Systematic experimental comparison of particle filtration efficiency test methods for commercial respirators and face masks

Respirators, medical masks, and barrier face coverings all filter airborne particles using similar physical principles. However, they are tested for certification using a variety of standardized test methods, creating challenges for the comparison of differently certified products. We have performed systematic experiments to quantify and understand the differences between standardized test methods for N95 respirators (NIOSH TEB-APR-STP-0059 under US 42 CFR 84), medical face masks (ASTM F2299/F2100), and COVID-19-related barrier face coverings (ASTM F3502-21). Our experiments demonstrate the role of face velocity, particle properties (mean size, size variability, electric charge, density, and shape), measurement techniques, and environmental preconditioning. The measured filtration efficiency was most sensitive to changes in face velocity and particle charge. Relative to the NIOSH method, users of the ASTM F2299/F2100 method have commonly used non-neutralized (highly charged) aerosols as well as smaller face velocities, each of which may result in approximately 10% higher measured filtration efficiencies. In the NIOSH method, environmental conditioning at elevated humidity increased filtration efficiency in some commercial samples while decreasing it in others, indicating that measurement should be performed both with and without conditioning. More generally, our results provide an experimental basis for the comparison of respirators certified under various international methods, including FFP2, KN95, P2, Korea 1st Class, and DS2.Comment: 34 pages, 8 figures, 3 table

NOX2, p22phox and p47phox are targeted to the nuclear pore complex in ischemic cardiomyocytes colocalizing with local reactive oxygen species.

BACKGROUND: NADPH oxidases play an essential role in reactive oxygen species (ROS)-based signaling in the heart. Previously, we have demonstrated that (peri)nuclear expression of the catalytic NADPH oxidase subunit NOX2 in stressed cardiomyocytes, e.g. under ischemia or high concentrations of homocysteine, is an important step in the induction of apoptosis in these cells. Here this ischemia-induced nuclear targeting and activation of NOX2 was specified in cardiomyocytes. METHODS: The effect of ischemia, mimicked by metabolic inhibition, on nuclear localization of NOX2 and the NADPH oxidase subunits p22(phox) and p47(phox), was analyzed in rat neonatal cardiomyoblasts (H9c2 cells) using Western blot, immuno-electron microscopy and digital-imaging microscopy. RESULTS: NOX2 expression significantly increased in nuclear fractions of ischemic H9c2 cells. In addition, in these cells NOX2 was found to colocalize in the nuclear envelope with nuclear pore complexes, p22(phox), p47(phox) and nitrotyrosine residues, a marker for the generation of ROS. Inhibition of NADPH oxidase activity, with apocynin and DPI, significantly reduced (peri)nuclear expression of nitrotyrosine. CONCLUSION: We for the first time show that NOX2, p22(phox) and p47(phox) are targeted to and produce ROS at the nuclear pore complex in ischemic cardiomyocytes

Loss of DPP4 activity is related to a prothrombogenic status of endothelial cells: implications for the coronary microvasculature of myocardial infarction patients

Pro-coagulant and pro-inflammatory intramyocardial (micro)vasculature plays an important role in acute myocardial infarction (AMI). Currently, inhibition of serine protease dipeptidyl peptidase 4 (DPP4) receives a lot of interest as an anti-hyperglycemic therapy in type 2 diabetes patients. However, DPP4 also possesses anti-thrombotic properties and may behave as an immobilized anti-coagulant on endothelial cells. Here, we studied the expression and activity of endothelial DPP4 in human myocardial infarction in relation to a prothrombogenic endothelial phenotype. Using (immuno)histochemistry, DPP4 expression and activity were found on the endothelium of intramyocardial blood vessels in autopsied control hearts (n = 9). Within the infarction area of AMI patients (n = 73), this DPP4 expression and activity were significantly decreased, coinciding with an increase in Tissue Factor expression. In primary human umbilical vein endothelial cells (HUVECs), Western blot analysis and digital imaging fluorescence microscopy revealed that DPP4 expression was strongly decreased after metabolic inhibition, also coinciding with Tissue Factor upregulation. Interestingly, inhibition of DPP4 activity with diprotin A also enhanced the amount of Tissue Factor encountered and induced the adherence of platelets under flow conditions. Ischemia induces loss of coronary microvascular endothelial DPP4 expression and increased Tissue Factor expression in AMI as well as in vitro in HUVECs. Our data suggest that the loss of DPP4 activity affects the anti-thrombogenic nature of the endothelium

Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47(phox).

