Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells.

SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main challenges is the ongoing emergence of SARS-CoV-2 variants that carry mutations within the RBD, resulting in the constant need to design and optimise the production of new recombinant protein variants. We describe here the impact of naturally occurring RBD mutations on the secretion of a recombinant Fc-tagged RBD protein expressed in HEK 293 cells. We show that mutation E484K of the B.1.351 variant interferes with the proper disulphide bond formation and folding of the recombinant protein, resulting in its retention into the endoplasmic reticulum (ER) and reduced protein secretion. Accumulation of the recombinant B.1.351 RBD-Fc fusion protein in the ER correlated with the upregulation of endogenous ER chaperones, suggestive of the unfolded protein response (UPR). Overexpression of the chaperone and protein disulphide isomerase PDIA2 further impaired protein secretion by altering disulphide bond formation and increasing ER retention. This work contributes to a better understanding of the challenges faced in producing mutant RBD proteins and can assist in the design of optimisation protocols.

Comparative study of chikungunya Virus-Like Particles and Pseudotyped-Particles used for serological detection of specific immunoglobulin M.

The incidence of chikungunya virus (CHIKV) infection has increased dramatically in recent decades. Effective diagnostic methods must be available to optimize patient management. IgM-capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) is routinely used for the detection of specific CHIKV IgM. This method requires inactivated CHIKV viral lysate (VL). The use of viral bioparticles such as Virus-Like Particles (VLPs) and Pseudotyped-Particles (PPs) could represent an alternative to VL. Bioparticles performances were established by MAC-ELISA; physico-chemical characterizations were performed by field-flow fractionation (HF5) and confirmed by electron microscopy. Non-purified PPs give a detection signal higher than for VL. Results suggested that the signal difference observed in MAC-ELISA was probably due to the intrinsic antigenic properties of particles. The use of CHIKV bioparticles such as VLPs and PPs represents an attractive alternative to VL. Compared to VL and VLPs, non-purified PPs have proven to be more powerful antigens for specific IgM capture.

Expression, refolding and bio-structural analysis of a tetravalent recombinant dengue envelope domain III protein for serological diagnosis.

Dengue is a mosquito-borne disease caused by four genetically and serologically related viruses that affect several millions of people. Envelope domain III (EDIII) of the viral envelope protein contains dengue virus (DENV) type-specific and DENV complex-reactive antigenic sites. Here, we describe the expression in Escherichia coli, the refolding and bio-structural analysis of envelope domain III of the four dengue serotypes as a tetravalent dengue protein (EDIIIT2), generating an attractive diagnostic candidate. In vitro refolding of denatured EDIIIT2 was performed by successive dialysis with decreasing concentrations of chaotropic reagent and in the presence of oxidized glutathione. The efficiency of refolding was demonstrated by protein mobility shifting and fluorescent visualization of labeled cysteine in non-reducing SDS-PAGE. The identity and the fully oxidized state of the protein were verified by mass spectrometry. Analysis of the structure by fluorescence, differential scanning calorimetry and circular dichroism showed a well-formed structural conformation mainly composed of ß-strands. A label-free immunoassay based on biolayer interferometry technology was subsequently used to evaluate antigenic properties of folded EDIIIT2 protein using a panel of dengue IgM positive and negative human sera. Our data collectively support the use of an oxidatively refolded EDIIIT2 recombinant chimeric protein as a promising antigen in the serological diagnosis of dengue virus infections.

Educational inequality in stillbirth: temporal trends in Québec from 1981 to 2009.

OBJECTIVE: Educational inequality in stillbirth has been documented in high-income countries and the province of Québec, Canada, but temporal trends are poorly understood. Our objective was to determine time trends in inequality related to maternal education for all-cause and cause-specific stillbirth over the past three decades in Québec. METHODS: We included 2,397,971 live births and 9,983 stillbirths from 1981 through 2009 using Québec vital statistics. For each decade, we computed measures of inequality capturing relative (relative index of inequality, RII) and absolute (slope index of inequality, SII) differences between the least- and most-educated mothers for all-cause and cause-specific stillbirth, adjusting for maternal characteristics. RESULTS: Stillbirth rates decreased over time for all education levels. Absolute educational inequality (SII 2.5 per 1000 births, 95% CI 2.1-2.8; all periods combined) was stable over time, whereas relative inequality increased (RII(1981-1989) 1.8 vs. RII(2000-2009) 2.3). Absolute inequality decreased for stillbirths caused by placental abruption (SII(1981-1989) 0.6 vs. SII(2000-2009) 0.3), but increased for unspecified causes (SII(1981-1989) 0.2 vs. SII(2000-2009) 0.7). CONCLUSIONS: Absolute educational inequality in stillbirth persisted and relative inequality increased over the past three decades, despite an overall decrease in stillbirth rates. The decrease in absolute inequality for placental abruption was countered by an increase for unspecified causes. A better understanding of the underlying components of unspecified causes is needed to further address educational inequality in stillbirth.

Mitigating effect of immigration on the relation between income inequality and mortality: a prospective study of 2 million Canadians.

