Search

Abstract Eosinophils were previously reported to play a role in intestinal inflammation and fibrosis. Whether this is as a bystander or as an active participant is still up for debate. Moreover, data describing a causal relationship between eosinophils and intestinal fibrosis are scarce. We here aimed to elucidate the role of eosinophils in the pathogenesis of intestinal inflammation and fibrosis. Therefore, we stimulated fibroblasts with (active) eosinophils or with Eosinophil Cationic Protein (ECP), and assessed fibroblast activation via flow cytometry and immunocytochemistry. We observed decreased fibroblast activation when fibroblasts were co-cultured with active eosinophils or after stimulation with ECP in comparison to monoculture conditions, but not in case of co-culturing with inactivated eosinophils. Furthermore, eosinophil depletion in a RAG−/− chronic DSS colitis model resulted in decreased inflammation, but increased development of fibrosis. In this model, we could show increased expression of the anti-inflammatory protein IL-10 and the pro-fibrotic factors IL-1β, FGF-21 and TGF-β3 in the eosinophil-depleted mice compared to the control mice. In conclusion, our in vitro data revealed an anti-fibrotic role for eosinophils. In line, in a chronic murine colitis model, we observed a pro-inflammatory, but an anti-fibrotic, role for eosinophils. Furthermore, we identified an increased presence of anti-inflammatory and pro-fibrotic cytokines in the eosinophil depleted group.

We identified a cluster of mpox exposures among key populations in Kenya through retrospective serologic screening. We identified strong seropositivity among sex workers and gay, bisexual, and other men who have sex with men. These findings demonstrate the need for increased mpox surveillance among mpox-endemic and mpox-endemic–adjacent regions in Africa.

Abstract Cobas EGFR mutation Test v2 was FDA-approved as qualitative liquid biopsy for actionable EGFR variants in non-small cell lung cancer (NSCLC). It generates semiquantitative index (SQI) values that correlate with mutant allele levels, but decision thresholds for clinical use in NSCLC surveillance are lacking. We conducted long-term ctDNA monitoring in 20 subjects with EGFR-mutated NSCLC; resulting in a 155 on-treatment samples. We defined optimal SQI intervals to predict/rule-out progression within 12 weeks from sampling and performed orthogonal calibration versus deep-sequencing and digital PCR. SQI showed significant diagnostic power (AUC 0.848, 95% CI 0.782–0.901). SQI below 5 (63% of samples) had 93% (95% CI 87–96%) NPV, while SQI above 10 (25% of samples) had 69% (95% CI 56–80%) PPV. Cobas EGFR showed perfect agreement with sequencing (Kappa 0.860; 95% CI 0.674–1.00) and digital PCR. SQI values strongly (r: 0.910, 95% 0.821–0.956) correlated to mutant allele concentrations with SQI of 5 and 10 corresponding to 6–9 (0.2–0.3%) and 64–105 (1.1–1.6%) mutant allele copies/mL (VAF) respectively. Our dual-threshold classifier of SQI 0/5/10 yielded informative results in 88% of blood draws with high NPV and good overall clinical utility for patient-centric surveillance of metastatic NSCLC.

Abstract Background Since 2011 when the Canadian Institutes of Health Research launched the Strategy for Patient Oriented Research, there has been a growing expectation to embed patient-oriented research (POR) in the health research community in Canada. To meet this expectation and build capacity for POR in the field of neurodevelopmental disability and child health, in 2017 researchers and family leaders at CanChild Centre for Childhood Disability Research, McMaster University partnered with Kids Brain Health Network and McMaster Continuing Education to develop and implement a 10-week online Family Engagement in Research (FER) Course. Main text From its inception, the FER Course has been delivered in partnership with family leaders and researchers. The FER Course is innovative in its co-learning and community building approach. The course is designed to bring family partners and researchers together to co-learn and connect, and to develop competency and confidence in both the theory and practice of family engagement in research. Coursework involves four live online group discussions, individual review of course materials, weekly group activities, and a final group project and presentation. Upon completion of the FER Course, graduates earn a McMaster University micro-credential. Conclusions To meet a need in building capacity in POR, a novel course in the field of neurodevelopmental disability and child health has been co-created and delivered. Over six years (2018–2023), the FER Course has trained more than 430 researchers and family partners across 20 countries. A unique outcome of the FER Course is that graduates expressed the wish to stay connected and continue to collaborate well beyond the course in turn creating an international FER Community Network that continues to evolve based on need. The FER Course is creating a growing international community of researchers, trainees, self-advocates, and family partners who are championing the implementation of meaningful engagement in neurodevelopmental disability and child health research and beyond. The course is internationally recognized with an established record of building capacity in POR. Its uptake, sustainability, and scalability to date has illustrated that training programs like the FER Course are necessary for building capacity and leadership in family engagement in research.

Abstract Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system and a leading cause of neurological disability in young adults. Clinical presentation and disease course are highly heterogeneous. Typically, disease progression occurs over time and is characterized by the gradual accumulation of disability. The risk of developing MS is driven by complex interactions between genetic and environmental factors, including the gut microbiome. How the commensal gut microbiota impacts disease severity and progression over time remains unknown. In a longitudinal study, disability status and associated clinical features in 58 MS patients were tracked over 4.2 ± 0.98 years, and the baseline fecal gut microbiome was characterized via 16S amplicon sequencing. Progressor status, defined as patients with an increase in Expanded Disability Status Scale (EDSS), were correlated with features of the gut microbiome to determine candidate microbiota associated with risk of MS disease progression. We found no overt differences in microbial community diversity and overall structure between MS patients exhibiting disease progression and non-progressors. However, a total of 41 bacterial species were associated with worsening disease, including a marked depletion in Akkermansia, Lachnospiraceae, and Oscillospiraceae, with an expansion of Alloprevotella, Prevotella-9, and Rhodospirillales. Analysis of the metabolic potential of the inferred metagenome from taxa associated with progression revealed enrichment in oxidative stress-inducing aerobic respiration at the expense of microbial vitamin K2 production (linked to Akkermansia), and a depletion in SCFA metabolism (linked to Oscillospiraceae). Further, as a proof of principle, statistical modeling demonstrated that microbiota composition and clinical features were sufficient to predict disease progression. Additionally, we found that constipation, a frequent gastrointestinal comorbidity among MS patients, exhibited a divergent microbial signature compared with progressor status. These results demonstrate a proof of principle for the utility of the gut microbiome for predicting disease progression in MS in a small well-defined cohort. Further, analysis of the inferred metagenome suggested that oxidative stress, vitamin K2, and SCFAs are associated with progression, warranting future functional validation and mechanistic study.

Abstract Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.

Abstract Childhood adversity, a prevalent experience, is related to a higher risk for externalizing and internalizing psychopathology. Alterations in the development of cognitive processes, for example in the attention-interference domain may link childhood adversity and psychopathology. Interfering stimuli can vary in their salience, i.e. ability to capture attentional focus, and valence. However, it is not known if interference by salience or valence is associated with self-reported adversity. In two independent study samples of healthy men (Study 1: n = 44; mean age [standard deviation (SD)] = 25.9 [3.4] years; Study 2: n = 37; 43.5 [9.7] years) we used the attention modulation task (AMT) that probed interference by two attention-modulating conditions, salience and valence separately across repeated target stimuli. The AMT measures the effects of visual distractors (pictures) on the performance of auditory discrimination tasks (target stimuli). We hypothesized that participants reporting higher levels of childhood adversity, measured with the childhood trauma questionnaire, would show sustained interference in trials with lower salience. Due to conflicting reports on the valence-modulation, we tested the valence condition in an exploratory manner. Linear mixed models revealed an interaction between reported childhood adversity and the salience condition across tone presentations in both study samples (Sample 1: p = .03; Sample 2: p = .04), while there were no effects for the valence condition across both studies. Our study suggests that higher self-reported childhood adversity is related to faster processing of target cues during high salience, but slower during low salience conditions. These results hint to the mechanisms linking childhood adversity and psychopathological symptoms in the attentional domain.

