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Microfluidics cell encapsulation offers a way to mimic a 3D microenvironment that supports cell growth and proliferation, while also protecting cells from environmental stress. This technique has found extensive applications in tissue engineering and cell therapies. Several studies have demonstrated the advantages of graphene oxide (GO) as an osteogenic inducer; however, the significance of GO on stem cell fate in the single-cell state is still unclear. Here, a microfluidics-based approach is developed for continuous encapsulation of mesenchymal stem cells (MSCs) at the single-cell level using alginate microgels. So, single-layer graphene oxide (slGO) nanosheet is used to be encapsulated inside the alginate droplets. The results of AFM and SEM show that slGO can increase the roughness and reduce the stiffness of alginate hydrogels. The Young's modulus of the alginate and alginate-slGO was obtained as 1414 kPa and 985.9 kPa, respectively. Live/dead assay and fluorescence microscopy images illustrate that slGO can maintain the viability and proliferation of microencapsulated hBMSCs. The obtained results show that slGO increases the mineralization of the encapsulated hBMSCs, so that microgels containing hBMSCs gradually became opaque during 21 days of culture. RT-qPCR results indicate that the expression of OCN, Runx2, and ALP in the alginate-slGO microgels is significantly higher than in the alginate microgels. The expression of OCN and Runx2 in the alginate-slGO microgels is 4.27 and 5.87-fold higher than in the alginate microgels, respectively. It can be concluded that low doses of slGO nanosheets have the potential to be utilized in the development of tissue engineering and bone regeneration. This finding offers a new method for creating injectable tissue transplants that are minimally invasive. [ABSTRACT FROM AUTHOR]

Total-internal reflection fluorescence (TIRF) microscope is a unique technique for selective excitation of only those fluorophore molecules in a cellular environment, which are located at the sub-diffraction axial distance of a cell's contact-area. Despite this prominent feature of the TIRF microscope, making quantitative use of this technique has been a challenge, since the excitation intensity strongly depends on the axial position of a fluorophore molecule. Here, we present an easy-implemented data analysis method to quantitatively characterize the fluorescent signal, without considering the intensity-value. We use F-actin patches in single-melanoma cells as an example and define two quantities of elongation and surface density for F-actin patches at the contact-area of a melanoma cell. The elongation parameter can evaluate the dispersion of F-actin patches at the contact-area of a cell and is useful to classify the attaching, spreading, and expanding stages of a cell. Following that, we present the profile of the surface density of F-actin patches as a quantity to probe the spatio-temporal distribution of the F-actin patches at the contact-area of a cell. The data analysis methods that are proposed here will also be applicable in the image analysis of the other advanced optical microscopic methods. [ABSTRACT FROM AUTHOR]

The simulated liposome models provide events in molecular biological science and cellular biology. These models may help to understand the cell membrane mechanisms, biological cell interactions, and drug delivery systems. In addition, the liposomes model may resolve specific issues such as membrane transports, ion channels, drug penetration in the membrane, vesicle formation, membrane fusion, and membrane protein function mechanism. One of the approaches to investigate the lipid membranes and the mechanism of their formation is by molecular dynamics (MD) simulations. In this study, we used the coarse-grained MD simulation approach and designed a liposome model system. To simulate the liposome model, we used phospholipids that are present in the structure of natural cell membranes (1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)). Simulation conditions such as temperature, ions, water, lipid concentration were performed based on experimental conditions. Our results showed a liposome model (ellipse vesicle structure) during the 2100 ns was formed. Moreover, the analysis confirmed that the stretched and ellipse structure is the best structure that could be formed. The eukaryotic and even the bacterial cells have elliptical and flexible structures. Usually, an elliptical structure is more stable than other assembled structures. The results indicated the assembly of the lipids is directed through short-range interactions (electrostatic interactions and, van der Waals interactions). Total energy (Van der Waals and electrostatic interaction energy) confirmed the designed elliptical liposome structure has suitable stability at the end of the simulation process. Our findings confirmed that phospholipids DOPC and DOPE have a good tendency to form bilayer membranes (liposomal structure) based on their geometric shapes and chemical-physical properties. Finally, we expected the simulated liposomal structure as a simple model to be useful in understanding the function and structure of biological cell membranes. Furthermore, it is useful to design optimal, suitable, and biocompatible liposomes as potential drug carriers. [ABSTRACT FROM AUTHOR]

