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Noroviruses are major pathogens associated with acute gastroenteritis worldwide. Their RNA genomes are diverse, with two major genogroups (GI and GII) comprised of at least 28 genotypes associated with human disease. To elucidate mechanisms underlying norovirus diversity and evolution, we used a large-scale genomics approach to analyze human norovirus sequences. Comparison of over 2000 nearly full-length ORF2 sequences representing most of the known GI and GII genotypes infecting humans showed a limited number (≤5) of distinct intra-genotypic variants within each genotype, with the exception of GII.4. The non-GII.4 genotypes were comprised of one or more intra-genotypic variants, with each variant containing strains that differed by only a few residues over several decades (remaining "static") and that have co-circulated with no clear epidemiologic pattern. In contrast, the GII.4 genotype presented the largest number of variants (>10) that have evolved over time with a clear pattern of periodic variant replacement. To expand our understanding of these two patterns of diversification ("static" versus "evolving"), we analyzed using NGS the nearly full-length norovirus genome in healthy individuals infected with GII.4, GII.6 or GII.17 viruses in different outbreak settings. The GII.4 viruses accumulated mutations rapidly within and between hosts, while the GII.6 and GII.17 viruses remained relatively stable, consistent with their diversification patterns. Further analysis of genetic relationships and natural history patterns identified groupings of certain genotypes into larger related clusters designated here as "immunotypes". We propose that "immunotypes" and their evolutionary patterns influence the prevalence of a particular norovirus genotype in the human population.

Abstract Norovirus is a leading cause of acute gastroenteritis (AGE) globally. AGE resulting from norovirus causes significant morbidity and mortality in countries of all income levels, particularly among young children and older adults. Prevention of norovirus AGE represents a unique challenge as the virus is genetically diverse with multiple genogroups and genotypes cocirculating globally and causing disease in humans. Variants of the GII.4 genotype are typically the most common genotype, and other genotypes cause varying amounts of disease year-to-year, with GII.2, GII.3, and GII.6 most prevalent in recent years. Noroviruses are primarily transmitted via the fecal-oral route and only a very small number of virions are required for infection, which makes outbreaks of norovirus extremely difficult to control when they occur. Settings like long-term care facilities, daycares, and hospitals are at high risk of outbreaks and can have very high attack rates resulting in substantial costs and disease burden. Severe cases of norovirus AGE are most common in vulnerable patient populations, such as infants, the elderly, and immunocompromised individuals, with available treatments limited to rehydration therapies and supportive care. To date, there are no FDA-approved norovirus vaccines; however, several candidates are currently in development. Given the substantial human and economic burden associated with norovirus AGE, a vaccine to prevent morbidity and mortality and protect vulnerable populations could have a significant impact on global public health.

Background: Surgeon-prescribed opioids contribute to 11% of prescription drug overdoses in the United States (US). With prescription opioids involved in 24% of all opioid-related overdose deaths in 2020, the US Centers for Disease Control and Prevention (CDC) recommends naloxone co-prescribing to patients at high-risk of overdose and death as a harm reduction strategy. We sought to 1) examine naloxone co-prescribing rates to surgical patients (using common post-surgical prescribing amounts) and those with potential risk factors for opioid-related overdoses or adverse events, and 2) identify the factors associated with patients receiving naloxone co-prescriptions. Methods: We conducted a single-institution, retrospective study using the electronic medical records of all patients undergoing surgery at an academic institution between August 2020 and May 2021. We included post-surgical adults prescribed opioids that were sent to a pharmacy in our health system. The primary outcome was the percentage of co-prescribed naloxone in patients prescribed opioids. Results: The overall naloxone co-prescription rate was low (1.7%). Only 14.6% of patients prescribed ≥350 morphine milligram equivalents (MME, equivalent to 46.7 oxycodone 5 mg tablets) and 8.6% of patients using illicit drugs were co-prescribed naloxone. On multivariable analysis, patients who were prescribed >350 MME, used illicit drugs or tobacco, underwent an elective or emergent general surgery procedure, self-identified as Hispanic, or had ASA scores of 2-4 were more likely to receive a naloxone co-prescription. Conclusions: Naloxone co-prescribing after surgery remains low, even for high-risk patients. Harm reduction strategies such as naloxone, safe storage, and disposal of leftover opioids could reduce surgeons’ iatrogenic contributions to the worsening US opioid crisis.

