Real-time evaluation of signal accuracy in wastewater surveillance of pathogens with high rates of mutation.

Wastewater surveillance of coronavirus disease 2019 (COVID-19) commonly applies reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater over time. In most applications worldwide, maximal sensitivity and specificity of RT-qPCR has been achieved, in part, by monitoring two or more genomic loci of SARS-CoV-2. In Ontario, Canada, the provincial Wastewater Surveillance Initiative reports the average copies of the CDC N1 and N2 loci normalized to the fecal biomarker pepper mild mottle virus. In November 2021, the emergence of the Omicron variant of concern, harboring a C28311T mutation within the CDC N1 probe region, challenged the accuracy of the consensus between the RT-qPCR measurements of the N1 and N2 loci of SARS-CoV-2. In this study, we developed and applied a novel real-time dual loci quality assurance and control framework based on the relative difference between the loci measurements to the City of Ottawa dataset to identify a loss of sensitivity of the N1 assay in the period from July 10, 2022 to January 31, 2023. Further analysis via sequencing and allele-specific RT-qPCR revealed a high proportion of mutations C28312T and A28330G during the study period, both in the City of Ottawa and across the province. It is hypothesized that nucleotide mutations in the probe region, especially A28330G, led to inefficient annealing, resulting in reduction in sensitivity and accuracy of the N1 assay. This study highlights the importance of implementing quality assurance and control criteria to continually evaluate, in near real-time, the accuracy of the signal produced in wastewater surveillance applications that rely on detection of pathogens whose genomes undergo high rates of mutation.

Many mouths to feed: the control of food intake during lactation.

Providing nutrients to their developing young is perhaps the most energetically demanding task facing female mammals. In this paper we focus primarily on studies carried out in rats to describe the changes in the maternal brain that enable the dam to meet the energetic demands of her offspring. In rats, providing milk for their litter is associated with a dramatic increase in caloric intake, a reduction in energy expenditure and changes in the pattern of energy utilization as well as storage. These behavioral and physiological adaptations result, in part, from alterations in the central pathways controlling energy balance. Differences in circulating levels of metabolic hormones such as leptin, ghrelin and insulin as well as in responsiveness to these signals between lactating and nonlactating animals, contribute to the modifications in energy balance pathways seen postpartum. Suckling stimulation from the pups both directly, and through the hormonal state that it induces in the mother, plays a key role in facilitating these adaptations.

Energetic demands of lactation produce an increase in the expression of growth hormone secretagogue receptor in the hypothalamus and ventral tegmental area of the rat despite a reduction in circulating ghrelin.

Lactating rats show changes in the secretion of hormones and brain signals that promote hyperphagia and facilitate the production of milk. Little is known, however, about the role of ghrelin in the mechanisms sustaining lactational hyperphagia. Here, we used Wistar female rats that underwent surgery to sever the galactophores to prevent milk delivery (GC rats) and decrease the energetic drain of milk delivery. We compared plasma acyl-ghrelin concentrations and growth hormone secretagogue receptor (GHSR) mRNA expression in different brain regions of GC rats with those of sham operated lactating and nonlactating rats. Additional lactating and nonlactating rats were implanted with cannulae aimed at the lateral ventricles and were used to compare feeding responses to central ghrelin or GHSR antagonist infusions to those of nonlactating rats receiving similar infusions on day 14-16 postpartum (pp). Results show lower plasma acyl-ghrelin concentrations on day 15 pp sham operated lactating rats compared to GC or nonlactating rats. These changes occur in association with increased GHSR mRNA expression in the hypothalamic arcuate nucleus (ARC) and ventral tegmental area (VTA) of sham operated lactating rats. Despite lactational hyperphagia, infusions of ghrelin (0.25 or 1 µg) resulted in similar increases in food intake in lactating and nonlactating rats. In addition, infusions of the GHSR antagonist JMV3002 (4 µg in 1 µl of vehicle) produced greater suppression of food intake in lactating rats than in nonlactating rats. These data suggest that, despite lower plasma ghrelin, the energetic drain of lactation increases sensitivity to the orexigenic effects of ghrelin in brain regions important for food intake and energy balance, and these events are associated with lactational hyperphagia.

Rats with a truncated ghrelin receptor (GHSR) do not respond to ghrelin, and show reduced intake of palatable, high-calorie food.

Ghrelin, a peptide hormone produced by the stomach, is the endogenous ligand for the Growth Hormone Secretagogue Receptor (GHSR). Ghrelin acts on the GHSR to increase food intake, appetitive behaviors, and adiposity. Recently, a rat model with a null mutation to the GHSR gene (FHH-GHSR(m1/Mcwi)) was generated and used in behavioral studies, but the basic metabolic phenotype of this strain as well as that of the background strain (Fawn Hooded Hypertensive, FHH) has not been characterized in detail. Here we compared male FHH-GHSR(m1/Mcwi) rats with their wild-type littermates (FHH-WT) in a number of metabolic parameters. In the 24h of recovery following an acute overnight fast, FHH-GHSR(m1/Mcwi) rats consumed less food than FHH-WT animals, and relative to their body weights, adult FHH-GHSR(m1/Mcwi) rats consumed fewer calories when placed on a high-fat diet. Despite this, FHH-GHSR(m1/Mcwi) rats did not show a difference in diet-induced obesity or weight gain. Fasted FHH-GHSR(m1/Mcwi) rats exhibited increased Agouti-Related Peptide (AgRP) and Neuropeptide Y (NPY) expression in the Arcuate Nucleus (ARC), indicative of altered central regulation of feeding and energy balance. FHH-GHSR(m1/Mcwi) rats exhibited lower levels of home cage locomotor behavior over the entire light/dark cycle, and reduced levels of food anticipatory activity when placed on a restricted feeding schedule. Finally, FHH-GHSR(m1/Mcwi) rats consumed less of a palatable dessert (cookie dough) given after the completion of the scheduled meal. Altogether, our data show that rats lacking a functional GHSR tend to eat less than their wild-type counterparts in the face of acute fasts, chronic high-fat diet exposure, and exposure to a palatable dessert, despite not showing differences in body weight and glucose homeostasis that are characteristic of GHSR null mice. These data indicate that many, but not all responses to GHSR ablation are conserved between rats and mice. The FHH-GHSR(m1/Mcwi) rat thus represents a novel and useful model for studying GHSR function in rats.

