Treatment of Bacillus licheniformis α-amylase (BLA) with guanidine hydrochloride (GdnHCl) produced both denatured and aggregated forms of the enzyme as studied by circular dichroism, fluorescence, UV difference spectroscopy, size exclusion chromatography (SEC), and enzymatic activity. The presence of CaCl(2) in the incubation mixture produced significant recovery in spectral signals, being complete in presence of 10 mM CaCl(2), as well as in enzymatic activity, which is indicative of protein stabilization. However, the SEC results obtained with GdnHCl-denatured BLA both in the absence and the presence of 10 mM CaCl(2) suggested significant aggregation of the protein in the absence of CaCl(2) and disaggregation in its presence. Although partial structural stabilization with significant retention of enzymatic activity was observed in the presence of calcium, it was far from the native state, as reflected by spectral probes. Hence, spectral results as to BLA stabilization should be treated with caution in the presence of aggregation.
A HPLC method was validated for quantification of (+)-calanolide A (1), a novel anti-HIV agent, in rat, dog and human plasma. The synthetic intermediate (+/-)-12-oxocalanolide A (2) was found to be a suitable internal standard. Compounds were extracted from plasma using a solid-phase C(18) cartridge and quantified over the assay range of 12.5 to 800 ng/ml. The method was utilized to determine (+)-calanolide A pharmacokinetics in rats, dogs and humans. This is the first report of a validated HPLC assay for determination of (+)-calanolide A concentrations in rat and dog plasma as well as human plasma obtained from clinical trials. There was no evidence of in vivo epimerization of (+)-calanolide A to its inactive epimer (+)-calanolide B (3).
miRNA (miR)-155 is a potential biomarker for breast cancers. We aimed at developing a nanosensor for miR-155 detection by integrating hybridization chain reaction (HCR) and silver nanoclusters (AgNCs). HCR serves as an enzyme-free and isothermal amplification method, whereas AgNCs provide a built-in fluorogenic detection probe that could simplify the downstream analysis. The two components were integrated by adding a nucleation sequence of AgNCs to the hairpin of HCR. The working principle was based on the influence of microenvironment towards the hosted AgNCs, whereby unfolding of hairpin upon HCR has manipulated the distance between the hosted AgNCs and cytosine-rich toehold region of hairpin. As such, the dominant emission of AgNCs changed from red to yellow in the absence and presence of miR-155, enabling a ratiometric measurement of miR with high sensitivity. The limit of detection (LOD) of our HCR-AgNCs nanosensor is 1.13 fM in buffered solution. We have also tested the assay in diluted serum samples, with comparable LOD of 1.58 fM obtained. This shows the great promise of our HCR-AgNCs nanosensor for clinical application.
Interaction of an anticancer drug, vandetanib (VDB) with a ligand transporter, lysozyme (LYZ) was explored using multispectroscopic techniques, such as fluorescence, absorption and circular dichroism along with computational analysis. Fluorescence data and absorption results confirmed VDB-LYZ complexation. VDB-induced quenching was characterized as static quenching based on inverse correlation of KSV with temperature as well as kq values. The complex was characterized by the weak binding constant (Ka=4.96-3.14×103M-1). Thermodynamic data (ΔS=+12.82Jmol-1K-1; ΔH=-16.73kJmol-1) of VDB-LYZ interaction revealed participation of hydrophobic and van der Waals forces along with hydrogen bonds in VDB-LYZ complexation. Microenvironmental perturbations around tryptophan and tyrosine residues as well as secondary and tertiary structural alterations in LYZ upon addition of VDB were evident from the 3-D fluorescence, far- and near-UV CD spectral analyses, respectively. Interestingly, addition of VDB to LYZ significantly increased protein's thermostability. Molecular docking results suggested the location of VDB binding site near the LYZ active site while molecular dynamics simulation results suggested stability of VDB-LYZ complex. Presence of Mg2+, Ba2+ and Zn2+ was found to interfere with VDB-LYZ interaction.
