The separation of ribonucleic acids from Escherichia coli on lysin-agarose.

Lysine-agarose provides a simple means of separating RNA species of different moleecular weight. When RNA from Escherichia coli is added to a small column of lysine-agarose and elution is carried out at neutral pH with a shallow linear gradient of NaCl the RNA species are eluted according to size; 4S and 5S RNA species are completely separated and after a delay the 16S and 23S species are eluted as separate peaks. The process is very reproducible and the different species are eluted at a fixed salt concentration even when changes are made in the gradient, provided other conditions, under which the column is run remain constant.     

Geometry of the human erythrocyte. I. Effect of albumin on cell geometry.

The effects of albumin on the geometry of human erythrocytes have been studied. Individual red cells, hanging on edge from coverslips were photographed. Enlarged cell profiles were digitized using a Gradicon digitizer (Instronics Ltd., Stittsville, Ontario). Geometric parameters including diameter, area, volume, minimum cylindrical diameter, sphericity index, swelling index, maximum and minimum cell thickness, were calculated for each cell using a CDC 6400 computer. Maximum effect of human serum albumin was reached at about 1 g/liter. Studies of cell populations showed decreases in mean cell diameter of up to 6%, area 6%, and volume 15%, varying from sample to sample. The thickness of the rim was increased while that at the dimple was decreased. Studies of single cells showed that area and volume changes do not occur equally in all cells. Cells with lower sphericity indices showed larger effects. In the presence of albumin, up to 50% of the cells assumed cup-shapes (stomatocytes). These cells had smaller volumes but the same area as biconcave cells. Mechanical agitation could reversibly induce biconcave cells to assume cup shapes without area or volume changes. Experiments with de-fatted human albumins showed that the presence of bound fatty acids in varying concentrations does not alter the observed effects. Bovine serum albumin has similar effects on human erythrocytes as human serum albumin.     

200 injuries caused by playground equipment.

Two-hundred children with injuries caused by playground equipment were studied. Whereas only about 9% of the total casualty attendances are for fractures, 26-5% (53) of these children had fractures. The climbing frame and the slide seemed to be associated with more severe injuries than the swing or other equipment, but more cases need to be studied to confirm this. The youngest children were at particular risk on equipment such as the wooden rocking horse or roundabout, when the speed of operation could be controlled by older children. Many of the injuries to the very youngest children occurred when they were walking behind a moving swing. Faulty equipment did not seem to be a major factor in causing accidents, but the use by older children of apparatus designed for young ones led to accidents. There was supervision, either at home, in a school playground, or in a park, in 62% of the cases. Many of the accidents were the result of the normal desire of children for experimentation and adventure.     

Association between long-term exposure to ambient air pollution and COVID-19 severity: a prospective cohort study

Background:The tremendous global health burden related to COVID-19 means that identifying determinants of COVID-19 severity is important for prevention and intervention. We aimed to explore long-term exposure to ambient air pollution as a potential contributor to COVID-19 severity, given its known impact on the respiratory system.Methods:We used a cohort of all people with confirmed SARS-CoV-2 infection, aged 20 years and older and not residing in a long-term care facility in Ontario, Canada, during 2020. We evaluated the association between long-term exposure to fine particulate matter (PM_2.5), nitrogen dioxide (NO₂) and ground-level ozone (O₃), and risk of COVID-19-related hospital admission, intensive care unit (ICU) admission and death. We ascertained individuals’ long-term exposures to each air pollutant based on their residence from 2015 to 2019. We used logistic regression and adjusted for confounders and selection bias using various individual and contextual covariates obtained through data linkage.Results:Among the 151 105 people with confirmed SARS-CoV-2 infection in Ontario in 2020, we observed 8630 hospital admissions, 1912 ICU admissions and 2137 deaths related to COVID-19. For each interquartile range increase in exposure to PM_2.5 (1.70 μg/m³), we estimated odds ratios of 1.06 (95% confidence interval [CI] 1.01–1.12), 1.09 (95% CI 0.98–1.21) and 1.00 (95% CI 0.90–1.11) for hospital admission, ICU admission and death, respectively. Estimates were smaller for NO₂. We also estimated odds ratios of 1.15 (95% CI 1.06–1.23), 1.30 (95% CI 1.12–1.50) and 1.18 (95% CI 1.02–1.36) per interquartile range increase of 5.14 ppb in O₃ for hospital admission, ICU admission and death, respectively.Interpretation:Chronic exposure to air pollution may contribute to severe outcomes after SARS-CoV-2 infection, particularly exposure to O₃.

