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Understanding the incidence of disease is often crucial for public policy decision-making, as observed during the COVID-19 pandemic. Estimating incidence is challenging, however, when the definition of incidence relies on tests that imperfectly measure disease, as in the case when assays with variable performance are used to detect the SARS-CoV-2 virus. To our knowledge, there are no pragmatic methods to address the bias introduced by the performance of labs in testing for the virus. In the setting of a longitudinal study, we developed a maximum likelihood estimation-based approach to estimate laboratory performance-adjusted incidence using the expectation-maximization algorithm. We constructed confidence intervals (CIs) using both bootstrapped-based and large-sample interval estimator approaches. We evaluated our methods through extensive simulation and applied them to a real-world study (TrackCOVID), where the primary goal was to determine the incidence of and risk factors for SARS-CoV-2 infection in the San Francisco Bay Area from July 2020 to March 2021. Our simulations demonstrated that our method converged rapidly with accurate estimates under a variety of scenarios. Bootstrapped-based CIs were comparable to the large-sample estimator CIs with a reasonable number of incident cases, shown via a simulation scenario based on the real TrackCOVID study. In more extreme simulated scenarios, the coverage of large-sample interval estimation outperformed the bootstrapped-based approach. Results from the application to the TrackCOVID study suggested that assuming perfect laboratory test performance can lead to an inaccurate inference of the incidence. Our flexible, pragmatic method can be extended to a variety of disease and study settings.

Metalloproteins with dinuclear cores are known to bind and activate dioxygen, with a subclass of these proteins having active sites containing FeMn cofactors and activities ranging from long-range proton-coupled electron transfer (PCET) to post-translational peptide modification. While mechanistic studies propose that these metallocofactors access FeIIIMnIV intermediates, there is a dearth of related synthetic analogs. Herein, the first well-characterized synthetic FeIII-(μ-O)-MnIV complex is reported; this complex shows similar spectroscopic features as the catalytically competent FeIIIMnIV intermediate X found in Class Ic ribonucleotide reductase and demonstrates PCET function towards phenolic substrates. This complex is prepared from the oxidation of the isolable FeIII-(μ-O)-MnIII species, whose stepwise assembly is facilitated by a tripodal ligand containing phosphinic amido groups. Structural and spectroscopic studies found proton movement involving the FeIIIMnIII core, whereby the initial bridging hydroxido ligand is converted to an oxido ligand with concomitant protonation of one phosphinic amido group. This series of FeMn complexes allowed us to address factors that may dictate the preference of an active site for a heterobimetallic cofactor over one that is homobimetallic: comparisons of the redox properties of our FeMn complexes with those of the di-Fe analogs suggested that the relative thermodynamic ease of accessing an FeIIIMnIV core can play an important role in determining the metal ion composition when the key catalytic steps do not require an overly potent oxidant. Moreover, these complexes allowed us to demonstrate the effect of the hyperfine interaction from non-Fe nuclei on 57Fe Mössbauer spectra which is relevant to MnFe intermediates in proteins.

