Viroporin activity from rotavirus nonstructural protein 4 induces intercellular calcium waves that contribute to pathogenesis.

Acute gastroenteritis remains the second leading cause of death among children under the age of 5 worldwide. While enteric viruses are the most common etiology, the drivers of their virulence remain incompletely understood. We recently found that cells infected with rotavirus, the most prevalent enteric virus in infants and young children, initiate hundreds of intercellular calcium waves that enhance both fluid secretion and viral spread. Understanding how rotavirus triggers intercellular calcium waves may allow us to design safer, more effective vaccines and therapeutics, but we still lack a mechanistic understanding of this process. In this study, we used existing virulent and attenuated rotavirus strains, as well as reverse engineered recombinants, to investigate the role of rotavirus nonstructural protein 4 (NSP4) in intercellular calcium wave induction using in vitro , organoid, and in vivo model systems. We found that the capacity to induce purinergic intercellular calcium waves (ICWs) segregated with NSP4 in both simian and murine-like rotavirus backgrounds, and NSP4 expression alone was sufficient to induce ICWs. NSP4's ability to function as a viroporin, which conducts calcium out of the endoplasmic reticulum, was necessary for ICW induction. Furthermore, viroporin activity and the resulting ICWs drove transcriptional changes indicative of innate immune activation, which were lost upon attenuation of viroporin function. Multiple aspects of RV disease severity in vivo correlated with the generation of ICWs, identifying a critical link between viroporin function, intercellular calcium waves, and enteric viral virulence.

Purinergic Signaling Drives Multiple Aspects of Rotavirus Pathophysiology.

Rotavirus causes life-threatening diarrhea in children, resulting in ∼200,000 deaths/year. The current treatment during infection is Oral Rehydration Solution which successfully replenishes fluids but does not alleviate diarrhea volume or severity. As a result, there is an urgent need to better understand rotavirus pathophysiology and develop more effective pediatric therapeutics. Rotavirus primarily infects the tips of small intestinal villi, yet has far-reaching effects on cell types distant from infected cells. We recently identified that rotavirus infected cells release the purinergic signaling molecule ADP, which activates P2Y1 receptors on nearby uninfected cells in vitro . To elucidate the role of purinergic signaling via P2Y1 receptors during rotavirus infection in vivo , we used the mouse-like rotavirus strain D6/2 which generates a severe infection in mice. C57BL/6J mouse pups were given an oral gavage of D6/2 rotavirus and assessed over the course of 5-7 days. Beginning at day 1 post infection, infected pups were treated daily by oral gavage with saline or 4 mg/kg MRS2500, a selective P2Y1 antagonist. Mice were monitored for diarrhea severity, diarrhea incidence, and viral shedding. Neonatal mice were euthanized at days 3 and 5 post-infection and small intestine was collected to observe infection. MRS2500 treatment decreased the severity, prevalence, and incidence of rotavirus diarrhea. Viral stool shedding, assessed by qPCR for rotavirus gene levels, revealed that MRS2500 treated pups had significantly lower viral shedding starting at day 4 post infection compared to saline treated pups, which suggests P2Y1 signaling may enhance rotavirus replication. Finally, we found that inhibition of P2Y1 with MRS2500 limited transmitted rotavirus diarrhea to uninfected pups within a litter. Together, these results suggest that P2Y1 signaling is involved in the pathogenesis of a homologous murine rotavirus strain, making P2Y1 receptors a promising anti-diarrheal, anti-viral therapeutic target to reduce rotavirus disease burden.

Espousing the innocence of paediatric patients: an innocent act?

Since the 19th century, innocence has been a hallmark of childhood. The innocence of children is seen as both a sanctity worth defending and a feature that excuses the unavoidable mistakes of adolescence. While beneficial in many settings, notions of childhood innocence are often entangled with values judgements. Inherent in innocence is the notion that that which we are innocent of is undesirable. Further, attributing innocence to some implies the tolerability of blame for others. This has unique implications in a medical setting. This essay explores the implications of espousing the innocence of paediatric patients. Ultimately, because attribution of innocence is both prone to bias and rooted in the same framework as blame, it degrades patient-centred care and compromises the patient-provider relationship. I argue that avoiding such characterisations may allow providers to more effectively promote paediatric health.

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators.

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

Live-Cell Fluorescence Imaging for Virus-Host Interactions.

