Effect of area-level factors on heat-associated morbidity and mortality in the Eastern Townships

Les épisodes de chaleur extrême sont une cause importante de morbidité et de mortalité au Québec. Des outils de cartographie permettent d’identifier les secteurs géographiques ayant une plus grande prévalence de facteurs de vulnérabilité à la chaleur (économique, sociale, biologique, relative aux caractéristiques du logement ou à l’environnement urbain), mais nous ne connaissons pas leur capacité à prédire correctement où surviennent les problèmes de santé causés par la chaleur dans la région de l’Estrie. Nous avons évalué la distribution territoriale des troubles de santé liés à la chaleur selon les facteurs de vulnérabilité. Des modèles de régression linéaire mixte de Poisson ont été utilisés pour mesurer la modification d’effet causée par treize facteurs de vulnérabilité sur l’association entre la température moyenne horaire sur 24h (Tmoy) et quatre indicateurs sanitaires, séparément : les transports ambulanciers, les consultations à l’urgence, les hospitalisations et les décès, à l’échelle des aires de diffusion (AD) du recensement de la population canadienne de 2016. Trois facteurs agissent à titre de modificateur d’effet aggravant la relation entre la température et les transports ambulanciers ou les hospitalisations : l’indice de maladies chroniques, le pourcentage de population âgée de 65 ans et plus et le pourcentage de population âgée de 65 ans et plus et vivant seul. Deux autres facteurs ont une action de modificateur d’effet : le pourcentage de population consacrant plus de 30 % du revenu au logement (hospitalisations) et le pourcentage de population sous le seuil de faible revenu (consultations à l’urgence). À Tmoy = 27,7°C, l’indice de maladies chroniques est le facteur causant le plus haut rapport de taux d’incidence entre le 5e et le 1er quintile pour les hospitalisations (RTI = 5,24 [I.C. 95% 4,13 – 6,65]) et les transports ambulanciers (RTI = 3,66 [I.C. 95% 3,01 – 4,45]). Un RTI supérieur à 2 a été observé avec onze facteurs de vulnérabilité pour les hospitalisations, sept facteurs pour les transports ambulanciers et deux facteurs pour les consultations à l’urgence. Il n’y avait pas d’association entre la mortalité et la température. Cette étude permettra de mieux cibler les interventions de santé publique pour prévenir la morbidité associée à la chaleur en Estrie.Abstract : Extreme heat episodes cause significant mortality and morbidity in the province of Quebec, Canada. Mapping tools allow us to locate areas with a higher prevalence of specific risk factors for heat-related health problems, which can be of different nature: economic, social, biological, related to housing characteristics or related to the physical urban environment. We sought to quantify the predictive capacity of these area-level risk factors to identify areas with higher heat-related morbidity or mortality in the Eastern Townships region of Quebec. We used Poisson linear mixed regression models to examine effect modification caused by 13 separate area-level risk factors on the association between daily 24h average temperature (Tavg) and daily counts of all-cause ambulance transports, all-cause emergency department visits, heat-related hospitalisations, or all-cause deaths, at the diffusion area (DA) level from the 2016 Canadian census of population. Three factors aggravate the effect of temperature on hospitalisations and ambulance transports: the chronic disease index, the percentage of elderly population and the percentage of elderly population living alone. Two more factors act as effect modifiers for a single health indicator: the percentage of population spending more than 30% of income on shelter costs (for hospitalisations) and the percentage of population below the low income cut-off (for emergency department visits). When Tavg = 27.7°C, the chronic diseases index is the risk factor for which we observed the highest incidence rate ratio (IRR) for hospitalisations (IRR = 5.24 [95 % CI: 4.13 – 6.65]) and ambulance transports (IRR = 3.66 [95 % CI: 3.01 – 4.45]) between the 5th and the 1st quintile of that risk factor. We observed an IRR greater than 2.00 for hospitalisations between the 5th and the 1st quintile for 11 out of 13 factors, while 7 factors caused an IRR greater than 2.0 for ambulance transports and only 2 for emergency department visits. This study will help better target public health interventions to prevent heat-related morbidity in the Eastern Townships region

Defining the role of different KRAS effectors in the initiation and progression of lung cancer

