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Neutrophils are the first line of defense of the innate immune system. In response to methicillin-resistant Staphylococcus aureus infection in the skin, hematopoietic stem, and progenitor cells (HSPCs) traffic to wounds and undergo extramedullary granulopoiesis, producing neutrophils necessary to resolve the infection. This prompted the engineering of a gelatin methacrylate (GelMA) hydrogel that encapsulates HSPCs within a matrix amenable to subcutaneous delivery. The authors study the influence of hydrogel mechanical properties to produce an artificial niche for granulocyte-monocyte progenitors (GMPs) to efficiently expand into functional neutrophils that can populate infected tissue. Lin-cKIT+ HSPCs, harvested from fluorescent neutrophil reporter mice, are encapsulated in GelMA hydrogels of varying polymer concentration and UV-crosslinked to produce HSPC-laden gels of specific stiffness and mesh sizes. Softer 5% GelMA gels yield the most viable progenitors and effective cell-matrix interactions. Compared to suspension culture, 5% GelMA results in a twofold expansion of mature neutrophils that retain antimicrobial functions including degranulation, phagocytosis, and ROS production. When implanted dermally in C57BL/6J mice, luciferase-expressing neutrophils expanded in GelMA hydrogels are visualized at the site of implantation for over 5 days. They demonstrate the potential of GelMA hydrogels for delivering HSPCs directly to the site of skin infection to promote local granulopoiesis.

Recognition of integrins by CD62P has not been reported and this motivated a docking simulation using integrin αvβ3 as a target. We predicted that the C-type lectin domain of CD62P functions as a potential integrin ligand and observed that it specifically bound to soluble β3 and β1 integrins. Known inhibitors of the interaction between CD62P-PSGL-1 did not suppress the binding, whereas the disintegrin domain of ADAM-15, a known integrin ligand, suppressed recognition by the lectin domain. Furthermore, an R16E/K17E mutation in the predicted integrin-binding interface located outside of the glycan-binding site within the lectin domain, strongly inhibited CD62P binding to integrins. In contrast, the E88D mutation that strongly disrupts glycan binding only slightly affected CD62P-integrin recognition, indicating that the glycan and integrin-binding sites are distinct. Notably, the lectin domain allosterically activated integrins by binding to the allosteric site 2. We conclude that CD62P-integrin binding may function to promote a diverse set of cell-cell adhesive interactions given that β3 and β1 integrins are more widely expressed than PSGL-1 that is limited to leukocytes.

AbstractThe American Institute for Medical and Biological Engineering (AIMBE) hosted a virtual symposium titled “Diversifying Paths to Academic Leadership” on January 27 and 28, 2022. The symposium sought to educate the community on the opportunities for and impact of leadership by biomedical engineering faculty, to encourage and invite women faculty, especially women of color, to consider and prepare to pursue leadership roles, to educate faculty on the expectations and duties of these roles, and to highlight experiences and paths to leadership of women engineering leaders. Here we review the main outcomes of the symposium to provide perspective on (1) personal visioning and positioning for leadership, (2) negotiating for success in leadership positions, and (3) leadership strategies for success specific to women faculty and where applicable, faculty of color.

Abstract Background Tumor-associated macrophages (TAMs) are a prominent immune subpopulation in the tumor microenvironment that could potentially serve as therapeutic targets for breast cancer. Thus, it is important to characterize this cell population across different tumor subtypes including patterns of association with demographic and prognostic factors, and breast cancer outcomes. Methods We investigated CD163+ macrophages in relation to clinicopathologic variables and breast cancer outcomes in the Women’s Circle of Health Study and Women’s Circle of Health Follow-up Study populations of predominantly Black women with breast cancer. We evaluated 611 invasive breast tumor samples (507 from Black women, 104 from White women) with immunohistochemical staining of tissue microarray slides followed by digital image analysis. Multivariable Cox proportional hazards models were used to estimate hazard ratios for overall survival (OS) and breast cancer-specific survival (BCSS) for 546 cases with available survival data (median follow-up time 9.68 years (IQR: 7.43–12.33). Results Women with triple-negative breast cancer showed significantly improved OS in relation to increased levels of tumor-infiltrating CD163+ macrophages in age-adjusted (Q3 vs. Q1: HR = 0.36; 95% CI 0.16–0.83) and fully adjusted models (Q3 vs. Q1: HR = 0.30; 95% CI 0.12–0.73). A similar, but non-statistically significant, association was observed for BCSS. Macrophage infiltration in luminal and HER2+ tumors was not associated with OS or BCSS. In a multivariate regression model that adjusted for age, subtype, grade, and tumor size, there was no significant difference in CD163+ macrophage density between Black and White women (RR = 0.88; 95% CI 0.71–1.10). Conclusions In contrast to previous studies, we observed that higher densities of CD163+ macrophages are independently associated with improved OS and BCSS in women with invasive triple-negative breast cancer. Trial registration Not applicable.

Abstract Ecological analyses typically involve many interacting variables. Ecologists often specify lagged interactions in community dynamics (i.e. vector‐autoregressive models) or simultaneous interactions (e.g. structural equation models), but there is less familiarity with dynamic structural equation models (DSEM) that can include any simultaneous or lagged effect in multivariate time‐series analysis. We propose a novel approach to parameter estimation for DSEM, which involves constructing a Gaussian Markov random field (GMRF) representing simultaneous and lagged path coefficients, and then fitting this as a generalized linear mixed model to missing and/or non‐normal data. We provide a new R‐package dsem, which extends the ‘arrow interface’ from path analysis to represent user‐specified lags when constructing the GMRF. We also outline how the resulting nonseparable precision matrix can generalize existing separable models, for example, for time‐series and species interactions in a vector‐autoregressive model. We first demonstrate dsem by simulating a two‐species vector‐autoregressive model based on wolf–moose interactions on Isle Royale. We show that DSEM has improved precision when data are missing relative to a conventional dynamic linear model. We then demonstrate DSEM via two contrasting case studies. The first identifies a trophic cascade where decreased sunflower starfish has increased urchin and decreased kelp densities, while sea otters have a simultaneous positive effect on kelp in the California Current from 1999 to 2018. The second estimates how declining sea ice has decreased cold‐water habitats, driving a decreased density for fall copepod predation and inhibiting early‐life survival for Alaska pollock from 1963 to 2023. We conclude that DSEM can be fitted efficiently as a GLMM involving missing data, while allowing users to specify both simultaneous and lagged effects in a time‐series structural model. DSEM then allows conceptual models (developed with stakeholder input or from ecological expertise) to be fitted to incomplete time series and provides a simple interface for granular control over the number of estimated time‐series parameters. Finally, computational methods are sufficiently simple that DSEM can be embedded as component within larger (e.g. integrated population) models. We therefore recommend greater exploration and performance testing for DSEM relative to familiar time‐series forecasting methods.

Abstract Triple-negative breast cancer (TNBC) is one of the most aggressive types of cancer. Despite decades of intense investigation, treatment options remain limited, and rapid recurrence with distant metastases remains a significant challenge. Cancer cell-intrinsic production of cytokines such as type I interferons (IFN-I) is a known potent modulator of response to therapy in many cancers, including TNBC, and can influence therapeutic outcome. Here, we report that, in TNBC systems, the aryl hydrocarbon receptor (AhR) suppresses IFN-I expression via inhibition of STImulator of Interferon Genes (STING), a key mediator of interferon production. Intratumoral STING activity is essential in mediating the efficacy of PARP inhibitors (PARPi) which are used in the treatment of cancers harboring BRCA1 deficiency. We find that, in TNBC cells, PARPi treatment activates AhR in a BRCA1 deficiency-dependent manner, thus suggesting the presence of a negative feedback loop aimed at modulating PARPi efficacy. Importantly, our results indicate that the combined inhibition of PARP and AhR is superior in elevating IFN-I expression as compared to PARPi-alone. Thus, AhR inhibition may allow for enhanced IFN-I production upon PARPi in BRCA1-deficient breast cancers, most of which are of TNBC origin, and may represent a therapeutically viable strategy to enhance PARPi efficacy.

