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Oxytetracycline (OTC) as the frequently applied antibiotics present in human and animal diets and its residue may results to various toxic effects. So, there is a crucial need for developing sensitive method for detection of OTC. Aptamer‐based biosensors (aptasensors) are frequently used in recent applied detection strategies with high affinity and specificity. In the present study, a colorimetric aptasensor was developed for detection of oxytetracycline (OTC) based on aggregation of aptamer functionalized AuNRs. The synthesized AuNRs were characterized and functionalized with specific DNA oligonucleotide as the aptamer to generate an optical signal after binding to the OTC antibiotic. The sensitivity of optical changes in spectroscopic analysis was improved under ambient temperature that resulted in aptamer folded structure and subsequent aggregation state of AuNRs. The longitudinal surface plasmon resonance (LSPR) of AuNRs was decreased in the linear range of applied OTC concentration from 0.1 nM to 100 nM with a detection limit of 0.04 nM. The proposed method was facile and showed sensitive and selective results, that would be consider as an alternative and efficient approach toward rapid detection of OTC. [ABSTRACT FROM AUTHOR]

The glycolysis is a common characteristic of cancer and it is responsible for providing enough energy to ensure growth. The glycolysis suppression is beneficial in tumor growth reduction. The stimulation/inhibition of glycolysis in cancer is tightly regulated by ncRNAs. The regulation of glycolysis by ncRNAs can influence proliferation and therapy response of tumor. The miRNAs are capable of inactivating enzymes responsible for glycolysis and suppressing signaling networks resulting in glycolysis induction. By regulation of glycolysis, miRNAs can affect therapy response. The lncRNAs and circRNAs follow a same pathway and by targeting glycolysis, they affect progression and therapy response of tumor. Noteworthy, lncRNAs and circRNAs sponge miRNAs in glycolysis mechanism control in tumor cells. Furthermore, ncRNA-mediated regulation of glycolysis mechanism can influence metastasis to organs of body. The ncRNAs regulating glycolysis are reliable biomarkers in cancer patients and more importantly, exosomal ncRNAs due to their presence in body fluids, are minimally-invasive biomarkers. [Display omitted] [ABSTRACT FROM AUTHOR]

Low oxygen level at tumor microenvironment leads to a condition, known as hypoxia that is implicated in cancer progression. Upon hypoxia, HIF-1α undergoes activation and due to its oncogenic function and interaction with other molecular pathways, promotes tumor progression. The HIF-1α role in regulating breast cancer progression is described, Overall, HIF-1α has upregulation in breast tumor and due to its tumor-promoting function, its upregulation is in favor of breast tumor progression. HIF-1α overexpression prevents apoptosis in breast tumor and it promotes cell cycle progression. Silencing HIF-1α triggers cycle arrest and decreases growth. Migration of breast tumor enhances by HIF-1α signaling and it mainly induces EMT in providing metastasis. HIF-1α upregulation stimulates drug resistance and radio-resistance in breast tumor. Furthermore, HIF-1α signaling induces immune evasion of breast cancer. Berberine and pharmacological intervention suppress HIF-1α signaling in breast tumor and regulation of HIF-1α by non-coding RNAs occurs. Furthermore, HIF-1α is a biomarker in clinic. [Display omitted] [ABSTRACT FROM AUTHOR]

DNA self-assembled hybrid nanostructures are widely used in recent research in nanobiotechnology. Combination of DNA with carbon based nanoparticles such as single-walled carbon nanotube (SWNT), multi-walled carbon nanotube (MWNT) and carbon quantum dot were applied in important biological applications. Many examples of biosensors, nanowires and nanoelectronic devices, nanomachine and drug delivery systems are fabricated by these hybrid nanostructures. In this study, a new hybrid nanostructure has been fabricated by noncovalent interactions between single or double stranded DNA and SWNT nanoparticles and biophysical properties of these structures were studied comparatively. Biophysical properties of hybrid nanostructures studied by circular dichroism, UV–vis and fluorescence spectroscopy techniques. Also, electrochemical properties studied by cyclic voltammetry, linear sweep voltammetry, square wave voltammetry, choronoamperometry and impedance spectroscopy (EIS). Results revealed that the biophysical and electrochemical properties of SWNT/DNA hybrid nanostructures were different compare to ss-DNA, ds-DNA and SWNT singly. Circular dichroism results showed that ss-DNA wrapped around the nanotubes through π-π stacking interactions. The results indicated that after adding SWNT to ss-DNA and ds-DNA intensity of CD and UV–vis spectrum peaks were decreased. Electrochemical experiments indicated that the modification of single-walled carbon nanotubes by ss-DNA improves the electron transfer rate of hybrid nanostructures. It was demonstrated SWNT/DNA hybrid nanostructures should be a good electroactive nanostructure that can be used for electrochemical detection or sensing. [ABSTRACT FROM AUTHOR]

Acute myocardial infarction (AMI) is one of the leading causes of death throughout the world. Usual methods for detecting AMI are expensive, time-consuming and using blood samples as biological samples. Therefore, creating an ultra-fast, sensitive and non-invasive diagnostic test is necessary. Herein, a novel ultra-sensitive, fluorescent, plasmon-exciton coupling hybrid of a gold nanoparticle-quantum dot (PQ)-based aptamer nanobiosensor is presented for the detection of human cardiac troponin I (cTnI), the golden biomarker of AMI, and a preclinical test is performed within saliva. The binding of the cTnI protein to aptamer leads to a fluorescence enhancement of the plexcitonic hybrid system. The response range of this nanobiosensor is 0.4-2500 fM and the limit of detection is 0.3 fM. It seems that this novel design of nanobiosensor in the form of the PQ plexcitonic hybrid system can presents new opportunities for nanobiosensor progress. [ABSTRACT FROM AUTHOR]

