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BackgroundRemote patient management may improve prognosis in heart failure. Daily review of transmitted data for early recognition of patients at risk requires substantial resources that represent a major barrier to wide implementation. An automated analysis of incoming data for detection of risk for imminent events would allow focusing on patients requiring prompt medical intervention.MethodsWe analysed data of the Telemedical Interventional Management in Heart Failure II (TIM-HF2) randomized trial that were collected during quarterly in-patient visits and daily transmissions from non-invasive monitoring devices. By application of machine learning, we developed and internally validated a risk score for heart failure hospitalisation within seven days following data transmission as estimate of short-term patient risk for adverse heart failure events. Score performance was assessed by the area under the receiver-operating characteristic (ROCAUC) and compared with a conventional algorithm, a heuristic rule set originally applied in the randomized trial.ResultsThe machine learning model significantly outperformed the conventional algorithm (ROCAUC 0.855 vs. 0.727, p

The thymus is the central organ involved with T-cell development and the production of naïve T cells. During normal aging, the thymus undergoes marked involution, reducing naïve T-cell output and resulting in a predominance of long-lived memory T cells in the periphery. Outside of aging, systemic stress responses that induce corticosteroids (CS), or other insults such as radiation exposure, induce thymocyte apoptosis, resulting in a transient acute thymic involution with subsequent recovery occurring after cessation of the stimulus. Despite the increasing utilization of immunostimulatory regimens in cancer, effects on the thymus and naïve T cell output have not been well characterized. Using both mouse and human systems, the thymic effects of systemic immunostimulatory regimens, such as high dose IL-2 (HD IL-2) with or without agonistic anti-CD40 mAbs and acute primary viral infection, were investigated. These regimens produced a marked acute thymic involution in mice, which correlated with elevated serum glucocorticoid levels and a diminishment of naïve T cells in the periphery. This effect was transient and followed with a rapid thymic “rebound” effect, in which an even greater quantity of thymocytes was observed compared to controls. Similar results were observed in humans, as patients receiving HD IL-2 treatment for cancer demonstrated significantly increased cortisol levels, accompanied by decreased peripheral blood naïve T cells and reduced T-cell receptor excision circles (TRECs), a marker indicative of recent thymic emigrants. Mice adrenalectomized prior to receiving immunotherapy or viral infection demonstrated protection from this glucocorticoid-mediated thymic involution, despite experiencing a substantially higher inflammatory cytokine response and increased immunopathology. Investigation into the effects of immunostimulation on middle aged (7-12 months) and advance aged (22-24 months) mice, which had already undergone significant thymic involution and had a diminished naïve T cell population in the periphery at baseline, revealed that even further involution was incurred. Thymic rebound hyperplasia, however, only occurred in young and middle-aged recipients, while advance aged not only lacked this rebound hyperplasia, but were entirely absent of any indication of thymic restoration. This coincided with prolonged deficits in naïve T cell numbers in advanced aged recipients, further skewing the already memory dominant T cell pool. These results demonstrate that, in both mice and humans, systemic immunostimulatory cancer therapies, as well as immune challenges like subacute viral infections, have the potential to induce profound, but transient, glucocorticoid-mediated thymic involution and substantially reduced thymic output, resulting in the reduction of peripheral naive T cells. This can then be followed by a marked rebound effect with naïve T cell restoration, events that were shown not to occur in advanced-aged mice.

IntroductionInterprofessional collaboration in healthcare involves diverse professionals working together to address complex patient needs. Interprofessional training wards offer workplace-based interprofessional education in real healthcare settings, fostering collaborative learning among students. While their educational value is widely recognized, debates persist regarding their cost-effectiveness due to limited research. This study assesses the cost efficiency of the interprofessional training ward Regensburg (A-STAR) within the Department of Internal Medicine I at the University Hospital Regensburg, compared to conventional wards.MethodsFrom October 2019 to December 2022, 7,244 patient cases were assigned to A-STAR or conventional wards by case managers, with a comprehensive analysis of all associated revenues and costs.ResultsA-STAR treated 1,482 patients, whereas conventional wards treated 5,752 patients, with more males and younger patients at A-STAR. A-STAR achieved higher profit per case (€1,508.74) attributed to increased revenues and reduced material costs. It generated an average of €1,366.54 more Diagnosis Related Groups (DRG) revenue per case annually than conventional wards, due to greater medical complexity reflected in a higher case-mix index (CMI: 2.4 vs. 2.2). The increased case complexity led to longer patient stays (9.0 vs. 8.1 days) and fewer cases treated annually at A-STAR (27.4 cases/year vs. 37.8 cases/year). The higher CMI did not result in a higher proportion of patients requiring isolation. A-STAR exhibited a higher capacity utilization rate (87.1% vs. 83.9%). Personnel costs per case at A-STAR were initially elevated due to enhanced observation by the senior physician but were gradually mitigated by expanding A-STAR’s bed capacity. Material costs were consistently lower on a per-case basis at A-STAR (€1512.02 vs. €1577.12), particularly in terms of medication expenses, indicating more resource-efficient operations. From the A-STAR graduates, 18 individuals were recruited for permanent positions as doctors or nurses over 2 years.ConclusionA-STAR demonstrates economic efficiency and stability even during the COVID-19 pandemic. The substantial personnel acquisition is likely influenced by high levels of satisfaction with education and work and is economically relevant in medical staff shortages. These findings provide a compelling rationale for the broader implementation of interprofessional training wards, establishing them as vital platforms for nurturing future professionals.

BackgroundSince the onset of the COVID-19 pandemic, global healthcare systems have faced unprecedented challenges, leading to significant psychological distress among healthcare professionals. Recognizing the importance of enhanced interprofessional collaboration in alleviating this burden, as emphasized by the World Health Organization in 2020, we investigated whether such collaboration could mitigate staff psychological distress during crises. To our knowledge, no study has yet explored the role of interprofessional collaboration as a resilience factor in crises.MethodsFor this monocentric cross-sectional study at a German university hospital, we examined the relationship between the quality of interprofessional collaboration and the psychological distress of healthcare professionals during the initial pandemic wave. We employed validated mental health instruments, such as the GAD-7 and PHQ-2, to assess anxiety and depressive symptoms. Additionally, custom-designed questionnaires evaluated “Pandemic-Associated Burden and Anxiety (PAB; PAA)” and interprofessional crisis management experiences. A novel “Interprofessional collaboration and communication (IPC)” assessment tool was developed based on international competency frameworks, demonstrating strong reliability.ResultsThe study involved 299 healthcare professionals (78.6% in direct contact with COVID-19 patients). Moderate levels of PAB/PAA were reported. However, a significant proportion experienced clinically relevant anxiety, as indicated by GAD-7. Negative IPC perceptions correlated with higher levels of psychological distress. Linear regression analysis showed associations between interprofessional collaboration and anxious and depressive symptoms, and pandemic-related burden.ConclusionOur findings highlight the vital role of enhanced interprofessional collaboration in strengthening the psychological well-being of healthcare professionals during crises. The study underscores the need to foster a collaborative environment and integrate interprofessional education for resilience.