Diabetes mellitus is a chronic disease characterized by metabolic dysregulation which is frequently associated with diabetic foot ulcers that result from a severely compromised innate immune system. The high levels of blood glucose characteristic of diabetes cause an increase in circulating inflammatory mediators, which accelerate cellular senescence and dampen antimicrobial activity within dermal tissue. In diabetic wounds, bacteria and fungi proliferate in a protective biofilm forming a structure that a compromised host defense system cannot easily penetrate, often resulting in chronic infections that require antimicrobial intervention to promote the healing process. The designed host defense peptide (dHDP) RP557 is a synthesized peptide whose sequence has been derived from naturally occurring antimicrobial peptides (AMPs) that provide the first line of defense against invading pathogens. AMPs possess an amphipathic α-helix or β-sheet structure and a net positive charge that enables them to incorporate into pathogen membranes and perturb the barrier function of Gram-positive and Gram-negative bacteria along with fungi. The capacity of skin to resist infections is largely dependent upon the activity of endogenous AMPs that provided the basis for the design and testing of RP557 for the resolution of wound infections. In the current study, the topical application of RP557 stopped bacterial growth in the biofilm of methicillin-resistant Staphylococcus aureus (MRSA) USA300 infected wounds on the flanks of clinically relevant diabetic TALLYHO mice. Topical application of RP557 reduced bacterial load and accelerated wound closure, while wound size in control diabetic mice continued to expand. These studies demonstrate that RP557 reduces or eliminates an infection in its biofilm and restores wound-healing capacity.

IntroductionResearch is needed to understand the frequency of e-cigarette impressions in scripted television and movies, especially in scripted content with characters and storylines that may appeal to young adults. This study aimed to determine the extent of e-cigarette-related imagery and dialogue in Netflix content popular with young adults. We also determine the demographics and character qualities of actors shown holding e-cigarettes.MethodsNielsen ratings data were used to compile a list of the most popular Netflix original films and TV shows among U.S. viewers 18-24 years old between June 1, 2020, and May 31, 2021. We used a sample of 12 films and 113 TV episodes from 12 series. Three coders were trained to analyze a total of 101 h of content for the presence of e-cigarettes, level of use, type of characters holding e-cigarettes, brand visibility, and the presence of vaping-related dialogue. Twenty percent of all episodes/films were double coded to ensure reliability.ResultsOut of 125 titles, 16 (13%) had e-cigarette-related content. Thirteen titles (10%) showed at least one character holding an e-cigarette, and three others mentioned vaping without showing e-cigarettes. The total time of e-cigarettes onscreen amounted to 399 s and the average screen time for e-cigarettes was 31 s. Ninety-nine percent of the time an e-cigarette appeared on screen it was being held by a character.ConclusionThis study documented recent e-cigarette imagery found on Netflix and demonstrates the need for health communication campaigns to denormalize e-cigarette use, particularly among susceptible populations, such as young adults.

IntroductionSpinal cord injury (SCI) often leads to neuropathic pain that negatively affects quality of life. Several qualitative research studies in individuals with SCI who experience neuropathic pain indicate the lack of adequate information about pain. We previously developed an educational resource, the SeePain, based on scientific literature and a series of qualitative interviews of people with SCI, their significant others/family members, and SCI healthcare providers.MethodsHowever, to quantitatively evaluate the utility of this educational resource in a larger sample, we examined the agreement and usefulness ratings of statements regarding clarity/comprehensibility, content, and format of the SeePain, derived from the thematic analysis of our previous qualitative interviews. Participants completed a survey that provided a digital version of the SeePain and then rated their agreement/usefulness with the statements using numerical rating scales.ResultsThere were overall high perceived agreement and usefulness ratings regarding the SeePain’s clarity, content, and format. A factor analysis reduced the agreement and usefulness ratings into 4 components (content, clarity, format, and delivery medium). Group comparisons showed that individuals with higher education were more likely to endorse electronic and website formats, and the usefulness of a shorter version of the SeePain; females and younger individuals showed greater endorsement for clarity. Finally, higher pain intensity ratings were associated with greater agreement and usefulness of the content of the SeePain.DiscussionOverall, these results support the utility of the SeePain as a source of information regarding pain that may facilitate communication about pain and its management following SCI.

ImportanceExposure to tobacco-related content on social media may foster positive attitudes toward tobacco products and brands, and influence the likelihood of initiating or continuing use of tobacco, especially among adolescents and young adults.ObjectiveTo perform the first systematic review and meta-analysis, to our knowledge, on studies that examined the association between exposure to tobacco content on social media and lifetime tobacco use, past 30-day tobacco use, and susceptibility to use tobacco among never users.Data sourcesTobacco, social media, and marketing search terms were entered into online databases, including MEDLINE, ISI Web of Science, Scopus, and PsychINFO. Study characteristics, including research design and methods, sampling strategy, and demographics, were assessed for each study.Study selectionStudies reporting odds ratios (ORs) for self-reported exposure to, or experimentally manipulated, tobacco content on social media and lifetime tobacco use, past 30-day tobacco, and susceptibility to use tobacco among never users. The systematic search produced 897 independent articles, of which 29 studies met inclusion criteria.Data extraction and synthesisA 3-level random-effects meta-analysis was used to estimate ORs, 95% CIs, and heterogeneity (I2) for each tobacco use outcome. Study quality and publication bias were assessed.Main outcomes and measuresLifetime tobacco use, past 30-day tobacco use, and susceptibility to use tobacco among never users. Tobacco use included e-cigarettes, cigarettes, and other (cigar, hookah, smokeless tobacco).ResultsThe total sample size across the 24 included datasets was 139 624, including 100 666 adolescents (72%), 20 710 young adults (15%), and 18 248 adults (13%). Participants who were exposed to tobacco content on social media, compared with those who were not exposed, had greater odds of reporting lifetime tobacco use (OR, 2.18; 95% CI, 1.54-3.08; I2 = 94%), past 30-day tobacco use (OR, 2.19; 95% CI, 1.79-2.67; I2 = 84%), and susceptibility to use tobacco among never users (OR, 2.08; 95% CI, 1.65-2.63; I2 = 73%). Subgroup analyses showed similar associations for tobacco promotions, active engagement, passive engagement, lifetime exposure to tobacco content, exposure to tobacco content on more than 2 platforms, and exposure to tobacco content among adolescents and young adults.Conclusions and relevanceFindings suggest that a comprehensive strategy to reduce the amount of tobacco content on social media should be developed by federal regulators. Such actions may have downstream effects on adolescent and young adult exposure to protobacco content, and ultimately tobacco use behaviors.

Clinically used inhibitors of mammalian target of rapamycin (mTOR) negatively impacts endothelial-dependent vasodilatation (EDD) through unidentified mechanisms. Here we show that either the endothelium-specific deletion of Mtor to inhibit both mTOR complexes, or depletion of Raptor or Rictor to disrupt mTORC1 or mTORC2, causes impaired EDD, accompanied by reduced NO in the serum of mice. Consistently, inhibition of mTOR decreases NO production by human and mouse EC. Specifically, inhibition of mTORC1 suppresses eNOS gene expression, due to impairment in p70S6K-mediated posttranscriptional regulation of the transcription factor KLF2 expression. In contrast to mTORC1 inhibition, a positive-feedback between MAPK (p38 and JNK) activation and Nox2 upregulation contributes to the excessive generation of reactive oxygen species (ROS), which causes eNOS uncoupling and decreased NO bioavailability in mTORC2-inhibited EC. Adeno-associated virus-mediated EC-specific overexpression of KLF2 or suppression of Nox2 restores EDD function in endothelial mTORC1- or mTORC2-inhibited mice.

