N95 mask reuse in a major urban hospital: COVID-19 response process and procedure.

BACKGROUND: The shortage of single-use N95 respirator masks (NRMs) during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted consideration of NRM recycling to extend limited stocks by healthcare providers and facilities. AIM: To assess potential reuse via autoclaving of NRMs worn daily in a major urban Canadian hospital. METHODS: NRM reusability was assessed following collection from volunteer staff after 2-8 h use, sterilization by autoclaving and PortaCount fit testing. A workflow was developed for reprocessing hundreds of NRMs daily. FINDINGS: Used NRMs passed fit testing after autoclaving once, with 86% passing a second reuse/autoclave cycle. A separate cohort of used masks pre-warmed before autoclaving passed fit testing. To recycle 200-1000 NRMs daily, procedures for collection, sterilization and re-distribution were developed to minimize particle aerosolization risk during NRM handling, to reject NRM showing obvious wear, and to promote adoption by staff. NRM recovery ranged from 49% to 80% across 12 collection cycles. CONCLUSION: Reuse of NRMs is feasible in major hospitals and other healthcare facilities. In sharp contrast to studies of unused NRMs passing fit testing after 10 autoclave cycles, we show that daily wear substantially reduces NRM fit, limiting reuse to a single cycle, but still increasing NRM stocks by ∼66%. Such reuse requires development of a comprehensive plan that includes communication across staffing levels, from front-line workers to hospital administration, to increase the collection, acceptance of and adherence to sterilization processes for NRM recovery.

Hydrogen peroxide inhibition of nuclear protein import is mediated by the mitogen-activated protein kinase, ERK2.

H(2)O(2) alters gene expression in many cell types. Alterations in nuclear import of transcription factors or similar key proteins may be responsible for these changes. To investigate this possibility, a cytosolic nuclear import cocktail was treated with varying ¿H(2)O(2) and used in import assays. H(2)O(2) caused a dose- and time-dependent inhibition of import at concentrations as low as 100 microM. Catalase reversed this effect. H(2)O(2) treatment of permeablized cells did not affect import, suggesting that H(2)O(2) was acting on a cytosolic factor. Treatment of import cocktail with two different free radical generating systems had no effect, but treatment of permeablized cells inhibited import, suggesting H(2)O(2) works via a distinct process from hydroxyl or superoxide radicals. Pretreatment of import cocktail with genistein reversed the effect of H(2)O(2) on import. Western blotting revealed that H(2)O(2) activated ERK2. The specific MEK1/2 inhibitor, PD98059, completely blocked the effects of H(2)O(2) on import. Activated ERK2 mimicked H(2)O(2)'s effect on import. Immunocytochemistry revealed that H(2)O(2) treatment of whole cells increased cytosolic Ran/TC4 levels, an effect reversible by catalase or PD98059. These data demonstrate that H(2)O(2) inhibits nuclear protein import and that this effect is mediated by mitogen-activated protein (MAP) kinase activation, possibly by altering Ran/TC4 function.

The nuclear protein import assay in vascular smooth muscle cells.

Nuclear protein import plays a critical role in both proliferation and apoptosis. Both actions are of great importance in vascular development and pathology. Consequently, a technique that would allow us to characterize import and identify novel cofactors important in modulating the process in smooth muscle cells is of great applicability. In this article, we describe a simple and reliable nuclear protein import assay that we have modified for use on aortic smooth muscle cells in culture. Briefly, the procedure permeabilizes the cells and analyzes the nuclear retention of a fluorescent import marker. Using this method, we are able to analyze the effect of agents on nuclear protein import. Most importantly, we are able to treat the cytosol, nucleus or the whole cell independently. This technique will allow for the identification and development of drugs to inhibit or stimulate the process and, potentially, to identify mechanistic insight into disease processes.

A model of low-flow ischemia and reperfusion in single, beating adult cardiomyocytes.

The present study was undertaken to comprehensively characterize low-flow ischemia and reperfusion in single adult cardiomyocytes and to determine whether it is important to control contractile activity. The ischemia-mimetic solution was hypoxic, acidic (pH 6.0), and deficient in glucose but contained elevated KCl. Cardiomyocytes were stimulated to contract throughout ischemia and during reperfusion with control perfusate. After the ischemia-reperfusion insult, cells exhibited poor recovery of active cell shortening, a decrease in passive cell length, increased frequency of necrosis, lower ATP content, and evidence of the generation of oxygen-derived free radicals within the cells. Intracellular lactate concentration increased, pH decreased, and Ca(2+) transients were depressed during the ischemic insult, but the latter two parameters recovered partially on reperfusion. Basal intracellular Ca(2+) concentration was elevated during ischemia and early into reperfusion. Recovery was attenuated in cells that were electrically stimulated to contract throughout ischemia. The duration of ischemia, stimulation frequency, and composition of the ischemia-mimetic solution were important variables. The inclusion of 10 mM lactate in the ischemia-mimetic solution significantly aggravated all the parameters examined above. Our data demonstrate that 1) an ischemia-mimetic solution administered to single, isolated adult cardiomyocytes can reproduce many of the responses observed in whole hearts, 2) caution should be used in adding lactate to an ischemic solution, and 3) it is important to stimulate contractile activity throughout ischemia to reproduce the effects of ischemia in whole hearts.