IntroductionThe emergence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is an urgent and alarming One Health problem. This study aimed to investigate duplications of plasmid-encoded ESBL genes and their impact on antimicrobial resistance (AMR) phenotypes in clinical and screening isolates.MethodsMulti-drug-resistant bacteria from hospitalized patients were collected during routine clinical surveillance from January 2022 to June 2023, and their antimicrobial susceptibility patterns were determined. Genotypes were extracted from long-read whole-genome sequencing data. Furthermore, plasmids and other mobile genetic elements associated with ESBL genes were characterized, and the ESBL genes were correlated to ceftazidime minimal inhibitory concentration (MIC).ResultsIn total, we identified four cases of plasmid-encoded ESBL gene duplications that match four genetically similar plasmids during the 18-month surveillance period: five Escherichia coli and three Klebsiella pneumoniae isolates. As the ESBL genes were part of transposable elements, the surrounding sequence regions were duplicated as well. In-depth analysis revealed insertion sequence (IS)-mediated transposition mechanisms. Isolates with duplicated ESBL genes exhibited a higher MIC for ceftazidime in comparison to isolates with a single gene copy (3–256 vs. 1.5–32 mg/L, respectively).ConclusionESBL gene duplications led to an increased phenotypic resistance against ceftazidime. Our data suggest that ESBL gene duplications by an IS-mediated transposition are a relevant mechanism for how AMR develops in the clinical setting and is part of the microevolution of plasmids.

Cardiac rehabilitation (CR) represents an important steppingstone for many cardiac patients into a more heart-healthy lifestyle to prevent premature death and improve quality of life years. However, CR is underutilized worldwide. In order to support the development of targeted digital health interventions, this narrative review (I) provides understandings of factors influencing CR utilization from a behavioral perspective, (II) discusses the potential of digital health technologies (DHTs) to address barriers and reinforce facilitators to CR, and (III) outlines how DHTs could incorporate shared decision-making to support CR utilization. A narrative search of reviews in Web of Science and PubMed was conducted to summarize evidence on factors influencing CR utilization. The factors were grouped according to the Behaviour Change Wheel. Patients' Capability for participating in CR is influenced by their disease knowledge, awareness of the benefits of CR, information received, and interactions with healthcare professionals (HCP). The Opportunity to attend CR is impacted by healthcare system factors such as referral processes and HCPs' awareness, as well as personal resources including logistical challenges and comorbidities. Patients' Motivation to engage in CR is affected by emotions, factors such as gender, age, self-perception of fitness and control over the cardiac condition, as well as peer comparisons. Based on behavioral factors, this review identified intervention functions that could support an increase of CR uptake: Future DHTs aiming to support CR utilization may benefit from incorporating information for patients and HCP education, enabling disease management and collaboration along the patient pathway, and enhancing social support from relatives and peers. To conclude, considerations are made how future innovations could incorporate such functions.

IntroductionInterprofessional teamwork is pivotal in modern healthcare, prompting the establishment of interprofessional training wards since 1996. While these wards serve as hubs for optimizing healthcare professional collaboration and communication, research into patient outcomes remains notably sparse and geographically limited, predominantly examining patient satisfaction and sparingly exploring other metrics like mortality or self-discharge rates. This study seeks to bridge this gap, comparing patient outcomes in interprofessional training wards and conventional wards under the hypothesis that the former offers no disadvantage to patient outcomes.Materials and methodsWe explored patient outcomes within an interprofessional student ward called A-STAR at a University Hospital from October 2019 to December 2022. Engaging with patients discharged between May 2021 and April 2022, we utilized digital and paper-based anonymous questionnaires, catering to patient preference, to gather pertinent data.ResultsAnalysis of outcomes for 1,482 A-STAR (interprofessional student ward) and 5,752 conventional ward patients revealed noteworthy findings. A-STAR patients tended to be younger (59 vs. 61 years, p

IntroductionHorse clinics are hotspots for the accumulation and spread of clinically relevant and zoonotic multidrug-resistant bacteria, including extended-spectrum β-lactamase producing (ESBL) Enterobacterales. Although median laparotomy in cases of acute equine colic is a frequently performed surgical intervention, knowledge about the effects of peri-operative antibiotic prophylaxis (PAP) based on a combination of penicillin and gentamicin on the gut microbiota is limited.MethodsWe collected fecal samples of horses from a non-hospitalized control group (CG) and from horses receiving either a pre-surgical single-shot (SSG) or a peri-operative 5-day (5DG) course of PAP. To assess differences between the two PAP regimens and the CG, all samples obtained at hospital admission (t0), on days three (t1) and 10 (t2) after surgery, were screened for ESBL-producing Enterobacterales and subjected to 16S rRNA V1–V2 gene sequencing.ResultsWe included 48 samples in the SSG (n = 16 horses), 45 in the 5DG (n = 15), and 20 in the CG (for t0 and t1, n = 10). Two samples of equine patients receiving antibiotic prophylaxis (6.5%) were positive for ESBL-producing Enterobacterales at t0, while this rate increased to 67% at t1 and decreased only slightly at t2 (61%). Shannon diversity index (SDI) was used to evaluate alpha-diversity changes, revealing there was no significant difference between horses suffering from acute colic (5DG, SDImean of 5.90, SSG, SDImean of 6.17) when compared to the CG (SDImean of 6.53) at t0. Alpha-diversity decreased significantly in both PAP groups at t1, while at t2 the onset of microbiome recovery was noticed. Although we did not identify a significant SDImean difference with respect to PAP duration, the community structure (beta-diversity) was considerably restricted in samples of the 5DG at t1, most likely due to the ongoing administration of antibiotics. An increased abundance of Enterobacteriaceae, especially Escherichia, was noted for both study groups at t1.ConclusionColic surgery and PAP drive the equine gut microbiome towards dysbiosis and reduced biodiversity that is accompanied by an increase of samples positive for ESBL-producing Enterobacterales. Further studies are needed to reveal important factors promoting the increase and residency of ESBL-producing Enterobacterales among hospitalized horses.

BackgroundMethanogenic archaea represent a less investigated and likely underestimated part of the intestinal tract microbiome in swine.Aims/MethodsThis study aims to elucidate the archaeome structure and function in the porcine intestinal tract of healthy and H1N1 infected swine. We performed multi-omics analysis consisting of 16S rRNA gene profiling, metatranscriptomics and metaproteomics.Results and discussionWe observed a significant increase from 0.48 to 4.50% of archaea in the intestinal tract microbiome along the ileum and colon, dominated by genera Methanobrevibacter and Methanosphaera. Furthermore, in feces of naïve and H1N1 infected swine, we observed significant but minor differences in the occurrence of archaeal phylotypes over the course of an infection experiment. Metatranscriptomic analysis of archaeal mRNAs revealed the major methanogenesis pathways of Methanobrevibacter and Methanosphaera to be hydrogenotrophic and methyl-reducing, respectively. Metaproteomics of archaeal peptides indicated some effects of the H1N1 infection on central metabolism of the gut archaea.Conclusions/Take home messageFinally, this study provides the first multi-omics analysis and high-resolution insights into the structure and function of the porcine intestinal tract archaeome during a non-lethal Influenza A virus infection of the respiratory tract, demonstrating significant alterations in archaeal community composition and central metabolic functions.