Changes in the microbiota composition are associated with many human diseases, but factors that govern strain abundance remain poorly defined. We show that a commensal Escherichia coli strain and a pathogenic Salmonella enterica serovar Typhimurium isolate both utilize nitrate for intestinal growth, but each accesses this resource in a distinct biogeographical niche. Commensal E. coli utilizes epithelial-derived nitrate, whereas nitrate in the niche occupied by S. Typhimurium is derived from phagocytic infiltrates. Surprisingly, avirulent S. Typhimurium was shown to be unable to utilize epithelial-derived nitrate because its chemotaxis receptors McpB and McpC exclude the pathogen from the niche occupied by E. coli. In contrast, E. coli invades the niche constructed by S. Typhimurium virulence factors and confers colonization resistance by competing for nitrate. Thus, nutrient niches are not defined solely by critical resources, but they can be further subdivided biogeographically within the host into distinct microhabitats, thereby generating new niche opportunities for distinct bacterial species.

Summary: Tissue-resident macrophages (TRMs) are abundant immune cells within pre-metastatic sites, yet their functional contributions to metastasis remain incompletely understood. Here, we show that alveolar macrophages (AMs), the main TRMs of the lung, are susceptible to downregulation of the immune stimulatory transcription factor IRF8, impairing anti-metastatic activity in models of metastatic breast cancer. G-CSF is a key tumor-associated factor (TAF) that acts upon AMs to reduce IRF8 levels and facilitate metastasis. Translational relevance of IRF8 downregulation was observed among macrophage precursors in breast cancer and a CD68hiIRF8loG-CSFhi gene signature suggests poorer prognosis in triple-negative breast cancer (TNBC), a G-CSF-expressing subtype. Our data highlight the underappreciated, pro-metastatic roles of AMs in response to G-CSF and identify the contribution of IRF8-deficient AMs to metastatic burden. AMs are an attractive target of local neoadjuvant G-CSF blockade to recover anti-metastatic activity.

BackgroundThe cannabis product and regulatory landscape is changing in the United States. Against the backdrop of these changes, there have been increasing reports on health-related motives for cannabis use and adverse events from its use. The use of social media data in monitoring cannabis-related health conversations may be useful to state- and federal-level regulatory agencies as they grapple with identifying cannabis safety signals in a comprehensive and scalable fashion.ObjectiveThis study attempted to determine the extent to which a medical dictionary-the Unified Medical Language System Consumer Health Vocabulary-could identify cannabis-related motivations for use and health consequences of cannabis use based on Twitter posts in 2020.MethodsTwitter posts containing cannabis-related terms were obtained from January 1 to August 31, 2020. Each post from the sample (N=353,353) was classified into at least 1 of 17 a priori categories of common health-related topics by using a rule-based classifier. Each category was defined by the terms in the medical dictionary. A subsample of posts (n=1092) was then manually annotated to help validate the rule-based classifier and determine if each post pertained to health-related motivations for cannabis use, perceived adverse health effects from its use, or neither.ResultsThe validation process indicated that the medical dictionary could identify health-related conversations in 31.2% (341/1092) of posts. Specifically, 20.4% (223/1092) of posts were accurately identified as posts related to a health-related motivation for cannabis use, while 10.8% (118/1092) of posts were accurately identified as posts related to a health-related consequence from cannabis use. The health-related conversations about cannabis use included those about issues with the respiratory system, stress to the immune system, and gastrointestinal issues, among others.ConclusionsThe mining of social media data may prove helpful in improving the surveillance of cannabis products and their adverse health effects. However, future research needs to develop and validate a dictionary and codebook that capture cannabis use-specific health conversations on Twitter.

Despite the discovery of several closely related viruses in bats, the direct evolutionary progenitor of SARS-CoV-2 has not yet been identified. In this study, we investigated potential animal sources of SARS-related coronaviruses using archived specimens from Sunda pangolins (Manis javanica) and Chinese pangolins (Manis pentadactyla) confiscated from the illegal wildlife trade, and from common palm civets (Paradoxurus hermaphroditus) raised on wildlife farms in Viet Nam. A total of 696 pangolin and civet specimens were screened for the presence of viral RNA from five zoonotic viral families and from Sarbecoviruses using primers specifically designed for pangolin coronaviruses. We also performed a curated data collection of media reports of wildlife confiscation events involving pangolins in Viet Nam between January 2016 and December 2020, to illustrate the global pangolin supply chain in the context of Viet Nam where the trade confiscated pangolins were sampled for this study. All specimens from pangolins and civets sampled along the wildlife supply chains between February 2017 and July 2018, in Viet Nam and tested with conventional PCR assays designed to detect flavivirus, paramyxovirus, filovirus, coronavirus, and orthomyxovirus RNA were negative. Civet samples were also negative for Sarbecoviruses, but 12 specimens from seven live pangolins confiscated in Hung Yen province, northern Viet Nam, in 2018 were positive for Sarbecoviruses. Our phylogenetic trees based on two fragments of the RdRp gene revealed that the Sarbecoviruses identified in these pangolins were closely related to pangolin coronaviruses detected in pangolins confiscated from the illegal wildlife trade in Yunnan and Guangxi provinces, China. Our curated data collection of media reports of wildlife confiscation events involving pangolins in Viet Nam between January 2016 and December 2020, reflected what is known about pangolin trafficking globally. Pangolins confiscated in Viet Nam were largely in transit, moving toward downstream consumers in China. Confiscations included pangolin scales sourced originally from Africa (and African species of pangolins), or pangolin carcasses and live pangolins native to Southeast Asia (predominately the Sunda pangolin) sourced from neighboring range countries and moving through Viet Nam toward provinces bordering China.

Homozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the transfer RNA (tRNA) splicing endonuclease complex and the cleavage and polyadenylation machinery, but its function remains unclear. We generated two mouse models of PCH10: one homozygous for the disease-associated Clp1 mutation, R140H, and one heterozygous for this mutation and a null allele. Both models exhibit loss of lower motor neurons and neurons of the deep cerebellar nuclei. To explore whether Clp1 mutation impacts tRNA splicing, we profiled the products of intron-containing tRNA genes. While mature tRNAs were expressed at normal levels in mutant mice, numerous other products of intron-containing tRNA genes were dysregulated, with pre-tRNAs, introns, and certain tRNA fragments up-regulated, and other fragments down-regulated. However, the spatiotemporal patterns of dysregulation do not correlate with pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on precursor messenger RNA (pre-mRNA) cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1R140H/- spinal cord. PAS usage was shifted from proximal to distal sites in the mutant mouse, particularly in short and closely spaced genes. Many such genes were also expressed at lower levels in the Clp1R140H/- mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that impaired mRNA 3' processing may contribute to pathogenesis in PCH10.

Approximately 60% of individuals with a spinal cord injury (SCI) experience neuropathic pain, which often persists despite the use of various pharmacological treatments. Increasingly, the potential analgesic effects of cannabis and cannabinoid products have been studied; however, little research has been conducted among those with SCI-related neuropathic pain. Therefore, the primary objective of the study was to investigate the perceived effects of cannabis and cannabinoid use on neuropathic pain among those who were currently or had previously used these approaches. Additionally, the study aimed to determine if common pain medications are being substituted by cannabis and cannabinoids. Participants (N = 342) were recruited from existing opt-in listserv sources within the United States. Of those, 227 met the inclusion criteria and were enrolled in the study. The participants took part in an anonymous online survey regarding past and current use of cannabis and their perceived effects on neuropathic pain, including the use of pain medication. Those in the sample reported average neuropathic pain intensity scores over the past week of 6.8 ± 2.1 (0 to 10 scale), reflecting a high moderate to severe level of pain. Additionally, 87.9% noted that cannabis reduced their neuropathic pain intensity by more than 30%, and 92.3% reported that cannabis helped them to better deal with their neuropathic pain symptoms. Most participants (83.3%) also reported substituting their pain medications with cannabis, with the most substituted medication categories being opioids (47.0%), gabapentinoids (42.8%) and over-the-counter pain medications (42.2%). These preliminary results suggest that cannabis and cannabinoids may be effective in reducing neuropathic pain among those with SCI and may help to limit the need for certain pain medications.