Biomechanical and morphological analysis of the cells is a novel approach for monitoring the environmental features, drugs, and toxic compounds' effects on cells. Graphene oxide (GO) has a broad range of medical applications such as tissue engineering and drug delivery. However, the effects of GO nanosheets on biological systems have not been completely understood. In this study, we focused on the biophysical characteristics of cells and their changes resulting from the effect of GO nanosheets. The biophysical properties of the cell population were characterized as follows: cell stiffness was calculated by atomic force microscopy, cell motility and invasive properties were characterized in the microfluidic chip in which the cells are able to visualize cell migration at a single-cell level. Intracellular actin was stained to establish a quantitative picture of the intracellular cytoskeleton. In addition, to understand the molecular interaction of GO nanosheets and actin filaments, coarse-grained (CG) molecular dynamics (MD) simulations were carried out. Our results showed that GO nanosheets can reduce cell stiffness in MCF7 cells and MDA-MB-231 cell lines and highly inhibited cell migration (39.2%) in MCF-7 and (38.6%) in MDA-MB-231 cell lines through the GO nanosheets-mediated disruption of the intracellular cytoskeleton. In the presence of GO nanosheets, the cell migration of both cell lines, as well as the cell stiffness, significantly decreased. Moreover, after GO nanosheets treatment, the cell actin network dramatically changed. The experimental and theoretical approaches established a quantitative picture of changes in these networks. Our results showed the reduction of the order parameter in actin filaments was 23% in the MCF7 cell line and 20.4% in the MDA-MB-231 cell line. The theoretical studies also showed that the GO nanosheet–actin filaments have stable interaction during MD simulation. Moreover, the 2D free energy plot indicated the GO nanosheet can induce conformational changes in actin filaments. Our findings showed that the GO nanosheets can increase the distance of actin-actin subunits from 3.22 to 3.5 nm and in addition disrupt native contacts between two subunits which lead to separate actin subunits from each other in actin filaments. In this study, the biomechanical characteristics were used to explain the effect of GO nanosheets on cells which presents a novel view of how GO nanosheets can affect the biological properties of cells without cell death. These findings have the potential to be applied in different biomedical applications. [ABSTRACT FROM AUTHOR]

A combination of light microscopy and image processing was applied to investigate morphology of label-free primary-melanocytes and melanoma cells. A novel methodological approach based on morphology of nuclear body was used to find those single cells, which were at the same phase of cell cycle. The area and perimeter of melanocytes and melanoma cells were quantified. We found that there was a significant difference between area and perimeter of adendritic-shaped melanocytes with melanoma cells and the reason(s) of this finding was speculated. Finally, a theoretical model based on losing dendrites was proposed, which was in agreement with our experimental data. [ABSTRACT FROM AUTHOR]

Lung cancer is a leading cause of cancer death in both men and women worldwide. The high mortality rate in lung cancer is in part due to late-stage diagnostics as well as spread of cancer-cells to organs and tissues by metastasis. Automated lung cancer detection and its sub-types classification from cell's images play a crucial role toward an early-stage cancer prognosis and more individualized therapy. The rapid development of machine learning techniques, especially deep learning algorithms, has attracted much interest in its application to medical image problems. In this study, to develop a reliable Computer-Aided Diagnosis (CAD) system for accurately distinguishing between cancer and healthy cells, we grew popular Non-Small Lung Cancer lines in a microfluidic chip followed by staining with Phalloidin and images were obtained by using an IX-81 inverted Olympus fluorescence microscope. We designed and tested a deep learning image analysis workflow for classification of lung cancer cell-line images into six classes, including five different cancer cell-lines (P-C9, SK-LU-1, H-1975, A-427, and A-549) and normal cell-line (16-HBE). Our results demonstrate that ResNet18, a residual learning convolutional neural network, is an efficient and promising method for lung cancer cell-lines categorization with a classification accuracy of 98.37% and F1-score of 97.29%. Our proposed workflow is also able to successfully distinguish normal versus cancerous cell-lines with a remarkable average accuracy of 99.77% and F1-score of 99.87%. The proposed CAD system completely eliminates the need for extensive user intervention, enabling the processing of large amounts of image data with robust and highly accurate results. [ABSTRACT FROM AUTHOR]

