Excess deaths in China during SARS-CoV-2 viral waves in 2022-2023.

Background: The extent to which the Omicron variant of SARS-CoV-2 raised death rates in China during its viral wave of December 2022-January 2023 remains largely undocumented. Methods: We worked with an established national survey organization to survey 8,004 adults in all 31 administrative areas of China to ask about deaths in families since January 2020. We examined age-specific death rates, focusing on deaths above age 60 years, and at 15-59 years. We compared these to the United Nations (UN) estimates of age-specific mortality in 2019. Findings: The survey participants were broadly similar to the 2020 census and other national surveys in age, sex, region, and smoking status, but had lower SARS-CoV-2 vaccination rates and higher education levels. There were no differences in reporting of deaths during the Omicron period (after November 2021) versus earlier. The survey captured 456 deaths, of which 329 occurred at ages 60+ years and 212 were of women. At ages 60+ years, death rates approximately doubled during December 2022-January 2023. Deaths at ages 15-59 years did not rise appreciably. The UN estimates approximately 675,000 deaths per month at ages 60+ years in 2019. If rates doubled nationally as in our survey, China had approximately 1.35 million excess deaths from December 2022-January 2023. Interpretation: China experienced a sharp but short increase in excess deaths among its elderly during the Omicron wave. If death registry data corroborate our estimates of substantial excess deaths in China, the worldwide estimates of excess deaths due to SARS-CoV-2 in 2022-2023 may need upward adjustment.

Identification of the Core Promoter and Variants Regulating Chicken CCKAR Expression.

Decreased expression of chicken cholecystokinin A receptor (CCKAR) attenuates satiety, which contributes to increased food intake and growth for modern broilers. The study aims to define the core promoter of CCKAR, and to identify variants associated with expression activity. A 21 kb region around the CCKAR was re-sequenced to detect sequence variants. A series of 5'-deleted promoter plasmids were constructed to define the core promoter of CCKAR. The effects of sequence variants located in promoter (PSNP) and conserved (CSNP) regions on promoter activity were analyzed by comparing luciferase activity between haplotypes. A total of 182 variants were found in the 21 kb region. There were no large structural variants around CCKAR. pNL-328/+183, the one with the shortest insertion, showed the highest activity among the six promoter constructs, implying that the key cis elements regulating CCKAR expression are mainly distributed 328 bp upstream. We detected significant activity differences between high- and low-growth associated haplotypes in four of the six promoter constructs. The high-growth haplotypes of constructs pNL-1646/+183, pNL-799/+183 and pNL-528/+183 showed lower activities than the low-growth haplotypes, which is consistent with decreased expression of CCKAR in high-growth chickens. Lower expression of the high-growth allele was also detected for the CSNP5-containing construct. The data suggest that the core promoter of CCKAR is located the 328 bp region upstream from the transcription start site. Lower expression activities shown by the high-growth haplotypes in the reporter assay suggest that CSNP5 and variants located between 328 bp and 1646 bp upstream form a promising molecular basis for decreased expression of CCKAR and increased growth in chickens.

Gene expression of sex steroid metabolizing enzymes and receptors in the skeletal muscle of migrant and resident subspecies of white-crowned sparrow (Zonotrichia leucophrys).

Circulating sex steroid concentrations vary dramatically across the year in seasonally breeding animals. The ability of circulating sex steroids to effect muscle function can be modulated by changes in intracellular expression of steroid metabolizing enzymes (e.g., 5α-reductase type 2 and aromatase) and receptors. Together, these combined changes in plasma hormones, metabolizing enzymes and receptors allow for seasonally appropriate changes in skeletal muscle function. We tested the hypothesis that gene expression of sex steroid metabolizing enzymes and receptors would vary seasonally in skeletal muscle and these changes would differ between a migrant and resident life history strategy. We quantified annual changes in plasma testosterone and gene expression in pectoralis and gastrocnemius skeletal muscles using quantitative polymerase chain reaction (qPCR) in free-living migrant (Zonotrichia leucophrys gambelii) and resident (Z. l. nuttalli) subspecies of white-crowned sparrow during breeding, pre-basic molt, and wintering life history stages. Pectoralis muscle profile was largest in migrants during breeding, while residents maintained large muscle profiles year-round. Circulating testosterone peaked during breeding in both subspecies. Pectoralis muscle androgen receptor mRNA expression was lower in females of both subspecies during breeding. Estrogen receptor-α expression was higher in the pectoralis muscle, but not gastrocnemius, of residents throughout the annual cycle when compared to migrants. Pectoralis aromatase expression was higher in resident males compared to migrant males. No differences were observed for 5α-reductase 2. Between these two subspecies, patterns of plasma testosterone and androgen receptors appear to be conserved, however estrogen receptor gene expression appears to have diverged.