PURPOSE: Contact lens wear can induce corneal parainflammation involving CD11c+ cell responses (24 hours), γδ T cell responses (24 hours and 6 days), and IL-17-dependent Ly6G+ cell responses (6 days). Topical antibiotics blocked these CD11c+ responses. Because corneal CD11c+ responses to bacteria require transient receptor potential (TRP) ion-channels (TRPA1/TRPV1), we determined if these channels mediate lens-induced corneal parainflammation. METHODS: Wild-type mice were fitted with contact lenses for 24 hours or 6 days and compared to lens wearing TRPA1 (-/-) or TRPV1 (-/-) mice or resiniferatoxin (RTX)-treated mice. Contralateral eyes were not fitted with lenses. Corneas were examined for major histocompatibility complex (MHC) class II+, CD45+, γδ T, or TNF-α+ cell responses (24 hours) or Ly6G+ responses (6 days) by quantitative imaging. The quantitative PCR (qPCR) determined cytokine gene expression. RESULTS: Lens-induced increases in MHC class II+ cells after 24 hours were abrogated in TRPV1 (-/-) but not TRPA1 (-/-) mice. Increases in CD45+ cells were unaffected. Increases in γδ T cells after 24 hours of wear were abrogated in TRPA1 (-/-) and TRPV1 (-/-) mice, as were 6 day Ly6G+ cell responses. Contralateral corneas of TRPA1 (-/-) and TRPV1 (-/-) mice showed reduced MHC class II+ and γδ T cells at 24 hours. RTX inhibited lens-induced parainflammatory phenotypes (24 hours and 6 days), blocked lens-induced TNF-α and IL-18 gene expression, TNF-α+ cell infiltration (24 hours), and reduced baseline MHC class II+ cells. CONCLUSIONS: TRPA1 and TRPV1 mediate contact lens-induced corneal parainflammation after 24 hours and 6 days of wear and can modulate baseline levels of resident corneal immune cells.

BackgroundMusculoskeletal (MSK) disorders are one of the main causes of disability among adults globally. The burden of MSK disorders varies greatly between different regions and is the highest in low- and middle income- countries. This study sought to investigate trends in the burden of MSK disorders across the MENA region, utilizing the GBD 2019 dataset.MethodsThis ecological study utilized data from the Global Burden of Disease (GBD) to report on the burden of musculoskeletal (MSK) disorders in The Middle East and North Africa (MENA) region between 1990 and 2019. Our analysis involved descriptive statistics and sociodemographic trends and did not employ any specific statistical analyses. Using age-standardized rates of prevalence and disability-adjusted life-years (DALYs), we reported trends in the burden of MSK disorders, as well as national variation between different countries. Furthermore, we analyzed trends in risk factors contributing to MSK disorders by age and gender.ResultsThe longitudinal analysis from 1990 to 2019 showed an increase in the age-standardized rate for prevalence and DALYs of MSK disorders by 5% and 4.80%, respectively. Low back pain continued to be the most prevalent MSK condition, while RA and other MSK disorders had the largest percentage increase for DALYs between 1990 and 2019. The study found that Afghanistan had the lowest age standardized DALYs rate attributed to MSK disorders, while Iran, Turkey, and Jordan had the highest. Further, Syria showed the most dramatic decrease while Saudi Arabia had the most notable increase in age standardized DALY rates from 1990 to 2019. In 2019, occupational risks, high body mass index, and tobacco smoking were the main risk factors for MSK disorders, with occupational risks being the largest contributor, and between 1990 and 2019, there was a decrease in the contribution of occupational risks but an increase in the contribution of high body mass index as a risk factor.ConclusionThis study highlights the significant burden of MSK disorders in the MENA region, with various risk factors contributing to its increasing prevalence in recent decades. Further research is needed to better understand the underlying factors and potential interventions that could improve health outcomes. Addressing MSK disorders should be a public health priority in the region, and efforts should be made to develop effective strategies to prevent and manage this debilitating condition.

PURPOSE: Previously, using a murine model, we reported that contact lens (CL) wear induced corneal parainflammation involving CD11c+ cells after 24 h and Ly6G+ cells (neutrophils) after 5-6 days. Here, we investigated the role of IL-17 and γδ T cells in the CL-induced neutrophil response. METHODS: CL-wearing C57BL/6 wild-type (WT) mice were compared to lens-wearing IL-17A/F single or double gene knock-out mice, or mice treated with UC7-13D5 monoclonal antibody to functionally deplete γδ T cells. Contralateral eyes served as no lens wear controls. Corneal Ly6G+ and γδ T cell responses were quantified as was expression of genes encoding pro-inflammatory cytokines IL-17A/F, IL-β, IL-18 and expression of IL-17A/F protein. RESULTS: After 6 days lens wear, WT corneas showed Ly6G+ cell infiltration while remaining free of visible pathology. In contrast, lens-wearing corneas of IL-17AF (-/-), IL-17A (-/-) mice and γδ T cell-depleted mice showed little or no Ly6G+ cell infiltration. No Ly6G+ cell infiltration was detected in contralateral eye controls. Lens-wearing WT corneas also showed a significant increase in γδ T cells after 24 h that was maintained after 6 days of wear, and significantly increased cytokine gene expression after 6 days versus contralateral controls: IL-18 & IL-17A (∼3.9 fold) and IL-23 (∼6.5-fold). Increased IL-17A protein (∼4-fold) was detected after 6 days lens wear. γδ T cell-depletion abrogated these lens-induced changes in cytokine gene and protein expression. CONCLUSION: Together, these data show that IL-17A and γδ T cells are required for Ly6G+ cell (neutrophil) infiltration of the cornea during contact lens-induced parainflammation.