Two nickel(II) complexes with formula NiL1 and NiL2 (HL1 = S-allyl-4-methoxybenzylidene hydrazinecarbodithioate, HL2 = S-allyl-1-napthylidenehydrazinecarbodithioate) have been synthesized and characterized by elemental analysis, FT-IR, NMR, UV-vis spectroscopy and ESI mass spectrometry. The crystal structure of complex 1 has been determined by single crystal X-ray diffractometry. Both HL1 and HL2 ligands are coordinated to the metal in thiolate form. In complexes, squareplanar geometry of the nickel is coordinated with two bidentate ligand units acting through azomethine nitrogen and thiolato sulfur atoms. To explore the potential medicinal value of the complexes with calf thymus DNA and bovine serum albumin (BSA) were studied at normal physiological conditions using fluorescence spectral techniques. The DNA binding constant values of the complexes were found in the range from 5.02 × 10(4), 3.54 × 10(4), and the binding affinities are in the following order 1 > 2. In addition, nickel complexes 1 and 2 shows better binding propensity to the bovine serum albumin (BSA) protein, giving a Ksv value 5.8 × 10(4), 4.47 × 10(4) respectively. From the oxidative cleavage of the complexes with pBR322 DNA, it is inferred that the effects of cleavage are dose-dependent. In addition, in vitro cytotoxicity of the complexes assayed against Vero and HeLa cell lines have shown higher cytotoxic activity with the lower IC50 values indicating their efficiency in killing cancer cells even at various concentrations.
New derivatives of thiosemicarbazone Schiff base with isatin moiety were synthesized L1-L6. The structures of these compounds were characterized based on the spectroscopic techniques. Compound L6 was further characterized by XRD single crystal. The interaction of these compounds with calf thymus (CT-DNA) exhibited high intrinsic binding constant (k(b)=5.03-33.00×10(5) M(-1)) for L1-L3 and L5 and (6.14-9.47×10(4) M(-1)) for L4 and L6 which reflect intercalative activity of these compounds toward CT-DNA. This result was also confirmed by the viscosity data. The electrophoresis studies reveal the higher cleavage activity of L1-L3 than L4-L6. The in vitro anti-proliferative activity of these compounds against human colon cancer cell line (HCT 116) revealed that the synthesized compounds (L3, L6 and L2) exhibited good anticancer potency.
Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB-HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92-6.89 × 10(3 )M(-1) at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB-HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive ΔS (52.76 J mol(-1) K(-1)) and negative ΔH (-6.57 kJ mol(-1)) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB-HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow's site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca(2+), Zn(2+), Cu(2+), Ba(2+), Mg(2+), and Mn(2+) in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA.
The study and applications of in vivo skin optics have been openly documented as early as the year 1954, or possibly earlier. To date, challenges in analyzing the complexities of this field remain, with wide scopes requiring more scrutiny. Recent advances in spectroscopic research and multivariate analytics allow a closer look into applications potentially for detecting or monitoring diseases. One of the challenges in this field is in establishing a reference for applications which correspond to certain bandwidths. This article reviews the scope on past research on skin spectroscopy, and the clinical aspects which have or may have applications on disease detection or enhancing diagnostics. A summary is supplied on the technicalities surrounding the measurements reported in literature, focused towards the wavelength-dependent applications in themes central to the respective research. Analytics on the topology of the papers' data cited in this work is also provided for a statistical perspective. In short, this paper strives to immediately inform the reader with possible applications via the spectroscopic devices at hand. Graphical Abstract .
Abraxane, an albumin-bound paclitaxel nanoparticle formulation, is superior to conventional paclitaxel preparations because it has better efficacy against unresectable pancreatic cancer. Previous reports suggest that this better efficacy of Abraxane than conventional paclitaxel preparation is probably due to its transport through Gp60, an albumin receptor on the surface of vascular endothelial cells. The increased tumor accumulation of Abraxane is also caused by the secreted protein acid and rich in cysteine in the tumor stroma. However, the uptake mechanism of Abraxane remains poorly understood. In this study, we demonstrated that the delivery of Abraxane occurred via different receptor pathways from that of endogenous albumin. Our results showed that the uptake of endogenous albumin was inhibited by a Gp60 pathway inhibitor in the process of endocytosis through endothelial cells or tumor cells. In contrast, the uptake of Abraxane-derived HSA was less affected by the Gp60 pathway inhibitor but significantly reduced by denatured albumin receptor inhibitors. In conclusion, these data indicate that Abraxane-derived HSA was taken up into endothelial cells or tumor cells by a mechanism different from normal endogenous albumin. These new data on distinct cellular transport pathways of denatured albumin via gp family proteins different from those of innate albumin shed light on the mechanisms of tumor delivery and antitumor activity of Abraxane and provide new scientific rationale for the development of a novel albumin drug delivery strategy via a denatured albumin receptor.
