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Background: Blood transfusions, crucial in managing anemia and coagulopathy in intensive care unit (ICU) settings, require accurate prediction for effective resource allocation and patient risk assessment. However, existing clinical decision support systems have primarily targeted a particular patient demographic with unique medical conditions and focused on a single type of blood transfusion. This study aims to develop an advanced machine learning-based model to predict the probability of transfusion necessity over the next 24 h for a diverse range of non-traumatic ICU patients. Methods: We conducted a retrospective cohort study on 72,072 non-traumatic adult ICU patients admitted to a high-volume US metropolitan academic hospital between 2016 and 2020. We developed a meta-learner and various machine learning models to serve as predictors, training them annually with 4-year data and evaluating on the fifth, unseen year, iteratively over 5 years. Results: The experimental results revealed that the meta-model surpasses the other models in different development scenarios. It achieved notable performance metrics, including an area under the receiver operating characteristic curve of 0.97, an accuracy rate of 0.93, and an F1 score of 0.89 in the best scenario. Conclusion: This study pioneers the use of machine learning models for predicting the likelihood of blood transfusion receipt in a diverse cohort of critically ill patients. The findings of this evaluation confirm that our model not only effectively predicts transfusion reception but also identifies key biomarkers for making transfusion decisions., Competing Interests: Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Alireza Rafiei et al.)

Aquatic ecosystems are often negatively affected by invasive species. However, biotic resistance by native species, either by competition or predation, can reduce the impacts of invasions by non-native species. The Western Mosquitofish ( Gambusia affinis ) is one of the most impactful invasive species of freshwater fish and cause declines in native fish populations. Using two mesocosm experiments conducted in different years, we examined the ecological interactions between juveniles of the native fish, Bluegill Sunfish ( Lepomis macrochirus ), and adults of the invasive fish, G. affinis . We found evidence for interactions between L. macrochirus and G. affinis . However, interactions did not appear symmetric, with L. macrochirus generally more affected by intraspecific interactions than interspecific interactions whereas G. affinis was more affected by interspecific interactions than intraspecific interactions. The presence of either species of fish led to a decrease in the number of large zooplankton and a tendency for a decrease in the total number of zooplankton. Based on these results, native L. macrochirus appear to be able to reduce the ability of non-native G. affinis to establish or maintain populations through both competition and predation (i.e., acting as an intraguild predator). The consistency of our results across both experiments, with their different designs and their occurring in different years, gives weight to these conclusions. The reduction of or prevention of establishment of populations of invasive G. affinis would likely benefit the aquatic communities of ponds with fish, especially small-bodied native fish., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.)

Hyperpigmented melanistic lesions (HPMLs) are a visual anomaly documented on the skin of smallmouth bass Micropterus dolomieu in the Susquehanna River Basin, Pennsylvania and in numerous other geographical locations. Currently, there is a lack of information on environmental and fish characteristics that may influence the prevalence of HPMLs associated with a recently described Adomavirus. The goal of this study was to understand potential drivers associated with HPMLs in socioeconomically and ecologically important riverine smallmouth bass populations. A total of 16,220 smallmouth bass were collected and examined for HPMLs between 2012 and 2022 in the Susquehanna River Basin. Overall, HPMLs were documented on 2.9% of fish collected. The interaction between temperature and fish size suggested differing relationships between shorter and longer fish with respect to temperature. Predicted probability of HPML prevalence ranged from 1.1% (95% CI = 0.3, 3.2) at 4°C to 0.01% (CI = 0.00, 0.04) at 26°C for an age-0 (125 mm) fish. In contrast, predicted probability of HPML prevalence ranged from 10.5% (95% CI = 5.8, 18.9) at 4°C to 0.8% (CI = 0.4, 1.5) at 26°C for an adult (322 mm) fish. Overall, HPMLs were more common in longer fish during cooler temperature periods which also corresponds to key life history periods for smallmouth bass (e.g., pre-spawn and overwintering) and could represent different exposure histories for juvenile and adult fish., (© 2024 The Author(s). Journal of Fish Diseases published by John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

The Transcriptional Enhanced Associated Domain (TEAD) family of transcription factors are key components of the Hippo signalling family which play a crucial role in the regulation of cell proliferation, differentiation and apoptosis. The identification of inhibitors of the TEAD transcription factors are an attractive strategy for the development of novel anticancer therapies. A HTS campaign identified hit 1, which was optimised using structure-based drug design, to deliver potent TEAD1 selective inhibitors with both a reversible and covalent mode of inhibition. The preference for TEAD1 could be rationalised by steric differences observed in the lower pocket of the palmitoylation-site between subtypes, with TEAD1 having the largest available volume to accommodate substitution in this region., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Monitoring wild fish health and exposure effects in impacted rivers and streams with differing land use has become a valuable research tool. Smallmouth bass (Micropterus dolomieu) are a sensitive, indicator species that exhibit signs of immunosuppression and endocrine disruption in response to water quality changes and contaminant exposure. To determine the impact of agriculture and development on smallmouth bass health, two sites (a developed/agriculture site and a forested site) in the Susquehanna River watershed, Pennsylvania were selected where bass and water chemistry were sampled from 2015 to 2019. Smallmouth bass were sampled for histopathology to assess parasite and macrophage aggregate density in the liver and spleen, condition factor (Ktl), hepatic gene transcript abundance, hepatosomatic index (HSI), and a health assessment index (HAI). Land use at the developed/agriculture site included greater pesticide application rates and phytoestrogen crop cover and more detections and higher concentrations of pesticides, wastewater-associated contaminants, hormones, phytoestrogens, and mycotoxins than at the forested site. Additionally, at the developed/agriculture site, indicators of molecular changes, including oxidative stress, immune/inflammation, and lipid metabolism-related hepatic gene transcripts, were associated with more contaminants and land use variables. At both sites, there were multiple associations of contaminants with liver and/or spleen macrophage aggregate density, indicating that changes at the molecular level seemed to be a better indicator of exposures unique to each site. The findings illustrate the importance of timing for land management practices, the complex mixtures aquatic animals are exposed to, and the temporal changes in contaminant concentration. Agricultural practices that affect hepatic gene transcripts associated with immune function and disease resistance were demonstrated which could negatively affect smallmouth bass populations., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The World Health Organization (WHO) diagnostic criteria for malignant phyllodes tumor (MPT) may miss a significant number of MPTs with metastatic potential. New refined diagnostic criteria (Refined Criteria) for MPT were recently proposed. The aim of this study is to validate the Refined Criteria. This validation study included 136 borderline (borderline phyllodes tumor [BoPT]) and MPT cases that were not included in the initial study. We evaluated tumor classifications based on both the Refined Criteria and the WHO criteria. The Refined Criteria defines MPT when these criteria are met (1) stromal overgrowth with ≥ 1 feature(s) of marked stromal cellularity, marked stromal cytologic atypia, or ≥10 mitoses per 10 high-power fields (10 mitoses/10 HPFs) or (2) marked stromal cellularity with ≥1 feature(s) of marked stromal cytologic atypia, ≥10 mitoses/10 HPFs or permeative border. The WHO criteria require all 5 morphologic features (stromal overgrowth, permeative border, marked stromal cellularity, marked stromal cytologic atypia, and ≥10 mitoses/10 HPFs) for an MPT diagnosis. Using the Refined Criteria, none of the 61 BoPTs developed metastasis and 40.0% of the 75 MPTs developed metastases; local recurrence was seen in 11.5% BoPTs and 25.3% MPTs. Using the WHO criteria, 9.6% of the 94 BoPTs developed metastases and 50.0% of the 42 MPTs developed metastases; 14.9% of the BoPTs had local recurrence and 28.6% of the MPTs had local recurrence. Nine (30.0%) of the 30 tumors that developed distant metastases were diagnosed as BoPTs by the WHO criteria. When we combined the 75 MPTs from this validation cohort with the 65 MPT cases from the published data using the Refined Criteria, 50 (35.7%) of the 140 MPTs developed metastases, whereas 8 cases with metastases were <5 cm. In the univariate analysis with log-rank test, stromal overgrowth, marked stromal cellularity, marked stromal cytologic atypia, ≥10 mitoses/10 HPFs, presence of heterologous components other than liposarcomatous component, and presence of stromal necrosis were significantly associated with the risk of metastasis (all with P < 0.05). In multivariate analysis with Cox proportional hazard regression, stromal overgrowth and marked stromal cellularity were significantly associated with metastasis (both with P < 0.001). The Refined Criteria are superior to the WHO criteria in predicting the clinical outcomes of BoPTs and MPTs. Using the Refined Criteria, 35.7% of 140 patients with MPT developed metastases, whereas none (0%) of the patients with BoPT developed metastases. Patients with MPT have a high metastatic rate; these patients may benefit from systemic chemotherapy or targeted therapies. In contrast, patients with BoPT may be managed with complete local excision alone without chemotherapy., Competing Interests: Conflicts of Interest and Source of Funding: X.L. has served as an advisor for Astra Zeneca, Roche, Eli Lilly, and Onviv, and Champions Oncology has funded research in Xiaoxian Li’ lab. S.W. has served as Breast Pathology Faculty Advisory Board for Daiichi Sankyo Inc. and AstraZeneca. For the remaining authors, none were declared., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The Orthopoxvirus (OPXV) genus of the Poxviridae includes human pathogens variola virus (VARV), monkeypox virus (MPXV), vaccinia virus (VACV), and a number of zoonotic viruses. A number of Bcl-2-like proteins of VACV are involved in escaping the host innate immunity. However, little work has been devoted to the evolution and function of their orthologues in other OPXVs. Here, we found that MPXV protein P2, encoded by the P2L gene, and P2 orthologues from other OPXVs, such as VACV protein N2, localize to the nucleus and antagonize interferon (IFN) production. Exceptions to this were the truncated P2 orthologues in camelpox virus (CMLV) and taterapox virus (TATV) that lacked the nuclear localization signal (NLS). Mechanistically, the NLS of MPXV P2 interacted with karyopherin α-2 (KPNA2) to facilitate P2 nuclear translocation, and competitively inhibited KPNA2-mediated IRF3 nuclear translocation and downstream IFN production. Deletion of the NLS in P2 or orthologues significantly enhanced IRF3 nuclear translocation and innate immune responses, thereby reducing viral replication. Moreover, deletion of NLS from N2 in VACV attenuated viral replication and virulence in mice. These data demonstrate that the NLS-mediated translocation of P2 is critical for P2-induced inhibition of innate immunity. Our findings contribute to an in-depth understanding of the mechanisms of OPXV P2 orthologue in innate immune evasion.

