Reentry into the cell cycle of differentiated skeletal myocytes

The activation of muscle-specific myosin synthesis and its relationship to withdrawal from the cell cycle have been examined in cycle-synchronized myoblasts under growth-restrictive, fusion-impermissive (low Ca²⁺) culture conditions. Under these conditions, embryonic quail skeletal myoblasts, collected in mitosis by mechanical shake-off, complete one normal cycle and arrest in G₁. The presence of skeletal muscle myosin is first detected, by indirect immunofluorescence, 8 hr into this protracted G₁. Within the next 10–11 hr the percentage myosin positive (Myo+) cells increases with good synchrony, reaching approximately 95%. Refeeding with a proliferation-stimulating, low Ca²⁺ medium when approximately 50% of the cells are Myo+ induces reentry into S. Applying a 15-min pulse with [³H]TdR immediately preceding fixation at regular intervals following refeeding, cells can be detected which are Myo+ and whose nuclei have incorporated [³H]TdR. The numbers of such doubly labeled cells are small but consistent with the fraction of cells in S (by time-lapse analysis) at the postfeeding times sampled. These cinematographic studies also indicate that progression to mitosis following stimulation occurs slowly and asynchronously. The kinetics of progression of the stimulated cells suggest that they reenter S from a different compartment in G₁ than do log-phase myoblasts. We conclude that in fusion-blocked quail myocytes irreversible withdrawal from the cell cycle is neither an obligate precondition for, nor an immediate consequence of the activation of the muscle-specific contractile gene set.     

Stable recombinant production of codon-scrambled lubricin and mucin in human cells

Widespread therapeutic and commercial interest in recombinant mucin technology has emerged due to the unique ability of mucin glycoproteins to hydrate, protect, and lubricate biological surfaces. However, recombinant production of the large, highly repetitive domains that are characteristic of mucins remains a challenge in biomanufacturing likely due, at least in part, to the inherent instability of DNA repeats in the cellular genome. To overcome this challenge, we exploit codon redundancy to encode desired mucin polypeptides with minimal nucleotide repetition. The codon-scrambling strategy was applied to generate synonymous genes, or “synDNAs,” for two mucins of commercial interest: lubricin and mucin 1. Stable, long-term recombinant production in suspension-adapted human 293-F cells was demonstrated for the synonymous lubricin complementary DNA (cDNA), which we refer to as SynLubricin. Under optimal conditions, a 293-F subpopulation produced recombinant SynLubricin at more than 200 mg/L of media and was stable throughout 2 months of continuous culture. Functionality tests confirmed that the recombinant lubricin could effectively inhibit cell adhesion and lubricate cartilage explants. Together, our work provides a viable workflow for cDNA design and stable mucin production in mammalian host production systems.     

The mobility gap: estimating mobility thresholds required to control SARS-CoV-2 in Canada

BACKGROUND:Nonpharmaceutical interventions remain the primary means of controlling severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) until vaccination coverage is sufficient to achieve herd immunity. We used anonymized smartphone mobility measures to quantify the mobility level needed to control SARS-CoV-2 (i.e., mobility threshold), and the difference relative to the observed mobility level (i.e., mobility gap).METHODS:We conducted a time-series study of the weekly incidence of SARS-CoV-2 in Canada from Mar. 15, 2020, to Mar. 6, 2021. The outcome was weekly growth rate, defined as the ratio of cases in a given week versus the previous week. We evaluated the effects of average time spent outside the home in the previous 3 weeks using a log-normal regression model, accounting for province, week and mean temperature. We calculated the SARS-CoV-2 mobility threshold and gap.RESULTS:Across the 51-week study period, a total of 888 751 people were infected with SARS-CoV-2. Each 10% increase in the mobility gap was associated with a 25% increase in the SARS-CoV-2 weekly case growth rate (ratio 1.25, 95% confidence interval 1.20–1.29). Compared to the prepandemic baseline mobility of 100%, the mobility threshold was highest in the summer (69%; interquartile range [IQR] 67%–70%), and dropped to 54% in winter 2021 (IQR 52%–55%); a mobility gap was present in Canada from July 2020 until the last week of December 2020.INTERPRETATION:Mobility strongly and consistently predicts weekly case growth, and low levels of mobility are needed to control SARS-CoV-2 through spring 2021. Mobility measures from anonymized smartphone data can be used to guide provincial and regional loosening and tightening of physical distancing measures.