Technological and methodological advances in multi-omics data generation and integration approaches help elucidate genetic features of complex biological traits and diseases such as prostate cancer. Due to its heterogeneity, the identification of key functional components involved in the regulation and progression of prostate cancer is a methodological challenge. In this study, we identified key regulatory interactions responsible for primary to metastasis transitions in prostate cancer using network inference approaches by integrating patient derived transcriptomic and miRomics data into gene/miRNA/transcription factor regulatory networks. One such network was derived for each of the clinical states of prostate cancer based on differentially expressed and significantly correlated gene, miRNA and TF pairs from the patient data. We identified key elements of each network using a network analysis approach and validated our results using patient survival analysis. We observed that HOXD10, BCL2 and PGR are the most important factors affected in primary prostate samples, whereas, in the metastatic state, STAT3, JUN and JUNB are playing a central role. Benefiting integrative networks our analysis suggests that some of these molecules were targeted by several overexpressed miRNAs which may have a major effect on the dysregulation of these molecules. For example, in the metastatic tumors five miRNAs (miR-671-5p, miR-665, miR-663, miR-512-3p and miR-371-5p) are mainly responsible for the dysregulation of STAT3 and hence can provide an opportunity for early detection of metastasis and development of alternative therapeutic approaches. Our findings deliver new details on key functional components in prostate cancer progression and provide opportunities for the development of alternative therapeutic approaches. [ABSTRACT FROM AUTHOR]

Gold nanorods have been nominated as propitious candidates for nanobiodiagnostic applications. Herein, a technique has been introduced for rapid visual detection of lysozyme, as its high level of excretion in biological fluids is a characteristic sign of leukemia and kidney disorders. Gold nanorods were biofunctionalized with lysozyme aptamer and characterized with UV–Visible and FTIR spectroscopy, zeta potential analyzer and transmission electron microscopy. Exposure of the nanoprobe to nano molar levels of lysozyme (20 nmol l −1 ) lead to dictated aggregation of the nanostructures at ambient temperature; which was significantly improved by heat induced morphological perturbations and rapid detection by the naked eye (down to pico molar level). Qualitative analysis of Acute myeloid leukemia, Acute lymphocytic leukemia and Lymphoma blood serums showed sensitivity and specificity of the fabricated aptasensor under both temperature conditions. This report encourages utilization of heat-induced aggregation of gold nanorods as a promising nanodiagnostic technique for the emerging nanotechnologies. [ABSTRACT FROM AUTHOR]

G-quadruplexes are supramolecular structures of G-rich nucleic acid, formed by non-canonical base pairing in the presence of specific environmental inducers. These structures have been vastly considered in diagnostic and therapeutic applications. However, detailed information on structure, optical properties and thermal stability of G-quadruplex potent oligonucleotides is scarce. Herein, optical properties and thermodynamic stability of AS1411 quadruplex is reported for various concentrations of potassium and lead ions. Circular dichroism showed that AS1411 ss-DNA folds into parallel conformation in the presence of metal ions and molecular crowding condition. UV–vis spectroscopy indicated formation of quadruplex and fluorescent spectroscopy revealed intercalation of PicoGreen in its structure, with enhancement of emission intensity upon increment of metal ion concentration. This investigation also proposes high-throughput and reliable analysis of AS1411 quadruplex's thermal stability by real-time PCR technique, which can be further applied for other quadruplex structures. [ABSTRACT FROM AUTHOR]

Plasmonic circular dichroism (CD) responses of hybrid nanostructures containing noble metal nanoparticles and chiral molecules have received increasing interest with various applications in nanophotonics. Chiral biomolecules show strong CD signals typically found in the ultraviolet region, whereas, in the visible range, they produce a weak signal. Strengthening the CD signal in the visible region is of high importance, which could be achieved through fabrication of novel hybrid nanostructures. Herein, gold nanoparticles (GNPs) have been assembled via DNA linker to investigate the possibility of enhancing plasmonic CD signal in the visible range. DNA-linked assemblies with pre- and postannealed conditions were characterized by ultraviolet-visible spectroscopy, dynamic light scattering (DLS), and CD spectropolarimetry. In the presence of DNA linker with sticky ends, the aggregation phenomenon was traced by red shifts of surface plasmon resonance of nanoparticles. Time-dependent hybridization of single-stranded 'sticky ends' with DNA-conjugated GNPs and increased probability of hydrogen bond formation lead to enhancement of CD signals in the ultraviolet region. Complexation of biomolecule and nanoparticle assemblies induced enhanced CD signals in the visible range, which was noticed both before and after purification. DLS characterization of the assemblies also confirmed the difference in the size of aggregates, which could be controlled by the linker molecules. This investigation encourages possibility of utilizing plasmonic CD technique as a tool for tracing fabricated nanostructure assemblies with enhanced characterization possibility. [ABSTRACT FROM AUTHOR]

Horseradish peroxidase is an all alpha-helical enzyme, which widely used in biochemistry applications mainly because of its ability to enhance the weak signals of target molecules. This monomeric heme-containing plant peroxidase is also used as a reagent for the organic synthesis, biotransformation, chemiluminescent assays, immunoassays, bioremediation, and treatment of wastewaters as well. Accordingly, enhancing stability and catalytic activity of this protein for biotechnological uses has been one of the important issues in the field of biological investigations in recent years. In this study, pH-induced structural alterations of native (HRP), and modified (MHRP) forms of Horseradish peroxidase have been investigated. Based on the results, dramatic loss of the tertiary structure and also the enzymatic activity for both forms of enzymes recorded at pH values lower than 6 and higher than 8. Ellipticiy measurements, however, indicated very slight variations in the secondary structure for MHRP at pH 5. Spectroscopic analysis also indicated that melting of the tertiary structure of MHRP at pH 5 starts at around 45° C, which is associated to the pKa of His 42 that has a serious role in keeping of the heme prostethic group in its native position through natural hydrogen bond network in the enzyme structure. According to our data, a molten globule like structure of a chemically modified form of Horseradish peroxidase at pH 5 with initial steps of conformational transition in tertiary structure with almost no changes in the secondary structure has been detected. Despite of some conformational changes in the tertiary structure of MHRP at pH 5, this modified form still keeps its catalytic activity to some extent besides enhanced thermal stability. These findings also indicated that a molten globular state does not necessarily preclude efficient catalytic activity. [ABSTRACT FROM AUTHOR]

Abstract: The effect of proline on refolding and unfolding kinetics as well as activity of lipase from Pseudomonas fluorescens was determined using stopped-flow fluorescence and UV/Vis absorbance spectroscopy. Enzyme assay at different concentrations of proline revealed that activity of enzyme reaches maximum at 0.6M concentration of proline. Kinetic measurements showed that refolding rate is considerably accelerated in the presence of 0.6M proline. Unfolding kinetic traces were fitted to double exponential function, and it was revealed that lipase molecules were unfolded via two different pathways. Fast unfolding rate constant decreased, while slow one did not change significantly upon addition of proline. [Copyright &y& Elsevier]