Translation-dependent quality control pathways such as no-go decay (NGD), non-stop decay (NSD), and nonsense-mediated decay (NMD) govern protein synthesis and proteostasis by resolving non-translating ribosomes and preventing the production of potentially toxic peptides derived from faulty and aberrant mRNAs. However, how translation is altered and the in vivo defects that arise in the absence of these pathways are poorly understood. Here, we show that the NGD/NSD factors Pelo and Hbs1l are critical in mice for cerebellar neurogenesis but expendable for survival of these neurons after development. Analysis of mutant mouse embryonic fibroblasts revealed translational pauses, alteration of signaling pathways, and translational reprogramming. Similar effects on signaling pathways, including mTOR activation, the translatome and mouse cerebellar development were observed upon deletion of the NMD factor Upf2. Our data reveal that these quality control pathways that function to mitigate errors at distinct steps in translation can evoke similar cellular responses.

Extremity reconstruction surgery is increasingly performed rather than amputation for patients with large-segment pathologic bone loss. Debate persists as to the optimal void filler for this "limb salvage" surgery, whether metal or allograft bone. Clinicians focus on optimizing important functional gains for patients, and the risk of devastating implant infection has been thought to be similar regardless of implant material. Recent insights into infection pathophysiology are challenging this equipoise, however, with both basic science data suggesting a novel mechanism of infection of Staphylococcus aureus (the most common infecting agent) into the host lacunar-canaliculi network, and also clinical data revealing a higher rate of infection of allograft over metal. The current translational study was therefore developed to bridge the gap between these insights in a longitudinal murine model of infection of allograft bone and metal. Real-time Staphylococci infection characteristics were quantified in cortical bone vs metal, and both microarchitecture of host implant and presence of host immune response were assessed. An orders-of-magnitude higher bacterial burden was established in cortical allograft bone over both metal and cancellous bone. The establishment of immune-evading microabscesses was confirmed in both cortical allograft haversian canal and the submicron canaliculi network in an additional model of mouse femur bone infection. These study results reveal a mechanism by which Staphylococci evasion of host immunity is possible, contributing to elevated risks of infection in cortical bone. The presence of this local infection reservoir imparts massive clinical implications that may alter the current paradigm of osteomyelitis and bulk allograft infection treatment.

