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Background. Preformed donor-specific HLA antibodies (DSA) are a well-known risk factor in kidney transplantation. There is still considerable debate, however, about the optimal risk stratification among patients with preformed DSA. Additionally, data on the prognostic value of different crossmatch assays in DSA-positive patients are scarce. Methods. DSA-positive living kidney transplant recipients were selected from a multicenter study examining 4233 consecutive renal transplants. An additional 7 patients from 2 further centers were included. Flow cytometric crossmatches (FXM), Luminex-based crossmatches, and virtual crossmatches based on C1q- and C3d-binding antibodies (C1qXM and C3dXM) were performed retrospectively using pretransplant sera and lymphocytes isolated from fresh samples. These samples were obtained from 44 donor and recipient pairs from 12 centers. Clinical outcome data and the control group without DSA were compiled from the previous study and were supplemented by data on 10-y death-censored graft survival (10yGS). Results. Between 19% (C3dXM) and 46% (FXM) of crossmatches were positive. Crossmatch-positive patients showed high incidences of antibody-mediated rejection (AMR) within 6 mo (up to 60% in B-cell FXM+ patients). The incidence of AMR in crossmatch-negative patients ranged between 5% (FXM−) and 13% (C1qXM−). 10yGS was significantly impaired in patients with positive T-cell FXM and total FXM compared with both patients without DSA and those with DSA with negative FXM. Conclusions. Especially FXM are useful for risk stratification, as the outcome of DSA-positive, FXM-negative patients is similar to that of DSA-negative patients, whereas FXM-positive patients have both more AMR and decreased 10yGS. Because of their lower sensitivity, the significance of Luminex-based crossmatches, C1qXM, and C3dXM would have to be examined in patients with stronger DSA.

Abstract Background Impaired intestinal barrier function can enable passage of enteric microorganisms into the bloodstream and lead to nosocomial bloodstream infections during critical illness. We aimed to determine the relative importance of gut translocation as a source for ICU-acquired enterococcal bacteremia of unknown origin. Methods We conducted a nested case–control study in two mixed medical-surgical tertiary ICUs in the Netherlands among patients enrolled between 2011 and 2018. We selected 72 cases with ICU-acquired bacteremia due to enterococci (which are known gastrointestinal tract commensals) and 137 matched controls with bacteremia due to coagulase-negative staphylococci (CoNS) (which are of non-intestinal origin). We measured intestinal fatty acid-binding protein, trefoil factor-3, and citrulline 48 h before bacteremia onset. A composite measure for Gut Barrier Injury (GBI) was calculated as the sum of standardized z-scores for each biomarker plus a clinical gastrointestinal failure score. Results No single biomarker yielded statistically significant differences between cases and controls. Median composite GBI was higher in cases than in controls (0.58, IQR − 0.36–1.69 vs. 0.32, IQR − 0.53–1.57, p = 0.33) and higher composite measures of GBI correlated with higher disease severity and ICU mortality (p

Abstract Burkitt lymphoma (BL) is responsible for many childhood cancers in sub-Saharan Africa, where it is linked to recurrent or chronic infection by Epstein-Barr virus or Plasmodium falciparum. However, whether human leukocyte antigen (HLA) polymorphisms, which regulate immune response, are associated with BL has not been well investigated, which limits our understanding of BL etiology. Here we investigate this association among 4,645 children aged 0-15 years, 800 with BL, enrolled in Uganda, Tanzania, Kenya, and Malawi. HLA alleles are imputed with accuracy >90% for HLA class I and 85-89% for class II alleles. BL risk is elevated with HLA-DQA1*04:01 (adjusted odds ratio [OR] = 1.61, 95% confidence interval [CI] = 1.32-1.97, P = 3.71 × 10−6), with rs2040406(G) in HLA-DQA1 region (OR = 1.43, 95% CI = 1.26-1.63, P = 4.62 × 10−8), and with amino acid Gln at position 53 versus other variants in HLA-DQA1 (OR = 1.36, P = 2.06 × 10−6). The associations with HLA-DQA1*04:01 (OR = 1.29, P = 0.03) and rs2040406(G) (OR = 1.68, P = 0.019) persist in mutually adjusted models. The higher risk rs2040406(G) variant for BL is associated with decreased HLA-DQB1 expression in eQTLs in EBV transformed lymphocytes. Our results support the role of HLA variation in the etiology of BL and suggest that a promising area of research might be understanding the link between HLA variation and EBV control.

