Alcohol-free hand sanitizer and other quaternary ammonium disinfectants quickly and effectively inactivate SARS-CoV-2.

BACKGROUND: SARS-CoV-2 is the virus responsible for the current global pandemic, COVID-19. Because this virus is novel, little is known about its sensitivity to disinfection. METHODS: We performed suspension tests against SARS-CoV-2 using three commercially available quaternary ammonium compound (Quat) disinfectants and one laboratory-made 0.2% benzalkonium chloride solution. FINDINGS: Three of the four formulations completely inactivated the virus within 15 s of contact, even in the presence of a soil load or when diluted in hard water. CONCLUSION: Quats rapidly inactivate SARS-CoV-2, making them potentially useful for controlling SARS-CoV-2 spread in hospitals and the community.

Protein degradation in cultured cells. II. The uptake of chloroquine by rat fibroblasts and the inhibition of cellular protein degradation and cathepsin B1.

The degradation of cellular proteins in fibroblasts, both those of rapid and those of slow turnover rates, was inhibited by low concentrations of chloroquine or neutral red in the medium. Cells inhibited by chloroquine can be inhibited further by fluoride. Chloroquine was taken up by the fibroblasts and the concentration in the cells reached several hundred times that in the medium. Isopycnic fractionation studies showed that within the cells the chloroquine was concentrated in the lysosomes, and that these chloroquine-containing lysosomes had a lower equilibrium density than the lysosomes of untreated cells. Chloroquine, at concentrations attained inside the lysosomes, inhibited cathepsin B(1) but not cathepsin D. It is concluded that chloroquine impairs the breakdown of cellular proteins after these have entered the lysosome system, probably through inhibition of cathepsin B(1).

Cytoplasmic vacuolation of mouse peritoneal macrophages and the uptake into lysosomes of weakly basic substances.

With few exceptions, weakly basic compounds that are sufficiently lipophilic in their neutral forms and sufficiently hydrophilic in their protonated forms accumulate in lysosomes. When the concentration within the lysosomes becomes sufficiently high, osmotic swelling occurs. The cells than take on a vacuolated appearance. The concentrations at which different weak bases cause lysosomal vacuolation vary over almost three orders of magnitude. For any particular weak base, it is the concentration of the neutral form that determines the extent of uptake and the degree of vacuolation. Chloroquine is anomalous in that concentrations greater than approximately 30 microM cause less uptake and less vacuolation than do lower concentrations.

Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages.

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.

The effects of basic substances and acidic ionophores on the digestion of exogenous and endogenous proteins in mouse peritoneal macrophages.

Basic substances and acidic ionophores that increase the lysosomal pH in cultured macrophages (Ohkuma, S., and B. Poole, 1978, Proc. Natl. Acad. Sci. USA., 75:3327-3331; Poole, B., and S. Ohkuma, 1981, J. Cell Biol., 90:665-669) inhibited the digestion of heat-denatured acetylated bovine serum albumin (BSA) taken up by the cells. For several substances, the shift in pH sufficed to explain the inhibition of proteolysis. Additional effects, presumably on enzyme activities, have to be postulated for tributylamine, amantadine, and chloroquine. Sodium fluoride (10 mM) had no significant effect on the breakdown of BSA by macrophages. The breakdown of endogenous macrophage proteins, whether short lived or long lived, was inhibited approximately 40% by 10 mM NaF and 30%, or sometimes less in the case of long-lived proteins, by 100 microM chloroquine. When the cells were supplied with BSA, a mixture of cell proteins, or even inert endocytosible materials, the breakdown of endogenous long-lived proteins and the inhibitory effect of chloroquine on this process were selectively reduced. Inhibition of endocytosis by cytochalasins B or D did not affect the chloroquine-sensitive breakdown of endogenous proteins, indicating that the proteins degraded by this process were truly endogenous and not taken in from the outside by cellular cannibalism. On the other hand, when macrophage proteins were supplied extracellularly, their breakdown occurred at the same rate for short-lived and long-lived proteins, and it was strongly inhibited by chloroquine and not by NaF. It is concluded from these results that the breakdown of endogenous proteins, both short-lived and long-lived, probably takes place partly (approximately 30%) in lysosomes and partly through one or more nonlysosomal mechanism(s) unaffected by chloroquine and presumably susceptible to inhibition by fluoride. A difference must exist between short-lived and long-lived proteins in the manner in which they reach lysosomes or are handled by these organelles; this difference would account for the selective effect of the supply of endocytosible materials on the lysosomal processing of long-lived proteins.