Background Heavy menstrual bleeding affects one in four women and negatively impacts quality of life. The levonorgestrel-releasing intrauterine system is an effective long-term treatment but is discontinued by many due to unpredictable bleeding, or adverse effects. The selective progesterone receptor modulator ulipristal acetate is used to treat symptomatic fibroids but long-term efficacy for the symptom of heavy menstrual bleeding, irrespective of presence of fibroids, is unknown. Objectives To determine whether ulipristal acetate is more effective at reducing the burden of heavy menstrual bleeding than levonorgestrel-releasing intrauterine system after 12 months of treatment in women with and without fibroids. We investigated mechanism of action of ulipristal acetate in a subset of 20 women. Design Randomised, open-label, parallel group, multicentre trial with embedded mechanistic study. Setting Ten UK hospitals. Participants Women with heavy menstrual bleeding aged 18 and over with no contraindications to levonorgestrel-releasing intrauterine system or ulipristal acetate. Interventions Three 12-week treatment cycles of 5 mg ulipristal acetate daily, separated by 4-week treatment-free intervals, or continuous levonorgestrel-releasing intrauterine system following allocation in a 1 : 1 ratio using a web-based minimisation procedure. Main trial outcome measures Primary outcome was quality-of-life measured by menorrhagia multi-attribute scale at 12 months. Secondary outcomes included menstrual bleeding and patient satisfaction. Impact on fibroid size, endometrial appearance and liver function was also collected. Mechanistic study outcome Cellular markers for endometrial cell structure and function, determined from endometrial biopsies; volume of uterus and fibroids and microcirculation parameters were determined from magnetic resonance images. Results Sample size was increased from 220 to 302 as a result of temporary halt to recruitment due to concerns of ulipristal acetate hepatoxicity. Subsequent withdrawal of ulipristal acetate and the COVID-19 pandemic led to a premature closure of recruitment, with 118 women randomised to each treatment and 103 women completing 12-month menorrhagia multi-attribute scale scores prior to this point. Primary outcome scores substantially improved in both arms, but at 12 months there was no evidence of a difference between those receiving three cycles of ulipristal acetate [median score category: 76–99, interquartile range (51–75 to 100), n = 53] and levonorgestrel-releasing intrauterine system [median score category: 76–99, interquartile range (51–75 to 100), n = 50; adjusted odds ratio 0.55, 95% confidence interval 0.26 to 1.17; p = 0.12]. Rates of amenorrhoea were much higher in those allocated ulipristal acetate compared with the levonorgestrel-releasing intrauterine system (12 months: 64% vs. 25%, adjusted odds ratio 7.12, 95% confidence interval 2.29 to 22.2). There was no evidence of a difference in other participant-reported outcomes. There were no cases of endometrial malignancy and no hepatotoxicity due to ulipristal acetate use. Mechanistic study results Ulipristal acetate produced a reversible reduction in endometrial cell proliferation, as well as reversible alteration of other endometrial cellular markers. Ulipristal acetate did not produce a reduction in the volume of the uterus irrespective of coexisting fibroids, nor an effect on uterine microvascular blood flow. Limitations The urgent safety measures and premature closure of recruitment impacted final sample size. Conclusions We found no evidence of a difference in quality of life between the two treatments, but ulipristal acetate was superior to levonorgestrel-releasing intrauterine system at inducing amenorrhoea. Ulipristal acetate currently has restricted availability due to concerns regarding hepatotoxicity. Future work There is a need to develop new, safe, effective and fertility-sparing medical treatments for heavy menstrual bleeding. The observed acceptability and effectiveness of ulipristal acetate warrants further research into the selective progesterone receptor modulator class of pharmacological agents. Study registration This trial is registered as ISRCTN 20426843. Plain language summary What is the problem? Heavy menstrual bleeding is a common condition that affects the lives of many women. A hormone-releasing coil, fitted inside the womb, is effective in making periods lighter but can make them less regular. A medicine called ulipristal acetate or UPA, taken as a pill, has been shown to reduce rapidly menstrual bleeding in women with large, non-cancerous tumours in the womb, known as fibroids. It was not known whether UPA is effective in women who have heavy periods but do not have fibroids of any significant size. What did we plan to do? To find out which treatment was better at controlling heavy periods, 236 women were enrolled in a clinical trial where they received either the hormone coil or UPA. The choice of treatment was made at random by a computer rather than the wishes of researchers or patients, to ensure a fair comparison. Participants completed questionnaires about their symptoms and life quality at intervals up to 1 year. Twice during the trial, medicines regulatory authorities raised safety concerns about UPA causing liver problems. This resulted in the introduction of regular blood tests. The second time, recruitment to the trial stopped early. What did we find? Both treatments improved the symptoms of heavy menstrual bleeding in the majority of women. We found no evidence that UPA was better overall after 1 year of treatment, compared with the hormone coil, although fewer women on UPA continued to have periods. Laboratory studies on samples taken from the lining of the womb showed temporary changes due to UPA, which disappeared after treatment stopped. What does this mean? Both treatments improve the symptoms of menstrual bleeding and general wellbeing. Because of safety concerns UPA is not available for all women with heavy menstrual bleeding and new, safer medical treatments are needed. Scientific summary Background Heavy menstrual bleeding (HMB) is the most common gynaecological problem in women of reproductive age, affecting one in four women, and has adverse profound impact on health-related quality of life. Common causes of HMB include structural abnormalities such as uterine fibroids, adenomyosis and dysfunction of the endometrium. The levonorgestrel-releasing intrauterine system (LNG-IUS) is a proven, effective long-term treatment but about one-third of women cease use by two years due to unpredictable bleeding, hormonal adverse effects or lack of effectiveness. Furthermore, fibroids can make the LNG-IUS less effective. Alternative medical options for HMB exist, but are either less effective or associated with unacceptable adverse effects. Surgical interventions are effective at inducing bleeding control and improving quality of life but are typically incompatible with future fertility. Effective long-term medical treatments for women with HMB are needed. A class of drugs called selective progesterone receptor modulators (SPRMs) have potential to provide an effective oral treatment for HMB. SPRMs bind with progesterone receptors, resulting in tissue-specific effects in both myometrial and overlying endometrial tissue as well as shrinking uterine fibroids. The SPRM ulipristal acetate (UPA) has been successfully used to treat fibroids, but we do not know how effective UPA is for the treatment of women with HMB who do not have fibroids. Furthermore, there are uncertainties regarding the mechanism and location of action of UPA, as well as its longer-term safety. SPRMs induce distinctive, non-physiological endometrial changes, which can be confused with endometrial hyperplasia. More recently there has been concern regarding the potential for UPA to cause drug-induced liver injury (DILI). Post marketing surveillance reports resulted in a temporary halt in UPA use in 2018 and 2020. Use of UPA has since been reinstated since January 2021, albeit in a restricted context, reflecting the paucity of existing alternatives for HMB. Given these uncertainties, we designed the UCON trial to evaluate the safety, tolerability and effectiveness of UPA on HMB and to understand its mechanism of action. Clinical objectives Primary objective: to determine whether UPA is more effective at reducing the burden of HMB symptoms than LNG-IUS after 12 months of treatment. Secondary objectives: Ascertain whether UPA use beyond 3 months’ and up to 12 months’ duration is associated with histological changes to the endometrium and, if so, whether this compromises safety. Ascertain whether UPA is more effective than LNG-IUS in relation to menstrual blood loss, sexual activity, generic quality of life, satisfaction with treatment, patient-reported adverse events, and compliance at 3, 6 and 12 months. Determine the response to UPA and LNG-IUS treatment difference in the presence of uterine fibroids in terms of (1) alleviation of HMB and (2) change in uterine/fibroid volume. Collect data on liver function in women taking UPA, once safety concerns were raised. Mechanism of action study objectives To understand how UPA causes a reduction in menstrual bleeding and uterine/fibroid volume in women with HMB, we determined whether UPA administration: Alters endometrial cell function (e.g., and not limited to, proliferation, apoptosis, expression of steroid receptors, tumour suppressors and inflammatory mediators). Reduces blood plasma flow in the endometrium, uterine myometrium and fibroid tissue. Alters the volume fraction of the extracellular matrix in these tissues. Reduces uterine and fibroid volume. Design This was a randomised, open-label, parallel group, multicentre trial with embedded mechanistic study. Methods Setting The trial recruited participants in 10 sites in NHS hospital settings across the UK between 2015 and 2020. The mechanism of action study was conducted solely at the Edinburgh site. Participants For the main trial, informed consent was sought from premenopausal women (aged 18–50 years) with self-reported HMB, no contraindications to LNG-IUS or UPA. Those with uterine size greater than equivalent 14-week size or with submucosal fibroids >2cm were excluded. Other exclusion criteria relating to use of other treatments and current health status were applied, including history of severe hepatic impairment. Screening and randomisation Participants were recruited in gynaecology clinics by research nurses who screened patient referral letters. Following consent, haemoglobin and circulating estradiol levels were assessed, clinical history elicited and transvaginal and/or abdominal ultrasound and endometrial biopsy were obtained if not previously performed. Following this, and confirmation of eligibility, randomisation was via a web-based central service based at Birmingham Clinical Trials Unit to allocate women in a 1 : 1 ratio using a minimisation algorithm. Screened patients in Edinburgh were offered the opportunity to participate in the mechanistic study. Interventions and follow-up Those allocated to UPA received three courses of treatment, each course comprising a daily 5-mg oral dose for 12 weeks followed by a four-week break. Those allocated to the LNG-IUS had it fitted in hospital or primary care. Participants allocated to UPA returned to hospital to collect their repeat prescription at 3 and 6 months, and may have been seen by a member of the care team if required. They were then seen in clinic at 12 months for ultrasound scan (USS) and haemoglobin/serum estradiol measurement. Those allocated to the LNG-IUS group attended USS at 12 months. Follow-up at interim time points was conducted by postal questionnaire. Those partaking in the mechanism of action study underwent magnetic resonance imaging (MRI) following randomisation and at the end of treatment cycles two and three. An additional endometrial biopsy was obtained at the end of treatment cycle two. Outcome measures Primary Condition-specific quality of life score as measured by the menorrhagia multi-attribute scale (MMAS) questionnaire at 12 months. Summary scores range from 0 (worst affected) to 100 (not affected). Secondary Condition-specific quality of life score as measured by MMAS at 3 and 6 months Menstrual bleeding (pictorial blood loss assessment chart)* Cycle regularity (ordinal four-point scale)* Duration of period (ordinal three option scale)* Pelvic pain during periods, intercourse and at other times (visual analogue scales; 0 = best outcome, 10 = worse outcome)* Uterine fibroid symptom and quality of life instrument (only given to women diagnosed with fibroids)* Sexual function (sexual activity questionnaire)* Generic quality of life (EQ-5D-5L)* Satisfaction with treatment outcome (five-point Likert scale) Participant rating of effect of treatment on HMB over 12 months (four-point Likert scale) Whether participant is willing to recommend the treatment to a friend (yes/no) Surgical intervention Adherence to trial treatments and reasons for changing treatment, as reported by the participant Serious adverse events and reactions Uterine volume, evidence of adenomyosis, presence of fibroids, largest fibroid volume, endometrial thickness, endometrial appearance, evidence of ovarian cysts at 12 months (USS) Endometrial biopsy at 12 months (UPA group only) Liver function tests, from 20 March 2018 every four weeks (UPA group only) Haemoglobin and serum estradiol at 12 months * assessed at 3, 6 and 12 months Mechanism of action A: Effects on cellular markers of endometrial steroid receptors and metabolising enzymes (governing local endometrial steroid [ligand] availability), cell proliferation, cell survival (apoptosis); detection of genes implicated in control of proliferation in endometrium; B: Effects on uterine/fibroid structure addressed by obtaining volume measurements for the whole uterus, and for the total volume of fibroids when present, by using high resolution structural MRI and stereology; and C: Uterine vascularity using dynamic contrast-enhanced MRI (DCE-MRI). Urgent safety measures In November 2017, the European Medicines Agency (EMA) issued an urgent drug alert for UPA due to a small number of reports of serious liver injury. A detailed investigation by the regulatory authorities was undertaken and it was found that eight reports of serious liver injury were reported in Europe from an estimated 740,000 women using UPA for uterine fibroids. Restrictions on prescribing UPA were subsequently issued and the trial sponsor implemented an urgent safety measure (USM) in February 2018, which halted recruitment. Those allocated UPA were allowed to complete their current course of UPA treatment but not commence any further outstanding courses. In addition, they commenced monthly assessment of LFTs (as well as a post treatment test approximately 2 weeks after the last course of UPA). In August 2018, the halt on UPA prescribing was lifted and recruitment to UCON resumed in October 2018 with additional safety measures in place, including exclusion of those with any history of liver disease [defined as levels of alanine transaminase (ALT) or aspartate aminotransferase (AST) of more than two times the upper limit of normal] and LFT monitoring as described above. UPA was stopped if women had an ALT or AST more than three times the upper limit of normal and a hepatology opinion was sought. In March 2020, the EMA temporarily suspended use of UPA a second time due to ongoing concerns regarding hepatotoxicity and a further USM was issued. All treatment courses of UPA were immediately stopped. In view of the second USM, the investigators, in discussion with the funder, chose premature closure of recruitment to the study but planned follow-up actions continued as per protocol. Statistical considerations The study was powered to detect a clinically useful difference in MMAS score (13 points) between the two groups at twelve months. To detect a difference of this size [0.5 standard deviations (SDs)] with 90% power (p = 0.05) would require 86 women in each group (172 in total). To allow for a 20% loss to follow-up or pregnancy, the sample size was inflated to 220 women. Following the initial USM, this figure was inflated to 302 women to ensure that there were adequate responses in the primary analysis population (defined below) to detect the same size of difference. The original planned primary analysis population comprised all participants, regardless of adherence to treatment, employing suitable regression models to estimate difference between groups. The enforced non-compliance as a result of the withdrawal of UPA had substantial implications for the validity of the data reported by participants. It was therefore necessary to redefine analysis populations, considering the restrictions that prevented women taking their courses of UPA might influence their responses and any other new potential biases that may be apparent in either group due to, for example, knowledge of the safety concerns around UPA. The primary analysis population would now comprise participants with questionnaire responses received prior to the first USM (12 February 2018), along with questionnaire responses from participants recruited following the study restart (18 October 2018) provided that the responses were returned before the second USM (17 March 2020). Results Main trial A total of 4471 women were approached for the study, with 236 consented and randomised, of whom 181 (77%) returned primary outcome data at 12 months (103 within the primary analysis population). Baseline data were comparable between groups; 92% were white, 34% had fibroids and 8% adenomyosis. In the primary analysis population, MMAS scores substantially improved in both arms, but at 12 months there was no evidence of a difference between the UPA [median score category: 76–99, IQR (51–75 to 100), n = 53] and LNG-IUS [median score category: 76–99, IQR (51–75 to 100), n = 50] groups (adjusted OR 0.55, 95% CI 0.26 to 1.17; p = 0.12). Rates of amenorrhoea where much higher in those allocated UPA compared with LNG-IUS at each time point (3 months: 56% vs. 5%, adjusted OR 29.3, 95% CI 7.37 to 116; 6 months 53% vs. 10%, adjusted OR 11.7 95% CI 3.78 to 36.0; 12 months: 64% vs. 25%, adjusted OR 7.12, 95% CI 2.29 to 22.2). There was no evidence of a difference in the other patient-reported outcomes although there was considerable uncertainty. In those with uterine fibroids, there were no changes in fibroid or uterine volume in either treatment group at 12 months. On endometrial biopsy, seven participants (8%) had evidence of progesterone receptor modulator associated endometrial changes (PAEC) at 12 months, although none was observed at a further 6 months post treatment; there were no cases of endometrial malignancy. Rates of serious adverse events were low, and no patients required admission to hospital for management of deranged liver function tests due to UPA use. Mechanism of action study Effects of UPA administration on the uterus: UPA produced a reduction in cell proliferation in the endometrium, as well as alteration of other local endometrial cellular markers (steroid receptor and steroid metabolising enzyme expression) creating a local endometrial oestrogenic environment. The effects on endometrial cellular markers were reversed upon withdrawal of UPA treatment. Stereological analysis in 19 patients showed that UPA did not produce a reduction in the volume of the uterus, irrespective of coexisting fibroids or adenomyosis. DCE-MRI in 15 patients showed that UPA appears not to have an effect on uterine blood flow. If adenomyosis was present in the uterus, there was a significant increase in plasma volume in the endometrium. However, one of the five women with adenomyosis also had fibroids. Effects of UPA administration on uterine fibroids: DCE-MRI studies showed that UPA produced an average reduction in plasma volume in 11 fibroids, which may be interpreted as being due to a reduction in extracellular matrix components. This finding was not supported by stereological analysis, which failed to show a reduction in the total volume of fibroids in eight patients. However, it should be noted that the number of subjects studied is small. Conclusions Both UPA and LNG-IUS alleviated the adverse impact of heavy menstrual bleeding on quality of life but we found no evidence of a difference between groups over 12 months. UPA was evidently superior to LNG-IUS in terms of inducing amenorrhoea. We observed no difference in reduction in the volume of the uterus, whether or not fibroids were present and no difference in change in the volume of fibroids was observed. Analysis of selected markers of endometrial cellular function demonstrated UPA modulation of the progesterone receptor, resulting in molecular and cellular alteration in steroid receptors within the endometrium, consistent with the development of a local (endometrial) oestrogenic microenvironment. Despite this, there is no evidence of pathological endometrial changes. We demonstrated that alteration in the endometrial microenvironment reverses on cessation of UPA treatment, a key factor for a medical treatment of HMB, particularly for those who wish to preserve fertility. UPA now has restricted availability due to concerns regarding hepatotoxicity. Findings from this study may offer insights into mechanism of action of other SPRM class members. New, effective and acceptable oral medical treatment options are needed to address an important unmet clinical need. Recommendations for research Further studies of medical treatments for HMB Developing other SPRMs, not associated with DILI Other hormonal/non-hormonal medical treatments for HMB Patient populations that encompass both the symptoms of HMB and underlying aetiologies, including structurally normal uterus, adenomyosis and small fibroids Study design with outcome measures impact on menstrual bleeding pattern, pelvic pain and impact on haemoglobin and iron-deficiency, as well as quality of life Qualitative studies to determine what are the most important outcomes to women who suffer HMB Study registration This trial is registered as ISRCTN 20426843. Funding This project was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation Programme and will be published in full in Efficacy and Mechanism Evaluation; Vol. 10, No. 8. See the NIHR Journals Library website for further project information.