In the Canadian prairies, pulse crops such as field pea (Pisum sativum L.) and lentil (Lens culinaris L.) are economically important and widely grown. However, in recent years, root rot, caused by a variety of fungal and oomycete pathogens, including Aphanomyces euteiches, has become a limiting factor on yield. In this study, we examined the impacts of nitrogen (N) fertilization and a commercial arbuscular mycorrhizal fungal (AMF) inoculant on pea and lentil plant health and agronomic production at three locations in Saskatchewan: Swift Current, Indian Head and Melfort. The AMF inoculation had no impact on root rot severity, and therefore is not considered a reliable method to manage root rot in pea and lentil. In contrast, N fertilization led to reductions in root rot in Swift Current, but not the other two sites. However, N fertilization did reduce nodulation. When both pea and lentil are considered, the abundance of A. euteiches in soil increased from pre-seeding to mid-bloom. A negative correlation between soil pH and disease severity was also observed. The high between-site variability highlights the importance of testing root rot mitigation strategies under multiple soil conditions to develop site-specific recommendations. Use of N fertilizer as a root rot management strategy merits further exploration, including investigation into its interactions with other management strategies, soil properties, and costs and benefits.

PurposeThough a subgroup analysis has shown improved survival for patients suffering severely reduced ventricular function undergoing coronary artery bypass grafting, RCTs were not able to demonstrate overall beneficial effects of perioperative Levosimendan in cardiac surgery. This might be due to Levosimendan’s pharmacokinetics reaching a steady-state concentration only 4–8 h after administration. Thus, this study now analysed the influence of timing of Levosimendan administration on perioperative outcome in cardiac surgery patients preoperatively presenting with severely reduced ventricular function and therefore considered at high-risk for intra- or postoperative low cardiac output syndrome. We hypothesized that prolonged preoperative Levosimendan administration (“preconditioning”) would reduce mortality.MethodsAll adult patients undergoing cardiac surgery between 2006 and 2018 perioperatively receiving Levosimendan were included in this retrospective, observational cohort study (n = 498). Patients were stratified into 3 groups: Levosimendan started on the day prior to surgery (“preop”), Levosimendan started on the day of surgery (“intraop”) or post ICU admission (“postop”). After propensity score matching (PSM) was performed, outcomes defined according to proposed standard definitions for perioperative outcome research were compared between groups.ResultsAfter PSM, there were no significant differences in patients’ characteristics, comorbidities and type/priority of surgery between groups. Compared to intraop or postop Levosimendan treatment, preop treated patients had significantly lower in-hospital-mortality (preop vs. intraop. vs. postop = 16,7% vs. 33,3% vs. 42,3%), duration of mechanical ventilation and rate of continuous renal replacement therapy.ConclusionsProlonged preoperative treatment with Levosimendan of cardiac surgery patients preoperatively presenting with severely reduced left ventricular function might be beneficial in terms of postoperative outcome. Our results are in line with recent experts’ recommendations concerning the prolonged perioperative use of Levosimendan. We strongly recommend that future randomized trials include this “preconditioning” treatment as an experimental arm.

There is currently great interest in photocatalytic degradation technologies of pollutants in industrial effluents. This is due to the need to reduce the environmental pollution generated by the textile industry’s high demand of clothing for fast fashion; in addition to severe environmental problems, this also generates social problems. Since the catalysts of this type of processes are usually nanoparticles of metal oxides such as zinc and titanium, it is necessary to promote research into the synthesis and evaluation of photocatalysts. Therefore, this article describes three free basic access tools for the academic analysis of nanoparticles, from experimental design to representation, using the study of kinetics and particle size analysis. After pre-selecting easily accessible software, it was found that RStudio, J-Image, and Vesta are very useful programs for the analysis of nanoparticles in the respective areas of statistical processing, image analysis, and three-dimensional representation.

IntroductionSoil at the Roman site of Vindolanda (Northumberland, UK) provides excellent preservation of wooden artefacts including Roman writing tablets.MethodsIn this study we examined chemical and microbial signature changes within varied occupation contexts of archaeological soil. Analysis included investigating elemental composition, sterol biomarkers, bacterial diversity and community structures from excavation trenches at Vindolanda using pXRF, GC-MS and 16S rRNA gene amplicon sequencing. Samples were taken from varying depths starting at topsoil and working down through layers of Roman occupation including one cavalry stable floor, two infantry barracks and a cook house, and layers which contained Roman writing tablets.Results and DiscussionThe chemical results indicate that areas where wooden artefacts were found had increased soil moisture which was also correlated with specific chemical conditions including shifts in iron, sulphur and phosphorous concentration. Steroid biomarkers indicate the presence of faecal matter in layers, supporting occupation descriptions. Overall microbial diversity did not change across the depth profile but was correlated with soil moisture. Anaerobic soils associated with more optimal preservation differed to other soils with increases in Firmicutes, Proteobacteria, Campilobacterota and Bacteroidota observed. Microbial community structure and putative function as revealed by PICRUSt2 is linked to occupation usage rather than depth of samples with laminated floor layers differing from turf structures. Understanding the complex processes within archaeological soil can help us to understand dynamics of decomposition and preservation. In addition, the apparent preservation of the environmental microbial community as well as the artefacts themselves allows us to understand the microbial environments of the past, how they relate to the present and what this means for our changing environments in the future.

While affordance detection and Human-Object interaction (HOI) detection tasks are related, the theoretical foundation of affordances makes it clear that the two are distinct. In particular, researchers in affordances make distinctions between J. J. Gibson's traditional definition of an affordance, “the action possibilities” of the object within the environment, and the definition of a telic affordance, or one defined by conventionalized purpose or use. We augment the HICO-DET dataset with annotations for Gibsonian and telic affordances and a subset of the dataset with annotations for the orientation of the humans and objects involved. We then train an adapted Human-Object Interaction (HOI) model and evaluate a pre-trained viewpoint estimation system on this augmented dataset. Our model, AffordanceUPT, is based on a two-stage adaptation of the Unary-Pairwise Transformer (UPT), which we modularize to make affordance detection independent of object detection. Our approach exhibits generalization to new objects and actions, can effectively make the Gibsonian/telic distinction, and shows that this distinction is correlated with features in the data that are not captured by the HOI annotations of the HICO-DET dataset.

This work considers the estimation of impulsive time series pertaining to biomedical systems and, in particular, to endocrine ones. We assume a signal model in the form of the output of a continuous linear time-invariant system driven by a sequence of instantaneous impulses, which concept is utilized here, in particular, for modeling of the male reproductive hormone axis. An estimation method to identify the impulsive sequence and the continuous system dynamics from sampled measurements of the output is proposed. Hinging on thorough mathematical analysis, the method improves upon a previously developed least-squares algorithm by resolving the trade-off between model fit and input sparsity, thus removing the need for manual tuning of user-defined estimation algorithm parameters. Experiments with synthetic data and Markov chain Monte-Carlo estimation demonstrate the viability of the proposed method, but also indicate that measurement noise renders the estimation problem ill-posed, as multiple estimates along a curve in the parameter space yield similar fits to data. The method is furthermore applied to clinical luteinizing hormone data collected from healthy males and, for comparability, one female, with similar results. Comparison between the estimated and theoretical elimination rates, as well as simulation of the estimated models, demonstrate the efficacy of the method. The sensitivity of the impulse distribution to the estimated elimination rates is investigated on a subject-specific data subset, revealing that the input sequence and elimination rate estimates can be interdependent. The dose-dependent effect of a selective gonadotropin releasing hormone receptor antagonist on the frequency and weights of the estimated impulses is also analyzed; a significant impact of the medication on the impulse weights is confirmed. To demonstrate the feasibility of the estimation approach for other hormones with pulsatile secretion, the modeling of cortisol data sets collected from three female adolescents was performed.

