Role of the Yersinia pestis phospholipase D (Ymt) in the initial aggregation step of biofilm formation in the flea.

Transmission of Yersinia pestis by fleas depends on the formation of condensed bacterial aggregates embedded within a gel-like matrix that localizes to the proventricular valve in the flea foregut and interferes with normal blood feeding. This is essentially a bacterial biofilm phenomenon, which at its end stage requires the production of a Y. pestis exopolysaccharide that bridges the bacteria together in a cohesive, dense biofilm that completely blocks the proventriculus. However, bacterial aggregates are evident within an hour after a flea ingests Y. pestis, and the bacterial exopolysaccharide is not required for this process. In this study, we characterized the biochemical composition of the initial aggregates and demonstrated that the yersinia murine toxin (Ymt), a Y. pestis phospholipase D, greatly enhances rapid aggregation following infected mouse blood meals. The matrix of the bacterial aggregates is complex, containing large amounts of protein and lipid (particularly cholesterol) derived from the flea's blood meal. A similar incidence of proventricular aggregation occurred after fleas ingested whole blood or serum containing Y. pestis, and intact, viable bacteria were not required. The initial aggregation of Y. pestis in the flea gut is likely due to a spontaneous physical process termed depletion aggregation that occurs commonly in environments with high concentrations of polymers or other macromolecules and particles such as bacteria. The initial aggregation sets up subsequent binding aggregation mediated by the bacterially produced exopolysaccharide and mature biofilm that results in proventricular blockage and efficient flea-borne transmission. IMPORTANCE: Yersinia pestis, the bacterial agent of plague, is maintained in nature in mammal-flea-mammal transmission cycles. After a flea feeds on a mammal with septicemic plague, the bacteria rapidly coalesce in the flea's digestive tract to form dense aggregates enveloped in a viscous matrix that often localizes to the foregut. This represents the initial stage of biofilm development that potentiates transmission of Y. pestis when the flea later bites a new host. The rapid aggregation likely occurs via a depletion-aggregation mechanism, a non-canonical first step of bacterial biofilm development. We found that the biofilm matrix is largely composed of host blood proteins and lipids, particularly cholesterol, and that the enzymatic activity of a Y. pestis phospholipase D (Ymt) enhances the initial aggregation. Y. pestis transmitted by flea bite is likely associated with this host-derived matrix, which may initially shield the bacteria from recognition by the host's intradermal innate immune response.

Plasma SOMAmer proteomics of postoperative delirium.

BACKGROUND: Postoperative delirium is prevalent in older adults and has been shown to increase the risk of long-term cognitive decline. Plasma biomarkers to identify the risk for postoperative delirium and the risk of Alzheimer's disease and related dementias are needed. METHODS: This biomarker discovery case-control study aimed to identify plasma biomarkers associated with postoperative delirium. Patients aged ≥65 years undergoing major elective noncardiac surgery were recruited. The preoperative plasma proteome was interrogated with SOMAmer-based technology targeting 1433 biomarkers. RESULTS: In 40 patients (20 with vs. 20 without postoperative delirium), a preoperative panel of 12 biomarkers discriminated patients with postoperative delirium with an accuracy of 97.5%. The final model of five biomarkers delivered a leave-one-out cross-validation accuracy of 80%. Represented biological pathways included lysosomal and immune response functions. CONCLUSION: In older patients who have undergone major surgery, plasma SOMAmer proteomics may provide a relatively non-invasive benchmark to identify biomarkers associated with postoperative delirium.

Early immune factors associated with the development of post-acute sequelae of SARS-CoV-2 infection in hospitalized and non-hospitalized individuals.

