Synergistic antibacterial effect of the pistachio green hull extract-loaded porphysome decorated with 4-nitroimidazole against bacteria.

'Active targeting' refers to modifying a nanocarrier's surface with targeting ligands. This study introduced an efficient approach for immobilizing imidazole-based drugs onto the metallated-porphyrin complex within the porphysome nanocarrier. To enhance cellular and bacterial uptake, a Ni-porphyrin with a fatty acid tail was synthesized and placed in the bilayer center of DPPC, facilitating receptor-mediated endocytosis. The Ni-porphyrin in the head group of the Ni-porphyrin-tail was placed superficially in the polar region of the membrane. Spherical unilamellar vesicle formation (DPPC: Ni-porphyrin-tail 4:1 mole ratio), as metallo-porphysome, was achieved through supramolecular self-assembly in an aqueous buffer. These vesicles exhibited a diameter of 279 ± 7 nm and a zeta potential of -15.3 ± 2.5 mV, showcasing their unique cytocompatibility. Nitroimidazole was decorated on the surface of metallo-porphysomes and pistachio green hull extract (PGHE) was loaded into the carrier for synergistic activity against (E. coli) and (S. aureus) bacteria strains. The physicochemical properties of Nitroimidazole-porphysome-PGHE, including size, zeta potential, morphology, loading efficiency, and release profile under various pH and temperature conditions in simulated gastrointestinal fluids were characterized. This combination therapy prevented bacterial cell attachment and biofilm formation in Caco-2 cells, as colon epithelial cells. The remarkable benefit of this system is that it does not affect cell viability even at 0.5 mg/ml. This study demonstrates the potential of a new co-delivery system using biocompatible metallo-porphysomes to decrease bacterial infections.

Porphysome Engineered With Specific Protein Binding Sites as a Multimodal Theranostic Nanocarrier for Targeted Protein Delivery.

The immobilization of proteins on the surface of carriers is challenging due to the loss of protein structure and function in this process. Here, we report the development of the protein immobilization on the surface of the metallated-porphyrin complex in the porphysome nanocarrier. The conjugated Ni-porphyrin to fatty acid (as a tail) has been synthesized and independently placed at the depth of the bilayer center of Dipalmitoylphosphatidylcholine (DPPC) in which the Ni-porphyrin was at the polar region of the membrane and is thus superficial. This porphysome (DPPC: Ni-porphyrin, 4 : 1 mole ratio) was formed by supramolecular self-assembly with a diameter of 173±7 nm and zeta potential -8.5±3.4 mv, which exhibited no significant toxicity at the experimental concentrations and acceptable cellular uptake on MCF-7 cells. The physicochemical properties and specific protein binding sites of the firefly luciferase as a model protein into the porphysome (1 : 2 mole ratio) show the conjugation efficiency about 80 % and the conformation of protein was completely maintained. Furthermore, bioluminescence assay and SDS-PAGE confirmed the preservation of protein function. The stabilized platform of porphyrin-lipid structure can potentially improve the efficacy of protein functionality for a particular display, shifting porphysomes from a simple carrier to a therapeutic agent.

The application of nano-hydrogels and hydrogels in wound dressings.

Wounds and the healing process are one of the main concerns of medical science today. A wound is any loss of integrity, or rupture of the layers of skin (epidermis, dermis, and hypodermis) or subcutaneous tissue caused by physical factors (surgical incision, trauma, pressure, and gunshot wounds) or chemical factors (acid burns). It is observed that soft tissue, muscle, or bone is involved in occurrences of wounds. Lesions and fractures of the skin surface necessitate medical attention, wherein dressings expedite the healing process by establishing a physical barrier between the wound and the external environment, thereby preventing further injury or infection. Hydrogel dressings create a moist environment that facilitates common healing steps, such as granulation hyperplasia, epidermal repair, and removal of excess dead tissue. The limited adhesion of the hydrogel and the hydrated wound bed allows for easy removal of the dressing without secondary damage, thereby significantly reducing the discomfort and risk of infection during dressing changes. These modern, wet dressings foster a moist healing environment by absorbing excess inflammatory secretions and allowing proper passage of steam and air, which expedites the healing process. In this analysis, the utilization of hydrogels as wound dressings is briefly presented.

