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Concept: Hypochlorous acid

137

Hypochlorous acid (HOCl) is produced naturally by neutrophils and other cells to kill conventional microbes in vivo. Synthetic preparations containing HOCl can also be effective as microbial disinfectants. Here we have tested whether HOCl can also inactivate prions and other self-propagating protein amyloid seeds. Prions are deadly pathogens that are notoriously difficult to inactivate, and standard microbial disinfection protocols are often inadequate. Recommended treatments for prion decontamination include strongly basic (pH ≥~12) sodium hypochlorite bleach, ≥1 N sodium hydroxide, and/or prolonged autoclaving. These treatments are damaging and/or unsuitable for many clinical, agricultural and environmental applications. We have tested the anti-prion activity of a weakly acidic aqueous formulation of HOCl (BrioHOCl) that poses no apparent hazard to either users or many surfaces. For example, BrioHOCl can be applied directly to skin and mucous membranes and has been aerosolized to treat entire rooms without apparent deleterious effects. Here, we demonstrate that immersion in BrioHOCl can inactivate not only a range of target microbes, including spores of Bacillus subtilis, but also prions in tissue suspensions and on stainless steel. Real-time quaking-induced conversion (RT-QuIC) assays showed that BrioHOCl treatments eliminated all detectable prion seeding activity of human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, cervine chronic wasting disease, sheep scrapie and hamster scrapie; these findings indicated reductions of ≥103- to 106-fold. Transgenic mouse bioassays showed that all detectable hamster-adapted scrapie infectivity in brain homogenates or on steel wires was eliminated, representing reductions of ≥~105.75-fold and >104-fold, respectively. Inactivation of RT-QuIC seeding activity correlated with free chlorine concentration and higher order aggregation or destruction of proteins generally, including prion protein. BrioHOCl treatments had similar effects on amyloids composed of human α-synuclein and a fragment of human tau. These results indicate that HOCl can block the self-propagating activity of prions and other amyloids.

Concepts: Acid, Prion, Chlorine, Hypochlorous acid, Scrapie, Transmissible spongiform encephalopathy, Bovine spongiform encephalopathy, Sodium hypochlorite

28

To evaluate the antimicrobial effect of a diode laser irradiation, photo-activated disinfection (PAD), conventional and sonic activated irrigation with 2.5% sodium hypochlorite (NaOCl) on Enterococcus faecalis.

Concepts: Hydrogen peroxide, Laser diode, Chlorine, Disinfectant, Hypochlorous acid, Sodium hypochlorite, Enterococcus faecalis, Bleach

28

To the best of our knowledge, there was little information available on pathogen removal using low level disinfectant followed by free chlorine in sequential disinfection (SD). This study investigated Escherichia coli inactivation by four types of disinfection: single step disinfection (SSD), SD, traditional sequential disinfection (TSD) and mixed disinfectant disinfection (MDD). Results indicated that SD had higher ability to inactivate E. coli than the others, indicating there was a positive synergistic effect on chlorine disinfection by prior dosing with a low level of chlorine dioxide (ClO(2)). The ONPG assay suggested that the permeability of cell wall rather than the viability of E. coli were changed under 0.02 mg/l ClO(2) treatment. The coexistence of residual ClO(2) and free chlorine also plays an active synergistic effect. Additionally, temperature had a positive effect on E. coli inactivation in SD, while inactivation was reduced in alkaline compared to neutral and acidic conditions.

Concepts: Bacteria, Escherichia coli, Enterobacteriaceae, Chlorine, Disinfectant, Hypochlorous acid, Chlorine dioxide, Bleach

27

Aloe vera (Syn Aloe barbadensis Mill.), a medicinal plant, has a great potential in cosmetic and drug industry due to presence of more than 200 bioactive compounds. Natural propagation of Aloe vera, by means of suckers, is very slow and insufficient to meet the increasing demand of pharmaceutical and cosmetic industries. Shoot tip was used as an explant for in vitro regeneration of Aloe vera. Explants were disinfested with the use of 0.1% mercuric chloride and 0.5% sodium hypochlorite, and washed thoroughly with autoclaved distilled water. Solid MS medium was used with addition of different concentrations of 6-benzyl aminopurine and α-naphthalene acitic acid. After 7 weeks of inoculation, greatest number of shoots (11.18) and highest shoot length (12.15cm) were found in MS medium supplemented with 0.5 mg l-1 6-benzylaminopurine (BAP) along with same concentration of α-naphthalene acitic acid (NAA). Best rooting (84.67%) was found in medium supplemented with 1.5 mg l-1 of indole butyric acid (IBA). The rooted explants were then gradually acclimatized and shifted to green house.

