Molecular Biotechnology (MOL BIOTECHNOL )

Publisher: Springer Verlag

Description

This practical periodical is devoted to the rapid issuance of essential step-by-step laboratory protocols for molecular biology techniques (both protein and nucleic acid based), review articles, and original papers on the application of these techniques in both basic and applied biotechnology.

  • Impact factor
    2.26
  • 5-year impact
    2.23
  • Cited half-life
    6.10
  • Immediacy index
    0.66
  • Eigenfactor
    0.01
  • Article influence
    0.64
  • Website
    Molecular Biotechnology website
  • Other titles
    Molecular biotechnology
  • ISSN
    1559-0305
  • OCLC
    29487200
  • Material type
    Periodical, Internet resource
  • Document type
    Journal / Magazine / Newspaper, Internet Resource

Publisher details

Springer Verlag

  • Pre-print
    • Author can archive a pre-print version
  • Post-print
    • Author can archive a post-print version
  • Conditions
    • Authors own final version only can be archived
    • Publisher's version/PDF cannot be used
    • On author's website or institutional repository
    • On funders designated website/repository after 12 months at the funders request or as a result of legal obligation
    • Published source must be acknowledged
    • Must link to publisher version
    • Set phrase to accompany link to published version (The original publication is available at www.springerlink.com)
    • Articles in some journals can be made Open Access on payment of additional charge
  • Classification
    ​ green