The synthesis and turnover of rat liver peroxisomes. II. Turnover of peroxisome proteins.

After preliminary experiments had established that the injection of Triton WR-1339 necessary for the separation of lysosomes and peroxisomes did not affect the turnover rate of catalase, the decay of (3)H-leucine incorporated into peroxisomes was studied in whole particles and in protein subfractions. It was shown that peroxisomes are destroyed in a completely random way, probably as wholes since the apparent half-life was the same for all subfractions, about 3(1/2) days. In agreement with the results of Price et al. (11), the half-life of catalase derived from the rate of recovery from aminotriazole inhibition was about 11(1/2) days, as was the apparent half-life of the heme prosthetic groups measured with (14)C-alpha-aminolevulinic acid. Guanidino-labeled arginine gave an apparent half-life of 2(1/2) days with large statistical uncertainty. Either the leucine label was reutilized very extensively in our animals and the true half-life of peroxisomes is 1(1/2) days, or the prosthetic groups of catalase turn over more rapidly than the protein part of the molecule.

The synthesis and turnover of rat liver peroxisomes. 3. The size distribution of peroxisomes and the incorporation of new catalase.

Rat liver peroxisomes have been separated according to size by zonal sedimentation. A method is described for calculating the size of the particles from their final position in the gradient. Peroxisomes seem biochemically homogeneous throughout their size distribution. 3 hr after injection of tritiated leucine, the specific radioactivity of catalase is the same in peroxisomes of different sizes, and it remains so for up to 1 wk after administration of the precursor. This observation rules out the possibility that peroxisomes have an extended period of independent growth. If individual particles maintain an independent existence, they must be formed very rapidly. The other possible explanation is that peroxisomes exchange material within the liver cell.

The synthesis and turnover of rat liver peroxisomes. I. Fractionation of peroxisome proteins.

Rat liver peroxisomes isolated by density gradient centrifugation were disrupted at pH 9, and subdivided into a soluble fraction containing 90% of their total proteins and virtually all of their catalase, D-amino acid oxidase, L-alpha-hydroxy acid oxidase and isocitrate dehydrogenase activities, and a core fraction containing urate oxidase and 10% of the total proteins. The soluble proteins were chromatographed on Sephadex G-200, diethylaminoethyl (DEAE)-cellulose, hydroxylapatite, and sulfoethyl (SE)-Sephadex. None of these methods provided complete separation of the protein components, but these could be distributed into peaks in which the specific activities of different enzymes were substantially increased. Catalase, D-amino acid oxidase, and L-alpha-hydroxy acid oxidase contribute a maximum of 16, 2, and 4%, respectively, of the protein of the peroxisome. The contribution of isocitrate dehydrogenase could be as much as 25%, but is probably much less. After dissolution of the cores at pH 11 , no separation between their urate oxidase activity and their protein was achieved by Sephadex G-200 chromatography.

The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions.