ABSTRACTNoroviruses are a major cause of acute gastroenteritis worldwide and can establish chronic infection in immunocompromised individuals. To investigate the mechanisms of norovirus evolution during chronic infection, we selected seven representative patients from a National Institutes of Health study cohort who sustained norovirus infection for periods ranging from 73 to 1,492 days. Six patients shed viruses belonging to a single genotype (GII.2[PNA], GII.4 New Orleans[P4], GII.4 Den Haag[P4], GII.3[P21], GII.6[P7], or GII.14[P7]) over the period examined, while one patient sequentially shed two genotypes (GII.6[P7] followed by GII.4 Sydney[P31]). Norovirus genomes from consecutive stool samples were sequenced at high resolution (>3,300 reads/nucleotide position) using the Illumina platform and subjected to bioinformatics analysis. Norovirus sequences could be resolved into one or more discrete clonal RNA genomes that persisted within these patients over time. Phylogenetic analyses inferred that clonal populations originated from a single founder virus and not by reinfection with community strains. Estimated evolutionary rates of clonal populations during persistent infection were similar to those of noroviruses from acute infection in the global database, suggesting that inherently higher RNA-dependent polymerase error rates were not associated with the ability to persist. The high-resolution analysis of norovirus diversity and evolution at the population level described here should allow a better understanding of adaptive mutations sustained during chronic infection.IMPORTANCENoroviruses are an important cause of chronic diarrhea in patients with compromised immune systems. Presently, there are no effective therapies to clear the virus, which can persist for years in the intestinal tract. The goal of our study was to develop a better understanding of the norovirus strains that are associated with these long-term infections. With the remarkable diversity of norovirus strains detected in the immunocompromised patient cohort we studied, it appears that most, if not all, noroviruses circulating in nature may have the capacity to establish a chronic infection when a person is unable to mount an effective immune response. Our work is the most comprehensive genetic data set generated to date in which near full-length genomes from noroviruses associated with chronic infection were analyzed by high-resolution next-generation sequencing. Analysis of this data set led to our discovery that certain patients in our cohort were shedding noroviruses that could be subdivided into distinct haplotypes or populations of viruses that were co-evolving independently. The ability to track haplotypes of noroviruses during chronic infection will allow us to fine-tune our understanding of how the virus adapts and maintains itself in the human host, and how selective pressures such as antiviral drugs can affect these distinct populations.

Human norovirus pathogenesis is incompletely understood due to a lack of appropriate animal disease models. Here, Green et al. show norovirus replication in chromogranin A-positive enteroendocrine cells and other epithelial cells in tissue from a pediatric intestinal transplant recipient with severe gastroenteritis.