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Alternaria blight caused by Alternaria brassicicola (Schwein.) Wiltshire and A. brassicae (Berk.) Sacc., is one of the most important disease of rapeseed–mustard, characterized by the formation of spots on leaves, stem, and siliquae with premature defoliation and stunting of growth. These two species are very difficult to differentiate based on disease symptoms or spore morphology. Therefore, the aim of present investigation was to identify and characterize transferable microsatellite loci from A. brassicicola to A. brassicae for the development of diagnostic marker. A total of 8,457 microsatellites were identified from transcript sequences of A. brassicicola. The average density of microsatellites was one microsatellite per 1.94 kb of transcript sequence screened. The most frequent repeat was tri-nucleotide (74.03 %), whereas penta-nucleotide (1.14 %) was least frequent. Among amino acids, arginine (13.11 %) showed maximum abundance followed by lysine (10.11 %). A total of 32 alleles were obtained across the 31 microsatellite loci for the ten isolates of A. brassicicola. In cross-species amplifications, 5 of the 31 markers amplified the corresponding microsatellite regions in twenty isolates of A. brassicae and showed monomorphic banding pattern. Microsatellite locus ABS28 was highly specific for A. brassicicola, as no amplification was observed from twenty-nine other closely related taxa. Primer set, ABS28F/ABS28R, amplified a specific amplicon of 380 bp from all A. brassicicola isolates. Standard curves were generated for A. brassicicola isolate using SYBR Green I fluorescent dye for detection of amplification in real-time PCR assay. The lowest detection limit of assay was 0.01 ng. Thus, the primer set can be used as diagnostic marker to discriminate and diagnose A. brassicicola from synchronously occurring fungus, A. brassicae associated with rapeseed and mustard.
    Molecular Biotechnology 07/2014;
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    ABSTRACT: The development and function of the central nervous system (CNS) are realized through interactions between many neurons. To investigate cellular and molecular mechanisms of the development and function of the CNS, it is thus crucial to be able to manipulate the gene expression of single neurons in a complex cell population. We recently developed a technique for gene silencing by introducing small interfering RNA into single neurons in primary CNS cultures using single-cell electroporation. However, we had not succeeded in forced gene expression by introducing expression plasmids using single-cell electroporation. In the present study, we optimized the experimental conditions to enable the forced expression of green fluorescent protein (GFP) in cultured cerebellar Purkinje neurons using single-cell electroporation. We succeeded in strong GFP expression in Purkinje neurons by increasing the inside diameter of micropipettes or by making the size of the original plasmid smaller by digestion and cyclizing it by ligation. Strong GFP expression in Purkinje neurons electroporated under the optimal conditions continued to be observed for more than 25 days after electroporation. Thus, this technique could be used for forced gene expression in single neurons to investigate cellular and molecular mechanisms of the development, function, and disease of the CNS.
    Molecular Biotechnology 05/2014;
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    ABSTRACT: The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases.
    Molecular Biotechnology 05/2014;
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    ABSTRACT: Collar rot disease caused by Sclerotium rolfsii is an economically important disease prevailing in all Amorphophallus growing areas. The pathogen propagules surviving in soil and planting material are the major sources of inoculum. A nested PCR assay has been developed for specific detection of S. rolfsii in soil and planting material. The PCR detection limit was 10 pg in conventional assay whereas 0.1 pg in nested assay. The primers designed were found to be highly specific and could be used for accurate identification of pathogen up to species level. The protocol was standardized for detection of the pathogen in artificially and naturally infected field samples.
    Molecular Biotechnology 05/2014;
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    ABSTRACT: Terminal moieties of most proteins are long known to be disordered and flexible. To unravel the functional role of these regions on the structural stability and biochemical properties of AT2 lipase, four C-terminal end residues, (Ile-Thr-Arg-Lys) which formed a flexible, short tail-like random-coil segment were targeted for mutation. Swapping of the tail-like region had resulted in an improved crystallizability and anti-aggregation property along with a slight shift of the thermostability profile. The lipolytic activity of mutant (M386) retained by 43 % compared to its wild-type with 18 % of the remaining activity at 45 °C. In silico analysis conducted at 25 and 45 °C was found to be in accordance to the experimental findings in which the RMSD values of M386 were more stable throughout the total trajectory in comparison to its wild-type. Terminal moieties were also observed to exhibit large movement and flexibility as denoted by high RMSF values at both dynamics. Variation in organic solvent stability property was detected in M386 where the lipolytic activity was stimulated in the presence of 25 % (v/v) of DMSO, isopropanol, and diethyl ether. This may be worth due to changes in the surface charge residues at the mutation point which probably involve in protein-solvent interaction.
    Molecular Biotechnology 04/2014;
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    ABSTRACT: Four novel basic peroxidases, named AaP-1, AaP-2, AaP-3, and AaP-4, were purified from Asparagus acutifolius L. seeds by cation-exchange and gel filtration chromatographies. The four proteins showed a similar electrophoretic mobility of 46 kDa while, by MALDI-TOF MS, different Mr values of 42758.3, 41586.9, 42796.3, and 41595.5 were determined for AaP-1, AaP-2, AaP-3, and AaP-4, respectively. N-terminal sequences of AaPs 1-4 up to residue 20 showed a high percentage of identity with the peroxidase from Glycine max. In addition, AaP-1, AaP-2, AaP-3, and AaP-4 were found to be glycoproteins, containing 21.75, 22.27, 25.62, and 18.31 % of carbohydrates, respectively. Peptide mapping and MALDI-TOF MS analysis of AaPs 1-4 showed that the structural differences between AaP-1 and AaP-2 and AaP-3 and AaPs-4 were mainly due to their glycan content. We also demonstrate that AaPs were able to remove phenolic compounds from olive oil mill wastewaters with a higher catalytic efficiency with respect to horseradish peroxidase, thus representing candidate enzymes for potential biotechnological applications in the environmental field.