We investigated the light-absorption properties of brown carbon (BrC) as part of the Georgia Wildland-Fire Simulation Experiment. We constructed fuel beds representative of three ecoregions in the Southeastern U.S. and varied the fuel-bed moisture content to simulate either prescribed fires or drought-induced wildfires. Based on decreasing fire radiative energy normalized by fuel-bed mass loading (FRE norm ), the combustion conditions were grouped into wildfire (Wild), prescribed fire (Rx), and wildfire involving duff ignition (WildDuff). The emitted BrC ranged from weakly absorbing (WildDuff) to moderately absorbing (Rx and Wild) with the imaginary part of the refractive index ( k ) values that were well-correlated with FRE norm . We apportioned the BrC into water-soluble (WSBrC) and water-insoluble (WIBrC). Approximately half of the WSBrC molecules detected using electrospray-ionization mass spectrometry were potential chromophores. Nevertheless, k of WSBrC was an order of magnitude smaller than k of WIBrC. Furthermore, k of WIBrC was well-correlated with FRE norm while k of WSBrC was not, suggesting different formation pathways between WIBrC and WSBrC. Overall, the results signify the importance of combustion conditions in determining BrC light-absorption properties and indicate that variables in wildland fires, such as moisture content and fuel-bed composition, impact BrC light-absorption properties to the extent that they influence combustion conditions., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Population genetic analysis of invasive populations can provide valuable insights into the source of introductions, pathways for expansion, and their demographic histories. Flathead catfish (Pylodictis olivaris) are a prolific invasive species with high fecundity, long-distance dispersal, and piscivorous feeding habits that can lead to declines in native fish populations. In this study, we analyse the genetics of invasive P. olivaris in the Mid-Atlantic region to assess their connectivity and attempt to reconstruct the history of introduced populations. Based on an assessment across 13 microsatellite loci, P. olivaris from the Susquehanna River system (N = 537), Schuylkill River (N = 33), and Delaware River (N = 1) have low genetic diversity (global H obs  = 0.504), although we detected no evidence of substantial inbreeding (F IS  = -0.083 to 0.022). P. olivaris from these different river systems were genetically distinct, suggesting separate introductions. However, population structure was much weaker within each river system and exhibited a pattern of high connectivity, with some evidence of isolation by distance. P. olivaris from the Susquehanna and Schuylkill rivers showed evidence for recent genetic bottlenecks, and demographic models were consistent with historical records, which suggest that populations were established by recent founder events consisting of a small number of individuals. Our results show the risk posed by small introductions of P. olivaris, which can spread widely once a population is established, and highlight the importance of prevention and sensitive early detection methods to prevent the spread of P. olivaris in the future., (© 2024 The Author(s). Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

We report measurements of the absorption Ångström exponent (AAE) and single scattering albedo (SSA) of biomass burning aerosol from the combustion of fuel beds representing three eco-regions of the Southeast U.S. (Piedmont, Coastal Plain, and Blue Ridge Mountains) with moisture content representative of wildfires and prescribed fires. We find a strong correlation between the AAE and SSA for both simulated wildfires (low fuel moisture) and prescribed fires (higher fuel moisture). For wildfires, the AAE and SSA are strongly dependent on the eco-region of the fuel bed and span a much wider range (AAE = 1.3-4.2, SSA = 0.75-0.97) than they do for prescribed fires (AAE = 2.4-3.1, SSA = 0.88-0.96). The AAE and SSA are also found to be correlated with the fraction of total carbon that is elemental carbon ( f EC ) for both wildfires and prescribed fires, but the range of f EC observed (0.02-0.14) from the fuel beds is much smaller than that reported previously from laboratory studies using individual fuels. The observations from the present study suggest that fuel-bed composition and moisture content are significant factors in determining the relative amount of organic material in biomass burning aerosols and, consequentially, their optical properties., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: The shoulder microbiome is an emerging field in orthopedic research. Large synovial joints which were typically considered to be sterile now have strong evidence demonstrating the presence of native organisms colonizing the joint. Many of the conditions that surgeons see and treat in the shoulder, including osteoarthritis, rotator cuff tears, and adhesive capsulitis, have unclear etiology. The shoulder microbiome is a potential source of pathology in dysbiosis states, or source of protection against pathology in normobiosis states. The purpose of this review is to characterize the published research detailing the microbiome of the native shoulder joint., Methods: We conducted a systematic review and meta-analysis of data searches on Medline, Embase, Cochrane Central Register of Controlled Trials, and SCOPUS. The following search terms were used with various permutations; shoulder, skin, microbiome, infection, colonization, commensal, microbiota, flora. The terms 'shoulder' and 'shoulder AND skin' were combined with the other 6 terms for a total of 12 searches per database. Two independent reviewers conducted the review with a third reviewer available to resolve differences of interpretation. Studies were eligible if they were human studies of patients undergoing shoulder surgery (or surgical preparation therapy) with a shoulder that had not previously been operated on, where microbial samples were reported. Studies were excluded if they were systematic reviews and meta-analyses, animal studies, cadaveric studies, studies of patients under the age of 18 and studies including postoperative shoulders., Results: The search methodology yielded 47 eligible studies for analysis, with a total of 3283 patients. Native shoulder sampling yielded positive organisms in 50.8% of all samples (95% confidence interval [CI] = 41.8%-59.8%, I 2  = 97.9%, P  = .000). The predominate organism reported was C. acnes , with 29% of studies specific for C. acnes , and a total of 52 other organisms detected in the native shoulder. Skin preparation solutions reduced the skin colonization rate of 55.7% (95% CI = 32.7%-78.6%, I 2  = 98.2%, P  = .000) to 43.5% (95% CI = 29.5%-57.5%, I 2  = 98.2%, P  = .000). Despite the high rates of colonization detected under sterile conditions, of 42 studies and 3083 patients reporting postoperative outcomes, the infection rate was 1.8% (95% CI = 0.5%-3.2%, I 2  = 0.0%, P  = .612)., Conclusion: There is strong evidence to support the existence of a native shoulder microbiome. Further research is required to characterize this microbiome and correlate it to disease and health states., (Crown Copyright © 2024 Published by Elsevier Inc. on behalf of American Shoulder and Elbow Surgeons.)