Improved, largely automated methods are described for the purification and analysis o peroxisomes, lysosomes, and mitochondria from the livers of rats injected with Triton WR-1339. With these new methods, it has become possible to obtain, in less than 6 hr and with reliable reproducibility, mitochondria practically free of contaminants, as well as the rarer cytoplasmic particles in amounts (about 100 mg of protein) and in a state of purity (95%) that make them suitable for detailed biochemical studies. The results obtained so far on these preparations have made more conclusive and precise previous estimates of the biochemical and morphological properties of the three groups of cytoplasmic particles. In addition, peroxisomes were found to contain essentially all the L-alpha-hydroxy acid oxidase of the liver, as well as a small, but significant fraction of its NADP-linked isocitrate dehydrogenase activity. Another small fraction of the latter enzyme is present in the mitochondria, the remainder being associated with the cell sap. The mitochondrial localization of the metabolically active cytoplasmic DNA could be verified. The relative content of the fractions in mitochondria, whole peroxisomes, peroxisome cores, lysosomes, and endoplasmic reticulum was estimated independently by direct measurements on electron micrographs, and by linear programming (based on the assumption that the particles are biochemically homogeneous) of the results of enzyme assays. The two types of estimates agreed very well, except for one fraction in which low cytochrome oxidase activity was associated with mitochondrial damage.

A compact linac for intensity modulated proton therapy based on a dielectric wall accelerator.

A novel compact CT-guided intensity modulated proton radiotherapy (IMPT) system is described. The system is being designed to deliver fast IMPT so that larger target volumes and motion management can be accomplished. The system will be ideal for large and complex target volumes in young patients. The basis of the design is the dielectric wall accelerator (DWA) system being developed at the Lawrence Livermore National Laboratory (LLNL). The DWA uses fast switched high voltage transmission lines to generate pulsed electric fields on the inside of a high gradient insulating (HGI) acceleration tube. High electric field gradients are achieved by the use of alternating insulators and conductors and short pulse times. The system will produce individual pulses that can be varied in intensity, energy and spot width. The IMPT planning system will optimize delivery characteristics. The system will be capable of being sited in a conventional linac vault and provide intensity modulated rotational therapy. Feasibility tests of an optimization system for selecting the position, energy, intensity and spot size for a collection of spots comprising the treatment are underway. A prototype is being designed and concept designs of the envelope and environmental needs of the unit are beginning. The status of the developmental new technologies that make the compact system possible will be reviewed. These include, high gradient vacuum insulators, solid dielectric materials, SiC photoconductive switches and compact proton sources.

Fractionation of the rat liver enzymes that hydrolyze benzoyl-arginine-2-naphthylamide.

1. The enzyme activity in the particulate fraction from rat liver that hydrolyzes alpha-N-benzoyl-DL-arginine-2-naphthylamide (Bz-Arg-NNap) has been separated into two approximately equal components by chromatography on DEAE-cellulose. One component (peak II) is completely retained by the column at low ionic strength while the other component (peak I) passes through. 2. In contrast to the enzyme in peak I, the enzyme in peak II is extremely sensitive to inhibition by leupeptin, it will hydrolyze carbobenzoxy-alanylarginylarginyl-4-methoxy-2-naphthylamine, and it will inactivate aldolase. 3. There appears to be also a minor high molecular weight component of the alpha-N-benzoyl-DL-arginyl-2-naphthylamine-hydrolyzing activity that is retained by the DEAE-cellulose but which has properties similar to those of the peak I enzyme.

The effect of canavanine on protein synthesis and protein degradation in IMR-90 fibroblasts.

Proteins of IMR-90 fibroblasts incorporating [35S]methionine during a 1 h labelling period in the presence of the arginine analogue canavanine were degraded twice as rapidly in the cells as were proteins similarly made in the presence of arginine. Using both isoelectric focusing and SDS-polyacrylamide gel electrophoretic analyses, the banding patterns of proteins labelled in the presence of canavanine and arginine were found to differ. This banding difference was detected as early as 15 min after canavanine treatment. With the exception of one minor band in isoelectric focusing gel, the relative intensity of labelled protein bands for the control samples remained unchanged during the 2 h period of protein degradation being investigated. This was also true for the proteins labelled in the presence of canavanine, despite the increase in their rate of degradation. Banding difference between canavanine and arginine treatment was also detected in an in vitro reticulocyte lysate translation system dependent on fibroblast mRNA. Proteins labelled in the presence of a different analogue, p-fluorophenylalanine instead of phenylalanine, however, had similar banding patterns as the control both in the lysate system and in intact cells.