Concepts: Pharmacology, Water, Mercury, Chlorine, Hypochlorous acid, Butyric acid, Aloe vera, Aloe

27

Abstract Myeloperoxidase (MPO), a major constituent of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide (H(2)O(2)) and chloride anion. We have previously reported that MPO-deficient (MPO(-/-)) neutrophils produce greater amount of macrophage inflammatory protein-2 (MIP-2) in vitro than do wild-type when stimulated with zymosan. In this study, we investigated the molecular mechanisms governing the up-regulation of MIP-2 production in the mutant neutrophils. Interestingly, we found that zymosan-induced production of MIP-2 was blocked by pre-treatment with U0126, an inhibitor of mitogen-activated protein kinase/extracellular-signal regulated kinase (ERK), and with BAY11-7082, an inhibitor of nuclear factor (NF)-κB. Western blot analysis indicated that U0126 also inhibited the phosphorylation of p65 subunit of NF-κB (p65), indicating that MIP-2 was produced via the ERK/NF-κB pathway. Intriguingly, we found that ERK1/2, p65, and alpha subunit of inhibitor of κB (IκBα) in the MPO(-/-) neutrophils were phosphorylated more strongly than in the wild-type when stimulated with zymosan. Exogenous H(2)O(2) treatment in addition to zymosan stimulation enhanced the phosphorylation of ERK1/2 without affecting the zymosan-induced MIP-2 production. In contrast, exogenous HOCl inhibited the production of MIP-2 as well as IκBα phosphorylation without affecting ERK activity. The zymosan-induced production of MIP-2 in the wild-type neutrophils was enhanced by pre-treatment of the MPO inhibitor 4-Aminobenzoic acid hydrazide. Collectively, these results strongly suggest that both lack of HOCl and accumulation of H(2)O(2 )due to MPO deficiency contribute to the up-regulation of MIP-2 production in mouse neutrophils stimulated with zymosan.

Concepts: Oxygen, Acid, Hydrogen, Western blot, Chlorine, Hypochlorous acid, Myeloperoxidase, Myeloperoxidase deficiency

27

A BODIPY-based fluorescent probe, HCSe, has been successfully developed for the rapid detection of hypochlorous acid based on the specific HOCl-promoted oxidation of diphenyl selenide in response to the amount of HOCl. Confocal fluorescence microscopy imaging using RAW264.7 cells showed that the new probe HCSe could be used as an effective fluorescent probe for detecting HOCl in living cells.

Concepts: Protein, Fluorescence, Acid, Microscope, Chlorine, Hypochlorous acid, Fluorescence microscope

25

We aimed to evaluate the effectiveness of low-concentration hypochlorous acid (HOCl) nasal irrigation compared to isotonic normal saline for pediatric chronic rhinosinusitis.

Concepts: Acid, Sinusitis, Chlorine, Hypochlorous acid, Saline, Nasal irrigation

24

Chlorine is a public health concern and potential threat due to its high reactivity, ease and scale of production, widespread industrial use, bulk transportation, massive stockpiles and history as a chemical weapon. This work describes a new, sensitive and rapid stable isotope dilution method for the retrospective detection and quantitation of two chlorine adducts. The biomarkers 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (Cl2-Tyr) were isolated from the pronase digest of chlorine exposed whole blood, serum or plasma by solid-phase extraction (SPE), separated by reversed-phase HPLC and detected by tandem mass spectrometry (MS-MS). The calibration range is 2.50-1,000 ng/mL (R(2) ≥ 0.998) with a lowest reportable limit (LRL) of 2.50 ng/mL for both analytes, an accuracy of ≥93% and an LOD of 0.443 ng/mL for Cl-Tyr and 0.396 ng/mL for Cl2-Tyr. Inter- and intra-day precision of quality control samples had coefficients of variation of ≤10% and ≤7.0%, respectively. Blood and serum samples from 200 healthy individuals and 175 individuals with chronic inflammatory disease were analyzed using this method to assess background levels of chlorinated tyrosine adducts. Results from patients with no known inflammatory disease history (healthy) showed baseline levels of