Laboratory studies have shown that photolytic mass loss can be a significant sink for secondary organic aerosol (SOA). Here, we use a quartz crystal microbalance to measure mass loss of Suwannee River Humic Acid (SRHA) and Suwannee River Fulvic Acid (SRFA), surrogates for SOA, exposed to 254, 300, and 405 nm radiation over the course of 24 h. We find that the photolytic mass loss rates of these materials are comparable to those for laboratory-generated limonene and toluene SOA material from the study of Baboomian et al, ACS Earth Space Chem . 2020, 4, 1078. Scaling our results to ambient conditions, we estimate that humic substances in aerosols can lose as much as 8% by mass in the first day of exposure in the atmosphere, equivalent to 0.025% of J NO 2 , the photolysis rate of nitrogen dioxide. By using zero air instead of nitrogen, we also find that the presence of oxygen accelerates the photolytic mass loss rate by a factor of 2 to 4 at all wavelengths suggesting a potential role for reactive oxygen species. UV photolysis of an aqueous SRFA solution demonstrated both photobleaching at UV wavelengths and photoenhancement at visible wavelengths. Ultrahigh-resolution mass spectrometric analysis showed that condensed-phase SRFA photolysis led to decreased intensity in the 100-300 m / z range while aqueous SRFA photolysis resulted in an increase in intensity in the same range. This work reaffirms that photolytic mass loss is a potentially significant sink for SOA, but only on the time scale of a day or two and demonstrates that SRHA and SRFA are suitable surrogates for atmospheric SOA with respect to photolytic mass loss., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

This work, as part of the Georgia Wildland fire Simulation Experiment (G-WISE) campaign, explores the aqueous photolysis of water-soluble brown carbon (W-BrC) in biomass burning aerosols from the combustion of fuel beds collected from three distinct ecoregions in Georgia: Piedmont, Coastal Plain, and Blue Ridge. Burns were conducted under conditions representative of wildfires, which are common unplanned occurrences in Southeastern forests (low fuel moisture content), and prescribed fires, which are commonly used in forest management (higher fuel moisture content). Upon exposure to radiation from UV lamps equivalent to approximately 5 h in the atmosphere, the absorption spectra of all six samples exhibited up to 40% photobleaching in the UV range (280-400 nm) and as much as 30% photo-enhancement in the visible range (400-500 nm). Together, these two effects reduced the absorption Ångström exponent (AAE), a measure of the wavelength dependence of the spectrum, from 6.0-7.9 before photolysis to 5.0-5.7 after. Electrospray ionization ultrahigh-resolution mass spectrometry analysis shows the potential formation of oligomeric chromophores due to aqueous photolysis. This work provides insight into the impacts that aqueous photolysis has on W-BrC in biomass burning aerosols and its dependence on fuel bed composition and moisture content., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Reproduction, although absolutely essential to a species' persistence, is in itself challenging. As anthropogenic change increasingly affects every landscape on Earth, it is critical to understand how specific pressures impact the reproductive efforts of individuals, which directly contribute to the success or failure of populations. However, organisms rarely encounter a single burden at a time, and the interactions of environmental challenges can have compounding effects. Understanding environmental and physiological pressures is difficult because they are often context-dependent and not generalizable, but long-term monitoring across variable landscapes and weather patterns can improve our understanding of these complex interactions. We tested the effects of urbanization, climate, and individual condition on the reproductive investment of wild side-blotched lizards ( Uta stansburiana ) by measuring physiological/reproductive metrics from six populations in urban and rural areas over six consecutive years of variable precipitation. We observed that reproductive stage affected body condition, corticosterone concentration, and oxidative stress. We also observed that reproductive patterns differed between urban and rural populations depending on rainfall, with rural animals increasing reproductive investment during rainier years compared to urban conspecifics, and that reproductive decisions appeared to occur early in the reproductive process. These results demonstrate the plastic nature of a generalist species optimizing lifetime fitness under varying conditions., Competing Interests: There are no conflicts of interests, financial or otherwise, for any of the authors., (© 2024 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Context.—: Urinalysis instrument-specific dip strips offer physicians qualitative results for actionable analytes (protein, glucose, leukocyte esterase, nitrates, hemoglobin, and ketones)., Objective.—: To explain a strategy implemented to support clinical decision-making by providing urine quantification of protein, glucose, white blood cells (WBCs), and red blood cells because of urine strip shortages., Design.—: During shortages, we implemented an automated algorithm that triggered sending urine samples to the automation line for quantification of protein and glucose and ensured that urine microscopy was performed to obtain WBC and red blood cell counts. The algorithm printed 2 labels so nursing staff would collect 2 specimens. We monitored the turnaround time from the specimen being received in the laboratory to result verification, ensured that the culture reflex order was triggered, and tracked complaints by physicians regarding not having usual urinalysis results. Prior to implementation, correlation between sample types for protein and glucose measurement was found acceptable., Results.—: The algorithm was put in place twice during 2022. The turnaround time of urine microscopic study was identical to that obtained when the urinalysis was done with the strips; however, the quantification of glucose and protein took approximately 30 minutes more. Urine reflex cultures were triggered correctly with the algorithm, as they were derived entirely from a WBC count higher than 10 per high-power field. During the shortage period we had only 1 complaint, by a physician wanting to have results of nitrates., Conclusions.—: During urine strip shortages, we successfully implemented a diversion algorithm that provided actionable urinalysis analytes in a timely manner with minimal provider complaints., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Histone deacetylases (HDACs) regulate gene expression and innate immunity. Previously, we showed that HDAC5 is degraded during Vaccinia virus (VACV) infection and is a restriction factor for VACV and herpes simplex virus type 1. Here, we report that HDAC5 promotes interferon regulatory factor 3 (IRF3) activation downstream of Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 or Sendai virus-mediated stimulation without requiring HDAC activity. Loss of HDAC5-mediated IRF3 activation is restored by re-introduction of HDAC5 but not HDAC1 or HDAC4. The antiviral activity of HDAC5 is antagonized by VACV protein C6 and orthologs from the orthopoxviruses cowpox, rabbitpox, camelpox, monkeypox, and variola. Infection by many of these viruses induces proteasomal degradation of HDAC5, and expression of C6 alone can induce HDAC5 degradation. Mechanistically, C6 binds to the dimerization domain of HDAC5 and prevents homodimerization and heterodimerization with HDAC4. Overall, this study describes HDAC5 as a positive regulator of IRF3 activation and provides mechanistic insight into how the poxviral protein C6 binds to HDAC5 to antagonize its function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Vaccinia virus (VACV) is a large DNA virus that encodes scores of proteins that modulate the host immune response. VACV protein C4 is one such immunomodulator known to inhibit the activation of both the NF-κB signaling cascade and the DNA-PK-mediated DNA sensing pathway. Here, we show that the N-terminal region of C4, which neither inhibits NF-κB nor mediates interaction with DNA-PK, still contributes to virus virulence. Furthermore, this domain interacts directly and with high affinity to the C-terminal domain of filamin B (FLNB). FLNB is a large actin-binding protein that stabilizes the F-actin network and is implicated in other cellular processes. Deletion of FLNB from cells results in larger VACV plaques and increased infectious viral yield, indicating that FLNB restricts VACV spread. These data demonstrate that C4 has a new function that contributes to virulence and engages the cytoskeleton. Furthermore, we show that the cytoskeleton performs further previously uncharacterized functions during VACV infection., Importance: Vaccinia virus (VACV), the vaccine against smallpox and monkeypox, encodes many proteins to counteract the host immune response. Investigating these proteins provides insights into viral immune evasion mechanisms and thereby indicates how to engineer safer and more immunogenic VACV-based vaccines. Here, we report that the N-terminal domain of VACV protein C4 interacts directly with the cytoskeletal protein filamin B (FLNB), and this domain of C4 contributes to virus virulence. Furthermore, VACV replicates and spreads better in cells lacking FLNB, thus demonstrating that FLNB has antiviral activity. VACV utilizes the cytoskeleton for movement within and between cells; however, previous studies show no involvement of C4 in VACV replication or spread. Thus, C4 associates with FLNB for a different reason, suggesting that the cytoskeleton has further uncharacterized roles during virus infection., Competing Interests: The authors declare no conflict of interest.