Objective: Phosphatidylethanolamine methyltransferase (PEMT) generates phosphatidylcholine (PC), the most abundant phospholipid in the mitochondria and an important acyl chain donor for cardiolipin (CL) biosynthesis. Mice lacking PEMT (PEMTKO) are cold-intolerant when fed a high-fat diet (HFD) due to unclear mechanisms. The purpose of this study was to determine whether PEMT-derived phospholipids are important for the function of uncoupling protein 1 (UCP1) and thus for maintenance of core temperature. Methods: To test whether PEMT-derived phospholipids are important for UCP1 function, we examined cold-tolerance and brown adipose (BAT) mitochondria from PEMTKO mice with or without HFD feeding. We complemented these studies with experiments on mice lacking functional CL due to tafazzin knockdown (TAZKD). We generated several conditional mouse models to study the tissue-specific roles of PEMT, including mice with BAT-specific knockout of PEMT (PEMT-BKO). Results: Chow- and HFD-fed PEMTKO mice completely lacked UCP1 protein in BAT, despite a lack of difference in mRNA levels, and the mice were accordingly cold-intolerant. While HFD-fed PEMTKO mice exhibited reduced mitochondrial CL content, this was not observed in chow-fed PEMTKO mice or TAZKD mice, indicating that the lack of UCP1 was not attributable to CL deficiency. Surprisingly, the PEMT-BKO mice exhibited normal UCP1 protein levels. Knockout of PEMT in the adipose tissue (PEMT-AKO), liver (PEMT-LKO), or skeletal muscle (PEMT-MKO) also did not affect UCP1 protein levels, suggesting that lack of PEMT in other non-UCP1-expressing cells communicates to BAT to suppress UCP1. Instead, we identified an untranslated UCP1 splice variant that was triggered during the perinatal period in the PEMTKO mice. Conclusions: PEMT is required for UCP1 splicing that yields functional protein. This effect is derived by PEMT in nonadipocytes that communicates to BAT during embryonic development. Future research will focus on identifying the non-cell-autonomous PEMT-dependent mechanism of UCP1 splicing. Keywords: Brown adipose tissue, UCP1, PEMT, Phosphatidylcholine, Cardiolipin, Alternative splicing

Localization of the components of the cardiac conduction system (CCS) is essential for many therapeutic procedures in cardiac surgery and interventional cardiology. While histological studies provided fundamental insights into CCS localization, this information is incomplete and difficult to translate to aid in intraprocedural localization. To advance our understanding of CCS localization, we set out to establish a framework for quantifying nodal region morphology. Using this framework, we quantitatively analyzed the sinoatrial node (SAN) and atrioventricular node (AVN) in ovine with postmenstrual age ranging from 4.4 to 58.3 months. In particular, we studied the SAN and AVN in relation to the epicardial and endocardial surfaces, respectively. Using anatomical landmarks, we excised the nodes and adjacent tissues, sectioned those at a thickness of 4 μm at 100 μm intervals, and applied Masson's trichrome stain to the sections. These sections were then imaged, segmented to identify nodal tissue, and analyzed to quantify nodal depth and superficial tissue composition. The minimal SAN depth ranged between 20 and 926 μm. AVN minimal depth ranged between 59 and 1192 μm in the AVN extension region, 49 and 980 μm for the compact node, and 148 and 888 μm for the transition to His Bundle region. Using a logarithmic regression model, we found that minimal depth increased logarithmically with age for the AVN (R2 = 0.818, P = 0.002). Also, the myocardial overlay of the AVN was heterogeneous within different regions and decreased with increasing age. Age associated alterations of SAN minimal depth were insignificant. Our study presents examples of characteristic tissue patterns superficial to the AVN and within the SAN. We suggest that the presented framework provides quantitative information for CCS localization. Our studies indicate that procedural methods and localization approaches in regions near the AVN should account for the age of patients in cardiac surgery and interventional cardiology.

Total knee arthroplasty (TKA) is generally considered a successful surgery, although 5–20% of individuals are unhappy with their outcome following surgery (1, 2), and approximately 5–10% will eventually go on to revision surgery within 10 years (3, 4). There are reports of up to 23% of patients experiencing of anterior knee pain (AKP) postoperatively (5, 6, 7). Loss of motion, decreased function, and pain are three causes of post-TKA AKP that are believed to be attributable to the anatomy and function of the patellofemoral joint (PFJ) (8, 9, 10, 11, 12, 13, 14, 15, 16). The dimensions of the PFJ are affected by the femoral implant position and size, as well as the position and size of the patellar prosthesis after resurfacing is performed (15, 17). PFJ overstuffing occurs as the amount of bone and cartilage resected is less than the thickness of the prosthesis replacing it (11, 12, 18). The anteroposterior PFJ size is altered by a combination of changes in the anterior patellar displacement (APD), anterior-posterior femur diameter (APFD), anterior femoral offset (AFO), and posterior femoral offset (PFO) (11, 12, 17, 18). There have been several biomechanical (19, 20, 21, 22, 23, 24, 25) and clinical papers suggesting the negative effects of PFJ overstuffing (11, 26, 27, 28, 29, 30, 31). In contrast, there have also been a number of biomechanical (20, 32) and clinical studies suggesting no effect on clinical outcomes when the amount of overstuffing is within reason (7, 15, 16, 18, 33, 34, 35). This study aims to systematically review all of the available literature to determine if PFJ overstuffing in TKA affects clinical outcomes.