Abstract: Like many other bacterial lipases Pseudomonas aeruginosa lipase has a very strong structure–function relationship. Herein, the effect of structural compactness of lipase has been investigated on its activity. P. aeruginosa HR59 was isolated from burn infection as a new and inexpensive source. Bacterial lipase was extracted from this strain and purified by ion-exchange chromatography. Two divalent metal ions (calcium and magnesium) and three polar organic solvents (methanol, ethanol and iso-propanol) were used to study the structure–function relationship. Results of this study revealed that these additives activated enzyme by reducing the helix content of enzyme. Moreover, thermo stability of the enzyme decreased in the presence of calcium and magnesium ions; whereas it increased upon interaction with polar organic solvents under heat-induced denaturation. Results of this investigation encourage utilization of these activity enhancers for various medical and industrial applications. [Copyright &y& Elsevier]

Candida antarctica lipase B (CALB) belongs to psychrophilic lipases which hydrolyze carboxyl ester bonds at low temperatures. There have been some features reported about cold-activity of the enzyme through experimental methods, whereas there is no detailed information on its mechanism of action at molecular level. Herein, a comparative molecular dynamics simulation and essential dynamics analysis have been carried out at three temperatures (5, 35 and 50°C) to trace the dominant factors in the psychrophilic properties of CALB under cold condition. The results clearly describe the effect of temperature on CALB with meaningful differences in the flexibility of the lid region (a5 helix), covering residues 141-147. Open- closed conformations have been obtained from different sets of long-term simulations (60 ns) at 5°C gave two reproducible distinct forms of CALB. The starting open conformation became closed immediately at 35 and 50°C during 60 ns of simulation, while a sequential open-closed form was observed at 5°C. These structural alterations were resulted from α5 helical movements, where the closed conformation of active site cleft was formed by displacement of both helix and its side chains. Analysis of normal mode showed concerted motions that are involved in the movement of both α5 and α10 helices. It is suggested that the functional motions needed for lypolytic activity of CALB is constructed from short-range movement of α5, accompanied by long-range movement of the domains connected to the lid region. [ABSTRACT FROM AUTHOR]

Abstract: The unique morphology of anisotropic rod-shaped gold nanostructures has offered new prospects for biomedical and biosensing applications. This study investigates the interaction of two types of rod-shaped nanostructures, gold nanorods and gold nanorices with lysozyme as a model protein, comparing the probable structural, activity and kinetic stability alterations. Circular dichroism spectropolarimeter revealed that lysozyme retains high fraction of its native conformation in the presence of both nanostructures, with a slight increase in the helical and beta content. Upon the protein adsorption on both types of nanorods, kinetic studies showed maintenance of enzymatic activity, together with increase in the enzymatic affinity and kinetic stability at high temperature. Comparatively, gold nanorice induced better effect on the activity and stability of enzyme than that of gold nanorod. This study might open new insight into potential applications of gold nanorods as nanocarriers for genes and drugs; provided that the toxicological aspect of cationic surfactant-coated nanostructure is taken into consideration. [Copyright &y& Elsevier]

Abstract: The stability of enzymes with no reduction in their catalytic activity still remains a critical issue in industrial applications. Naturally occurring osmolytes are commonly used as protein stabilizers. In this study we have investigated the effects of sorbitol and trehalose on the structural stability and activity of Pseudomonas cepacia lipase (PCL), using UV–visible, circular dichroism (CD) and fluorescence spectroscopy. Surface plasmon resonance (SPR) technique was used to trace changes in the refractive index and dielectric constant of the environment. The results revealed that catalytic activity and intrinsic fluorescence intensity of PCL increased in the presence of both osmolytes. Far-UV CD spectra indicated that the protein has undergone some conformational changes upon interacting with these osmolytes. Increasing the concentration of sorbitol led to changes in the refractive index and consequently the dielectric constant of environment; whereas in the case of trehalose, such changes were not significant. Unfavorable interactions of trehalose with protein surface induced higher preferential exclusion from the enzyme–water interface than that of sorbitol. Results of this report could give further insights about the stabilization mechanism of osmolytes. [Copyright &y& Elsevier]

Abstract: Gold nanorods, with their unique morphology and optical properties have offered new prospects for biomedical and biosensing applications. Herein, the interaction between gold nanorods and a model protein has been monitored using spectroscopic techniques. The enzyme retains high fraction of its native structure with a slight increase in the helical content at the expense of β-turns. Kinetic investigations revealed notable increase of enzyme affinity for substrate without significant decrease in the V max. Emission spectra of tryptophan residues in the presence of chaotropic agent highlighted the maintenance of internal quenching due to the induced compactness. Comparison of the gold nanorod treated lysozyme with free enzyme revealed higher thermodynamic stability under denaturing condition. Results from this study encourage the possibility of utilizing gold nanorods as promising nanocarrier candidates for a new generation of drug delivery applications. [Copyright &y& Elsevier]

Abstract: Firefly luciferase is widely used in many analytical techniques. However, the enzyme is unstable, so that its relative inactivation results in low sensitivity of those techniques. In this study, we have investigated the effects of MgSO4 and trehalose on the structural stability and function of luciferase from Photinus pyralis using circular dichroism (CD), conventional and stopped-flow fluorescence spectroscopy and bioluminescence assay. The secondary structural content, compactness and its melting temperature are also studied, which showed that the stability of luciferase increased in the presence of additives. Measurements of refolding rate constants under conditions that favor folding, show that MgSO4 accelerates the folding of enzyme, on the contrary, refolding rate constant decreases in the presence of trehalose which can be attributed to its high viscosity. Finally, combined with remaining activity assay we concluded that magnesium sulfate and trehalose can be used for short- and long-term stabilization, respectively. [Copyright &y& Elsevier]

