Nanoscale zerovalent iron particles induce differential cytotoxicity,genotoxicity, oxidative stress and hemolytic responses in human lymphocytes and erythrocytes in vitro

The growing usage of nanoscale zerovalent iron particles (nZVI) in the remediation of soil, ground/surface water has elicited large‐scale environmental release triggering human exposure. The size of nanomaterials is a key regulator of toxicity. However, the effect of a variable size of nZVI on genotoxicity is unexplored in human cells. To the best of our knowledge, in this study, the cytotoxic, genotoxic and hemolytic potential of nZVI‐1 (15 nm) and nZVI‐2 (50 nm) at concentrations of 5, 10 and 20 μg/mL was evaluated for the first time in human lymphocytes and erythrocytes treated for 3 hours. In erythrocytes, spherocytosis and echinocytosis occurred upon exposure to nZVI‐1 and nZVI‐2, respectively, leading to hemolysis. Lymphocytes treated with 20 μg/mL nZVI‐2 and 10 μg/mL nZVI‐1, incurred maximum DNA damage, although nZVI‐2 induced higher cyto‐genotoxicity than nZVI‐1. This can be attributed to higher Fe ion dissolution and time/concentration‐dependent colloidal destabilization (lower zeta potential) of nZVI‐2. Although nZVI‐1 showed higher uptake, its lower genotoxicity can be due to lesser Fe content, Fe ion dissolution and superior colloidal stability (higher zeta potential) compared with nZVI‐2. Substantial accumulation of Ca²⁺, superoxide anions, hydroxyl radicals and H₂O₂ leading to mitochondrial impairment and altered antioxidant enzyme activity was noted at the same concentrations. Pre‐treatment with N‐acetyl‐cysteine modulated these parameters indicating the indirect action of reactive oxygen species in nZVI‐induced DNA damage. The morphology of diffused nuclei implied the possible onset of apoptotic cell death. These results validate the synergistic role of size, ion dissolution, colloidal stability and reactive oxygen species on cyto‐genotoxicity of nZVI and unlock further prospects in its environmental nano‐safety evaluation.     

Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating

In K⁺ channels, the selectivity filter, pore helix, and outer vestibule play a crucial role in gating mechanisms. The outer vestibule is an important structurally extended region of KcsA in which toxins, blockers, and metal ions bind and modulate the gating behavior of K⁺ channels. Despite its functional significance, the gating-related structural dynamics at the outer vestibule are not well understood. Under steady-state conditions, inactivating WT and noninactivating E71A KcsA stabilize the nonconductive and conductive filter conformations upon opening the activation gate. Site-directed fluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule of open/conductive conformation is highly dynamic compared with the motional restriction experienced by the outer vestibule during inactivation gating. A wavelength-selective fluorescence approach shows a change in hydration dynamics in inactivated and noninactivated conformations, and supports a possible role of restricted/bound water molecules in C-type inactivation gating. Using a unique restrained ensemble simulation method, along with distance measurements by EPR, we show that, on average, the outer vestibule undergoes a modest backbone conformational change during its transition to various functional states, although the structural dynamics of the outer vestibule are significantly altered during activation and inactivation gating. Taken together, our results support the role of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gating mechanisms of K⁺ channels.The functional behavior of K⁺ channels is defined by a series of structural rearrangements associated with the processes of activation and inactivation gating (1–6). In response to a prolonged stimulus and in the absence of an N-terminal inactivating particle, most K⁺ channels become nonconductive through a process known as C-type inactivation (7). This C-type inactivation is crucial in controlling the firing patterns in excitable cells and is fundamental in determining the length and frequency of the cardiac action potential (8). C-type inactivation is inhibited by high extracellular K⁺ (9, 10), and the blocker tetraethylammonium (TEA) (11) can also be slowed down in the presence of permeant ions with a long residence time in the selectivity filter (Rb⁺, Cs⁺, and NH⁴⁺) (10).The prokaryotic pH-gated K⁺ channel KcsA shares most of the mechanistic properties of C-type inactivation in voltage-dependent K⁺ channels (5, 6, 12–16). Recent crystal structures of open/inactivated KcsA reveal that there is a remarkable correlation between the degree of opening at the activation gate and the conformation and ion occupancy of the selectivity filter (5). In KcsA, the selectivity filter is stabilized by a hydrogen bond network, with key interactions between residues Glu71, Asp80, and Trp67 and a bound water molecule (17). Disrupting this hydrogen bond network favors the conductive conformation of the selectivity filter (12, 13, 15).Early electrophysiological experiments have suggested that the outer vestibule (around T449 residue in Shaker and Y82 residue in KcsA) undergoes significant conformational rearrangement during C-type inactivation gating (16, 18, 19). However, comparison of the WT KcsA crystal structure, where the filter is in its conductive conformation, with either the structure obtained with low K⁺ (collapsed filter) (17) or the crystal structure of open-inactivated KcsA with maximum opening (inactivated filter) (5) does not show major conformational changes in the outer vestibule that would explain these results (Fig. 1A). We have suggested that this apparent discrepancy can be understood if we take into consideration the potential differences in the dynamic behavior of the outer vestibule changes as the K⁺ channel undergoes its gating cycle (16).Open in a separate windowFig. 1.Comparison of outer vestibule conformation in KcsA structures with conductive and collapsed/inactivated filters. (A) High-K⁺ KcsA structure [Protein Data Bank (PDB) ID code 1K4C; yellow] is compared with a low-K⁺ KcsA structure (PDB ID code 1K4D; blue) in the closed state (Left) and open/inactivated conformation (PDB ID code 3F5W; green) (Right). The outer vestibule residues are depicted as red spheres, and relevant residues are labeled. (B) Schematic representation of typical macroscopic currents elicited by pH-jump experiments in WT (inactivating) and E71A (noninactivating) KcsA channels at a depolarizing membrane potential is shown. Conditions that stabilize the closed, open/inactivated, and open/conductive conformations at the steady state are indicated with a black circle. (C) Effect of opening the lower gate on the mobility of spin-labeled outer vestibule residues in palmitoyloleoylphosphatidyl choline/palmitoyloleoylphosphatidyl glycerol (POPC/POPG) (3:1, moles/moles) reconstituted WT (Left) and noninactivating mutant E71A (Right) backgrounds for the closed (pH 7, red) and open (pH 4, black) states of KcsA, as determined by continuous wave (CW) EPR. The spectra shown are amplitude-normalized. Details are provided in SI Materials and Methods.We have probed the gating-induced structural dynamics at the outer vestibule of KcsA using site-directed fluorescence and site-directed spin labeling and pulsed EPR approaches in combination with a recently developed computational method, restrained ensemble (RE) simulations. RE simulation was used to constrain the outer vestibule using experimentally derived distance histograms in different functional states (closed, open/inactivated, and open/conductive) and to monitor the extent of backbone conformational changes during gating. To this end, we took advantage of our ability to stabilize both the open/conductive (E71A mutant) and the open/inactivated (WT) conformations of KcsA upon opening the activation gate under steady-state conditions (Fig. 1B).Our data show that the outer vestibule in the open/conductive conformation is highly dynamic. In addition, the red edge excitation shift (REES) points to a change in hydration dynamics between conductive and nonconductive outer vestibule conformations, suggesting a role of restricted water molecules in C-type inactivation gating. We suggest that, on average, the backbone conformation of the outer vestibule does not change significantly between different functional states but that local dynamics change significantly, underlining the importance of the hydrogen bond network behind the selectivity filter and the microscopic observables (e.g., dynamics of hydration) in K⁺ channel gating and C-type inactivation.     