Abstract Dark-blood late gadolinium enhancement (LGE) has been shown to improve the visualization and quantification of areas of ischemic scar compared to standard bright-blood LGE. Recently, the performance of various semi-automated quantification methods has been evaluated for the assessment of infarct size using both dark-blood LGE and conventional bright-blood LGE with histopathology as a reference standard. However, the impact of this sequence on different quantification strategies in vivo remains uncertain. In this study, various semi-automated scar quantification methods were evaluated for a range of different ischemic and non-ischemic pathologies encountered in clinical practice. A total of 62 patients referred for clinical cardiovascular magnetic resonance (CMR) were retrospectively included. All patients had a confirmed diagnosis of either ischemic heart disease (IHD; n = 21), dilated/non-ischemic cardiomyopathy (NICM; n = 21), or hypertrophic cardiomyopathy (HCM; n = 20) and underwent CMR on a 1.5 T scanner including both bright- and dark-blood LGE using a standard PSIR sequence. Both methods used identical sequence settings as per clinical protocol, apart from the inversion time parameter, which was set differently. All short-axis LGE images with scar were manually segmented for epicardial and endocardial borders. The extent of LGE was then measured visually by manual signal thresholding, and semi-automatically by signal thresholding using the standard deviation (SD) and the full width at half maximum (FWHM) methods. For all quantification methods in the IHD group, except the 6 SD method, dark-blood LGE detected significantly more enhancement compared to bright-blood LGE (p

Abstract Several recumbirostran ‘microsaurs’ are known from early Permian sites across Germany, including the Tambach Formation in Thuringia, central Germany. The only ‘microsaur’ thus far described from the Tambach Formation was the ostodolepid recumbirostran Tambaroter carrolli. However, there is also the documented presence of an undescribed recumbirostran ‘microsaur’ at the well-known Bromacker locality. The Bromacker locality is highly recognized and best known for its very diverse and extremely well-preserved terrestrial tetrapod assemblage combined with the co-occurrence of an exceptional vertebrate ichnofossil record. Here we describe a second new recumbirostran taxon from the Tambach Formation, which is also the first from the Bromacker locality itself. Phylogenetic analysis indicates that the new taxon, Bromerpeton subcolossus gen. et sp. nov., is a brachystelechid recumbirostran, a group also known elsewhere in Germany. The following features differentiate Bromerpeton from the other members of the clade: the presence of 13 maxillary teeth, narrow postorbitals that do not substantially contribute to the postorbital region of the skull, the frontal does not contribute to the orbital margin, and the presence of five manual digits. This new recumbirostran ‘microsaur’ further adds to the unique ecosystem that is preserved at the Bromacker locality, granting us a better understanding of what was living underfoot the larger more well-known animals at the locality. Likewise, it expands our understanding of the evolution of recumbirostran ‘microsaurs’, especially with regards to digit and limb reduction within the clade.

Abstract Background Interferon-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. We studied CXCL10(1–73), lacking the four endmost C-terminal amino acids, which was previously identified in supernatant of cultured human fibroblasts and keratinocytes. Methods Relative levels of CXCL10(1–73) and intact CXCL10(1–77) were determined in synovial fluids of patients with rheumatoid arthritis (RA) through tandem mass spectrometry. The production of CXCL10(1–73) was optimized through Fmoc-based solid phase peptide synthesis (SPPS) and a strategy to efficiently generate human CXCL10 proteoforms was introduced. CXCL10(1–73) was compared to intact CXCL10(1–77) using surface plasmon resonance for glycosaminoglycan (GAG) binding affinity, assays for cell migration, second messenger signaling downstream of CXCR3, and flow cytometry of CHO cells and primary human T lymphocytes and endothelial cells. Leukocyte recruitment in vivo upon intraperitoneal injection of CXCL10(1–73) was also evaluated. Results Natural CXCL10(1–73) was more abundantly present compared to intact CXCL10(1–77) in synovial fluids of patients with RA. CXCL10(1–73) had diminished affinity for GAG including heparin, heparan sulfate and chondroitin sulfate A. Moreover, CXCL10(1–73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1–73) incited significantly less primary human T lymphocyte chemotaxis in vitro and peritoneal ingress of CXCR3+ T lymphocytes in mice. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on migration, proliferation, wound closure, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. Conclusion Our study shows that the C-terminal residues Lys74-Pro77 of CXCL10 are important for GAG binding, signaling through CXCR3A, T lymphocyte chemotaxis, but dispensable for angiostasis. Graphical Abstract

Background: Reduced cardiac energy is a hallmark feature of heart failure and is common in cardiac amyloidosis (CA) and can be aggravated by the presence of iron deficiency. Methods: Retrospective analysis of a single tertiary care center CA registry. Prevalence of iron deficiency was determined based on two definitions: (1) Classic definition, ferritin < 100 µg/L irrespective of transferin saturation (TSAT) or ferritin between 100 and 300 µg/L with a TSAT < 20%, and (2) TSAT-based definition, TSAT < 20%. Results: Out of a total of 393 CA patients who had a full set of iron indices (44% light chain [AL]-CA, 50% transthyretin [ATTR]-CA, remainder other or unspecified CA subtype), 56% had iron deficiency according to the classic definition and 58% according to the TSAT definition, with similar prevalence in AL-CA vs ATTR-CA (p = .135). Per both definitions 58% had anemia. Only the TSAT-based definition was associated with worse functional status (p = .039) and worse cardiac function. CA patients with a TSAT < 20% illustrated features of more pronounced right ventricular (RV) failure including lower TAPSE on echocardiography, lower RV ejection fraction and RV stroke volume index on CMR, increased right-sided filling pressures, lower pulmonary artery pulsatility index, and higher RAP/PCWP ratio by right heart catheterization. Neither the classic nor the TSAT-based definition was associated with a higher risk of all-cause mortality after covariate adjustment. Conclusion: Iron deficiency is common in cardiac amyloidosis and, when identified with a TSAT < 20%, is associated with worse functional status and more pronounced RV disease, but not with a higher risk of all-cause mortality.

Summary: Background: The principal barrier to an HIV cure is the presence of the latent viral reservoir (LVR), which has been understudied in African populations. From 2018 to 2019, Uganda instituted a nationwide rollout of ART consisting of Dolutegravir (DTG) with two NRTI, which replaced the previous regimen of one NNRTI and the same two NRTI. Methods: Changes in the inducible replication-competent LVR (RC-LVR) of ART-suppressed Ugandans with HIV (n = 88) from 2015 to 2020 were examined using the quantitative viral outgrowth assay. Outgrowth viruses were examined for viral evolution. Changes in the RC-LVR were analyzed using three versions of a Bayesian model that estimated the decay rate over time as a single, linear rate (model A), or allowing for a change at time of DTG initiation (model B&C). Findings: Model A estimated the slope of RC-LVR change as a non-significant positive increase, which was due to a temporary spike in the RC-LVR that occurred 0–12 months post-DTG initiation (p

Abstract Paludiculture, the productive use of wet or rewetted peatlands, offers an option for continued land use by farmers after rewetting formerly drained peatlands, while reducing the greenhouse gas emissions from peat soils. Biodiversity conservation may benefit, but research on how biodiversity responds to paludiculture is scarce. We conducted a multi-taxon study investigating vegetation, breeding bird and arthropod diversity at six rewetted fen sites dominated by Carex or Typha species. Sites were either unharvested, low- or high-intensity managed, and were located in Mecklenburg-Vorpommern in northeastern Germany. Biodiversity was estimated across the range of Hill numbers using the iNEXT package, and species were checked for Red List status. Here we show that paludiculture sites can provide biodiversity value even while not reflecting historic fen conditions; managed sites had high plant diversity, as well as Red Listed arthropods and breeding birds. Our study demonstrates that paludiculture has the potential to provide valuable habitat for species even while productive management of the land continues.