Wnt/β-catenin signaling, a highly evolutionary conserved pathway, is abnormally regulated in many human cancers. This pathway is one of the proposed mechanisms of atrial natriuretic peptide (ANP) anti-cancer effect. ANP which at first reported as a cardio hormone, inhibits proliferation of different cancer cell lines and tumor growth in vitro and in vivo respectively. Previous studies have shown a possibility of direct interaction between ANP and Frizzled (FZD), the main extracellular receptor of the pathway, and so a competition between ANP and Wnt for binding to this receptor. Here, using a molecular dynamics approach, we investigated this hypothesis validity and also the probable mechanism involved. We found three overlapping binding regions between ANP and Wnt3a carboxyl-terminal domain (CTD) on FZD7, but there is not any overlap with the large amino-terminal domain (NTD) of this protein. Based on the results derived from our study and the previous report on the intrinsic inhibitory potential of NTD subdomain against Wnt signalling and the conserved structure of Wnt-FZD complex architecture, we concluded that ANP is able to compete with Wnt CTD for binding to FZD that it can lead to incompletion of complex formation procedure between Wnt3a and FZD7. Finally, we introduce this peptide as a potential scaffold to design selective inhibitors against FZD-dependant cancers. [ABSTRACT FROM AUTHOR]

This study is aimed at extracting and purifying bioactive peptides from white grub larvae (Polyphylla adspersa) hydrolysate and examining their antioxidant effects on lung cancer cells (A549). Bioactive peptides from white grub larvae hydrolysate (WGLH) were produced using papain, pancreatin, proteinase-K and trypsin. The results showed that papain hydrolysate had the most 1, 1-diphenyl-2-pycryl-hydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging. This hydrolysate was fractionated by reverse-phase liquid chromatography (HPLC) and two most antioxidant fractions were identified by tandem mass spectrometry that had the sequence of YPQSLRWRAK (1304.6 Da, hereby called YK-10) and LPLFFYDVRP (1266.6 Da, hereby called LP-10). Cytotoxicity effect of YK-10 and LP-10 peptides on A549 cells, human umbilical vein endothelial cells (HUVECs) and human red blood cells were examined. The two peptides induced the specific activity of catalase, superoxide dismutase, glutathione peroxidase, and total level of thiol in A549 cells. Reactive oxygen species in treated A549 cells were significantly lower than that of controls. In conclusion, our results suggest that YK-10 and LP-10 peptides from WGLH have antioxidant activities and can protect A549 cells against free radicals' damages. [ABSTRACT FROM AUTHOR]

The low membrane permeability of lipophilic drugs was resolved using liposomes as a solubilizing agent and the precise size control of them is a significant parameter in drug carrier technology. Here, we have established a microfluidic octanol-assisted liposome assembly method to produce a surfactant-assisted liposome which has merged by the cytoskeleton drug (Taxotere) encapsulation in a single process step, then a complete microfluidic cellular analysis was performed in trapping cell device with an optofluidic assay for quantifying drug permeability. The optimization of process variables resulted in the formation of liposomes with particle size 6.75 ± 0.5 µm and monodispersity 6.2%, representing encapsulation efficiency and loading capacity of 65.49 ± 3.08% and 10.16 ± 0.32%, respectively. Qualitative and quantitative studies of cellular uptake in MCF-7 cell line that was cultured in the cell trapping chip indicated a significant increase in cellular uptake of carboxyfluorescein-loaded liposomes, suggesting endocytic mechanisms. The drug-loaded liposomes with an IC50 value of 0.55 ± 0.04 μg mL−1 have shown a higher level of cellular inhibition and apoptosis in cells than free Taxotere (2.48 ± 0.01). Furthermore, real-time analysis of the dynamic labeling assay for live/dead cells was investigated. Our data revealed that lab-on-a-chip platforms for the time-lapse fluorescence imaging were applied for drug screening routines. Cytoskeleton drugs encapsulated in liposome using microfluidic approaches and investigation of programmed cell death assay. [ABSTRACT FROM AUTHOR]