This paper reviews the best known techniques using circular dichroism spectroscopy such as conventional circular dichroism (i.e. electronic circular dichroism), magnetic circular dichroisms (magnetic vibrational circular dichroism, x-ray magnetic circular dichroism), fluorescence detected circular dichroism, near-infrared circular dichroism, vibrational circular dichroism, Fourier transform infrared circular dichroism, high pressure liquid chromatography circular dichroism, stopped-flow circular dichroism, and synchrotron radiation circular dichroism. Also, we have described here the most important applications of circular dichroism spectroscopy in structural biochemistry and nanoscience. [ABSTRACT FROM AUTHOR]

Abstract: A facile method for the formation of covalent bonds between protein molecules is zero length cross-linking. This method enables the formation of cross-links without use of any chemical reagents. Here, we report a cross-linking method for lysozyme and some structural studies as well as catalytic activity assay was performed on lysozyme dimer. The results showed that catalytic activity of lysozyme dimer was the same as monomer. Also, the GdnCl-induced equilibrium unfolding of hen egg-white lysozyme monomer and dimer at pH 2 was studied over a temperature range of 290.7–303.2 K by means of CD spectroscopy. The lack of coincidence between two unfolding curves at 222 and 289 nm in lysozyme dimer was observed, which suggested the existence of intermediate state in unfolding process, while lysozyme monomer showed a single cooperative transition. Thus, the thermodynamic parameters were estimated on the basis of two-state mechanism for lysozyme monomer and three-state one for lysozyme dimer. These results indicated that zero length cross-linking can stabilize the intermediate, so the population of intermediate increased. Our results offer a special opportunity to study the role of intermediates in protein folding mechanisms. In addition thermal unfolding of monomer and dimer in 222 nm was achieved. [Copyright &y& Elsevier]

Abstract: Horseradish peroxidase is an important heme-containing plant enzyme with enormous medical diagnostic, biosensing, bioremediation and biotechnological applications. Any improvement in the stability of the enzyme will greatly enhance its application in mentioned areas. In the present study, the stabilizing effects of certain additives and chemical modification by citraconic anhydride on the thermal behavior of HRP were investigated. Both strategies brought about dramatic enhancement of the thermostability of the enzyme. Results obtained on T m, changes in the circular dichroism (CD) spectra and kinetic parameters of HRP and its modified form are discussed in terms of contributions to the mechanism of the thermal stability and the activity enhancement. Polyols were very effective in providing protection against the irreversible thermoinactivation of the native and the modified forms of the enzyme. Our results reveal that a combination of medium change and surface modification may provide an effective strategy for the enhancement of the thermodynamic and the kinetic stability of the enzyme. [Copyright &y& Elsevier]

Chemical modification of lysine residues in glucose oxidase was carried out using citraconic anhydride. Modification brought about changes in the kinetic properties of the enzyme as evident by substantial lowering of Vmax and Km. Enhancement of tryptophan fluorescence was observed with a dramatic change in its pH dependence due to modification. Near- and far-UV circular dichroism spectra of the native and modified forms suggested formation of molten globule-like structures, further supported by 8-anilino-1-naphthalenesulfonic acid fluorescence which indicated higher exposure of hydrophobic residues as a result of chemical modification. [Copyright &y& Elsevier]

Dodecyl sulfate doped polypyrrole (PPy-DS) was prepared electrochemically by anodic polymerization of pyrrole in the presense of dodecyl sulfate (DS−) ions in aqueous solution and used as a DS− ion selective electrode. This surfactant selective electrode was used to study the effect of DS− ion on the conformation changes of lysozyme in binary ethanol–water mixtures. The binding of DS− ion to lysozyme have been studied by electrochemical measurements at 293 and 310 K. DS− ion interact cooperatively with lysozyme in different composition of ethanol. The binding isotherms are discussed in terms of the binding potential concept of Wyman and Gibbs free energy, enthalpy and entropy of binding were calculated. The thermodynamic parameters (ΔHν¯ and ΔSν¯) were evaluated from the temperature dependence of equilibrium binding constants. The binding process in 0, 5 and 10% ethanol are entropy controlled and with increasing the percent of ethanol (15%), both enthalpy and entropy are favored. The values of ΔSν¯ being more positive with decease in the content of the mixture. The resulting T ΔSν¯ against ΔHν¯ plot showed a fairly good linear correlation, indicating the existence of an enthalpy–entropy compensation in the binding process. [Copyright &y& Elsevier]

Antimicrobial resistance occurs mostly through the ineffective and unauthorized use of antibiotics in both the environment and health care. Biofilms are a new target in the search for new antibacterial agents. Here, the sol‐gel method is used to produce zinc oxide quantum dots (ZnO‐QDs). The ZnO‐QDs show yellow emission at 526 nm under 360 nm excitation. After that, the ability of ZnO‐QDs as an antibacterial and antibiofilm agent against methicillin‐resistant Staphylococcus aureus (MRSA) is examined. The 100 samples are collected from patients with hospital‐acquired infections between December 2020 and October 2021 from Imamen Kadhmiyan Teaching Hospital in Baghdad (Iraq). Out of these 100 samples, 63 are S. aureus and 20 out of 63 are MRSA. The minimum inhibitory concentration assay demonstrates the antibacterial activity of ZnO‐QDs on MRSA strains. Also, results show that 80, 5, and 15% of isolated MRSA strains produce strong, moderate, and weak or very weak biofilm, respectively. The effect of ZnO‐QDs on strong and moderate biofilms reveal that QDs could also inhibit and destroy biofilm produced by MRSA. In addition, the antibiofilm effect of ZnO‐QDs is greater than their antibacterial effect. Generally, this work offers new insights into the development of antibacterial and antibiofilm nanomaterials. [ABSTRACT FROM AUTHOR]

Studies the role of electrostatic mutations in the conformation and thermal denaturation of apocalmodulin. Evidence for the major role of the electrostatic potential in the stability and flexibility of synthetic calmodulin; Parameters of thermal denaturation.

