The elimination of infected or cancerous cells by CD8+ cytotoxic T lymphocytes (CTL) is a crucial effector mechanism of the immune system. Upon antigen recognition, CTL stop migrating, establish a tight contact with target cells and deliver cytotoxic molecules such as perforin and granzymes that lead to target cell apoptosis. The ability of CTL to control a population of infected cells or a tumor depends on multiple parameters, such as the relative numbers of CTL and target cells, the intrinsic cytotoxic activity of CTL, the intrinsic resistance of target cells and the repertoire of immune checkpoints tuning cytotoxic activity at the CTL:target cell interface. In this context, in vitro assays to precisely measure CTL:target cell interactions and cytotoxic activity over time are required to monitor natural or therapeutic responses. We here present an image-based method that allows recording of positions and survival of CTL and target cells over time in a high-throughput format. The protocol relies on the staining of CTL and target cells with fluorescent dyes and the automated imaging of cells deposited in wells of a 384-well plate with an automated cell imaging device. We discuss potential applications offered by the kinetic assessment of CTL cytotoxic activity in a high-throughput format.

Methylammonium lead iodide bromides MAPb(Brx I1-x )3 are a class of mixed halide lead perovskites, materials that offer high-power conversion efficiencies and bandgap tunability. For these reasons, they are a promising absorber material for future solar cells, although their use is still limited due to several factors. The reversible phase segregation under even low light intensities is one of them, lowering the effective bandgap due to local segregation into iodide-rich and bromide-rich phases. While several studies have been done to illuminate the mechanism and suppression of phase segregation, challenges remain to understand its kinetics. We obtained dynamic ellipsometric measurements from x=0.5 mixed halide lead perovskite thin films protected by a polystyrene layer under green laser light with a power density of ∼11 W/cm2 . Time constants between 1.7(±0.7)×10-3  s-1 for the segregation and 1.5(±0.6)×10-4  s-1 for recovery were calculated. The phase segregation rate constants are surprisingly two orders of magnitude slower than and the recovery rate is consistent with those measured using photoluminescence methods under similar conditions. These results confirm a concern in the literature about the complexity in the phase separation kinetics measured from photoluminescence. We expect ellipsometry to serve as a complementary technique to other spectroscopies in studying mixed-halide lead perovskites phase segregation in the future.

Studying the catalytic behavior of biocatalysts under different conditions including temperature, buffer conditions, and cofactor concentrations is an important tool to understand their reaction mechanism. We describe two protocols that allow for the investigation of the catalysis of RNA-cleaving DNAzymes. The techniques include the use of FRET-labeled RNA substrates for studying the RNA-cleavage reaction in real-time under high throughput as well as RNA substrates labeled with a fluorescein molecule at the 5\' end for gel-based assays. Both methods allow for an accurate determination of rate constants given a reaction model.

The NEET proteins constitute a unique class of [2Fe-2S] proteins. The metal ions bind to three cysteines and one histidine. The proteins\' clusters exist in two redox states; the oxidized protein (containing two FeIII ions) can transfer the cluster to apo-acceptor protein(s), while the reduced form (containing one ferrous ion) remains bound to the protein frame. Here, we perform in silico and in vitro studies on human NEET proteins in both reduced and oxidized forms. Quantum chemical calculations on all available human NEET proteins structures suggest that reducing the cluster weakens the Fe-NHis and Fe-SCys bonds, similar to what is seen in other Fe-S proteins (e.g., ferredoxin and Rieske protein). We further show that the extra electron in the [2Fe-2S]+ clusters of one of the NEET proteins (mNT) is localized on the His-bound iron ion, consistently with our previous spectroscopic studies. Kinetic measurements demonstrate that the mNT [2Fe-2S]+ is released only by an increase in temperature. Thus, the reduced state of human NEET proteins [2Fe-2S] cluster is kinetically inert. This previously unrecognized kinetic inertness of the reduced state, along with the reactivity of the oxidized state, is unique across all [2Fe-2S] proteins. Finally, using a coevolutionary analysis, along with molecular dynamics simulations, we provide insight on the observed allostery between the loop L2 and the cluster region. Specifically, we show that W75, R76, K78, K79, F82 and G85 in the latter region share similar allosteric characteristics in both redox states.

This work presents an in situ nanoscale structural characterization of a SrCl2-expanded natural graphite (ENG) composite during ammonia absorption and desorption using small-angle neutron scattering (SANS) together with X-ray powder diffraction and sorption measurements. For the processing of the composite material SANS patterns, we developed and implemented two methods, which showed comparable results. The study allowed following the evolution of the SrCl2 particles and the nanopores inside the particles during five sorption cycles. The structural changes were compared to the absorption and desorption kinetic measurements, allowing us to make qualitative analysis of the impact of the structural changes on the material properties, such as thermal conductivity and permeability. It was shown that the structural evolution of the composite material did not affect the desorption rate but significantly influenced the absorption rate after the first cycle. We also observed a significant improvement of the absorption kinetics due to the formation of nanopores in the fully deammoniated sample. In addition, the ENG matrix was shown to hinder the agglomeration of the SrCl2 particles during sorption processes, which is in contrast to literature findings reported for a nonsupported metal halide. The findings presented in this study can be of great interest in the research areas where SrCl2-ENG composites are widely studied, i.e., heat storage, heat pumps/refrigerators, deNOx removal, and solid-state ammonia storage.