Lung cancer is currently the most deadly malignancy in Canada, accounting for 27% of all cancer-related deaths. Over 70% of patients with non-small cell lung cancer (NSCLC) are diagnosed at a late stage, with a 5-year survival below 10%. In NSCLC, the two oncogenes that are most frequently mutated are the EGFR and KRAS genes. While targeted therapies have been developed for patients with EGFR mutations, oncogenic KRAS mutations are so far not druggable. KRAS is a small GTPase that acts as an on/off switch to activate multiple signalling pathways, including the PI3K/Akt pathway, the Raf-Mek-Erk pathway and the RalGDS/Ral pathway. In the BrafCA mouse model of lung tumourigenesis, it was shown that the Cre-mediated expression of BrafV600E, activating the Raf-Mek-Erk pathway, causes the formation of adenomas that undergo widespread senescence at the benign stage. However, oncogenic KRAS mutations in mice cause adenocarcinomas, which suggests that other pathways activated by KRAS cooperate with sustained RAF-MEK-ERK signalling to bypass the oncogene-induced senescence proliferation arrest. To elucidate which pathways may cooperate with the Raf-Mek-Erk pathway to lead to lung adenocarcinomas, I created four effector domain mutants of KRASV12 (S35, G37, E38 and C40). The S35 and E38 mutants bind to Raf proteins but not PI3K or RalGDS; the G37 mutant binds to RalGDS and not Raf or PI3K and the C40 mutant is specific to PI3K. I designed lentiviral vectors that code for the KRAS mutants (V12, V12/S35, V12/G37, V12/E38 or V12/C40), or eGFP as a negative control, bicistronically with the Cre recombinase. These lentiviruses were used to infect BrafCA/+ and wild-type mice. The biggest tumours seen in BrafCA/+ mice received the KRASV12 virus, followed closely by KRASV12/C40, suggesting that the PI3K and Raf-Mek-Erk pathways cooperate to increase tumour growth. There was a significant decrease in tumour penetrance in all conditions where any KRAS mutant was present compared to the eGFP control, suggesting that KRAS may directly activate effectors with tumour suppressive functions. Moreover, tumours in wild-type mice were only seen with KRASV12 expression.Le cancer du poumon est le cancer affichant la plus grande mortalité au Canada, comptant pour 27% des mortalités liées au cancer. Plus de 70% des patients atteints du cancer du poumon non à petites cellules (CPNPC) sont diagnostiqués à un stade tardif, avec une survie à 5 ans en-dessous de 10%. Les deux oncogènes les plus fréquemment mutés dans le CPNPC sont les gènes KRAS et EGFR. Des thérapies ont été développées pour les patients dont le cancer a le gène EGFR muté, mais aucune médicamentation ciblée n'existe pour les mutations de KRAS. KRAS est une petite protéine GTPase qui agit comme interrupteur marche/arrêt pour l'activation de plusieurs voies de signalisation en aval, dont les voies PI3K/Akt, Raf-Mek-Erk et RalGDS/Ral. Dans le modèle murin de tumorigenèse pulmonaire BrafCA, il a été démontré que la mutation BrafV600E, activant la voie Raf-Mek-Erk, cause la formation d'adénomes qui subissent une induction de sénescence au stade bénin. Des mutations du gène KRAS peuvent causer la formation d'adénocarcinomes, suggérant que d'autres voies de signalisation activées par KRAS coopèrent avec une signalisation soutenue de la voie Raf-Mek-Erk pour contourner l'apparition de sénescence d'induction oncogénique.Pour faire la lumière sur quelles voies de signalisation peuvent coopérer avec la voie Raf-Mek-Erk pour mener aux adénocarcinomes pulmonaires, j'ai créé quatre mutants de KRASV12 (S35, G37, E38, C40). Les mutants S35 et E38 interagissent avec les protéines Raf mais pas PI3K ou RalGDS; le mutant G37 peux lier RalGDS mais pas Raf et PI3K; le mutant C40 est spécifique pour PI3K. J'ai conçu des vecteurs lentiviraux codant pour ces mutants de KRAS (V12, V12/S35, V12/G37, V12/E38, V12/C40), ou l'eGFP comme contrôle négatif, de manière bicistronique avec la recombinase Cre. Des souris BrafCA/+ et d'autres de type sauvage ont été infectées avec ces lentivirus. Les plus grosses tumeurs observées étaient dans des souris BrafCA/+ ayant reçu le virus codant pour KRASV12, suivi de près par le mutant V12/C40, ce qui suggère que les voies PI3K et Raf-Mek-Erk coopèrent positivement dans la croissance tumorale. Il y avait une diminution significative dans le nombre de tumeurs observées dans toutes les conditions où un mutant de KRAS était présent comparativement au contrôle eGFP, suggérant que KRAS pourrait activer directement des effecteurs avec des fonctions suppresseurs de tumeur. De plus, dans les souris de type sauvage, des tumeurs n'ont été observées qu'avec l'expression de KRASV12

