Relationship between variations in pathogenicity and lag phase at 37°C of Listeria monocytogenes previously stored at 4°C

s. BUNCIC AND S.M. AVERY. 1996. Three haemolytic, pathogenic strains of Listeria monocytogenes (a reference strain, a food-derived strain and a human strain) were held at 4°C for 4 weeks in phosphate-buffered saline pH 5.5 or 7.0, with and without 0.2% potassium sorbate or 0.3% sodium acetate. The number of viable cells did not change significantly during this storage. Pathogenicity of non-growing L. monocytogenes cells for 14-d-old chick embryos was determined before and after storage. Storage at 4°C resulted in decreased pathogenicity, but effects were strain-, pH- and substrate-dependent. After 4 weeks storage at 4°C non-growing bacterial cells were transferred to Brain Heart Infusion broth and growth characteristics were determined during incubation at 37°C. Strains that showed decreased pathogenicity had significantly longer lag phases at 37°C than strains that maintained pathogenicity. It is concluded that decreased pathogenicity of L. monocytogenes stored without growth at 4°C for 4 weeks and subsequent long lag phase at 37°C are correlated.     

A novel approach for in vitro production of bovine embryos: use of the Oxoid atmosphere generating system

The importance of the incubator type is often overlooked when protocols for in vitro production of embryos are evaluated. In this study the ability of a standard CO2 Heraeus incubator and the Oxoid CO2Gen atmosphere-generating system to support bovine in vitro oocyte maturation, fertilization and embryo development is described for the first time. The Oxoid CO2Gen gas generating system, originally designed for the growth of bacteria, is based on the chemical reaction of ascorbic acid and air. When the sachet with ascorbic acid is placed in the confined volume of the airtight AnaeroJar, an atmosphere of 6% CO2 in 15% O2 is created, which is comparable to the 5% CO2 and 20% O2 used for standard in vitro production of bovine embryos. In the first set of experiments oocyte in vitro maturation (IVM), fertilization (IVF) and embryo culture (IVC) were allocated to one or the other of the culture systems. In the second set of experiments IVM and IVF took place in the Heraeus incubator, while IVC was allocated either to the Heraeus or to the AnaeroJar. During experiments the AnaeroJar was placed in the Heraeus incubator to ensure identical incubation temperatures of 38.8 degrees C. A standard protocol was used for production of embryos: 23 h of IVM in TCM-199, 20 h of IVF with frozen-thawed washed spermatozoa in TALP medium and 7 days of IVC (8 days after insemination) in B2 medium with bovine oviduct epithelial cells. In the first set of experiments, based on a total of 766 inseminated oocytes, the Day 8 blastocyst rates were the same in the Heraeus incubator and the AnaeroJar: 30% vs. 30% with oviduct cell coculture, and 21% vs. 18% without coculture. In the second set of experiments, based on 1963 inseminated oocytes, the average blastocyst rates were 27% vs. 32% from the Heraeus incubator and the AnaeroJar. In 2 of 6 replicates blastocyst rates were lower in the Heraeus incubator than in the jar; in the remaining replicates they were alike. No differences were noted in blastocyst kinetics or morphology. In conclusion, the Oxoid gas generating system seems to be a cheap, convenient and stable alternative to expensive CO2 incubators, not only for the growth of bacteria, but also for in vitro production of bovine embryos.     

Uncovering SARS-COV-2 vaccine uptake and COVID-19 impacts among First Nations,Inuit and Métis Peoples living in Toronto and London,Ontario

Background:First Nations, Inuit and Métis Peoples across geographies are at higher risk of SARS-CoV-2 infection and COVID-19 because of high rates of chronic disease, inadequate housing and barriers to accessing health services. Most Indigenous Peoples in Canada live in cities, where SARS-CoV-2 infection is concentrated. To address gaps in SARS-CoV-2 information for these urban populations, we partnered with Indigenous agencies and sought to generate rates of SARS-CoV-2 testing and vaccination, and incidence of infection for First Nations, Inuit and Métis living in 2 Ontario cities.Methods:We drew on existing cohorts of First Nations, Inuit and Métis adults in Toronto (n = 723) and London (n = 364), Ontario, who were recruited using respondent-driven sampling. We linked to ICES SARS-CoV-2 databases and prospectively monitored rates of SARS-CoV-2 testing, diagnosis and vaccination for First Nations, Inuit and Métis, and comparator city and Ontario populations.Results:We found that SARS-CoV-2 testing rates among First Nations, Inuit and Métis were higher in Toronto (54.7%, 95% confidence interval [CI] 48.1% to 61.3%) and similar in London (44.5%, 95% CI 36.0% to 53.1%) compared with local and provincial rates. We determined that cumulative incidence of SARS-CoV-2 infection was not significantly different among First Nations, Inuit and Métis in Toronto (7364/100 000, 95% CI 2882 to 11 847) or London (7707/100 000, 95% CI 2215 to 13 200) compared with city rates. We found that rates of vaccination among First Nations, Inuit and Métis in Toronto (58.2%, 95% CI 51.4% to 64.9%) and London (61.5%, 95% CI 52.9% to 70.0%) were lower than the rates for the 2 cities and Ontario.Interpretation:Although Ontario government policies prioritized Indigenous populations for SARS-CoV-2 vaccination, vaccine uptake was lower than in the general population for First Nations, Inuit and Métis Peoples in Toronto and London. Ongoing access to culturally safe testing and vaccinations is urgently required to avoid disproportionate hospital admisson and mortality related to COVID-19 in these communities.