    Molecular Biotechnology 04/2014;
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    ABSTRACT: The low economic profits of feather recycling lead that the large amount of feathers is currently discarded in China. To convert feather hydrolysates into GSH with high values, surface display of the bifunctional glutathione synthetase encoded by gcsgs from Streptococcus thermophilus on Saccharomyces cerevisiae and the potential in glutathione (GSH) production from feather hydrolysates were studied. The surface-displayed GCSGS could be used to convert feather hydrolysates into GSH. Results showed that 10 g/l of feather was converted into 321.8 mg/l GSH by the Trichoderma atroviride F6 and surface-displayed GCSGS in the study. Compared with production of intracellular GSH by S. cerevisiae from amino acids or feather hydrolysate, the concentration of GSH in the study was higher, and purification of GSH was more feasible. Due to the glycolytic pathway, the S. cerevisiae was used to generate ATP and cheap feather hydrolysate as precursors, the process for GSH production based on surface-displayed GCSGS is cheap and feasible. The process showed the potential to convert feather hydrolysates into GSH on an industrial scale.
    Molecular Biotechnology 04/2014;
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    ABSTRACT: Previously, we reported that an artificial zinc-finger protein (AZP)-staphylococcal nuclease (SNase) hybrid (designated AZP-SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP-SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP-SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP-scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP-SNase. Then, by linker-mediated PCR analysis, we confirmed that AZP-scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP-scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP-scFokI hybrid is expected to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects.
    Molecular Biotechnology 03/2014;
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    ABSTRACT: Protein E (PE) of Haemophilus influenzae is a highly conserved ubiquitous surface protein involved in adhesion to and activation of epithelial cells. The host proteins-vitronectin, laminin, and plasminogen are major targets for PE-dependent interactions with the host. To identify novel inhibitory molecules of PE, we used an in vitro selection method based on systematic evolution of ligands by exponential enrichment known as SELEX in order to select 2'F-modified RNA aptamers that specifically bind to PE. Fourteen selection cycles were performed with decreasing concentrations of PE. Sequencing of clones from the 14th selection round revealed the presence of semiconserved sequence motifs in loop regions of the RNA aptamers. Among these, three aptamers showed the highest affinity to PE in electrophoretic mobility shift assays and in dot blots. These three aptamers also inhibited the interaction of PE with vitronectin as revealed by ELISA. Moreover, pre-treatment of H. influenzae with the aptamers significantly inhibited binding of vitronectin to the bacterial surface. Biacore experiments indicated that one of the aptamers had a higher binding affinity for PE as compared to the other aptamers. Our results show that it is possible to select RNA inhibitors against bacterial adhesins using SELEX in order to inhibit interactions with target proteins.
    Molecular Biotechnology 03/2014;
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    ABSTRACT: Mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs), a valuable tool for in vitro disease modeling and regenerative medicine. These applications demand for iPSCs devoid of reprogramming factor transgenes, but current procedures for the derivation of transgene-free iPSCs are inefficient and cumbersome. Here, we describe a new approach for the simple derivation of transgene-free iPSCs by the sequential use of two DNA recombinases, C31 Integrase and Cre, to control the genomic insertion and excision of a single, non-viral reprogramming vector. We show that such transgene-free iPSCs exhibit gene expression profiles and pluripotent developmental potential comparable to genuine, blastocyst-derived embryonic stem cells. As shown by a reporter iPSC line for the differentiation into midbrain dopaminergic neurons, the dual recombinase approach offers a simple and efficient way to derive transgene-free iPSCs for studying disease mechanisms and cell replacement therapies.
    Molecular Biotechnology 03/2014;
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    ABSTRACT: Tropical theileriosis is a disease caused by infection with an apicomplexan parasite, Theileria annulata, and giving rise to huge economic losses. In recent years, parasite resistance has been reported against the most effective antitheilerial drug used for the treatment of this disease. This emphasizes the need for alternative methods of treatment. Enolase is a key glycolytic enzyme and can be selected as a macromolecular target of therapy of tropical theileriosis. In this study, an intron sequence present in T. annulata enolase gene was removed by PCR-directed mutagenesis, and the gene was first cloned into pGEM-T Easy vector and then subcloned into pLATE31 vector, and expressed in Escherichia coli cells. The enzyme was purified by affinity chromatography using Ni-NTA agarose column. Steady-state kinetic parameters of the enzyme were determined using GraFit 3.0. High quantities (~65 mg/l of culture) of pure recombinant T. annulata enolase have been obtained in a higly purified form (>95 %). Homodimer form of purified protein was determined from the molecular weights obtained from a single band on SDS-PAGE (48 kDa) and from size exclusion chromatography (93 kDa). Enzyme kinetic measurements using 2-PGA as substrate gave a specific activity of ~40 U/mg, K m: 106 μM, kcat: 37 s(-1), and k cat/K m: 3.5 × 10(5) M(-1) s(-1). These values have been determined for the first time from this parasite enzyme, and availability of large quantities of enolase enzyme will facilitate further kinetic and structural characterization toward design of new antitheilerial drugs.
    Molecular Biotechnology 03/2014;