The use of standard protocols in studies supports consistent data collection, improves data quality, and facilitates cross-study analyses. Funded by the National Institutes of Health, the PhenX (consensus measures for Phenotypes and eXposures) Toolkit is a catalog of recommended measurement protocols that address a wide range of research topics and are suitable for inclusion in a variety of study designs. In 2020, a PhenX Working Group of smoking cessation experts followed a well-established consensus process to identify and recommend measurement protocols suitable for inclusion in smoking cessation and smoking harm reduction studies. The broader scientific community was invited to review and provide feedback on the preliminary recommendation of the Working Group. Fourteen selected protocols for measuring smoking cessation, harm reduction, and biomarkers research associated with smoking cessation were released in the PhenX Toolkit (https://www.phenxtoolkit.org) in February 2021. These protocols complement existing PhenX Toolkit content related to tobacco regulatory research, substance use and addiction research, and other measures of smoking-related health outcomes. Adopting well-established protocols enables consistent data collection and facilitates comparing and combining data across studies, potentially increasing the scientific impact of individual studies.

Intermediate monocytes (iMo; CD14+CD16+) increase in number in the circulation of patients with unstable coronary artery disease (CAD), and their recruitment to inflamed arteries is implicated in events leading to mortality following MI. Monocyte recruitment to inflamed coronary arteries is initiated by high affinity β2-integrin (CD11c/CD18) that activates β1-integrin (VLA-4) to bind endothelial VCAM-1. How integrin binding under shear stress mechanosignals a functional shift in iMo toward an inflammatory phenotype associated with CAD progression is unknown. Whole blood samples from patients treated for symptomatic CAD including non-ST elevation MI, along with healthy age-matched subjects, were collected to assess chemokine and integrin receptor levels on monocytes. Recruitment on inflamed human aortic endothelium or rVCAM-1 under fluid shear stress was assessed using a microfluidic-based artery on a chip (A-Chip). Membrane upregulation of high affinity CD11c correlated with concomitant activation of VLA-4 within focal adhesive contacts was required for arrest and diapedesis across inflamed arterial endothelium to a greater extent in non-ST elevation MI compared with stable CAD patients. The subsequent conversion of CD11c from a high to low affinity state under fluid shear activated phospho-Syk- and ADAM17-mediated proteolytic cleavage of CD16. This marked the conversion of iMo to an inflammatory phenotype associated with nuclear translocation of NF-κB and production of IL-1β+ We conclude that CD11c functions as a mechanoregulator that activates an inflammatory state preferentially in a majority of iMo from cardiac patients but not healthy patients.