Concepts: Mass spectrometry, Chlorine, Hypochlorous acid, Isotope, Chemical warfare, Sodium hypochlorite, Chlorination

20

In June 2015, personnel from California’s Contra Costa Health Services Environmental Health and Hazardous Materials (hazmat) divisions were alerted to a possible chemical release at a swimming pool in an outdoor municipal water park. Approximately 50 bathers were in the pool when symptoms began; 34 (68%) experienced vomiting, coughing, or eye irritation. Among these persons, 17 (50%) were treated at the scene by Contra Costa’s Emergency Medical Services (EMS) and released, and 17 (50%) were transported to local emergency departments; five patients also were evaluated later at an emergency department or by a primary medical provider. Environmental staff members determined that a chemical controller malfunction had allowed sodium hypochlorite and muriatic acid (hydrochloric acid) solutions to be injected into the main pool recirculation line while the recirculation pump was off; when the main recirculation pump was restarted, toxic chlorine gas (generated by the reaction of concentrated sodium hypochlorite and muriatic acid) was released into the pool. A review of 2008-2015 California pesticide exposure records identified eight additional such instances of toxic chlorine gas releases at public aquatic venues caused by equipment failure or human error that sickened 156 persons. Chemical exposures at public aquatic venues can be prevented by proper handling, storage, and monitoring of pool chemicals; appropriate equipment operation and maintenance; training of pool operators and staff members on pool chemical safety; and reporting of chemical exposures.

Concepts: Hydrogen, Sodium chloride, Chlorine, Hypochlorous acid, Hydrogen chloride, Hydrochloric acid, Swimming pool, Sodium carbonate

14

To prevent Ebola transmission, frequent handwashing is recommended in Ebola Treatment Units and communities. However, little is known about which handwashing protocol is most efficacious. We evaluated six handwashing protocols (soap and water, alcohol-based hand sanitizer (ABHS), and 0.05% sodium dichloroisocyanurate, high-test hypochlorite, and stabilized and non-stabilized sodium hypochlorite solutions) for 1) efficacy of handwashing on the removal and inactivation of non-pathogenic model organisms and, 2) persistence of organisms in rinse water. Model organisms E. coli and bacteriophage Phi6 were used to evaluate handwashing with and without organic load added to simulate bodily fluids. Hands were inoculated with test organisms, washed, and rinsed using a glove juice method to retrieve remaining organisms. Impact was estimated by comparing the log reduction in organisms after handwashing to the log reduction without handwashing. Rinse water was collected to test for persistence of organisms. Handwashing resulted in a 1.94-3.01 log reduction in E. coli concentration without, and 2.18-3.34 with, soil load; and a 2.44-3.06 log reduction in Phi6 without, and 2.71-3.69 with, soil load. HTH performed most consistently well, with significantly greater log reductions than other handwashing protocols in three models. However, the magnitude of handwashing efficacy differences was small, suggesting protocols are similarly efficacious. Rinse water demonstrated a 0.28-4.77 log reduction in remaining E. coli without, and 0.21-4.49 with, soil load and a 1.26-2.02 log reduction in Phi6 without, and 1.30-2.20 with, soil load. Chlorine resulted in significantly less persistence of E. coli in both conditions and Phi6 without soil load in rinse water (p<0.001). Thus, chlorine-based methods may offer a benefit of reducing persistence in rinse water. We recommend responders use the most practical handwashing method to ensure hand hygiene in Ebola contexts, considering the potential benefit of chlorine-based methods in rinse water persistence.

Concepts: Escherichia coli, Sodium, Chlorine, Hypochlorous acid, Hygiene, Hand washing, Sodium hypochlorite, Hand sanitizer