Surveying the local public health response to COVID-19 in Canada: Study protocol.

BACKGROUND: Public health services and systems research is under-developed in Canada and this is particularly the case with respect to research on local public health unit operational functioning and capacity. The purpose of this paper is to report on a study that will collect retrospective information on the local public health response to COVID-19 throughout Canada between 2020 and 2021. METHODS/DESIGN: The goal of the study is to develop and implement a study framework that will collect retrospective information on the local public health system response to the COVID-19 pandemic in Canada. This study will involve administering a mixed-method survey to Medical Health Officers/Medical Officers of Health in every local and regional public health unit across the country, followed by a process of coding and grouping these responses in a consistent and comparable way. Coded responses will be assessed for patterns of divergent or convergent roles and approaches of local public health across the country with respect to interventions in their response to COVID-19. The Framework Method of thematic analysis will be applied to assess the qualitative answers to the open-ended questions that speak to public health policy features. DISCUSSION: The strengths of the study protocol include the engagement of Medical Health Officers/Medical Officers of Health as research partners and a robust integrated knowledge translation approach to further public health services and systems research in Canada.

Cadmium disrupts melanocortin 2 receptor signaling in rainbow trout.

Cadmium is an endocrine disruptor and inhibits corticosteroid production, but the mechanisms are far from clear. We tested the hypothesis that sublethal exposure to environmentally realistic levels of cadmium impairs cortisol production by disrupting the melanocortin 2 receptor (MC2R) signaling in rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of cadmium (0.75 or 2.0 µg/L) in a flow-through system for 7 d and subjected to an acute secondary stressor to evoke a cortisol response. Cadmium exposure for 7 d did not affect plasma cortisol concentrations, but head kidney mc2r mRNA levels were higher than in control fish. The cortisol stress performance to a secondary-stressor was attenuated in the cadmium groups, and this corresponded with transient reduction in transcript abundance of mc2r and the gene encoding its accessory protein MRAP1 but not MRAP2 in the head kidney. Furthermore, in vivo cadmium exposure attenuated the adrenocorticotropic hormone (ACTH)-, but not 8-br-cAMP-stimulated cortisol production in head kidney slices ex vivo. This corresponded with reduced transcript abundance of mc2r and mrap1, but not mrap2 in these tissue slices. Also, reporter assays with CHO cells transiently transfected with rainbow trout mc2r and zebrafish mrap1 revealed a dose-independent inhibition in ACTH-stimulated luciferase activity by cadmium. Collectively, waterborne exposure to environmentally realistic concentration of cadmium compromises the stressor-induced cortisol response, and a mode of action involves the disruption of MC2R signaling in rainbow trout.

Utility of Transient Elastography in Estimating Hepatic Iron Concentration in Comparison to Magnetic Resonance Imaging in Patients Who are Transfusion-Dependent: A Canadian Center Experience.

Transfusion-dependent hereditary anemias such as ß-thalassemia (ß-thal), predispose patients to iron overload and its numerous clinical sequelae. Accurate assessment of overall iron status and prompt initiation of chelation therapy to prevent irreversible end-organ damage can be achieved using magnetic resonance imaging (MRI) to measure liver iron concentration (LIC) as a surrogate marker of total body iron; however, its access may be associated with long wait times and delay in treatment. We report an observational cohort study at a single tertiary care center assessing the theoretical role of transient elastography (TE), which measures liver stiffness, in estimating LIC compared to other established diagnostic measures. While regression analyses confirm a moderate correlation between LIC per R2 MRI and serum ferritin level (pooled estimate of correlation = 0.55), there was no significant correlation between TE reading and LIC based on R2 MRI (pooled estimate of correlation = -0.06), and only a weak correlation was observed with serum ferritin level (pooled estimate of correlation = 0.45). These results suggest TE may not be sensitive enough to detect subtle changes in the hepatic parenchymal stiffness associated with liver iron deposition.

Identifying the activation motif in the N-terminal of rainbow trout and zebrafish melanocortin-2 receptor accessory protein 1 (MRAP1) orthologs.

The activation of mammalian melanocortin-2 receptor (MC2R) orthologs is dependent on a four-amino acid activation motif (LDYL/I) located in the N-terminal of mammalian MRAP1 (melanocortin-2 receptor accessory protein). Previous alanine substitution analysis had shown that the Y residue in this motif appears to be the most important for mediating the activation of mammalian MC2R orthologs. Similar, but not identical amino acid motifs were detected in rainbow trout MRAP1 (YDYL) and zebrafish MRAP1 (YDYV). To determine the importance of these residues in the putative activation motifs, rainbow trout and zebrafish MRAP1 orthologs were individually co-expressed in CHO cells with rainbow trout MC2R, and the activation of this receptor with either the wild-type MRAP1 ortholog or alanine-substituted analogs of the two teleost MRAP1s was analyzed. Alanine substitutions at all four amino acid positions in rainbow trout MRAP1 blocked activation of the rainbow trout MC2R. Single alanine substitutions of the D and Y residues in rainbow trout and zebrafish MRAP1 indicate that these two residues play a significant role in the activation of rainbow trout MC2R. These observations indicate that there are subtle differences in the way that teleost and mammalian MRAPs are involved in the activation of their corresponding MC2R orthologs.

Structure/function studies on the activation of the rainbow trout melanocortin-2 receptor.

