High-content and high-throughput identification of macrophage polarization phenotypes

Macrophages are plastic cells of the innate immune system that perform a wide range of immune- and homeostasis-related functions. Due to their plasticity, macrophages can polarize into a spectrum of activated phenotypes. Rapid identification of macrophage polarization states provides valuable information for drug discovery, toxicological screening, and immunotherapy evaluation. The complexity associated with macrophage activation limits the ability of current biomarker-based methods to rapidly identify unique activation states. In this study, we demonstrate the ability of a 2-element sensor array that provides an information-rich 5-channel output to successfully determine macrophage polarization phenotypes in a matter of minutes. The simple and robust sensor generates a high dimensional data array which enables accurate macrophage evaluations in standard cell lines and primary cells after cytokine treatment, as well as following exposure to a model disease environment.

Phenotyping macrophage activation states using an array-based sensor. FRET complex assembly selectively interacts with the macrophage surface, generating a fingerprint for each polarization state that is further used to identify the activation state.     

Mechanistic Insights into the Inhibition of Endo-β 1,4 Xyloglucan Hydrolase by a Classical Aspartic Protease Inhibitor

This is the first report of inactivation of xyloglucanase from Thermomonospora sp by pepstatin A, a specific inhibitor towards aspartic proteases. The steady state kinetics revealed a reversible, competitive, two-step inhibition mechanism with IC ₅₀ and K _i values of 3.5?±?0.5 μM and 1.25?±?0.5 μM respectively. The rate constants determined for the isomerization of EI to EI^* and the dissociation of EI* were 14.5?±?1.5?×?10^?5?s^?1 and 2.85?±?1.2?×?10^?8?s^?1 respectively, whereas the overall inhibition constant K _i ^* was 27?±?1 nM. The conformational changes induced upon inhibitor binding to xyloglucanase were monitored by fluorescence analysis and the rate constants derived were in agreement with the kinetic data. The abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled xyloglucanase and far UV analysis suggested that pepstatin binds to the active site of the enzyme. Our results revealed that the inactivation of xyloglucanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis.     

Mössbauer studies of after-effects of auger ionization following electron capture in cobalt complexe

Abstract

The electron-capture decay of a cobalt-57 atom triggers an Auger event resulting in the loss of several electrons from the molecule in which it is incorporated. The 14.4 keV Mössbauer emission conveys information regarding the chemical forms in which the daughter iron-57 is ‘stabilized’ within 10^?7 sec following electron capture. During this time the electronic relaxation occurs completely and several tens of electron volt energy is deposited in the molecule as a result of neutralization. We find that the ethylenediamine tetra-acetate, bis-salicylaldehyde tri-ethylenetetramine, acetylacetone, and indenyl chelates fragment in a large majority of events, resulting in the formation of degraded ionic Fe²⁺ and Fe³⁺ in the former cases and C₉H₇Fe⁺ in the latter. On the other hand, highly conjugated compounds such as cobalt phthalocyanine and Vitamin B₁₂ escape fragmentation in 100 per cent of the Auger events. Tris-dipyridyl Co(III) perchlorate also escapes fragmentation in a majority of events. Apparently, the large amount of excitation energy deposited in the molecule as a consequence of charge neutralization is very rapidly (in less than 10^?13sec) and efficiently dispersed through neighboring molecules. It is a novel phenomenon.

When the dipyridyl chelate molecules are dispersed in a foreign matrix, the dissipation of charge and energy is no longer rapid and efficient and the probability of fragmentation is considerably enhanced.

We also find that part of the coordinated parent species, in the case of labeled dipyridyl chelate, arises through fragmentation followed by interaction of the electronically excited degraded iron species with a neighboring chelate molecule resulting in replacement of cobalt with an iron atom. This finding was made possible by using the chelate doped with ‘carrier-free’ ⁵⁷Co citrate.

Emission spectroscopy yields values for the isomer shifts and quadrupole splittings which differ somewhat from those obtained by absorption spectroscopy. The apparent disparities are attributed to the dissimilarities of the matrices in which the Mössbauer emitter or absorber is situated.     

Defining Cdk5 ligand chemical space with small molecule inhibitors of tau phosphorylation

Cyclin-dependent kinase 5 (Cdk5) is widely viewed as a possible target for a wide variety of neurological disorders. One pathological role attributed to Cdk5 is the abnormal phosphorylation of tau that may lead to the neuronal inclusions known as neurofibrillary tangles. A high through-put screen for inhibitors of Cdk5-mediated phosphorylation of tau resulted in three compounds with distinct mechanisms of action. One compound is competitive with ATP and has a high affinity for the Cdk5 ATP binding pocket. The second compound also competes with ATP, is noncompetitive with tau, and (uniquely among this class of inhibitors) displaces adjacent amino acid residues to make room for the nitrophenyl group. A third compound did not compete with ATP, but did compete with tau at low concentrations of tau. The SAR and charge optimization derived from cocrystals of the two ATP competitors along with cocrystals of three other ATP competitors map out the importance of filling and properly charging different regions of the ATP binding pocket. Taken together, this analysis shows how the structure of Cdk5 constrains the space of potential inhibitors and reveals a pocket unfilled in all of the structures. These leads could be a starting point for structure-based drug design of more potent and selective inhibitors.     

Control of catastrophic bifurcations of SSR in a hybrid series compensated system

Nonlinear Dynamics - Series compensation of the transmission line increases the power flow capability of the system. Hybrid series compensation is a combination of active and passive series...     

Unsteady Natural Convection Flow in a Square Cavity Filled with a Porous Medium Due to Impulsive Change in Wall Temperature

Unsteady natural convection flow in a two-dimensional square cavity filled with a porous material has been studied. The flow is initially steady where the left-hand vertical wall has temperature T _h and the right-hand vertical wall is maintained at temperature T _c (T _h > T _c) and the horizontal walls are insulated. At time t > 0, the left-hand vertical wall temperature is suddenly raised to which introduces unsteadiness in the flow field. The partial differential equations governing the unsteady natural convection flow have been solved numerically using a finite control volume method. The computation has been carried out until the final steady state is reached. It is found that the average Nusselt number attains a minimum during the transient period and that the time required to reach the final steady state is longer for low Rayleigh number and shorter for high Rayleigh number.