Abstract A20 serves as a critical brake on NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been linked to various inflammatory disorders, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Experimental gene knockout studies in mice have confirmed A20 as a susceptibility gene for SLE and RA. Here, we examine the significance of protein citrullination and NET formation in the autoimmune pathology of A20 mutant mice because autoimmunity directed against citrullinated antigens released by neutrophil extracellular traps (NETs) is central to the pathogenesis of RA and SLE. Furthermore, genetic variants impairing the deubiquitinase (DUB) function of A20 have been shown to contribute to autoimmune susceptibility. Our findings demonstrate that genetic disruption of A20 DUB function in A20 C103R knockin mice does not result in autoimmune pathology. Moreover, we show that PAD4 deficiency, which abolishes protein citrullination and NET formation, does not prevent the development of autoimmunity in A20 deficient mice. Collectively, these findings provide experimental confirmation that PAD4-dependent protein citrullination and NET formation do not serve as pathogenic mechanisms in the development of RA and SLE pathology in mice with A20 mutations.

Understanding variables that influence antibody responses to COVID-19 vaccination within a population can provide valuable information on future vaccination strategies. In this population-based study, we examined the antibody responses to COVID-19 vaccination in Manitoba using residual serum specimens collected between January 2021 and March 2022 (n = 20,365). Samples were tested for spike and nucleocapsid IgG against SARS-CoV-2 using clinically validated assays. We assessed the impacts of multiple factors on post-vaccination antibody titres including type of vaccine, age, sex, geographic location, number of doses received, and timing of vaccination. Our investigation demonstrated that vaccination with one dose of Moderna mRNA-1273 elicited higher anti-spike IgG titres overall compared to Pfizer BNT162b2 vaccination, while one dose of Pfizer BNT162b2 followed by a second dose of Moderna mRNA-1273 exhibited higher titres than two doses of Pfizer BNT162b2 or Moderna mRNA-1273, irrespective of age. Age and time post-vaccination had considerable effects on antibody responses, with older age groups exhibiting lower anti-spike IgG titres than younger ages, and titres of those vaccinated with Pfizer BNT162b2 waning faster than those vaccinated with Moderna mRNA-1273 or a combination of Pfizer BNT162b2 and Moderna mRNA-1273. Antibody titres did not appear to be affected by sex or geographic location. Our results identify how factors such as age and type of vaccine can influence antibody responses to vaccination, and how antibody titres wane over time. This information highlights the importance of tailoring vaccine regimens to specific populations, especially those at increased risk of severe COVID-19 and can be used to inform future vaccination strategies, scheduling of booster doses, and public health measures.

Abstract The aim of this randomized clinical trial was to evaluate the impact of all-trans retinoic acid (ATRA) in combination with non-intensive chemotherapy in older unfit patients (> 60 years) with newly diagnosed NPM1-mutated acute myeloid leukemia. Patients were randomized (1:1) to low-dose chemotherapy with or without open-label ATRA 45 mg/m2, days 8–28; the dose of ATRA was reduced to 45 mg/m2, days 8–10 and 15 mg/m2, days 11–28 after 75 patients due to toxicity. Up to 6 cycles of cytarabine 20 mg/day s.c., bid, days 1–7 and etoposide 100 mg/day, p.o. or i.v., days 1–3 with (ATRA) or without ATRA (CONTROL) were intended. The primary endpoint was overall survival (OS). Between May 2011 and September 2016, 144 patients (median age, 77 years; range, 64–92 years) were randomized (72, CONTROL; 72, ATRA). Baseline characteristics were balanced between the two study arms. The median number of treatment cycles was 2 in ATRA and 2.5 in CONTROL. OS was significantly shorter in the ATRA compared to the CONTROL arm (p = 0.023; median OS: 5 months versus 9.2 months, 2-years OS rate: 7% versus 10%, respectively). Rates of CR/CRi were not different between treatment arms; infections were more common in ATRA beyond treatment cycle one. The addition of ATRA to low-dose cytarabine plus etoposide in an older, unfit patient population was not beneficial, but rather led to an inferior outcome. The clinical trial is registered at clinicaltrialsregister.eu (EudraCT Number: 2010-023409-37, first posted 14/12/2010).

IntroductionToday, modern technology is used to diagnose and treat cardiovascular disease. These medical devices provide exact measures and raw data such as imaging data or biosignals. So far, the Broad Integration of These Health Data into Hospital Information Technology Structures—Especially in Germany—is Lacking, and if data integration takes place, only non-Evaluable Findings are Usually Integrated into the Hospital Information Technology Structures. A Comprehensive Integration of raw Data and Structured Medical Information has not yet Been Established. The aim of this project was to design and implement an interoperable database (cardio-vascular-information-system, CVIS) for the automated integration of al medical device data (parameters and raw data) in cardio-vascular medicine.MethodsThe CVIS serves as a data integration and preparation system at the interface between the various devices and the hospital IT infrastructure. In our project, we were able to establish a database with integration of proprietary device interfaces, which could be integrated into the electronic health record (EHR) with various HL7 and web interfaces.ResultsIn the period between 1.7.2020 and 30.6.2022, the data integrated into this database were evaluated. During this time, 114,858 patients were automatically included in the database and medical data of 50,295 of them were entered. For technical examinations, more than 4.5 million readings (an average of 28.5 per examination) and 684,696 image data and raw signals (28,935 ECG files, 655,761 structured reports, 91,113 x-ray objects, 559,648 ultrasound objects in 54 different examination types, 5,000 endoscopy objects) were integrated into the database. Over 10.2 million bidirectional HL7 messages (approximately 14,000/day) were successfully processed. 98,458 documents were transferred to the central document management system, 55,154 materials (average 7.77 per order) were recorded and stored in the database, 21,196 diagnoses and 50,353 services/OPS were recorded and transferred. On average, 3.3 examinations per patient were recorded; in addition, there are an average of 13 laboratory examinations.DiscussionFully automated data integration from medical devices including the raw data is feasible and already creates a comprehensive database for multimodal modern analysis approaches in a short time. This is the basis for national and international projects by extracting research data using FHIR.

It remains poorly understood how SARS-CoV-2 infection influences the physiological host factors important for aerosol transmission. We assessed breathing pattern, exhaled droplets, and infectious virus after infection with Alpha and Delta variants of concern (VOC) in the Syrian hamster. Both VOCs displayed a confined window of detectable airborne virus (24–48 hr), shorter than compared to oropharyngeal swabs. The loss of airborne shedding was linked to airway constriction resulting in a decrease of fine aerosols (1–10 µm) produced, which are suspected to be the major driver of airborne transmission. Male sex was associated with increased viral replication and virus shedding in the air. Next, we compared the transmission efficiency of both variants and found no significant differences. Transmission efficiency varied mostly among donors, 0–100% (including a superspreading event), and aerosol transmission over multiple chain links was representative of natural heterogeneity of exposure dose and downstream viral kinetics. Co-infection with VOCs only occurred when both viruses were shed by the same donor during an increased exposure timeframe (24–48 hr). This highlights that assessment of host and virus factors resulting in a differential exhaled particle profile is critical for understanding airborne transmission.