Lung Cancer Cell-Derived Exosome Detection Using Electrochemical Approach towards Early Cancer Screening.

Lung cancer is one of the deadliest cancers worldwide due to the inability of existing methods for early diagnosis. Tumor-derived exosomes are nano-scale vesicles released from tumor cells to the extracellular environment, and their investigation can be very useful in both biomarkers for early cancer screening and treatment assessment. This research detected the exosomes via an ultrasensitive electrochemical biosensor containing gold nano-islands (Au-NIs) structures. This way, a high surface-area-to-volume ratio of nanostructures was embellished on the FTO electrodes to increase the chance of immobilizing the CD-151 antibody. In this way, a layer of gold was first deposited on the electrode by physical vapor deposition (PVD), followed by thermal annealing to construct primary gold seeds on the surface of the electrode. Then, gold seeds were grown by electrochemical deposition through gold salt. The cell-derived exosomes were successfully immobilized on the FTO electrode through the CD-151 antibody, and cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods were used in this research. In the CV method, the change in the current passing through the working electrode is measured so that the connection of exosomes causes the current to decrease. In the EIS method, surface resistance changes were investigated so that the binding of exosomes increased the surface resistance. Various concentrations of exosomes in both cell culture and blood serum samples were measured to test the sensitivity of the biosensor, which makes our biosensor capable of detecting 20 exosomes per milliliter.

Hybrid Microfluidic Device for High Throughput Isolation of Cells Using Aptamer Functionalized Diatom Frustules.

Circulating tumor cells (CTCs), secreted from primary and metastatic malignancies, hold a wealth of essential diagnostic and prognostic data for multiple cancers. Significantly, the information contained within these cells may hold the key to understanding cancer metastasis, both individually and fundamentally. Accordingly, developing ways to identify, isolate and interrogate CTCs plays an essential role in modern cancer research. Unfortunately, CTCs are typically present in the blood in vanishingly low titers and mixed with other blood components, making their isolation and analysis extremely challenging. Herein, we report the design, fabrication and optimization of a microfluidic device capable of automatically isolating CTCs from whole blood. This is achieved in two steps, via the passive viscoelastic separation of CTCs and white blood cells (WBCs) from red blood cells (RBCs), and subsequent active magnetophoretic separation of CTCs from WBCs. We detail the specific geometries required to balance the elastic and inertial forces required for successful passive separation of RBCs, and the use of computational fluid dynamics (CFD) to optimize active magnetophoretic separation. We subsequently describe the use of magnetic biosilica frustules, extracted from Chaetoceros sp. diatoms, to fluorescently tag CTCs and facilitate magnetic isolation. Finally, we use our microfluidic platform to separate HepG2-derived CTCs from whole blood, demonstrating exceptional CTC recovery (94.6%) and purity (89.7%).

Reaction mechanism of the bioluminescent protein mnemiopsin1 revealed by X-ray crystallography and QM/MM simulations.

Bioluminescence of a variety of marine organisms, mostly cnidarians and ctenophores, is carried out by Ca2+-dependent photoproteins. The mechanism of light emission operates via the same reaction in both animal families. Despite numerous studies on the ctenophore photoprotein family, the detailed catalytic mechanism and arrangement of amino acid residues surrounding the chromophore in this family are a mystery. Here, we report the crystal structure of Cd2+-loaded apo-mnemiopsin1, a member of the ctenophore family, at 2.15 Å resolution and used quantum mechanics/molecular mechanics (QM/MM) to investigate its reaction mechanism. The simulations suggested that an Asp-156-Arg-39-Tyr-202 triad creates a hydrogen-bonded network to facilitate the transfer of a proton from the 2-hydroperoxy group of the chromophore coelenterazine to bulk solvent. We identified a water molecule in the coelenteramide-binding cavity that forms a hydrogen bond with the amide nitrogen atom of coelenteramide, which, in turn, is hydrogen-bonded via another water molecule to Tyr-131. This observation supports the hypothesis that the function of the coelenteramide-bound water molecule is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion, thereby triggering the bioluminescence reaction in the ctenophore photoprotein family.

