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We used a large national cohort in Canada to assess the incidence of acute myocardial infarction (AMI) and stroke hospitalizations in association with long-term exposure to fine particulate matter (PM 2.5 ), nitrogen dioxide (NO 2 ), and ozone (O 3 ). The study population comprised 2.7 million respondents from the 2006 Canadian Census Health and Environment Cohort (CanCHEC), followed for incident hospitalizations of AMI or stroke between 2006 and 2016. We estimated 10-year moving average estimates of PM 2.5 , NO 2 , and O 3 , annually. We used Cox proportional hazards models to examine the associations adjusting for various covariates. For AMI, each interquartile range (IQR) increase in exposure was found to be associated with a hazard ratio of 1.026 (95% CI: 1.007-1.046) for PM 2.5 , 1.025 (95% CI: 1.001-1.050) for NO 2 , and 1.062 (95% CI: 1.041-1.084) for O 3 , respectively. Similarly, for stroke, an IQR increase in exposure was associated with a hazard ratio of 1.078 (95% CI: 1.052-1.105) for PM 2.5 , 0.995 (95% CI: 0.965-1.030) for NO 2 , and 1.055 (95% CI: 1.028-1.082) for O 3 , respectively. We found consistent evidence of positive associations between long-term exposures to PM 2.5 , and O 3 , and to a lesser degree NO 2 , with incident AMI and stroke hospitalizations., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Owing to their greater outdoor activity and ongoing lung development, children are particularly vulnerable to the harmful effects of exposure to fine particulate matter (PM 2.5 ) . However, the effects of PM 2.5 components are poorly understood. This study aimed to use a longitudinal birth cohort of children with physician-diagnosed incident asthma to investigate the effect of PM 2.5 components at birth on morbidity measured by health services utilization. Of 1277 Toronto Child Health Evaluation Questionnaire (T-CHEQ) participants, the study population included 362 children diagnosed with asthma who were followed for a mean of 13 years from birth until March 31, 2016, or loss-to-follow-up. Concentrations of PM 2.5 and its components were assigned based on participants' postal codes at birth. Study outcomes included counts of asthma, asthma-related, and all-cause health services use. Poisson regression in single-, two-, and multi-pollutant models was used to estimate rate ratios (RR) per interquartile range (IQR) increase of exposures. Covariates were included in all models to further adjust for potential confounding. The adjusted RR for sulfate (SO 4 ) and all-cause hospitalizations was statistically significant with RR = 2.23 (95% confidence interval [CI]: 1.25-3.96) in a multi-pollutant model with nitrogen dioxide (NO 2 ) and ozone (O 3 ). In multi-pollutant models with oxidants, the adjusted RRs for SO 4 of all-cause hospitalizations and emergency department (ED) visits were also statistically significant with RR = 2.31 (95% CI: 1.32-4.03) and RR = 1.39 (95% CI: 1.02-1.90), respectively. While unadjusted single-pollutant RRs for asthma-specific and asthma-related health services use with the SO 4 component of PM 2.5 were above one, none were statistically significant. This study found significant associations with exposure to SO 4 in PM 2.5 and all-cause acute care, chiefly for hospitalizations, in children with asthma., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Individual and neighbourhood-scale socioeconomic characteristics modify associations between exposure to air pollution and mortality. The role of stress, which may integrate effects of social and environmental exposures on health, is unknown. We examined whether an individual's perspective on their own well-being, as assessed using self-rated measures of stress and health, modifies the pollutant-mortality relationship., Methods: The Canadian Community Health Survey (CCHS)-mortality cohort includes respondents from surveys administered between 2001 and 2012 linked to vital statistics and postal codes from 1981 until 2016. Annual fine particulate matter (PM 2.5 ), nitrogen dioxide (NO 2 ), and ozone (O 3 ) exposure estimates were attached to a sample of cohort members aged 30-89 years (n = 398,300 respondents/3,848,400 person-years). We examined whether self-rated stress, distress, mental health, and general health modified associations between long-term exposure to each pollutant (three-year moving average with one-year lag) and non-accidental mortality using Cox survival models, adjusted for individual- (i.e. socioeconomic and behavioural) and neighbourhood-scale covariates., Results: In fully-adjusted models, the relationship between exposure to pollutants and mortality was stronger among those with poor self-rated mental health, including a significant difference for NO 2 (hazard ratio (HR) = 1.15, 95% CI 1.06-1.25 per IQR) compared to those with very good/excellent mental health (HR = 1.05, 95% CI 1.01-1.08; Cochran's Q = 4.01; p < 0.05). Poor self-rated health was similarly associated with higher pollutant-associated HRs, but only in unadjusted models. Stress and distress did not modify pollutant-mortality associations., Conclusions: Poor self-rated mental and general health were associated with increased mortality attributed to exposure to ambient pollutants., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Plasmodium sporozoites are able to migrate through host cells by breaching their plasma membrane and gliding inside their cytoplasm. This migratory activity, called cell traversal (CT), was studied in vivo mainly using mutant sporozoites lacking the ability to wound host cells, and thus to perform CT. However, direct evidence of CT activity in host tissues by wild-type sporozoites remains scarce. Here, we describe a double-wounding assay to dynamically image CT activity in vivo and monitor cell membrane integrity over time. Based on the incorporation kinetics of a first live cell-impermeant dye, propidium iodide, we could determine whether traversed cells repair their wounded membranes or not. A second impermeant dye, SYTOX Green, was used to confirm the transient or the permanent loss of membrane integrity of traversed cells. This assay allowed, for the first time, the direct observation of sporozoites wounding and traversing host skin cells and showed that, while some traversed cells resealed their membrane, most became irreversibly permeable to these live cell-impermeant dyes. In combination with the study of CT-deficient sporozoites and the use of specific host cell markers, this intravital assay will provide the means to identify the nature of the cells traversed by sporozoites and will thus contribute to elucidating the role of CT by apicomplexan parasites in the vertebrate host., (© 2013.)