Herein, we explore the generation and characterization of the radical cations of glycylphenylalanylglycine, or [GFG]•+, formed via dissociative electron-transfer reaction from the tripeptide to copper(II) within a ternary complex. A comprehensive investigation employing isotopic labeling, infrared multiple-photon dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations elucidated the details and energetics in formation of the peptide radical cations as well as their dissociation products. Unlike conventional aromatic-containing peptide radical cations that primarily form canonical π-radicals, our findings reveal that 75% of the population of the experimentally produced [GFG]•+ precursors are [GFα•G]+, where the radical resides on the middle α-carbon of the phenylalanyl residue. This unexpected isomeric ion has an enthalpy of 6.8 kcal/mol above the global minimum, which has an N-terminal captodative structure, [Gα•FG]+, comprising 25% of the population. The [b₂-H]•+ product ions are also present in a ratio of 75/25 from [GFα•G]+/ [Gα•FG]+, the results of which are obtained from matches between the IRMPD action spectrum and predicted IR absorption spectra of the [b₂-H]•+ candidate structures, as well as from IRMPD isomer population analyses.

[This corrects the article DOI: 10.1371/journal.pone.0254880.].

ObjectiveTo determine the association between infant non-nutritive suck (NNS) dynamics at 3 months and developmental outcomes at 12 months of age in full-term infants. We hypothesized that infants with more mature NNS at 3 months, as evidence by shorter burst duration, fewer cycles per burst, cycles per minute, higher amplitude, and more bursts, would have higher (better) scores on the developmental outcomes at 12 months.MethodsThis was a prospective study that utilized objective and self-report measures. A five-minute NNS sample was collected from 67 infants (54% male) at 3 months of age (average age 2.99 (0.27) months). At 12 months (average age 11.91 (0.26) months), the Development Profile-3 was administered through caregiver interview.ResultsInfant NNS burst duration, cycles per burst, and cycles per minute were significantly negatively associated with the Development Profile-3 cognitive domain and general scores at 12 months. This is consistent with our hypothesis that infants who have more efficient NNS (fewer bursts and cycles) at 3 months would have higher (better) scores on the Development Profile-3 at 12 months.ConclusionsFindings from this work complement emerging research linking infant NNS with subsequent neurodevelopmental outcomes. This is the first time that these associations have been examined using a quantitative and physiologic-based measure of NNS. These results seem to indicate that specific NNS metrics, which demonstrate maturation of this complex skill, may be useful predictors of neurodevelopment later in life.

An integrated botanical and chemical approach is used to study surface residues on Funnel Beaker ceramics from the site of Oldenburg LA 77, in northern Germany. Organic residues were discovered adhering to fragments of thick-walled, undecorated ceramic vessels (n = 19) and ceramic discs (n = 2). The surface residues were studied using scanning electron microscopy (SEM), to examine remains of cereals and other plant tissues that survived food preparation and cooking, and using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and direct time-resolved mass spectrometry (DTMS), to chemically identify specific food components. The SEM results show a reoccurring presence of cereal grain (emmer and barley) and one case of co-occurrence of emmer and fat-hen seeds. The SEM evidence for the use of sprouted emmer grain and milk-ripe barley from the Oldenburg residues greatly enhances our understanding of Neolithic foodways in northwestern Europe. The ATR-FTIR results showed that roughly a third of the surface residues contain traces of the original foods prepared or processed and DTMS results confirm that most of the residues primarily contain polysaccharides and a minimal amount of plant protein and that they lack lipids. Only one residue presents minor indications for a (partly) animal origin. The ceramic vessels were thus used almost exclusively for the processing or cooking of cereal grains. This study offers an intimate view of the cuisine and cooking practices (and in some cases their seasonal timing) in an early agricultural village located in a marginal farming region on the south coast of the Baltic Sea.

Abstract Next generation sequencing of cell-free DNA (cfDNA) is a promising method for treatment monitoring and therapy selection in metastatic breast cancer (MBC). However, distinguishing tumor-specific variants from sequencing artefacts and germline variation with low false discovery rate is challenging when using large targeted sequencing panels covering many tumor suppressor genes. To address this, we built a machine learning model to remove false positive variant calls and augmented it with additional filters to ensure selection of tumor-derived variants. We used cfDNA of 70 MBC patients profiled with both the small targeted Oncomine breast panel (Thermofisher) and the much larger Qiaseq Human Breast Cancer Panel (Qiagen). The model was trained on the panels’ common regions using Oncomine hotspot mutations as ground truth. Applied to Qiaseq data, it achieved 35% sensitivity and 36% precision, outperforming basic filtering. For 20 patients we used germline DNA to filter for somatic variants and obtained 245 variants in total, while our model found seven variants, of which six were also detected using the germline strategy. In ten tumor-free individuals, our method detected in total one (potentially germline) variant, in contrast to 521 variants detected without our model. These results indicate that our model largely detects somatic variants.

Abstract Biological treatment of cereal straw for ruminant nutrition purposes might present an environmentally friendly option of valorizing a widely available by-product of grain production for farming systems with low external input. Several strains of white-rot fungi have been selected in the past under mostly controlled laboratory conditions for their capacity of lignin degradation. The study adapted to conditions on farm for upscaling purposes. The development of the in vitro straw digestibility with two different moistening pre-treatments and inoculated with three different fungi species, namely Pleurotus ostreatus, Ceriporiopsis subvermispora and Volvariella volvacea, was determined up to 42 days of fermentation with five sampling times. The effect of physical straw pre-treatments on nutritional parameters was evaluated. The neutral detergent fiber digestibility (NDFD30h), enzymatically soluble organic substance (ELOS) and the gas production (Hohenheim Feed value Test, HFT) as indicators for in vitro ruminal degradability decreased over time independent of the fungus: HFT, ELOS and NDFD30h by up to 50, 35 and 30% of the original straw. Remoistening and autoclaving the straw increased the gas production significantly by 2.6 mL/200 g dry matter (DM), and ELOS and NDFD30h by 45 and 51 g/kg DM compared to the original straw (34.9 mL/200 mg DM, 342 g/kg DM, 313 g/kg NDF).

Abstract The potential of sponge-derived chemicals for pharmaceutical applications remains largely unexploited due to limited available biomass. Although many have attempted to culture marine sponge cells in vitro to create a scalable production platform for such biopharmaceuticals, these efforts have been mostly unsuccessful. We recently showed that Geodia barretti sponge cells could divide rapidly in M1 medium. In this study we established the first continuous marine sponge cell line, originating from G. barretti. G. barretti cells cultured in OpM1 medium, a modification of M1, grew more rapidly and to a higher density than in M1. Cells in OpM1 reached 1.74 population doublings after 30 min, more than twofold higher than the already rapid growth rate of 0.74 population doublings in 30 min in M1. The maximum number of population doublings increased from 5 doublings in M1 to at least 98 doublings in OpM1. Subcultured cells could be cryopreserved and used to inoculate new cultures. With these results, we have overcome a major obstacle that has blocked the path to producing biopharmaceuticals with sponge cells at industrial scale for decades.

The recent development of single-cell techniques is essential to unravel complex biological systems. By measuring the transcriptome and the accessible genome on a single-cell level, cellular heterogeneity in a biological environment can be deciphered. Transcription factors act as key regulators activating and repressing downstream target genes, and together they constitute gene regulatory networks that govern cell morphology and identity. Dissecting these gene regulatory networks is crucial for understanding molecular mechanisms and disease, especially within highly complex biological systems. The gene regulatory network analysis software ANANSE and the motif enrichment software GimmeMotifs were both developed to analyse bulk datasets. We developed scANANSE, a software pipeline for gene regulatory network analysis and motif enrichment using single-cell RNA and ATAC datasets. The scANANSE pipeline can be run from either R or Python. First, it exports data from standard single-cell objects. Next, it automatically runs multiple comparisons of cell cluster data. Finally, it imports the results back to the single-cell object, where the result can be further visualised, integrated, and interpreted. Here, we demonstrate our scANANSE pipeline on a publicly available PBMC multi-omics dataset. It identifies well-known cell type-specific hematopoietic factors. Importantly, we also demonstrated that scANANSE combined with GimmeMotifs is able to predict transcription factors with both activating and repressing roles in gene regulation.