Context.—: Genetic profiling data of prostatic adenocarcinoma are derived from predominantly White patients. In African Americans, prostatic adenocarcinoma has a poorer prognosis, raising the possibility of distinct genetic alterations., Objective.—: To investigate the genomic alterations of prostatic adenocarcinoma metastatic to regional lymph nodes in African American patients, with an emphasis on SPOP mutation., Design.—: We retrospectively reviewed African American patients with pN1 prostatic adenocarcinoma managed with radical prostatectomy and lymph node dissection. Comprehensive molecular profiling was performed, and androgen receptor signaling scores were calculated., Results.—: Nineteen patients were included. The most frequent genetic alteration was SPOP mutations (5 of 17; 29.4% [95% CI: 10.3-56.0]). While most alterations were associated with a high androgen receptor signaling score, mutant SPOP was exclusively associated with a low median and interquartile range (IQR) androgen receptor signaling score (0.788 [IQR 0.765-0.791] versus 0.835 [IQR 0.828-0.842], P = .003). In mutant SPOP, mRNA expression of SPOP inhibitor G3BP1 and SPOP substrates showed a significantly decreased expression of AR (33.40 [IQR 28.45-36.30] versus 59.53 [IQR 53.10-72.83], P = .01), TRIM24 (3.95 [IQR 3.28-5.03] versus 9.80 [IQR 7.39-11.70], P = .008), and NCOA3 (15.19 [IQR 10.59-15.93] versus 21.88 [IQR 18.41-28.33], P = .046)., Conclusions.—: African American patients with metastatic prostate adenocarcinoma might have a higher prevalence of mutant SPOP (30%), compared to ∼10% in unselected cohorts with lower expressions of SPOP substrates. In our study, in patients with mutant SPOP, the mutation was associated with decreased SPOP substrate expression and androgen receptor signaling, raising concern for suboptimal efficacy of androgen deprivation therapy in this subset of patients., (© 2024 College of American Pathologists.)

Trastuzumab deruxtecan (T-DXd) has been approved by the US Food and Drug Administration (FDA) to treat patients with metastatic HER2-positive and HER2-low breast cancer, and clinical trials are examining its efficacy against early-stage breast cancer. Current HER2 immunohistochemical (IHC) assays are suboptimal in evaluating HER2-low breast cancers and identifying which patients would benefit from T-DXd. HER2 expression in 526 breast cancer tissue microarray (TMA) cores was measured using the FDA-approved PATHWAY and HercepTest IHC assays, and the corresponding RNA levels were evaluated by RNAscope. HER2 protein levels by regression analysis using a quantitative immunofluorescence score against cell line arrays with known HER2 protein levels determined by mass spectrometry were available in 48 of the cores. RNAscope was also performed in 32 metastatic biopsies from 23 patients who were subsequently treated with T-DXd, and the results were correlated with response rate. HER2 RNA levels by RNAscope strongly correlated with HER2 protein levels (P < .0001) and with HER2 IHC H-scores from the PATHWAY and HercepTest assays (P < .0001). However, neither protein levels nor RNA levels significantly differed between cases scored 0, ultralow, and 1+ by PATHWAY and HercepTest. The RNA levels were significantly higher (P = .030) in responders (6.4 ± 8.2 dots/cell, n = 12) than those in nonresponders (2.6 ± 2.2, n = 20) to T-DXd. RNAscope is a simple assay that can be objectively quantified and is a promising alternative to current IHC assays in evaluating HER2 expression in breast cancers, especially HER2-low cases, and may identify patients who would benefit from T-DXd., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Modified vaccinia Ankara (MVA) virus does not replicate in human cells and is the vaccine deployed to curb the current outbreak of mpox. Here, we conduct a multiplexed proteomic analysis to quantify >9000 cellular and ~80% of viral proteins throughout MVA infection of human fibroblasts and macrophages. >690 human proteins are down-regulated >2-fold by MVA, revealing a substantial remodelling of the host proteome. >25% of these MVA targets are not shared with replication-competent vaccinia. Viral intermediate/late gene expression is necessary for MVA antagonism of innate immunity, and suppression of interferon effectors such as ISG20 potentiates virus gene expression. Proteomic changes specific to infection of macrophages indicate modulation of the inflammatory response, including inflammasome activation. Our approach thus provides a global view of the impact of MVA on the human proteome and identifies mechanisms that may underpin its abortive infection. These discoveries will prove vital to design future generations of vaccines., (© 2023. The Author(s).)

Many environments present some degree of seasonal water limitations; organisms that live in such environments must be adapted to survive periods without permanent water access. Often this involves the ability to tolerate dehydration, which can have adverse physiological effects and is typically considered a physiological stressor. While having many functions, the hormone corticosterone (CORT) is often released in response to stressors, yet increasing plasma CORT while dehydrated could be considered maladaptive, especially for species that experience predictable bouts of dehydration and have related coping mechanisms. Elevating CORT could reduce immunocompetence and have other negative physiological effects. Thus, such species likely have CORT and immune responses adapted to experiencing seasonal droughts. We evaluated how dehydration affects CORT and immune function in eight squamate species that naturally experience varied water limitation. We tested whether hydric state affected plasma CORT concentrations and aspects of immunocompetence (lysis, agglutination, bacterial killing ability and white blood cell counts) differently among species based on how seasonally water limited they are and whether this is constrained by phylogeny. The species represented four familial pairs, with one species of each pair inhabiting environments with frequent access to water and one naturally experiencing extended periods (>30 days) with no access to standing water. The effects of dehydration on CORT and immunity varied among species. Increases in CORT were generally not associated with reduced immunocompetence, indicating CORT and immunity might be decoupled in some species. Interspecies variations in responses to dehydration were more clearly grouped by phylogeny than by habitat type., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Background: We have been documenting the biological responses to low levels of radiation (natural background) and very low level radiation (below background), and thus these studies are testing mild external stimuli to which we would expect relatively mild biological responses. We recently published a transcriptome software comparison study based on RNA-Seqs from a below background radiation treatment of two model organisms, E. coli and C. elegans (Thawng and Smith, BMC Genomics 23:452, 2022). We reported DNAstar-D (Deseq2 in the DNAstar software pipeline) to be the more conservative, realistic tool for differential gene expression compared to other transcriptome software packages (CLC, Partek and DNAstar-E (using edgeR). Here we report two follow-up studies (one with a new model organism, Aedes aegypti and another software package (Azenta) on transcriptome responses from varying dose rates using three different sources of natural radiation., Results: When E. coli was exposed to varying levels of K40, we again found that the DNAstar-D pipeline yielded a more conservative number of DEGs and a lower fold-difference than the CLC pipeline and DNAstar-E run in parallel. After a 30 read minimum cutoff criterion was applied to the data, the number of significant DEGs ranged from 0 to 81 with DNAstar-D, while the number of significant DEGs ranged from 4 to 117 and 14 to 139 using DNAstar-E and the CLC pipelines, respectively. In terms of the extent of expression, the highest foldchange DEG was observed in DNAstar-E with 19.7-fold followed by 12.5-fold in CLC and 4.3-fold in DNAstar-D. In a recently completed study with Ae. Aegypti and using another software package (Azenta), we analyzed the RNA-Seq response to similar sources of low-level radiation and again found the DNAstar-D pipeline to give the more conservative number and fold-expression of DEGs compared to other softwares. The number of significant DEGs ranged 31-221 in Azenta and 31 to 237 in CLC, 19-252 in DNAstar-E and 0-67 in DNAStar-D. The highest fold-change of DEGs were found in CLC (1,350.9-fold), with DNAstar-E (5.9 -fold) and Azenta (5.5-fold) intermediate, and the lowest levels of expression (4-fold) found in DNAstar-D., Conclusions: This study once again highlights the importance of choosing appropriate software for transcriptome analysis. Using three different biological models (bacteria, nematode and mosquito) in four different studies testing very low levels of radiation (Van Voorhies et al., Front Public Health 8:581796, 2020; Thawng and Smith, BMC Genomics 23:452, 2022; current study), the CLC software package resulted in what appears to be an exaggerated gene expression response in terms of numbers of DEGs and extent of expression. Setting a 30-read cutoff diminishes this exaggerated response in most of the software tested. We have further affirmed that DNAstar-Deseq2 gives a more conservative transcriptome expression pattern which appears more suitable for studies expecting subtle gene expression patterns., (© 2023. The Author(s).)