Recent technological advances in microscopy have facilitated novel approaches to investigate host-pathogen interactions. In particular, improvements in both microscope hardware and engineered biosensors have helped to overcome barriers to live-cell imaging with fluorescence microscopy. Live fluorescent microscopy allows for the detection of discrete signaling events and protein localization, improving our ability to assess the effects of pharmacologic agents, microbes, or infection with high temporal resolution. Here we describe a protocol for long-term live-cell fluorescence imaging of virus infected cell lines.

Turning good intentions into good outcomes: ethical dilemmas at a student-run clinic and a rubric for reflective action.

Student-run clinics represent a unique medical education and healthcare delivery model powered largely by good intentions. These good intentions may produce questionable results, however, when juxtaposed with intense academic pressure to fill one's curriculum vitae with personal achievements, leadership roles and peer-reviewed publications. It becomes a legitimate ethical question whether student-run clinics consistently and materially enrich the care of underserved communities, or merely inspire a litany of rushed, short-term and low-quality projects that sidestep patient welfare or even cause brazen harm. As co-directors of HOMES Clinic, a student-managed clinic which offers free health and social services to people experiencing housing insecurity, we routinely encounter such well-intentioned but ethically questionable proposals. Here, we present four short case studies that dissect apart some of these common yet suspect assumptions underpinning student-run clinics. We then conclude with a rubric for reflective, calibrated action.

Distribution of P2Y and P2X purinergic receptor expression within the intestine.

Nucleotides are potent extracellular signaling molecules during homeostasis, infection, and injury due to their ability to activate purinergic receptors. The nucleotide ATP activates P2X receptors (P2RXs), whereas the nucleotides ADP, ATP, UTP, and UDP-glucose selectively activate different P2Y receptors (P2RYs). Several studies have established crucial roles for P2 receptors during intestinal inflammatory and infectious diseases, yet the most extensive characterization of purinergic signaling has focused on immune cells and the central and enteric nervous systems. As epithelial cells serve as the first barrier against irritants and infection, we hypothesized that the gut epithelium may express multiple purinergic receptors that respond to extracellular nucleotide signals. Using the Human Protein Atlas and Gut Cell Survey, we queried single-cell RNA sequencing (RNAseq) data for the P2 purinergic receptors in the small and large intestines. In silico analysis reveals robust mRNA expression of P2RY1, P2RY2, P2RY11, and P2RX4 throughout the gastrointestinal tract. Human intestinal organoids exhibited a similar expression pattern with a prominent expression of P2RY1, P2RY2, and P2RX4, but this purinergic receptor repertoire was not conserved in T84, Caco2, and HT29 intestinal epithelial cell lines. Finally, P2YR1 and P2YR2 agonists elicited robust calcium responses in human intestinal organoids, but calcium responses were weaker or absent in the cell lines. These findings suggest that the gastrointestinal epithelia respond to extracellular purinergic signaling via P2RY1, P2RY2, P2RY11, and P2RX4 receptors and highlight the benefit of using intestinal organoids as a model of intestinal purinergic signaling.NEW & NOTEWORTHY Several studies have revealed crucial roles for P2 receptors during inflammatory and infectious diseases, however, these have largely been demonstrated in immune cells and the enteric nervous system. Although epithelial cells serve as the first barrier against infection and inflammation, the role of purinergic signaling within the gastrointestinal tract remains largely unknown. This work expands our knowledge of purinergic receptor distribution and relative expression along the intestine.

Gastrointestinal organoids in the study of viral infections.

Viruses are among the most prevalent enteric pathogens. Although virologists historically relied on cell lines and animal models, human intestinal organoids (HIOs) continue to grow in popularity. HIOs are nontransformed, stem cell-derived, ex vivo cell cultures that maintain the cell type diversity of the intestinal epithelium. They offer higher throughput than standard animal models while more accurately mimicking the native tissue of infection than transformed cell lines. Here, we review recent literature that highlights virological advances facilitated by HIOs. We discuss the variations and limitations of HIOs, how HIOs have allowed for the cultivation of previously uncultivatable viruses, and how they have offered insight into tropism, entry, replication kinetics, and host-pathogen interactions. In each case, we discuss exemplary viruses and archetypal studies. We discuss how the speed and flexibility of HIO-based studies contributed to our knowledge of SARS-CoV-2 and antiviral therapeutics. Finally, we discuss the current limitations of HIOs and future directions to overcome these.

Associations of Incomplete SARS-CoV-2 Vaccination among Patients with Unstable Housing in Houston.