IntroductionMalnutrition in cancer patients often remains undetected and underestimated in clinical practice despite studies revealing prevalences from 20 to 70%. Therefore, this study aimed to identify patient groups exposed to an increased nutritional risk in a university oncological outpatient center.MethodsBetween May 2017 and January 2018 we screened oncological patients there using the malnutrition universal screening tool (MUST). Qualitative data were collected by a questionnaire to learn about patients’ individual information needs and changes in patients’ diets and stressful personal nutrition restrictions.ResultsWe included 311 patients with various cancers. 20.3% (n = 63) were found to be at high risk of malnutrition, 16.4% (n = 51) at moderate risk despite a mean body mass index (BMI) of 26.5 ± 4.7 kg/m2. The average age was 62.7 (± 11.8) with equal gender distribution (52% women, n = 162). In 94.8% (n = 295) unintended weight loss led to MUST scoring. Patients with gastrointestinal tumors (25%, n = 78) and patients >65 years (22%, n = 68) were at higher risk. Furthermore, there was a significant association between surgery or chemotherapy within six months before survey and a MUST score ≥2 (OR = 3.6). Taste changes, dysphagia, and appetite loss were also particular risk factors (OR = 2.3–3.2). Young, female and normal-weight patients showed most interest in nutrition in cancer. However, only 38% (n = 118) had a nutritional counseling.ConclusionThis study confirms that using the MUST score is a valid screening procedure to identify outpatients at risk of developing malnutrition. Here one in five was at high risk, but only 1% would have been detected by BMI alone. Therefore, an ongoing screening procedure with meaningful parameters should be urgently implemented into the clinical routine of cancer outpatients as recommended in international guidelines.

Influenza A Virus (IAV) infection followed by bacterial pneumonia often leads to hospitalization and death in individuals from high risk groups. Following infection, IAV triggers the process of viral RNA replication which in turn disrupts healthy gut microbial community, while the gut microbiota plays an instrumental role in protecting the host by evolving colonization resistance. Although the underlying mechanisms of IAV infection have been unraveled, the underlying complex mechanisms evolved by gut microbiota in order to induce host immune response following IAV infection remain evasive. In this work, we developed a novel Maximal-Clique based Community Detection algorithm for Weighted undirected Networks (MCCD-WN) and compared its performance with other existing algorithms using three sets of benchmark networks. Moreover, we applied our algorithm to gut microbiome data derived from fecal samples of both healthy and IAV-infected pigs over a sequence of time-points. The results we obtained from the real-life IAV dataset unveil the role of the microbial families Ruminococcaceae, Lachnospiraceae, Spirochaetaceae and Prevotellaceae in the gut microbiome of the IAV-infected cohort. Furthermore, the additional integration of metaproteomic data enabled not only the identification of microbial biomarkers, but also the elucidation of their functional roles in protecting the host following IAV infection. Our network analysis reveals a fast recovery of the infected cohort after the second IAV infection and provides insights into crucial roles of Desulfovibrionaceae and Lactobacillaceae families in combating Influenza A Virus infection. Source code of the community detection algorithm can be downloaded from https://github.com/AniBhar84/MCCD-WN.

Background and aimsLiver diseases are frequent causes of morbidity and mortality worldwide. Liver diseases can lead to cirrhosis, with the risk of acute-on-chronic liver failure (ACLF). For the detection of changes in hepatic hemodynamics, Doppler ultrasonography is a well-established method. We investigated hepatic hemodynamics via serial Doppler ultrasonography to determine the predictive value of changes in hepatic perfusion for the outcome in patients with severe liver diseases compared to established prognostic models such as the MELD (Model for End-Stage Liver Disease) or CLIF-C (Chronic Liver Failure-Consortium) ACLF score.MethodsIn this prospective cohort study, hepatic perfusion was quantified at baseline before the initiation of treatment and every third day by means of serial measurements of the hepatic artery resistance index (HARI) and the maximum portal vein velocity (PVv) using Doppler ultrasonography in 50 consecutive patients with severe liver diseases admitted to a medical intensive care unit (MICU). The recorded hemodynamic parameters were compared to the MELD score, and the CLIF-C ACLF score to analyze their utility for the prediction of the outcome of patients with severe liver diseases, liver cirrhosis, and ACLF.ResultsThe changes (delta) obtained by serial measurements of the MELD score, HARI, and PVv were analyzed through scatter plots. Bivariate correlation analysis yielded a new positive linear correlation between the delta-HARI and the delta-MELD score (r = 0.469; p < 0.001). In addition, our data revealed a new negative linear correlation between delta-PVv and the delta-MELD score (r = −0.279, p = 0.001). The leading cause of MICU mortality was acute-on-chronic liver failure (ACLF). A subgroup analysis of patients with liver cirrhosis revealed a positive linear correlation between the delta-HARI and the delta-CLIF-C-ACLF score (r = 0.252, p = 0.005). Of clinical relevance, non-survivors of ACLF exhibited a significantly higher mean value for the delta-HARI (0.010 vs. −0.005; p = 0.015) and a lower mean value for the delta-PVv (−0.7 vs. 1.9 cm/s; p = 0.037) in comparison to survivors of ACLF.ConclusionThis study shows the prognostic value of the assessment of hepatic perfusion in critical care patients with severe liver diseases by bedside Doppler ultrasound examination and its utility as an accurate predictor of the outcome in patients with ACLF. Increasing HARI and a decreasing PVv are predictors of an adverse outcome. Delta-HARI and delta-PVv are new biomarkers of prognosis and ACLF-related mortality in patients with liver diseases. Delta-HARI and delta-PVv may be helpful in guiding clinical decision-making, especially in catecholamine and fluid management.

The COVID−19 pandemic not only resulted in a global crisis, but also accelerated vaccine development and antibody discovery. Herein we report a synthetic humanized VHH library development pipeline for nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) isolation. Trinucleotide-based randomization of CDRs by Kunkel mutagenesis with the subsequent rolling-cycle amplification resulted in more than 1011 diverse phage display library in a manageable for a single person number of electroporation reactions. We identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) by screening a novel synthetic humanized antibody library. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, these affinities are weakly influenced by most of SARS-CoV-2 VoC mutations and they retain moderate binding to BA.4\5. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\ml and 9.6 ng\ml for H7-Fc and G12x3-Fc antibodies, respectively, for the emerging Omicron BA.1 variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants.