Ribosome-associated quality control pathways respond to defects in translational elongation to recycle arrested ribosomes and degrade aberrant polypeptides and mRNAs. Loss of a tRNA gene leads to ribosomal pausing that is resolved by the translational GTPase GTPBP2, and in its absence causes neuron death. Here, we show that loss of the homologous protein GTPBP1 during tRNA deficiency in the mouse brain also leads to codon-specific ribosome pausing and neurodegeneration, suggesting that these non-redundant GTPases function in the same pathway to mitigate ribosome pausing. As observed in Gtpbp2-/- mice (Ishimura et al., 2016), GCN2-mediated activation of the integrated stress response (ISR) was apparent in the Gtpbp1-/- brain. We observed decreased mTORC1 signaling which increased neuronal death, whereas ISR activation was neuroprotective. Our data demonstrate that GTPBP1 functions as an important quality control mechanism during translation elongation and suggest that translational signaling pathways intricately interact to regulate neuronal homeostasis during defective elongation.

AimsMammalian target of rapamycin (mTOR) inhibitors used in drug-eluting stents (DES) to control restenosis have been found to delay endothelialization and increase incidence of late-stent thrombosis through mechanisms not completely understood. We revealed that mTOR inhibition (mTORi) upregulated the expression of cell growth suppressor IRF-1 in primary human arterial endothelial cells (HAEC). This study aimed to examine how mTOR-regulated IRF-1 expression contributes to the suppressive effect of mTORi on arterial endothelial proliferation.Methods and resultsWestern blotting, quantitative PCR, and a dual-luciferase reporter assay indicated that mTOR inhibitors rapamycin and torin 1 upregulated IRF-1 expression and increased its transcriptional activity. IRF-1 in turn contributed to the suppressive effect of mTORi by mediating HAEC apoptosis and cell cycle arrest in part through upregulation of caspase 1 and downregulation of cyclin D3, as revealed by CCK-8 assay, Annexin V binding assay, measurement of activated caspase 3, BrdU incorporation assay, and matrigel tube formation assay. In a mouse model of femoral artery wire injury, administration of rapamycin inhibited EC recovery, an effect alleviated by EC deficiency of IRF-1. Chromatin immunoprecipitation assay with HAEC and rescue expression of wild type or dominant-negative IRF-1 in EC isolated from Irf1-/- mice confirmed transcriptional regulation of IRF-1 on the expression of CASP1 and CCND3. Furthermore, mTORi activated multiple PKC members, among which PKCζ was responsible for the growth-inhibitory effect on HAEC. Activated PKCζ increased IRF1 transcription through JAK/STAT-1 and NF-κB signaling. Finally, overexpression of wild type or mutant raptor incapable of binding mTOR indicated that mTOR-free raptor contributed to PKCζ activation in mTOR-inhibited HAEC.ConclusionsThe study reveals an IRF-1-mediated mechanism that contributes to the suppressive effects of mTORi on HAEC proliferation. Further study may facilitate the development of effective strategies to reduce the side effects of DES used in coronary interventions.

Outbreaks of emerging coronaviruses in the past two decades and the current pandemic of a novel coronavirus (SARS-CoV-2) that emerged in China highlight the importance of this viral family as a zoonotic public health threat. To gain a better understanding of coronavirus presence and diversity in wildlife at wildlife-human interfaces in three southern provinces in Viet Nam 2013-2014, we used consensus Polymerase Chain Reactions to detect coronavirus sequences. In comparison to previous studies, we observed high proportions of positive samples among field rats (34.0%, 239/702) destined for human consumption and insectivorous bats in guano farms (74.8%, 234/313) adjacent to human dwellings. Most notably among field rats, the odds of coronavirus RNA detection significantly increased along the supply chain from field rats sold by traders (reference group; 20.7% positivity, 39/188) by a factor of 2.2 for field rats sold in large markets (32.0%, 116/363) and 10.0 for field rats sold and served in restaurants (55.6%, 84/151). Coronaviruses were also detected in rodents on the majority of wildlife farms sampled (60.7%, 17/28). These coronaviruses were found in the Malayan porcupines (6.0%, 20/331) and bamboo rats (6.3%, 6/96) that are raised on wildlife farms for human consumption as food. We identified six known coronaviruses in bats and rodents, clustered in three Coronaviridae genera, including the Alpha-, Beta-, and Gammacoronaviruses. Our analysis also suggested either mixing of animal excreta in the environment or interspecies transmission of coronaviruses, as both bat and avian coronaviruses were detected in rodent feces on wildlife farms. The mixing of multiple coronaviruses, and their apparent amplification along the wildlife supply chain into restaurants, suggests maximal risk for end consumers and likely underpins the mechanisms of zoonotic spillover to people.

Background Ferumoxytol is approved for use in the treatment of iron deficiency anemia, but it can serve as an alternative to gadolinium-based contrast agents. On the basis of postmarketing surveillance data, the Food and Drug Administration issued a black box warning regarding the risks of rare but serious acute hypersensitivity reactions during fast high-dose injection (510 mg iron in 17 seconds) for therapeutic use. Whereas single-center safety data for diagnostic use have been positive, multicenter data are lacking. Purpose To report multicenter safety data for off-label diagnostic ferumoxytol use. Materials and Methods The multicenter ferumoxytol MRI registry was established as an open-label nonrandomized surveillance databank without industry involvement. Each center monitored all ferumoxytol administrations, classified adverse events (AEs) using the National Cancer Institute Common Terminology Criteria for Adverse Events (grade 1-5), and assessed the relationship of AEs to ferumoxytol administration. AEs related to or possibly related to ferumoxytol injection were considered adverse reactions. The core laboratory adjudicated the AEs and classified them with the American College of Radiology (ACR) classification. Analysis of variance was used to compare vital signs. Results Between January 2003 and October 2018, 3215 patients (median age, 58 years; range, 1 day to 96 years; 1897 male patients) received 4240 ferumoxytol injections for MRI. Ferumoxytol dose ranged from 1 to 11 mg per kilogram of body weight (≤510 mg iron; rate ≤45 mg iron/sec). There were no systematic changes in vital signs after ferumoxytol administration (P > .05). No severe, life-threatening, or fatal AEs occurred. Eighty-three (1.9%) of 4240 AEs were related or possibly related to ferumoxytol infusions (75 mild [1.8%], eight moderate [0.2%]). Thirty-one AEs were classified as allergiclike reactions using ACR criteria but were consistent with minor infusion reactions observed with parenteral iron. Conclusion Diagnostic ferumoxytol use was well tolerated, associated with no serious adverse events, and implicated in few adverse reactions. Registry results indicate a positive safety profile for ferumoxytol use in MRI. © RSNA, 2019 Online supplemental material is available for this article.

Abstract The origin of fecal floatation phenomenon remains poorly understood. Following our serendipitous discovery of differences in buoyancy of feces from germ-free and conventional mice, we characterized microbial and physical properties of feces from germ-free and gut-colonized (conventional and conventionalized) mice. The gut-colonization associated differences were assessed in feces using DNA, bacterial-PCR, scanning electron microscopy, FACS, thermogravimetry and pycnometry. Based on the differences in buoyancy of feces, we developed levô in fimo test (LIFT) to distinguish sinking feces (sinkers) of germ-free mice from floating feces (floaters) of gut-colonized mice. By simultaneous tracking of microbiota densities and gut colonization kinetics in fecal transplanted mice, we provide first direct evidence of causal relationship between gut microbial colonization and fecal floatation. Rare discordance in LIFT and microbiota density indicated that enrichment of gasogenic gut colonizers may be necessary for fecal floatation. Finally, fecal metagenomics analysis of ‘floaters’ from conventional and syngeneic fecal transplanted mice identified colonization of > 10 gasogenic bacterial species including highly prevalent B. ovatus, an anaerobic commensal bacteria linked with flatulence and intestinal bowel diseases. The findings reported here will improve our understanding of food microbial biotransformation and gut microbial regulators of fecal floatation in human health and disease.