Item does not contain fulltextWe previously found that homocysteine (Hcy) induced plasma membrane flip-flop, apoptosis, and necrosis in cardiomyocytes. Inactivation of flippase by Hcy induced membrane flip-flop, while apoptosis was induced via a NOX2-dependent mechanism. It has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in hyperhomocysteinemia (HHC)-induced pathogenesis of cardiovascular disease. Therefore, we evaluated whether the observed cytotoxic effect of Hcy in cardiomyocytes is SAH dependent. Rat cardiomyoblasts (H9c2 cells) were treated under different conditions: (1) non-treated control (1.5 nM intracellular SAH with 2.8 muM extracellular L -Hcy), (2) incubation with 50 muM adenosine-2,3-dialdehyde (ADA resulting in 83.5 nM intracellular SAH, and 1.6 muM extracellular L -Hcy), (3) incubation with 2.5 mM D, L -Hcy (resulting in 68 nM intracellular SAH and 1513 muM extracellular L -Hcy) with or without 10 muM reactive oxygen species (ROS)-inhibitor apocynin, and (4) incubation with 100 nM, 10 muM, and 100 muM SAH. We then determined the effect on annexin V/propodium iodide positivity, flippase activity, caspase-3 activity, intracellular NOX2 and p47(phox) expression and localization, and nuclear ROS production. In contrast to Hcy, ADA did not induce apoptosis, necrosis, or membrane flip-flop. Remarkably, both ADA and Hcy induced a significant increase in nuclear NOX2 expression. However, in contrast to ADA, Hcy additionally induced nuclear p47(phox) expression, increased nuclear ROS production, and inactivated flippase. Incubation with SAH did not have an effect on cell viability, nor on flippase activity, nor on nuclear NOX2-, p47phox expression or nuclear ROS production. HHC-induced membrane flip-flop and apoptosis in cardiomyocytes is due to increased Hcy levels and not primarily related to increased intracellular SAH, which plays a crucial role in nuclear p47(phox) translocation and subsequent ROS production.1 december 201

Homocysteine Induces Phosphatidylserine Exposure in Cardiomyocytes through Inhibition of Rho Kinase and Flippase Activity.

AIMS: Increased levels of homocysteine (Hcy) form an independent risk factor for cardiovascular disease. In a previous study we have shown that Hcy induced phosphatidylserine (PS) exposure to the outer leaflet of the plasma membrane in cardiomyocytes, inducing a pro-inflammatory phenotype. In the present study the mechanism(s) involved in Hcy-induced PS exposure were analyzed. METHODS: H9c2 rat cardiomyoblasts were subjected to 2.5 mM D,L-Hcy and analyzed for RhoA translocation and activity, Rho Kinase (ROCK) activity and expression and flippase activity. In addition, the effect of ROCK inhibition with Y27632 on Hcy-induced PS exposure and flippase activity was analyzed. Furthermore, GTP and ATP levels were determined. RESULTS: Incubation of H9c2 cells with 2.5 mM D,L-Hcy did not inhibit RhoA translocation to the plasma membrane. Neither did it inhibit activation of RhoA, even though GTP levels were significantly decreased. Hcy did significantly inhibit ROCK activation, but not its expression, and did inhibit flippase activity, in advance of a significant decrease in ATP levels. ROCK inhibition via Y27632 did not have significant added effects on this. CONCLUSION: Hcy induced PS exposure in the outer leaflet of the plasma membrane in cardiomyocytes via inhibition of ROCK and flippase activity. As such Hcy may induce cardiomyocytes vulnerable to inflammation in vivo in hyperhomocysteinaemia patients

Ischemia induces nuclear NOX2 expression in cardiomyocytes and subsequently activates apoptosis

In previous work we have demonstrated increased expression of NOX2 in cardiomyocytes of infarcted human hearts. In the present manuscript we investigated the functional role of NOX2 in ischemically challenged H9c2 cells, a rat cardiomyoblast cell line, and adult rat cardiomyocytes. Expression of NOX2 in H9c2 cells was confirmed by RT-PCR. In Western-blot experiments, increased NOX2 expression was detected during ischemia, which was inhibited by transcription and translation inhibitors. Surprisingly, under ischemia, in addition to an increased cytosolic expression, NOX2 was localized mainly in the nucleus of apoptotic cardiomyocytes, where it colocalized with nitrotyrosine residues and activated caspase 3. Inhibition of reactive-oxygen-species generation with the flavoenzyme inhibitor diphenylene iodonium (DPI) and the NADPH-oxidase inhibitor apocynin led to a significantly decreased induction of apoptosis as assessed by quantification of caspase-3 activity and by TUNEL analysis. These results demonstrate that NOX2 is expressed in the nucleus of cardiomyocytes during apoptosis and that it likely participates in proapoptotic signaling. To the best of our knowledge, this is the first demonstration of nuclear NOX2 expression and its involvement in cardiomyocyte apoptosi