A cyclic peptide reproducing the α1 helix of VEGF-B binds to VEGFR-1 and VEGFR-2 and inhibits angiogenesis and tumor growth.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are pivotal regulators of angiogenesis. The VEGF-VEGFR system is therefore an important target of anti-angiogenesis therapy. Based on the X-ray structure of VEGF-B/VEGFR-1 D2, we designed a cyclic peptide (known as VGB1) reproducing the α1 helix and its adjacent region to interfere with signaling through VEGFR-1. Unexpectedly, VGB1 bound VEGFR-2 in addition to VEGFR-1, leading to inhibition of VEGF-stimulated proliferation of human umbilical vein endothelial cells and 4T1 murine mammary carcinoma cells, which express VGEFR-1 and VEGFR-2, and U87 glioblastoma cells that mostly express VEGFR-2. VGB1 inhibited different aspects of angiogenesis, including proliferation, migration and tube formation of endothelial cells stimulated by VEGF-A through suppression of extracellular signal-regulated kinase 1/2 and AKT (Protein Kinase B) phosphorylation. In a murine 4T1 mammary carcinoma model, VGB1 caused regression of tumors without causing weight loss in association with impaired cell proliferation (decreased Ki67 expression) and angiogenesis (decreased CD31 and CD34 expression), and apoptosis induction (increased TUNEL staining and p53 expression, and decreased Bcl-2 expression). According to far-UV circular dichroism (CD) and molecular dynamic simulation data, VGB1 can adopt a helical structure. These results, for the first time, demonstrate that α1 helix region of VEGF-B recognizes both VEGFR-1 and VEGFR-2.

Imidazolium-based ionic liquid functionalized mesoporous silica nanoparticles as a promising nano-carrier: response surface strategy to investigate and optimize loading and release process for Lapatinib delivery.

Imidazolium-based ionic liquid functionalized PEGylated mesoporous silica nanoparticles MCM-41 (denoted as [ImIL-PEGylated@MCM-41] NPs) is synthesized and evaluated as an efficient and reliable pH-sensitive nano-carrier for controlled release of cationic Lapatinib (Lap) drug. This nano-DDS was fully characterized by dynamic light scattering, scanning electron microscopy, X-ray powder diffraction, Fourier transform infrared spectroscopy, N2 adsorption-desorption measurement, and differential scanning calorimeter. Furthermore, the drug loading content and in-vitro drug release profile were studied. The entrapment and loading efficiency of the optimized formulation for Lap were 91 ± 2.0% and 32.21 ± 2.70%, respectively. The results of cytotoxicity assay demonstrated that ImIL-PEG@MCM-41 has no significant toxicity on both cancerous and normal cell lines and the anticancer activity of Lap@ImIL-PEG@MCM-41 was comparable to free drug in case of human breast cells (SKBR3) and human embryonic kidney 293 cells (HEK-293). Meanwhile, three-dimensional (3D) cell culture was performed by multicellular tumor spheroids for understanding of cell response to drugs in physiologically 3D microenvironments. The results of Lap@ImIL-PEG@MCM-41 uptake during 48 hours showed a gradual release of the Lap through the multicellular tumor spheroids. This showed that the pH-responsive controlled release of Lapatinib leads to the satisfactory results in the in vitro breast cancer therapy.

Tuning fluorophore excitation in a total-internal-reflection-fluorescence microscopy.

In a total-internal-reflection-fluorescence-microscopy method, there is anisotropy in the polarized evanescent wave. Since the evanescent wave is used as an excitation field, the mentioned anisotropy is a disadvantage in using the total-internal-reflection-fluorescence-microscopy technique. Therefore, by theoretical and analytical approaches, and based on the Fresnel coefficients, the effect of three dielectrics media on the anisotropy of the evanescent wave is investigated. Following that, a proper combination of the cover glass, oil immersion, and prism for both living and non-living samples is suggested that not only enhances the intensity of the evanescent wave, but also and importantly, decreases the essential anisotropy of the evanescent wave.

Bio-active molecules modified surfaces enhanced mesenchymal stem cell adhesion and proliferation.