Toxicology has been an active research field for many decades, with academic, industrial and government involvement. Modern omics and computational approaches are changing the field, from merely disease-specific observational models into target-specific predictive models. Traditionally, toxicology has strong links with other fields such as biology, chemistry, pharmacology, and medicine. With the rise of synthetic and new engineered materials, alongside ongoing prioritisation needs in chemical risk assessment for existing chemicals, early predictive evaluations are becoming of utmost importance to both scientific and regulatory purposes. ELIXIR is an intergovernmental organisation that brings together life science resources from across Europe. To coordinate the linkage of various life science efforts around modern predictive toxicology, the establishment of a new ELIXIR Community is seen as instrumental. In the past few years, joint efforts, building on incidental overlap, have been piloted in the context of ELIXIR. For example, the EU-ToxRisk, diXa, HeCaToS, transQST, and the nanotoxicology community have worked with the ELIXIR TeSS, Bioschemas, and Compute Platforms and activities. In 2018, a core group of interested parties wrote a proposal, outlining a sketch of what this new ELIXIR Toxicology Community would look like. A recent workshop (held September 30th to October 1st, 2020) extended this into an ELIXIR Toxicology roadmap and a shortlist of limited investment-high gain collaborations to give body to this new community. This Whitepaper outlines the results of these efforts and defines our vision of the ELIXIR Toxicology Community and how it complements other ELIXIR activities.

Abstract Telomere length is associated with longevity and survival in multiple species. In human population-based studies, multiple prenatal factors have been described to be associated with a newborn’s telomere length. In the present study, we measured relative leukocyte telomere length in 210 Holstein Friesian heifers, within the first ten days of life. The dam’s age, parity, and milk production parameters, as well as environmental factors during gestation were assessed for their potential effect on telomere length. We found that for both primi- and multiparous dams, the telomere length was 1.16% shorter for each day increase in the calf’s age at sampling (P = 0.017). The dam’s age at parturition (P = 0.045), and the median temperature-humidity index (THI) during the third trimester of gestation (P = 0.006) were also negatively associated with the calves’ TL. Investigating multiparous dams separately, only the calf’s age at sampling was significantly and negatively associated with the calves’ TL (P = 0.025). Results of the present study support the hypothesis that in cattle, early life telomere length is influenced by prenatal factors. Furthermore, the results suggest that selecting heifers born in winter out of young dams might contribute to increased longevity in dairy cattle.

Abstract Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER⍺/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER⍺/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively (including known GRHL2 targets such as ErbB3, CLDN4/7) or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells. Video Abstract

Objective To assess whether the risk of cardiovascular complications of covid-19 differ between the sexes and to determine whether any sex differences in risk are reduced in individuals with pre-existing cardiovascular disease.Design Registry based observational study.Setting 74 hospitals across 13 countries (eight European) participating in CAPACITY-COVID (Cardiac complicAtions in Patients With SARS Corona vIrus 2 regisTrY), from March 2020 to May 2021Participants All adults (aged ≥18 years), predominantly European, admitted to hospital with highly suspected covid-19 disease or covid-19 disease confirmed by positive laboratory test results (n=11 167 patients).Main outcome measures Any cardiovascular complication during admission to hospital. Secondary outcomes were in-hospital mortality and individual cardiovascular complications with ≥20 events for each sex. Logistic regression was used to examine sex differences in the risk of cardiovascular outcomes, overall and grouped by pre-existing cardiovascular disease.Results Of 11 167 adults (median age 68 years, 40% female participants) included, 3423 (36% of whom were female participants) had pre-existing cardiovascular disease. In both sexes, the most common cardiovascular complications were supraventricular tachycardias (4% of female participants, 6% of male participants), pulmonary embolism (3% and 5%), and heart failure (decompensated or de novo) (2% in both sexes). After adjusting for age, ethnic group, pre-existing cardiovascular disease, and risk factors for cardiovascular disease, female individuals were less likely than male individuals to have a cardiovascular complication (odds ratio 0.72, 95% confidence interval 0.64 to 0.80) or die (0.65, 0.59 to 0.72). Differences between the sexes were not modified by pre-existing cardiovascular disease; for the primary outcome, the female-to-male ratio of the odds ratio in those without, compared with those with, pre-existing cardiovascular disease was 0.84 (0.67 to 1.07).Conclusions In patients admitted to hospital for covid-19, female participants were less likely than male participants to have a cardiovascular complication. The differences between the sexes could not be attributed to the lower prevalence of pre-existing cardiovascular disease in female individuals. The reasons for this advantage in female individuals requires further research.

Abstract Background Increasing evidence supports the concept of prenatal programming as an early factor in the aging process. DNA methylation age (DNAm age), global genome-wide DNA methylation (global methylation), telomere length (TL), and mitochondrial DNA content (mtDNA content) have independently been shown to be markers of aging, but their interrelationship and determinants at birth remain uncertain. Methods We assessed the inter-correlation between the aging biomarkers DNAm age, global methylation, TL and mtDNA content using Pearson's correlation in 190 cord blood samples of the ENVIRONAGE birth cohort. TL and mtDNA content was measured via qPCR, while the DNA methylome was determined using the human 450K methylation Illumina microarray. Subsequently, DNAm age was calculated according to Horvath's epigenetic clock, and mean global, promoter, gene-body, and intergenic DNA methylation were determined. Path analysis, a form of structural equation modeling, was performed to disentangle the complex causal relationships among the aging biomarkers and their potential determinants. Results DNAm age was inversely correlated with global methylation (r = -0.64, p

Background: While biological age in adults is often understood as representing general health and resilience, the conceptual interpretation of accelerated biological age in children and its relationship to development remains unclear. We aimed to clarify the relationship of accelerated biological age, assessed through two established biological age indicators, telomere length and DNA methylation age, and two novel candidate biological age indicators, to child developmental outcomes, including growth and adiposity, cognition, behavior, lung function and the onset of puberty, among European school-age children participating in the HELIX exposome cohort. Methods: The study population included up to 1173 children, aged between 5 and 12 years, from study centres in the UK, France, Spain, Norway, Lithuania, and Greece. Telomere length was measured through qPCR, blood DNA methylation, and gene expression was measured using microarray, and proteins and metabolites were measured by a range of targeted assays. DNA methylation age was assessed using Horvath’s skin and blood clock, while novel blood transcriptome and ‘immunometabolic’ (based on plasma proteins and urinary and serum metabolites) clocks were derived and tested in a subset of children assessed six months after the main follow-up visit. Associations between biological age indicators with child developmental measures as well as health risk factors were estimated using linear regression, adjusted for chronological age, sex, ethnicity, and study centre. The clock derived markers were expressed as Δ age (i.e. predicted minus chronological age). Results: Transcriptome and immunometabolic clocks predicted chronological age well in the test set (r=0.93 and r=0.84 respectively). Generally, weak correlations were observed, after adjustment for chronological age, between the biological age indicators. Conclusions: In children, as in adults, biological aging appears to be a multi-faceted process and adiposity is an important correlate of accelerated biological aging. Patterns of associations suggested that accelerated immunometabolic age may be beneficial for some aspects of child development while accelerated DNA methylation age and telomere attrition may reflect early detrimental aspects of biological aging, apparent even in children. Funding: UK Research and Innovation (MR/S03532X/1); European Commission (grant agreement numbers: 308333; 874583).