Gold nanoparticles (AuNPs) with unique optical properties, such as easy operation and visualized assay, have a great ability to detect different types of analytes. In the present study, a novel AuNPs based aptasensor strategy for the detection of trinitrotoluene (TNT) was used. For this purpose, two different batches of AuNPs were functionalized by thiol modified DNA probes (P1 and P2) complementary to 5' and 3' ends of anti-TNT aptamer. Then, two batches of gold nanoprobes were mixed and the addition of anti-TNT aptamer induced the assembly and aggregation of nanoparticles. However, according to this strategy, in the presence of TNT, aptamer can bind to its target molecule (TNT) and prevent nanoparticles aggregation. The results indicate that the produced aptasensor can detect TNT molecule with high specificity (detection limit of 6 pM). In conclusion, this gold nanoparticle-based colorimetric anti-TNT aptasensor can be used in the next investigations with high efficiency. [ABSTRACT FROM AUTHOR]

A hybrid scaffold containing gold nanorods and lysozyme amyloid fibrils has been fabricated, and the effect of surface Modification on improving nanostructure assembly on the biological template has been investigated. The nanohybrid system was characterized by monitoring surface plasmon resonance bands, dynamic light scattering spectroscopy, Thiofavin-T assay, and transmission electron microscopy. Surface of gold nanorods (GNRs) was modifed with polystyrene sulfonate (PSS), and possible difference in assembly of the pristine and modified nanostructures was compared upon interaction with amyloid fibrils. Analysis of transmission electron microscopy showed that changing the surface charge of GNRs with biocompatible polymer improved electrostatic interactions between the nanostructures and amyloid fibril templates. Analysis of cell viability assays also showed that surface functionalization of GNRs remarkably improved bio- compatibility of the nanoscaffold. Results of this study encourage utilization of modification strategies to fabricate a new generation of nanoscaffolds with fruitful applications in regenerative medicine. [ABSTRACT FROM AUTHOR]

Gold nanoparticles are metallic nanostructures with wide range of applications in biomedicine and biosensing. Among diverse morphologies of gold nanostructures, gold nanorods have attracted much more attention for their outstanding structure, optical property and function. The unique characteristic of these nanostructures encourages their integration with biomolecules of interest for versatile biomedical proposes. In this effort, amyloid fibrils with well-known mechanical strength and chemical stability have been used as biotemplates for assembly of gold nanorods to exploit dual property of both nano and bio counterparts in a hybrid nanoscaffold. To maximize the interaction of the nanostructures with the biotemplate, surface of the gold nanorods was modified with biocompatible polystyrene sulfonate (PSS). Spectroscopic and microscopic techniques including UV–Vis, CD, ThT and TEM were used to characterize formation of insulin amyloid fibrils, gold nanorods and their assemblies on the biotemplate. Morphology of gold nanorods was also monitored upon interaction with amyloid fibrils. Electrochemical impedance spectroscopy of the hybrid nanoscaffold showed that increase in the concentration of the assembled nanostructures on the biotemplate has clearly led to enhancement of conductivity. Results of this investigation highlight gold nanorods as promising candidates in design and fabrication of novel hybrid nanoscaffolds of enhanced conductivity, especially for biosensing and tissue engineering purposes. A hybrid nanoscaffold has been fabricated via assembly of Gold Nanorods on Insulin Amyloid Fibrils. The assembly process has been facilitated by coating of the nanostructures with Polystyrenesulfonate (PSS) for inducing electrostatic interaction with the biotemplate as well as dictating biocompatibility. The increment of the concentration of nanostructures enhanced the conductivity of the overall nanohybrid system. This study paves the way for the development of a new generation of nanoscaffolds with improved conductivity for future biosensing and tissue engineering platforms. [ABSTRACT FROM AUTHOR]

An abstract of the article "Binding site locations and interaction of the isolated middle loop of snake neurotoxin I with muscle type nicotinic acetylcholine receptor: molecular dynamics and docking simulation study," by Adak Nasiripourdori, Bijan Ranjbar and Hossein Naderi-Manesh is presented.

Linker mediated self-assembly of gold nanoparticles is emerging as an interesting strategy for construction of hybrid nanoscale systems with enhanced plasmonic circular dichroism (PCD) signals. Herein, gold nanorods were assembled via DNA linker to investigate the possibility of signal enhancement. Assemblies were characterized by UV–visible, fluorescence, dynamic light scattering and circular dichroism (CD) spectroscopy. Hybridization of complementary strands resulted in PCD signal enhancement, which was further monitored by the increase of real time PicoGreen fluorescence intensity. Impressively, such changes in the real time fluorescence and plasmonic CD responses could be used as a new detection method for ultrasensitive sensors. [ABSTRACT FROM AUTHOR]

Aptamers have been used as recognition elements in various sensors, called aptasensors. In this study, a facile colorimetric aptasensor was designed based on only two un-labeled oligonucleotides including carcinoembryonic antigen (CEA) aptamer and split peroxidase-mimicking DNAzyme. The halves of DNAzyme were connected by the complementary sequence of aptamer. In the absence of CEA, aptamer and its complementary hybridized and helped to pull DNAzyme halves apart which could decrease catalytic reaction. When CEA was recognized by aptamer, the duplex of oligonucleotide was not formed so the peroxidation rate increased. In the first step, three types of CEA aptamer were applied to select the more applicable one. In addition, UV–vis spectroscopy, CD technique and fluorescence spectroscopy were also used for monitoring structural changes. The cost-effective aptasensor enables to detect CEA over a range from 1 to 50 ng ml −1 and the detection limit of CEA was 1 ng ml −1 . The controlled experiments demonstrated that CEA signal was not interfered with other existing proteins. Moreover, it was revealed that the aptasensor could detect CEA in saliva. It is noticeable that the CEA level in saliva increases much more than blood in patients with oral and salivary malignant tumors. [ABSTRACT FROM AUTHOR]

We had previously investigated the role of a loop on the activity and conformational stability of chondroitinase ABC Ι (cABC Ι) by constructing some representative mutants in which a network interaction around Asp 689 was manipulated. Here we extended our study by measuring the proteolytic resistance, long term and thermal stability as well as unfolding kinetics of these variants. Long term stability data at 4 and 25 °C for 3 weeks indicates that all mutants remain considerably active at 4 °C. Thermoinactivation rates for all variants shows that the wild type (WT) enzyme retained 50% of its activity after 2 min keeping at 40 °C, while L701T, H700N and H700N/L701T as conformationally stabilized variants, have slower inactivation rate. It was also found that compact and thermodynamically stabilized variants are more resistant to tryptolytic digestion. Also, kinetic curves of chemical unfolding of the enzyme variants from stopped-flow fluorescence measurements were best fitted into a three-exponential function with three rate constants and corresponding amplitudes. We found that the energy barrier of the fast unfolding phase is lower in stabilized variants; while the amplitude of this phase to the whole amplitude of the unfolding reaction is lower than that of destabilized variants, indicating more population of stabilized mutants unfold via slower unfolding phase. We concluded that the rate of local conformational change alone is not the same that is expected from global thermodynamic stability; however the corresponding amplitude can compensate the rate constant toward thermodynamic stability. [ABSTRACT FROM AUTHOR]