Background: Speech and language disorders in childhood have the potential to affect every aspect of a child's day-to-day life and can potentially have negative long-term impacts. Aim: This scoping review seeks to collate the existing evidence to identify the long-term effects of childhood speech and language disorders. Methods: A systematic search of speechBITE, ERIC (Education Resources Information Center), Linguistics and Language Behaviour Abstracts, PubMed, MEDLINE, PsycINFO, SocINDEX and the Cochrane Library was conducted. Peer-reviewed English language publications reporting on the long-term (2+-year) outcomes of individuals with a childhood history of speech or language disorders were included. Data were extracted and the study quality assessed using a modified Newcastle-Ottawa scale. Results: Fifty-one studies met the inclusion criteria. These studies reported mixed results, the most common of which were suboptimal mental health, social and academic outcomes for persons with a history of speech or language disorders. We found an association between childhood speech or language disorders and psychiatric disability, behavioural problems, lower socio-economic status, relationship and living difficulties, and lower academic achievement compared to the general population. Conclusion: Individuals with a history of childhood speech or language disorders may experience long-term difficulties in mental health, social well-being and academic outcomes.

ABSTRACT Vitamin B12 (B12) serves as a critical cofactor within mycobacterial metabolism. While some pathogenic strains can synthesize B12 de novo, others rely on host-acquired B12. In this investigation, we studied the transport of vitamin B12 in Mycobacterium marinum using B12-auxotrophic and B12-sensitive strains by deleting metH or metE, respectively. These two enzymes rely on B12 in different ways to function as methionine synthases. We used these strains to select mutants affecting B12 scavenging and confirmed their phenotypes during growth experiments in vitro. Our analysis of B12 uptake mechanisms revealed that membrane lipids and cell wall integrity play an essential role in cell envelope transport. Furthermore, we identified a potential transcription regulator that responds to B12. Our study demonstrates that M. marinum can take up exogenous B12 and that altering mycobacterial membrane integrity affects B12 uptake. Finally, during zebrafish infection using B12-auxotrophic and B12-sensitive strains, we found that B12 is available for virulent mycobacteria in vivo.IMPORTANCEOur study investigates how mycobacteria acquire essential vitamin B12. These microbes, including those causing tuberculosis, face challenges in nutrient uptake due to their strong outer layer. We focused on Mycobacterium marinum, similar to TB bacteria, to uncover its vitamin B12 absorption. We used modified strains unable to produce their own B12 and discovered that M. marinum can indeed absorb it from the environment, even during infections. Changes in the outer layer composition affect this process, and genes related to membrane integrity play key roles. These findings illuminate the interaction between mycobacteria and their environment, offering insights into combatting diseases like tuberculosis through innovative strategies. Our concise research underscores the pivotal role of vitamin B12 in microbial survival and its potential applications in disease control.

Abstract Pulmonary artery pseudoaneurysm (PAP) is a rare cause of hemoptysis. Potential causes include trauma, infection, or medical interventions. There is a risk of rupture, which is associated with a high mortality rate. We describe a 72‐year‐old patient, with a past medical history of a lung carcinoma for which she was treated with chemoradiotherapy 6 years prior, who presented with hemoptysis. She was hemodynamically stable and there were no other complaints. CT angiography of the thorax showed a PAP originating from a branch of the right pulmonary artery in the previously irradiated area. The patient was successfully treated by an embolization with plugs. Treatment of lung carcinoma with chemoradiotherapy can result in the development of a PAP. Clinicians should be aware of this complication, even years after the therapy. In literature, only a few cases of PAP in patients treated with (chemo)radiotherapy for lung cancer are described, with a maximum interval up to 7 years.

Abstract The intestinal epithelium is a single cell layer that is constantly renewed and acts as a physical barrier that separates intestinal microbiota from underlying tissues. In inflammatory bowel disease (IBD) in humans, as well as in experimental mouse models of IBD, this barrier is impaired, causing microbial infiltration and inflammation. Deficiency in OTU deubiquitinase with linear linkage specificity (OTULIN) causes OTULIN-related autoinflammatory syndrome (ORAS), a severe inflammatory pathology affecting multiple organs including the intestine. We show that mice with intestinal epithelial cell (IEC)-specific OTULIN deficiency exhibit increased susceptibility to experimental colitis and are highly sensitive to TNF toxicity, due to excessive apoptosis of OTULIN deficient IECs. OTULIN deficiency also increases intestinal pathology in mice genetically engineered to secrete excess TNF, confirming that chronic exposure to TNF promotes epithelial cell death and inflammation in OTULIN deficient mice. Mechanistically we demonstrate that upon TNF stimulation, OTULIN deficiency impairs TNF receptor complex I formation and LUBAC recruitment, and promotes the formation of the cytosolic complex II inducing epithelial cell death. Finally, we show that OTULIN deficiency in IECs increases susceptibility to Salmonella infection, further confirming the importance of OTULIN for intestinal barrier integrity. Together, these results identify OTULIN as a major anti-apoptotic protein in the intestinal epithelium and provide mechanistic insights into how OTULIN deficiency drives gastrointestinal inflammation in ORAS patients.

Abstract Purely technical interventions aimed at enhancing evidence-informed decision-making (EIDM) have rarely translated into organizational institutionalization or systems change. A panel of four presentations at the Health Systems Global 2020 conference provides a basis for inference about contextual factors that influence the establishment and sustainability of institutional platforms to support EIDM. These cases include local structures such as citizen panels in Uganda, regional knowledge translation structures such as the West African Health Organization, global multilateral initiatives such as the “One Health” Quadrapartite and regional public health networks in South-East Asia. They point to the importance of political economy as well as technical capability determinants of evidence uptake and utilization at institutional, organizational and individual levels. The cases also lend support to evidence that third-party (broker and intermediary) supportive institutions can facilitate EIDM processes. The involvement of third-party supranational organizations, however, poses challenges in terms of legitimacy and accountability.

BackgroundThe use of research evidence in health policy-making is a popular line of inquiry for scholars of public health and policy studies, with qualitative methods constituting the dominant strategy in this area. Research on this subject has been criticized for, among other things, disproportionately focusing on high-income countries; overemphasizing ‘barriers and facilitators’ related to evidence use to the neglect of other, less descriptive concerns; relying on descriptive, rather than in-depth explanatory designs; and failing to draw on insights from political/policy studies theories and concepts. We aimed to comprehensively map the global, peer-reviewed qualitative literature on the use of research evidence in health policy-making and to provide a descriptive overview of the geographic, temporal, methodological, and theoretical characteristics of this body of literature. MethodsWe conducted a systematic review following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. We searched nine electronic databases, hand-searched 11 health- and policy-related journals, and systematically scanned the reference lists of included sudies and previous reviews. No language, date or geographic limitations were imposed. ResultsThe review identified 319 qualitative studies on a diverse array of topics related to the use of evidence in health policy-making, spanning 72 countries and published over a nearly 40 year period. A majority of these studies were conducted in high-income countries, but a growing proportion of the research output in this area is now coming from low- and middle-income countries, especially from sub-Saharan Africa. While over half of all studies did not use an identifiable theory or framework, and only one fifth of studies used a theory or conceptual framework drawn from policy studies or political science, we found some evidence that theory-driven and explanatory (eg, comparative case study) designs are becoming more common in this literature. Investigations of the barriers and facilitators related to evidence use constitute a large proportion but by no means a majority of the work in this area. ConclusionThis review provides a bird’s eye mapping of the peer reviewed qualitative research on evidence-to-policy processes, and has identified key features of – and gaps within – this body of literature that will hopefully inform, and improve, research in this area moving forward.

Abstract Ecologically specialized plants are expected to be at greater risk of extinction than generalists due to climate change. Such risk is greatest in biodiversity hotspots such as the Greater Cape Floristic Region (GCFR), which accommodates both ecological specialists and generalists. Thesium L., a genus with the highest number of species in Santalaceae and the most diverse in Africa, offers an appropriate system for evaluating both the correlates of range extent and specialization and the relative extinction risks associated with both. We hypothesized that range size, ecological specialization, and consequent climatically modulated extinction risks are all phylogenetically structured, such that climate change will precipitate a disproportionate loss of phylogenetic diversity in the GCFR Thesium. Past and future species distribution ranges were predicted using MaxEnt models based on present‐day occurrences and environmental conditions. Of the 101 Thesium species modeled, 70% have had large range sizes during the Last Glacial Maximum (LGM), 50% currently have a large range size, and future conditions are predicted to allow 40% to obtain large range sizes. Between the LGM and the present, 17% of species are thought to have undergone a contraction of available range space in the present time whereas 37% are expected to expand their ranges into the future, while 51% of species will experience range contractions. Of the 65 species currently ranked as Least Concern in the South African Red List, 24% will likely shift into higher extinction risk categories. Interestingly, 8.5% of ecological specialists, although having experienced a range reduction from the LGM to the present, are predicted to persist in the face of future climate change. However, the range extent, ecological specialization, and extinction risk are phylogenetically random and therefore should have a negligible impact on the phylogenetic diversity of the GCFR Thesium.

Background: Systematic reviews cataloguing the barriers to and facilitators of various outcomes are increasingly popular, despite criticisms of this type of review on philosophical, methodological, and practical grounds. The aims of this review are to appraise, analyze, and discuss the reporting and synthesis practices used in recently published barriers and facilitators reviews in health services research. Methods: All English-language peer-reviewed systematic reviews that synthesized research on barriers and facilitators in a health services context were eligible for inclusion. We searched 11 databases over a 13-month period (1 November 2017-30 November 2018) using an exhaustive list of search terms for "barrier(s)," "facilitator(s)," and "systematic review." Results: One hundred reviews were included. We found a high degree of variation in the synthesis practices used in these reviews, with the majority employing aggregative (rather than interpretive) approaches. The findings echo common critiques of this review type, including concerns about the reduction of complex phenomena to simplified, discrete factors. Although several reviews highlighted the "complexity" of barriers and facilitators, this was usually not analyzed systematically. Analysis of the subsample of reviews that explicitly discussed the barriers and facilitators approach revealed some common issues. These tended to be either conceptual/definitional (eg, ideas about interrelationships and overlap between factors) and methodological/practical (eg, challenges related to aggregating heterogeneous research). Conclusion: Barriers and facilitators reviews should (a) clearly operationally define "barrier" and "facilitator," (b) explicitly describe how factors are extracted and subsequently synthesized, and (c) provide critical reflection on the contextual variability and reliability of identified factors.

OTULIN is a deubiquitinase for linear ubiquitin chains. Here the authors show, using genetic mouse models and single-cell RNA-sequencing, that deficiency of OTULIN in keratinocytes causes skin inflammation and verrucous carcinoma via the induction of keratinocyte death, thereby implicating a function of OTULIN in keratinocyte homeostasis.

Abnormalities at any stage of trophoblast development may result in pregnancy-related complications. Many of these adverse outcomes are discovered later in pregnancy, but the underlying pathomechanisms are constituted during the first trimester. Acquiring developmentally relevant material to elucidate the disease mechanisms is difficult. Human pluripotent stem cell (hPSC) technology can provide a renewable source of relevant cells. BMP4, A83-01, and PD173074 (BAP) treatment drives trophoblast commitment of hPSCs toward syncytiotrophoblast (STB), but lacks extravillous trophoblast (EVT) cells. EVTs mediate key functions during placentation, remodeling of uterine spiral arteries, and maintenance of immunological tolerance. We optimized the protocol for a more efficient generation of HLA-Gpos EVT-like trophoblasts from primed hiPSCs. Increasing the concentrations of A83-01 and PD173074, while decreasing bulk cell density resulted in an increase in HLA-G of up to 71%. Gene expression profiling supports the advancements of our treatment regarding the generation of trophoblast cells. The reported differentiation protocol will allow for an on-demand access to human trophoblast cells enriched for HLA-Gpos EVT-like cells, allowing for the elucidation of placenta-related disorders and investigating the immunological tolerance toward the fetus, overcoming the difficulties in obtaining primary EVTs without the need for a complex differentiation pathway via naïve pluripotent or trophoblast stem cells.

Objectives: Evaluation of the feasibility of SARS-CoV-2-specific T cell manufacturing for adoptive T cell transfer in COVID-19 patients at risk to develop severe disease.Methods: Antiviral SARS-CoV-2-specific T cells were detected in blood of convalescent COVID-19 patients following stimulation with PepTivator SARS-CoV-2 Select using Interferon-gamma Enzyme-Linked Immunospot (IFN-γ ELISpot), SARS-CoV-2 T Cell Analysis Kit (Whole Blood) and Cytokine Secretion Assay (CSA) and were characterized with respect to memory phenotype, activation state and cytotoxic potential by multicolor flow cytometry, quantitative real-time PCR and multiplex analyses. Clinical-grade SARS-CoV-2-specific T cell products were generated by stimulation with MACS GMP PepTivator SARS-CoV-2 Select using CliniMACS Prodigy and CliniMACS Cytokine Capture System (IFN-gamma) (CCS). Functionality of enriched T cells was investigated in cytotoxicity assays and by multiplex analysis of secreted cytotoxic molecules upon target recognition.Results: Donor screening via IFN-γ ELISpot allows for pre-selection of potential donors for generation of SARS-CoV-2-specific T cells. Antiviral T cells reactive against PepTivator SARS-CoV-2 Select could be magnetically enriched from peripheral blood of convalescent COVID-19 patients by small-scale CSA resembling the clinical-grade CCS manufacturing process and showed an activated and cytotoxic T cell phenotype. Four clinical-grade SARS-CoV-2-specific T cell products were successfully generated with sufficient cell numbers and purities comparable to those observed in donor pretesting via CSA. The T cells in the generated products were shown to be capable to replicate, specifically recognize and kill target cells in vitro and secrete cytotoxic molecules upon target recognition. Cell viability, total CD3+ cell number, proliferative capacity and cytotoxic potential remained stable throughout storage of up to 72 h after end of leukapheresis.Conclusion: Clinical-grade SARS-CoV-2-specific T cells are functional, have proliferative capacity and target-specific cytotoxic potential. Their function and phenotype remain stable for several days after enrichment. The adoptive transfer of partially matched, viable human SARS-CoV-2-specific T lymphocytes collected from convalescent individuals may provide the opportunity to support the immune system of COVID-19 patients at risk for severe disease.

Abstract The consequences of bird mortality caused by collisions with wind turbines are increasingly receiving attention. So‐called acceptable mortality limits of populations, that is, those that assume that 1%–5% of additional mortality and the potential biological removal (PBR), provide seemingly clear‐cut methods for establishing the reduction in population viability. We examine how the application of these commonly used mortality limits could affect populations of the Common Starling, Black‐tailed Godwit, Marsh Harrier, Eurasian Spoonbill, White Stork, Common Tern, and White‐tailed Eagle using stochastic density‐independent and density‐dependent Leslie matrix models. Results show that population viability can be very sensitive to proportionally small increases in mortality. Rather than having a negligible effect, we found that a 1% additional mortality in postfledging cohorts of our studied populations resulted in a 2%–24% decrease in the population level after 10 years. Allowing a 5% mortality increase to existing mortality resulted in a 9%–77% reduction in the populations after 10 years. When the PBR method is used in the density‐dependent simulations, the proportional change in the resulting growth rate and carrying capacity was species‐independent and largely determined by the recovery factor (Fr). When Fr = 1, a value typically used for robust populations, additional mortality resulted in a 50%–55% reduction in the equilibrium density and the resulting growth rate. When Fr = 0.1, used for threatened populations, the reduction in the equilibrium density and growth rate was about 5%. Synthesis and applications. Our results show that by allowing a mortality increase from wind farm collisions according to both criteria, the population impacts of these collisions can still be severe. We propose a simple new method as an alternative that was able to estimate mortality impacts of age‐structured stochastic density‐dependent matrix models.

Subcutaneous emphysema, pneumothorax and pneumomediastinum are well-known complications of invasive ventilation in patients with acute hypoxemic respiratory failure. We determined the incidences of air leaks that were visible on available chest images in a cohort of critically ill patients with acute hypoxemic respiratory failure due to coronavirus disease of 2019 (COVID-19) in a single-center cohort in the Netherlands. A total of 712 chest images from 154 patients were re-evaluated by a multidisciplinary team of independent assessors; there was a median of three (2–5) chest radiographs and a median of one (1–2) chest CT scans per patient. The incidences of subcutaneous emphysema, pneumothoraxes and pneumomediastinum present in 13 patients (8.4%) were 4.5%, 4.5%, and 3.9%. The median first day of the presence of an air leak was 18 (2–21) days after arrival in the ICU and 18 (9–22)days after the start of invasive ventilation. We conclude that the incidence of air leaks was high in this cohort of COVID-19 patients, but it was fairly comparable with what was previously reported in patients with acute hypoxemic respiratory failure in the pre-COVID-19 era.

BackgroundAs chronic cardiac and pulmonary diseases often coexist, there is a need for combined physical home-based rehabilitation programs, specifically addressing older patients with advanced disease stages. ObjectiveThe primary aim of this study is to evaluate the completion and adherence rates of an 8-week, home-based exercise program for patients with advanced cardiopulmonary disease. The secondary end points include patient satisfaction; adverse events; and program efficacy in terms of change in functional capacity, level of dyspnea, and health-related quality of life. MethodsThe participants received a goal-oriented, home-based exercise program, and they used a wrist-worn activity tracker to record their exercise sessions. Activity tracker data were made visible on a digital platform, which was also equipped with several other features such as short instruction videos on how to perform specific exercises. The participants received weekly coaching by a physiotherapist and an occupational therapist through video communication. ResultsIn all, 10 patients with advanced combined cardiopulmonary disease participated (median age 71, IQR 63-75 years), and 50% (5/10) were men. Of the 10 participants, 9 (90%) completed the 8-week program. Median adherence to the exercise prescription was 75% (IQR 37%-88%), but it declined significantly when the program was divided into 2-week periods (first 2 weeks: 86%, IQR 51%-100%, and final 2 weeks: 57%, IQR 8%-75%; P=.03). The participants were highly satisfied with the program (Client Satisfaction Questionnaire: median score 29, IQR 26-32, and Purpose-Designed Questionnaire: median score 103, IQR 92-108); however, of the 9 participants, 4 (44%) experienced technical issues. The Patient-Specific Complaints Instrument scores declined, indicating functional improvement (from median 7.5, IQR 6.1-8.9, to median 5.7, IQR 3.8-6.7; P=.01). Other program efficacy metrics showed a trend toward improvement. ConclusionsHome-based cardiopulmonary telerehabilitation for patients with severe combined cardiopulmonary disease is feasible in terms of high completion and satisfaction rates. Nevertheless, a decrease in adherence during the program was observed, and some of the participants reported difficulties with the technology, indicating the importance of the integration of behavior change techniques, using appropriate technology. Trial RegistrationNetherlands Trial Register NL9182; https://www.trialregister.nl/trial/9182

A biodiversity survey on three corallivorous snails (Mollusca: Gastropoda) was performed at 28 sites around the island of Bonaire to assess their distribution patterns and associated host corals. The snails and their hosts were identified and counted in three depth zones: 5–10, 10–20, and 20–30 m. The snails were Coralliophila galea and C. salebrosa (Muricidae: Coralliophilinae), and Cyphoma gibbosum (Ovulidae: Simniinae). All three species were widespread around the island without apparent interspecific geographical variation. Coralliophila galea was found exclusively on scleractinian corals, Coralliophila salebrosa almost exclusively on octocorals, and Cyphoma gibbosum only on octocorals. Coralliophila salebrosa showed more dietary overlap with Cyphoma gibbosum than with Coralliophila galea. Coralliophila galea was the most commonly encountered species with the largest number of host species. Owing to its hosts distribution, this species also showed a greater maximum depth and a wider bathymetrical range than the other two snails. The other two snails were shallower and their depth ranges did not differ significantly. Host-coral size did not seem to have influence on the number of snails per host. Coral damage caused by the snails was visible but appeared to be low, causing no mortality in Bonaire, which suggests that the relation with their hosts is more parasitic than predatory. Because these three corallivores have occasionally been reported to occur as outbreaks in other Caribbean localities and may act as vectors in the dispersal of coral diseases, it is recommended that future studies should focus on their population dynamics.

Abstract Background Early recognition of sepsis is challenging, and diagnostic criteria have changed repeatedly. We assessed the robustness of sepsis-3 criteria in intensive care unit (ICU) patients. Methods We studied the apparent incidence and associated mortality of sepsis-3 among patients who were prospectively enrolled in the Molecular Diagnosis and Risk Stratification of Sepsis (MARS) cohort in the Netherlands, and explored the effects of minor variations in the precise definition and timing of diagnostic criteria for organ failure. Results Among 1081 patients with suspected infection upon ICU admission, 648 (60%) were considered to have sepsis according to prospective adjudication in the MARS study, whereas 976 (90%) met sepsis-3 criteria, yielding only 64% agreement at the individual patient level. Among 501 subjects developing ICU-acquired infection, these rates were 270 (54%) and 260 (52%), respectively (yielding 58% agreement). Hospital mortality was 234 (36%) vs 277 (28%) for those meeting MARS-sepsis or sepsis-3 criteria upon presentation (p < 0.001), and 121 (45%) vs 103 (40%) for those having sepsis onset in the ICU (p < 0.001). Minor variations in timing and interpretation of organ failure criteria had a considerable effect on the apparent prevalence of sepsis-3, which ranged from 68 to 96% among those with infection at admission, and from 22 to 99% among ICU-acquired cases. Conclusion The sepsis-3 definition lacks robustness as well as discriminatory ability, since nearly all patients presenting to ICU with suspected infection fulfill its criteria. These should therefore be specified in greater detail, and applied more consistently, during future sepsis studies. Trial registration The MARS study is registered at ClinicalTrials.gov (identifier NCT 01905033).

Abstract Background Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis. Results We evaluated two suitable RNA isolation kits (the RNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5× dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results. Conclusions The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells.

BackgroundChronic heart failure (CHF) and chronic obstructive pulmonary disease (COPD) often coexist and are associated with a high morbidity and reduced quality of life (QoL). Although these diseases share similarities in symptoms and clinical course, and exacerbations of both diseases often overlap, care pathways for both conditions are usually not integrated. This results in frequent outpatient consultations and suboptimal treatment during exacerbations, leading to frequent hospital admissions. Therefore, we propose an integrated care pathway for both diseases, using telemonitoring to detect deterioration at an early stage and a single case manager for both diseases. ObjectiveThis study aims to investigate whether an integrated care pathway using telemonitoring in patients with combined CHF and COPD results in a higher general health-related QoL (HRQoL) as compared with the traditional care pathways. Secondary end points include disease-specific HRQoL, level of self-management, patient satisfaction, compliance to the program, and cost-effectiveness. MethodsThis is a monocenter, prospective study using a quasi-experimental interrupted time series design. Thirty patients with combined CHF and COPD are included. The study period of 2.5 years per patient is divided into a preintervention phase (6 months) and a postintervention phase (2 years) in which end points are assessed. The intervention consists of an on-demand treatment strategy based on monitoring symptoms related to CHF/COPD and vital parameters (weight, blood pressure, heart rate, oxygen saturation, temperature), which are uploaded on a digital platform. The monitoring frequency and the limit values of the measurements to detect abnormalities are determined individually. Monitoring is performed by a case manager, who has the opportunity for a daily multidisciplinary meeting with both the cardiologist and the pulmonologist. Routine appointments at the outpatient clinic are cancelled and replaced by telemonitoring-guided treatment. ResultsFollowing ethical approval of the study protocol, the first patient was included in May 2018. Inclusion is expected to be complete in May 2021. ConclusionsThis study is the first to evaluate the effects of a novel integrated care pathway using telemonitoring for patients with combined CHF and COPD. Unique to this study is the concept of remote on-demand disease management by a single case manager for both diseases, combined with multidisciplinary meetings. Moreover, modern telemonitoring technology is used instead of, rather than as an addition to, regular care. Trial RegistrationNetherlands Trial Register NL6741; https://www.trialregister.nl/trial/6741 International Registered Report Identifier (IRRID)DERR1-10.2196/20571