Objectives To examine the extent of the impact of the COVID-19 pandemic on the mental health and well-being of mental health professionals (MHPs) in the Netherlands and understand their needs during the COVID-19 pandemic.Design and setting A cross-sectional, mixed-methods study was conducted with MHPs from the Netherlands from June 2020 to October 2020, consisting of an online survey and three online focus group discussions.Participants Participants were MHPs from various occupational groups (psychologists, social workers, mental health nurses, developmental education workers, etc).Primary and secondary outcome measures The online survey included questions about work-related changes due to COVID-19 perceived resilience to stress, changes in lifestyle behaviours and mental health symptoms. The focus group discussions focused mostly on work experiences during the first wave of the COVID-19 pandemic.Results MHP’s reported an increase in experience workload during the pandemic (mean score 8.04 based on a scale of 1-10) compared to before the pandemic (mean score of 7). During the first wave of the pandemic, 50% of respondents reported increased stress, 32% increased sleeping problems and 24% increased mental health problems. Adverse occupational (eg, increased workload OR 1.72, 95% CI 1.28–2.32), psychological (eg, life satisfaction OR 0.63, 95% CI 0.52–0.75), lifestyle (eg, increased sleep problems OR 2.80, 95% CI 2.07–3.80) and physical factors (decline in physical health OR 3.56, 95% CI 2.61–4.85) were associated with a decline in mental health. Participants expressed significant concern in the focus group discussions about the duration of the pandemic, the high workload, less work-life balance and lack of contact with colleagues. Suggestions to improve working conditions included ensuring clear communication about guidelines and facilitating worker contact and support via peer-to-peer coaching where experiences can be shared.Conclusions The current study indicates that MHP experienced a decline in mental health status during the first wave of the COVID-19 pandemic, which should be taken into consideration by employers, policymakers and researchers.

Introduction Obtaining level 1 evidence on efficacy of glycosaminoglycan (GAG) therapy is difficult, due to low incidence of bladder pain syndrome/interstitial cystitis (BPS/IC) and heterogeneous symptoms experienced by patients with BPS/IC. Currently, because of a lack of high-grade evidence, the recommendation for applying GAG therapy in most guidelines is ‘low grade’. An aggregated N-of-1 trial is a multicrossover design that yields similar level 1 evidence as a traditional randomised controlled trial (RCT), while requiring far less patients. The goal of this study is to investigate the efficacy of intravesical GAG therapy (Ialuril) for patients with BPS/IC with Hunner lesions using a dual RCT and aggregated N-of-1 trial design to obtain level 1 evidence.Methods and analysis The GETSBI study is a double-blind multidesign multicentre randomised placebo-controlled study to assess the short-term and long-term efficacy of hyaluronic acid (1.6%) + chondroitin sulfate (2%) therapy (Ialuril Prefill, IBSA, Goodlife) in patients with symptomatic BPS/IC with Hunner lesions. It starts as a standard RCT (n=80), but continues as an aggregated N-of-1 trial. There are three parallel arms, receiving blinded treatment for three periods (1 x/week for 6 weeks, ratio placebo to intervention in periods of 2:1). Followed by an open prospective part for the long-term efficacy. The primary study outcome is the maximum bladder pain experienced in the last 3 days measured using the visual analogue pain scale (0–10).This study is a collaboration with the Dutch government and will deliver evidence for the decision to reimburse the therapy. Furthermore, this multidesign study will allow us to compare the two main methods to evaluate applicability for future study designs for BPS/IC research.Ethics and dissemination Ethical approval was given by METC Oost-Nederland, file number: 2020-7265, NL-number: NL76290.091.20. Findings from this study will be disseminated via publication, reports and conference presentations.Trial registration number ClinicalTrials.gov identifier (NCT number): NCT05518864.

Abstract Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and increase in blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency. We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

Dill (Anethum graveolens L.) is an aromatic herb widely used in the food industry, with several commercial cultivars available with different qualitative characteristics. Commercial cultivars are usually preferred over landraces due to their higher yield and also the lack of improved landraces than can be commercialized. In Greece, however, traditional dill landraces are cultivated by local communities. Many are conserved in the Greek Gene Bank and the aim here was to investigate and compare the morphological, genetic, and chemical biodiversity of twenty-two Greek landraces and nine modern/commercial cultivars. Multivariate analysis of the morphological descriptors, molecular markers, and essential oil and polyphenol composition revealed that the Greek landraces were clearly distinguished compared with modern cultivars at the level of phenological, molecular and chemical traits. Landraces were typically taller, with larger umbels, denser foliage, and larger leaves. Plant height, density of foliage, density of feathering as well as aroma characteristics were desirable traits observed for some landraces, such as T538/06 and GRC-1348/04, which were similar or superior to those of some commercial cultivars. Polymorphic loci for inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) molecular markers were 76.47% and 72.41% for landraces, and 68.24% and 43.10% for the modern cultivars, respectively. Genetic divergence was shown, but not complete isolation, indicating that some gene flow may have occurred between landraces and cultivars. The major constituent in all dill leaf essential oils was α-phellandrene (54.42–70.25%). Landraces had a higher α-phellandrene and dill ether content than cultivars. Two dill landraces were rich in chlorogenic acid, the main polyphenolic compound determined. The study highlighted for the first-time Greek landraces with desirable characteristics regarding quality, yield, and harvest time suitable for breeding programs to develop new dill cultivars with superior features.

Many microbiota-based therapeutics rely on our ability to introduce a microbe of choice into an already-colonized intestine. In this study, we used genetically barcoded Bacteroides thetaiotaomicron (B. theta) strains to quantify population bottlenecks experienced by a B. theta population during colonization of the mouse gut. As expected, this reveals an inverse relationship between microbiota complexity and the probability that an individual wildtype B. theta clone will colonize the gut. The polysaccharide capsule of B. theta is important for resistance against attacks from other bacteria, phage, and the host immune system, and correspondingly acapsular B. theta loses in competitive colonization against the wildtype strain. Surprisingly, the acapsular strain did not show a colonization defect in mice with a low-complexity microbiota, as we found that acapsular strains have an indistinguishable colonization probability to the wildtype strain on single-strain colonization. This discrepancy could be resolved by tracking in vivo growth dynamics of both strains: acapsular B.theta shows a longer lag phase in the gut lumen as well as a slightly slower net growth rate. Therefore, as long as there is no niche competitor for the acapsular strain, this has only a small influence on colonization probability. However, the presence of a strong niche competitor (i.e., wildtype B. theta, SPF microbiota) rapidly excludes the acapsular strain during competitive colonization. Correspondingly, the acapsular strain shows a similarly low colonization probability in the context of a co-colonization with the wildtype strain or a complete microbiota. In summary, neutral tagging and detailed analysis of bacterial growth kinetics can therefore quantify the mechanisms of colonization resistance in differently-colonized animals.

Gait is a large component and indicator of health. Many factors affect gait including age, disease, and even mood disorders. Few studies have looked at the influence of emotional states on gait. This study aimed to investigate the influence of emotional states on walking performance to understand whether an emotional state may be an important factor to consider when evaluating gait. Thirty-six young adults were recruited (23F, 13M) and performed a neutral baseline condition of walking which included six passes of walking across an 8m walkway (a total of 48m of walking). Participants then completed 6 pseudo-randomized emotional state induction conditions while immersive 360-degree videos were used to induce the following emotional conditions: happiness, excitement, sadness, fear, and anger. Participants viewed the emotion elicitation videos using a virtual reality head-mounted display (HMD), then rated their emotional state using self-assessment manikins and walked (without the HMD) over a pressure sensor walkway. One-way repeated measures ANOVA and pairwise comparisons were used to examine differences in gait parameters across the emotional conditions. Participants walked with significantly reduced step length and speed during the sadness condition compared to the other emotional conditions and the neutral condition. Furthermore, participants adjusted the timing of their walking during the sadness condition and walked with significantly increased step, stance, and swing times compared to other emotional conditions, but not the neutral condition. Step time was significantly reduced during the conditions of excitement and fear compared to the neutral condition. Emotions may impact variety of gait parameters involving pace and rhythm, however have little influence on gait variability and postural control. These results indicate that perhaps the emotions of sadness and excitement should be taken into account as potential confounds for future gait analysis.