Ras Effector Mutant Expression Suggest a Negative Regulator Inhibits Lung Tumor Formation

<div><p>Lung cancer is currently the most deadly malignancy in industrialized countries and accounts for 18% of all cancer-related deaths worldwide. Over 70% of patients with non-small cell lung cancer (NSCLC) are diagnosed at a late stage, with a 5-year survival below 10%. KRAS and the EGFR are frequently mutated in NSCLC and while targeted therapies for patients with EGFR mutations exist, oncogenic KRAS is thus far not druggable. KRAS activates multiple signalling pathways, including the PI3K/Akt pathway, the Raf-Mek-Erk pathway and the RalGDS/Ral pathway. Lung-specific expression of BrafV600E, the most prevalent BRAF mutation found in human tumors, results in Raf-Mek-Erk pathway activation and in the formation of benign adenomas that undergo widespread senescence in a Cre-activated Braf mouse model (Braf^CA). However, oncogenic KRAS expression in mice induces adenocarcinomas, suggesting additional KRAS-activated pathways cooperate with sustained RAF-MEK-ERK signalling to bypass the oncogene-induced senescence proliferation arrest.</p><p>To determine which KRAS effectors were responsible for tumor progression, we created four effector domain mutants (S35, G37, E38 and C40) in G12V-activated KRAS and expressed these alone or with BrafV600E in mouse lungs… The S35 and E38 mutants bind to Raf proteins but not PI3K or RalGDS; the G37 mutant binds to RalGDS and not Raf or PI3K and the C40 mutant is specific to PI3K. We designed lentiviral vectors to code for Cre recombinase along with KRAS mutants (V12, V12/S35, V12/G37, V12/E38 or V12/C40) or EGFP as a negative control.. These lentiviruses were used to infect <i>Braf^CA</i> and wild-type mice. Surprisingly there was a significant decrease in tumor number and penetrance with each KRAS effector domain mutant relative to controls, suggesting that KRAS directly activates effectors with tumor suppressive functions.</p></div

Plasmids used for lentivirus production and subsequent mouse infections/Lentiviral vectors.

<p><b>A</b>) Schematic of a generalized two-plasmid LR recombination reaction between a generalize entry vector containing a cDNA and gLEX-iCL, a Gateway compatible lentivirus encoding Cre(T2A)Luc <b>B</b>) The resulting recombinant lentiviral expression vector when integrated contains a single CMV-driven bicistronic transcript encoding a cDNA (EGFP, KRasG12, or effector domain mutants) and downstream of an ires, Cre(2a)Luc fusion to induce Braf^V600E expression in <i>Braf^CA</i> mice.</p

Median tumor size.

<p>Tumor size was measured by section area at 16 weeks post-infection along with Cre-mediated activation of the <i>Braf^CA</i> allele. Distributions of tumor size for all the KRAS mutants were compared with the LEX-EGFP-iCL negative control with the Mann-Whitney U test (* p<0.05; *** p<0.001; **** p<0.0001). Note median tumor size was significantly larger in KRAS^V12 and KRAS^V12/C40 expressors.</p

KRas expression inhibits tumor formation in after Cre-mediated expression of Braf^V600E in <i>Braf^CA</i>^/+ mice.

<p>Sections of lungs from <i>Braf^CA/+</i> mice infected with LEX-iCL lentiviruses expressing EGFP, KRAS^V12 or the indicated KRAS^V12/ED mutants. Slides were stained with haematoxylin and eosin and are shown at low (upper panels) and high (lower panels) magnification. The box in the upper panels depicts region shown at higher power. All images are representative for each lentiviral construct. Bars, 3 mm.</p

Distribution of size and proliferation of tumors.

<p><i>Braf^CA/+</i> mice were infected with as indicated and tumor size vs. proliferation as measured by Ki67 staining at 16 weeks post-infection. Each dot on the graphs represents a single tumor, for which the surface area and the percentage of KI67-positive cell was determined. The cDNA encoded in each LEX-iCL lentivirus used to infect mice is indicated on each graph.</p

A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo.

The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems