Underestimation of SARS-CoV-2 in wastewater due to single or double mutations in the N1 qPCR probe binding region.

Wastewater surveillance using RT-qPCR has now been widely adopted to track circulating levels of SARS-CoV-2 virus in many sewersheds. The CDC qPCR assays targeting two regions (N1 and N2) within the N gene are commonly used, but a discrepancy between the two biomarkers has been noticed by independent studies using these methods since late 2021. The reason is presumed to be due to mutations in regions targeted by the N1 qPCR probe. In this study, we systematically investigated and unequivocally confirmed that the underlying reason for this discrepancy was mutations in the N1 probe target, and that a single mutation could cause a significant drop in signal. We first confirmed the proportion of related mutations in wastewater samples (Jan 2021-Dec 2022) using nested PCR and LC-MS. Based on relative proportions of N1 alleles, we separated the wastewater data into four time periods corresponding to different variant waves: Period I (Alpha and Delta waves with 0 mutation), Period II (BA.1/BA.2 waves with a single mutation found in all Omicron strains), Period III (BA.5.2* wave with two mutations), and Period IV (BQ.1* wave with two mutations). Significantly lower N1 copies relative to N2 copies in samples from Periods II-IV compared to those from Period I was observed in wastewater. To further pinpoint the extent to which each mutation impacted N1 quantification, we compared the qPCR response among different synthetic oligomers with corresponding mutations. This study highlighted the impact of even just one or two mutations on qPCR-based wastewater surveillance of SARS-CoV-2.

Sex- and Estrus-Dependent Differences in Rat Basolateral Amygdala.

Depression and anxiety are diagnosed almost twice as often in women, and the symptomology differs in men and women and is sensitive to sex hormones. The basolateral amygdala (BLA) contributes to emotion-related behaviors that differ between males and females and across the reproductive cycle. This hints at sex- or estrus-dependent features of BLA function, about which very little is known. The purpose of this study was to test whether there are sex differences or estrous cyclicity in rat BLA physiology and to determine their mechanistic correlates. We found substantial sex differences in the activity of neurons in lateral nuclei (LAT) and basal nuclei (BA) of the BLA that were associated with greater excitatory synaptic input in females. We also found strong differences in the activity of LAT and BA neurons across the estrous cycle. These differences were associated with a shift in the inhibition-excitation balance such that LAT had relatively greater inhibition during proestrus which paralleled more rapid cued fear extinction. In contrast, BA had relatively greater inhibition during diestrus that paralleled more rapid contextual fear extinction. These results are the first to demonstrate sex differences in BLA neuronal activity and the impact of estrous cyclicity on these measures. The shift between LAT and BA predominance across the estrous cycle provides a simple construct for understanding the effects of the estrous cycle on BLA-dependent behaviors. These results provide a novel framework to understand the cyclicity of emotional memory and highlight the importance of considering ovarian cycle when studying the BLA of females.SIGNIFICANCE STATEMENT There are differences in emotional responses and many psychiatric symptoms between males and females. This may point to sex differences in limbic brain regions. Here we demonstrate sex differences in neuronal activity in one key limbic region, the basolateral amygdala (BLA), whose activity fluctuates across the estrous cycle due to a shift in the balance of inhibition and excitation across two BLA regions, the lateral and basal nuclei. By uncovering this push-pull shift between lateral and basal nuclei, these results help to explain disparate findings about the effects of biological sex and estrous cyclicity on emotion and provide a framework for understanding fluctuations in emotional memory and psychiatric symptoms.

Abnormalities in the Structure and Function of Cerebellar Neurons and Neuroglia in the Lc/+ Chimeric Mouse Model of Variable Developmental Purkinje Cell Loss.

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders characterized by impaired and disordered language, decreased social interactions, stereotyped and repetitive behaviors, and impaired fine and gross motor skills. It has been well established that cerebellar abnormalities are one of the most common structural changes seen in the brains of people diagnosed with autism. Common cerebellar pathology observed in autistic individuals includes variable loss of cerebellar Purkinje cells (PCs) and increased numbers of reactive neuroglia in the cerebellum and cortical brain regions. The Lc/+ mutant mouse loses 100 % of cerebellar PCs during the first few weeks of life and provided a valuable model to study the effects of developmental PC loss on underlying structural and functional changes in cerebellar neural circuits. Lurcher (Lc) chimeric mice were also generated to explore the link between variable cerebellar pathology and subsequent changes in the structure and function of cerebellar neurons and neuroglia. Chimeras with the most severe cerebellar pathology (as quantified by cerebellar PC counts) had the largest changes in cFos expression (an indirect reporter of neural activity) in cerebellar granule cells (GCs) and cerebellar nucleus (CN) neurons. In addition, Lc chimeras with the fewest PCs also had numerous reactive microglia and Bergmann glia located in the cerebellar cortex. Structural and functional abnormalities observed in the cerebella of Lc chimeras appeared to be along a continuum, with the degree of pathology related to the number of PCs in individual chimeras.