Background: Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to post-acute sequelae of SARS-CoV-2 (PASC) that can persist for weeks to years following initial viral infection. Clinical manifestations of PASC are heterogeneous and often involve multiple organs. While many hypotheses have been made on the mechanisms of PASC and its associated symptoms, the acute biological drivers of PASC are still unknown. Methods: We enrolled 494 patients with COVID-19 at their initial presentation to a hospital or clinic and followed them longitudinally to determine their development of PASC. From 341 patients, we conducted multi-omic profiling on peripheral blood samples collected shortly after study enrollment to investigate early immune signatures associated with the development of PASC. Results: During the first week of COVID-19, we observed a large number of differences in the immune profile of individuals who were hospitalized for COVID-19 compared to those individuals with COVID-19 who were not hospitalized. Differences between individuals who did or did not later develop PASC were, in comparison, more limited, but included significant differences in autoantibodies and in epigenetic and transcriptional signatures in double-negative 1 B cells, in particular. Conclusions: We found that early immune indicators of incident PASC were nuanced, with significant molecular signals manifesting predominantly in double-negative B cells, compared with the robust differences associated with hospitalization during acute COVID-19. The emerging acute differences in B cell phenotypes, especially in double-negative 1 B cells, in PASC patients highlight a potentially important role of these cells in the development of PASC.

Skin muscle is the initial site of viral replication for arboviral bunyavirus infection.

The first step in disease pathogenesis for arboviruses is the establishment of infection following vector transmission. For La Crosse virus (LACV), the leading cause of pediatric arboviral encephalitis in North America, and other orthobunyaviruses, the initial course of infection in the skin is not well understood. Using an intradermal (ID) model of LACV infection in mice, we find that the virus infects and replicates nearly exclusively within skin-associated muscle cells of the panniculus carnosus (PC) and not in epidermal or dermal cells like most other arbovirus families. LACV is widely myotropic, infecting distal muscle cells of the peritoneum and heart, with limited infection of draining lymph nodes. Surprisingly, muscle cells are resistant to virus-induced cell death, with long term low levels of virus release progressing through the Golgi apparatus. Thus, skin muscle may be a key cell type for the initial infection and spread of arboviral orthobunyaviruses.

Assessing patterns of delirium symptoms reveals a novel subtype among elective surgical patients with postoperative delirium.

OBJECTIVES: Prior studies reported incidence of hypoactive and hyperactive subtypes of postoperative delirium, but did not consider cognitive symptoms of delirium which are highlighted in the DSM-5 criteria for delirium. This study aims to address this gap in the literature by classifying cases of delirium according to their constellation of cognitive and motoric symptoms of delirium using a statistical technique called Latent Class Analysis (LCA). METHODS: Data were from five independent study cohorts (N = 1968) of patients who underwent elective spine, knee/hip, or elective gastrointestinal and thoracic procedures, between 2001 and 2017. Assessments of delirium symptoms were conducted using the long form of the Confusion Assessment Method (CAM) pre- and post-surgery. Latent class analyses of CAM data from the first 2 days after surgery were conducted to determine subtypes of delirium based on patterns of cognitive and motoric symptoms of delirium. We also determined perioperative patient characteristics associated with each latent class of delirium and assessed whether the length of delirium for each of the patterns of delirium symptoms identified by the latent class analysis. RESULTS: The latent class model from postoperative day 1 revealed three distinct patterns of delirium symptoms. One pattern of symptoms, denoted as the Hyperalert class, included patients whose predominant symptoms were being hyperalert or overly sensitive to environmental stimuli and having a low level of motor activity. Another pattern of symptoms, denoted as the Hypoalert class, included patients whose predominant symptom was being hypoalert (lethargic or drowsy). A third pattern of symptoms, denoted as the Cognitive Changes class, included patients who experienced new onset of disorganized thinking, memory impairment, and disorientation. Among 352 patients who met CAM criteria for delirium on postoperative day 1, 34% had symptoms that fit within the Hyperalert latent class, 39% had symptoms that fit within the Hypoalert latent class, and 27% had symptoms that fit within the Cognitive Changes latent class. Similar findings were found when latent class analysis was applied to those who met CAM criteria for delirium on postoperative day 2. Multinomial regression analyses revealed that ASA class, surgery type, and preoperative cognitive status as measured by the Telephone Interview for Cognitive Status (TICS) scores were associated with class membership. Length of delirium differed between the latent classes with the Cognitive Changes latent class having a longer duration compared to the other two classes. CONCLUSIONS: Older elective surgery patients who did not have acute events or illnesses or a diagnosis of dementia prior to surgery displayed varying symptoms of delirium after surgery. Compared to prior studies that described hypoactive and hyperactive subtypes of delirium, we identified a novel subtype of delirium that reflects cognitive symptoms of delirium. The three subtypes of delirium reveal distinct patterns of delirium symptoms which provide insight into varying risks and care needs of patients with delirium, indicating the necessity of future research on reducing risk for cognitive symptoms of delirium.

Cytoarchitecture of ex vivo midgut cultures of unfed Ixodes scapularis infected with a tick-borne flavivirus.

A bite from an infected tick is the primary means of transmission for tick-borne flaviviruses (TBFV). Ticks ingest the virus while feeding on infected blood. The traditional view is that the virus first replicates in and transits the tick midgut prior to dissemination to other organs, including salivary glands. Thus, understanding TBFV infection in the tick midgut is a key first step in identifying potential countermeasures against infection. Ex vivo midgut cultures prepared from unfed adult female Ixodes scapularis ticks were viable and remained morphologically intact for more than 8 days. The midgut consisted of two clearly defined cell layers separated by a basement membrane: an exterior network of smooth muscle cells and an internal epithelium composed of digestive generative cells. The smooth muscle cells were arranged in a stellate circumferential pattern spaced at regular intervals along the long axis of midgut diverticula. When the cultures were infected with the TBFV Langat virus (LGTV), virus production increased by two logs with a peak at 96 hours post-infection. Infected cells were readily identified by immunofluorescence staining for the viral envelope protein, nonstructural protein 3 (NS3) and dsRNA. Microscopy of the stained cultures suggested that generative cells were the primary target for virus infection in the midgut. Infected cells exhibited an expansion of membranes derived from the endoplasmic reticulum; a finding consistent with TBFV infected cell cultures. Electron microscopy of infected cultures revealed virus particles in the basolateral region between epithelial cells. These results demonstrated LGTV replication in midgut generative cells of artificially infected, ex vivo cultures of unfed adult female I. scapularis ticks.

Distinct temporal trajectories and risk factors for Post-acute sequelae of SARS-CoV-2 infection.

Background: The understanding of Post-acute sequelae of SARS-CoV-2 infection (PASC) can be improved by longitudinal assessment of symptoms encompassing the acute illness period. To gain insight into the various disease trajectories of PASC, we assessed symptom evolution and clinical factors associated with the development of PASC over 3 months, starting with the acute illness period. Methods: We conducted a prospective cohort study to identify parameters associated with PASC. We performed cluster and case control analyses of clinical data, including symptomatology collected over 3 months following infection. Results: We identified three phenotypic clusters associated with PASC that could be characterized as remittent, persistent, or incident based on the 3-month change in symptom number compared to study entry: remittent (median; min, max: -4; -17, 3), persistent (-2; -14, 7), or incident (4.5; -5, 17) (p = 0.041 remittent vs. persistent, p < 0.001 remittent vs. incident, p < 0.001 persistent vs. incident). Despite younger age and lower hospitalization rates, the incident phenotype had a greater number of symptoms (15; 8, 24) and a higher proportion of participants with PASC (63.2%) than the persistent (6; 2, 9 and 52.2%) or remittent clusters (1; 0, 6 and 18.7%). Systemic corticosteroid administration during acute infection was also associated with PASC at 3 months [OR (95% CI): 2.23 (1.14, 4.36)]. Conclusion: An incident disease phenotype characterized by symptoms that were absent during acute illness and the observed association with high dose steroids during acute illness have potential critical implications for preventing PASC.

Multi-omic profiling reveals early immunological indicators for identifying COVID-19 Progressors.