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localizes to mitochondria, where it inhibits innate immunity by restricting IFN-β production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterized mechanism of innate immune evasion by this important human pathogen., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Pike killifish Belonesox belizanus is an established non-native fish species in Florida, USA, that was first documented in south Florida in 1957 and then in Tampa Bay tributaries in 1994. Decreases in small-bodied fish abundances have been linked to the introduction of B. belizanus in both of these regions. Increases in the range and abundance of B. belizanus in the Tampa Bay area and overlap in habitat usage have led to concerns about potential competition with, and predation on, early-juvenile common snook Centropomus undecimalis [≤100 mm standard length (SL)]. Stomach contents of B. belizanus (N = 422; 14-127 mm SL) and early-juvenile C. undecimalis (N = 1132; 5-119 mm SL) were collected to examine the dietary overlap of these two species and potential differences in the diet of early-juvenile C. undecimalis from locations with and without B. belizanus co-occurring. Prey resources were collected by seine to assess prey resource limitation and prey selectivity. Stomach content analysis indicated that there was low overlap in the diet of early-juvenile C. undecimalis and B. belizanus (C ≤ 0.40). Early-juvenile C. undecimalis had a wider diet breadth, consuming many organisms that are not consumed by B. belizanus and which make up a large portion of the early-juvenile C. undecimalis diet. Analysis of prey resources indicated that some prey groups may have lower abundances in locations where B. belizanus are present, with some of these differences reflected in the diet of early-juvenile C. undecimalis. Despite these differences, there was minimal difference in the diet overlap of early-juvenile C. undecimalis from locations with and without B. belizanus co-occurring. Currently B. belizanus appear to be competing minimally with early-juvenile C. undecimalis for prey resources, with no substantial impacts being detected., (© 2023 Fisheries Society of the British Isles.)

Entry of antigen-specific T cells into human tumors is critical for immunotherapy, but the underlying mechanisms are poorly understood. Here, we combined high-dimensional spatial analyses with in vitro and in vivo modeling to study the mechanisms underlying immune infiltration in human multiple myeloma (MM) and its precursor monoclonal gammopathy of undetermined significance (MGUS). Clustered tumor growth was a feature of MM but not MGUS biopsies, and this growth pattern was reproduced in humanized mouse models. MM biopsies exhibited intralesional as well as spatial heterogeneity, with coexistence of T cell-rich and T cell-sparse regions and the presence of areas of T cell exclusion. In vitro studies demonstrated that T cell entry into MM clusters was regulated by agonistic signals and CD2-CD58 interactions. Upon adoptive transfer, antigen-specific T cells localized to the tumor site but required in situ DC-mediated antigen presentation for tumor entry. C-type lectin domain family 9 member A-positive (CLEC9A+) DCs appeared to mark portals of entry for gradients of T cell infiltration in MM biopsies, and their proximity to T cell factor 1-positive (TCF1+) T cells correlated with disease state and risk status. These data illustrate a role for tumor-associated DCs and in situ activation in promoting the infiltration of antigen-specific T cells in MM and provide insights into spatial alterations in tumor/immune cells with malignant evolution.

Human tripartite motif protein 5α (TRIM5α) is a well-characterized restriction factor for some RNA viruses, including HIV 1-5 ; however, reports are limited for DNA viruses 6,7 . Here we demonstrate that TRIM5α also restricts orthopoxviruses and, via its SPRY domain, binds to the orthopoxvirus capsid protein L3 to diminish virus replication and activate innate immunity. In response, several orthopoxviruses, including vaccinia, rabbitpox, cowpox, monkeypox, camelpox and variola viruses, deploy countermeasures. First, the protein C6 binds to TRIM5 via the RING domain to induce its proteasome-dependent degradation. Second, cyclophilin A (CypA) is recruited via interaction with the capsid protein L3 to virus factories and virions to antagonize TRIM5α; this interaction is prevented by cyclosporine A (CsA) and the non-immunosuppressive derivatives alisporivir and NIM811. Both the proviral effect of CypA and the antiviral effect of CsA are dependent on TRIM5α. CsA, alisporivir and NIM811 have antiviral activity against orthopoxviruses, and because these drugs target a cellular protein, CypA, the emergence of viral drug resistance is difficult. These results warrant testing of CsA derivatives against orthopoxviruses, including monkeypox and variola., (© 2023. The Author(s).)

Urbanization can cause innumerable abiotic and biotic changes that have the potential to influence the ecology, behavior, and physiology of native resident organisms. Relative to their rural conspecifics, urban Side-blotched Lizard (Uta stansburiana) populations in southern Utah have lower survival prospects and maximize reproductive investment via producing larger eggs and larger clutch sizes. While egg size is an important predictor of offspring quality, physiological factors within the egg yolk are reflective of the maternal environment and can alter offspring traits, especially during energetically costly processes, such as reproduction or immunity. Therefore, maternal effects may represent an adaptive mechanism by which urban-dwelling species can persist within a variable landscape. In this study, we assess urban and rural differences in egg yolk bacterial killing ability (BKA), corticosterone (CORT), oxidative status (d-ROMs), and energy metabolites (free glycerol and triglycerides), and their association with female immune status and egg quality. Within a laboratory setting, we immune challenged urban lizards via lipopolysaccharide injection (LPS) to test whether physiological changes associated with immune system activity impacted egg yolk investment. We found urban females had higher mite loads than rural females, however mite burden was related to yolk BKA in rural eggs, but not urban eggs. While yolk BKA differed between urban and rural sites, egg mass and egg viability (fertilized vs. unfertilized) were strong predictors of yolk physiology and may imply tradeoffs exist between maintenance and reproduction. LPS treatment caused a decrease in egg yolk d-ROMs relative to the control treatments, supporting results from previous research. Finally, urban lizards laid a higher proportion of unfertilized eggs, which differed in egg yolk BKA, CORT, and triglycerides in comparison to fertilized eggs. Because rural lizards laid only viable eggs during this study, these results suggest that reduced egg viability is a potential cost of living in an urban environment. Furthermore, these results help us better understand potential downstream impacts of urbanization on offspring survival, fitness, and overall population health., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations., Competing Interests: Declaration of interests A.J.P. is chair of the DHSC Joint Committee on Vaccination and Immunisation (JCVI) but does not take part in the JCVI COVID-19 committee. Oxford University has entered into a partnership with AstraZeneca for development of a COVID-19 vaccine. A.J.P. has provided advice to Shionogi & Co., Ltd., on the development of a COVID-19 vaccine., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

Poxviridae is a family of enveloped, brick-shaped or ovoid viruses. The genome is a linear molecule of dsDNA (128-375 kbp) with covalently closed ends. The family includes the sub-families Entomopoxvirinae , whose members have been found in four orders of insects, and Chordopoxvirinae, whose members are found in mammals, birds, reptiles and fish. Poxviruses are important pathogens in various animals, including humans, and typically result in the formation of lesions, skin nodules, or disseminated rash. Infections can be fatal. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Poxviridae , which is available at ictv.global/report/poxviridae.

Pathology text mining is a challenging task given the reporting variability and constant new findings in cancer sub-type definitions. However, successful text mining of a large pathology database can play a critical role to advance 'big data' cancer research like similarity-based treatment selection, case identification, prognostication, surveillance, clinical trial screening, risk stratification, and many others. While there is a growing interest in developing language models for more specific clinical domains, no pathology-specific language space exist to support the rapid data-mining development in pathology space. In literature, a few approaches fine-tuned general transformer models on specialized corpora while maintaining the original tokenizer, but in fields requiring specialized terminology, these models often fail to perform adequately. We propose PathologyBERT - a pre-trained masked language model which was trained on 347,173 histopathology specimen reports and publicly released in the Huggingface 1 repository 2 . Our comprehensive experiments demonstrate that pre-training of transformer model on pathology corpora yields performance improvements on Natural Language Understanding (NLU) and Breast Cancer Diagnose Classification when compared to nonspecific language models., (©2022 AMIA - All rights reserved.)

Temporally separated species are often thought to have limited competition over a shared resource. However, early arriving species may consume a limited resource such that later-arriving species have access to fewer resources and thus experience competitive effects, even if they are temporally separated (i.e., they experience legacy effects from the early species). The presence of a predator might affect potential legacy effects by influencing the behavior or survivorship of the early species. Using a mesocosm experiment, I examined whether the presence of nonnative Western Mosquitofish ( Gambusia affinis ) mediated legacy effects in the interaction of two temporally separated species of tadpoles, early arriving American Toads ( Anaxyrus americanus ) and late-arriving Bullfrogs ( Rana catesbeiana ). Anaxyrus americanus tadpoles reduced R. catesbeiana tadpole growth despite all A. americanus tadpoles metamorphosing 8 days before the introduction of R. catesbeiana tadpoles into the mesocosms (i.e., legacy effects). Gambusia affinis had limited effects on A. americanus (1 day delay in metamorphosis but no effect on survivorship or size at metamorphosis) and positive effects on R. catesbeiana (increased growth). There were no significant interactions between the A. americanus tadpole density and G. affinis treatments. In conclusion, I found evidence of significant legacy effects of A. americanus tadpoles on R. catesbeiana tadpoles, but no evidence that G. affinis mediated the legacy effects., Competing Interests: The author declares that he has no competing interests., (© 2023 The Author. Ecology and Evolution published by John Wiley & Sons Ltd.)