Type III secretion systems (T3SS) are present in many pathogenic gram-negative bacteria and mediate the translocation of bacterial effector proteins into host cells. Here, we report the phenotypic characterization of S. flexneri ipgB1 and ipgB2 mutants, in which the genes encoding the IpgB1 and IpgB2 effectors have been inactivated, either independently or simultaneously. Like IpgB1, we found that IpgB2 is secreted by the T3SS and its secretion requires the Spa15 chaperone. Upon infection of semi-confluent HeLa cells, the ipgB2 mutant exhibited the same invasive capacity as the wild-type strain and the ipgB1 mutant was 50% less invasive. Upon infection of polarised Caco2-cells, the ipgB2 mutant did not show a significant defect in invasion and the ipgB1 mutant was slightly more invasive than the wild-type strain. Entry of the ipgB1 ipgB2 mutant in polarized cells was reduced by 70% compared to the wild-type strain. Upon infection of the cornea in Guinea pigs, the ipgB2 mutant exhibited a wild-type phenotype, the ipgB1 mutant was hypervirulent and elicited a more pronounced proinflammatory response, while the ipgB1 ipgB2 mutant was highly attenuated. The attenuated phenotype of the ipgB1 ipgB2 mutant was confirmed using a murine pulmonary model of infection and histopathology and immunochemistry studies.

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) carries out N-terminal processing of the viral replicase polyprotein, and also exhibits Lys48-linked polyubiquitin chain debranching and ISG15 precursor processing activities in vitro. Here, we used SDS-PAGE and fluorescence-based assays to demonstrate that ISG15 derivatives are the preferred substrates for the deubiquitinating activity of the PLpro. With k(cat)/K(M) of 602,000 M(-1)s(-1), PLpro hydrolyzes ISG15-AMC 30- and 60-fold more efficiently than Ub-AMC and Nedd8-AMC, respectively. Data obtained with truncated ISG15 and hybrid Ub/ISG15 substrates indicate that both the N- and C-terminal Ub-like domains of ISG15 contribute to this preference. The enzyme also displays a preference for debranching Lys48- over Lys63-linked polyubiquitin chains. Our results demonstrate that SARS-CoV PLpro can differentiate between ubiquitin-like modifiers sharing a common C-terminal sequence, and that the debranching activity of the PLpro is linkage type selective. The potential structural basis for the demonstrated specificity of SARS-CoV PLpro is discussed.

Cathepsin X is a papain-like cysteine protease with restricted positional specificity, acting primarily as a carboxy-monopeptidase. We mapped the specificities at the S2, S1, and S1' subsites of human cathepsin X by systematically and independently substituting the P2, P1, and P1' positions of the carboxy-monopeptidase substrate Abz-FRF(4NO(2)) with natural amino acids. Human cathepsin X has broad S2, S1, and S1' specificities within two orders of magnitude in k(cat)/K(M), excluding proline that is not tolerated at these subsites. Glycine is not favored in S2, but is among the preferred residues in S1 and S1', which highlights S2 as the affinity-determinant subsite. The presence of peculiar residues at several binding site positions (Asp76, His234, Asn75, and Glu72) does not translate into a markedly different sequence specificity profile relative to other human cathepsins. These findings suggest that a specific function of human cathepsin X is unlikely to result from sequence specificity, but rather from a combination of its unique positional specificity and the co-localization of enzyme and substrate in a specific cellular environment.