We sought to better understand the immune response during the immediate post-diagnosis phase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by identifying molecular associations with longitudinal disease outcomes. Multi-omic analyses identified differences in immune cell composition, cytokine levels, and cell subset-specific transcriptomic and epigenomic signatures between individuals on a more serious disease trajectory (Progressors) as compared to those on a milder course (Non-progressors). Higher levels of multiple cytokines were observed in Progressors, with IL-6 showing the largest difference. Blood monocyte cell subsets were also skewed, showing a comparative decrease in non-classical CD14-CD16+ and intermediate CD14+CD16+ monocytes. In lymphocytes, the CD8+ T effector memory cells displayed a gene expression signature consistent with stronger T cell activation in Progressors. These early stage observations could serve as the basis for the development of prognostic biomarkers of disease risk and interventional strategies to improve the management of severe COVID-19. BACKGROUND: Much of the literature on immune response post-SARS-CoV-2 infection has been in the acute and post-acute phases of infection. TRANSLATIONAL SIGNIFICANCE: We found differences at early time points of infection in approximately 160 participants. We compared multi-omic signatures in immune cells between individuals progressing to needing more significant medical intervention and non-progressors. We observed widespread evidence of a state of increased inflammation associated with progression, supported by a range of epigenomic, transcriptomic, and proteomic signatures. The signatures we identified support other findings at later time points and serve as the basis for prognostic biomarker development or to inform interventional strategies.

N4 -Hydroxycytidine/Molnupiravir Inhibits RNA-Virus Induced Encephalitis by Producing Mutated Viruses with Reduced Fitness.

A diverse group of RNA viruses including Rabies, Polio, La Crosse, West Nile, Zika, Nipah, Eastern and Western equine encephalitis, Venezuelan equine encephalitis, Japanese encephalitis, and tick-borne encephalitis viruses have the ability to gain access to and replicate in the central nervous system (CNS), causing severe neurological disease. Current treatment for these patients is generally limited to supportive care. To address the need for a generalizable antiviral, we utilized a strategy of mutagenesis to limit virus replication. We evaluated ribavirin (RBV), favipiravir (FAV) and N 4 -hydroxycytidine (NHC) against La Crosse virus (LACV) which is the primary cause of pediatric arboviral encephalitis cases in North America. NHC was more potent than RBV or FAV in neuronal cells. Oral administration of molnupiravir (MOV), the 5'-isobutyryl prodrug of NHC, decreased neurological disease development by 32% following intraperitoneal (IP) infection of LACV. MOV also reduced disease by 23% when virus was administered intranasally (IN). NHC and MOV produced less fit viruses by incorporating predominantly G-to-A or C-to-U mutations. Furthermore, NHC also inhibited two other orthobunyaviruses, Jamestown Canyon virus and Cache Valley virus. Collectively, these studies indicate that NHC/MOV has therapeutic potential to inhibit virus replication and subsequent neurological disease caused by this neurotropic RNA virus.

Genetically diverse mouse models of SARS-CoV-2 infection reproduce clinical variation in type I interferon and cytokine responses in COVID-19.

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18-hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.

Multi-omic Profiling Reveals Early Immunological Indicators for Identifying COVID-19 Progressors.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a rapid response by the scientific community to further understand and combat its associated pathologic etiology. A focal point has been on the immune responses mounted during the acute and post-acute phases of infection, but the immediate post-diagnosis phase remains relatively understudied. We sought to better understand the immediate post-diagnosis phase by collecting blood from study participants soon after a positive test and identifying molecular associations with longitudinal disease outcomes. Multi-omic analyses identified differences in immune cell composition, cytokine levels, and cell subset-specific transcriptomic and epigenomic signatures between individuals on a more serious disease trajectory (Progressors) as compared to those on a milder course (Non-progressors). Higher levels of multiple cytokines were observed in Progressors, with IL-6 showing the largest difference. Blood monocyte cell subsets were also skewed, showing a comparative decrease in non-classical CD14-CD16+ and intermediate CD14+CD16+ monocytes. Additionally, in the lymphocyte compartment, CD8+ T effector memory cells displayed a gene expression signature consistent with stronger T cell activation in Progressors. Importantly, the identification of these cellular and molecular immune changes occurred at the early stages of COVID-19 disease. These observations could serve as the basis for the development of prognostic biomarkers of disease risk and interventional strategies to improve the management of severe COVID-19.

Fine structure and molecular characterization of two new parabasalid species that naturally colonize laboratory mice, Tritrichomonas musculus and Tritrichomonas casperi.