BackgroundDiagnosing concomitant pulmonary embolism (PE) in COVID-19 patients remains challenging. As such, PE may be overlooked. We compared the diagnostic yield of systematic PE-screening based on the YEARS-algorithm to PE-screening based on clinical gestalt in emergency department (ED) patients with COVID-19.MethodsWe included all ED patients who were admitted because of COVID-19 between March 2020 and February 2021. Patients already receiving anticoagulant treatment were excluded. Up to April 7, 2020, the decision to perform CT-pulmonary angiography (CTPA) was based on physician's clinical gestalt (clinical gestalt cohort). From April 7 onwards, systematic PE-screening was performed by CTPA if D-dimer level was ≥1000 ug/L, or ≥500 ug/L in case of ≥1 YEARS-item (systematic screening cohort).Results1095 ED patients with COVID-19 were admitted. After applying exclusion criteria, 289 were included in the clinical gestalt and 574 in the systematic screening cohort. The number of PE diagnoses was significantly higher in the systematic screening cohort compared to the clinical gestalt cohort: 8.2% vs. 1.0% (3/289 vs. 47/574; p100 mg/L (OR 2.78, 95%CI 1.37-5.66, p = 0.005) were independently associated with PE.ConclusionIn ED patients with COVID-19, the number of PE diagnosis was significantly higher in the cohort that underwent systematic PE screening based on the YEARS-algorithm in comparison with the clinical gestalt cohort, with a number needed to test of 7.1 CTPAs to detect one PE.

Standardized Inka tunics, or unku, were created under the auspices of the state as symbolic expressions of its expansionist power. To ensure these textiles acquired the status of effective insignias of power and territorial control, the Inka established and imposed technical and stylistic canons for their production (techne) by means of highly-skilled state weavers. In the provinces, social groups that came under imperial rule, local expert weaving agents adopted the conventions of the state and included meaningful symbolic elements of the idiosyncrasies, traditions, and experiential knowledge of the local community (metis). We therefore propose that this was not a unidirectional process and that the Caleta Vitor Inka unku (hereon referred to as the CV unku), presented here, reflects a syncretism promoted by local weavers. In terms of methods, we have developed a decoding tool for the unku, with the aim of distinguishing state from local hallmarks, thereby revealing the syncretic complexity of these iconic tunics. This methodological tool is based on a series of standard analytical parameters and attributes linked to morphological, technological, and stylistic features, which we applied to the CV unku. Unlike others, this unku does come from a looted tomb but was scientifically excavated in a cemetery located in the Caleta Vitor Bay in northern Chile. By deconstructing the CV unku we determined the steps in the chaîne opératoire at which local technical and stylistic elements were incorporated, thus affecting or transforming, in part, its emblematic imperial imagery. This study also marks a step forward in our understanding of a syncretic landscape that combines the state worldview and organized production system (imperial Inka) with craft-production practices that were rooted in provincial and local communities (provincial Inka).

Introduction Integrated care interventions for type 2 diabetes (T2D) and hypertension (HT) are effective, yet challenges exist with regard to their implementation and scale-up. The ‘SCale-Up diaBetes and hYpertension care’ (SCUBY) Project aims to facilitate the scale-up of integrated care for T2D and HT through the co-creation and implementation of contextualised scale-up roadmaps in Belgium, Cambodia and Slovenia. We hereby describe the plan for the process and scale-up evaluation of the SCUBY Project. The specific goals of the process and scale-up evaluation are to (1) analyse how, and to what extent, the roadmap has been implemented, (2) assess how the differing contexts can influence the implementation process of the scale-up strategies and (3) assess the progress of the scale-up.Methods and analysis A comprehensive framework was developed to include process and scale-up evaluation embedded in implementation science theory. Key implementation outcomes include acceptability, feasibility, relevance, adaptation, adoption and cost of roadmap activities. A diverse range of predominantly qualitative tools—including a policy dialogue reporting form, a stakeholder follow-up interview and survey, project diaries and policy mapping—were developed to assess how stakeholders perceive the scale-up implementation process and adaptations to the roadmap. The role of context is considered relevant, and barriers and facilitators to scale-up will be continuously assessed.Ethics and dissemination Ethical approval has been obtained from the Institutional Review Board (ref. 1323/19) at the Institute of Tropical Medicine (Antwerp, Belgium). The SCUBY Project presents a comprehensive framework to guide the process and scale-up evaluation of complex interventions in different health systems. We describe how implementation outcomes, mechanisms of impact and scale-up outcomes can be a basis to monitor adaptations through a co-creation process and to guide other scale-up interventions making use of knowledge translation and co-creation activities.

An animal model that fully recapitulates severe COVID-19 presentation in humans has been a top priority since the discovery of SARS-CoV-2 in 2019. Although multiple animal models are available for mild to moderate clinical disease, models that develop severe disease are still needed. Mink experimentally infected with SARS-CoV-2 developed severe acute respiratory disease, as evident by clinical respiratory disease, radiological, and histological changes. Virus was detected in nasal, oral, rectal, and fur swabs. Deep sequencing of SARS-CoV-2 from oral swabs and lung tissue samples showed repeated enrichment for a mutation in the gene encoding nonstructural protein 6 in open reading frame 1ab. Together, these data indicate that American mink develop clinical features characteristic of severe COVID-19 and, as such, are uniquely suited to test viral countermeasures.

Background: Pathogenic variants in MFN2 cause Charcot-Marie-Tooth disease (CMT) type 2A (CMT2A) and are the leading cause of the axonal subtypes of CMT. CMT2A is characterized by predominantly distal motor weakness and muscle atrophy, with highly variable severity and onset age. Notably, some MFN2 variants can also lead to other phenotypes such as optic atrophy, hearing loss and lipodystrophy. Despite the clear link between MFN2 and CMT2A, our mechanistic understanding of how dysfunction of the MFN2 protein causes human disease pathologies remains incomplete. This lack of understanding is due in part to the multiple cellular roles of MFN2. Though initially characterized for its role in mediating mitochondrial fusion, MFN2 also plays important roles in mediating interactions between mitochondria and other organelles, such as the endoplasmic reticulum and lipid droplets. Additionally, MFN2 is also important for mitochondrial transport, mitochondrial autophagy, and has even been implicated in lipid transfer. Though over 100 pathogenic MFN2 variants have been described to date, only a few have been characterized functionally, and even then, often only for one or two functions. Method: Several MFN2-mediated functions were characterized in fibroblast cells from a patient presenting with cerebellar ataxia, deafness, blindness, and diffuse cerebral and cerebellar atrophy, who harbours a novel homozygous MFN2 variant, D414V, which is found in a region of the HR1 domain of MFN2 where few pathogenic variants occur. Results: We found evidence for impairment of several MFN2-mediated functions. Consistent with reduced mitochondrial fusion, patient fibroblasts exhibited more fragmented mitochondrial networks and had reduced mtDNA copy number. Additionally, patient fibroblasts had reduced oxygen consumption, fewer mitochondrial-ER contacts, and altered lipid droplets that displayed an unusual perinuclear distribution. Conclusion: Overall, this work characterizes D414V as a novel variant in MFN2 and expands the phenotypic presentation of MFN2 variants to include cerebellar ataxia.