Multigenerational colonial policies that aimed to assimilate First Nations, Inuit and Métis Peoples and appropriate land and resources have led to inequities across most major health outcomes for First Nations, Inuit and Métis living in urban, rural and remote geographies compared with non-Indigenous people in Canada, as well as striking gaps in access to equitable and culturally safe health care.^1,2More than half of Indigenous Peoples in Canada live in urban areas.³ In cities, jurisdictional complexities, including structured exclusion from potentially beneficial government programs, combined with persistent and growing inequities in the distribution of urban health and social resources, have exacerbated pre-existing Indigenous compared to non-Indigenous health inequities during the COVID-19 pandemic.⁴ Dense and multigenerational social networks; barriers in access to culturally safe health care; and a disproportionate burden of poverty, chronic disease and inadequate housing^5–7 create conditions for the spread of SARS-CoV-2 among First Nations, Inuit and Métis living in urban areas in Canada.The quality, comprehensiveness and accessibility of First Nations, Inuit and Métis health and social statistics in Canada, particularly for those living in urban and related homelands, is a critical problem.^4,8 A lack of accurate, inclusive and culturally safe identification processes for First Nations, Inuit and Métis in health service and public health data systems,⁸ and inadequate engagement of Indigenous leadership in the governance and management of their health information, which is essential,^8,9 contribute to this problem. As a result, Indigenous health policy and service responses are commonly implemented without accurate and reliable population-based sociodemographic and health outcomes data.Although First Nations health authorities mobilized quickly to document SARS-CoV-2 incidence and COVID-19 morbidity and mortality in First Nations communities in the early pandemic period, and vaccination campaigns led by First Nations were successful,^10,11 published reports of SARS-CoV-2 outcomes for First Nations, Inuit and Métis living in urban and related homelands remain unavailable more than 2 years into the COVID-19 pandemic.Since 2008, our research team has partnered with urban Indigenous health service providers to address gaps in health and social information for First Nations, Inuit and Métis living in urban and related homelands to produce representative, population-based, community-controlled health information for urban First Nations, Inuit and Métis,^5,6,12 by successfully applying respondentdriven sampling (RDS) methods to generate valid, population-representative cohorts of First Nations, Inuit and Métis adults.¹³ Drawing on 2 of these cohorts (Our Health Counts Toronto and Our Health Counts London), which had existing linkages to health care databases at ICES, we sought to generate accurate and valid rates of SARS-CoV-2 testing and vaccination, and incidence of infection for First Nations, Inuit and Métis living in Toronto and London, Ontario, and to compare these rates with those in the general populations in each city and Ontario.     

Acute effects of different warm-up protocols on fitness performance in children

The purpose of this study was to compare the acute effects on youth fitness of 3 different warm-up protocols utilizing static stretching or dynamic exercise performance. Sixty children (mean age 11.3 +/- 0.7 years) performed 3 different warm-up routines in random order on nonconsecutive days. The warm-up protocols consisted of 5 minutes of walking and 5 minutes of static stretching (SS), 10 minutes of dynamic exercise (DY), or 10 minutes of dynamic exercise plus 3 drop jumps from 15-cm boxes (DYJ). Following each warm-up session, subjects were tested on the vertical jump, long jump, shuttle run, and v-sit flexibility. Analysis of the data revealed that vertical-jump and shuttle-run performance declined significantly following SS as compared to DY and DYJ, and long-jump performance was significantly reduced following SS as compared to DYJ (p < 0.05). There were no significant differences in flexibility following the 3 warm-up treatments. The results of this study suggest that it may be desirable for children to perform moderate- to high-intensity dynamic exercises prior to the performance of activities that require a high power output.     