Tritrichomonas muris is a common flagellated protist isolated from the cecum of wild rodents. This commensal protist has been shown previously to alter immune phenotypes in laboratory mice. Other trichomonads, referred to as Tritrichomonas musculis and Tritrichomonas rainier, also naturally colonize laboratory mice and cause immune alterations. This report formally describes two new trichomonads, Tritrichomonas musculus n. sp., and Tritrichomonas casperi n. sp., at the ultrastructural and molecular level. These two protists were isolated from laboratory mice and were differentiated by their size and the structure of their undulating membrane and posterior flagellum. Analysis at the 18S rRNA and trans-ITS genetic loci supported their designation as distinct species, related to T. muris. To assess the true extent of parabasalid diversity infecting laboratory mice, 135 mice bred at the National Institutes of Health (NIH) were screened using pan-parabasalid primers that amplify the trans-ITS region. Forty-four percent of mice were positive for parabasalids, encompassing a total of eight distinct sequence types. Tritrichomonas casperi and Trichomitus-like protists were dominant. T. musculus and T. rainier were also detected, but T. muris was not. Our work establishes a previously underappreciated diversity of commensal trichomonad flagellates that naturally colonize the enteric cavity of laboratory mice.

Presence of Preoperative Neurodegeneration Biofluid Markers in Patients with Postoperative Delirium.

BACKGROUND: The pathophysiology of delirium is incompletely understood, including what molecular pathways are involved in brain vulnerability to delirium. This study examined whether preoperative plasma neurodegeneration markers were elevated in patients who subsequently developed postoperative delirium through a retrospective case-control study. METHODS: Inclusion criteria were patients of 65 yr of age or older, undergoing elective noncardiac surgery with a hospital stay of 2 days or more. Concentrations of preoperative plasma P-Tau181, neurofilament light chain, amyloid ß1-42 (Aß42), and glial fibrillary acidic protein were measured with a digital immunoassay platform. The primary outcome was postoperative delirium measured by the Confusion Assessment Method. The study included propensity score matching by age and sex with nearest neighbor, such that each patient in the delirium group was matched by age and sex with a patient in the no-delirium group. RESULTS: The initial cohort consists of 189 patients with no delirium and 102 patients who developed postoperative delirium. Of 291 patients aged 72.5 ± 5.8 yr, 50.5% were women, and 102 (35%) developed postoperative delirium. The final cohort in the analysis consisted of a no-delirium group (n = 102) and a delirium group (n = 102) matched by age and sex using the propensity score method. Of the four biomarkers assayed, the median value for neurofilament light chain was 32.05 pg/ml for the delirium group versus 23.7 pg/ml in the no-delirium group. The distribution of biomarker values significantly differed between the delirium and no-delirium groups (P = 0.02 by the Kolmogorov-Smirnov test) with the largest cumulative probability difference appearing at the biomarker value of 32.05 pg/ml. CONCLUSIONS: These results suggest that patients who subsequently developed delirium are more likely to be experiencing clinically silent neurodegenerative changes before surgery, reflected by changes in plasma neurofilament light chain biomarker concentrations, which may identify individuals with a preoperative vulnerability to subsequent cognitive decline.

Sleep Loss the night before surgery and incidence of postoperative delirium in adults 65-95 years of age.

STUDY OBJECTIVES: To describe the association between preoperative sleep disruption and postoperative delirium. METHODS: Prospective cohort study with six time points (3 nights pre-hospitalization and 3 nights post-surgery). The sample included 180 English-speaking patients ≥65 years old scheduled for major non-cardiac surgery and anticipated minimum hospital stay of 3 days. Six days of wrist actigraphy recorded continuous movement to estimate wake and sleep minutes during the night from 22:00 to 05:59. Postoperative delirium was measured by a structured interview using the Confusion Assessment Method. Sleep variables for patients with (n = 32) and without (n = 148) postoperative delirium were compared using multivariate logistic regression. RESULTS: Participants had a mean age of 72 ± 5 years (range 65-95 years). The incidence of postoperative delirium during any of the three postoperative days was 17.8%. Postoperative delirium was significantly associated with surgery duration (OR = 1.49, 95% CI 1.24-1.83) and sleep loss >15% on the night before surgery (OR = 2.64, 95% CI 1.10-6.62). Preoperative symptoms of pain, anxiety and depression were unrelated to preoperative sleep loss. CONCLUSIONS: In this study of adults ≥65 years of age, short sleep duration was more severe preoperatively in the patients who experienced postoperative delirium as evidenced by sleep loss >15% of their normal night's sleep. However, we were unable to identify potential reasons for this sleep loss. Further investigation should include additional factors that may be associated with preoperative sleep loss to inform potential intervention strategies to mitigate preoperative sleep loss and reduce risk of postoperative delirium.

Genetically diverse mouse models of SARS-CoV-2 infection reproduce clinical variation in type I interferon and cytokine responses in COVID-19.

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18- hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.

Lidocaine Infusion for the Management of Postoperative Pain and Delirium (LIMPP): protocol for a randomised control trial.

INTRODUCTION: Postoperative delirium is a frequent adverse event following elective non-cardiac surgery. The occurrence of delirium increases the risk of functional impairment, placement to facilities other than home after discharge, cognitive impairment at discharge, as well as in-hospital and possibly long-term mortality. Unfortunately, there is a dearth of effective strategies to minimise the risk from modifiable risk factors, including postoperative pain control and the analgesic regimen. Use of potent opioids, currently the backbone of postoperative pain control, alters cognition and has been associated with an increased risk of postoperative delirium. Literature supports the intraoperative use of lidocaine infusions to decrease postoperative opioid requirements, however, whether the use of postoperative lidocaine infusions is associated with lower opioid requirements and subsequently a reduction in postoperative delirium has not been investigated. METHODS AND ANALYSIS: The Lidocaine Infusion for the Management of Postoperative Pain and Delirium trial is a randomised, double-blinded study of a postoperative 48-hour infusion of lidocaine at 1.33 mg/kg/hour versus placebo in older patients undergoing major reconstructive spinal surgery at the University of California, San Francisco. Our primary outcome is incident delirium measured daily by the Confusion Assessment Method in the first three postoperative days. Secondary outcomes include delirium severity, changes in cognition, pain scores, opioid use, incidence of opioid related side effects and functional benefits including time to discharge and improved recovery from surgery. Lidocaine safety will be assessed with daily screening questionnaires and lidocaine plasma levels. ETHICS AND DISSEMINATION: This study protocol has been approved by the ethics board at the University of California, San Francisco. The results of this study will be published in a peer-review journal and presented at national conferences as poster or oral presentations. Participants wishing to know the results of this study will be contacted directly on data publication. TRIAL REGISTRATION NUMBER: NCT05010148.

Postoperative delirium prediction using machine learning models and preoperative electronic health record data.

BACKGROUND: Accurate, pragmatic risk stratification for postoperative delirium (POD) is necessary to target preventative resources toward high-risk patients. Machine learning (ML) offers a novel approach to leveraging electronic health record (EHR) data for POD prediction. We sought to develop and internally validate a ML-derived POD risk prediction model using preoperative risk features, and to compare its performance to models developed with traditional logistic regression. METHODS: This was a retrospective analysis of preoperative EHR data from 24,885 adults undergoing a procedure requiring anesthesia care, recovering in the main post-anesthesia care unit, and staying in the hospital at least overnight between December 2016 and December 2019 at either of two hospitals in a tertiary care health system. One hundred fifteen preoperative risk features including demographics, comorbidities, nursing assessments, surgery type, and other preoperative EHR data were used to predict postoperative delirium (POD), defined as any instance of Nursing Delirium Screening Scale ≥2 or positive Confusion Assessment Method for the Intensive Care Unit within the first 7 postoperative days. Two ML models (Neural Network and XGBoost), two traditional logistic regression models ("clinician-guided" and "ML hybrid"), and a previously described delirium risk stratification tool (AWOL-S) were evaluated using the area under the receiver operating characteristic curve (AUC-ROC), sensitivity, specificity, positive likelihood ratio, and positive predictive value. Model calibration was assessed with a calibration curve. Patients with no POD assessments charted or at least 20% of input variables missing were excluded. RESULTS: POD incidence was 5.3%. The AUC-ROC for Neural Net was 0.841 [95% CI 0. 816-0.863] and for XGBoost was 0.851 [95% CI 0.827-0.874], which was significantly better than the clinician-guided (AUC-ROC 0.763 [0.734-0.793], p < 0.001) and ML hybrid (AUC-ROC 0.824 [0.800-0.849], p < 0.001) regression models and AWOL-S (AUC-ROC 0.762 [95% CI 0.713-0.812], p < 0.001). Neural Net, XGBoost, and ML hybrid models demonstrated excellent calibration, while calibration of the clinician-guided and AWOL-S models was moderate; they tended to overestimate delirium risk in those already at highest risk. CONCLUSION: Using pragmatically collected EHR data, two ML models predicted POD in a broad perioperative population with high discrimination. Optimal application of the models would provide automated, real-time delirium risk stratification to improve perioperative management of surgical patients at risk for POD.

A pigtailed macaque model of Kyasanur Forest disease virus and Alkhurma hemorrhagic disease virus pathogenesis.

Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.

The Impact of Surgery duration and Surgery End Time on Postoperative Sleep in Older Adults.

OBJECTIVES/BACKGROUND: Sleep disruption is prevalent in older patients. No previous studies have considered the impact of surgery duration or surgery end time of day on postoperative sleep disruption. Accordingly, we examined the duration of surgery and surgery end times for associations with postoperative sleep disruption. METHODS: Inclusion criteria were patients ≥ 65 years of age undergoing major, non-cardiac surgery. Sleep disruption was measured by wrist actigraphy and defined as wake after sleep onset (WASO) during the night, or inactivity/sleep time during the day. The sleep opportunity window was set from 22:00 to 06:00 which coincided with "lights off and on" in the hospital. WASO during this 8-hour period on the first postoperative day was categorized into one of three groups: ≤ 15%, 15-25%, and > 25%. Daytime sleep (inactivity) during the first postoperative day was categorized as ≤ 20%, 20-40%, and > 40%. Statistical analyses were conducted to test for associations between surgery duration, surgery end time and sleep disruption on the first postoperative day and following night. RESULTS: For this sample of 156 patients, surgery duration ≥ 6 hours and surgery end time after 19:00 were not associated with WASO groups (p = 0.17, p = 0.94, respectively). Furthermore, daytime sleep was also not affected by surgery duration or surgery end time (p = 0.07, p = 0.06 respectively). CONCLUSION: Our hypothesis that patients with longer duration or later-ending operations have increased postoperative sleep disruption was not supported. Our results suggest the pathophysiology of postoperative sleep disruption needs further investigation.

Rottlerin inhibits La Crosse virus-induced encephalitis in mice and blocks release of replicating virus from the Golgi body in neurons.

La Crosse virus (LACV) is a mosquito-borne orthobunyavirus that causes approximately 60 to 80 hospitalized pediatric encephalitis cases in the United States yearly. The primary treatment for most viral encephalitis, including LACV, is palliative care, and specific antiviral therapeutics are needed. We screened the National Center for Advancing Translational Sciences library of 3,833 FDA-approved and bioactive small molecules for the ability to inhibit LACV-induced death in SH-SY5Y neuronal cells. The top three hits from the initial screen were validated by examining their ability to inhibit virus-induced cell death in multiple neuronal cell lines. Rottlerin consistently reduced LACV-induced death by 50% in multiple human and mouse neuronal cell lines with an effective concentration of 0.16-0.69 µg ml-1 depending on cell line. Rottlerin was effective up to 12 hours post-infection in vitro and inhibited virus particle trafficking from the Golgi apparatus to trans-Golgi vesicles. In human inducible pluripotent stem cell-derived cerebral organoids, rottlerin reduced virus production by one log and cell death by 35% compared with dimethyl sulfoxide-treated controls. Administration of rottlerin in mice by intraperitoneal or intracranial routes starting at 3 days post-infection decreased disease development by 30-50%. Furthermore, rottlerin also inhibited virus replication of other pathogenic California serogroup orthobunyaviruses (Jamestown Canyon and Tahyna virus) in neuronal cell lines.