A new approach has been developed to improve sensing performances of electrochemically grown Au nanostructures (AuNSs) based on the pre-seeding of the electrode. The pre-seeding modification is simply carried out by vacuum thermal deposition of 5 nm thin film of Au on the substrate followed by thermal annealing at 500 °C. The electrochemical growth of AuNSs on the pre-seeded substrates leads to impressive electrochemical responses of the electrode owing to the seeding modification. The dependence of the morphology and the electrochemical properties of the AuNSs on various deposition potentials and times have been investigated. For the positive potentials, the pre-seeding leads to the growth of porous and hole-possess networks of AuNSs on the surface. For the negative potentials, AuNSs with carved stone ball shapes are produced. The superior electrode was achieved from AuNSs developed at 0.1 V for 900 s with pre-seeding modification. The sensing properties of the superior electrode toward glucose detection show a high sensitivity of 184.9 µA mM−1 cm−2, with a remarkable detection limit of 0.32 µM and a wide range of linearity. The excellent selectivity and reproducibility of the sensors propose the current approach as a large-scale production route for non-enzymatic glucose detection. [ABSTRACT FROM AUTHOR]

Due to increasing application of nanofibers in many research fields, comprehensive knowledge of the electrospinning process as the most popular method of fiber production is essential. Modeling techniques are valuable tools for managing contributing factors in the electrospinning process, prior to the more expensive experimental techniques. In the present research, effective parameters on the diameter of electrospun polycaprolactone (PCL) nanofibers are analyzed using artificial neural networks (ANN) and response surface methodology (RSM). The assessed parameters include polymer concentration, voltage, and nozzle-to-collector distance. Response surface methodology based on the Box-Behnken design is utilized to develop a mathematical model as well as to determine the optimum condition for production of nanofiber with minimum diameter. In addition, multilayer perceptron neural networks are designed and trained by the sets of input-output patterns using the Levenberg-Marquardt backpropagation algorithm. The high regression coefficient value (R2 ≥ 0.97) and low root-mean-square error (RMSE ≤3.81) of the two models indicate that both models performed well in predicting PCL fiber diameter, although the RSM model slightly outperformed the ANN model in accuracy. The represented models could assist researchers in fabricating electrospun scaffolds with a defined fiber diameter, thus specializing such scaffolds in particular applications. [ABSTRACT FROM AUTHOR]

Fabrication of electrospun chitosan nanofibers is still a controversial issue in publications. Although regarding the lots of reports, mixtures of chitosan with a hydrophilic synthetic polymer such as polyethylene oxide (PEO) have been electrospun successfully, abundance of partly contradictory protocols in which one variable has been surveyed in each study is unfortunately baffling. In the present study, influence of three considerable parameters including the average molecular weight of chitosan, chitosan solution concentration and the mass ratio of polyethylene oxide to chitosan at the mixtures on electrospinning possibility as well as the quality of as-spun fibers is investigated. Eventually, the necessities for obtaining the best results are introduced followed by further analysis of optimized nanofibers using atomic force microscopy. According to our results, the blend solutions prepared from the low molecular weight (LMW) chitosan and PEO are efficient for reproducible production of bead-free electrospun nanofibers even in low proportion of polyethylene oxide. [ABSTRACT FROM AUTHOR]

The authors describe a novel water-soluble, stable, biocompatible, and highly fluorescent probe consisting of iron quantum clusters incorporated into human adult hemoglobin (Hb-FeQCs). The Hb-FeQCs were characterized by various spectroscopic techniques. The probe displays strong absorption and yellow fluorescence with a peak centered at 567 nm (photo-excited at 460 nm). The Hb-FeQCs show excellent photostability over a wide range of pH values (5-12), even in the presence of high electrolyte concentrations. A colorimetric and a fluorometric method were worked out for the quantitation Zn(II) and cysteine in aqueous solution. Zinc ions induce a visible color change from brown to yellow. The sensitivity of Hb-FeQCs towards other metal ions was negligible, with the exception of Co2+1 and Cu2+, which caused a modest interference. The Hb-FeQCs were exploited in a sensitive and selective turn-on fluorescence assay for Zn2+. It is also found that cysteine quenches the fluorescence of the Hb-FeQCs/Zn(II) complex. Under the optimized conditions, the probe has a linear response in the 0.04 to 2.2 µM Zn(II) concentration range, with a 48 nM detection limit. Response to cysteine is linear in the 1-60 µM concentration range, with a 0.25 µM limit of detection. This fluorescent probe undergoes fluorescent emission intensity enhancement upon binding to zinc ions in living normal human fibroblast cells under visible lamp. The cellular imaging capability and very low cytotoxicity of this soluble iron quantum clusters can be potentially extended as an exciting sub-nanoplatform with promising biomaterial applications. [ABSTRACT FROM AUTHOR]

