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Gram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against the physical and chemical environmental challenges. To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein-channel. Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the β-barrel assembly machinery (BAM). Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG-SecDF-YidC – aka the holo-translocon (HTL). Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. We propose that this immediate interaction with a recruited chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane.

The epoch-making proliferation of Internet of Things (IoT) networks in recent years has brought connectivity to homes, cities, farms, and many other industries. ISM bands are accommodating most of these networks around the world. However, our experience from several global deployments has shown that such networks are bottlenecked by communication range and bandwidth. With these IoT deployment constraints in mind, we propose a new connectivity solution, Whisper [1], where IoT devices can opportunistically transmit data in the TV White Space (TVWS) spectrum, while protecting incumbents from receiving harmful interference.

Flow cytometry of intestinal lymphocytes is discussed to be a stronger predictor of enteropathy-associated T-cell lymphoma development in refractory celiac disease than T-cell clonality analysis.To investigate possible associations between clinical characteristics of refractory celiac disease patients and aberrant intraepithelial lymphocytes and to evaluate the accuracy of immunophenotyping for the identification of high-risk refractory celiac disease.Flow cytometry of isolated lymphocytes from duodenal biopsies of controls and celiac disease patients was performed and results were compared to clinical data.Flow cytometry analysis was performed on 42 controls, 37 non-complicated celiac disease and 30 refractory celiac disease cases with or without T-cell receptor clonality. Elevated aberrant intraepithelial lymphocyte counts were significantly associated with severe malabsorption. A 15% cut-off (aberrant lymphocytes among all lymphocytes) had the best discriminatory ability to identify high-risk patients. However, this technique failed to identify some high-risk cases (sensitivity 63%, specificity 100%). The severity of malabsorption was added to the criteria for high-risk refractory celiac disease, improving the correct patients' allocation (sensitivity 100%, specificity 96%).Immunophenotyping of aberrant intraepithelial lymphocytes is a good predictor for high-risk refractory celiac disease. Furthermore, adding the evaluation of malabsorption to the diagnostic assessment of refractory celiac disease optimizes accuracy.

Strength of fiber reinforced polymers (FRPs) under compression loads is typically limited by a shear localization failure mode called microbuckling which is highly sensitive to fiber misalignment. In addition to the magnitude of fiber misalignment, the dimensionality of fiber misalignment also plays a prominent role in the prediction of compression strength. Therefore, a comparison between 1D, 2D, and 3D fiber misalignment is carried out in a finite element setting with a homogenized representation of fiber and matrix materials. In real FRP structures, fiber misalignment is spread in a correlated random manner throughout the material volume resulting in a distribution of compression strength. Spectral representation method is used for developing the volumetric representation of fiber misalignment in numerical models, thus preserving the spatial correlations of fiber misalignment. As an input to the spectral representation method, two different functional forms of spectral density of the fiber misalignment are considered. The results of model series based on functional forms of spectral density are also compared against a reference model series based on experimental measurements of the fiber misalignment. Finally, a simple relation is proposed for prediction of compression strength at different percentiles of distribution of failure strengths with regard to scaling of mean square spectral density.