Age- and sex-related differences in nuclear lipid content and nucleoside triphosphatase activity in the JCR:LA-cp corpulent rat.

The putative role of the nuclear nucleoside triphosphatase (NTPase) is to provide energy to the nuclear pore complex for poly A(+) mRNA export. Previous work has demonstrated that liver nuclear NTPase activity is greater in 6 month old corpulent (cp/cp) female JCR:LA rats, a hyperlipidemic rat model, compared to lean (+/?) animals. This increase appeared to be related to increases in nuclear membrane cholesterol content. The current study extended these initial data to compare NTPase activity as a function of age and sex in isolated JCR:LA-cp rat liver nuclei, to further test the hypothesis that nuclear membrane cholesterol may modulate NTPase activity. NTPase activity was increased in cp/cp female animals compared to +/? females at all ages studied, with Vmax values increased by 60-176%. Membrane integrity of cp/cp female nuclei was reduced compared to +/? female nuclei. Nuclear membrane cholesterol levels increased linearly with age by 50, 150 and 250% in 3, 6 and 9 month old cp/cp females over leans. In contrast, nuclei from cp/cp males exhibited only minor, isolated changes in NTPase activity. Furthermore, there were no significant changes in nuclear cholesterol content or membrane integrity in the less hyperlipidemic male animals at any age. These data suggest that altered lipid metabolism may lead to changes in nuclear membrane structure, which in turn may alter NTPase activity and functioning of the nuclear pore complex.

The nuclear membrane integrity assay.

A novel technique is described for the evaluation of membrane integrity in isolated nuclei. Membrane integrity is assessed by measuring nuclear fragility in response to high salt conditions. Salt-induced disruption of the nuclear membrane is followed by spectrophotometric monitoring of released nucleotides. The assay is based on determining the amount of salt necessary to induce release of 50% of the total pool of releasable nucleotides. This allows semiquantitative comparison of relative nuclear membrane strength or integrity of different samples in response to high salt conditions. In this manner, the effects of altered nuclear membrane composition or metabolism on membrane integrity may be monitored.

Oxidation of nuclear membrane cholesterol inhibits nucleoside triphosphatase activity.

Oxygen derived free radicals can oxidize membrane cholesterol. We have previously shown that cholesterol in the nuclear membrane can modulate nuclear nucleoside triphosphatase (NTPase) activity. Nucleocytoplasmic transport of peptides and mRNA via the nuclear pore complex may be regulated by the NTPase. The purpose of the present study was to determine if oxidation of nuclear cholesterol could alter NTPase activity. Nuclear membrane cholesterol was oxidized in situ with cholesterol oxidase (to selectively oxidize cholesterol) and NTPase activity measured. HPLC analysis confirmed the formation of cholesterol oxides. The activity of the NTPase was strikingly inhibited by cholesterol oxidase treatment. The Vmax of the NTPase was significantly decreased after cholesterol oxidase treatment but the Km value was unchanged. The sensitivity of NTPase activity to varying cholesterol oxidase concentrations also suggested that cholesterol located in the inner leaflet of the nuclear membrane appeared to be more important in the modulation of NTPase activity than that in the cytoplasmic leaflet. Our results indicate that oxidation of nuclear membrane cholesterol inhibits NTPase activity. These results have implications for peptide and mRNA flux across the nuclear membrane during conditions where lipid oxidation may be expected.

Nuclear membrane cholesterol can modulate nuclear nucleoside triphosphatase activity.

Previous work has suggested that changes in nuclear membrane cholesterol may induce a stimulation in nuclear nucleoside triphosphatase (NTPase) activity. The purpose of the present study was to directly investigate if nuclear membrane cholesterol can stimulate nuclear NTPase activity. The cholesterol content of nuclei was altered with a liposomal methodology. The cholesterol content of nuclei isolated from hepatic tissue was relatively low in comparison to that typically exhibited by other membrane fractions. Because of this, it was difficult to further deplete the nuclear membrane of cholesterol, but we could successfully increase the cholesterol content after exposure to cholesterol-enriched liposomes. Nuclear NTPase activity was potently stimulated (approximately 150-200% of control) by an increase in the nuclear membrane cholesterol content. The Vmax of the NTPase activity in the presence of ATP or GTP was significantly increased after cholesterol enrichment without altering the affinity of the enzyme for these moieties. Mg2+ dependency of NTPase activity was also altered by cholesterol incorporation into the nuclear membrane. Cholesterol enrichment of the nuclear membrane also left the nuclei more susceptible to damage by salt-induced lysis than control nuclei. Our results clearly demonstrate that the cholesterol content of the nuclear membrane will have significant, direct effects on nuclear integrity and NTPase activity.