Natural killer (NK) cells have an established role in controlling poxvirus infection and there is a growing interest to exploit their capabilities in the context of poxvirus-based oncolytic therapy and vaccination. How NK cells respond to poxvirus-infected cells to become activated is not well established. To address this knowledge gap, we studied the NK cell response to vaccinia virus (VACV) in vivo , using a systemic infection murine model. We found broad alterations in NK cells transcriptional activity in VACV-infected mice, consistent with both direct target cell recognition and cytokine exposure. There were also alterations in the expression levels of specific NK surface receptors (NKRs), including the Ly49 family and SLAM receptors, as well as upregulation of memory-associated NK markers. Despite the latter observation, adoptive transfer of VACV-expercienced NK populations did not confer protection from infection. Comparison with the NK cell response to murine cytomegalovirus (MCMV) infection highlighted common features, but also distinct NK transcriptional programmes initiated by VACV. Finally, there was a clear overlap between the NK transcriptional response in humans vaccinated with an attenuated VACV, modified vaccinia Ankara (MVA), demonstrating conservation between the NK response in these different host species. Overall, this study provides new data about NK cell activation, function, and homeostasis during VACV infection, and may have implication for the design of VACV-based therapeutics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Depierreux, Smith and Ferguson.)

The pathologic diagnosis of bone marrow disorders relies in part on the microscopic analysis of bone marrow aspirate (BMA) smears and the manual counting of marrow nucleated cells to obtain a differential cell count (DCC). This manual process has significant limitations, including the analysis of only a small subset of optimal slide areas and nucleated cells, as well as interobserver variability due to differences in cell selection and classification. To address these shortcomings, we developed an automated machine learning-based pipeline for obtaining 11-component DCCs on whole-slide BMAs. This pipeline uses a sequential process of identifying optimal BMA regions with high proportions of marrow nucleated cells, detecting individual cells within these optimal areas, and classifying these cells into 1 of 11 DCC components. Convolutional neural network models were trained on 396,048 BMA region, 28,914 cell boundary, and 1,510,976 cell class images from manual annotations. The resulting automated pipeline produced 11-component DCCs that demonstrated a high statistical and diagnostic concordance with manual DCCs among a heterogeneous group of testing BMA slides with varying pathologies and cellularities. Additionally, we demonstrated that an automated analysis can reduce the intraslide variance in DCCs by analyzing the whole slide and marrow nucleated cells within all optimal regions. Finally, the pipeline outputs of region classification, cell detection, and cell classification can be visualized using whole-slide image analysis software. This study demonstrates the feasibility of a fully automated pipeline for generating DCCs on scanned whole-slide BMA images, with the potential for improving the current standard of practice for utilizing BMA smears in the laboratory analysis of hematologic disorders., (Copyright © 2022 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Genome-wide CRISPR-based screening is a powerful tool in forward genetics, enabling biologic discovery by linking a desired phenotype to a specific genetic perturbation. However, hits from a genome-wide screen require individual validation to reproduce and accurately quantify their effects outside of a pooled experiment. Here, we describe a step-by-step protocol to rapidly assess the effects of individual sgRNAs from CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems. All steps, including cloning, lentivirus generation, cell transduction, and phenotypic readout, can be performed entirely in 96-well plates. The system is highly flexible in both cell type and selection system, requiring only that the phenotype(s) of interest be read out via flow cytometry. We expect that this protocol will provide researchers with a rapid way to sift through potential screening hits, and prioritize them for deeper analysis in more complex in vitro or even in vivo systems. Graphical abstract., Competing Interests: Competing interests JSC has received consulting fees from Gilde Healthcare and Eko, unrelated to the published work. The other authors have no disclosures., (Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC.)

Supplemental material is available for this article. Keywords: Mammography, Breast, Machine Learning © RSNA, 2023., Competing Interests: Disclosures of conflicts of interest: J.J.J. No relevant relationships. B.L.V. No relevant relationships. A.B. No relevant relationships. T.K. No relevant relationships. T.S. No relevant relationships. R.C. No relevant relationships. R.D. No relevant relationships. M.M. No relevant relationships. G.O. No relevant relationships. G.S. No relevant relationships. M.W. No relevant relationships. C.R.M. No relevant relationships. M.S.N. No relevant relationships. I.B. No relevant relationships. J.G. NSF and NIH grants; Nightingale Foundation grant; ACR AI Advisory group; SIIM director at large; HL7 board member; trainee editorial board member lead and associate editor for Radiology: Artificial Intelligence. H.T. Kheiron Medical Technologies, academic-industry collaboration; consultant for Arterys, Sirona Medical, and BioData Consortium; owner of Lightbox AI., (© 2023 by the Radiological Society of North America, Inc.)

Elevated total plasma homocysteine (hyperhomocysteinemia) is a marker of cardiovascular, thrombotic, and neuropsychological disease. It has multiple causes, including the common nutritional vitamin B12 or folate deficiency. However, some rare but treatable, inborn errors of metabolism (IEM) characterized by hyperhomocysteinemia can be missed due to variable presentations and the lack of awareness. The aim of this study is to identify undiagnosed IEM in adults with significantly elevated homocysteine using key existing clinical data points, then IEM specific treatment can be offered to improve outcome. We conducted a retrospective study with data mining and chart review of patients with plasma total homocysteine >30 μmol/L over a two-year period. We offer biochemical and genetic testing to patients with significant hyperhomocysteinemia without a clear explanation to diagnose IEM. We identified 22 subjects with significant hyperhomocysteinemia but no clear explanation. Subsequently, we offered genetic testing to seven patients and diagnosed one patient with classic homocystinuria due to cystathionine beta-synthase deficiency. With treatment, she lowered her plasma homocysteine and improved her health. This study stresses the importance of a thorough investigation of hyperhomocysteinemia in adults to identify rare but treatable IEM. We propose a metabolic evaluation algorithm for elevated homocysteine levels., (© 2022 Wiley Periodicals LLC.)

Background: The purpose of this study was to define the features of scapular morphology that are associated with changes in the critical shoulder angle (CSA) by developing the best predictive model for the CSA based on multiple potential explanatory variables, using a completely 3D assessment., Methods: 3D meshes were created from CT DICOMs using InVesalius (Vers 3.1.1, RTI [Renato Archer Information Technology Centre], Brazil) and Meshmixer (3.4.35, Autodesk Inc., San Rafael, CA). The analysis included 17 potential angular, weighted linear and area measurements. The correlation of the explanatory variables with the CSA was investigated with the Pearson's correlation coefficient. Using multivariable linear regression, the approach for predictive model-building was leave-one-out cross-validation and best subset selection., Results: Fifty-three meshes were analysed. Glenoid inclination (GI) and coronal plane angulation of the acromion (CPAA) [Pearson's r: 0.535; -0.502] correlated best with CSA. The best model (adjusted R-squared value 0.67) for CSA prediction contained 10 explanatory variables including glenoid, scapular spine and acromial factors. CPAA and GI were the most important based on their distribution, estimate of coefficients and loss in predictive power if removed., Conclusions: The relationship between scapular morphology and CSA is more complex than the concept of it being dictated solely by GI and acromial horizontal offset and includes glenoid, scapular spine and acromial factors of which CPAA and GI are most important. A further investigation in a closely defined cohort with rotator cuff tears is required before drawing any clinical conclusions about the role of surgical modification of scapular morphology., Level of Evidence: Level 4 retrospective observational cohort study with no comparison group., (© 2022. The Author(s).)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic, induces an unbalanced immune response in the host. For instance, the production of type I interferon (IFN) and the response to it, which act as a front-line defense against virus invasion, are inhibited during SARS-CoV-2 infection. In addition, tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine, is upregulated in COVID-19 patients with severe symptoms. Studies on the closely related betacoronavirus, SARS-CoV, showed that viral proteins such as Nsp1, Orf6 and nucleocapsid protein inhibit IFN-β production and responses at multiple steps. Given the conservation of these proteins between SARS-CoV and SARS-CoV-2, it is not surprising that SARS-CoV-2 deploys similar immune evasion strategies. Here, we carried out a screen to examine the role of individual SARS-CoV-2 proteins in regulating innate immune signaling, such as the activation of transcription factors IRF3 and NF-κB and the response to type I and type II IFN. In addition to established roles of SARS-CoV-2 proteins, we report that SARS-CoV-2 proteins Nsp6 and Orf8 inhibit the type I IFN response but at different stages. Orf6 blocks the translocation of STAT1 and STAT2 into the nucleus, whereas ORF8 inhibits the pathway in the nucleus after STAT1/2 translocation. SARS-CoV-2 Orf6 also suppresses IRF3 activation and TNF-α-induced NF-κB activation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lu, Michel, Wang and Smith.)