A noninvasive cell-based assay has been developed to monitor the proteolytic activity of cathepsin L within a specific subcellular compartment, the lysosome. The green fluorescent protein (GFP) of Aequorea victoria was selected as a substrate. Targeting to lysosomes was achieved by fusing GFP to preprocathepsin L, which also ensures colocalization of the enzyme and the substrate. Stably transfected HeLa-rtTA (reverse tetracycline-controlled transactivator) cells were induced with doxycycline and cultured in the presence of various concentrations of cysteine protease inhibitors for 48 h. In the absence of inhibitor, proteolytic degradation of GFP leads to loss of fluorescence, which is due almost exclusively to the action of recombinant cathepsin L. However, a dose-dependent increase of GFP fluorescence is observed for cells treated with the potent cathepsin L inhibitor benzyloxycarbonyl-LeuLeuTyr-CHN(2). Fluorescence is also observed when GFP is fused to an inactive preprocathepsin L (C25A mutant). Targeting of GFP to an acidic cellular compartment can destabilize the protein and render it susceptible to proteolytic degradation. The approach should be generally applicable for proteases localized in acidic environments. Such an assay can be of great value in validating the participation of a specific enzyme in a given process or in testing the ability of putative inhibitors to reach their intracellular target.

Malaria is transmitted to a mammalian host when the sporozoite stage of the Plasmodium parasite is injected by a mosquito vector. Sporozoites are unique in being able to interact with both hosts. Formed and released in the mosquito midgut, sporozoites bind to the salivary glands and invade their secretory cells. Once injected into the mammalian host, they home to the liver and invade hepatocytes. Recent work has shown that two sporozoite surface proteins, CS and TRAP, act in both hosts, perform multiple functions, and are each essential for the parasite at more than one step of its life cycle.

Human cathepsin X is one of many proteins discovered in recent years through the mining of sequence databases. Its sequence shows clear homology to cysteine proteases from the papain family, containing the characteristic residue patterns, including the active site. However, the proregion of cathepsin X is only 38 residues long, the shortest among papain-like enzymes, and the cathepsin X sequence has an atypical insertion in the regions proximal to the active site. This protein was recently expressed and partially characterized biochemically. Unlike most other cysteine proteases from the papain family, procathepsin X is incapable of autoprocessing in vitro but can be processed under reducing conditions by exogenous cathepsin L. Atypically, the mature enzyme is primarily a carboxypeptidase and has extremely poor endopeptidase activity. We have determined the three-dimensional structure of the procathepsin X at 1.7 A resolution. The overall structure of the mature enzyme is characteristic for enzymes of the papain superfamily, but contains several novel features. Most interestingly, the short proregion binds to the enzyme with the aid of a covalent bond between the cysteine residue in the proregion (Cys10p) and the active site cysteine residue (Cys31). This is the first example of a zymogen in which the inhibition of enzyme's proteolytic activity by the proregion is achieved through a reversible covalent modification of the active site nucleophile. Such mode of binding requires less contact area between the proregion and the enzyme than observed in other procathepsins, and no auxiliary binding site on the enzyme surface is used. A three-residue insertion in a highly conserved region, just prior to the active site cysteine residue, confers a significantly different shape on the S' subsites, compared to other proteases from papain family. The 3D structure provides an explanation for the rather unusual carboxypeptidase activity of this enzyme and confirms the predictions based on homology modeling. Another long insertion in the cathepsin X amino acid sequence forms a beta-hairpin pointing away from the active site. This insertion, thought to be an equivalent of cathepsin B occluding loop, is located on the side of the protein, distant from the substrate binding site., (Copyright 2000 Academic Press.)

The aim of this study was to optimize by micellar electrokinetic chromatography the separation of four glucosinolates, i.e. sinigrin, glucobrassicin and methoxyglucobrassicin involved in Cruciferae resistance mechanisms and glucotropaeolin used as an internal standard. The separation borate buffer which contained sodium dodecyl sulphate, tetramethylammonium hydroxide and methanol was firstly optimized by using a three variable Doehlert experimental design. The optimum concentrations found enabled, for the first time, to obtain an acceptable resolution between the two indole glucosinolates, glucobrassicin and methoxyglucobrassicin. Modifications of the method such as a capillary pre-rinse with pure borate buffer and a step change in voltage during experiment were performed to improve the resolutions between glucosinolates and to reduce the analysis time. This method was validated by a statistical analysis and showed good linearity, repeatability and reproducibility.

A novel human cDNA encoding a cysteine protease of the papain family named cathepsin F is reported. The mature part of the predicted protease precursor displays between 26% and 42% identity to other human cysteine proteases while the proregion is unique by means of length and sequence. The very long proregion of the cathepsin F precursor (251 amino acid residues) can be divided into three regions: a C-terminal domain similar to the pro-segment of cathepsin L-like enzymes, a 50 residue flexible linker peptide, and an N-terminal domain predicted to adopt a cystatin-like fold. Cathepsin F would therefore be the first cysteine protease zymogen containing a cystatin-like domain., (Copyright 1999 Academic Press.)