The effects of feed restriction, time of day, and time since feeding on behavioral and physiological indicators of hunger in broiler breeder hens.

Broiler breeder chickens are commercially feed restricted to slow their growth and improve their health and production, however, there is research demonstrating that this leads to chronic hunger resulting in poor welfare. A challenge in these studies is to account for possible daily rhythms or the effects of time since last meal on measures relating hunger. To address this, we used 3 feed treatments: AL (ad libitum fed), Ram (restricted, fed in the morning), and Rpm (restricted, fed in the afternoon) to control for diurnal effects. We then conducted foraging motivation tests and collected home pen behavior and physiological samples at 4 times relative to feeding throughout a 24-h period. The feed treatment had the largest influence on the data, with AL birds weighing more, having lower concentrations of plasma NEFA, and mRNA expression of AGRP and NPY alongside higher expression of POMC in the basal hypothalamus than Ram or Rpm birds (P < 0.001). R birds were more successful at and had a shorter latency to complete the motivation test, and did more walking and less feeding than AL birds in the home pen (P < 0.01). There was little effect of time since last meal on many measures (P > 0.05) but AGRP expression was highest in the basal hypothalamus shortly after a meal (P < 0.05), blood plasma NEFA was higher in R birds just before feeding (P < 0.001) and glucose was higher in Ram birds just after feeding (P < 0.001), and the latency to complete the motivation test was shortest before the next meal (P < 0.05). Time of day effects were mainly found in the difference in activity levels in the home pen when during lights on and lights off periods. In conclusion, many behavioral and physiological hunger measures were not significantly influenced by time of day or time since the last meal. For the measures that do change, future studies should be designed so that sampling is balanced in such a way as to minimize bias due to these effects.

The expression of the gastrin/cholecystokinin (GAST/CCK) family and their receptors (CCKAR/CCKBR) in the chicken changes in response to quantitative restriction and reveals a functional role of CCK in the crop.

Gastrin and cholecystokinin peptides bind a common G-protein coupled receptor, cholecystokinin receptor B (CCKBR) whilst cholecystokinin receptor A (CCKAR) is preferentially bound by CCK. Gastrin and cholecystokinin mediate signalling from the gastrointestinal tract to regulate appetite and digestive function. In this study, expression of the cholecystokinin/gastrin family and distribution of their receptors expression was measured to understand the target organs for the peptides and how expression responds to changes in food intake. We confirmed the restricted expression of gastrin in the antrum and the abundant expression of cholecystokinin in the hypothalamus. The expression of gastrin in the antrum was significantly elevated in broiler breeders when released from feed restriction. CCKBR was most abundant in the hypothalamus and proventriculus. CCKAR was most abundant in the pancreas and crop, more than tenfold greater than the gastrointestinal tract. Cholecystokinin expression in the pancreas increased after removal of food restriction. CCKAR in the gastrointestinal tract peaks around the distal ileum, distal to the peak of cholecystokinin expression. There was virtually no cholecystokinin expression in the caecum but CCKAR expression was high. The CCKAR expression in the crop was unexpected, supporting a role of cholecystokinin in mediating crop emptying which was supported by the observation of in-vitro contraction after cholecystokinin administration. The response to changes in food intake and the expression pattern of the cholecystokinin/gastrin family and their receptors will stimulate and inform new hypotheses on their role in growth in poultry.

Assessment of SARS-CoV-2 Seropositivity During the First and Second Viral Waves in 2020 and 2021 Among Canadian Adults.

