Green synthesis and investigation of antioxidant and antibacterial activity of new derivatives of chromenoazepines employing CuO/TiO2@MWCNTs.

The synthesis of novel, high-yield derivatives of chromenoazepine was investigated in this work. CuO/TiO2@MWCNTs was used as a nanocatalyst in a multicomponent reaction involving 4-aminocumarine, activated acetylenic chemicals, and alkyl bromide in room temperature water to create these novel compounds. Using MCRs of 4-aminocumarine, isothiocyanate, and alkyl bromide in the presence of CuO/TiO2@MWCNTs as nanocatalysts in room-temperature water, chromenothiazepines were synthesized under comparable conditions. The freshly synthesized azepine exhibits antioxidant activity since its NH group has undergone two evaluation processes. Additionally, using two types of Gram-negative bacteria in a disk distribution procedure, the antibacterial activity of recently developed azepines was evaluated, and these compounds also inhibited the growth of Gram-positive bacteria. This method's benefits include quick reaction times, large product yields, and straightforward catalyst and product separation through easy steps.

Developing a circularly permuted variant of Renilla luciferase as a bioluminescent sensor for measuring Caspase-9 activity in the cell-free and cell-based systems.

Biosensors and whole cell biosensors consisting of biological molecules and living cells can sense a special stimulus on a living system and convert it to a measurable signal. A major group of them are the bioluminescent sensors derived from luciferases. This type of biosensors has a broad application in molecular biology and imaging systems. In this project, a luciferase-based biosensor for detecting and measuring caspase-9 activity is designed and constructed using the circular permutation strategy. The spectroscopic method results reveal changes in the biosensor structure. Additionally, its activity is examined in a cell-free coupled assay system. Afterward, the biosensor is utilized for measuring the cellular caspase-9 activity upon apoptosis induction in a cancer cell line. In following the gene of biosensor is sub-cloned into a eukaryotic vector and transfected to HEK293T cell line and then its activity is measured upon apoptosis induction in the presence and absence of a caspase-9 inhibitor. The obtained results show that the designed biosensor detects the caspase-9 activity in the cell-free and cell-based systems.

Bio-barcode technology for detection of Staphylococcus aureus protein A based on gold and iron nanoparticles.

S. aureus is one of important causes of disease, food poisoning in humans and animals. The generally methods for detection of S. aureus is time consuming. Therefore, a new method is necessary for rapid, sensitive and specific diagnosis of S. aureus. In the present study, two probes and a Bio-barcode DNA were designed for detection of S. aureus (Protein A). Firstly, magnetic nanoparticle (MNPs) and gold nanoparticle (AuNPs) were synthesized at 80 °C and 100 °C, respectively. The AuNPs and the MNPs were functionalized with probe1, Bio-barcode DNA and probe2, respectively. Target DNA was added into the nanomaterial's system containing bio-barcode DNA-AuNPs-probe1 and probe2-MNPs to formed bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex. The bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex was separated with magnetic field. Finally, the bio-barcode DNA was released from surface of complex using DTT (0.8 M) and there was isolated of nanoparticles by magnetic field and centrifuge. The fluorescence intensity of bio-barcode DNA was measured in different concentrations of S. aureus (101 to 108 CFU mL-1) by fluorescence spectrophotometry. The results showed that standard curve was linearly from 102 to 107 CFU mL-1. Limit of detection of bio-barcode assay for both PBS and real samples was 86 CFU mL-1.

Milk caseins as useful vehicle for delivery of dipyridamole drug.

The interaction of bovine milk α- and ß-caseins as an efficient drug carrier system with Dipyridamole (DIP) was investigated using spectroscopy and molecular docking studies at different temperatures (20-37 °C). FTIR, CD, and fluorescence spectroscopy methods demonstrated that α- and ß-caseins interact with DIP molecule mainly via hydrophobic and hydrophilic interactions and change in secondary structure of α- and ß-caseins. DIP showed a higher quenching efficiency and binding constant of α-casein than ß-casein. There was only one binding site for DIP and it was located on the surface of the protein molecule. The thermodynamic parameters of calculation showed that the binding process occurs spontaneously and demonstrated that α- and ß-caseins provide very good binding and entrapment to DIP via hydrogen bonds, Van der Waals forces, and hydrophobic interactions. Fluorescence resonance energy transfer, synchronous fluorescence spectroscopy, and docking study showed that DIP binds to the Trp residues of α- and ß-casein molecules with short distances. Docking study showed that DIP molecule made several hydrogen bonds and van der Waals interactions with α- and ß-caseins. The study of cell culture and micellar solubility of DIP demonstrated α- and ß-caseins relatively the same helping in delivery of DIP. Milk α- and ß-caseins are considered as a useful vehicle for the solublization and stabilization of DIP in aqueous solution at natural pH.

A study of the oxidation-induced conformational and functional changes in neuroserpin.