Quantitative trait locus-specific genotype x alcoholism interaction on linkage for evoked electroencephalogram oscillations

We explored the evidence for a quantitative trait locus (QTL)-specific genotype x alcoholism interaction for an evoked electroencephalogram theta band oscillation (ERP) phenotype on a region of chromosome 7 in participants of the US Collaborative Study on the Genetics of Alcoholism. Among 901 participants with both genotype and phenotype data available, we performed variance component linkage analysis (SOLAR version 2.1.2) in the full sample and stratified by DSM-III-R and Feighner-definite alcoholism categories. The heritability of the ERP phenotype after adjusting for age and sex effects in the combined sample and in the alcoholism classification sub-groups ranged from 40% to 66%. Linkage on chromosome 7 was identified at 158 cM (LOD = 3.8) in the full sample and at 108 in the non-alcoholic subgroup (LOD = 3.1). Further, we detected QTL-specific genotype x alcoholism interaction at these loci. This work demonstrates the importance of considering the complexity of common complex traits in our search for genes that predispose to alcoholism.     

Probing hydrogen-bonding interactions in the active site of medium-chain acyl-CoA dehydrogenase using Raman spectroscopy

The role of the oxyanion hole in the reaction catalyzed by pig medium-chain acyl-CoA dehydrogenase (pMCAD) has been investigated using enzyme reconstituted with 2'-deoxy-FAD. The k(cat) (18.8 +/- 0.5 s(-1)) and K(m) (2.5 +/- 0.4 microM) values for the oxidation of n-octanoyl-CoA (C(8)-CoA) by WT pMCAD recombinantly expressed in Escherichia coli are similar to those of native pMCAD isolated from pig kidney. In agreement with previous studies [Engst et al. (1999) Biochemistry 38, 257-267], reconstitution of the WT enzyme with 2'-deoxy-FAD causes a large (400-fold) decrease in k(cat) but has little effect on K(m). To investigate the molecular basis for the alterations in activity resulting from changes in hydrogen bonding between the substrate and the enzyme's oxyanion hole, the structure of the product analogue hexadienoyl-CoA (HD-CoA) bound to the 2'-deoxy-FAD-reconstituted enzyme has been probed by Raman spectroscopy. Importantly, while WT pMCAD causes a 27 cm(-1) decrease in the vibrational frequency of the HD enone band, from 1595 to 1568 cm(-1), the enone band is only shifted 10 cm(-1) upon binding HD-CoA to 2'-deoxy-FAD pMCAD. Thus, removal of the 2'-ribityl hydroxyl group results in a substantial reduction in the ability of the enzyme to polarize the ground state of the ES complex. On the basis of an analysis of a similar system, it is estimated that ground state destabilization is reduced by up to 17 kJ mol(-1), while the activation energy for the reaction is raised 15 kJ mol(-1). In addition, removal of the 2'-ribityl hydroxyl reduces the redox potential shift that is induced by HD-CoA binding from 18 to 11 kJ mol(-1). Consequently, while ligand polarization caused by hydrogen bonding in the oxyanion hole is intimately linked to substrate turnover, additional factors must be responsible for ligand-induced changes in redox potential. Finally, while replacement of the catalytic base E376 with Gln abolishes the ability of the enzyme to catalyze substrate oxidation and to catalyze the exchange of the C(8)-CoA alpha-protons with solvent deuterium, the 2'-deoxy-FAD-reconstituted enzyme catalyzes alpha-proton exchange at a rate (k(exc)) of 0.085 s(-1), which is only 4-fold slower than k(exc) for WT pMCAD (0.35 s(-1)). Thus, either the oxyanion hole plays only a minor role in stabilizing the transition state for alpha-proton exchange, in contrast to its role in substrate oxidation, or the value of k(exc) for WT pMCAD reflects a process such as exchange of the E376 COOH proton with solvent.     

Homology modeling of falcipain-2: validation, de novo ligand design and synthesis of novel inhibitors

Increasing resistance of malaria parasites, in particular Plasmodium falciparum, demands a serious search for novel targets. Cysteine protease in P. falciparum, encoded by a previously unidentified gene falcipain 2, provides one such target to design chemotherapeutic agents for treatment of malaria. In fact, a few cysteine protease inhibitors have been shown to inhibit growth of cultured malarial parasites. In absence of a crystal structure for this enzyme, homology modeling proved to be a reasonable alternative to study binding requirements of the enzyme. A homology model for falcipain 2 was developed and validated by docking of known vinyl sulfone inhibitors. Further, based on the observations of these studies, novel isoquinoline inhibitors were designed and synthesized, which exhibited in vitro enzyme inhibition at micromolar concentrations.     

Biotransformation of 10-deoxoartemisinin to its 7β-hydroxy derivative by Mucor ramannianus

10-Deoxoartemisinin at 0.2 mg ml^–1 medium was transformed to 7-hydroxy deoxoartemisinin by Mucor ramannianus growing on sucrose, 20 g l^–1, and peptone, 10 g l^–1, at pH 4 and 26 °C. The yield of product was increased from 16% to 45% by selecting optimal culture conditions using a 2^5–2 factorial design.