Furopyridine III, namely 1-(3-amino-4-(4-(tert-butyl)phenyl)-6-(p-tolyl)furo[2,3-b]pyridin-2-yl)ethan-1-one, synthesized from 4-(4-(tert-butyl)phenyl)-2-oxo-6-(p-tolyl)-1,2-dihydropyridine-3-carbonitrile I in two steps. The title compound is characterized by NMR, MS and its X-ray structure. The molecular structure consists of planar furopyridine ring with both phenyl rings being inclined from the furopyridine scaffold to a significant different extent. There are three intramolecular hydrogen bonds within the structure. The lattice is stabilized by N-H…O, H2C-H …π and π…π intermolecular interactions leading to three-dimensional network. Compound III exhibits fluorescent properties, which are investigated. Antimicrobial potential and antioxidant activity screening studies for the title compound III and the heterocyclic derivatives, I and II, show no activity towards neither bacterial nor fungal strains, while they exhibited weak to moderate antioxidant activity compared to reference.
In this study, we synthesized a multifunctional nanoparticulate system with specific targeting, imaging, and drug delivering functionalities by following a three-step protocol that operates at room temperature and solely in aqueous media. The synthesis involves the encapsulation of luminescent Mn:ZnS quantum dots (QDs) with chitosan not only as a stabilizer in biological environment, but also to further provide active binding sites for the conjugation of other biomolecules. Folic acid was incorporated as targeting agent for the specific targeting of the nanocarrier toward the cells overexpressing folate receptors. Thus, the formed composite emits orange-red fluorescence around 600 nm and investigated to the highest intensity at Mn(2+) doping concentration of 15 at.% and relatively more stable at low acidic and low alkaline pH levels. The structural characteristics and optical properties were thoroughly analyzed by using Fourier transform infrared, X-ray diffraction, dynamic light scattering, ultraviolet-visible, and fluorescence spectroscopy. Further characterization was conducted using thermogravimetric analysis, high-resolution transmission electron microscopy, field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray fluorescence, and X-ray photoelectron spectroscopy. The cell viability and proliferation studies by means of MTT assay have demonstrated that the as-synthesized composites do not exhibit any toxicity toward the human breast cell line MCF-10 (noncancer) and the breast cancer cell lines (MCF-7 and MDA-MB-231) up to a 500 µg/mL concentration. The cellular uptake of the nanocomposites was assayed by confocal laser scanning microscope by taking advantage of the conjugated Mn:ZnS QDs as fluorescence makers. The result showed that the functionalization of the chitosan-encapsulated QDs with folic acid enhanced the internalization and binding affinity of the nanocarrier toward folate receptor-overexpressed cells. Therefore, we hypothesized that due to the nontoxic nature of the composite, the as-synthesized nanoparticulate system can be used as a promising candidate for theranostic applications, especially for a simultaneous targeted drug delivery and cellular imaging.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive target for cancer therapy due to its ability to selectively induce apoptosis in cancer cells, without causing significant toxicity in normal tissues. We previously reported that galactoxyloglucan (PST001) possesses significant antitumor and immunomodulatory properties. However, the exact mechanism in mediating this anticancer effect is unknown. This study, for the first time, indicated that PST001 sensitizes non-small cell lung cancer (A549) and nasopharyngeal (KB) cells to TRAIL-mediated apoptosis. In vitro studies suggested that PST001 induced apoptosis primarily via death receptors and predominantly activated caspases belonging to the extrinsic apoptotic cascade. Microarray profiling of PST001 treated A549 and KB cells showed the suppression of survivin (BIRC5) and anti-apoptotic Bcl-2, as well as increased cytochrome C. TaqMan low density array analysis of A549 cells also confirmed that the induction of apoptosis by the polysaccharide occurred through the TRAIL-DR4/DR5 pathways. This was finally confirmed by in silico analysis, which revealed that PST001 binds to TRAIL-DR4/DR5 complexes more strongly than TNF and Fas ligand-receptor complexes. In summary, our results suggest the potential of PST001 to be developed as an anticancer agent that not only preserves innate biological activity of TRAIL, but also sensitizes cancer cells to TRAIL-mediated apoptosis.