Background: Speech and language disorders in childhood have the potential to affect every aspect of a child’s day-to-day life and can potentially have negative long-term impacts. Aim: This scoping review seeks to collate the existing evidence to identify the long-term effects of childhood speech and language disorders. Methods: A systematic search of speechBITE, ERIC (Education Resources Information Center), Linguistics and Language Behaviour Abstracts, PubMed, MEDLINE, PsycINFO, SocINDEX and the Cochrane Library was conducted. Peer-reviewed English language publications reporting on the long-term (2+-year) outcomes of individuals with a childhood history of speech or language disorders were included. Data were extracted and the study quality assessed using a modified Newcastle–Ottawa scale. Results: Fifty-one studies met the inclusion criteria. These studies reported mixed results, the most common of which were suboptimal mental health, social and academic outcomes for persons with a history of speech or language disorders. We found an association between childhood speech or language disorders and psychiatric disability, behavioural problems, lower socio-economic status, relationship and living difficulties, and lower academic achievement compared to the general population. Conclusion: Individuals with a history of childhood speech or language disorders may experience long-term difficulties in mental health, social well-being and academic outcomes.

Summary: Inflammatory signaling pathways are tightly regulated to avoid chronic inflammation and the development of disease. OTULIN is a deubiquitinating enzyme that controls inflammation by cleaving linear ubiquitin chains generated by the linear ubiquitin chain assembly complex. Here, we show that ablation of OTULIN in liver parenchymal cells in mice causes severe liver disease which is characterized by liver inflammation, hepatocyte apoptosis, and compensatory hepatocyte proliferation, leading to steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). Genetic ablation of Fas-associated death domain (FADD) completely rescues and knockin expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) significantly protects mice from developing liver disease, demonstrating that apoptosis of OTULIN-deficient hepatocytes triggers disease pathogenesis in this model. Finally, we demonstrate that type I interferons contribute to disease in hepatocyte-specific OTULIN-deficient mice. Our study reveals the critical importance of OTULIN in protecting hepatocytes from death, thereby preventing the development of chronic liver inflammation and HCC. : Hepatocellular carcinoma (HCC) develops as a result of chronic liver inflammation. Verboom et al. identify OTULIN as a critical liver-protective protein, essential in preventing hepatocyte apoptosis, which could trigger compensatory hepatocyte proliferation, chronic liver inflammation, fibrosis, and HCC.

While biodiversity hotspots are typically identified on the basis of species number per unit area, their exceptional richness is often attributed, either implicitly or explicitly, to high diversification rates. High species concentrations, however, need not reflect rapid diversification, with the diversity of some hotspots accumulating at modest rates over long timespans. Here we explore the relationship between diversification in time vs. diversification in space and develop the concept of diversification density to describe the spatial scale of species accumulation in a clade. We investigate how plant height is associated with both aspects of diversification in Alooideae, a large plant subfamily with its center of diversity in the Greater Cape Floristic Region. We first reconstruct a time-calibrated phylogeny for Alooideae and demonstrate an evolutionary tendency towards reduced plant height. While plant height does not correlate with diversification rate across Alooideae it does so with diversification per unit space: clades of small plants tend to have the highest diversification densities. Furthermore, we find that diversification in time vs. space are uncorrelated. Our results show that diversification rate and density can be decoupled, and suggest that while some biodiversity hotspots might have been generated by high diversification rates, others are the product of high diversification density.

In high-risk neuroblastoma cases, gains in chromosome 17q are common. Here, the authors investigate the epigenomics and transcriptomics of neuroblastoma, identifying TBX2 as a core regulatory circuitry component enhancing the reactivation of DREAM targets by MYCN/FOXM1.

In this paper we present the continued development of the miniDOAS, an active differential optical absorption spectroscopy (DOAS) instrument used to measure ammonia concentrations in ambient air. The miniDOAS has been adapted for use in the Dutch National Air Quality Monitoring Network. The miniDOAS replaces the life-expired continuous-flow denuder ammonia monitor (AMOR). From September 2014 to December 2015, both instruments measured in parallel before the change from AMOR to miniDOAS was made. The instruments were deployed at six monitoring stations throughout the Netherlands. We report on the results of this intercomparison. Both instruments show a good uptime of ca. 90 %, adequate for an automatic monitoring network. Although both instruments produce 1 min values of ammonia concentrations, a direct comparison on short timescales such as minutes or hours does not give meaningful results because the AMOR response to changing ammonia concentrations is slow. Comparisons between daily and monthly values show good agreement. For monthly averages, we find a small average offset of 0.65 ± 0.28 µg m−3 and a slope of 1.034 ± 0.028, with the miniDOAS measuring slightly higher than the AMOR. The fast time resolution of the miniDOAS makes the instrument suitable not only for monitoring but also for process studies.