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    ABSTRACT: HPV prophylactic vaccination based on VLPs was implemented 7 years ago and has now shown a high degree of efficiency to reduce HPV-induced lesions. Moreover, it was shown that HPV-derived virus-like particles or pseudovirions could be used as gene therapy vectors. As a consequence, characterization of the antigenic structure of HPV capsids is crucial for designing future HPV vaccines with better or broader efficacy and for the design of HPV-derived gene therapy vectors with reduced immunogenicity or vaccination escaping. In this study, we have generated 10 HPV16 FG loop L1 protein mutants and analyzed their ability to self-assemble into VLP, their immunogenicity, and their ability to transduce cells when used as pseudovirions. Most of the mutants had lost their ability to transduce cells at the exception of two chimeric HPV16/31 L1 protein FG loop mutants. Sera from mice immunized with HPV16 L1 wt VLPs very weakly neutralized pseudovirions derived from these two HPV16/31 L1 protein FG loop mutants. These findings suggest that only a few point substitutions within the FG loop are sufficient to generate a new serotype escaping vaccination. As a consequence, derived pseudovirions might be suitable as gene therapy vectors in vaccinated subjects.
    Molecular Biotechnology 03/2014;
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    ABSTRACT: Members of the genus Aglaia have been reported to contain bioactive phytochemicals. The genus, belonging to the Meliaceae family, is represented by at least 120 known species of woody trees or shrubs in the tropical rain forest. As some of these species are very similar in their morphology, taxonomic identification can be difficult. A reliable and definitive molecular method which can identify Aglaia to the level of the species will hence be useful in comparing the content of specific bioactive compounds between the species of this genus. Here, we report the analysis of DNA sequences in the internal transcribed spacer (ITS) of the nuclear ribosomal DNA and the observation of a unique nucleotide signature in the ITS that can be used for the identification of Aglaia stellatopilosa. The nucleotide signature consists of nine bases over the length of the ITS sequence (654 bp). This uniqueness was validated in 37 samples identified as Aglaia stellatopilosa by an expert taxonomist, whereas the nucleotide signature was lacking in a selection of other Aglaia species and non-Aglaia genera. This finding suggests that molecular typing could be utilized in the identification of Aglaia stellatopilosa.
    Molecular Biotechnology 03/2014;
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    ABSTRACT: Protein antigens have drawn a lot of attention from investigators working on tuberculosis vaccines. These proteins can be used to improve the immunogenicity of the new generation BCG vaccines or even replace them completely. Recombinant technology is used to insure the production of pure mycobacterial antigens in high quantities. Mycolyl transferase 85B (Ag85B) is a potent, mycobacterial antigen that significantly stimulates immune responses. Since Ag85B is an apolar protein, production of the water-soluble antigen is of interest. In this work, we report a systematic optimization strategy concerning cloning systems and purification methods, aiming at increasing the yield of recombinant Ag85B. Our optimized method resulted in a yield of 8 mg of recombinant Ag85B from 1 liter of induced culture (400 μg/ml) by using pET32a(+), Escherichia coli Rosseta-gami™(DE3) pLysS and a Ni-NTA agarose-based procedure and on-column re-solubilization. The purified recombinant Ag85B showed strong immunostimulating properties by inducing high levels of TNF-α, IFN-γ, IL-12, and IgG2a in immunized mice, therefore it can effectively be applied in TB vaccine researches.
    Molecular Biotechnology 03/2014;
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    ABSTRACT: Adipose-derived stem cells (ADSCs) are a desirable stem cell source in neurodegenerative diseases treatment due to their ability to differentiate into different cell lineages. In this study, we transplanted human ADSCs (hADSCs) into a lysophosphatidylcholine (lysolecithin) model of multiple sclerosis (MS) and determined the efficiency of these cells in remyelination process. Forty adult rats were randomly divided into control, lysolecithin, vehicle, and transplantation groups, and focal demyelination was induced by lysolecithin injection into spinal cord. To assess motor performance, all rats were examined weekly with a standard EAE scoring scale. Four weeks after cell transplantation, to assess the extent of demyelination and remyelination, Luxol Fast Blue staining was used. In addition, immunohistochemistry technique was used for assessment of the presence of oligodendrocyte phenotype cells in damaged spinal cord. Our results indicated that hADSCs had ability to differentiate into oligodendrocyte phenotype cells and improved remyelination process. Moreover, the evaluation of rat motor functions showed that animals which were treated with hADSC compared to other groups had significant improvement (P < 0.001). Our finding showed that hADSCs transplantation for cell-based therapies may play a proper cell source in the treatment of neurodegenerative diseases such as MS.
    Molecular Biotechnology 02/2014;
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    ABSTRACT: The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber- or opal-suppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11-16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIII-induced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine.
    Molecular Biotechnology 02/2014;
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    ABSTRACT: Fluorescence biosensors are indispensable tools for understanding protein behavior and function in cells. Recent advancements utilize fluorogen-activating-proteins (FAPs) that form complexes with small organic molecules (fluorogens) and result in their fluorescence activation. The technology has found multiple uses in protein discovery applications; however, the current method of detection requires the expression of FAPs as gene fusion tags in cells-a process that is time- and labor-intensive. In this report, we present an alternate method that utilizes FAPs as affinity reagents. Accordingly, we isolated soluble reagents based on FAP fusions with streptavidin (Strep) or avidin proteins, both highly selective for biotin. When tested in vitro, the reagents displayed bi-functional activity, fluorogen activation, and biotin affinity. For live-cell protein discovery, surface targets were biotinylated via biotin-tagged immunoglobulins or a genetically encoded biotin acceptor peptide. As a result, when the cells were labeled with FAP-Strep or FAP-avidin reagent, the in vivo fluorescence measurements indicated high target specificity, minimal background, and bright signal detection. In summary, we present a novel FAP reagent platform that offers a rapid and efficient approach for cell surface protein detection.
    Molecular Biotechnology 02/2014;

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