Background Cancer immunotherapies can produce complete therapeutic responses, however, outcomes in ovarian cancer (OC) are modest. While adoptive T-cell transfer (ACT) has been evaluated in OC, durable effects are rare. Poor therapeutic efficacy is likely multifactorial, stemming from limited antigen recognition, insufficient tumor targeting due to a suppressive tumor microenvironment (TME), and limited intratumoral accumulation/persistence of infused T cells. Importantly, host T cells infiltrate tumors, and ACT approaches that leverage endogenous tumor-infiltrating T cells for antitumor immunity could effectively magnify therapeutic responses.Methods Using retroviral transduction, we have generated T cells that secrete a folate receptor alpha (FRα)-directed bispecific T-cell engager (FR-B T cells), a tumor antigen commonly overexpressed in OC and other tumor types. The antitumor activity and therapeutic efficacy of FR-B T cells was assessed using FRα+ cancer cell lines, OC patient samples, and preclinical tumor models with accompanying mechanistic studies. Different cytokine stimulation of T cells (interleukin (IL)-2+IL-7 vs IL-2+IL-15) during FR-B T cell production and the resulting impact on therapeutic outcome following ACT was also assessed.Results FR-B T cells efficiently lysed FRα+ cell lines, targeted FRα+ OC patient tumor cells, and were found to engage and activate patient T cells present in the TME through secretion of T cell engagers. Additionally, FR-B T cell therapy was effective in an immunocompetent in vivo OC model, with response duration dependent on both endogenous T cells and FR-B T cell persistence. IL-2/IL-15 preconditioning prior to ACT produced less differentiated FR-B T cells and enhanced therapeutic efficacy, with mechanistic studies revealing preferential accumulation of TCF-1+CD39−CD69− stem-like CD8+ FR B T cells in the peritoneal cavity over solid tumors.Conclusions These findings highlight the therapeutic potential of FR-B T cells in OC and suggest FR-B T cells can persist in extratumoral spaces while actively directing antitumor immunity. As the therapeutic activity of infused T cell therapies in solid tumor indications is often limited by poor intratumoral accumulation of transferred T cells, engager-secreting T cells that can effectively leverage endogenous immunity may have distinct mechanistic advantages for enhancing therapeutic responses rates.

Summary: Background: Heavy menstrual bleeding affects one in four women and negatively impacts quality of life. Ulipristal acetate is prescribed to treat symptoms associated with uterine fibroids. We compared the effectiveness of ulipristal acetate and the levonorgestrel-releasing intrauterine system at reducing the burden of heavy menstrual bleeding, irrespective of the presence of fibroids. Methods: This randomised, open-label, parallel group phase III trial enrolled women over 18 years with heavy menstrual bleeding from 10 UK hospitals. Participants were centrally randomised, in a 1:1 ratio, to either three, 12-week treatment cycles of 5 mg ulipristal acetate daily, separated by 4-week treatment-free intervals, or a levonorgestrel-releasing intrauterine system. The primary outcome, analysed by intention-to-treat, was quality of life measured by the Menorrhagia Multi-Attribute Scale at 12 months. Secondary outcomes included menstrual bleeding and liver function. The trial is registered with ISRCTN, 20426843. Findings: Between June 5th, 2015 and February 26th, 2020, 236 women were randomised, either side of a recruitment suspension due to concerns of ulipristal acetate hepatoxicity. Subsequent withdrawal of ulipristal acetate led to early cessation of recruitment but the trial continued in follow-up. The primary outcome substantially improved in both groups, and was 89, (interquartile range [IQR] 65 to 100, n = 53) and 94, (IQR 70 to 100, n = 50; adjusted odds ratio 0.55, 95% confidence interval [CI] 0.26–1.17; p = 0.12) in the ulipristal and levonorgestrel-releasing intrauterine system groups. Rates of amenorrhoea at 12 months were higher in those allocated ulipristal acetate compared to levonorgestrel-releasing intrauterine system (64% versus 25%, adjusted odds ratio 7.12, 95% CI 2.29–22.2). Other outcomes were similar between the two groups and there were no cases of endometrial malignancy or hepatotoxicity due to ulipristal acetate use. Interpretation: Our findings suggested that both treatments improved quality of life. Ulipristal was more effective at inducing amenorrhoea. Ulipristal has been demonstrated to be an effective medical therapeutic option but currently its use has restrictions and requires liver function monitoring. Funding: UK Medical Research Council and National Institute of Health Research EME Programme (12/206/52).

Elevated triglyceride-rich lipoproteins (TGRL) in circulation is a risk factor for atherosclerosis. TGRL from subjects consuming a high saturated fat test meal elicited a variable inflammatory response in TNFα-stimulated endothelial cells (EC) that correlated strongly with the polyunsaturated fatty acid (PUFA) content. This study investigates how the relative abundance of oxygenated metabolites of PUFA, oxylipins, is altered in TGRL postprandially, and how these changes promote endothelial inflammation. Human aortic EC were stimulated with TNFα and treated with TGRL, isolated from subjects' plasma at fasting and 3.5 hrs postprandial to a test meal high in saturated fat. Endothelial VCAM-1 surface expression stimulated by TNFα provided a readout for atherogenic inflammation. Concentrations of esterified and non-esterified fatty acids and oxylipins in TGRL were quantified by mass spectrometry. Dyslipidemic subjects produced TGRL that increased endothelial VCAM-1 expression by ≥35%, and exhibited impaired fasting lipogenesis activity and a shift in soluble epoxide hydrolase and lipoxygenase activity. Pro-atherogenic TGRL were enriched in eicosapentaenoic acid metabolites and depleted in esterified C18-PUFA-derived diols. Abundance of these metabolites was strongly predictive of VCAM-1 expression. We conclude the altered metabolism in dyslipidemic subjects produces TGRL with a unique oxylipin signature that promotes a pro-atherogenic endothelial phenotype.

T cell cytokines contribute to immunity against Staphylococcus aureus, but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL-17 response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1β, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4 However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6+ T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6+Vδ4+ T cells in immunity against S. aureus skin infections.

IntroductionThe glycocalyx is a layer of glycoproteins, proteoglycans and glycosaminoglycans that coats the luminal surface of most blood vessels. It effectively regulates adhesive interactions between leukocytes in flowing blood and the endothelium, where during inflammation, binding to E- and P-selectins and intercellular adhesion molecule-1 (ICAM-1) promotes cell tethering and arrest under shear flow.MethodsIn this study, we examine the targeting of E-selectin by an engineered peptide moiety bound to a dermatan sulfate backbone. We further investigate this conjugate, denoted as EC-SEAL, by observing its binding to inflamed endothelium, and quantifying its ability to modulate neutrophil-endothelium interactions.ResultsBinding data reveal that EC-SEAL recognizes domains on E-selectin, and to a lesser degree on P- and L-selectin, and ICAM-1. Further, EC-SEAL increases neutrophil rolling velocity, and decreases neutrophil arrest and migration on inflamed human microvascular endothelial cells under physiologically relevant flow conditions.ConclusionsWe conclude that simple targeting strategies can mimic glycocalyx function under inflammatory conditions, effectively reducing neutrophil recruitment.

Staphylococcus aureus (S. aureus) infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP×MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.