Fabrication of nanofibers with a defined diameter is a primary purpose of the electrospinning process. The diameter of nanofiber is directly related to its individual features, such as mechanical property and porosity. The motivation to conduct the current study was to explore the diameter of hybrid nanofibers of polycaprolactone-gelatin (PCL-GT) as one of the most attractive scaffolds employed in various research fields, such as tissue engineering and industrial fields. We have developed two predictive models describing the electrospinning process of PCL-GT using response surface methodology (RSM) and artificial neural network (ANN). The effect of 4 variables on diameter was analyzed, including total polymer concentration, ratio of PCL to Gel, voltage, and tip-to-collector distance. The individual and interactive effects of the mentioned factors were analyzed using RSM. The total polymer concentration had the most significant individual effect on the diameter of PCL-Gel nanofiber, whereas the other three factors showed less strong individual effects, although, the interactive effects of these factors were more remarkable. It was demonstrated that both models, especially the ANN model, could accurately predict the diameter of PCL-GT nanofiber (regression coefficient > 0.92, mean absolute percentage error < 5.7). The represented predictive models could facilitate construction of electrospun nanofibers from PCL-Gel with wellcontrolled diameter required for any intended purpose. [ABSTRACT FROM AUTHOR]

Preserving wounds from bacterial and fungal infections and retaining optimum moist environment over damaged tissue are major challenges in wound care management. Application of wound dressings with antimicrobial activity and appropriate wound exudates handling ability is of particular significance for promoting wound healing. To this end, preparation and evaluation of novel wound dres1sings made from polyurethane/siloxane network containing graphene oxide (GO) nanoplatelets are described. The particular sol-gel hydrolysis/condensation procedure applied for the preparation of dressings leads to an appropriate distribution of GO nanoplatelets in the dressing membranes. The crosslinked siloxane domains and the presence of GO nanoplatelets within polymeric chains offered necessary mechanical strength for dressings. Meanwhile, a combination of hydrophilic and hydrophobic moieties in dressing backbone enabled suitable wound exudate management. Therefore, both of physical protection from external forces and preservation of moist environment over wound were attained by using the designed dressings. Widespread antimicrobial activity against gram-positive, gram-negative and fungal strains was recorded for the dressing with the optimum amount of GO, meanwhile, very good cytocompatibility against fibroblast cells was noted for these dressings. In vivo assay of the GO containing dressing on rat animal model reveals that the dressing can promote wound healing by complete re-epithelization, enhanced vascularization and collagen deposition on healed tissue. Graphical Abstract: [ABSTRACT FROM AUTHOR]

Objectives: To investigate the role of microRNA-145, that regulates gene expression of genes related to differentiation, proliferation and the phenotype of smooth muscle cells (SMCs), in the differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) to SMCs. Results: Real-time PCR analysis indicated significant upregulation of SMC markers, including SM-α-actin, calponin, caldesmon and SMMHC, in SMCs compared to hBM-MSCs. Conversely, Krüppel-like factor 4, the direct target of microRNA-145 and the suppressor of smooth muscle differentiation, was suppressed in hBM-MSC-derived SMCs. Western blot analysis and immunocytochemistry also confirmed that the introduction of microRNA-145 into hBM-MSCs induced mature contractile SMCs. The functionality of hBM-MSC-derived SMCs was assessed by proliferation assay using PDGF-BB and contractility assay using carbachol. The results showed that the produced SMCs contracted in response to carbachol stimulation. Conclusion: Overexpression of microRNA-145 in undifferentiated hBM-MSCs results in functionally mature contractile SMCs that can be used in drug discovery and cell therapy in SMC disorders such as vascular disease. [ABSTRACT FROM AUTHOR]