Vaccinia virus (VACV) protein N1 is an intracellular immunomodulator that contributes to virus virulence via inhibition of NF-κB. Intradermal infection with a VACV lacking gene N1L (vΔN1) results in smaller skin lesions than infection with wild-type virus (WT VACV), but the impact of N1 deletion on the local microbiota as well as the innate and cellular immune responses in infected ear tissue is mostly uncharacterized. Here, we analysed the bacterial burden and host immune response at the site of infection and report that the presence of protein N1 correlated with enhanced expansion of skin microbiota, even before lesion development. Furthermore, early after infection (days 1-3), prior to lesion development, the levels of inflammatory mediators were higher in vΔN1-infected tissue compared to WT VACV infection. In contrast, infiltration of ear tissue with myeloid and lymphoid cells was greater after WT VACV infection and there was significantly greater secondary bacterial infection that correlated with greater lesion size. We conclude that a more robust innate immune response to vΔN1 infection leads to better control of virus replication, less bacterial growth and hence an overall reduction of tissue damage and lesion size. This analysis shows the potent impact of a single viral immunomodulator on the host immune response and the pathophysiology of VACV infection in the skin.

Introduction: FLT3 internal tandem duplicate (ITD) is associated with unfavorable prognosis of acute myeloid leukemia; targeted therapy improves clinical outcome. We propose that FLT3-ITD detected by next generation sequencing (NGS) should be reported with the same nomenclature pattern as single nucleotide variants so that the mutation can be better interpreted clinically., Methods: A Python-based web application was developed to generate FLT3-ITD nomenclature as recommended by the Human Genome Variation Society (HGVS). Assembled FLT3-ITD sequences from 84 patients and 11 artificially created ITD sequences were used for the validation of this web-based application. Each sequence was inspected manually to confirm that the nomenclature was accurate., Results: Accurate nomenclatures were generated for 113 of 114 sequencing results and 7 artificial sequences. One assembled sequence and four artificial sequences were not named accurately; warning statements were automatically generated to alert further inspection. Of the 105 unique FLT3-ITDs, the ITD lengths range from 18 to 300 bp. Depending whether the ITD involves intron or extends into exon 15, three patterns were recognized. Only 44 (42%) ITDs were pure duplications, and three types of variants were identified at the 5' of ITD. When ITD involves intronic sequence, the protein may comprise inserted amino acids encoded by the intron, due to disrupted RNA splicing., Conclusion: The web application generates accurate FLT3-ITD nomenclature from NGS results except in rare situations. The HGVS nomenclatures provide information on the molecular architecture of FLT3-ITDs and reveal details of complex insertions with partial duplications., (© 2022 John Wiley & Sons Ltd.)

Vaccinia virus (VACV) encodes scores of proteins that suppress host innate immunity and many of these target intracellular signalling pathways leading to activation of inflammation. The transcription factor NF-κB plays a critical role in the host response to infection and is targeted by many viruses, including VACV that encodes 12 NF-κB inhibitors that interfere at different stages in this signalling pathway. Here we report that VACV proteins C2 and F3 are additional inhibitors of this pathway. C2 and F3 are BTB-Kelch proteins that are expressed early during infection, are non-essential for virus replication, but affect the outcome of infection in vivo . Using reporter gene assays, RT-qPCR analyses of endogenous gene expression, and ELISA, these BTB-Kelch proteins are shown here to diminish NF-κB activation by reducing translocation of p65 into the nucleus. C2 and F3 are the 13 th and 14 th NF-κB inhibitors encoded by VACV. Remarkably, in every case tested, these individual proteins affect virulence in vivo and therefore have non-redundant functions. Lastly, immunisation with a VACV strain lacking C2 induced a stronger CD8 + T cell response and better protection against virus challenge.

The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current therapies and known causes of familial hypercholesterolemia. We found that cold shock domain-containing protein E1 (CSDE1) enhanced hepatic LDLR messenger RNA (mRNA) decay via its 3' untranslated region and regulated atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens in a tissue culture model, we identified 40 specific regulators of the LDLR that were not previously identified by observational human genetic studies. Among these, we demonstrated that, in HepG2 cells, CSDE1 regulated the LDLR at least as strongly as statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. In addition, we showed that hepatic gene silencing of Csde1 treated diet-induced dyslipidemia in mice to a similar degree as Pcsk9 silencing. These results suggest the therapeutic potential of targeting CSDE1 to manipulate the posttranscriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. Our approach of modeling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, may serve as a generalizable template for the identification of therapeutic targets in other human disease states.

Introduction: This study compares an entirely patient reported modification of the Constant-Murley Score (CS) (the Multiple Assessment Subjective Shoulder Score [MASSS]), with the CS in symptomatic patients with non-instability shoulder pathology., Hypothesis: The hypothesis is that the sub scores and overall score of the MASSS will be well correlated and in high agreement to the corresponding scores of the CS., Materials and Methods: The MASSS, the CS and adjusted-CS (a-CS) were administered to 74 patients. The MASSS replaced the strength and ROM domains of the CS with SANE scores. Correlations, differences, agreement, test-retest reliability, and internal consistency were assessed using Pearson's R test, paired t-test, Bland-Altman method, intraclass correlations (ICC) and Cronbach's alpha., Results: Correlation between MASSS and CS (0.834) and a-CS (0.824) were excellent. Total MASSS (54.0) was higher than CS (41.8) (p<0.001) and a-CS (47.8) (p<0.05). MASSS strength and ROM domains were higher than those of the CS (Strength: 13.4 v 5.3 [p<0.001], ROM: 24.8 v 20.8 [p<0.001]. There was a floor effect for the strength domain of the CS (46.7% minimum score), but not the MASSS (0% minimum score). MASSS internal consistency (0.626-0.734) was better than the CS (0.401-0.643). Test re-test reliability of the total MASSS (ICC 0.93) and the strength (ICC 0.90) and ROM (ICC 0.86) domains was excellent., Discussion: The MASSS has several advantages over the CS. Although the correlation of the total MASSS with the CS and a-CS was high, there was poor agreement in the strength and the ROM domains and therefore the MASSS value is not interchangeable with the CS, but rather should be considered as an alternative., Level of Evidence: II; Prospective cohort study., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Introduction: Serum protein electrophoresis (SPEP) is commonly used to detect monoclonal paraproteins to meet laboratory diagnostic criteria for plasma cell neoplasms. We propose an automated screening method for paraprotein detection that uses minimal computational resources for training and deployment., Methods: A model screening for paraproteins based on the presence of high-frequency components in the spatial frequency spectrum of the SPEP densitometry curve was calibrated on a set of 330 samples, and evaluated on representative (n=110) and external (n=1,321) test sets. The model takes as input a patient's serum densitometry curve and a standardized control curve and outputs a prediction of whether a paraprotein is present. We built an interactive web application allowing users to easily perform paraprotein screening given inputs for densitometry curves, as well as a macro-enabled spreadsheet for easy automated screening., Results: When tuned to maximize likelihood ratio with minimum sensitivity 0.90, the model achieved AUC 0.90, sensitivity 0.90, positive-predictive value 0.64, specificity 0.55, and accuracy 0.72 in the representative test set. In the external test set, the model achieved AUC 0.90, sensitivity 0.97, positive-predictive value 0.42, specificity 0.29, and accuracy 0.52. A subset analysis showed sensitivities of 0.90, 0.96, and 1.0 in detecting low (0.1-0.5 g/dL), medium (0.5-3.0 g/dL), and high paraprotein levels (≥3.0 g/dL), respectively. We have released a web service allowing users to score their own SPEP data, and also released the algorithm and application programming interface in an open-source package allowing users to customize the model to their needs., Conclusions: We developed a proof of concept for an automated method for paraprotein screening using only the characteristics of the SPEP curve. Future work should focus on testing the method with other laboratory data including immunofixation gels, as well as incorporation of outside data sources including clinical data., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

The interaction between immune cells and virus-infected targets involves multiple plasma membrane (PM) proteins. A systematic study of PM protein modulation by vaccinia virus (VACV), the paradigm of host regulation, has the potential to reveal not only novel viral immune evasion mechanisms, but also novel factors critical in host immunity. Here, >1000 PM proteins were quantified throughout VACV infection, revealing selective downregulation of known T and NK cell ligands including HLA-C, downregulation of cytokine receptors including IFNAR2, IL-6ST and IL-10RB, and rapid inhibition of expression of certain protocadherins and ephrins, candidate activating immune ligands. Downregulation of most PM proteins occurred via a proteasome-independent mechanism. Upregulated proteins included a decoy receptor for TRAIL. Twenty VACV-encoded PM proteins were identified, of which five were not recognised previously as such. Collectively, this dataset constitutes a valuable resource for future studies on antiviral immunity, host-pathogen interaction, poxvirus biology, vector-based vaccine design and oncolytic therapy., Competing Interests: The authors have declared that no competing interests exist.