BackgroundEndoscopic vacuum therapy (EVT) is an evidence-based option to treat anastomotic leakages of the upper gastrointestinal (GI) tract, but the technical challenges and clinical outcomes of patients with large defects remain poorly described.MethodsAll patients with leakages of the upper GI tract that were treated with endoscopic negative pressure therapy at our institution from 2012–2021 were analyzed. Patients with large defects (>30 mm) as an indicator of complex treatment were compared to patients with smaller defects (control group).ResultsNinety-two patients with postoperative anastomotic or staplerline leakages were identified, of whom 20 (21.7%) had large defects. Compared to the control group, these patients required prolonged therapy (42 vs. 14 days, p

We describe the identification of a methicillin-resistant, high-level mupirocin-resistant Staphylococcus argenteus. The isolate (1801221) was characterized as t6675-ST2250-SCCmecIVc, and whole-genome sequencing revealed that the isolate possessed two plasmids. One plasmid (34,870 bp), designated p1_1801221 with rep23, harboured the mupirocin resistance (mupA) gene. The second plasmid (20,644 bp), assigned as p2_1801221 with rep5a and rep16, carried the resistance determinants for penicillin (blaZ) and cadmium (cadD). Phylogenetic analysis revealed that the isolate clustered with the European ST2250 lineage. The overall high similarity of both plasmids in S. argenteus with published DNA sequences of Staphylococcus aureus plasmids strongly suggests an interspecies transfer. The pathogenic potential, community and nosocomial spread, and acquisition of antibiotic resistance gene determinants, including the mupA gene by S. argenteus, highlight its clinical significance and the need for its correct identification.

BackgroundSurgical ventricular restoration (SVR) leads to functional improvement by volume reduction and restoration of left ventricular (LV) geometry. Our purpose was to refine the planning for SVR using cardiac computed tomography (CCT).MethodsThe possibility to anticipate the postoperative residual LV volume was assessed using CCT in 205 patients undergoing SVR combined with coronary artery bypass grafting (77%), mitral valve repair/replacement (19%) and LV thrombectomy (19%). The potential of CCT to guide the procedure was evaluated. Additionally, the predictive value of CCT characteristics on survival was addressed.Results30-day, 1- and 5-year survival was 92.6, 82.7, and 72.1%, respectively, with a marked reduction of NYHA class III-IV quota after surgery (95.1% vs. 20.5% in the follow-up). Both pre- and postoperative LV end-systolic volume index (LVESVI) were predictive of all defined endpoints according to the following tertiles: preoperative: 114 ml/m2; postoperative: 82 ml/m2. On average, a 50 ml/m2 increase of preoperative LVESVI was associated with a 35% higher hazard of death (p = 0.043). Aneurysms limited to seven antero-apical segments (1–7) were associated with a lower death risk (n = 60, HR 0.52, CI 0.28–0.96, p = 0.038). LVESVI predicted by CCT was found to correlate significantly with effectively achieved LVESVI (r = 0.87 and r = 0.88, respectively, p < 0.0001).ConclusionsCCT-guided SVR can be performed with good mid-term survival and significant improvement in HF severity. CCT-based assessment of achievable postoperative LV volume helps estimate the probability of therapeutic success in individual patients.

Pangeo Forge is a new community-driven platform that accelerates science by providing high-level recipe frameworks alongside cloud compute infrastructure for extracting data from provider archives, transforming it into analysis-ready, cloud-optimized (ARCO) data stores, and providing a human- and machine-readable catalog for browsing and loading. In abstracting the scientific domain logic of data recipes from cloud infrastructure concerns, Pangeo Forge aims to open a door for a broader community of scientists to participate in ARCO data production. A wholly open-source platform composed of multiple modular components, Pangeo Forge presents a foundation for the practice of reproducible, cloud-native, big-data ocean, weather, and climate science without relying on proprietary or cloud-vendor-specific tooling.

BackgroundDue to an increasing life expectancy, more and more patients experience the failure of peripheral arterial revascularization. This study aims to investigate patients treated for the failure of infra-inguinal bypass grafts, and to investigate the interaction of different bypass materials [great saphenous vein (GSV) and polytetrafluoroethylene (PTFE)] and the further outcome.MethodsRetrospective single-center analysis of consecutive patients treated for acute or chronic occlusion of infra-inguinal bypasses at a university hospital was conducted. Hospitalizations from 1st January 2010 through 31st December 2019 were included. Perioperative parameters from the index operation including graft material (prosthetic vs. autologous) were assessed. After bypass occlusion, the grade of ischemia, as well as the distal landing zone of the redo bypass compared with the primary bypass was investigated.ResultsIn this study, 158 (65% men and 35% women with a m mean age of 70.5 years) eligible patients were included (57% vein and 42% prosthetic bypass grafts). After graft occlusion, 47% of the patients presented with symptoms of acute limb-threatening ischemia, 53% with symptoms of chronic leg ischemia. The rate of acute limb-threatening ischemia was significantly higher when prosthetic graft material was used during the index operation (p =0.016). Additionally, in case of reoperation, the landing zone of the redo bypass was significantly more distally located after occlusion of prosthetic bypass graft (p = 0.014)ConclusionOcclusions of prosthetic bypass grafts were associated with significantly higher rates of acute symptoms compared with vein grafts. Additionally, a shift to a more distal landing zone was recognized after the failure of a prosthetic bypass graft during the redo bypass operation.

The immunological response against Clostridioides difficile (C. difficile) is crucial for an improved understanding of disease mechanisms and the development of novel therapeutic strategies. From April 2014 to February 2015, adult patients with C. difficile infection (CDI) were recruited, and the clinical course and treatment response were carefully monitored. On day 1, 3, and 6 after diagnosis, patient plasma samples were screened for anti-GDH (glutamate dehydrogenase), anti-TcdA, anti-TcdB, and anti-CWP84 (cell-wall protein 84) antibodies by ELISA. Additionally, neutralization assays of toxins from conditioned media of clinical isolates (RT010, RT014, and RT027) were performed. Most patients with CDI (n = 46) had antibodies against GDH (85%) and CWP84 (61%), but only few had antibodies against TcdA (11%) and TcdB (28%). We found patients with neutralizing antibodies against C. difficile toxins (conditioned media) produced by RT027 (26%). A subgroup of these samples could neutralize both toxins from RT027 and RT014 [11%, (5/46)]; however, no single sample neutralized only RT014. Overall, neutralizing antibody titers were low (≤1:16). In a one week follow-up of acute infection, we never observed an early booster effect with seroconversion or antibody increases, irrespective of disease severity. No correlation was found between the presence of antigen-specific (ELISA) or neutralizing antibodies and the clinical course of disease. Anti-TcdB but not anti-TcdA antibodies correlated with the occurrence of neutralizing antibodies. In conclusion, natural antibody titers against C. difficile toxins were absent or low and were not associated with disease severity. The correlation between the anti-TcdB with toxin neutralization confirms the importance of TcdB for virulence of CDI. Alternative sensitization strategies, e.g., through vaccine development, are required to overcome the regular low-titer antibody production following natural intestinal C. difficile exposure.

Several studies have shown that “dry” immersion appears as a promising method of rehabilitation for Parkinson's disease. Still, little is known about the cardiovascular reaction in “dry” immersion (DI), especially in Parkinson's disease (PD). Therefore, this study was aimed to evaluate the effect of repeated 45-min DI sessions on autonomic function in subjects with PD. The study group consisted of 20 subjects with PD [13 men, seven women, aged 51–66 years old, Hoehn & Yahr (H&Y) staged 1–3] were enrolled in the study according to inclusion and non-inclusion criteria. The DI program was comprised of seven 45-min DI sessions, applied within 25–30 days. Blood pressure (BP), heart rate (HR), and electrocardiogram (ECG) in the standard lead II were recorded at 1st, 4th, and 7th DI, before, on the 15, 30, and 40th min of DI session. Autonomic function was assessed with analysis of heart rate variability (HRV) using Kubios Standard version 2 software. Linear (time- and frequency-domain) and non-linear (correlation dimension, entropies, DFA1 and DFA2, percent of determinism, and recurrence) were computed. At baseline condition, time- and frequency-domain HRV parameters showed low variability of HR, which indicates reduced autonomic neurogenic control of HR. Throughout the DI session, systolic and diastolic BP has decreased by 5–7 mm Hg (p < 0.001), and time- and frequency-domain parameters of HRV have significantly increased, what can be regarded as compensatory mechanisms of hemodynamics during DI. The structure of the regulatory input to the heart seen by HRV was characterized by low complexity and reduced autonomic neurogenic control of HR. Across the program of DI sessions, the hypotensive effect was documented, but no notable modification of the HRV-parameters was found. The absence of long-term modification of the studied parameters can be attributed both to deconditioning environmental effect of DI and limited adaptation of the organism due to neurodegeneration in PD. That should be taken into consideration when planning rehabilitation measures in subjects of older age and chronic somatic diseases with modeled microgravity.

Hyperthermophilic archaea of the genus Archaeoglobus are the subject of many fundamental and biotechnological researches. Despite their significance, the class Archaeoglobi is currently represented by only eight species obtained as axenic cultures and taxonomically characterized. Here, we report the isolation and characterization of a new species of Archaeoglobus from a deep-sea hydrothermal vent (Mid-Atlantic Ridge, TAG) for which the name Archaeoglobus neptunius sp. nov. is proposed. The type strain is SE56T (=DSM 110954T = VKM B-3474T). The cells of the novel isolate are motile irregular cocci growing at 50–85°C, pH 5.5–7.5, and NaCl concentrations of 1.5–4.5% (w/v). Strain SE56T grows lithoautotrophically with H2 as an electron donor, sulfite or thiosulfate as an electron acceptor, and CO2/HCO3− as a carbon source. It is also capable of chemoorganotrophic growth by reduction of sulfate, sulfite, or thiosulfate. The genome of the new isolate consists of a 2,115,826 bp chromosome with an overall G + C content of 46.0 mol%. The whole-genome annotation confirms the key metabolic features of the novel isolate demonstrated experimentally. Genome contains a complete set of genes involved in CO2 fixation via reductive acetyl-CoA pathway, gluconeogenesis, hydrogen and fatty acids oxidation, sulfate reduction, and flagellar motility. The phylogenomic reconstruction based on 122 conserved single-copy archaeal proteins supported by average nucleotide identity (ANI), average amino acid identity (AAI), and alignment fraction (AF) values, indicates a polyphyletic origin of the species currently included into the genus Archaeoglobus, warranting its reclassification.

Alloreactive regulatory T cells (arTregs) are more potent than polyclonal Tregs at suppressing immune responses to transplant antigens. Human arTregs can be expanded with allogeneic CD40L-stimulated B cells (sBcs) or stimulated-matured monocyte-derived dendritic cells (sDCs). Here, we compared the expansion efficiency and properties of arTregs stimulated ex vivo using these two types of antigen-presenting cells. Compared to sBcs, sDCs stimulated Tregs to expand two times more in number. The superior expansion-inducing capacity of sDCs correlated with their higher expression of CD80, CD86, and T cell-attracting chemokines. sBc- and sDC-arTregs expressed comparable levels of FOXP3, HELIOS, CD25, CD27, and CD62L, demethylated FOXP3 enhancer and in vitro suppressive function. sBc- and sDCs-arTregs had similar gene expression profiles that were distinct from primary Tregs. sBc- and sDC-arTregs exhibited similar low frequencies of IFN-γ, IL-4, and IL-17A-producing cells, and the cytokine-producing arTregs expressed high levels of FOXP3. Almost all sBc- and sDC-arTregs expressed CXCR3, which may enable them traffic to inflammatory sites. Thus, sDCs-arTregs that expand more readily, are phenotypically similar to sBc-arTregs, supporting sDCs as a viable alternative for arTreg production for clinical evaluation.

With ever growing automation of public transport automated shuttles offer an attractive alternative in areas where traffic regulations limit the deployment of large buses (city centres) or where low degrees of utilization renders the manual vehicles operation non-economical (last mile). The low capacities of shuttles in combination with the human factor (driver or conductor) makes capacity management a greater challenge for the user. Capacity management describes the allocation of available seats in a vehicle, e.g., when buying a ticket. In this paper, we present the results of series of studies where capacity management in automated shuttles has been tested via instruments that are currently available in public transit (audio announcements, in-shuttle displays, booking apps). We found that measures during and after boarding are not sufficient and that capacity management in automated shuttle requires a more detailed planning of pre-boarding stages; when boarding automated shuttles as opposed to non-automated public buses the flexibility is reduced. The paper concludes with discussion and recommendations for an optimal capacity management d.

BackgroundImmune checkpoint inhibitors (ICIs) have led to a paradigm shift in cancer therapy, improving outcomes in the treatment of various malignancies. However, not all patients benefit to the same extend from ICI. Reliable tools to predict treatment response and outcome are missing. Soluble urokinase plasminogen activator receptor (suPAR) is a marker of immune activation, whose levels are prognostic in various cancers. We evaluated circulating suPAR levels as a novel predictive and prognostic biomarker in patients receiving ICI therapy for solid tumors.MethodsA total of n = 87 patients receiving ICI therapy for different solid malignancies as well as 32 healthy controls were included into this study. Serum levels of suPAR were measured by ELISA prior to and sequentially at two time points during ICI therapy.ResultsBaseline suPAR serum levels were significantly higher in solid tumor patients compared to healthy controls. Importantly, patients with low suPAR levels both before or during ICI treatment were more likely to have a favorable response to treatment at three and six months, respectively. This finding was confirmed by multivariate binary logistic regression analysis including several clinicopathological parameters. Moreover, circulating suPAR levels before and during therapy were an independent prognostic factor for overall survival (OS). As such, patients with initial suPAR levels above our ideal prognostic cut-off value (4.86 ng/ml) had a median OS of only 160 days compared to 705 days for patients with suPAR levels below this cut-off value. Finally, low baseline suPAR levels identified a subgroup of patients who experienced ICI-related side effects which in turn were associated with favorable treatment response and outcome.ConclusionOur data suggest that measurements of suPAR serum levels are a previously unknown, easily accessible tool to predict individual treatment response and outcome to ICI therapy. Circulating suPAR might therefore be implemented into stratification algorithms to identify the ideal candidates for ICI treatment.