Nuclear cholesterol content and nucleoside triphosphatase activity are altered in the JCR:LA-cp corpulent rat.

A nuclear pore complex-associated nucleoside triphosphatase (NTPase) activity is believed to provide energy for nuclear export of poly(A)+ mRNA. This study was initiated to determine if nuclear membrane lipid composition is altered during chronic hyperlipidemia, and what effect this has on NTPase activity. The JCR:LA-cp corpulent rat model is characterized by severe hypertriglyceridemia and moderate hypercholesterolemia, and thus represents an ideal animal model in which to study nuclear cholesterol and NTPase activity. NTPase activity was markedly increased in purified hepatic nuclei from corpulent female JCR:LA-cp rats in comparison to lean control rats as a function of assay time, [GTP], [ATP], and [Mg2+]. Nuclear membrane cholesterol and phospholipid content were significantly elevated in the corpulent animals. Nuclei of corpulent animals were less resistant to salt-induced lysis than nuclei of lean animals, suggesting a change in relative membrane integrity. Together, these results indicate that altered lipid metabolism in a genetic corpulent animal model can lead to changes in nuclear membrane lipid composition, which in turn may alter nuclear membrane NTPase activity and integrity.

The presence and partitioning of calcium binding proteins in hepatic and cardiac nuclei.

Calcium is an important signal in key nuclear events, including cell cycle timing regulation of gene expression and activation of nuclear kinases and phosphatases. It is therefore important to identify calcium binding proteins in the nucleus which may play roles in these functions, and to determine whether these proteins are located in the nuclear envelope or in the nucleoplasm. Rat hepatic and pig cardiac nuclei were isolated and treated with a high salt solution to separate nucleoplasmic proteins from those associated with the nuclear envelope. The presence of calcium binding proteins was then revealed by Stains-All, staining of electrophoretic gels and 45Ca2+ overlays of Western blots. Four major calcium binding proteins were putatively identified in the pig cardiac nuclei, and another three in the rat hepatic nuclei. Proteins of 110, 93 and 35 kDa were observed in the pig cardiac nuclear envelope fraction, and another of 55 kDa in the pig cardiac high salt fraction. A 93-kDa protein was observed in the rat hepatic nuclear envelope fraction, and others of 120 and 110 kDa in the rat hepatic high salt fraction. A tentative identification has been made of the 93-kDa protein in each tissue type as calnexin, and of the cardiac 55 kDa protein as calsequestrin. This study, therefore, has putatively identified for the first time the presence of several calcium binding proteins which have distinct partitioning within hepatic and cardiac nuclei. This localization may play an important functional role within the nuclei.

The contribution of ionic imbalance to ischemia/reperfusion-induced injury.

Recent data from a number of independent laboratories have implicated ionic disturbances within the myocardium as being a critical contributory factor in injury to the heart during ischemia/reperfusion. The present treatise discusses the data supporting a role for Ca2+, Na+ and K+ in ischemic/reperfusion injury. The mechanism whereby intracellular ionic homeostasis becomes altered is the focus of the review. In particular, the evidence in support of an involvement of abnormal ion movements through Na+/H+ exchange, Na+ channels and the ATP-sensitive K+ channel in ischemic/reperfusion injury is advanced.

Calcium and calcium-binding proteins in the nucleus.

Calcium has long been known to play a role as a key cytoplasmic second messenger, but until relatively recently its possible involvement in nuclear signal transduction and the regulation of nuclear events has not been extensively studied. Evidence revealing the presence of transmembrane nuclear Ca2+ gradients and a variety of intranuclear Ca2+ binding proteins has fueled renewed interest in this key ion and its involvement in cell-cycle timing and division, gene expression, and protein activation. This review will offer an overview of the current state of knowledge and theory regarding calcium orchestration of nuclear functions and events and discuss possible future directions in this field of study.

Effect of chronic administration of verapamil on Ca++ channel density in rat tissue.

The purpose of this study was to determine if chronic administration of verapamil could alter the density of Ca++ channels in the heart as determined by [3H]PN 200-110 binding. Initially, we compared the effects of verapamil given by s.c. injection or via implantable, slow-release verapamil pellets. We found the majority of animals treated with the pellets died within 24 hr. Those that survived exhibited significantly depressed maximal binding and Kd values for PN 200-110 binding to cardiac membranes, but binding to brain membranes was unaffected. Quantitation of the serum levels of verapamil and its metabolites by high-performance liquid chromatography demonstrated that the verapamil pellets did not release the drug evenly over a 3-week period. Most of the drug was released in toxic quantities during the 1st day after implantation. Verapamil administered by injection (2.5-30 mg/kg/day) for up to 16 weeks raised plasma verapamil levels to 25 to 250 ng/ml, but had no effect on Ca++ channel characteristics in cardiac or brain tissue. The maximal binding and Kd values for skeletal muscle PN 200-110 binding were increased only at the highest dosage for 8 weeks duration. Our results demonstrate that implantable pellets are not a reliable administration method for verapamil and cardiac Ca++ channels are highly resistant to change during chronic verapamil administration.