Functional expression of the rainbow trout (rt) melanocortin-2 receptor (MC2R) in CHO cells requires co-expression with a teleost melanocortin-2 receptor accessory protein (MRAP) such as zebrafish (zf) MRAP. Transiently transfected rtMC2R/zfMRAP1 CHO cells were used to evaluate the efficacy of alanine substituted analogs of hACTH(1-24) in three motifs in the ligand: H(6)F(7)R(8)W(9), G(10)K(11)P(12)V(13)G(14), and K(15)K(16)R(17)R(18)P(19). Alanine substitution at all positions in each motif either completely blocked activation of the receptor (H(6)F(7)R(8)W(9) and K(15)K(16)R(17)R(18)P(19)) or resulted in just over 400 fold increase in EC50 value (G(10)K(11)P(12)V(13)G(14)). Single alanine substitutions in the H(6)F(7)R(8)W(9) motif indicated that substitution at either W(9) or R(8) resulted in a much larger increase in EC50 values as compared to substitutions at either F(7) or W(9). Alanine substitution at either K(15)K(16) or R(17)R(18)P(19) in the K(15)K(16)R(17)R(18)P(19) motif resulted in a statistically equivalent increase in EC50 value of at least 600 fold. Finally, alanine substitutions in the G(10)K(11)P(12)V(13)G(14) motif resulted in increases in EC50 values presumably as a result of altering the secondary structure of the ligand. However, truncated analogs of hACTH(1-24) in which either G(10)G(14) (ACTH(1-22), or K(11)P(12)V(13) (ACTH(1-21) were removed had no stimulatory activity. Finally, some of the hACTH(1-24) analogs were tested using rainbow trout head kidney pieces in vitro to confirm whether the response to analogs seen with the transient transfected rtMC2R CHO cells was similar to that of trout interrenal cells. The results of these alanine substitution analog studies are used to construct a multistep hypothetical model for the activation of teleost and tetrapod MC2Rs to account for the unique ligand selectivity of this receptor.

Exposure to environmental levels of waterborne cadmium impacts corticosteroidogenic and metabolic capacities, and compromises secondary stressor performance in rainbow trout.

The physiological responses to waterborne cadmium exposure have been well documented; however, few studies have examined animal performances at low exposure concentrations of this metal. We tested the hypothesis that longer-term exposure to low levels of cadmium will compromise the steroidogenic and metabolic capacities, and reduce the cortisol response to a secondary stressor in fish. To test this, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to 0 (control), 0.75 or 2.0 µg/L waterborne cadmium in a flow-through system and were sampled at 1, 7 and 28 d of exposure. There were only very slight disturbances in basal plasma cortisol, lactate or glucose levels in response to cadmium exposure over the 28 d period. Chronic cadmium exposure significantly affected key genes involved in corticosteroidogenesis, including melanocortin 2 receptor, steroidogenic acute regulatory protein and cytochrome P450 side chain cleavage enzyme. At 28 d, the high cadmium exposure group showed a significant drop in the glucocorticoid receptor and mineralocorticoid receptor protein expressions in the liver and brain, respectively. There were also perturbations in the metabolic capacities in the liver and gill of cadmium-exposed trout. Subjecting these fish to a secondary handling disturbance led to a significant attenuation of the stressor-induced plasma cortisol, glucose and lactate levels in the cadmium groups. Collectively, although trout appears to adjust to subchronic exposure to low levels of cadmium, it may be at the cost of impaired interrenal steroidogenic and tissue-specific metabolic capacities, leading to a compromised secondary stress performance in rainbow trout.

Cadmium-mediated disruption of cortisol biosynthesis involves suppression of corticosteroidogenic genes in rainbow trout.

Cadmium is widely distributed in the aquatic environment and is toxic to fish even at sublethal concentrations. This metal is an endocrine disruptor, and one well established role in teleosts is the suppression of adrenocorticotrophic hormone (ACTH)-stimulated cortisol biosynthesis by the interrenal tissue. However the mechanism(s) leading to this steroid suppression is poorly understood. We tested the hypothesis that cadmium targets genes encoding proteins critical for corticosteroid biosynthesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc), in rainbow trout (Oncorhynchus mykiss). To test this, head kidney slices (containing the interrenal tissues) were incubated in vitro with cadmium chloride (0, 10, 100 and 1000nM) for 4h either in the presence or absence of ACTH (0.5IU/mL). In the unstimulated head kidney slices, cadmium exposure did not affect basal cortisol secretion and the mRNA levels of MC2R and P450scc, while StAR gene expression was significantly reduced. Cadmium exposure significantly suppressed ACTH-stimulated cortisol production in a dose-related fashion. This cadmium-mediated suppression in corticosteroidogenesis corresponded with a significant reduction in MC2R, StAR and P450scc mRNA levels in trout head kidney slices. The inhibition of ACTH-stimulated cortisol production and suppression of genes involved in corticosteroidogenesis by cadmium were completely abolished in the presence of 8-Bromo-cAMP (a cAMP analog). Overall, cadmium disrupts the expression of genes critical for corticosteroid biosynthesis in rainbow trout head kidney slices. However, the rescue of cortisol production as well as StAR and P450scc gene expressions by cAMP analog suggests that cadmium impact occurs upstream of cAMP production. We propose that MC2R signaling, the primary step in ACTH-induced cortocosteroidogenesis, is a key target for cadmium-mediated disruption of cortisol production in trout.