Importance: The incidence of infection during SARS-CoV-2 viral waves, the factors associated with infection, and the durability of antibody responses to infection among Canadian adults remain undocumented. Objective: To assess the cumulative incidence of SARS-CoV-2 infection during the first 2 viral waves in Canada by measuring seropositivity among adults. Design, Setting, and Participants: The Action to Beat Coronavirus study conducted 2 rounds of an online survey about COVID-19 experience and analyzed immunoglobulin G levels based on participant-collected dried blood spots (DBS) to assess the cumulative incidence of SARS-CoV-2 infection during the first and second viral waves in Canada. A sample of 19 994 Canadian adults (aged ≥18 years) was recruited from established members of the Angus Reid Forum, a public polling organization. The study comprised 2 phases (phase 1 from May 1 to September 30, 2020, and phase 2 from December 1, 2020, to March 31, 2021) that generally corresponded to the first (April 1 to July 31, 2020) and second (October 1, 2020, to March 1, 2021) viral waves. Main Outcomes and Measures: SARS-CoV-2 immunoglobulin G seropositivity (using a chemiluminescence assay) by major geographic and demographic variables and correlation with COVID-19 symptom reporting. Results: Among 19 994 adults who completed the online questionnaire in phase 1, the mean (SD) age was 50.9 (15.4) years, and 10 522 participants (51.9%) were female; 2948 participants (14.5%) had self-identified racial and ethnic minority group status, and 1578 participants (8.2%) were self-identified Indigenous Canadians. Among participants in phase 1, 8967 had DBS testing. In phase 2, 14 621 adults completed online questionnaires, and 7102 of those had DBS testing. Of 19 994 adults who completed the online survey in phase 1, fewer had an educational level of some college or less (4747 individuals [33.1%]) compared with the general population in Canada (45.0%). Survey respondents were otherwise representative of the general population, including in prevalence of known risk factors associated with SARS-CoV-2 infection. The cumulative incidence of SARS-CoV-2 infection among unvaccinated adults increased from 1.9% in phase 1 to 6.5% in phase 2. The seropositivity pattern was demographically and geographically heterogeneous during phase 1 but more homogeneous by phase 2 (with a cumulative incidence ranging from 6.4% to 7.0% in most regions). The exception was the Atlantic region, in which cumulative incidence reached only 3.3% (odds ratio [OR] vs Ontario, 0.46; 95% CI, 0.21-1.02). A total of 47 of 188 adults (25.3%) reporting COVID-19 symptoms during phase 2 were seropositive, and the OR of seropositivity for COVID-19 symptoms was 6.15 (95% CI, 2.02-18.69). In phase 2, 94 of 444 seropositive adults (22.2%) reported having no symptoms. Of 134 seropositive adults in phase 1 who were retested in phase 2, 111 individuals (81.8%) remained seropositive. Participants who had a history of diabetes (OR, 0.58; 95% CI, 0.38-0.90) had lower odds of having detectable antibodies in phase 2. Conclusions and Relevance: The Action to Beat Coronavirus study found that the incidence of SARS-CoV-2 infection in Canada was modest until March 2021, and this incidence was lower than the levels of population immunity required to substantially reduce transmission of the virus. Ongoing vaccination efforts remain central to reducing viral transmission and mortality. Assessment of future infection-induced and vaccine-induced immunity is practicable through the use of serial online surveys and participant-collected DBS.

Seasonal differences in hypothalamic thyroid-stimulating hormone ß, gonadotropin-releasing hormone-I and deiodinase expression between migrant and resident subspecies of white-crowned sparrow (Zonotrichia leucophrys).

Across taxa, the seasonal transition between non-breeding and breeding states is controlled by localised thyroid hormone signalling in the deep brain via reciprocal switching of deiodinase enzyme expression from type 3 (DIO3) to type 2 (DIO2). This reciprocal switch is considered to be mediated by increasing thyroid-stimulating hormone ß (TSHß) release from the pars tuberalis, which occurs in response to a change in photoperiod. Although well characterised in a handful of model organisms in controlled laboratory settings, this pathway remains largely unexplored in free-living animals under natural environmental conditions. In this comparative gene expression study, we investigated hypothalamic thyroid hormone signalling in two seasonally breeding subspecies of white-crowned sparrow (Zonotrichia leucophrys), across the entirety of their annual cycles. The migratory Gambel's (Z. l. gambelii) and resident Nuttall's (Z. l. nuttalii) subspecies differ with respect to timing of reproduction, as well as life history stage and migratory strategies. Although DIO3 mRNA expression was elevated and DIO2 mRNA expression was reduced in the wintering period in both subspecies, DIO2 peaked in both subspecies prior to the onset of reproduction. However, there was differential timing between subspecies in peak DIO2 expression. Intriguingly, seasonal modulation of TSHß mRNA was only observed in migrants, where expression was elevated at the start of breeding, consistent with observations from other highly photoperiodic species. There was no correlation between TSHß, DIO2 and gonadotropin-releasing hormone-I mRNA or reproductive metrics in residents. Based on these observed differences, we discuss potential implications for our understanding of how changes in medial basal hypothalamic gene expression mediates initiation of seasonal reproduction.

