The effects of elongational stress exposure on the activation and aggregation of blood platelets.

Hemodynamic shear is known to stimulate blood and endothelial cells and induce platelet activation. Many studies of shear-induced platelet stimulation have employed rotational viscometers in which secondary flow effects are assumed to be negligible. Shear induced platelet activation occurs at elevated shear rates where secondary flows may contribute a significant percentage of the total hydrodynamic force experienced by the sample. Elongational stress, one component of this secondary flow, has been shown to alter transmembrane ion flux in intact cell and the permeability of synthetic membrane preparations. Elongational flow also occurs in the vasculature at sites of elevated shear stress. Secondary flow components may contribute to platelet activation induced during shear stress application in rotational viscometry. A unique 'constrained convergence' elongational flow chamber was designed and fabricated to study platelet response to elongational stress exposure. The elongational flow chamber was capable of producing an elongation rate of 2.1 s-1 with a corresponding volume averaged shear rate of 58.33 s-1. Significant changes were observed in the total platelet volume distribution and measured response to added chemical antagonists after elongational stress exposure. The total platelet volume histogram shifted toward larger particle sizes, suggesting the formation of large aggregates as a result of elongational stress exposure. Platelets exposed to elongational stress demonstrated a dose dependent decrease in added ADP-induced aggregation rate and extent of aggregation.     

A rapid method for counting cell nuclei using a particle sizer/counter

Summary We report here an improved method for nuclei counting utilizing Triton-X 100 to reduce the size of cell debris, thereby allowing the use of a particle sizer/counter. Furthermore, nuclei are completely released within 30 seconds, as compared to 1 hour using hypotonic solution. The method is accurate above 0.3 × 10⁶ cells/mL.     

Chromatographic and protein chemical analysis of the ubiquinol-cytochrome c2 oxidoreductase isolated from Rhodobacter sphaeroides

The ubiquinol-cytochrome c2 oxidoreductase (cytochrome bc1 complex) purified from chromatophores of Rhodobacter sphaeroides consists of four polypeptide subunits corresponding to cytochrome b, c1, and the Rieske iron-sulfur protein, as well as a 14-kDa polypeptide of unknown function, respectively. In contrast, the complex isolated from Rhodospirillum rubrum by the same procedure lacked a polypeptide corresponding to the 14-kDa subunit. Gel-permeation chromatography of the R. sphaeroides cytochrome bc1 complex in the presence of 200 mM NaCl removed the iron-sulfur protein, while the 14-kDa polypeptide remained tightly bound to the cytochromes; this is consistent with the possibility that the latter protein is an authentic component of the complex rather than an artifact of the isolation procedure. The individual polypeptides of the R. sphaeroides complex were purified to homogeneity by gel-permeation chromatography in the presence of 50% aqueous formic acid and their amino acid compositions determined. The 14-kDa polypeptide was found to be rich in charged and polar residues. Edman degradation analysis indicated that its N terminus is blocked and not rendered accessible by de-blocking procedures. Cyanogen bromide cleavage gave rise to a blocked N-terminal fragment as well as a C-terminal peptide comprising more than one-third of the protein. Gas-phase sequence analysis of this peptide established a sequence of 48 residues and identified a putative trans-membrane segment near the C terminus. The blocked N-terminal fragment was cleaved at tryptophan with BNPS-skatole. The resulting peptides, together with tryptic fragments derived from the intact protein, yielded additional sequence information; however, none of the sequences exhibited significant homologies to any known proteins. Tryptic fragments were also used to generate sequence information for cytochrome c1.     

Effectively Communicating the Uncertainties Surrounding Ebola Virus Transmission

The current Ebola virus outbreak has highlighted the uncertainties surrounding many aspects of Ebola virus virology, including routes of transmission. The scientific community played a leading role during the outbreak—potentially, the largest of its kind—as many of the questions surrounding ebolaviruses have only been interrogated in the laboratory. Scientists provided an invaluable resource for clinicians, public health officials, policy makers, and the lay public in understanding the progress of Ebola virus disease and the continuing outbreak. Not all of the scientific communication, however, was accurate or effective. There were multiple instances of published articles during the height of the outbreak containing potentially misleading scientific language that spurred media overreaction and potentially jeopardized preparedness and policy decisions at critical points. Here, we use articles declaring the potential for airborne transmission of Ebola virus as a case study in the inaccurate reporting of basic science, and we provide recommendations for improving the communication about unknown aspects of disease during public health crises.     