Background: Multimodal perioperative patient education and expectation-setting can reduce post-operative opioid use while maintaining pain control and satisfaction. As part of a quality-improvement project, we developed a standardized model for perioperative education built upon the American College of Surgeons (ACS) Safe and Effective Pain Control After Surgery (SEPCAS) brochure to improve perioperative education regarding opioid use and pain control. Material and methods: Our study was designed within the Define, Measure, Analyze, Improve, Control (DMAIC) quality-improvement framework. Patients were surveyed about the adequacy of their perioperative education regarding pain control and use of prescription opioid medication. After gathering baseline data, a multimodal educational intervention based on the SEPCAS brochure was implemented. Survey responses were then compared between groups. Results: Twenty-seven subjects were included from the pre-intervention period, and thirty-nine were included from the post-intervention period (n = 66). Those in the post-intervention period were more likely to report receiving the appropriate amount of education regarding recognizing the signs of opioid overdose and how to safely store and dispose of opioid medications. The majority of patients who received the SEPCAS brochure reported that it was useful in their post-operative recovery and that it should be given to every patient undergoing surgery. Conclusions: The ACS SEPCAS brochure is an effective tool for improving patient preparation to safely store and dispose of their opioid medication and recognize the signs of opioid overdose. The brochure was also well received by patients and perceived as an effective educational material.

Reactive oxygen species (ROS) accumulation is a cardinal feature of skeletal muscle atrophy. ROS refers to a collection of radical molecules whose cellular signals are vast, and it is unclear which downstream consequences of ROS are responsible for the loss of muscle mass and strength. Here, we show that lipid hydroperoxides (LOOH) are increased with age and disuse, and the accumulation of LOOH by deletion of glutathione peroxidase 4 (GPx4) is sufficient to augment muscle atrophy. LOOH promoted atrophy in a lysosomal-dependent, proteasomal-independent manner. In young and old mice, genetic and pharmacological neutralization of LOOH or their secondary reactive lipid aldehydes robustly prevented muscle atrophy and weakness, indicating that LOOH-derived carbonyl stress mediates age- and disuse-induced muscle dysfunction. Our findings provide novel insights for the role of LOOH in sarcopenia including a therapeutic implication by pharmacological suppression.

Objectives:. We examined how convenience and financial incentives influence patient willingness to dispose of leftover prescription opioids after surgery. We also identified additional barriers and facilitators to disposal. Background:. In the United States, up to 70% of surgical patients are prescribed opioids and up to 92% will have leftover tablets. Most do not dispose of leftover opioids, increasing the risk for opioid-related harm. Current interventions promoting opioid disposal have shown mixed success. Methods:. We conducted a mixed methods study using a standard gamble survey and semi-structured interviews. Participants estimated willingness to dispose in 16 scenarios with varying convenience (time requirements of

Background: Precursor T-cell acute lymphoblastic leukemia (pre-T-ALL) may cause ocular pathologies such as cotton-wool spots, retinal hemorrhage, and less commonly, retinal detachment or leukemic infiltration of the retina itself. However, these findings are typically accompanied by the pathognomonic hematological signs of acute leukemia. CasePresentation: In this case report and review of the literature, we describe a particularly unusual case of a 25-year-old man who presented to our hospital with bilateral exudative retinal detachments associated with posterior pole thickening without any hematological or neurological findings. The patient, who had a history of previously treated pre-T-ALL in complete remission, was found to have leukemia cell infiltration on retinal biopsy. Conclusion: Our case underscores the fact that the ophthalmologist may be the first provider to detect the relapse of previously treated leukemia, and that ophthalmic evaluation is critical for detecting malignant ocular infiltrates.