Acute restraint stress does not alter corticosteroid receptors or 11ß-hydroxysteroid dehydrogenase gene expression at hypothalamic-pituitary-adrenal axis regulatory sites in captive male white-crowned sparrows (Zonotrichia leucophrys gambelii).

Capture-restraint is often used to investigate the acute hypothalamic-pituitary-adrenal axis (HPA) response to stress in wild and captive animals through the production of glucocorticoids. Although this approach is useful for understanding changes in glucocorticoids, it overlooks potential changes in the complex regulatory systems associated with the glucocorticoid response, including genomic receptors, steroid metabolizing enzymes, carrier proteins, and downstream target proteins (e.g. gonadotropin-inhibitory hormone; GnIH). The present study in captive male white-crowned sparrows (Zonotrichia leucophrys) tests the hypothesis that corticosteroid receptors (mineralocorticoid - MR and glucocorticoid - GR), 11ß-hydroxysteroid dehydrogenase 1 (11ßHSD1) and 2 (11ßHSD2), corticosteroid binding globulin (CBG), and GnIH undergo rapid changes in expression to mediate the glucocorticoid response to acute stress. To determine dynamic changes in gene mRNA expression in the hippocampus, hypothalamus, pituitary gland, and liver, birds were sampled within 3 min of entering the room and after 10, 30, and 60 min of capture restraint stress in a cloth bag. Restraint stress handling increased CBG and decreased GnIH mRNA expression in the liver and hypothalamus, respectively. MR, GR, 11ßHSD1, and 11ßHSD2 mRNA expression in the brain, pituitary gland, and liver did not change. No correlations were found between gene expression and baseline or stress-induced plasma corticosterone levels. No rapid changes of MR, GR, 11ßHSD1, and 11ßHSD2 mRNA expression during a standardized acute restraint protocol suggests that tissue level sensitivity may remain constant during acute stressors. However, the observed rise in CBG mRNA expression could act to facilitate transport to target tissues or buffer the rise in circulating glucocorticoids. Further studies on tissue specific sensitivity are warranted.

Predictors of self-reported symptoms and testing for COVID-19 in Canada using a nationally representative survey.

Random population-based surveys to estimate prevalence of SARS-CoV2 infection causing coronavirus disease (COVID-19) are useful to understand distributions and predictors of the infection. In April 2020, the first-ever nationally representative survey in Canada polled 4,240 adults age 18 years and older about self-reported COVID experience in March, early in the epidemic. We examined the levels and predictors of COVID symptoms, defined as fever plus difficulty breathing/shortness of breath, dry cough so severe that it disrupts sleep, and/or loss of sense of smell; and testing for SARS-CoV-2 by respondents and/or household members. About 8% of Canadians reported that they and/or one or more household members experienced COVID symptoms. Symptoms were more common in younger than in older adults, and among visible minorities. Overall, only 3% of respondents and/or household members reported testing for SARS-CoV-2. Being tested was associated with having COVID symptoms, Indigenous identity, and living in Quebec. Periodic nationally representative surveys of symptoms, as well as SARS-CoV-2 antibodies, are required in many countries to understand the pandemic and prepare for the future.

Tissue-specific expression of 11ß-HSD and its effects on plasma corticosterone during the stress response.

The hypothalamic-pituitary-adrenal (HPA) axis is under complex regulatory control at multiple levels. Enzymatic regulation plays an important role in both circulating levels of glucocorticoids and target tissue exposure. Three key enzyme pathways are responsible for the immediate control of glucocorticoids. De novo synthesis of glucocorticoid from cholesterol involves a multistep enzymatic cascade. This cascade terminates with 11ß-hydroxylase, responsible for the final conversion of 11-deoxy precursors into active glucocorticoids. Additionally, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) controls regeneration of glucocorticoids from inactive metabolites, providing a secondary source of active glucocorticoids. Localized inactivation of glucocorticoids is under the control of Type 2 11ß-HSD (11ß-HSD2). The function of these enzymes is largely unexplored in wild species, particularly songbirds. Here, we investigated the contribution of both clearance and generation of glucocorticoids to regulation of the hormonal stress response via the use of pharmacological antagonists. Additionally, we mapped 11ß-HSD gene expression. We found 11ß-HSD1 primarily in liver, kidney and adrenal glands, although it was detectable across all tissue types. 11ß-HSD2 was predominately expressed in the adrenal glands and kidney with moderate gonadal and liver expression. Inhibition of glucocorticoid generation by metyrapone was found to decrease levels peripherally, while both peripheral and central administration of the 11ß-HSD2 inhibitor DETC resulted in elevated concentrations of corticosterone. These data suggest that during the stress response, peripheral antagonism of the 11ß-HSD system has a greater impact on circulating glucocorticoid levels than central control. Further studies should aim to elucidate the respective roles of the 11ß-HSD and 11ß-hydroxylase enzymes.