Excitotoxic Insult Results in a Long-Lasting Activation of CaMKIIα and Mitochondrial Damage in Living Hippocampal Neurons

Over-activation of excitatory NMDA receptors and the resulting Ca²⁺ overload is the main cause of neuronal toxicity during stroke. CaMKII becomes misregulated during such events. Biochemical studies show either a dramatic loss of CaMKII activity or its persistent autonomous activation after stroke, with both of these processes being implicated in cell toxicity. To complement the biochemical data, we monitored CaMKII activation in living hippocampal neurons in slice cultures using high spatial/temporal resolution two-photon imaging of the CaMKIIα FRET sensor, Camui. CaMKII activation state was estimated by measuring Camui fluorescence lifetime. Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma. Camui activation was either persistent (> 1–3 hours) or transient (~20 min) and, in general, correlated with its protein redistribution. After longer NMDA insult, however, Camui redistribution persisted longer than its activation, suggesting distinct regulation/phases of these processes. Mutational and pharmacological analysis suggested that persistent Camui activation was due to prolonged Ca²⁺ elevation, with little impact of autonomous states produced by T286 autophosphorylation and/or by C280/M281 oxidation. Cell injury was monitored using expressible mitochondrial marker mito-dsRed. Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation. The results suggest that in living neurons excitotoxic insult produces long-lasting Ca²⁺-dependent active state of CaMKII temporarily linked to cell injury. CaMKII function, however, is to be restricted due to strong clustering. The study provides the first characterization of CaMKII activation dynamics in living neurons during excitotoxic insults.     

Enhanced regional terrestrial carbon uptake over Korea revealed by atmospheric CO2 measurements from 1999 to 2017

Understanding changes in terrestrial carbon balance is important to improve our knowledge of the regional carbon cycle and climate change. However, evaluating regional changes in the terrestrial carbon balance is challenging due to the lack of surface flux measurements. This study reveals that the terrestrial carbon uptake over the Republic of Korea has been enhanced from 1999 to 2017 by analyzing long‐term atmospheric CO₂ concentration measurements at the Anmyeondo Station (36.53°N, 126.32°E) located in the western coast. The influence of terrestrial carbon flux on atmospheric CO₂ concentrations (ΔCO₂) is estimated from the difference of CO₂ concentrations that were influenced by the land sector (through easterly winds) and the Yellow Sea sector (through westerly winds). We find a significant trend in ΔCO₂ of ?4.75 ppm per decade (p < .05) during the vegetation growing season (May through October), suggesting that the regional terrestrial carbon uptake has increased relative to the surrounding ocean areas. Combined analysis with satellite measured normalized difference vegetation index and gross primary production shows that the enhanced carbon uptake is associated with significant nationwide increases in vegetation and its production. Process‐based terrestrial model and inverse model simulations estimate that regional terrestrial carbon uptake increases by up to 18.9 and 8.0 Tg C for the study period, accounting for 13.4% and 5.7% of the average annual domestic carbon emissions, respectively. Atmospheric chemical transport model simulations indicate that the enhanced terrestrial carbon sink is the primary reason for the observed ΔCO₂ trend rather than anthropogenic emissions and atmospheric circulation changes. Our results highlight the fact that atmospheric CO₂ measurements could open up the possibility of detecting regional changes in the terrestrial carbon cycle even where anthropogenic emissions are not negligible.     

Comparison of forest above‐ground biomass from dynamic global vegetation models with spatially explicit remotely sensed observation‐based estimates

Gaps in our current understanding and quantification of biomass carbon stocks, particularly in tropics, lead to large uncertainty in future projections of the terrestrial carbon balance. We use the recently published GlobBiomass data set of forest above‐ground biomass (AGB) density for the year 2010, obtained from multiple remote sensing and in situ observations at 100 m spatial resolution to evaluate AGB estimated by nine dynamic global vegetation models (DGVMs). The global total forest AGB of the nine DGVMs is 365 ± 66 Pg C, the spread corresponding to the standard deviation between models, compared to 275 Pg C with an uncertainty of ~13.5% from GlobBiomass. Model‐data discrepancy in total forest AGB can be attributed to their discrepancies in the AGB density and/or forest area. While DGVMs represent the global spatial gradients of AGB density reasonably well, they only have modest ability to reproduce the regional spatial gradients of AGB density at scales below 1000 km. The 95th percentile of AGB density (AGB₉₅) in tropics can be considered as the potential maximum of AGB density which can be reached for a given annual precipitation. GlobBiomass data show local deficits of AGB density compared to the AGB₉₅, particularly in transitional and/or wet regions in tropics. We hypothesize that local human disturbances cause more AGB density deficits from GlobBiomass than from DGVMs, which rarely represent human disturbances. We then analyse empirical relationships between AGB density deficits and forest cover changes, population density, burned areas and livestock density. Regression analysis indicated that more than 40% of the spatial variance of AGB density deficits in South America and Africa can be explained; in Southeast Asia, these factors explain only ~25%. This result suggests TRENDY v6 DGVMs tend to underestimate biomass loss from diverse and widespread anthropogenic disturbances, and as a result overestimate turnover time in AGB.