Summary: Background: Better understanding of the association between characteristics of patients hospitalized with coronavirus disease 2019 (COVID-19) and outcome is needed to further improve upon patient management. Methods: Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective, observational study of 1164 patients from 20 hospitals across the United States. Disease severity was assessed using a 7-point ordinal scale based on degree of respiratory illness. Patients were prospectively surveyed for 1 year after discharge for post-acute sequalae of COVID-19 (PASC) through quarterly surveys. Demographics, comorbidities, radiographic findings, clinical laboratory values, SARS-CoV-2 PCR and serology were captured over a 28-day period. Multivariable logistic regression was performed. Findings: The median age was 59 years (interquartile range [IQR] 20); 711 (61%) were men; overall mortality was 14%, and 228 (20%) required invasive mechanical ventilation. Unsupervised clustering of ordinal score over time revealed distinct disease course trajectories. Risk factors associated with prolonged hospitalization or death by day 28 included age ≥ 65 years (odds ratio [OR], 2.01; 95% CI 1.28–3.17), Hispanic ethnicity (OR, 1.71; 95% CI 1.13–2.57), elevated baseline creatinine (OR 2.80; 95% CI 1.63– 4.80) or troponin (OR 1.89; 95% 1.03–3.47), baseline lymphopenia (OR 2.19; 95% CI 1.61–2.97), presence of infiltrate by chest imaging (OR 3.16; 95% CI 1.96–5.10), and high SARS-CoV2 viral load (OR 1.53; 95% CI 1.17–2.00). Fatal cases had the lowest ratio of SARS-CoV-2 antibody to viral load levels compared to other trajectories over time (p=0.001). 589 survivors (51%) completed at least one survey at follow-up with 305 (52%) having at least one symptom consistent with PASC, most commonly dyspnea (56% among symptomatic patients). Female sex was the only associated risk factor for PASC. Interpretation: Integration of PCR cycle threshold, and antibody values with demographics, comorbidities, and laboratory/radiographic findings identified risk factors for 28-day outcome severity, though only female sex was associated with PASC. Longitudinal clinical phenotyping offers important insights, and provides a framework for immunophenotyping for acute and long COVID-19. Funding: NIH.

Ciliopathies are a class of genetic diseases resulting in cilia dysfunction in multiple organ systems, including the olfactory system. Currently, there are no available curative treatments for olfactory dysfunction and other symptoms in ciliopathies. The loss or shortening of olfactory cilia, as seen in multiple mouse models of the ciliopathy Bardet–Biedl syndrome (BBS), results in olfactory dysfunction. However, the underlying mechanism of the olfactory cilia reduction is unknown, thus limiting the development of therapeutic approaches for BBS and other ciliopathies. Here, we demonstrated that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a phosphoinositide typically excluded from olfactory cilia, aberrantly redistributed into the residual cilia of BBS mouse models, which caused F-actin ciliary infiltration. Importantly, PI(4,5)P2 and F-actin were necessary for olfactory cilia shortening. Using a gene therapeutic approach, the hydrolyzation of PI(4,5)P2 by overexpression of inositol polyphosphate-5-phosphatase E (INPP5E) restored cilia length and rescued odor detection and odor perception in BBS. Together, our data indicate that PI(4,5)P2/F-actin–dependent cilia disassembly is a common mechanism contributing to the loss of olfactory cilia in BBS and provide valuable pan-therapeutic intervention targets for the treatment of ciliopathies.

COVID-19 vaccination raised concerns about its potential effects on physical performance. To assess the impact of COVID-19 vaccination on the perceived change in physical performance, we conducted an online survey among elite athletes from Belgium, Canada, France and Luxembourg, with questions about socio-demographics, COVID-19 vaccination, perceived impact on physical performance and perceived pressure to get vaccinated. Full vaccination was defined as two doses of mRNA or vector vaccine or a heterologous vaccine scheme. Among 1106 eligible athletes contacted, 306 athletes answered the survey and were included in this study. Of these, 72% perceived no change in their physical performance, 4% an improvement and 24% a negative impact following full COVID-19 vaccination. For 82% of the included athletes, the duration of the negative vaccine reactions was ≤3 days. After adjustment for potential confounding variables, practicing an individual sport, a duration of vaccine reactions longer than 3 days, a high level of vaccine reaction and the perceived pressure to get vaccinated were independently associated with a perceived negative impact on physical performance of more than 3 days after the vaccination. The perceived pressure to get vaccinated appears to be a parameter associated with the negative perceived change in the physical performance and deserves further consideration.

Introduction To enhance health and prevent secondary consequences for patients with cardiovascular disease (CVD), maintenance of an active lifestyle following participation in cardiac rehabilitation (CR) is important. However, levels of physical activity often decrease after completion of a structured CR programme. Models that support long-term behaviour change with a sustained level of physical activity are imperative. The aim of this study is to evaluate the feasibility of a mobile health intervention based on the Health Action Process Approach theoretical model of behaviour change in patients with CVD for 3 months after completion of a CR programme.Methods and analysis In a feasibility trial design, we will recruit 40 participants from CR programmes at Slagelse Hospital, the City of Slagelse (municipality), or Holbæk Hospital. After completing the standard structured CR programme, each participant will create an action plan for physical activity together with a physiotherapist. Following that, participants are sent 2 weekly text messages for 3 months. The first text message prompts physical activity, and the second will check if the action plan has been followed. If requested by participants, a coordinator will call and guide the physical activities behaviour. The feasibility of this maintenance intervention is evaluated based on predefined progression criteria. Physical activity is measured with accelerometers at baseline and at 3 months follow-up.Ethics and dissemination Study approval was waived (EMN-2021-00020) by the Research Ethics Committee of Region Zealand, Denmark. Study results will be made public and findings disseminated to patients, health professionals, decision-makers, researchers and the public.Trial registration number NCT05011994.

BackgroundDespite optimal medical and surgical intervention, freezing of gait commonly occurs in people with Parkinson disease. Action observation via video self-modeling, combined with physical practice, has potential as a noninvasive intervention to reduce freezing of gait. ObjectiveThe aim of this study is to determine the feasibility and acceptability of a home-based, personalized video self-modeling intervention delivered via a virtual reality head-mounted display (HMD) to reduce freezing of gait in people with Parkinson disease. The secondary aim is to investigate the potential effect of this intervention on freezing of gait, mobility, and anxiety. MethodsThe study was a single-group pre-post mixed methods pilot trial for which 10 participants with Parkinson disease and freezing of gait were recruited. A physiotherapist assessed the participants in their homes to identify person-specific triggers of freezing and developed individualized movement strategies to overcome freezing of gait. 180° videos of the participants successfully performing their movement strategies were created. Participants watched their videos using a virtual reality HMD, followed by physical practice of their strategies in their own homes over a 6-week intervention period. The primary outcome measures included the feasibility and acceptability of the intervention. Secondary outcome measures included freezing of gait physical tests and questionnaires, including the Timed Up and Go Test, 10-meter walk test, Goal Attainment Scale, and Parkinson Anxiety Scale. ResultsThe recruitment rate was 24% (10/42), and the retention rate was 90% (9/10). Adherence to the intervention was high, with participants completing a mean of 84% (SD 49%) for the prescribed video viewing and a mean of 100% (SD 56%) for the prescribed physical practice. One participant used the virtual reality HMD for 1 week and completed the rest of the intervention using a flat-screen device because of a gradual worsening of his motion sickness. No other adverse events occurred during the intervention or assessment. Most of the participants found using the HMD to view their videos interesting and enjoyable and would choose to use this intervention to manage their freezing of gait in the future. Five themes were constructed from the interview data: reflections when seeing myself, my experience of using the virtual reality system, the role of the virtual reality system in supporting my learning, developing a deeper understanding of how to manage my freezing of gait, and the impact of the intervention on my daily activities. Overall, there were minimal changes to the freezing of gait, mobility, or anxiety measures from baseline to postintervention, although there was substantial variability between participants. The intervention showed potential in reducing anxiety in participants with high levels of anxiety. ConclusionsVideo self-modeling using an immersive virtual reality HMD plus physical practice of personalized movement strategies is a feasible and acceptable method of addressing freezing of gait in people with Parkinson disease.