Surface modification of the substrate as a component of in vitro cell culture and tissue engineering, using bio-active molecules including extracellular matrix (ECM) proteins or peptides derived ECM proteins can modulate the surface properties and thereby induce the desired signaling pathways in cells. The aim of this study was to evaluate the behavior of human bone marrow mesenchymal stem cells (hBM-MSCs) on glass substrates modified with fibronectin (Fn), collagen (Coll), RGD peptides (RGD) and designed peptide (R-pept) as bio-active molecules. The glass coverslips were coated with fibronectin, collagen, RGD peptide and R-peptide. Bone marrow mesenchymal stem cells were cultured on different substrates and the adhesion behavior in early incubation times was investigated using scanning electron microscopy (SEM) and confocal microscopy. The MTT assay was performed to evaluate the effect of different bio-active molecules on MSCs proliferation rate during 24 and 72 h. Formation of filopodia and focal adhesion (FA) complexes, two steps of cell adhesion process, were observed in MSCs cultured on bio-active molecules modified coverslips, specifically in Fn coated and R-pept coated groups. SEM image showed well adhesion pattern for MSCs cultured on Fn and R-pept after 2 h incubation, while the shape of cells cultured on Coll and RGD substrates indicated that they might experience stress condition in early hours of culture. Investigation of adhesion behavior, as well as proliferation pattern, suggests R-peptide as a promising bio-active molecule to be used for surface modification of substrate in supporting and inducing cell adhesion and proliferation.

Photoinactivation related dynamics of ctenophore photoproteins: Insights from molecular dynamics simulation under electric-field.

Photoinactivation is a common phenomenon in bioluminescence ctenophore photoproteins (e.g mnemiopsin, berovin and BfosPP) with still unknown mechanism. The activity of coelenterate photoproteins (e.g aequorin), which has high structural similarity with ctenophore photoproteins, is not affected by light. Recently, we have characterized the effects of light on ctenophore photoprotein mnemiopsin, in different conformations, which has demonstrated light induced structural changes, uniquely secondary structures, of both apo and holo mnemiopsin. This paper is further expansion of our previous work, by applying molecular dynamics simulations to investigate photoinactivation related dynamics of berovin at atomistic level, in comparison with aequorin, under the influence of electric component of electromagnetic field. The results have indicated that the intense electric filed could influence structure of both berovin and aequorin but in different manner, whereas moderate electric field only effects on berovin's structure remarkably. In this case, increased helicity of residues E180-M193 and decreased helical contents of L38-D46 and L125-D138 segments are considerable in berovin as well as flexibility elevation of calcium binding loops. These changes cause structural expansion of berovin, especially at N-terminal domain, in direction of electric field. In conclusion, the induced structural changes of mentioned helical parts together with elevated fluctuation of their adjacent segments, N26-D46 and M193-Y206, indicate the influence of light on substrate stabilizing residues, Arg41 and Y204. This condition could presumably leads to inactivation of bioluminescence reaction due to separation of substrate from the cavity of the protein.

Proteomic features of delayed ocular symptoms caused by exposure to sulfur mustard: As studied by protein profiling of corneal epithelium.

Exposure to mustard gas can lead to variations in the proteome of corneal epithelium cells and after a latency period produces delayed symptoms in the eyes of chemical victims. Hence, a comparative proteome analysis was conducted between the corneal epithelial cells of chemical victims from Iran-Iraq war (1980-1988) and healthy donors. To this end, corneal epithelium samples from victims and healthy individuals were collected, and the proteome of these samples were prepared for two-dimensional electrophoresis and the analysis of spots by statistical software. Selected spots were further analyzed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Twenty four proteins were identified of which eighteen proteins showed downregulation while six proteins were upregulated in the victims in comparison to the normal individuals. Also, six protein spots were confirmed by western-blot analysis. In conclusion, all the twenty-four identified proteins are involved in pathways which their up- or down-regulation leads to the accumulation of undesired substrates, cell death and apoptosis. Bioinformatics' tools indicated that these identified proteins were involved in various metabolic processes, DNA damage response, immune response and etc. The present study provides a suitable platform for further clinical studies.

Polyurethane/siloxane membranes containing graphene oxide nanoplatelets as antimicrobial wound dressings: in vitro and in vivo evaluations.

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.

Hemoglobin-incorporated iron quantum clusters as a novel fluorometric and colorimetric probe for sensing and cellular imaging of Zn(II) and cysteine.

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+ 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. Graphical abstract Schematic of yellow-emitting iron quantum clusters in hemoglobin matrix (Hb-FeQCs) were characterized and successfully applied for sensing zinc(II) and cysteine. The act as an on-off fluorescent probe and can be applied to image zinc ions in human fibroblast cells under visible light.