Carbon dots (CDs) are a strong alternative to conventional fluorescent probes for cell imaging due to their brightness, photostability, tunable fluorescence emission, low toxicity, inexpensive preparation, and chemical diversity. Improving the targeting efficiency by modulation of the surface functional groups and understanding the mechanisms of targeted imaging are the most challenging issues in cell imaging by CDs. Firstly, we briefly discuss important features of fluorescent CDs for live-cell imaging application in this review. Then, the newest modulated CDs for targeted live-cell imaging of whole-cell, cell organelles, pH, ions, small molecules, and proteins are elaborately discussed, and their challenges in these fields are explained. [ABSTRACT FROM AUTHOR]

In our previous study, we reported an increase in the thermal stability of bacterial laccase from Bacillus sp. HR03 using site-directed mutagenesis. Three-dimensional model of this enzyme showed a negative patch in the connecting loop between domains 1 and 2. In the present study, the stability of laccase in organic solvents was improved by introducing nonpolar (E188 → A, I, L, and V) and positively charged (E188 → K and R) residues in this region by site-directed mutagenesis. Irreversible thermoinactivation, C50 value (organic solvent concentration at which 50% of enzyme activity remains), change in transition-state stabilization energy, and kinetic parameters of the wild type and its variants were calculated in the presence and absence of various organic solvents (ethanol, methanol, and 1-propanol). All variants showed higher C50 values when compared to the wild type. Nonpolar amino acid substitutions were found to be the most efficient mutants for their remarkable increase in C50 value and a decrease in thermoinactivation rate in the presence of mentioned solvents. Data showed that replacing a negative residue with hydrophobic residues on the surface of a protein could enhance thermoresistance as well as solvent stability. The stability of the resulting enzymes was dependent on the length of the alkyl chain. Results demonstrated that solvent tolerance was positively correlated with thermal stability. [ABSTRACT FROM AUTHOR]

Highlights: [•] In vitro refolding of recombinant bacterial laccase from Bacillus HR03 is studied. [•] We have shown that the drop dilution as a powerful method for refolding of laccase. [•] Differences in kinetic parameters of soluble and refolded enzyme were also observed. [•] Structural changes during the refolding monitored using fluorescence spectroscopy [Copyright &y& Elsevier]

Abstract: Ionic liquids (ILs) have gained increasing attention as solvents in chemical and biotechnological applications. Since alcohol dehydrogenases are of prime interest in industrial field, the present study has been conducted to study the influence of imidazolium based ionic liquids on kinetics, structure and stability of the thermophilic Thermoanaerobacter brockii (TBADH) alcohol dehydrogenase. Our results exhibited that the ionic liquids could affect kinetic parameters and stability, but not the tertiary structure. Through the determination of inhibition profile, which revealed mixed inhibition, Ki (affinity of IL for the enzyme) and K I (affinity of IL for enzyme–substrate complex) values were calculated. Structural analysis using crystallographic data from protein data bank elucidated the structural details responsible for different responses of alcohol dehydrogenases toward ionic liquids. Finally, enhanced stability in [MIm][Cl] was discussed. [Copyright &y& Elsevier]

Abstract: Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are used in many biotechnological processes, including removal of polyphenols in beverages, decolorizing and detoxifying effluents, drug analysis and bioremediation. In the present work, we have tried to increase thermal stability of laccase from Bacillus HR03 using site directed point mutations. Glu188 was substituted with 2 positive (Lys and Arg) and one hydrophobic (Ala) residues. All mutations showed improved thermal stability. Thermal activation of laccase was also increased after introducing the mutations. Remarkably, the Glu188Lys variant showed 3-fold higher thermal activation and higher T 50 (5°C) with respect to the native enzyme. Furthermore steady-state k cat and K m values were influenced despite the distance between the mutated position and the catalytic site. In Glu188Arg mutation, the k cat was improved 3-fold and K m reduced by 25%. Interestingly, all three variants showed higher stability against urea as a chemical denaturant. Structural analyses of the native and mutated variants were carried out using fluorescence and far-UV circular dichroism. [Copyright &y& Elsevier]

Abstract: A thermophilic strain, L14, isolated from Iranian hot springs, produces an extracellular pullulanase upon growth on optimized liquid medium. According to 16S rDNA analysis and biochemical tests, L14 seems to be a Geobacillus stearothermophilus strain. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion-exchange and gel filtration chromatographies with a yield of 30%. The purified enzyme showed a single band on SDS-PAGE with an estimated molecular mass of about 100kDa for monomeric enzyme. Optimal conditions for the activity of the purified pullulanase were calculated as pH 5.5 and T opt =65°C. A reasonable stability was also observed at 60–70°C. The enzyme could hydrolyze pullulan, starch and malto-oligosaccharides and was unable to degrade α-cyclodextrin and panose. The apparent K m and V max for the enzyme activity on pullulan were 0.48g/l and 44μmolmin−1 and for soluble starch were 5g/l and 80μmolmin−1, respectively. The products of enzymatic reaction on pullulan were glucose, maltose and maltotriose, whereas conversion of starch produced glucose and maltose. This enzyme could also hydrolyze different types of malto-oligosaccharides like maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose. Consequently, the enzyme attacked both α-d-(1→4) and α-d-(1→6) glycosidic linkages of pullulan and branched polysaccharides like starch and produced glucose and maltose. Therefore, it is assumed that the newly purified amylopullulanase from Geobacillus sp. L14 is the first report on the presence of a specific debranching enzyme which can produce glucose from pullulan. [Copyright &y& Elsevier]