By November 2021, COVID-19 had caused more than 5 million deaths globally¹ and more than 29 400 in Canada.² The clinical manifestations of SARS-CoV-2 infection range from being asymptomatic to multiple organ failure and death. Identifying risk factors for COVID-19 severity is important to better understand etiological mechanisms and identify populations to prioritize for screening, vaccination and medical treatment. Risk factors for severity of COVID-19 include male sex, older age, pre-existing medical conditions and being from racialized communities.^3–5 More recently, ambient air pollution has been implicated as a potential driver of COVID-19 severity.^6–10Long-term exposure to ambient air pollution, a major contributor to global disease burden,¹¹ could increase the risk of severe COVID-19 outcomes by several mechanisms. Air pollutants can reduce individuals’ pulmonary immune responses and antimicrobial activities, boosting viral loads.⁸ Air pollution can also induce chronic inflammation and overexpression of the alveolar angiotensin-converting enzyme 2 (ACE) receptor,⁷ the key receptor that facilitates SARS-CoV-2 entry into cells.^12,13 Exposure to air pollution contributes to chronic conditions, such as cardiovascular disease, that are associated with unfavourable COVID-19 prognosis, possibly owing to persistent immune activation and excessive amplification of cytokine development.¹⁰ Thus, greater exposure to long-term air pollution may lead to severe COVID-19 outcomes.Reports exist of positive associations between long-term exposure to particulate matter with diameters equal to or smaller than 2.5 or 10 μm (PM_2.5 and PM₁₀), ground-level ozone (O₃) and nitrogen dioxide (NO₂), and metrics of COVID-19 severity (e.g., mortality and case fatality rate).^8–10 However, most studies to date have used ecological and cross-sectional designs, owing to limited access to individual data, which leads to ambiguity in interpreting the results, thus hindering their influence on policy. ^6,14 Ecological designs do not allow for disentangling the relative impacts of air pollution on individual susceptibility to infection and disease severity.¹⁴ Residual confounding by factors such as population mobility and social interactions is also problematic. Therefore, a cohort study with data on individuals with SARS-CoV-2 is a more appropriate design.^6,14 Studies that have used individual data were conducted in specific subpopulations^15,16 or populations with few severe cases,¹⁷ or had limited data on individual exposure to air pollutants.¹⁸ In Canada, 1 ecological study found a positive association between long-term exposure to PM_2.5 and COVID-19 incidence,¹⁹ but no published study has explored the association between air pollution and COVID-19 severity.We aimed to examine the associations between long-term exposure to 3 common air pollutants (PM_2.5, NO₂ and O₃) and key indicators of COVID-19 severity, including hospital admission, intensive care unit (ICU) admission and death, using a large prospective cohort of people with confirmed SARS-CoV-2 infection in Ontario, Canada, in 2020. The air contaminants PM_2.5, NO₂ and O₃ are regularly monitored by the Canadian government, and are key pollutants that are considered when setting air-quality policies. They originate from varying sources (NO₂ is primarily emitted during combustion of fuel, O₃ is primarily formed in air by chemical reactions of nitrogen oxides and volatile organic compounds, and PM_2.5 can be emitted during combustion or formed by reactions of chemicals like sulphur dioxide and nitrogen oxides in air) and they may affect human health differently.^20,21,22     

Optimization of a series of quinazolinone-derived antagonists of CXCR3

The evaluation of the CXCR3 antagonist AMG 487 in clinic trials was complicated due to the formation of an active metabolite. In this Letter, we will discuss the further optimization of the quinazolinone series that led to the discovery of compounds devoid of the formation of the active metabolite that was seen with AMG 487. In addition, these compounds also feature increased potency and good pharmacokinetic properties. We will also discuss the efficacy of the lead compound 34 in a mouse model of cellular recruitment induced by bleomycin.     

An assessment of macroinvertebrate functional feeding groups as water quality indicators in the Buffalo River, eastern Cape Province, South Africa

The aim of this paper was to investigate the potential for using functional feeding groups (FFGs) as indicators of water quality conditions in rivers, using the Buffalo River, South Africa, as a specific example. Multivariate classification and ordination techniques were used to investigate species and FFG distributions in relation to a number of physico-chemical variables at 16 sites from the headwaters to the estuary of the Buffalo River.Two-way indicator species analysis (TWINSPAN) of species composition ranked most of the sites sequentially down the river, irrespective of water quality conditions. Ordination of FFGs from a set of riffle samples collected in mid-late summer showed only weak relationships between FFG distribution and water quality changes, except where variables changed sequentially down the river (e.g. pH and temperature). Individual species responses to water quality gradients were examined for nine riffle-dwelling species representing diverse FFGs. Following correspondence analysis of a matrix of environmental variables and species frequencies, some species showed strong associations with defined ranges of some variables. In particular, Adenophlebia auriculata (Leptophlebiidae, Ephemeroptera) from the headwater sampling site, was associated with low pH and low temperature. Simulium damnosum occurred under conditions of high turbidity, while Afronurus harrisoni was found under high concentrations of potassium, ammonium and nitrite ions.We conclude that although there was a distinct headwaters fauna in the Buffalo River, and sequential downstream changes in species composition, most FFGs (apart from shredders) were represented down the whole length of the river. FFG classifications are therefore unlikely to provide useful indications of water quality conditions in the Buffalo River.Using a categorical approach to classifying water quality variables, and by applying correspondence analysis to the resulting matrix, we recognised nine species that could be used to define water quality. These indicator species can be used to define tolerance ranges of the fauna for water quality conditions in different parts of the Buffalo river.     