Centromere DNA Destabilizes H3 Nucleosomes to Promote CENP-A Deposition during the Cell Cycle.

Active centromeres are defined by the presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location, CENP-A chromatin and kinetochores are maintained at that location through a positive feedback loop where kinetochore proteins recruited by CENP-A promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences. Intrinsic properties of centromeric DNA may favor the assembly of CENP-A rather than H3 nucleosomes. Here we investigate histone dynamics on centromere DNA. We show that during S phase, histone H3 is deposited as a placeholder at fission yeast centromeres and is subsequently evicted in G2, when we detect deposition of the majority of new CENP-ACnp1. We also find that centromere DNA has an innate property of driving high rates of turnover of H3-containing nucleosomes, resulting in low nucleosome occupancy. When placed at an ectopic chromosomal location in the absence of any CENP-ACnp1 assembly, centromere DNA appears to retain its ability to impose S phase deposition and G2 eviction of H3, suggesting that features within centromere DNA program H3 dynamics. Because RNA polymerase II (RNAPII) occupancy on this centromere DNA coincides with H3 eviction in G2, we propose a model in which RNAPII-coupled chromatin remodeling promotes replacement of H3 with CENP-ACnp1 nucleosomes.

Gastrointestinal distribution of chicken gastrin-cholecystokinin family transcript expression and response to short-term nutritive state.

The related peptide hormones cholecystokinin (CCK) and gastrin are conserved throughout vertebrate clades and implicated in energy homeostasis. CCK is generally accepted as a satiety hormone in poultry, but the role of gastrin remains poorly studied. Functional dissection of these ligands is required to characterise the molecular control of growth & satiety in the domestic chicken, for which there is an increasingly pressing mandate. There are limited descriptions of physiological distributions for the two genes in birds, and these are mostly reliant on immunohistochemistry which can prove problematic due to the shared structure of the targets. Therefore, we have defined the tissue distributions of CCK and gastrin in the chicken, focussing on the gastrointestinal tract, by using transcript-dependent techniques to improve reliability by increasing specificity. Though considerably more highly expressed in the brain, gastrointestinal CCK transcripts were dispersed throughout the small intestine and particularly around the proximal ileum. Gastrin expression was strictly limited to the gastric antrum region of the intestinal tract, albeit very highly expressed. We demonstrate that CCK mRNA expression does not respond as expected for a short-term satiety hormone, and that the short-term response of gastrin expression is paradoxical compared to its role in mammals. These results partially corroborate previous peptide distribution studies and initiate exploration of the nutrient-responsive roles of these hormones in avian energy balance.

Pancreatic PYY but not PPY expression is responsive to short-term nutritional state and the pancreas constitutes the major site of PYY mRNA expression in chickens.

PP-fold peptides such as peptide YY (PYY) and pancreatic polypeptide (PPY) are known to play key roles in vertebrate energy homeostasis. Until recently, no gene sequence was available for avian PYY and therefore a gap in knowledge of regulation of its expression exists in avian species. Here we further evidence the mRNA sequence for chicken PYY and show that the pancreas is the major site of its mRNA expression, with a secondary peak of expression around the distal jejunum, in contrast to mammals where the large intestine is the major site of PYY expression. We also demonstrate that pancreatic PYY expression is responsive to short-term and long-term nutritional state, increasing within hours of feeding, in contrast to intestinal PYY which does not fluctuate to the same extent, and pancreatic PPY which appears to be primarily determined by long-term energy state. Both pancreatic PYY and PPY expression were found to exhibit ontogeny, being evenly distributed throughout the pancreas in young (2wk) chicks but having a decreasing splenic to duodenal gradient by adolescence (12wk).