The crystal structure of Bacillus amyloliquefaciensα-amylase (BAA) at 1.4 Å resolution revealed ambiguities in the thermal adaptation of homologous proteins in this family. The final model of BAA is composed of two molecules in a back-to-back orientation, which is likely to be a consequence of crystal packing. Despite a high degree of identity, comparison of the structure of BAA with those of other liquefying-type α-amylases indicated moderate discrepancies at the secondary-structural level. Moreover, a domain-displacement survey using anisotropic B-factor and domain-motion analyses implied a significant contribution of domain B to the total flexibility of BAA, while visual inspection of the structure superimposed with that of B. licheniformisα-amylase (BLA) indicated higher flexibility of the latter in the central domain A. Therefore, it is suggested that domain B may play an important role in liquefying α-amylases, as its rigidity offers a substantial improvement in thermostability in BLA compared with BAA. [ABSTRACT FROM AUTHOR]

Abstract: The industrial utilization of savinase is mainly constrained by its stability limitations. In the present study, the irreversible thermoinactivation of savinase has been evaluated at 70°C, and various possible mechanisms for irreversible thermoinactivation of savinase were examined. The main process seemed to be autodigestion of savinase at higher temperatures. To improve the thermal stability of the enzyme, the effect of two co-solvents (sorbitol and trehalose) on the enzyme''s activity and stability was investigated. Both osmolytes prevented the autolysis of savinase at 70°C without inactivating the enzyme; furthermore, the structural and kinetic stabilities of the enzyme increased in the presence of additives. [Copyright &y& Elsevier]

Abstract: 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase is an essential enzyme of the shikimate pathway and is the target for the herbicide, glyphosate. Several glyphosate-insensitive forms of Escherichia coli EPSP synthase had been reported in the literatures. In the present study the function and structure of wild type enzyme and three different mutated variants (G96A, A183T and G96A/A183T) were compared. Results showed that G96A and G96A/A183T variants are insensitive to glyphosate but display a 31- and 8-fold lower affinity for phosphoenolpyruvate (PEP) as substrate, respectively. In addition, chemical stability of the enzyme variants against Gdn-HCl revealed more stability of the wild type and G96A variant when compared to the G96A/A183T and A183T variants. Comparison of the enzymes containing Ala183Thr replacement with the wild type showed a lower resistance to digestion by the proteases. Moreover, with respect to fluorescence quenching by acrylamide, A183T and G96A/A183T variants were characterized by a higher structural flexibility and more exposure of tryptophan residues to the solvent. In addition, based on the results of circular dichroism and intrinsic fluorescence studies, these two variants represent a significant decrease of secondary structures and changes in the tertiary structure as compared to the wild type and the G96A variant. [Copyright &y& Elsevier]

The reaction of 2,2-oxydiethanethiol and 2-[2-mercaptoethyl) thio] ethanethiol with dichloroglyoxime (DCGO) in absolute EtOH led to crown compounds, oxadithia (5Z,6Z)-1,4,7-oxadithiadiononane-5,6-dionedioxime (1) and trithia (2Z,3Z)-1,4,7-trithionane-2,3-dionedioxime (2), respectively. The compounds 5,6,8,9-tetrahydro [1,4,7]oxadithionine[5,6-c][1,2,5]oxadiazole (3) and 5,6,8,9-tetrahydro[1,4,7]trithionino[2,3-c][1,2,5]oxadiazole (4) were prepared by dehydration of 1 and 2 in aqueous solution of potassium hydroxide at 170-180°C, respectively. [ABSTRACT FROM AUTHOR]

Abstract: Bacillus licheniformis α-amylase (BLA) is an industrially important extracellular enzyme with a number of applications. In the present work, an investigation was carried out on the tryptolytic digestion of BLA which produced two fragments, TF18K and TF38K, and no further fragments could be seen after 6h incubation of BLA with trypsin. The fragments were isolated by preparative gel electrophoresis and reverse phase HPLC. The N-terminal sequencing of fragments showed that trypsin attacks on Arg127–Val128 peptide bond in BLA. Intrinsic and acrylamide quenching fluorescence experiments and Far-UV circular dichroism studies showed that substantial changes in the secondary and tertiary structures of the TF18K and TF38K have occurred. Subsequently, polyclonal antibody was raised against TF18K. After purification of the antibody by protein A Sepharose, thermal stability of BLA in the presence of this antibody was determined. Results showed that the presence of antiTF18K leads to significant stabilization of BLA. For example, after 30min incubation at 90°C, residual activity of the enzyme in the presence of antibody (40μg/ml) was determined as 40% while the enzyme showed no activity in the absence of antibody after incubating in the same condition. In addition, it has been proved that calcium enhances the thermal stability of BLA and a synergistic stabilization of BLA has been seen with antiTF18K and calcium, simultaneously. [Copyright &y& Elsevier]

Abstract: The effect of organic solvents (n-propanol, isopropanol, dimethylformamide and dimethylsulfoxide) on the structure, activity and stability of thermolysin was the focus of this investigation. Results show the ability of the solvents to cause mixed inhibition of thermolysin, which was indicated by kinetic and structural studies (near-UV CD spectra and intrinsic fluorescence). Inhibitory effect of the solvents increased with increments in solvents log P. Thermoinactivation of thermolysin was studied at 80°C in 50% of solvents and showed that with the increase in solvent hydrophobicity, thermal stability of the enzyme decreased. For the stabilization of thermolysin at high temperature, additives such as glycerol, sorbitol and trehalose were employed. In the presence of DMF with a relatively low log P, trehalose was shown to be a good stabilizer, whereas glycerol had a marked stabilization effect in the presence of n-propanol and isopropanol with a relatively high log P. Consequently, it was concluded that the stabilizing effect of additives can be correlated with the log P of solvents. [Copyright &y& Elsevier]

Abstract: Structural properties of modified forms of glucose oxidase made by stepwise specific modification have been investigated. By a single step modification, one of the modified forms resulted in the conversion of native structure of glucose oxidase to molten globule like form [S. Hosseinkhani, B. Ranjbar, H. Naderi-Manesh, M. Nemat-Gorgani, FEBS Lett. 561 (2004) 213–216]. Chemical modification of lysine residues in glucose oxidase was carried out using different concentration of citraconic anhydride. Modification brought about changes in the tertiary structure with some degree of alteration in secondary structure. FTIR, far and near-UV CD spectropolarimetry, intrinsic and extrinsic fluorescence spectroscopy showed structural changes of glucose oxidase in a concentration dependent manner. This was supported by comparative study of secondary and tertiary structure. [Copyright &y& Elsevier]