Synthesis of β-Peptide Standards for Use in Model Prebiotic Reactions

A one-pot method was developed for the preparation of a series of β-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of β-alanine methyl ester, yielding β-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of β-alanine peptide methyl ester oligomers, producing a series of β-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of β-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of β-peptide standards for investigating differences between α- and β-peptides in the context of prebiotic chemistry.     

Molecular interactions in human T-cell-mediated cytotoxicity to Epstein-Barr virus. I. Blocking of effector cell function by monoclonal antibody OKT3

The ability of different anti-human T-cell lymphocyte monoclonal antibodies to inhibit the effector function of the cytotoxic T-cell response against autologous Epstein-Barr virus (EBV)-infected B-cell targets has been tested. It was found that monoclonal antibody, OKT3, which reacts with most human T cells, blocks the effector cell function in the absence of complement, an effect that was dose dependent. When monoclonal antibody OKT3 was tested at a concentration of 1 μg/ml, inhibition of cytotoxicity ranged between 50 and 80%. The F(ab′)₂ fragment of OKT3 inhibited as well as the intact IgG molecule, indicating that the Fc portion of the antibody is not necessary for the cytotoxicity blocking. The Fab fragment of OKT3 had lower blocking activity per microgram of protein tested. Antibodies SC1, OKT11 (anti-pan T cell), OKT8 (anti-cytotoxic/suppressor subset), and L368 (anti-HLA) did not have any discernible blocking effects. However, antibodies SC1, OKT8, and L368 could abrogate the cytotoxic activity in the presence of complement. Blocking by OKT3 was not due to its being present on the cell surface in higher concentrations than the other monoclonal antibodies since cytofluorographic analysis demonstrated that the amount of OKT8 or L368 antibodies bound on the cells was greater than OKT3. In addition, blocking was not due to antigenic modulation since incubation with antibody OKT3-F(ab′)₂ was not associated with a significant decrease in the amount of its reactive antigen. Under the conditions tested OKT3 did not affect cell viability or cause agglutination.     

Modeled microgravity disrupts collagen I/integrin signaling during osteoblastic differentiation of human mesenchymal stem cells

Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following 7 days culture in modeled microgravity (MMG). One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen (Col I)-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix (ECM) proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of MMG on integrin expression and function in hMSC. We demonstrate that 7 days of culture in MMG leads to reduced expression of the ECM protein, Col I. Conversely, MMG consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin subunit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt protein kinase (Akt) is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-mitogen activated protein kinase (MAPK) pathway is evidenced by a reduction in Ras and extracellular signal-related protein kinase (ERK) activation. Taken together, our findings indicate that MMG decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.     

SOS regulation of the type III secretion system of enteropathogenic Escherichia coli

Genomes of bacterial pathogens contain and coordinately regulate virulence-associated genes in order to cause disease. Enteropathogenic Escherichia coli (EPEC), a major cause of watery diarrhea in infants and a model gram-negative pathogen, expresses a type III secretion system (TTSS) that is encoded by the locus of enterocyte effacement (LEE) and is necessary for causing attaching and effacing intestinal lesions. Effector proteins encoded by the LEE and in cryptic prophage are injected into the host cell cytoplasm by the TTTS apparatus, ultimately leading to diarrhea. The LEE is comprised of multiple polycistronic operons, most of which are controlled by the global, positive regulator Ler. Here we demonstrated that the LEE2 and LEE3 operons also responded to SOS signaling and that this regulation was LexA dependent. As determined by a DNase I protection assay, purified LexA protein bound in vitro to a predicted SOS box located in the divergent, overlapping LEE2/LEE3 promoters. Expression of the lexA1 allele, encoding an uncleavable LexA protein in EPEC, resulted in reduced secretion, particularly in the absence of the Ler regulator. Finally, we obtained evidence that the cryptic phage-located nleA gene encoding an effector molecule is SOS regulated. Thus, we demonstrated, for the first time to our knowledge, that genes encoding components of a TTSS are regulated by the SOS response, and our data might explain how a subset of EPEC effector proteins, encoded in cryptic prophages, are coordinately regulated with the LEE-encoded TTSS necessary for their translocation into host cells.