A Simple Method of Electroelution of Individual Protein Bands from SDS Polyacrylamide Gels for Direct Study in Cellular Assays

A very simple and effective procedure which allows simultaneous electroelution of separated proteins from SDS polyacrylamide gel into small quantity of elution buffer is described. Elution parameters have been optimized for maximum possible recovery (50-60%). Protein fractions were collected in physiological buffer and an efficient removal of SDS have been obtained, thus fractions collected were suited for direct testing in cell cultures. Method was used to investigate human T-cell responses to purified secreted M. tuberculosis H37Rv proteins. Eight low molecular weight (M.w. range 10 kD to 25 kD) culture filtrate proteins were purified in quantities, sufficient for immunological characterization. Lymphocyte proliferative responses and cytokine release pattern from tuberculosis patients, healthy contacts and healthy controls were studied on stimulation with purified culture filtrate proteins. Immunologically important M. tuberculosis proteins were identified by using this method. This approach should be applicable to the rapid identification and characterization of any interesting T cell antigen.     

Developing an action plan for patient radiation safety in adult cardiovascular medicine. Proceedings from the Duke University Clinical Research Institute/American College of Cardiology Foundation/American Heart Association Think Tank Held on February 28, 2011

Technological advances and increased utilization of medical testing and procedures have prompted greater attention to ensuring the patient safety of radiation use in the practice of adult cardiovascular medicine. In response, representatives from cardiovascular imaging societies, private payers, government and nongovernmental agencies, industry, medical physicists, and patient representatives met to develop goals and strategies toward this end; this report provides an overview of the discussions. This expert "think tank" reached consensus on several broad directions including: the need for broad collaboration across a large number of diverse stakeholders; clarification of the relationship between medical radiation and stochastic events; required education of ordering and providing physicians, and creation of a culture of safety; development of infrastructure to support robust dose assessment and longitudinal tracking; continued close attention to patient selection by balancing the benefit of cardiovascular testing and procedures against carefully minimized radiation exposures; collation, dissemination, and implementation of best practices; and robust education, not only across the healthcare community but also to patients, the public, and media. Finally, because patient radiation safety in cardiovascular imaging is complex, any proposed actions need to be carefully vetted (and monitored) for possible unintended consequences.