Abstract: Interaction of N,N′,N″,N‴-tetramethyltetra-2,3-pyridinoporphyrazinatocopper(II), ([Cu(2,3-tmtppa)]4+) and N,N′,N″,N‴-tetramethyltetra-3,4-pyridinoporphyrazinatocopper(II), ([Cu(3,4-tmtppa)]4+) with calf thymus DNA was studied in 1mM phosphate buffer and low ionic strength (5mM NaCl) at various temperatures by UV-visible and circular dichroism (CD) spectroscopies and viscometric method. The binding constants were determined from the changes in the visible part of porphyrazine complexes spectra using SQUAD software. The values of K have been obtained (7.9±0.4)×104 and (2.2±0.1)×105 M−1 for [Cu(2,3-tmtppa)]4+ and [Cu(3,4-tmtppa)]4+, respectively at 27°C. The higher affinity of 3,4-isomer of Cu complex towards DNA with respect to the 2,3-isomer was attributed to favorable external positioning of the cationic charges in former, which enables superior interaction with the DNA duplex. The thermodynamic parameters (ΔG°, ΔH°, ΔS°) were calculated by van''t Hoff equation. The enthalpy and entropy changes were determined, +34.2±3.6kJmol−1 and +207.8±12.70Jmol−1 K−1 for [Cu(2,3-tmtppa)]4+ and +49.7±2.1kJmol−1 and +267.8±7.9Jmol−1 K−1 for [Cu(3,4-tmtppa)]4+. The existence of extensive hypochromicity, large red shift and negative CD in the visible part of [Cu(3,4-tmtppa)]4+ spectra suggested an intercalation binding mode. Analysis of the moderate hypochromicity, red shift and bisignate CD in the Q-band absorption region of [Cu(2,3-tmtppa)]4+ spectra possibly led us to the coexistence of intercalation and outside binding modes. The influence of the ionic strength on the binding parameters and binding modes was investigated. The results show that increase in ionic strength causes the decrease in the binding constants. It was also concluded that increase in ionic strength affects the binding characteristics of positively charged complexes with DNA. The increase in DNA viscosity in the presence of Cu–tmtppa complexes is attributed to the lengthening of DNA helix due to the intercalation. This result is consistent with conclusions obtained from the spectroscopic techniques. [Copyright &y& Elsevier]

Abstract: A comparative structure–function study was performed to establish possible roles of carbohydrates in stabilization of glycoproteins, using glucoamylase (GA) as a model system. In addition to kinetic properties, stability toward elevated temperatures, extremes of pH, high salt concentrations together with circular dichroism, intrinsic/extrinsic fluorescence studies, proteolysis and affinity for interaction with hydrophobic ligands were investigated. Related to all the main properties examined, with one exception, glycosylation provided improvement in functional characteristics of the enzyme, especially in relation to its thermostability. Results are explained in terms of provision of stabilizing intermolecular interactions by the sugar molecules. The improvement in protein rigidity together with reduction of surface hydrophobicity appear to be especially important in relation to prevention of aggregation, an important mechanism of irreversible thermoinactivation, occurring at elevated temperatures. [Copyright &y& Elsevier]

Detailed circular dichroism (CD), scattering and quenching studies, 1-anilinonaphthalene-8-sulfonate (ANS) binding, irreversible thermoinactivation, activity measurements and proteolytic digestion of bacterial α-amylases have been carried out to elucidate the effect of trifluoroethanol (TFE) on the structure of these enzymes. Under high concentrations of TFE both of the α-amylases, a thermostable α-amylase from Bacillus licheniformis (BLA) and its mesophilic counterpart from Bacillus amyloliquefaciens (BAA), acquire partially folded state characterized by an enhanced content of the secondary structure (helix) and reduced tertiary structures. According to ANS binding studies, we suggest that the TFE states induced by TFE/water mixture are not the molten globule state in the α-amylase folding pathway. In addition, data shows significant reversible aggregation of both enzymes in TFE/water mixtures with concentration between 10 and 60% (v/v). However, reversibility is more in case of BAA. As expected, in the absence of TFE, the thermophilic enzyme compared to mesophilic enzyme, shows a greater resistance to digestion by thermolysin. With respect to fluorescence quenching by acrylamide and potassium iodide, the thermophilic enzyme, BLA, is characterized by higher structural flexibility as compared to the BAA. On the other hand, in the presence of TFE, the enzymes are digested by protease to produce large protein fragments. It is proposed that highly helical secondary structures, acquired by BAA and BLA when dissolved in aqueous TFE, prevent binding and adaptation of the protein substrate at the active site of the protease. [Copyright &y& Elsevier]

Considering importance and several industrial applications of lysozyme, including natural antibiotic and preservative, identifier for the diagnosis of diseases, and extraction purposes, its reversibility and stability studies can be very important. In this paper, the role that buffer and osmolytes concentrations play on the thermodynamic stability of lysozyme denaturation process, that is a new simple and inexpensive method, was evaluated by Nano-DSC III, far- and near-UV CD and fluorescence techniques. In thermal denaturation study, RI and ΔG of protein increased from 25.62% to 58.82% and 48.87 to 63.63 kJ mol−1 with the increment of buffer and osmolytes concentrations, respectively. These changes showed a significant increase of 129.59% in RI and 28.16% in ΔG. The effect of buffer and osmolytes concentrations on the secondary and tertiary structures of protein was also investigated. The results indicated that increment of buffer and osmolytes concentrations increase rigidity and thermodynamic stability of protein. Also, structure of protein may be changed by its interaction with GNPs. Hence, interaction of lysozyme with GNPs was studied at the buffer and osmolytes concentrations that gives the maximum RI and ΔG, respectively. The results showed that molten globule-like state was formed by lysozyme in the presence of GNPs. • The effect of buffer, osmolytes and gold nanoparticles concentrations on lysozyme has been reported. • The role of buffer and osmolytes concentrations in thermodynamic stability has been studied. • Increment of buffer and osmolytes concentrations leads to increase of reversibility index and Gibbs free energy change. • Formation of molten globule-like state in the presence of gold nanoparticles has been investigated. [ABSTRACT FROM AUTHOR]