Growth Hormone Secretagogue Receptor Dimers: A New Pharmacological Target

The growth hormone secretagogue receptor (GHSR1a), the target of the ghrelin peptide, is widely distributed throughout the brain, and, while studies have often reported very low or absent levels of central ghrelin, it is now known that GHSR1a, even in the absence of a natural ligand, has physiological roles. Not only do these roles originate from the receptor's constitutive activity, but recent data indicate that GHSR1a dimerizes with a wide array of other receptors. These include the dopamine 1 receptor (D1R), the dopamine 2 receptor (D2R), the melanocortin-3 receptor (MC3R), the serotonin 2C receptor (5-HT2C), and possibly the cannabinoid type 1 receptor (CB1). Within these dimers, signaling of the protomers involved are modified through facilitation, inhibition, and even modification of signaling pathways resulting in physiological consequences not seen in the absence of these dimers. While in some cases the ghrelin peptide is not required for these modifications to occur, in others, the presence is necessary for these changes to take effect. These heterodimers demonstrate the broad array of roles and complexity of the ghrelin system. By better understanding how these dimers work, it is hoped that improved treatments for a variety of disorders, including Parkinson's disease, schizophrenia, addiction, obesity, diabetes, and more, can be devised. In this review, we examine the current state of knowledge surrounding GHSR heterodimers, and how we can apply this knowledge to various pharmacological treatments.

Knockdown of central ghrelin O-acyltransferase by vivo-morpholino reduces body mass of rats fed a high-fat diet.

The enzyme ghrelin O-acyltransferase (GOAT) activates the orexigenic peptide ghrelin by transferring an acyl group from fatty acids to the serine-3 residue of the ghrelin molecule. This allows ghrelin to bind to its only known receptor, the growth hormone secretagogue receptor type 1a (GHSR1a). While studies have examined the hypothalamic transcriptional response of GOAT to metabolic challenge in mice, little has been examined in the rat hypothalamus. Furthermore, it has not been possible to identify the role of central GOAT separate from that of the periphery, since previous studies either knocked out GOAT system-wide or administered a GOAT inhibitor intraperitoneally. To determine if central GOAT expression is modulated by changes in energy state, we subjected rats to either forty-eight hours of food deprivation or three weeks of food restriction and found that GOAT mRNA increases significantly in both the hypothalamus and the stomach fundus in response to both metabolic challenges. We also found increases in hypothalamic ghrelin mRNA and stomach GHSR1a mRNA in response to food deprivation, as well as increases in hypothalamic GHSR1a mRNA in response to food restriction. We then conducted a second study where we continuously infused amorpholino antisense oligonucleotide into the lateral ventricles of rats to knock-down GOAT centrally while the animals were exposed to a high fat diet. Our results show that rats receiving the GOAT antisense gained less weight, and decreased their caloric efficiency when eating a high fat diet compared to control animals. These data suggest that central GOAT plays a role in modulating metabolism in rats.

Novel Regulator of Acylated Ghrelin, CF801, Reduces Weight Gain, Rebound Feeding after a Fast, and Adiposity in Mice.

Ghrelin is a 28 amino acid hormonal peptide that is intimately related to the regulation of food intake and body weight. Once secreted, ghrelin binds to the growth hormone secretagogue receptor-1a, the only known receptor for ghrelin and is capable of activating a number of signaling cascades, ultimately resulting in an increase in food intake and adiposity. Because ghrelin has been linked to overeating and the development of obesity, a number of pharmacological interventions have been generated in order to interfere with either the activation of ghrelin or interrupting ghrelin signaling as a means to reducing appetite and decrease weight gain. Here, we present a novel peptide, CF801, capable of reducing circulating acylated ghrelin levels and subsequent body weight gain and adiposity. To this end, we show that IP administration of CF801 is sufficient to reduce circulating plasma acylated ghrelin levels. Acutely, intraperitoneal injections of CF801 resulted in decreased rebound feeding after an overnight fast. When delivered chronically, they decreased weight gain and adiposity without affecting caloric intake. CF801, however, did cause a change in diet preference, decreasing preference for a high-fat diet and increasing preference for regular chow diet. Given the complexity of ghrelin receptor function, we propose that CF801, along with other compounds that regulate ghrelin secretion, may prove to be a beneficial tool in the study of the ghrelin system, and potential targets for ghrelin-based obesity treatments without altering the function of ghrelin receptors.

Rats perinatally exposed to food restriction and high-fat diet show differences in adipose tissue gene expression under chronic caloric restriction.

The aim of this study is to analyze how maternal diet during the lactational period influences the adipose tissue response to chronic caloric restriction in offspring. Lactating dams were subjected to one of three treatments: 50% food restriction (FR), ad lib standard chow (AL), or ad lib high-fat diet (HF). Juveniles were first weaned onto standard chow, then in adulthood 50% calorically restricted and maintained at 90% of normal body weight for 60 d. HF animals showed increased percent body fat compared with AL and FR animals despite equivalent body weights. HF animals showed alterations in the balance of adipose tissue lipogenic (FAS, LPL) and lipolytic (HSL) gene expression that may underlie their propensity to maintain fat stores under caloric restriction.