Vaccination is a safe and effective way to protect against SARS-CoV-2. Two of the three authorized SARS-CoV-2 vaccines require two doses, presenting logistical challenges. Those with unstable housing face barriers that amplify these challenges. In this study, we utilized a database maintained by Healthcare for the Homeless-Houston to determine the rates of partial vaccination among those with unstable housing in Houston (n=294). We then performed post-hoc analyses to identify predictors of partial vaccination. Our key finding was that 30% of those with unstable housing missed their second dose, a proportion far higher than the national average. Those with permanent supportive housing and those who had a Harris County Gold Card (financial assistance for health care costs) were more likely to return for dose two, while those who were younger, living on the streets, or staying in a temporary homeless shelter were more likely to miss the second dose.

Rotavirus induces intercellular calcium waves through ADP signaling.

Rotavirus causes severe diarrheal disease in children by broadly dysregulating intestinal homeostasis. However, the underlying mechanism(s) of rotavirus-induced dysregulation remains unclear. We found that rotavirus-infected cells produce paracrine signals that manifested as intercellular calcium waves (ICWs), observed in cell lines and human intestinal enteroids. Rotavirus ICWs were caused by the release of extracellular adenosine 5'-diphosphate (ADP) that activated P2Y1 purinergic receptors on neighboring cells. ICWs were blocked by P2Y1 antagonists or CRISPR-Cas9 knockout of the P2Y1 receptor. Blocking the ADP signal reduced rotavirus replication, inhibited rotavirus-induced serotonin release and fluid secretion, and reduced diarrhea severity in neonatal mice. Thus, rotavirus exploited paracrine purinergic signaling to generate ICWs that amplified the dysregulation of host cells and altered gastrointestinal physiology to cause diarrhea.

Chronic pain in adolescence and internalizing mental health disorders: a nationally representative study.

Chronic pain in childhood and adolescence has been shown to heighten the risk for depressive and anxiety disorders in specific samples in adulthood; however, little is known about the association between a wider variety of chronic pains and internalizing mental health disorders. Using nationally representative data, the objectives of this study were to establish prevalence rates of internalizing mental health disorders (anxiety and depressive disorders) among cohorts with or without adolescent chronic pain, and to examine whether chronic pain in adolescence is associated with lifetime history of internalizing mental health disorders reported in adulthood. Data from the National Longitudinal Study of Adolescent to Adult Health (Add Health) was used (N = 14,790). Individuals who had chronic pain in adolescence subsequently reported higher rates of lifetime anxiety disorders (21.1% vs 12.4%) and depressive disorders (24.5% vs 14.1%) in adulthood as compared with individuals without a history of adolescent chronic pain. Multivariate logistic regression confirmed that chronic pain in adolescence was associated with an increased likelihood of lifetime history of anxiety disorders (odds ratio: 1.33; 95% confidence interval: 1.09-1.63, P = 0.005) and depressive disorders (odds ratio: 1.38; confidence interval: 1.16-1.64, P < 0.001) reported in adulthood. Future research is needed to examine neurobiological and psychological mechanisms underlying these comorbidities.

Trends in opioid prescriptions among children and adolescents in the United States: a nationally representative study from 1996 to 2012.

Prescription opioid misuse is a major public health concern in the United States, yet little is known about national prescription patterns. We aimed to assess trends in opioid prescriptions made to children and adolescents, to their families, and to adults in the United States from 1996 to 2012. The sample was drawn from nationally representative data, the Medical Expenditure Panel Surveys. We used survey design methods to examine trends in prescription opioid use over time and a logistic regression analysis to examine predictors associated with opioid use. Findings indicated that from 1996 to 2012 opioid prescriptions to children and adolescents remained stable and low. In 1996, 2.68% of children received an opioid prescription, and in 2012, 2.91% received an opioid prescription. In contrast, opioid prescriptions to family members of children and adolescents and adults in general significantly increased during this period. The most common opioid prescriptions to children and adolescents in 2012 were codeine, hydrocodone, and oxycodone. Using multivariate logistic regression models, the white non-Hispanic race, older age, health insurance, and parent-reported fair to poor general health were associated with higher rates of opioid prescriptions in children and adolescents. Our main finding was that although the rates of opioid prescriptions have increased among adults in the United States, the rates have not changed among children and adolescents. Recent epidemiologic association studies have identified a strong link between increased opioid prescriptions and increased rates of opioid misuse and abuse in adults. Future studies should assess the association between adult opioid prescriptions and children or adolescent opioid misuse.