Evidence for the selective release of lysosomal proteinases in fasted rabbits.

The enzyme responsible for the conversion of "neutral" to "alkaline" fructose 1,6-bisphosphatase (EC 3.1.3.11) by removal of a 7000 dalton peptide (converting enzyme, Proteinase I) has been shown to be localized in rat liverlysosomes. Lysosomes also contain a specific proteinase (Proteinase II) that catalyzes the release of a small peptide from the NH2-terminus of the native subunits. In fasted rabbits Proteinase II is released into the cytoplasm, together with Cathepsin A, but Proteinase I remains associated with the lysosomal fraction. Increased osmotic fragility of liver lysosomes in fasted rabbits has also been observed, but this increased fragility does not result in the release of Proteinase I. The appearance of Proteinase II in the cytoplasm may be due either to its selective release from the lysosomes, without release of Proteinase I, or its localization in a different lysosomal fraction. Changes in lysosomal structure induced by fasting may play a dual role in : 1) the mobilization of amino acids for gluconeogenesis and 2) the modulation of activity of gluconeogenic enzymes.

The comparative effects of the NOS inhibitor, Nomega-nitro-L-arginine, and the haemoxygenase inhibitor, zinc protoporphyrin IX, on tumour blood flow.

PURPOSE: To determine the relative effects of inhibiting nitric oxide synthase (NOS) and haemoxygenase (HO) on blood flow to the rat P22 carcinosarcoma. METHODS AND MATERIALS: HO is the enzyme responsible for in vivo production of carbon monoxide (CO). The vascular effects of zinc protoporphyrin IX (ZnPP), a competitive inhibitor of HO, were compared with those of copper protoporphyrin IX (CuPP), a poor inhibitor of HO, in isolated ex vivo perfusions of the P22 tumour and in intact tumour-bearing rats. In ex vivo perfusions, tumour vascular resistance was calculated from measurements of perfusion pressure at a known flow rate. In intact animals, blood flow to tumour and normal tissues was calculated using a radiotracer uptake method. The effects of ZnPP were compared with those of the NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA), and the combination of the two drugs. RESULTS: HO activity in the P22 tumour was reduced by 50% following administration of either ZnPP or CuPP directly to ex vivo perfused tumours, suggesting an indirect effect on the enzyme. Enzyme inhibition was not associated with any significant vasoactive effect. Neither ZnPP nor CuPP, at a dose of 45 micromol x kg(-1) administered i.p., inhibited tumour HO in vivo. However, they did significantly decrease tumour blood flow to 60-70% of control, with similar effects in skin and brain. Skeletal muscle blood flow was increased to 150% of control. L-NNA decreased both tumour and skeletal muscle blood flow to around 40% of control. These differences suggest that the nonspecific effects of ZnPP and CuPP were not mediated by NOS inhibition. The combination of ZnPP and L-NNA improved the selective reduction in tumour blood flow achieved with either agent alone. CONCLUSION: This suggests that the HO/CO pathway does not play a major vasodilatory role in this tumour. However, ZnPP and CuPP could be useful for inducing a relatively selective decrease in tumour blood flow via mechanisms unrelated to HO inhibition, especially when combined with NOS inhibition.

Peptidases from Plasmodium falciparum cultured in vitro.

An acid peptidase that degrades hemoglobin optimally at pH 3.5, a neutral aminopeptidase and an alkaline endopeptidase that acts on an alpha-N-blocked synthetic substrate have been demonstrated in Plasmodium falciparum in culture. The enzymes were shown to be distinct by anion exchange chromatography, gel filtration on Sephadex G-200 and isoelectric focusing. The activities of the acid peptidase and the aminopeptidase were inhibited by antimalarial compounds.

Evidence for an ester bond between thrombin and heparin cofactor.

