Determination of the location of fluorescent probes attached to fatty acids using parallax analysis of fluorescence quenching: effect of carboxyl ionization state and environment on depth.

In this report, parallax analysis of fluorescence quenching (see the preceding paper in this issue) was used to determine the location (depth) of anthroyloxy and carbazole probes attached to model membrane inserted fatty acids. A monotonic increase in depth was found as the number of carbon atoms between the attachment site of the probe and the fatty acyl carboxyl group is increased. It was also found that depth is sensitive to pH, with an increase in probe depth upon protonation of the fatty acid carboxyl group of around 0.5-2.5 A, depending on probe location and identity. This result shows that carboxyl protonation causes an increase in depth all along a fatty acid chain. In addition, it indicates that parallax analysis is very sensitive to small changes in depth. At a given pH, no significant change in probe depth was observed in vesicles containing anionic phospholipid or at various ionic strengths, suggesting these parameters do not strongly regulate fatty acyl chain location. It was also found that there is a decrease of the apparent depth of each of the fatty acyl attached probes both at longer excitation wavelengths and at longer emission wavelengths. This is consistent with there being a distribution of depth for each fluorophore, with shallower fluorophore dominating the fluorescence at red-shifted wavelengths. Solvent relaxation effects also appear to contribute to this wavelength dependence.     

Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol.     

Hyaluronic acid engrafted metformin loaded graphene oxide nanoparticle as CD44 targeted anti-cancer therapy for triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) is the most aggressive form of breast cancer with limited treatment modalities. It is associated with high propensity of cancer recurrence.MethodsUV Spectroscopy, FTIR, DLS, Zeta potential, TEM and SEM were employed to characterize nanoparticles. MTT assay, Wound healing assay, SEM, Immunocytochemistry analysis, Western blot, RT-PCR, mammosphere formation assay were employed to study apoptosis, cell migration and stemness. Tumor regression was studied in chick embryo xenograft and BALB/c mice model.ResultsHylaluronic acid engrafted metformin loaded graphene oxide (HA-GO-Met) nanoparticles exhibited an anti-cancer efficacy at much lower dosage as compared to metformin alone. HA-GO-Met nanoparticles induced apoptosis and inhibited cell migration of TNBC cells by targeting miR-10b/PTEN axis via NFkB-p65. Upregulation of PTEN affected pAKT(473) expression that induced apoptosis. Cell migration was inhibited by reduction of pFAK/integrinβ1 expressions. Treatment inhibited epithelial mesenchymal transition (EMT) and reduced stemness as evident from the increase in E-cadherin expression, inhibition of mammosphere formation and low expression levels of stemness markers including nanog, oct4 and sox2 as compared to control. Moreover, tumor regression was studied in chick embryo xenograft and BALB/c mice model. HA-GO-Met nanoparticle treatment reduced tumor load and nullified toxicity in peripheral organs imparted by tumor.ConclusionsHA-GO-Met nanoparticles exhibited an enormous anti-cancer efficacy in TNBC in vitro and in vivo.General significanceHA-GO-Met nanoparticles induced apoptosis and attenuated cell migration in TNBC. It nullified overall toxicity imparted by tumor load. It inhibited EMT and reduced stemness and thereby addressed the issue of cancer recurrence.     

Formation of a regular dissipative structure: a bifurcation and non-linear analysis.

A non-linear reaction diffusion model of a negative feedback epigenetic control system is presented. The model involves synthesis of the mitotic inducing and inhibiting proteins, simultaneously with intercellular self-diffusion and cross-diffusion of the latter only. The importance of negative cross-diffusion for creating a regular dissipative structure is shown. A bifurcation analysis of the non-linear diffusive system has been performed and it is concluded that bifurcation is supercritical. Lastly, using Liapunov's direct method, it is shown that the pattern evolved by the system is globally asymptotically stable.     

Solubilization of high affinity G-protein-coupled serotonin 1A receptors from bovine hippocampus using pre-micellar CHAPS at low concentration

The serotonin 1A (5-HT 1A ) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioural and cognitive functions. This paper reports an efficient strategy to solubilize 5-HT 1A receptors from bovine hippocampal membranes using the zwitterionic detergent CHAPS which is mild and non-denaturing. Since high concentration of CHAPS has earlier been shown to induce dissociation and depletion of G-protein sub-units, a low (pre-micellar) concentration of CHAPS was used for solubilizing 5-HT 1A receptors in the presence of NaCl followed by PEG precipitation. This results in solubilization of 5-HT 1A receptors with a high degree of efficiency and gives rise to high affinity, functionally active G-protein-sensitive solubilized receptors. Optimal solubilization of the receptor from the native source with high ligand binding affinity and intact signal transduction components may constitute the first step in the molecular characterization of the 5-HT 1A receptor in particular, and G-protein-coupled receptors in general.     

Cytokinin-like active principle associated with rice necrosis mosaic virus induced growth promotion in jute (Corchorus olitorius L.)

Rice necrosis mosaic virus-inoculated jute plants looked more juvenile with enhanced leaf size than control ones. Such enlarged leaves and those from control ones were collected separately and extracted in chilled methanol. The crude tissue extract in case of inoculated ones showed three times higher activity in bioassay for cytokinin over control. Biological assay with fractions of crude extract separated by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) was ascertained, and one fraction from inoculated sample showing higher activity in terms of growth over respective control was selected. Ultraviolet/visible absorbance spectra of TLC-separated fractions revealed the presence of different quantities of similar kind of compounds in both control and inoculated samples. A major compound with same m/z value of 217.1 was found in both cases as revealed through electrospray ionisation mass spectrophotometry analysis along with some other minor compounds. Different proton–proton coupling and a molecular structure of the highly substituted aliphatic groups were found in the material from inoculated sample having higher cytokinin-like activity than the control one, which was noticed through ¹H one-dimensional nuclear magnetic resonance analysis.     

Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

With advancements in high‐throughput generation of phenotypic data on mutant proteins, it has become important to individually characterize different proteins or their variants rapidly and with minimal sample consumption. We have made use of a nano differential scanning fluorimetric device, from NanoTemper technologies, to rapidly carry out isothermal chemical denaturation and measure folding/unfolding kinetics of proteins and compared these to corresponding data obtained from conventional spectrofluorimetry. We show that using sample volumes 10‐50‐fold lower than with conventional fluorimetric techniques, one can rapidly and accurately measure thermodynamic and kinetic stability, as well as folding/unfolding kinetics. This method also facilitates characterization of proteins that are difficult to express and purify.