Thymosin β4 (Tβ4) is one of the most promising thymosins for future clinical applications, and it is anticipated that commercial demand for Tβ4 will increase. In order to develop a new approach to produce recombinant Tβ4, a 168 bp DNA (termed Tβ4) was designed based on the Tβ4 protein sequence and used to express a 4 × Tβ4 concatemer (four tandem copies of Tβ4, termed 4 × Tβ4) together with a histidine tag (6 × His) in E. coli (strain BL21). SDS-PAGE and western blot analysis were used to confirm that a recombinant 4 × Tβ4 protein of the expected size (30.87 kDa) was produced following the induction of the bacterial cultures with isopropyl β-D-thiogalactoside (IPTG). The E. coli-derived 4 × Tβ4 was purified by Ni-NTA resin, and its activities were examined with regard to both stimulating proliferation of the mice spleen cells in vitro and in vivo wound healing. The results demonstrate that these activities of the E. coli-derived recombinant 4 × Tβ4 were similar or even better than existing commercially obtained Tβ4. This production strategy therefore represents a potentially valuable approach for future commercial production of recombinant Tβ4.
The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Stabilization of unstable steady states of a continuous stirred tank bioreactor with predator-prey kinetics
Source:Journal of Biotechnology
Author(s): Bolesław Tabiś , Szymon Skoneczny
Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium
Source:Journal of Biotechnology
Author(s): Laura Cervera , Sonia Gutiérrez , Marta Martínez , Julià Blanco , Francesc Gòdia , María Mercedes Segura
Source:Journal of Biotechnology, Volume 165, Issues 3–4
Cellulase activity mapping of Trichoderma reesei cultivated in sugar mixtures under fed-batch conditions
On-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions.
The effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold beta-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high beta-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars.
The sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode. Modelling can be used to predict cellulolytic activity based on the sugar composition of the culture-feeding solution, or to fine tune the substrate composition in order to produce a desired enzymatic cocktail.
During this last decade, nonlinear analyses have been used to characterize the irregularity that exists in the neuronal data stream of the basal ganglia. In comparison to linear parameters for disparity (i.e., rate, standard deviation, and oscillatory activities), nonlinear analyses focus on complex patterns that are composed of groups of interspike intervals with matching lengths but not necessarily contiguous in the data stream. In light of recent animal and clinical studies, we present a review and commentary on the basal ganglia neuronal entropy in the context of movement disorders.
Positron Emission Tomography as a Surrogate Marker for Evaluation of Treatment Response in Patients with Desmoid Tumors under Therapy with Imatinib
We used 2-deoxy-2-[18F] fluoro-D-glucose (FDG) positron emission tomography (PET) to evaluate patients with desmoid tumors undergoing therapy with imatinib. The study included 22 patients with progressive disease (PD) of a biopsy proven desmoid tumor treated orally with imatinib 800 mg daily. Patients were examined using PET prior to onset of therapy and during treatment. Restaging was performed in parallel using computed tomography (CT) and/or magnetic resonance imaging (MRI). Outcome of 22 evaluable patients was as follows: five patients with partial response (PR); twelve patients with stable disease (SD) accounting for 77% with non-progressive disease; five patients showed PD. A 30% decrease of the mean average standardized uptake value (SUV) of sequential PET examinations could be demonstrated; no patient demonstrated a substantial increase in SUV. Patients with PR/SD were matched to a group of nonprogressive disease and tested versus PD. The initial average SUV and seem to be candidates for a response prediction with an approximate -value of 0.06553 and 0.07785, respectively. This is the first larger series of desmoid patients monitored using PET showing that early SUV changes may help to discriminate responders from nonresponders and, thus, to decide whether imatinib therapy should be continued.
An Immunohistochemistry Study of Sox9, Runx2, and Osterix Expression in the Mandibular Cartilages of Newborn Mouse
The purpose of this study is to investigate the spacial expression pattern and functional significance of three key transcription factors related to bone and cartilage formation, namely, Sox9, Runx2, and Osterix in cartilages during the late development of mouse mandible. Immunohistochemical examinations of Sox9, Runx2, and Osterix were conducted in the mandibular cartilages of the 15 neonatal C57BL/6N mice. In secondary cartilages, both Sox9 and Runx2 were weakly expressed in the polymorphic cell zone, strongly expressed in the flattened cell zone and throughout the entire hypertrophic cell zone. Similarly, both transcriptional factors were weakly expressed in the uncalcified Meckel’s cartilage while strongly expressed in the rostral cartilage. Meanwhile, Osterix was at an extremely low level in cells of the flattened cell zone and the upper hypertrophic cell zone in secondary cartilages. Surprisingly, Osterix was intensely expressed in hypertrophic chondrocytes in the center of the uncalcified Meckel’s cartilage while moderately expressed in part of hypertrophic chondrocytes in the rostral process. Consequently, it is suggested that Sox9 is a main and unique positive regulator in the hypertrophic differentiation process of mandibular secondary cartilages, in addition to Runx2. Furthermore, Osterix is likely responsible for phenotypic conversion of Meckel’s chondrocytes during its degeneration.