Heparin cofactor, a thrombin inhibitor, is purified from human plasma by affinity chromatography on heparin-agarose. The nature of the binding between thrombin and the inhibitor is studied by treatment of the complex with 6 M guanidinium chloride, hydroxylamine, and dilute alkali. The complex is not dissociated during gel chromatography in 6 M guanidinium chloride. This result supports an earlier proposal that formation of the complex includes the formation of a covalent bond. Treatment of dodecyl sulfate-denatured complex with hydroxylamine results in dissociation of the complex to yield free thrombin and heparin cofactor. Hydroxylamine does not dissociate the complex unless it is denatured. The complex is also dissociated in dilute sodium hydroxide (pH 12) solutions. These results indicate that the covalent bond between thrombin and the inhibitor is a carboxylic ester.

Respiratory care protocol: an approach to in-hospital respiratory therapy.

The difficulty of delivering respiratory therapy according to currently accepted standards is an important problem in many hospitals. As a result of this problem in our hospital, we developed a new therapy delivery system--the Respiratory Care Protocol. In response to an order for Respiratory Care Protocol from an attending physician, a senior respiratory therapist evaluates the patient, prescribes specific respiratory therapy according to a protocol, and then daily re-evaluates the patient and makes appropriate therapeutic changes, including discontinuing respiratory therapy when appropriate. The Respiratory Care Protocol has been well-accepted by patients, physicians, and respiratory therapists, and by Joint Commission on Accreditation of Hospitals evaluation teams. We believe that our use of the Respiratory Care Protocol has led to improved quality and to the reduced cost of our in-hospital respiratory care.

Parasite recruitment by stocked walleye, Stizostedion vitreum vitreum (Mitchill), fry in a small boreal lake in central Canada.

Six species of parasites were recovered 4 mo after walleye fry were stocked in Heming Lake, Manitoba. The species of parasites acquired most rapidly were those that were non-host-specific and common to the indigenous populations of both walleye and yellow perch (Perca flavescens). Parasite species overlap (Jaccard's indices) was greatest within age groups of walleye and yellow perch, but was also high between older walleye and yellow perch. The higher numbers of parasites recruited by stocked walleye, particularly ones known to induce pathology, raises questions on the success of walleye introductions to aquatic systems with a diverse indigenous parasite fauna and a fish population with a large proportion of yellow perch.

Liver pathology of yellow perch, Perca flavescens (Mitchill), infected with larvae of the nematode Raphidascaris acus (Bloch, 1779).

Larvae of the nematode Raphidascaris acus were found free or encapsulated in the liver of yellow perch. Blood vessels were distorted or destroyed during larval migrations and larvae were eventually encapsulated in a thick-walled whitish nodule. Successful walling-off of the parasite resulted in the formation of a collagenous nodule and a complete loss of the worm. No mortality of perch was associated with larval R. acus but the introduction of susceptible fishes into a lake harboring this parasite may be important in some stocking programs.

Helminth parasites of pine marten, Martes americana (Turton), from Manitoba, Canada.

Five species of helminths were recovered during a survey of 139 North American pine marten (Martes americana) from three areas of Manitoba: Alaria taxideae in 75 marten; Taenia sp. (cf. martis martis) in 16; Taenia mustelae in nine; Baylisascaris devosi in one; Trichinella sp. larvae in one. Taenia mustelae and Taenia sp. (cf. martis martis) were found in two different areas of the province, Taenia sp. (cf. martis martis) being isolated from the more northerly regions. Alaria taxideae, the most prevalent parasite in the survey, was common to all three areas. The intensity of infection and prevalence level of A. taxideae was significantly higher (P less than 0.05) in the southern region of this study. Altogether, male marten had a significantly higher intensity of A. taxideae compared to females, although there was no significant difference in prevalence level. When data for A. taxideae was combined for sexes and for regions a significantly higher prevalence level in young-of-the-year marten was noted compared to juveniles or adults, but no significant difference in intensities among the three age classes was found. No significant differences were detected in the prevalence of A. taxideae, Taenia sp. (cf. martis martis), or T. mustelae between sexes or among age classes from any of the three areas.