The global toll of coronavirus disease 2019 (COVID-19) continues to grow, despite the promise of recently approved vaccines. A surge is occurring in many countries in the Northern Hemisphere, including Canada, that may take a considerable toll before vaccination is sufficiently widespread to achieve herd immunity. Nonpharmaceutical public health interventions, including physical distancing, remain the primary population-based means of controlling COVID-19.¹ Since early in the second wave, which started in September 2020, polling has suggested that most people in Canada have supported and adhered to government-directed restrictions,² and many favour strengthened measures to control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative viral agent of COVID-19.³SARS-CoV-2 is spread primarily through close contact with people who are infected.⁴ As with any infectious disease, contact rates are a primary driver of SARS-CoV-2 transmission.⁵ Mobility measures capturing human activity through anonymized tracking of smartphones are believed to be reasonable proxies of contact rates outside of one’s own home; these measures can provide more timely and reliable sources of information on contact rates compared with time-use surveys or contact tracing.^6–8Aggregated smartphone mobility data are provided by a number of software developers and have been used to quantify the impact of policy on mobility in Canada,⁹ the effectiveness of lockdowns aiming to reduce the spread of SARS-CoV-2^10–12 and loopholes from excessively localized measures.¹³ Mobility metrics are helpful for gauging the effect of restrictions on behaviour, but do not, on their own, show decision-makers whether restrictions in place at the time are sufficient to curtail the spread of SARS-CoV-2. In this study, we evaluated the association between smartphone mobility measures and the spread of SARS-CoV-2 in Canada, both nationally and provincially, between March 2020 and March 2021. We also sought to quantify the mobility level needed to control COVID-19 (i.e., the mobility threshold), and the difference between observed mobility levels and the threshold (i.e., the mobility gap). We hypothesized that lower mobility levels may be needed in provinces with larger urban populations in the winter compared with more rural provinces in the summer.¹⁴     

Uridine labeling of human lymphocytes: Differential uptake by T and B cells

Human peripheral blood lymphocytes were labeled with [³H]uridine and separated into immunoglobulin (Ig)-positive-enriched and Ig-negative populations by rosetting and Ficoll sedimentation. The Ig-negative (T cell-rich) fraction was found to be more heavily labeled than the B cell-enriched population, in agreement with previous results in rats. Combined autoradiography and rosetting confirmed the differential uridine labeling of T and B cells. Incorporation of cytidine and adenosine by T and B cell-enriched populations showed similar but less dramatic differential labeling.     

A marine protected area network does not confer community structure resilience to a marine heatwave across coastal ecosystems

Marine protected areas (MPAs) have gained attention as a conservation tool for enhancing ecosystem resilience to climate change. However, empirical evidence explicitly linking MPAs to enhanced ecological resilience is limited and mixed. To better understand whether MPAs can buffer climate impacts, we tested the resistance and recovery of marine communities to the 2014–2016 Northeast Pacific heatwave in the largest scientifically designed MPA network in the world off the coast of California, United States. The network consists of 124 MPAs (48 no-take state marine reserves, and 76 partial-take or special regulation conservation areas) implemented at different times, with full implementation completed in 2012. We compared fish, benthic invertebrate, and macroalgal community structure inside and outside of 13 no-take MPAs across rocky intertidal, kelp forest, shallow reef, and deep reef nearshore habitats in California's Central Coast region from 2007 to 2020. We also explored whether MPA features, including age, size, depth, proportion rock, historic fishing pressure, habitat diversity and richness, connectivity, and fish biomass response ratios (proxy for ecological performance), conferred climate resilience for kelp forest and rocky intertidal habitats spanning 28 MPAs across the full network. Ecological communities dramatically shifted due to the marine heatwave across all four nearshore habitats, and MPAs did not facilitate habitat-wide resistance or recovery. Only in protected rocky intertidal habitats did community structure significantly resist marine heatwave impacts. Community shifts were associated with a pronounced decline in the relative proportion of cold water species and an increase in warm water species. MPA features did not explain resistance or recovery to the marine heatwave. Collectively, our findings suggest that MPAs have limited ability to mitigate the impacts of marine heatwaves on community structure. Given that mechanisms of resilience to climate perturbations are complex, there is a clear need to expand assessments of ecosystem-wide consequences resulting from acute climate-driven perturbations, and the potential role of regulatory protection in mitigating community structure changes.     

Overview of the genetic toxicity of caprolactam and benzoin

DNA binding in vitro was measured with 2-amino-3- methylimidazo(4,5-f)[5-3H]quinoline (3H-IQ) in the presence of S9 and microsomes from Wistar rat liver. DNA binding of 3H-IQ was catalyzed by both microsomes and S9 and was increased 5-fold in Aroclor (PCB)-pretreated animals. DNA binding was reduced by the specific inhibitor of sulfotransferase 2,6-dichloro-4-nitrophenol and slightly increased by 3'-phosphoadenosine 5'-phosphosulfate. The incubation of IQ in media with increasing concentrations of sulfate produced a modest dose-dependent increase in DNA binding. Liver S9 obtained from rats which had been administered 1% sulfite in drinking water catalyzed a higher binding of IQ to DNA, and this effect was inhibited by pentachlorophenol. The data demonstrate that sulfotransferase has a role in the activation of IQ and that variations in the activity of this enzyme induced by dietary changes may vary the genotoxic activity of IQ.     

Human prothymosin α: Purification of a highly acidic nuclear protein by means of a phenol extraction

Human prothymosin a, virtually alone among proteins, is recovered from the aqueous phase of phenolextracted cell lysates prepared from human myeloma cells or COS cells that were transfected with the human prothymosin a gene. This observation forms the basis for purification of the protein to homogeneity in two steps—phenol extraction followed by electrophoresis steps—phenol extraction followed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels to remove residual contaminants consisting chiefly of carbohydrate and RNA.