Aims: Aortic valve replacement (AVR) may result in reverse cardiac remodeling. We aimed to assess long-term changes in the myocardium following AVR by Cardiac Magnetic Resonance Imaging (CMR).Methods: We prospectively observed the long-term left ventricular (LV) function and structure of 27 patients with AVR [n = 19 with aortic stenosis (AS); n = 8 with aortic regurgitation (AR)] by CMR. Patients underwent CMR before, as well as 1, 5, and 10 years after AVR. We evaluated clinical parameters, LV volumes, mass, geometry, ejection fraction (EF), global myocardial longitudinal strain (MyoGLS), global myocardial circular strain (MyoGCS), hemodynamic forces (HemForces), and Late Gadolinium Enhancement (LGE).Results: The median of LVMI, EDVI, and ESVI decreased in both groups. Patients with AR had higher initial values of EDVI and ESVI and showed a more prominent initial reduction. In AS, MyoGLS improved already after 1 year and remained constant afterward, whereas, in AR no improvement of MyoGLS was found. MyoGCS remained unchanged in the AS group but deteriorated in the AR group over 10 years. Ejection fraction (EF) was higher in AS patients compared to AR 10 years post-AVR. Late gadolinium enhancement (LGE) could be found more frequently in AS patients.Conclusion: CMR was well suited to investigate myocardial changes over a 10-year follow up period in patients with aortic valve disease. Regarding the long-term functional changes following AVR, patients with AR seemed to benefit less from AVR compared to AS patients. Fibrosis was more common in AS, but this did not reflect functional evolution in these patients. Close monitoring seems indispensable to avoid irreversible structural damage of the heart and to perform AVR at an appropriate stage.

The ongoing digitalization of educational resources and the use of the internet lead to a steady increase of potentially available learning media. However, many of the media which are used for educational purposes have not been designed specifically for teaching and learning. Usually, linguistic criteria of readability and comprehensibility as well as content-related criteria are used independently to assess and compare the quality of educational media. This also holds true for educational media used in economics. This article aims to improve the analysis of textual learning media used in economic education by drawing on threshold concepts. Threshold concepts are key terms in knowledge acquisition within a domain. From a linguistic perspective, however, threshold concepts are instances of specialized vocabularies, exhibiting particular linguistic features. In three kinds of (German) resources, namely in textbooks, in newspapers, and on Wikipedia, we investigate the distributive profiles of 63 threshold concepts identified in economics education (which have been collected from threshold concept research). We looked at the threshold concepts' frequency distribution, their compound distribution, and their network structure within the three kinds of resources. The two main findings of our analysis show that firstly, the three kinds of resources can indeed be distinguished in terms of their threshold concepts' profiles. Secondly, Wikipedia definitely shows stronger associative connections between economic threshold concepts than the other sources. We discuss the findings in relation to adequate media use for teaching and learning—not only in economic education.

De-novo designed proteins have received wide interest as potential platforms for nano-engineering and biomedicine. While much work is being done in the design of thermodynamically stable proteins, the folding process of artificially designed proteins is not well-studied. Here we used single-molecule force spectroscopy by optical tweezers to study the folding of ROSS, a de-novo designed 2x2 Rossmann fold. We measured a barrier crossing time in the millisecond range, much slower than what has been reported for other systems. While long transition times can be explained by barrier roughness or slow diffusion, we show that isotropic roughness cannot explain the measured transition path time distribution. Instead, this study shows that the slow barrier crossing of ROSS is caused by the population of three short-lived high-energy intermediates. In addition, we identify incomplete and off-pathway folding events with different barrier crossing dynamics. Our results hint at the presence of a complex transition barrier that may be a common feature of many artificially designed proteins.

We test the hypothesis that the extent to which one obtains information on a given topic through Wikipedia depends on the language in which it is consulted. Controlling the size factor, we investigate this hypothesis for a number of 25 subject areas. Since Wikipedia is a central part of the web-based information landscape, this indicates a language-related, linguistic bias. The article therefore deals with the question of whether Wikipedia exhibits this kind of linguistic relativity or not. From the perspective of educational science, the article develops a computational model of the information landscape from which multiple texts are drawn as typical input of web-based reading. For this purpose, it develops a hybrid model of intra- and intertextual similarity of different parts of the information landscape and tests this model on the example of 35 languages and corresponding Wikipedias. In the way it measures the similarities of hypertexts, the article goes beyond existing approaches by examining their structural and semantic aspects intra- and intertextually. In this way it builds a bridge between reading research, educational science, Wikipedia research and computational linguistics.

The purpose of the work is to develop an analytical method for calculating the effective ranges of structural parameters of the throttle part of the axial valve with the external location of the locking shell based on the proposed stochastic models of bubble formation at the initial stage of hydrodynamic cavitation. In contrast to the well-known engineering methods for choosing the conditional bore diameter for the designed control device constructed using the Pi-Buckingham theorem, this calculation method relies not only on gyrodynamic similarity criteria, but also on functional dependences for the laws of distribution of cavitation bubbles according to various characteristics (specific size bubbles, the degree of opening of the node “separator-external locking shell”). This takes into account a wide range of design and operating parameters, including the required valve capacity, as well as the physical and mechanical characteristics of the working medium. A block diagram of the calculation of the node “separator-external locking shell” for the specified axial valve is proposed. The influence of a set of design parameters for the “separator-external locking shell” assembly from the rational ranges of their variation obtained using the proposed analytical calculation algorithm on the size of cavitation bubbles is investigated. It has been established that when using the latest results in comparison with the values of parameters from irrational ranges of change, there is a tendency to reduce the ensemble average value of the diameter of the cavitation bubble by 1.95 times, when switching to a mode from 20% of the degree of separation of the separator to 80% of the opening of the throttle holes, the nominal valve capacity increases by 10.7 times. The results obtained illustrate the possibility of preventing an increase in the intensity of formation of cavities in the flow part of the control device already at the initial stage of the occurrence of cavitation.

BackgroundStaphylococcus aureus is one of the most isolated pathogens from the airways of cystic fibrosis (CF) patients. There is a lack of information about the clonal nature of S. aureus cultured from CF patients and their impact on disease. We hypothesized that patients would differ in their clinical status depending on S. aureus clonal carriage profiles during persistence.MethodsDuring a 21-months prospective observational multicenter study (Junge et al., 2016), 3893 S. aureus isolates (nose, oropharynx, and sputa) were cultured from 183 CF patients (16 German centers, 1 Austrian center) and subjected to spa-sequence typing to assess clonality. Data were associated to lung function, age, gender, and antibiotic treatment by multivariate regression analysis.ResultsTwo hundred and sixty-five different spa-types were determined with eight prevalent spa-types (isolated from more than 10 patients): t084, t091, t008, t015, t002 t012, t364, and t056. We observed different carriage profiles of spa-types during the study period: patients being positive with a prevalent spa-type, only one, a dominant or related spa-type/s. Patients with more antibiotic cycles were more likely to be positive for only one spa-type (p = 0.005), while older patients were more likely to have related (p = 0.006), or dominant spa-types (p = 0.026). Two percent of isolates were identified as methicillin-resistant S. aureus (MRSA) and evidence of transmission of clones within centers was low.ConclusionThere was a significant association of antibiotic therapy and age on S. aureus carriage profiles in CF patients indicating that antibiotic therapy prevents acquisition of new clones, while during aging of patients with persisting S. aureus, dominant clones were selected and mutations in the spa-repeat region accumulated.