Heme (Fe protoporphyrin IX) serves as a prosthetic group of numerous proteins implicated in oxidative metabolism. This molecule is abundantly present in the red blood cells where it serves as a cofactor of hemoglobin. As consequence of various pathological conditions, the membrane of red blood cells can be damaged and therefore large quantities of hemoglobin and subsequently heme released in the extracellular space. Since heme is a highly reactive compound, when released extracelluarly it can influence the functional activity of different plasma components. Thus, previous investigations have demonstrated that heme can interact with components of complement system and immunoglobulins, profoundly affecting their functions. Here we propose two basic protocols that can be used for characterization of interaction of free heme with complement proteins and immunoglobulins. The first technique is based on UV-Vis absorbance spectroscopy. It allows general characterization of the heme binding to the protein and estimation of the number of heme binding sites. The second protocol consists in the use of biosensor assay based on surface plasmon resonance. This protocol would be useful for evaluation of heme binding kinetics and equilibrium affinity. Besides for complement components and immunoglobulins, the presented protocols can be utilized for characterization of the interaction of heme with other proteins.

Intrathymic development of committed progenitor (pro)-T cells from multipotent hematopoietic precursors offers an opportunity to dissect the molecular circuitry establishing cell identity in response to environmental signals. This transition encompasses programmed shutoff of stem/progenitor genes, upregulation of T cell specification genes, proliferation, and ultimately commitment. To explain these features in light of reported cis-acting chromatin effects and experimental kinetic data, we develop a three-level dynamic model of commitment based upon regulation of the commitment-linked gene Bcl11b. The levels are (1) a core gene regulatory network (GRN) architecture from transcription factor (TF) perturbation data, (2) a stochastically controlled chromatin-state gate, and (3) a single-cell proliferation model validated by experimental clonal growth and commitment kinetic assays. Using RNA fluorescence in situ hybridization (FISH) measurements of genes encoding key TFs and measured bulk population dynamics, this single-cell model predicts state-switching kinetics validated by measured clonal proliferation and commitment times. The resulting multi-scale model provides a mechanistic framework for dissecting commitment dynamics.

Data provided with this article are relative to kinetic measures from standing posture trials in eye open and eye closed conditions of 15 healthy subjects, acquired from a dynamometric force plate and a Nintendo Wii Balance Board (NBB). Data have been originally collected for a research project aimed at evaluating the reliability of low-cost devices in clinical scenarios. Raw data from the force plate include three ground reaction force components, center of pressure trajectories and torque around the vertical axis. Raw data from the NBB consist of vertical component of the ground reaction force measured by each of the four device sensors. Processed data consist of synchronized center of pressure time-series from both devices, referred to the force plate reference frame. Data were acquired simultaneously from the devices, allowing a direct comparison between the kinetic measures provided by the gold-standard for posture analysis (dynamometric force plate) and a low-cost device (NBB). Utility of present data can be twofold: first they can be used to assess the overall quality of the NBB signals for posturographic analysis by a direct comparison with the same signals acquired from the gold-standard device for kinetic measurement. Secondly, data from the dynamometric force plate can be used per se to evaluate different kind of parameters useful to assess balance capabilities, also by comparing data from different sensorial conditions (eye open versus eye closed).

Functional selectivity of agonists has gained increasing interest in G protein-coupled receptor (GPCR) research, e.g. due to expectations of drugs with reduced adverse effects. Different agonist-dependent GPCR conformations are conceived to selectively activate a balanced or imbalanced intracellular signalling response, involving e.g. different Gα subtypes, Gβγ-subunits and β-arrestins. To discriminate between the different signalling pathways (bias), sensitive techniques are needed that do not interfere with signalling. We applied split luciferase complementation to the GPCR/β-arrestin2 interaction and thoroughly analysed the influence of its implementation on intracellular signalling. This led to an assay enabling the functional characterization of ligands at the hH1R, the hM1,5R and the hNTS1R in live HEK293T cells. As demonstrated at the hM1,5R, the assay was sensitive enough to identify iperoxo as a superagonist. Time-dependent analyses of the recruitment of β-arrestin2 became possible, allowing the identification of class A and class B GPCRs, due to the differential duration of their interaction with β-arrestin2 and their recycling to the cell membrane. The developed β-arrestin2 recruitment assay, which provides concentration- and time-dependent information on the interaction between GPCRs and β-arrestin2 upon stimulation of the receptor, should be broadly applicable and of high value for the analysis of agonist bias.

Interactions between oxygen and gold surfaces are fundamentally important in diverse areas of science and technology. In this work, an oxygen dimer structure was observed and identified on gold nanoparticles in catalytic decomposition of hydrogen peroxide to oxygen and water. This structure, which is different from isolated atomic or molecular oxygen surface structures, was observed with in situ surface-enhanced Raman spectroscopic measurements and identified with density functional theory calculations. The experimental measurements were performed using monodisperse 5, 50 and 400 nm gold particles supported on silica with liquid-phase hydrogen and deuterium peroxides at multiple pH values. The calculations show that on surfaces with coordinatively unsaturated gold atoms, two oxygen atoms preferentially share a gold atom with a bond distance of 0.194-0.196 nm and additionally bind to two other surface gold atoms with a larger bond distance of 0.203-0.213 nm, forming an Au-O-Au-O-Au structure. The formation of this structure depends on reaction rates and conditions.