. In this study, the evolutionary history of TvMP50 and the preliminary crystal structure of the recombinant enzyme determined at 3...
June 23, 2018: Molecular Biotechnology
Gustavo Torres de Souza, Rafaela Chitarra Rodrigues Hell, Jéssica Fernanda da Silva Souza, Luiz Sérgio de Almeida Camargo
The extensive growth in number and importance of experiments and clinical-aimed techniques based solely or majorly on the activity of RNA strands, e.g. CRSPR/Cas9 and siRNA, has put emphasis on the necessity of standardisation of experiments with RNA. Considering RNA degradation during its handling seems to be a major hindrance in all RNA-based tools, the assessment of its integrity is of utmost importance. Furthermore, evaluating whether the RNA to be transfected is intact requires time-consuming electrophoresis protocol...
October 2018: Molecular Biotechnology
Revathy Sankaran, Pau Loke Show, Yu-Shen Cheng, Yang Tao, Xia Ao, Thi Dong Phuong Nguyen, Dong Van Quyen
Microalgae are the most promising sources of protein, which have high potential due to their high-value protein content. Conventional methods of protein harnessing required multiple steps, and they are generally complex, time consuming, and expensive. Currently, the study of integration methods for microalgae cell disruption and protein recovery process as a single-step process is attracting considerable interest. This study aims to investigate the novel approach of integration method of electrolysis and liquid biphasic flotation for protein extraction from wet biomass of Chlorella sorokiniana CY-1 and obtaining the optimal operating conditions for the protein extraction...
October 2018: Molecular Biotechnology
Kavish Kumar Jain, Sandeep Kumar, Kailash N Bhardwaj, Ramesh Chander Kuhad
Thermostable cellulases offer several advantages like higher rates of substrate hydrolysis, lowered risk of contamination, and increased flexibility with respect to process design. In the present study, a thermostable native endoglucanase nEG (EC was purified and characterized from T. aurantiacus RCKK. Further, it was cloned in P. pastoris X-33 and processed for over expression. Expression of recombinant endoglucanase (rEG) of molecular size ~ 33 kDa was confirmed by SDS-PAGE and western blotting followed by in gel activity determination by zymogram analysis...
October 2018: Molecular Biotechnology
Christian Schröter, Jan Beck, Simon Krah, Stefan Zielonka, Achim Doerner, Laura Rhiel, Ralf Günther, Lars Toleikis, Harald Kolmar, Björn Hock, Stefan Becker
In this study, we present a multiparameter screening procedure for the identification of target-specific antibodies with prescribed properties. Based on B cell receptor gene repertoires from transgenic rats, yeast surface display libraries were generated, and high-affinity human antibodies were readily isolated. We demonstrate that specific desirable features, i.e., species' cross-reactivity and a broad epitope coverage can be integrated into the screening procedure using high-throughput fluorescence-activated cell sorting...
October 2018: Molecular Biotechnology
Anderson C Camargo, Svetoslav D Todorov, N E Chihib, D Drider, Luís A Nero
Bacteriocins are antimicrobial peptides produced by bacteria Gram-negative and Gram-positive, including lactic acid bacteria (LAB), organisms that are traditionally used in food preservation practices. Bacteriocins have been shown to have an aptitude as biofilm controlling agents in Listeria monocytogenes biofilms, a major risk for consumers and the food industry. Biofilms protect pathogens from sanitization procedures, allowing them to survive and persist in processing facilities, resulting in the cross-contamination of the end products...
September 2018: Molecular Biotechnology
Mohammad Pazhang, Fereshteh S Younesi, Faramarz Mehrnejad, Saeed Najavand, Alireza Tarinejad, Mehrnaz Haghi, Fatemeh Rashno, Khosro Khajeh
Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) has an Ig-like domain and the enzyme stability is dependent to calcium. In this study the effect of calcium on the structure and stability of the wild-type enzyme and the truncated form (the wild-type enzyme without Ig-like domain, AaCel9AΔN) was investigated. Fluorescence quenching results indicated that calcium increased and decreased the rigidity of the wild-type and truncated enzymes, respectively. RMSF results indicated that AaCel9A has two flexible regions (regions A and B) and deleting the Ig-like domain increased the truncated enzyme stability by decreasing the flexibility of region B probably through increasing the hydrogen bonds...
September 2018: Molecular Biotechnology
Yasmeen Yousif Ahmed Elyas, Kazusa Miyatani, Katsuhiko Shimizu, Jiro Arima
D-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2 has potential utility for the synthesis of D/L configuration dipeptides by an aminolysis reaction. Structural comparison of DAH with substrate-bound D-amino acid amidase revealed that three residues located in the active site pocket of DAH (Thr145, Ala267, and Gly271) might be involved in interactions with D-phenylalanine substrate. We substituted Ala267 and Gly271, which are located at the bottom of the hydrophobic pocket of DAH, with Phe and observed changes in the stereoselectivity and specific activity toward the free and acetylated forms of D/L-Phe-methyl esters...
September 2018: Molecular Biotechnology
Yue Zhang, Weiliang Dong, Ziyao Lv, Jiawei Liu, Wenmin Zhang, Jie Zhou, Fengxue Xin, Jiangfeng Ma, Min Jiang
Laccase CotA from Bacillus subtilis 168 was successfully displayed on the membrane of Escherichia coli cells using poly-γ-glutamate synthetase A protein (PgsA) from B. subtilis as an anchoring matrix. Further analyses demonstrated that the fusion protein PgsA/CotA efficiently translocates to the cell surface of E. coli with an enzymatic activity of 65 U/108 cells. Surface-displayed CotA was shown to possess improved enzymatic properties compared with those of the wild-type CotA, including higher thermal stability (above 90% activity at 70 °C and nearly 40% activity at 90 °C after 5-h incubation) and stronger inhibitor tolerance (approximately 80 and 65% activity when incubated with 200 and 400 mM NaCl, respectively)...
September 2018: Molecular Biotechnology
Melora Mackey, Alexander Kurosky, E Jane Robb, Ross N Nazar
Grafting vegetables for disease resistance has increased greatly in popularity over the past 10 years. Verticillium wilt of tomato is commonly controlled through grafting of commercial varieties on resistant rootstocks expressing the Ve1 R-gene. To mimic the grafted plant, proteomic analyses in tomato were used to identify a suitable root-specific promoter (TMVi), which was used to express the Ve1-allele in susceptible Craigella (Cs) tomato plants. The results indicate that when infected with Verticillim dahliae, race 1, the transformed plants are comparable to resistant cultivars (Cr) or grafted plants...
September 2018: Molecular Biotechnology
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