Staphylococcus aureus is one of the first and most prevalent pathogens cultured from the airways of cystic fibrosis (CF) patients, which can persist there for extended periods. Airway infections in CF patients are characterized by a strong inflammatory response of highly recruited neutrophils. One killing mechanism of neutrophils is the formation of neutrophil extracellular traps (NETs), which capture and eradicate bacteria by extracellular fibers of neutrophil chromatin decorated with antimicrobial granule proteins. S. aureus secretes nuclease, which can degrade NETs. We hypothesized, that S. aureus adapts to the airways of CF patients during persistent infection by escaping from NET-mediated killing via an increase of nuclease activity. Sputum samples of CF patients with chronic S. aureus infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, S. aureus bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting S. aureus isolates, as confirmed by whole genome sequencing, from an individual CF patient using a FRET-based nuclease activity assay. Additionally, some of these isolates were selected and analyzed by qRT-PCR to determine the expression of nuc1 and regulators of interest. NET-killing assays were performed with clinical S. aureus isolates to evaluate killing and bacterial survival depending on nuclease activity. To confirm the role of nuclease during NET-mediated killing, a clinical isolate with low nuclease activity was transformed with a nuclease expression vector (pCM28nuc). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease in vivo. In sputa, S. aureus was associated to extracellular DNA structures. Nuclease activity in clinical S. aureus isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of nuc1 was inversely correlated to the activity of agr and was independent of saeS. NET-mediated killing was significantly higher in S. aureus isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28nuc conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in in vivo sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that S. aureus adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.

It would be desirable to have an unambiguous scheme for the typing of Shiga toxin-producing Escherichia coli (STEC) isolates to subpopulations. Such a scheme should take the high genomic plasticity of E. coli into account and utilize the stratification of STEC into subgroups, based on serotype or phylogeny. Therefore, our goal was to identify specific marker combinations for improved classification of STEC subtypes. We developed and evaluated two bioinformatic pipelines for genomic marker identification from larger sets of bacterial genome sequences. Pipeline A performed all-against-all BLASTp analyses of gene products predicted in STEC genome test sets against a set of control genomes. Pipeline B identified STEC marker genes by comparing the STEC core proteome and the “pan proteome” of a non-STEC control group. Both pipelines defined an overlapping, but not identical set of discriminative markers for different STEC subgroups. Differential marker prediction resulted from differences in genome assembly, ORF finding and inclusion cut-offs in both workflows. Based on the output of the pipelines, we defined new specific markers for STEC serogroups and phylogenetic groups frequently associated with outbreaks and cases of foodborne illnesses. These included STEC serogroups O157, O26, O45, O103, O111, O121, and O145, Shiga toxin-positive enteroaggregative E. coli O104:H4, and HUS-associated sequence type (ST)306. We evaluated these STEC marker genes for their presence in whole genome sequence data sets. Based on the identified discriminative markers, we developed a multiplex PCR (mPCR) approach for detection and typing of the targeted STEC. The specificity of the mPCR primer pairs was verified using well-defined clinical STEC isolates as well as isolates from the ECOR, DEC, and HUSEC collections. The application of the STEC mPCR for food analysis was tested with inoculated milk. In summary, we evaluated two different strategies to screen large genome sequence data sets for discriminative markers and implemented novel marker genes found in this genome-wide approach into a DNA-based typing tool for STEC that can be used for the characterization of STEC from clinical and food samples.

Bats are economically important animals and serve as food sources in some African regions. They can be colonized with the Staphylococcus aureus complex, which includes Staphylococcus schweitzeri and Staphylococcus argenteus. Fecal carriage of S. aureus complex in the straw-colored fruit bat (Eidolon helvum) has been described. However, data on their transmission and adaptation in animals and humans are limited. The aim of this study was to investigate the population structure of the S. aureus complex in E. helvum and to assess the geographical spread of S. aureus complex among other animals and humans. Fecal samples were collected from E. helvum in Obafemi Awolowo University, Ile-Ife, Nigeria. The isolates were characterized by antimicrobial susceptibility testing, spa typing and multilocus sequence typing (MLST). Isolates were screened for the presence of lukS/lukF-PV and the immune evasion cluster (scn, sak, chp) which is frequently found in isolates adapted to the human host. A Neighbor-Joining tree was constructed using the concatenated sequences of the seven MLST genes. A total of 250 fecal samples were collected and 53 isolates were included in the final analysis. They were identified as S. aureus (n = 28), S. schweitzeri (n = 11) and S. argenteus (n = 14). Only one S. aureus was resistant to penicillin and another isolate was intermediately susceptible to tetracycline. The scn, sak, and chp gene were not detected. Species-specific MLST clonal complexes (CC) were detected for S. aureus (CC1725), S. argenteus (CC3960, CC3961), and S. schweitzeri (CC2463). STs of S. schweitzeri from this study were similar to STs from bats in Nigeria (ST2464) and Gabon (ST1700) or from monkey in Côte d'Ivoire (ST2058, ST2072). This suggests host adaptation of certain clones to wildlife mammals with a wide geographical spread in Africa. In conclusion, there is evidence of fecal carriage of members of S. aureus complex in E. helvum. S. schweitzeri from bats in Nigeria are closely related to those from bats and monkeys in West and Central Africa suggesting a cross-species transmission and wide geographical distribution. The low antimicrobial resistance rates and the absence of the immune evasion cluster suggests a limited exposure of these isolates to humans.

We performed a cohort study to analyze the virulome of Staphylococcus aureus from the Democratic Republic of the Congo using whole genome sequencing and to assess its impact on the course of S. aureus infections. Community-associated S. aureus from nasal colonization (n = 100) and infection (n = 86) were prospectively collected. Phenotypic susceptibility testing and WGS was done for each isolate. WGS data were used to screen for 79 different virulence factors and for genotyping purposes (spa typing, multilocus sequence typing). The majority of the 79 virulence factors were equally distributed among isolates from colonization and infection. Panton-Valentine leukocidin (PVL) and the non-truncated hemolysin β were associated with skin and soft tissue infection (SSTI) and recurrence of disease but did not influence the course of infection (i.e., mortality, surgical intervention). For the first time, we show that not only PVL but also hemolysin β could contribute to the development of SSTI in PVL-endemic areas such as Africa.

Melioribacter roseus, a representative of recently proposed Ignavibacteriae phylum, is a metabolically versatile thermophilic bacterium, inhabiting subsurface biosphere of the West-Siberian megabasin and capable of growing on various substrates and electron acceptors. Genomic analysis followed by inhibitor studies and membrane potential measurements of aerobically grown M. roseus cells revealed the activity of aerobic respiratory electron transfer chain comprised of respiratory complexes I and IV, and an alternative complex III. Phylogeny reconstruction revealed that oxygen reductases belonged to atypical cc(o/b)o3-type and canonical cbb3–type cytochrome oxidases. Also, two molybdoenzymes of M. roseus were affiliated either with Ttr or Psr/Phs clades, but not with typical respiratory arsenate reductases of the Arr clade. Expression profiling, both at transcripts and protein level, allowed us to assign the role of the terminal respiratory oxidase under atmospheric oxygen concentration for the cc(o/b)o3 cytochrome oxidase, previously proposed to serve for oxygen detoxification only. Transcriptomic analysis revealed the involvement of both molybdoenzymes of M. roseus in As(V) respiration, yet differences in the genomic context of their gene clusters allow to hypothesize about their distinct roles in arsenate metabolism with the ‘Psr/Phs’-type molybdoenzyme being the most probable candidate respiratory arsenate reductase. Basing on multi-omics data, the pathways for aerobic and arsenate respiration were proposed. Our results start to bridge the vigorously increasing gap between homology-based predictions and experimentally verified metabolic processes, what is especially important for understudied microorganisms of novel lineages from deep subsurface environments of Eurasia, which remained separated from the rest of the biosphere for several geological periods.