Background. Different patterns of plasma cytokines can be expected in the case of chronic active-antibody-mediated (cAMR) and acute cellular rejection (AR) after kidney transplantation (KTx). Methods. IL-2, 4, 6, 10, 17A, tumor necrosis factor alpha, and interferon gamma were measured in 51 pediatric KTx recipients at time of renal biopsy (17 AR, 14 cAMR, 20 normal). Patients were divided into a training (n = 30) and a validation (n = 21) set. Results. IL-6 was significantly higher in AR patients and significantly lower in the case of cAMR. In children with s-creatinine increase, IL-6 values were significantly different between AR and cAMR. IL-10 levels showed similar tendencies. For IL-2, 4, 17A, tumor necrosis factor alpha, and interferon gamma, no differences were found. In the independent validation cohort, the receiver operating characteristic area under the curve for IL-6 was 0.79 and 0.70 for AR and cAMR. In children with AR, an IL-6 721 fg/ml was associated with a specificity of 86%/76%, a sensitivity of 71%/80%, a positive predictive value of 56%/45%, and a negative predictive value of 92%/94%. Conclusions. In this pilot study, the plasma IL-6 level is a promising biomarker to identify pediatric kidney transplant recipients free from AR and cAMR and might help to distinguish between both entities, whereas there is only a nonsignificant trend toward the usability of IL-10. Validation in larger cohorts in combination with other biomarkers are warranted.

Trabectedin and ifosfamide are among the few cytostatic agents active in advanced soft tissue sarcomas (STSs). Trabectedin is most potent against so-called L-sarcomas (leiomyosarcoma and liposarcoma). The survival gain and cost-effectiveness of these agents in a second-line setting were analysed in the setting of advanced STS after failure of anthracyclines. A prospective observational trial had previously been performed to assess the use of trabectedin in a Dutch real-world setting. Data on ifosfamide monotherapy were acquired from previous studies, and an indirect comparison of survival was made. A state-transition economic model was constructed, in which patients could be in mutually exclusive states of being preprogression, postprogression, or deceased. The costs and quality-adjusted life years (QALYs) for both treatments were assessed from a Dutch health-care perspective. Separate analyses for the group of L-sarcomas and non-L-sarcomas were performed. Trabectedin treatment resulted in a median progression-free survival of 5.2 months for L-sarcoma patients, 2.0 months for non-L-sarcoma patients, and a median overall survival of 11.8 and 6.0 months, respectively. For L-sarcoma patients, trabectedin offered an increase of 0.368 life years and 0.251 QALYs compared to ifosfamide and €20,082 in additional costs, for an incremental cost-effectiveness ratio (ICER) of €80,000 per QALY gained. In the non-L-sarcoma patients, trabectedin resulted in 0.413 less life years and 0.266 less QALYs, at the increased cost of €4,698. The difference in survival between drugs and the acquisition costs of trabectedin were the main influences in these models. Trabectedin was shown to have antitumour efficacy in advanced L-sarcoma. From a health economics perspective, the costs per QALY gained compared to ifosfamide monotherapy that may be acceptable, considering what is currently regarded as acceptable in the Netherlands.

Depressive problems and academic performance, social well-being, and social problems in adolescents are strongly associated. However, longitudinal and bidirectional relations between the two remain unclear, as well as the role of gender. Consequently, this study focuses on the relation between depressive problems and three types of functioning in adolescents while testing gender differences. Depressive problems and functioning of 2,230 children were measured with structured questionnaires. The measurements took place biennially over 3 waves, from late childhood into adolescence (age range = 10-18 years). To examine the longitudinal relation between depression and functioning, path analyses with cross-lagged effects were conducted with structural equation modeling. Multigroup analyses were used to test for gender differences, which were only observed for academic performance. Other findings indicated substantial stability in depressive problems and functioning over time and within-wave correlations between depression and the 3 types of functioning. Poor social well-being was predicted by depressive problems but not the other way around. The relation between depressive and social problems was bidirectional, that is, they predicted each other. Finally, depressive problems and academic performance were bidirectionally related as well but only in girls.

The central nervous system (CNS) has specific barriers that protect the brain from potential threats and tightly regulate molecular transport. Despite the critical functions of the CNS barriers, the mechanisms underlying their development and function are not well understood, and there are very limited experimental models for their study. Claudin 5 is a tight junction protein required for blood brain barrier (BBB) and, probably, choroid plexus (CP) structure and function in vertebrates. Here, we show that the gene claudin 5a is the zebrafish orthologue with high fidelity expression, in the BBB and CP barriers, that demonstrates the conservation of the BBB and CP between humans and zebrafish. Expression of claudin 5a correlates with developmental tightening of the BBB and is restricted to a subset of the brain vasculature clearly delineating the BBB. We show that claudin 5a-expressing cells of the CP are ciliated ependymal cells that drive fluid flow in the brain ventricles. Finally, we find that CP development precedes BBB development and that claudin 5a expression occurs simultaneously with angiogenesis. Thus, our novel transgenic zebrafish represents an ideal model to study CNS barrier development and function, critical in understanding the mechanisms underlying CNS barrier function in health and disease.