A combination of light-microscopy and image processing has been applied to study naturally deformed Escherichia coli under in vivo condition and at the order of sub-pixel high-resolution accuracy. To classify deflagellated non-dividing E. coli cells to the rod-shape and bent-shape, a geometrical approach has been applied. From the analysis of the geometrical data which were obtained of image processing, we estimated the required effective energy for shaping a rod-shape to a bent-shape with the same size. We evaluated the energy of deformation in the naturally deformed bacteria with minimum cell manipulation, under in vivo condition, and with minimum influence of any external force, torque and pressure. Finally, we have also elaborated on the possible scenario to explain how naturally deformed bacteria are formed from initial to final-stage. [ABSTRACT FROM AUTHOR]

Nanoparticles can decrease the defects of usual drug and gene delivery systems. Chitosan is a low-toxicity, biodegradable, biocompatible, and safe polymer which is used in the production of nanoparticles. Nanoparticles including chitosan-tripolyphosphate (TPP) can affect the immune cells including dendritic cells (DCs) as the most potent antigen-presenting cells with a pivotal role in both humoral and cellular immunity. For efficient antigen presentation, DCs need to become mature and express high levels of class II major histocompatibility complex (MHC-II) as well as costimulatory molecules, including CD40 and CD86 molecules. In this study, we investigated the effects of chitosan-TPP nanoparticles on DC maturation and function. Chitosan-TPP was synthesized by ionotropic gelation methods from chitosan and TPP salt, and DCs were isolated from mouse spleen using the magnetic cell separation method. DCs were treated with chitosan-TPP nanoparticles during an overnight cell culture and phenotypic and functional characteristics of chitosan-TPP-treated DCs were evaluated by flow cytometric analysis. Our results showed that chitosan-TPP induced DC maturation. Moreover, chitosan-TPP-treated DCs were shown to be efficient inducers of T cell proliferation in allogenic mixed lymphocyte reaction. Indeed, chitosan-TPP as an adjuvant is able to induce DC maturation which is a vital step in efficient antigen presentation and activation of the immune system. Thus, chitosan-TPP nanoparticles can be used as a dual-function agent in clinical immunotherapy methods aiming at using them in drug and gene delivery as well as in potentiating immune responses. [ABSTRACT FROM AUTHOR]

Natural polymers and proteins such as chitosan (CS) and albumin (Alb) have recently attracted much attention both in drug delivery and gene delivery. The underlying rationale is their unique properties such as biodegradability, biocompatibility and controlled release. This study aimed to prepare novel albumin-chitosan-DNA (Alb-CS-DNA) core-shell nanoparticles as a plasmid delivery system and find the best conditions for their preparation. Phase separation method and ionic interaction were used for preparation of Alb nanoparticles and Alb-CS-DNA core-shell nanoparticles, respectively. The effects of three important independent variables (1) CS/Alb mass ratio, (2) the ratios of moles of the amine groups of cationic polymers to those of the phosphate groups of DNA (N/P ratio), and (3) Alb concentration, on the nanoparticle size and loading efficiency of the plasmid were investigated and optimized through Box-Behnken design of response surface methodology (RSM). The optimum conditions were found to be CS/Alb mass ratio = 3, N/P ratio = 8.24 and Alb concentration = 0.1 mg/mL. The most critical factors for the size of nanoparticles and loading efficiency were Alb concentration and N/P ratio. The optimized nanoparticles had an average size of 176 ± 3.4 nm and loading efficiency of 80 ± 3.9 %. Cytotoxicity experiments demonstrated that the prepared nanoparticles were not toxic. The high cellular uptake of nanoparticles (~85 %) was shown by flow cytometry and fluorescent microscopy. [ABSTRACT FROM AUTHOR]

Aurein1.2 is a cationic peptide with antimicrobial and anticancer activity. In order to find a novel peptide with this activity, aurein1.2 was utilized as a template and the sequences were designed by a RosettaDesign server with our proposed pattern. As a result, tempY was designed and synthesized, and its structural and biological activity was investigated. TempY has a sequence similar to that of the temporins group but with potent antibacterial properties against Gram-positive bacteria and effective anticancer activity (LC is 20-30 μg/ml). This latter activity may be due to the tyrosine at the C terminus of the peptide. Kar peptide is another model derived from the complete design of aurein1.2. Kar is only active against A549 cells. The net charge of Kar is four, and it is inactive against Gram-positive bacteria and MCF-7 cells, which may be due to the high positive-charge density on the surface of peptide. [ABSTRACT FROM AUTHOR]