The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.
A Comprehensive Tutorial on In Vitro Characterization of New Photosensitizers for Photodynamic Antitumor Therapy and Photodynamic Inactivation of Microorganisms
In vitro research performed on eukaryotic or prokaryotic cell cultures usually represents the initial step for characterization of a novel photosensitizer (PS) intended for application in photodynamic therapy (PDT) of cancer or photodynamic inactivation (PDI) of microorganisms. Although many experimental steps of PS testing make use of the wide spectrum of methods readily employed in cell biology, special aspects of working with photoactive substances, such as the autofluorescence of the PS molecule or the requirement of light protection, need to be considered when performing in vitro experiments in PDT/PDI. This tutorial represents a comprehensive collection of operative instructions, by which, based on photochemical and photophysical properties of a PS, its uptake into cells, the intracellular localization and photodynamic action in both tumor cells and microorganisms novel photoactive molecules may be characterized for their suitability for PDT/PDI. Furthermore, it shall stimulate the efforts to expand the convincing benefits of photodynamic therapy and photodynamic inactivation within both established and new fields of applications and motivate scientists of all disciplines to get involved in photodynamic research.
Purpose. Despite the latest technological advances in radiotherapy, cancer control is still challenging for several tumour sites. The survival rates for the most deadly cancers, such as ovarian and pancreatic, have not changed over the last decades. The solution to the problem lies in the change of focus: from local treatment to systemic therapy. The aim of this paper is to present the current status as well as the gaps in radiotherapy and, at the same time, to look into potential solutions to improve cancer control and survival. Methods. The currently available advanced radiotherapy treatment techniques have been analysed and their cost-effectiveness discussed. The problem of systemic disease management was specifically targeted. Results. Clinical studies show limited benefit in cancer control from hadron therapy. However, targeted therapies together with molecular imaging could improve treatment outcome for several tumour sites while controlling the systemic disease. Conclusion. The advances in photon therapy continue to be competitive with the much more expensive hadron therapy. To justify the cost effectiveness of proton/heavy ion therapy, there is a need for phase III randomised clinical trials. Furthermore, the success of systemic disease management lies in the fusion between radiation oncology technology and microbiology.
Mechanisms by Which Interleukin-6 Attenuates Cell Invasion and Tumorigenesis in Human Bladder Carcinoma Cells
Interleukin-6, a multifunctional cytokine, contributes to tumor cell proliferation and differentiation. However, the biological mechanisms that are affected by the expression of interleukin-6 in bladder cancer cells remain unclear. We evaluated the effects of interleukin-6 expression in human bladder carcinoma cells in vitro and in vivo. The results of interleukin-6-knockdown experiments in T24 cells and interleukin-6-overexpression experiments in HT1376 cells revealed that interleukin-6 reduced cell proliferation, migration, and invasion in vitro. Xenograft animal studies indicated that the overexpression of interleukin-6 downregulated tumorigenesis of bladder cells and that interleukin-6 knockdown reversed this effect. The results of RT-PCR, immunoblotting, and reporter assays indicated that the overexpression of interleukin-6 upregulated the expression of the mammary serine protease inhibitor (MASPIN), N-myc downstream gene 1 (NDRG1), and KAI1 proteins in HT1376 cells and that interleukin-6 knockdown reduced the expression of these proteins in T24 cells. In addition, results of immunoblotting assays revealed that interleukin-6 modulated epithelial-mesenchymal transitions by upregulating the expression of the E-cadherin, while downregulation N-cadherin and vimentin proteins. Our results suggest that the effects of interleukin-6 on the regulation of epithelial-mesenchymal transitions and the expressions of the MASPIN, NDRG1, and KAI1 genes attribute to the modulation of tumorigenesis in human bladder carcinoma cells.