Background: In this comparative study we evaluate the performance of four software tools: DNAstar-D (DESeq2), DNAstar-E (edgeR), CLC Genomics and Partek Flow for identification of differentially expressed genes (DEGs) using a transcriptome of E. coli. The RNA-seq data are from the effect of below-background radiation 5.5 nGy total dose (0.2nGy/hr) on E. coli grown shielded from natural radiation 655 m below ground in a pre-World War II steel vault. The gene expression response to three supplemented sources of radiation designed to mimic natural background, 1952 - 5720 nGy in total dose (71-208 nGy/hr), are compared to this "radiation-deprived" treatment. In addition, RNA-seq data of Caenorhabditis elegans nematode from similar radiation treatments was analyzed by three of the software packages., Results: In E. coli, the four software programs identified one of the supplementary sources of radiation (KCl) to evoke about 5 times more transcribed genes than the minus-radiation treatment (69-114 differentially expressed genes, DEGs), and so the rest of the analyses used this KCl vs "Minus" comparison. After imposing a 30-read minimum cutoff, one of the DNAStar options shared two of the three steps (mapping, normalization, and statistic) with Partek Flow (they both used median of ratios to normalize and the DESeq2 statistical package), and these two programs identified the highest number of DEGs in common with each other (53). In contrast, when the programs used different approaches in each of the three steps, between 31 and 40 DEGs were found in common. Regarding the extent of expression differences, three of the four programs gave high fold-change results (15-178 fold), but one (DNAstar's DESeq2) resulted in more conservative fold-changes (1.5-3.5). In a parallel study comparing three qPCR commercial validation software programs, these programs also gave variable results as to which genes were significantly regulated. Similarly, the C. elegans analysis showed exaggerated fold-changes in CLC and DNAstar's edgeR while DNAstar-D was more conservative., Conclusions: Regarding the extent of expression (fold-change), and considering the subtlety of the very low level radiation treatments, in E. coli three of the four programs gave what we consider exaggerated fold-change results (15 - 178 fold), but one (DNAstar's DESeq2) gave more realistic fold-changes (1.5-3.5). When RT-qPCR validation comparisons to transcriptome results were carried out, they supported the more conservative DNAstar-D's expression results. When another model organism's (nematode) response to these radiation differences was similarly analyzed, DNAstar-D also resulted in the most conservative expression patterns. Therefore, we would propose DESeq2 ("DNAstar-D") as an appropriate software tool for differential gene expression studies for treatments expected to give subtle transcriptome responses., (© 2022. The Author(s).)

Background: Oncotype DX Recurrence Score (RS) has been widely used to predict chemotherapy benefits in patients with estrogen receptor-positive breast cancer. Studies showed that the features used in Magee equations correlate with RS. We aimed to examine whether deep learning (DL)-based histology image analyses can enhance such correlations., Methods: We retrieved 382 cases with RS diagnosed between 2011 and 2015 from the Emory University and the Ohio State University. All patients received surgery. DL models were developed to detect nuclei of tumor cells and tumor-infiltrating lymphocytes (TILs) and segment tumor cell nuclei in hematoxylin and eosin (H&E) stained histopathology whole slide images (WSIs). Based on the DL-based analysis, we derived image features from WSIs, such as tumor cell number, TIL number variance, and nuclear grades. The entire patient cohorts were divided into one training set (125 cases) and two validation sets (82 and 175 cases) based on the data sources and WSI resolutions. The training set was used to train the linear regression models to predict RS. For prediction performance comparison, we used independent variables from Magee features alone or the combination of WSI-derived image and Magee features., Results: The Pearson's correlation coefficients between the actual RS and predicted RS by DL-based analysis were 0.7058 ( p -value = 1.32 × 10 -13 ) and 0.5041 ( p -value = 1.15 × 10 -12 ) for the validation sets 1 and 2, respectively. The adjusted R 2 values using Magee features alone are 0.3442 and 0.2167 in the two validation sets, respectively. In contrast, the adjusted R 2 values were enhanced to 0.4431 and 0.2182 when WSI-derived imaging features were jointly used with Magee features., Conclusion: Our results suggest that DL-based digital pathological features can enhance Magee feature correlation with RS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Wang, Li, Dababneh, Wang, Zhao, Smith, Teodoro, Li, Kong and Li.)

Poxvirus proteins remodel signaling throughout the cell by targeting host enzymes for inhibition and redirection. Recently, it was discovered that early in infection the vaccinia virus (VACV) B12 pseudokinase copurifies with the cellular kinase VRK1, a proviral factor, in the nucleus. Although the formation of this complex correlates with inhibition of cytoplasmic VACV DNA replication and likely has other downstream signaling consequences, the molecular mechanisms involved are poorly understood. Here, we further characterize how B12 and VRK1 regulate one another during poxvirus infection. First, we demonstrate that B12 is stabilized in the presence of VRK1 and that VRK1 and B12 coinfluence their respective solubility and subcellular localization. In this regard, we find that B12 promotes VRK1 colocalization with cellular DNA during mitosis and that B12 and VRK1 may be tethered cooperatively to chromatin. Next, we observe that the C-terminal tail of VRK1 is unnecessary for B12-VRK1 complex formation or its proviral activity. Interestingly, we identify a point mutation of B12 capable of abrogating interaction with VRK1 and which renders B12 nonrepressive during infection. Lastly, we investigated the influence of B12 on the host factor BAF and antiviral signaling pathways and find that B12 triggers redistribution of BAF from the cytoplasm to the nucleus. In addition, B12 increases DNA-induced innate immune signaling, revealing a new functional consequence of the B12 pseudokinase. Together, this study characterizes the multifaceted roles B12 plays during poxvirus infection that impact VRK1, BAF, and innate immune signaling. IMPORTANCE Protein pseudokinases comprise a considerable fraction of the human kinome, as well as other forms of life. Recent studies have demonstrated that their lack of key catalytic residues compared to their kinase counterparts does not negate their ability to intersect with molecular signal transduction. While the multifaceted roles pseudokinases can play are known, their contribution to virus infection remains understudied. Here, we further characterize the mechanism of how the VACV B12 pseudokinase and human VRK1 kinase regulate one another in the nucleus during poxvirus infection and inhibit VACV DNA replication. We find that B12 disrupts regulation of VRK1 and its downstream target BAF, while also enhancing DNA-dependent innate immune signaling. Combined with previous data, these studies contribute to the growing field of nuclear pathways targeted by poxviruses and provide evidence of unexplored roles of B12 in the activation of antiviral immunity.

Purpose: To compare the axial plane orientation and width and length of the acromial resections required to reduce the critical shoulder angle (CSA) using lateral acromioplasty (LA) techniques that are based on the lateral acromial border with an ideal resection that is oriented parallel to the glenoid., Methods: This was a retrospective observational cohort study of symptomatic patients that were investigated for shoulder pain, instability, or fracture with high-quality computed tomography (CT). The CT scan data were used to create 3-dimensional meshes, and a series of LA resection planes were mapped. The orientation, width, and length of each resection based on the lateral acromial border (lateral, anterolateral, posterolateral, and image guided) to reduce the measured CSA to 35° or 30° was compared with an ideal resection that was oriented parallel to the glenoid., Results: 23 models had CSA 30.1° to 35°, and 13 had CSA >35°. In the models with CSA >35°, there was no angular difference between the resection planes of the lateral, anterolateral, or image-guided resections compared with the ideal technique; there were differences in the required width and length of the resections to reduce the CSA to 35° (additional width/length: lateral, 3.2/14.8 mm; anterolateral, 2.8/10.6 mm; posterolateral, 6.9/19.2 mm; image guided, 2.4/10.3 mm). Width and length differences were also present in the models with CSA >30° when the resections aimed to reduce the CSA to 30° (additional width/length: lateral, 2.5/12.5 mm; anterolateral, 1.9/8.8 mm; posterolateral, 7.4/19.0 mm; image guided, 1.6/8.8 mm)., Conclusions: LA techniques based on the lateral acromial border did not replicate the ideal resection and may lead to excessive deltoid release which could adversely affect clinical results., Clinical Relevance: Our findings do not support LA techniques based on the lateral acromial border., (© 2022 The Authors.)