gene encoding methylglyoxal synthase from Thermus sp. GH5 (TMGS) was cloned, sequenced, overexpressed, and purified by Q-Sepharose. The TMGS gene was composed of 399 bp which encoded a polypeptide of 132 amino acids with a molecular mass of 14.3 kDa. The K and k values of TMGS were 0.56 mM and 325 (s), respectively. The enzyme exhibited its optimum activity at pH 6 and 75 °C. Comparing the amino acid sequences and Hill coefficients of Escherichia coli MGS and TMGS revealed that the loss of Arg 150 in TMGS has caused a decrease in the cooperativity between the enzyme subunits in the presence of phosphate as an allosteric inhibitor. Gel filtration experiments showed that TMGS is a hexameric enzyme, and its quaternary structure did not change in the presence of phosphate. [ABSTRACT FROM AUTHOR]

Successful industrial use of amylases requires that they are sufficiently stable and active at application conditions, e.g., at high temperature in starch-liquefaction process. In the present study, site-directed mutagenesis was used to enhance the thermal stability and calcium independency of a mesophilic α-amylase from Bacillus megaterium WHO. Mutations (A53S and H58I) were designed at the calcium-binding site based on the sequence alignment. Kinetic and thermostability parameters of the mutants were analyzed and compared with that of the wild type. In the presence of calcium, the affinity of the enzymes (wild type and mutants) toward starch was increased. In comparison to the wild type, calcium ion had more effect on the catalytic efficiency, kcat/ Km, and half-life (at 60 °C) of A53S mutant. In A53S, the dependence of half-life on calcium concentration showed that the enhanced calcium binding is likely to be responsible for the increased stability. In contrast, calcium-independent mutant (H58I) possessed high thermostability. In addition, thermodynamic parameters of amylolytic reaction exhibited an increase in the activation energy and the entropy of the system. Kinetics of irreversible thermal inactivation suggests that the activation energy increased by 1.4-fold in the most stable variant. [ABSTRACT FROM AUTHOR]

For the first time, a polygalacturonase from the culture broth of Tetracoccosporium sp. was isolated and incubated at 30°C in an orbital shaker at 160 rpm for 48h. The enzyme was purified by ammonium sulfate precipitation and two-step ion-exchange chromatography and had an apparent molecular mass of 36 kDa, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Its optimum activity was at pH 4.3 and 40°C, and the K m and Vmax values of this enzyme (for polygalacturonic acid) were 3.23 mg/mL and 0.15 μmol/min, respectively. Ag+, Co2+, EDTA, Tween-20, Tween-80, and Triton X-100 stimulated polygalacturonase activity whereas Al3+, Ba2+, Ca2+, Fe2+, Fe3+, Ni2+, Mg2+, Mn2+, and SDS inhibited it. In addition, iodoacetamide and iodoacetic acid did not inhibit enzyme activity at a concentration of 1 m M, indicating that cysteine residues are not part of the catalytic site of polygalacturonase. We studied the kinetic properties and thermal inactivation of polygalacturonase. This enzyme exhibited a t1/2 of 63 min at 60°C and its specific activity, turnover number, and catalytic efficiency were 6.17 U/mg, 113.64 min−1, and 35.18 mL/(min·mg), respectively. The activation energy (Δ E#) for heat inactivation was 5.341 kJ/mol, and the thermodynamic activation parameters Δ G#, Δ H#, and Δ S# were also calculated, revealing a potential application for the industry. [ABSTRACT FROM AUTHOR]

In this paper, ab initio calculated NQR parameters for some quinoline-containing derivatives are presented. The calculations are carried out in a search for the relationships between the charge distribution of these compounds and their ability to interact with haematin. On the basis of NQR parameters, π-electron density on the nitrogen atom of the quinoline ring plays a dominant role in determining the ability of quinolines to interact with haematin. This point was confirmed with investigation of Fe+3 cation-π quinoline ring interactions in 2- and 4-aminoquinoline. However, our results do not show any preference for those carbon atoms of the quinoline ring which previous reports have noted. In order to calculate the NQR parameters, the electric field gradient (EFG) should be evaluated at the site of a quadrupolar nucleus in each compound. EFGs are calculated by the Gaussian 98 program using the B3LYP/6-31G* level of theory. [ABSTRACT FROM AUTHOR]