In Vivo study of naturally deformed Escherichia coli bacteria.

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.

MicroRNA-145-based differentiation of human mesenchymal stem cells to smooth muscle cells.

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.

Increasing proteome coverage for gel-based human tear proteome maps: towards a more comprehensive profiling.

In situations where the molecular mechanism of many ocular disorders is unknown, owing to the difficulties associated with sampling from ocular tissues, human tear film can be a promising medium in ophthalmic research. The present study demonstrates an in-depth gel-based proteome optimization survey to approach more appropriate and efficient systems in various situations such as normal and dry-eye syndromes. Therefore, systematic and statistical evaluations were performed on different preparation methods, including acetone, acetone-methanol, chloroform-methanol-water, trichloroacetic acid (TCA)-acetone, tri-n-butylphosphate-acetone-methanol precipitations and ammonium sulfate fractionation at three different percentages of saturations (50, 70 and 90%). Methods were compared quantitatively on both one- and two-dimensional patterns. Some important parameters such as total protein recovery yield, densitometric analysis of some protein contaminants, banding patterns and total spot numbers along with statistical models for proper clustering were considered. Findings revealed noticeable impacts of preparation methods on all aspects of gel-based separations as well as recovery yield (ranging from 5.29 ± 0.96 to 22.56 ± 1.77 µg/mm) and banding and pattern resolution. In addition to all these, the most important point is that the total protein spot number on the final two-dimensional patterns (varied from 528.00 ± 19.00 to 657.00 ± 21.52 for different methods) were also noticeably increased in comparison with previously published reports (maximum of 250 spots), which is essential for a more comprehensive analysis. Increasing the proteome coverage in the present study is supposed to originate from improved solubility and effective rehydration during the sample application and isoelectric focusing (IEF) procedure along with proper sample preparation.

C-terminal amidation of an osteocalcin-derived peptide promotes hydroxyapatite crystallization.

Genesis of natural biocomposite-based materials, such as bone, cartilage, and teeth, involves interactions between organic and inorganic systems. Natural biopolymers, such as peptide motif sequences, can be used as a template to direct the nucleation and crystallization of hydroxyapatite (HA). In this study, a natural motif sequence consisting of 13 amino acids present in the first helix of osteocalcin was selected based on its calcium binding ability and used as substrate for nucleation of HA crystals. The acidic (acidic osteocalcin-derived peptide (OSC)) and amidic (amidic osteocalcin-derived peptide (OSN)) forms of this sequence were synthesized to investigate the effects of different C termini on the process of biomineralization. Electron microscopy analyses show the formation of plate-like HA crystals with random size and shape in the presence of OSN. In contrast, spherical amorphous calcium phosphate is formed in the presence of OSC. Circular dichroism experiments indicate conformational changes of amidic peptide to an open and regular structure as a consequence of interaction with calcium and phosphate. There is no conformational change detectable in OSC. It is concluded that HA crystal formation, which only occurred in OSN, is attributable to C-terminal amidation of a natural peptide derived from osteocalcin. It is also proposed that natural peptides with the ability to promote biomineralization have the potential to be utilized in hard tissue regeneration.

Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift.

Firefly bioluminescence reaction in the presence of Mg(2+), ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350-359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7Å and 2.2Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351-359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission.

Enhanced reproducibility of the human gel-based tear proteome maps in the presence of di-(2-hydroxyethyl) disulfide.

Patterns obtained in two-dimensional gel electrophoresis (2-DE) in the previously published articles suggest a varying number of proteins. To seek the cause of this variation, we investigated the effect of reduction power on the overall tear proteome maps. To this end, the buffers of two reducing agents, dithiothreitol (DTT) at nine different concentrations and di-(2-hydroxyethyl) disulfide (HED), were examined. The assay showed that HED clearly improved 2-DE resolution, increased the number of detectable protein spots, and offered well-resolved chain regions in comparison with those treated with DTT. Furthermore, this study introduced increasing the reduction power as a remedy to increase the reproducibility of two-dimensional human tear proteome maps. In addition, the results of our assessment showed that improved reduction efficiency was accompanied by increased procedure reproducibility from 42% to 89%.