BACKGROUND: Neuroserpin, a member of the Serine Proteinase Inhibitor (Serpin) superfamily, is known to be a neuroprotective factor in the focal ischemic stroke followed by reducing the microglial activation. Neuroserpin is a protein rich of methionine residues that can scavenge the free radical species which may increase its neuroprotective effect. On the other hand, the oxidative modifications of the amino acid residues in neuroserpin may lead to changes in its conformation and function. In this study, it was investigated the changes in the conformation and the function of the oxidized neuroserpin. METHODS: Neuroserpin expressed in E. coli, BL21 or M15 harboring plasmid pQE81L containing neuroserpin cDNA. Expressed neuroserpin was purified by resin sulfopropyl A50 precharged with 0.1 M NiSO4 under denaturing condition. Neuroserpin was oxidized under oxidative stress condition in the presence of different concentration of hydrogen peroxide. The oxidation of neuroserpin was conveniently detected by a carbonyl content assay using 2, 4 dinitrophenylhydrazine. Changes in tertiary structure of neuroserpin were monitored by spectrofluorimeter to study the alteration of intrinsic fluorescence and also fluorescence of 8-anilinonaphthalin-1 sulfonic acid (ANS) in native and oxidized form of neuroserpin. RESULTS: Total expressed neuroserpin was estimated 4-5 mg/lit in 2XYT culture media. SDS-PAGE analysis of purified neuroserpin showed a single band which reflects the efficiency of the resin SP A50 for purification of the proteins containing 6xHis tag. Carbonyl content of oxidized and native neuroserpin was estimated 12.3 +/- 0.3 and 0.45 +/- 0.05, respectively. The inhibitory activity of oxidized neuroserpin decreased up to 40-60% as compared with native form of neuroserpin. Intrinsic fluorescence and also the emission of ANS bind to the hydrophobic region of the protein altered from 380 to 85 and in the case of ANS from 105 to 150 in oxidized and native form of neuroserpin, respectively. CONCLUSION: The decreased intrinsic fluorescence intensity, an enhancement in the fluorescence of ANS, and loss of the inhibitory activity up to 40-60% in neuroserpin, all suggested a conformational modification in the protein under the oxidative stress condition. Remaining the inhibitory activity of neuroserpin reflects that the protein tolerates the oxidative stress condition effectively.

Intermolecular interaction of nickel (ii) phthalocyanine tetrasulfonic acid tetrasodium salt with bovine serum albumin: A multi-technique study.

The interaction of nickel (II) phthalocyanine tetrasulfonic acid tetrasodium salt with bovine serum albumin (BSA) has been investigated by combination of fluorescence, UV-vis absorption, Fourier transform infrared (FT-IR), and circular dichorism (CD) spectroscopies as well as through molecular docking. Fluorescence quenching and absorption spectra were investigated as a mean for estimating the binding parameters. Analysis of fluorescence quenching data at different temperatures was performed in order to specify the thermodynamics parameters for interactions of phthalocyanine complex with BSA. According to experimental data it was suggested that phthalocyanine had a significant binding affinity to BSA and the process was entropy driven. Based on the results of molecular docking it was indicated that the main active binding site for this phthalocyanine complex is site I in subdomain IIA of BSA. The results provide useful information for understanding the binding mechanism of anticancer drug-albumin and gives insight into the biological activity and metabolism of the drug in blood.

Spectroscopy and Molecular Modeling Study on Binding of Nickel Phthalocyanine to Human Serum Albumin.

The interaction of nickel tetra sulfunated phthalocyanine( NiTSPc) with human serum albumin (HSA), in 20 mM phosphate buffer pH 7.4 was investigated using advanced techniques including fluorescence, synchronous fluorescence, Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopy and molecular docking. The fluorescence quenching measurements showed a single binding site on HSA for NiTSPc with the binding constant (K_b) value equals to 1.26×106 at 25°C. The results showed that quenching mechanism of HSA by NiTSPc was of dynamic type. The results from FTIR and CD spectroscopies demonstrated that NiTSPc binds to amino acid residues of the main polypeptide chain in protein destroying the hydrogen bonding network. The corresponding thermodynamic parameters were then calculated by analysis of fluorescence data using van't Hoff plot. These data indicated that driving force for interaction was mainly hydrophobic in nature and the process was entropy driven. The information obtained from CD, FT-IR and synchronous fluorescence spectroscopies revealed that both microenvironment and conformation of HSA was changed. Molecular docking study confirmed the binding mode obtained by experimental data.

Interactions Between Sirolimus and Anti-Inflammatory Drugs: Competitive Binding for Human Serum Albumin.

PURPOSE: The aim of the present study was investigating the effects of three anti-inflammatory drugs, on Sirolimus protein biding. The binding site of Sirolimus on human serum albumin (HSA) was also determined. METHODS: Six different concentrations of Sirolimus were separately exposed to HSA at pH 7.4 and 37°C. Ultrafiltration method was used for separating free drug; then free drug concentrations were measured by HPLC. Finally, Sirolimus protein binding parameters was calculated using Scatchard plots. The same processes were conducted in the presence of NSAIDs at lower concentration of albumin and different pH conditions. To characterize the binding site of Sirolimus on albumin, the free concentration of warfarin sodium and Diazepam, site I and II specific probes, bound to albumin were measured upon the addition of increasing Sirolimus concentrations. RESULTS: Based on the obtained results presence of Diclofenac, Piroxicam and Naproxen, could significantly decrease the percentage of Sirolimus protein binding. The Binding reduction was the most in the presence of Piroxicam. Sirolimus-NSAIDs interactions were increased in higher pH values and also in lower albumin concentrations. Probe displacement study showed that Sirolimus may mainly bind to site I on albumin molecule. CONCLUSION: More considerations in co-administration of NSAIDs and Sirolimus is recommended.

Identification of a molten globule like state in HC-N fragment of botulinum neurotoxin A: shedding light on the poorly-known features of a conserved sub-domain.

C-terminal fragment of the Botulinum neurotoxin A comprises two sub-domains including H(C)-N and H(C)-C. Here, the conformational change of H(C)-N was studied by spectroscopic techniques. The results indicated that the partially unfolded state forms during unfolding of H(C)-N. This finding may shed light on poorly--known features of the protein.