Improving maltodextrin specificity for enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid by site-saturation engineering of subsite-3 in cyclodextrin glycosyltransferase from Paenibacillus macerans
Source:Journal of Biotechnology
Author(s): Long Liu , Qiaoyan Xu , Ruizhi Han , Hyun-dong Shin , Rachel R. Chen , Jianghua Li , Guocheng Du , Jian Chen
Extensive efforts have recently been devoted to developing noninvasive imaging tools capable of delineating brain tissue viability (penumbra) during acute ischemic stroke. These efforts could have profound clinical implications for identifying patients who may benefit from tPA beyond the currently approved therapeutic time window and/or patients undergoing neuroendovascular treatments. To date, the DWI/PWI MRI and perfusion CT have received the most attention for identifying ischemic penumbra. However, their routine use in clinical settings remains limited. Preclinical and clinical PET studies with [18F]-fluoro-2-deoxy-D-glucose (18F-FDG) have consistently revealed a decreased 18F-FDG uptake in regions of presumed ischemic core. More importantly, an elevated 18F-FDG uptake in the peri-ischemic regions has been reported, potentially reflecting viable tissues. To this end, this paper provides a comprehensive review of the literature on the utilization of 14C-2-DG and 18F-FDG-PET in experimental as well as human stroke studies. Possible cellular mechanisms and physiological underpinnings attributed to the reported temporal and spatial uptake patterns of 18F-FDG are addressed. Given the wide availability of 18F-FDG in routine clinical settings, 18F-FDG PET may serve as an alternative, non-invasive tool to MRI and CT for the management of acute stroke patients.
Quantification of Human and Animal Viruses to Differentiate the Origin of the Fecal Contamination Present in Environmental Samples
Many different viruses are excreted by humans and animals and are frequently detected in fecal contaminated waters causing public health concerns. Classical bacterial indicator such as E. coli and enterococci could fail to predict the risk for waterborne pathogens such as viruses. Moreover, the presence and levels of bacterial indicators do not always correlate with the presence and concentration of viruses, especially when these indicators are present in low concentrations. Our research group has proposed new viral indicators and methodologies for determining the presence of fecal pollution in environmental samples as well as for tracing the origin of this fecal contamination (microbial source tracking). In this paper, we examine to what extent have these indicators been applied by the scientific community. Recently, quantitative assays for quantification of poultry and ovine viruses have also been described. Overall, quantification by qPCR of human adenoviruses and human polyomavirus JC, porcine adenoviruses, bovine polyomaviruses, chicken/turkey parvoviruses, and ovine polyomaviruses is suggested as a toolbox for the identification of human, porcine, bovine, poultry, and ovine fecal pollution in environmental samples.
Shear Bond Strength of Orthodontic Brackets and Disinclusion Buttons: Effect of Water and Saliva Contamination
Purpose. The aim of this study was to assess the effect of water and saliva contamination on the shear bond strength and failure site of orthodontic brackets and lingual buttons. Materials and Methods. 120 bovine permanent mandibular incisors were randomly divided into 6 groups of 20 specimens each. Both orthodontic brackets and disinclusion buttons were tested under three different enamel surface conditions: (a) dry, (b) water contamination, and (c) saliva contamination. Brackets and buttons were bonded to the teeth and subsequently tested using a Instron universal testing machine. Shear bond strength values and adhesive failure rate were recorded. Statistical analysis was performed using ANOVA and Tukey tests (strength values) and Chi squared test (ARI Scores). Results. Noncontaminated enamel surfaces showed the highest bond strengths for both brackets and buttons. Under water and saliva contamination orthodontic brackets groups showed significantly lower shear strengths than disinclusion buttons groups. Significant differences in debond locations were found among the groups under the various enamel surface conditions. Conclusions. Water and saliva contamination of enamel during the bonding procedure lowers bond strength values, more with orthodontic brackets than with disinclusion buttons.
Biometric systems refer to biometric technologies which can be used to achieve authentication. Unlike cryptography-based technologies, the ratio for certification in biometric systems needs not to achieve 100% accuracy. However, biometric data can only be directly compared through proximal access to the scanning device and cannot be combined with cryptographic techniques. Moreover, repeated use, improper storage, or transmission leaks may compromise security. Prior studies have attempted to combine cryptography and biometrics, but these methods require the synchronization of internal systems and are vulnerable to power analysis attacks, fault-based cryptanalysis, and replay attacks. This paper presents a new secure cryptographic authentication method using biometric features. The proposed system combines the advantages of biometric identification and cryptographic techniques. By adding a subsystem to existing biometric recognition systems, we can simultaneously achieve the security of cryptographic technology and the error tolerance of biometric recognition. This method can be used for biometric data encryption, signatures, and other types of cryptographic computation. The method offers a high degree of security with protection against power analysis attacks, fault-based cryptanalysis, and replay attacks. Moreover, it can be used to improve the confidentiality of biological data storage and biodata identification processes. Remote biometric authentication can also be safely applied.
Interconversion of ketoprofen recognition in firefly luciferase-catalyzed enantioselective thioesterification reaction using from Pylocoeria miyako (PmL) and Hotaria parvura (HpL) just by mutating two amino acid residues
Source:Journal of Biotechnology
Author(s): Dai-ichiro Kato , Yoshihiro Hiraishi , Mika Maenaka , Keisuke Yokoyama , Kazuki Niwa , Yoshihiro Ohmiya , Masahiro Takeo , Seiji Negoro
Author(s): Behrooz Darbani , Jean-François Briat , Preben Bach Holm , Søren Husted , Shahin Noeparvar , Søren Borg