BACKGROUND: The relation between income inequality and mortality in Canada is unclear, and modifying effects of characteristics such as immigration have not been examined. METHODS: Using a cohort of 2 million Canadians followed for mortality from 1991-2001, we calculated HRs and 95% CIs for income inequality of 140 urban areas (Gini coefficient, Atkinson index, coefficient of variation; expressed as continuous variables) and working age (25-64 y) or post-working age (≥65 y) mortality in men and women according to immigration status, accounting for individual and neighbourhood income, and sociodemographic characteristics. Major causes of mortality were examined. RESULTS: Relative to low income inequality, high inequality was associated with greater working age mortality in male (HR(Gini) 1.08, 95% CI 1.04 to 1.13) and female (HR(Gini) 1.12, 95% CI 1.06 to 1.18) non-immigrants for all income inequality indictors. Results were similar for female post-working age mortality. There was no relation between income inequality and mortality in immigrants. Among Canadian-born individuals, associations were greater for alcohol-related mortality (both sexes) and smoking-related causes/transport injuries (women). CONCLUSION: Income inequality is associated with mortality in Canadian-born individuals but not immigrants.

The association between rural-urban continuum, maternal education and adverse birth outcomes in Québec, Canada.

CONTEXT: Rural relative to urban area and low socioeconomic status (SES) are associated with adverse birth outcomes. Whether a graded association of increasing magnitude is present across the urban-rural continuum, accounting for SES, is unclear. We examined the association between rural-urban continuum, SES and adverse birth outcomes. METHODS: Singleton births from 1999 to 2003 (n = 356,147) were linked to Québec municipalities ranked on a continuum of 3 urban and 4 rural areas based on population and economic base. Maternal education was used to represent SES. Odds ratios (OR) were calculated for preterm birth (PTB), low birth weight (LBW), and small-for-gestational-age (SGA) birth, accounting for municipality and individual-level covariates. We used stratified analyses to examine interaction between SES and rural-urban continuum. FINDINGS: Relative to metropolitan area residence, living in small urban or rural areas was associated with adverse birth outcomes. Living in rural areas was associated with SGA birth (OR 1.11, 95% CI 1.05-1.17) and LBW (OR 1.15, 95% CI 1.05-1.26), and living in small urban areas was associated with PTB (OR 1.14, 95% CI 1.08-1.20). Upon stratification by education, living in remote rural relative to metropolitan areas was associated with adverse birth outcomes among university educated mothers only, and living in small urban areas was associated with adverse birth outcomes among mothers with lesser but not higher education. An SES gradient was present in all rural-urban areas, particularly for SGA birth. CONCLUSION: Differences in perinatal health exist across the rural-urban continuum, and maternal education has a modifying influence.

Isoelectric point determination of cardiac troponin I forms present in plasma from patients with myocardial infarction.

BACKGROUND: Cardiac troponin I (cTnI) is a specific marker of myocardial injury. In blood of patients with cardiovascular diseases, cTnI is released as a mixture of free, complexed and post-translationally modified forms. METHODS: The cTnI forms present in the plasma from 8 patients with acute myocardial infarction (AMI) have been analysed by two-dimensional gel electrophoresis (2-DE) and Western Blot using anti-cTnI mAb 19C7 and anti-phosphorylated cTnI (Serines 22-23) mAb 5E6. RESULTS: After immunoextraction of cTnI in plasma samples by 19C7 and 2-DE separation, 4 different forms were detected by 19C7 in 7 out the 8 AMI plasma samples. Two 29 kDa spots corresponding to intact free cTnI forms were detected at pIs 5.2 and 5.4. However, spot with pI 5.4 was also recognized by mAb 5E6, and should be bis-phosphorylated cTnI. Two 55 kDa spots with pIs 6.6 and 6.7 could be IC complexes. CTnI forms with pIs lower than the theoretical pI were also found in free cTnI and phosphorylated cTnI purified materials. CONCLUSIONS: 2-DE analysis of AMI plasma showed the presence of acidic cTnI forms, one of them being phosphorylated. The clinical significance of these forms has to be further investigated.

Does living in rural areas make a difference for health in Québec?

This study offers an overview of the health status of rural populations and its major determinants in Québec. A wide range of indicators are examined along a spatial grid that allows rural-urban as well as intrarural comparisons. Differences between urban and rural populations mainly concern specific health problems and determinants, while notable variations on these are noted within rural areas. Some avenues for further research and public health policies in Québec are presented.

Deprivation and stroke mortality in Quebec.

Cerebrovascular accidents (CVAs) constitute an important cause of disability and death in Quebec. Among the primary CVA risk factors, certain socioeconomic characteristics of individuals and living environments appear to play a central role. The purpose of this article is to examine the links between material/social forms of deprivation and CVA mortality in a group of 4,339 individuals aged 25 to 74 years who died between 1994 and 1998. The socioeconomic profile of these persons was estimated on the basis of the enumeration area in which they resided. The Poisson regression technique was used to estimate the relative risk (RR) of mortality by deprivation level. Our results show the presence of a mortality gradient for both material and social forms of deprivation, where the relative risks of mortality of the most disadvantaged group and the most advantaged group are, respectively, 1.34 and 1.35. Despite the existence of a system of universal health care, inequalities in mortality persist and need to be taken into account when implementing intervention programs.