Summary: The maturation of genomic surveillance in the past decade has enabled tracking of the emergence and spread of epidemics at an unprecedented level. During the COVID-19 pandemic, for example, genomic data revealed that local epidemics varied considerably in the frequency of SARS-CoV-2 lineage importation and persistence, likely due to a combination of COVID-19 restrictions and changing connectivity. Here, we show that local COVID-19 epidemics are driven by regional transmission, including across international boundaries, but can become increasingly connected to distant locations following the relaxation of public health interventions. By integrating genomic, mobility, and epidemiological data, we find abundant transmission occurring between both adjacent and distant locations, supported by dynamic mobility patterns. We find that changing connectivity significantly influences local COVID-19 incidence. Our findings demonstrate a complex meaning of ‘local’ when investigating connected epidemics and emphasize the importance of collaborative interventions for pandemic prevention and mitigation.

GoalThis study explored the use of a Lean daily management system (DMS) for COVID-19 response and recovery in U.S. hospitals and health systems. Originally developed in manufacturing, Lean is an evidence-based approach to quality and process improvement in healthcare. Although Lean has been studied in individual hospital units and outpatient practices, it has not been examined as a whole system response to crisis events.MethodsWe conducted qualitative interviews with 46 executive leaders, clinical leaders, and frontline staff in four hospitals and health systems across the United States. We developed a semistructured interview guide to understand DMS implementation in these care delivery organizations. As interviews took place 6-8 months following the onset of the pandemic, a subset of our interview questions centered on DMS use to meet the demands of COVID-19. Based on a deductive approach to qualitative analysis, we identified clusters of themes that described how DMS facilitated rapid system response to the public health emergency.Principal findingsThere were many important ways in which U.S. hospitals and health systems leveraged their DMS to address COVID-19 challenges. These included the use of tiered huddles to facilitate rapid communication, the creation of standard work for redeployed staff, and structured problem-solving to prioritize new areas for improvement. We also discovered ways that the pandemic itself affected DMS implementation in all organizations. COVID-19 universally created greater DMS visibility by opening lines of communication among leadership, strengthening measurement and accountability, and empowering staff to develop solutions at the front lines. Many lessons learned using DMS for crisis management will carry forward into COVID-19 recovery efforts. Lessons include expanding telehealth, reactivating incident command systems as needed, and efficiently coordinating resources amid potential future shortages.Practical applicationsOverall, the Lean DMS functioned as a robust property that enabled quick organizational response to unpredictable events. Our findings on the use of DMS are consistent with organizational resilience that emphasizes collective sense-making and awareness of incident status, team decision-making, and frequent interaction and coordination. These features of resilience are supported by DMS practices such as tiered huddles for rapid information dissemination and alignment across organizational hierarchies. When used in conjunction with plan-do-study-act methodology, huddles provide teams with enhanced feedback that strengthens their ability to make changes as needed. Moreover, gaps between work-as-imagined (how work should be done) and work-as-done (how work is actually done) may be exacerbated in the initial chaos of emergency events but can be minimized through the development of standard work protocols. As a facilitator of resilience, the Lean DMS may be used in a variety of challenging situations to ensure high standards of care.

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases1-3. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing4,5. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Metalloproteins with active sites containing di-Fe cores exhibit diverse chemical reactivity that is linked to the precise transfer of protons and electrons which directly involve the di-Fe units. The redox conversions are commonly corroborated by spectroscopic methods, but the associated structural changes are often difficult to assess, particularly those related to proton movements. This report describes the development of di-Fe complexes in which the movements of protons and electrons are pinpointed during the stepwise oxidation of a di-FeII species to one with an FeIIIFeIV core. Complex formation was promoted using the phosphinic amido tripodal ligand [poat]3- (N,N',N″-[nitrilotris(ethane-2,1-diyl)]tris(P,P-diphenylphosphinic amido)) that provided dynamic coordination spheres that assisted in regulating both electron and proton transfer processes. Oxidation of an [FeII-(μ-OH)-FeIII] complex led to the corresponding di-FeIII species containing a hydroxido bridge that was not stable at room temperature and converted to a species containing an oxido bridging ligand and protonation of one phosphinic amido group to form [Hpoat]2-. Deprotonation led to a new species with an [FeIII-(μ-O)-FeIII] core that could be further oxidized to its FeIIIFeIV analogue. Reactions with phenols suggest homolytic cleavage of the O-H bond to give products that are consistent with the initial formation of a phenoxyl radical─spectroscopic studies indicated that the electron is transferred to the FeIV center, and the proton is initially transferred to the more sterically hindered oxido ligand but then relocates to [poat]3-. These findings offer new mechanistic insights related to the stability of and the reactions performed by di-Fe enzymes.

PurposeThe purpose of this study was to investigate factors impacting transplant-free survival among infants with biliary atresia.MethodsA multi-institutional, retrospective cohort study was performed at nine tertiary-level children's hospitals in the United States. Infants who underwent Kasai portoenterostomy (KP) from January 2009 to May 2017 were identified. Clinical characteristics included age at time of KP, steroid use, surgical approach, liver pathology, and surgeon experience. Likelihood of transplant-free survival (TFS) was evaluated using logistic regression, adjusting for patient and surgeon-level factors. Secondary outcomes at 1 year included readmission, cholangitis, reoperation, mortality, and biliary clearance.ResultsOverall, 223 infants underwent KP, and 91 (40.8%) survived with their native liver. Mean age at surgery was 63.9 days (± 24.7 days). At 1 year, 78.5% experienced readmission, 56.9% developed cholangitis, 3.8% had a surgical revision, and 5 died. Biliary clearance at 3 months was achieved in 76.6%. Controlling for patient and surgeon-level factors, each additional day of age toward operation was associated with a 2% decrease in likelihood of TFS (OR 0.98, 95% CI 0.97-0.99).ConclusionEarlier surgical intervention by Kasai portoenterostomy at tertiary-level centers significantly increases likelihood for TFS. Policy-level interventions to facilitate early screening and surgical referral for infants with biliary atresia are warranted to improve outcomes.

Cancers display significant heterogeneity with respect to tissue of origin, driver mutations, and other features of the surrounding tissue. It is likely that individual tumors engage common patterns of the immune system-here "archetypes"-creating prototypical non-destructive tumor immune microenvironments (TMEs) and modulating tumor-targeting. To discover the dominant immune system archetypes, the University of California, San Francisco (UCSF) Immunoprofiler Initiative (IPI) processed 364 individual tumors across 12 cancer types using standardized protocols. Computational clustering of flow cytometry and transcriptomic data obtained from cell sub-compartments uncovered dominant patterns of immune composition across cancers. These archetypes were profound insofar as they also differentiated tumors based upon unique immune and tumor gene-expression patterns. They also partitioned well-established classifications of tumor biology. The IPI resource provides a template for understanding cancer immunity as a collection of dominant patterns of immune organization and provides a rational path forward to learn how to modulate these to improve therapy.

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5' leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 "variants of concern" tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics.