2DE‐based approach for estimation of number of protein species in a cell

Insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. But to go further, we need at least to know the proteome size, or how many different protein species compose this proteome. This is the task that could be at least partially realized by the method described in this article. The approach used in our study is based on detection of protein spots in 2DE after staining by protein dyes with various sensitivities. As the different protein spots contain different protein species, counting the spots opens a way for estimation of number of protein species. The function representing the dependence of the number of protein spots on sensitivity or LOD of protein dyes was generated. And extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) allowed to counting the number of different molecules (polypeptide species) at the concentration level of a single polypeptide per proteome. Using this approach, it was estimated that the minimal numbers of protein species for model objects, Escherichia coli and Pirococcus furiosus, are 6200 and 3400, respectively. We expect a single human cell (HepG2) to contain minimum 70 000 protein species.     

Selective fluorescence and naked eye detection of histidine in aqueous medium via hydrogen bonding assisted Schiff base condensation

Visible light excitable rhodamine B derivative (TARDHD) has been developed for fluorescence and naked eye detection of histidine in aqueous medium. TARDHD shows 45 fold fluorescence enhancement in the presence of histidine. It forms Schiff base with histidine and stabilizes via intra-molecular H-bonding. TARDHD can efficiently detect intracellular histidine.     

Cathode catalysts degradation mechanism from liquid electrolyte to membrane electrode assembly

Single-cell and half-cell degradation test procedures were evaluated for carbon-supported Pt/C, PtCo/C and PtNi/C catalysts. Half-cell analyses were employed to understand the effect of the number of cycles and of the scan rate over the cathode catalysts degradation under potential cycling from 0.6 to 1.2 V. The data suggested a time-dependent degradation for all three catalytic systems. Single-cell measurements were used to evaluate the impact of catalyst degradation on fuel cell performance. The measurements in both setups showed similar ECSA and ORR mass activity losses. Specific degradation mechanisms related to Pt dissolution, Pt agglomeration, and transitional metal leaching were quantified and correlated with performance losses.     

Analysis of receptor-ligand binding by photoaffinity cross-linking

Photoaffinity cross-linking is a fast developing technology for biomolecular interactions,including receptor-ligand binding.The chemical mechanisms of the most commonly used photoactivatable probes and their respective photochemistry are summarized.This review focuses on the expanding utilities of this technology as a result of recent advances in the(i)identification of receptor contact sites,(ii)monitoring ligand-induced receptor conformational changes,(iii)identification of global binding surfaces,(iv)binding mode analysis using bifunctional photo-probes,(v)application of biosynthetic photo-probes,and(vi)examples of novel target discovery using this technology.Limitations and future potential of this approach are also discussed.     

A flow-through microfluidic chip for continuous dielectrophoretic separation of viable and non-viable human T-cells

Effective methods for rapid sorting of cells according to their viability are critical in T cells based therapies to prevent any risk to patients. In this context, we present a novel microfluidic device that continuously separates viable and non-viable T-cells according to their dielectric properties. A dielectrophoresis (DEP) force is generated by an array of castellated microelectrodes embedded into a microfluidic channel with a single inlet and two outlets; cells subjected to positive DEP forces are drawn toward the electrodes array and leave from the top outlet, those subjected to negative DEP forces are repelled away from the electrodes and leave from the bottom outlet. Computational fluid dynamics is used to predict the device separation efficacy, according to the applied alternative current (AC) frequency, at which the cells move from/to a negative/positive DEP region and the ionic strength of the suspension medium. The model is used to support the design of the operational conditions, confirming a separation efficiency, in terms of purity, of 96% under an applied AC frequency of 1.5 × 10⁶ Hz and a flow rate of 20 μl/h. This work represents the first example of effective continuous sorting of viable and non-viable human T-cells in a single-inlet microfluidic chip, paving the way for lab-on-a-chip applications at the point of need.     

Photoactivatable V-Shaped Bifunctional Quinone Methide Precursors as a New Class of Selective G-quadruplex Alkylating Agents

Combining the selectivity of G-quadruplex (G4) ligands with the spatial and temporal control of photochemistry is an emerging strategy to elucidate the biological relevance of these structures. In this work, we developed six novel V-shaped G4 ligands that can, upon irradiation, form stable covalent adducts with G4 structures via the reactive intermediate, quinone methide (QM). We thoroughly investigated the photochemical properties of the ligands and their ability to generate QMs. Subsequently, we analyzed their specificity for various topologies of G4 and discovered a preferential binding towards the human telomeric sequence. Finally, we tested the ligand ability to act as photochemical alkylating agents, identifying the covalent adducts with G4 structures. This work introduces a novel molecular tool in the chemical biology toolkit for G4s.     

Brassinin inhibits proliferation and induces cell cycle arrest and apoptosis in nasopharyngeal cancer C666-1 cells

BackgroundNasopharyngeal cancer is a tumor that occurs in the mucous epithelium of the nasopharynx. Due to its rapid growth and early metastatic nature, the successful treatment of nasopharyngeal cancer is highly challenging.ObjectiveHere, we intended to assess the in vitro anticancer property of brassinin against the nasopharyngeal cancer C666-1 cells.MethodologyThe in vitro free radical scavenging property of the brassinin was assessed by various free radical scavenging activities such as FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The cytotoxic level of the brassinin (1–50 µM) against the nasopharyngeal cancer C666-1 cells and normal Vero cells were assessed by the MTT cytotoxicity assay. The levels of TBARS, GSH, and the SOD activity was assessed using kits. The level of ROS generation, MMP, and apoptosis were investigated by the respective fluorescent staining techniques. The flow cytometry analysis was done to scrutinize the cell cycle arrest. The Bax/Bcl-2 level and caspase activities were examined using respective kits.ResultsThe brassinin treatment effectively scavenged the free radicals, which are assessed by the FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The proliferation of brassinin treated C666-1 cells were decreased remarkably, while the same concentration of brassinin did not disturbed the Vero cell viability. The 30 µM of brassinin effectively increased the ROS production, depleted the MMP, and stimulated the apoptosis in the C666-1 cells. The brassinin increased the TBARS and depleted the GSH and SOD in the C666-1 cells. The flow cytometry analysis revealed that the brassinin administration improved the G0/G1 ratio and decreased the proportion of cells with ‘S’ and ‘G2/M’ phase. The Bax, caspase-3 and ?9 were elevated and Bcl-2 level was decreased in the brassinin administered C666-1 cells.ConclusionOur findings discovered that the brassinin has the capacity to prevent the proliferation and stimulate the apoptotic cell death C666‐1 cells via blocking cell cycle and increasing oxidative stress and apoptotic markers. Hence, it can be a talented therapeutic agent to treat the nasopharyngeal cancer in the future.     

Spectrochemical analysis using LIBS and ICP-OES techniques of herbal medicine (Tinnevelly Senna leaves) and its anti-cancerous/antibacterial applications

Tinnevelly senna leaves are being applied to cure many diseases especially in developing countries and sub-Saharan region due to many bioactive compounds such as sennosides, phenols, and flavonoids. The conventional methods to isolate and analyze plant extracts biomolecules are not very effective as well cost effective as they require hazardous chemical solvents and reagents, which are time-consuming processes. The major objective of the present study is to investigate the feasibility of the Laser induced breakdown spectroscopy (LIBS) technique for rapid, eco-friendly, and multi-elemental analysis of Senna leaves extracts and study their antibacterial and anticancer potentials. The elegant LIBS technique was applied as a qualitative and quantitative method for Senna leaves sample’s elemental analysis and their biological activities were measured by evaluating anti-cancer and anti-bacterial analysis. The quantitative analysis of Senna leaves extracts was done using the calibration-free laser-induced breakdown spectroscopy (CF-LIBS) algorithm showing their appreciable content of several nutrient elements, and the obtained results were in close conformity with these achieved by using the standard analytical ICP OES technique. We studied the bactericidal efficacy of the Senna leaves extract against Staphylococcus aureus (S. aureus) by AWD assays and morphogenesis by scanning electron microscopy (SEM) and the anticancer activity was also investigated where different concentrations of Senna leaves extract were tested on cancer cells (HCT-116 and HeLa) and normal cells (HEK-293) using the cell metabolic activity MTT assay and Propidium iodide (PI) staining. We have also calculated the inhibitory concentration (IC50) value for the various extracts concentrations (25 µg/ml, 50 µg/ml, 100 µg/ml, 150 µg/ml, 200 µg/ml, and 225 µg/ml). We have found that IC₅₀ value for HCT-116 cells were 13.5 µg/ml, 17.5 µg/ml, 21.5 µg/ml, 22.5 µg/ml, 26 µg/ml and 33.5 µg/ml and for HeLa cells 15.25 µg/ml, 21.25 µg/ml, 23.5 µg/ml, 262.5 µg/ml, 36.25 µg/ml, and 39.50 µg/ml. The bactericidal efficacy of the Senna leaves extract showed significant inhibition against Gram-positive bacterium. Both MTT and PI analysis showed that Senna leaves extract induced profound inhibition on HCT-116 growth and proliferation. Additionally, Senna leaves extract did not exert an inhibitory influence on normal (HEK-293), which is non-cancerous cells. We suggest that the extract specifically targets the cancerous cells, which could be highly beneficial for the development of future safe anticancer and antibacterial drugs using these extracts.     

Double‐Wall Carbon Nanotube–Porphyrin Supramolecular Hybrid: Synthesis and Photophysical Studies

Double‐wall carbon nanotubes (DWCNTs) with pyridyl units covalently attached to the external wall through isoxazolino linkers and carboxylic groups that have been esterified by pentyl chains are synthesized. The properties of these modified DWCNTs are then compared with an analogous sample based on single‐wall carbon nanotubes (SWCNTs). Raman spectroscopy shows the presence of characteristic radial breathing mode vibrations, confirming that the samples partly retain the integrity of the nanotubes in the case of DWCNTs, including the internal and external nanotubes. Quantification of the pyridyl content for both samples (DWCNT and SWCNT derivatives) is based on X‐ray photoelectron spectroscopy and thermogravimetric profiles, showing very similar substituent load. Both pyridyl‐containing nanotubes (DWCNTs and SWCNTs) form a complex with zinc porphyrin (ZnP), as evidenced by the presence of two isosbestic points in the absorption spectra of the porphyrin upon addition of the pyridyl‐functionalized nanotubes. Supramolecular complexes based on pyridyl‐substituted DWCNTs and SWCNTs quench the emission and the triplet excited state identically, through an energy‐transfer mechanism based on pre‐assembly of the ground state. Thus, the presence of the intact inner wall in DWCNTs does not influence the quenching behavior, with respect to SWCNTs, for energy‐transfer quenching with excited ZnP. These results sharply contrast with previous ones referring to electron‐transfer quenching, in which the double‐wall morphology of the nanotubes has been shown to considerably reduce the lifetime of charge separation, owing to faster electron mobility in DWCNTs compared to SWCNTs.