The optimal treatment for patients with asymptomatic carotid artery stenosis is under debate. Since best medical treatment (BMT) has improved over time, the benefit of carotid endarterectomy (CEA) or carotid artery stenting (CAS) is unclear. Randomised data comparing the effect of CEA and CAS versus BMT alone are absent. We aimed to directly compare CEA plus BMT with CAS plus BMT and both with BMT only.SPACE-2 was a multicentre, randomised, controlled trial at 36 study centres in Austria, Germany, and Switzerland. We enrolled participants aged 50-85 years with asymptomatic carotid artery stenosis at the distal common carotid artery or the extracranial internal carotid artery of at least 70%, according to European Carotid Surgery Trial criteria. Initially designed as a three-arm trial including one group for BMT alone (with a randomised allocation ratio of 2·9:2·9:1), the SPACE-2 study design was amended (due to slow recruitment) to become two substudies with two arms each comparing CEA plus BMT with BMT alone (SPACE-2a) and CAS plus BMT with BMT alone (SPACE-2b); in each case in a 1:1 randomisation. Participants and clinicians were not masked to allocation. The primary efficacy endpoint was the cumulative incidence of any stroke or death from any cause within 30 days or any ipsilateral ischaemic stroke within 5 years. The primary safety endpoint was any stroke or death from any cause within 30 days after CEA or CAS. The primary analysis was by intention-to treat, which included all randomly assigned patients in SPACE-2, SPACE-2a, and SPACE-2b, analysed using meta-analysis of individual patient data. We did two-step hierarchical testing to first show superiority of CEA and CAS to BMT alone then to assess non-inferiority of CAS to CEA. Originally, we planned to recruit 3640 patients; however, the study had to be stopped prematurely due to insufficient recruitment. This report presents the primary analysis at 5-year follow-up. This trial is registered with ISRCTN, number ISRCTN78592017.513 patients across SPACE-2, SPACE-2a, and SPACE-2b were recruited and surveyed between July 9, 2009, and Dec 12, 2019, of whom 203 (40%) were allocated to CEA plus BMT, 197 (38%) to CAS plus BMT, and 113 (22%) to BMT alone. Median follow-up was 59·9 months (IQR 46·6-60·0). The cumulative incidence of any stroke or death from any cause within 30 days or any ipsilateral ischaemic stroke within 5 years (primary efficacy endpoint) was 2·5% (95% CI 1·0-5·8) with CEA plus BMT, 4·4% (2·2-8·6) with CAS plus BMT, and 3·1% (1·0-9·4) with BMT alone. Cox proportional-hazard testing showed no difference in risk for the primary efficacy endpoint for CEA plus BMT versus BMT alone (hazard ratio [HR] 0·93, 95% CI 0·22-3·91; p=0·93) or for CAS plus BMT versus BMT alone (1·55, 0·41-5·85; p=0·52). Superiority of CEA or CAS to BMT was not shown, therefore non-inferiority testing was not done. In both the CEA group and the CAS group, five strokes and no deaths occurred in the 30-day period after the procedure. During the 5-year follow-up period, three ipsilateral strokes occurred in both the CAS plus BMT and BMT alone group, with none in the CEA plus BMT group.CEA plus BMT or CAS plus BMT were not found to be superior to BMT alone regarding risk of any stroke or death within 30 days or ipsilateral stroke during the 5-year observation period. Because of the small sample size, results should be interpreted with caution.German Federal Ministry of Education and Research (BMBF) and German Research Foundation (DFG).

Background Personalized medicine requires the integration and analysis of vast amounts of patient data to realize individualized care. With Surgomics, we aim to facilitate personalized therapy recommendations in surgery by integration of intraoperative surgical data and their analysis with machine learning methods to leverage the potential of this data in analogy to Radiomics and Genomics. Methods We defined Surgomics as the entirety of surgomic features that are process characteristics of a surgical procedure automatically derived from multimodal intraoperative data to quantify processes in the operating room. In a multidisciplinary team we discussed potential data sources like endoscopic videos, vital sign monitoring, medical devices and instruments and respective surgomic features. Subsequently, an online questionnaire was sent to experts from surgery and (computer) science at multiple centers for rating the features’ clinical relevance and technical feasibility. Results In total, 52 surgomic features were identified and assigned to eight feature categories. Based on the expert survey (n = 66 participants) the feature category with the highest clinical relevance as rated by surgeons was “surgical skill and quality of performance” for morbidity and mortality (9.0 ± 1.3 on a numerical rating scale from 1 to 10) as well as for long-term (oncological) outcome (8.2 ± 1.8). The feature category with the highest feasibility to be automatically extracted as rated by (computer) scientists was “Instrument” (8.5 ± 1.7). Among the surgomic features ranked as most relevant in their respective category were “intraoperative adverse events”, “action performed with instruments”, “vital sign monitoring”, and “difficulty of surgery”. Conclusion Surgomics is a promising concept for the analysis of intraoperative data. Surgomics may be used together with preoperative features from clinical data and Radiomics to predict postoperative morbidity, mortality and long-term outcome, as well as to provide tailored feedback for surgeons. Graphical abstract

Phages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ffs have seen an extraordinary range of applications, yet the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. In this work, we use cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods to determine a structure of a filamentous virus including the tips. We show that structure combined with mutagenesis can identify phage domains that are important in bacterial attack and for release of new progeny, allowing new models to be proposed for the phage lifecycle.

Infant temperament is usually considered biologically driven and a precursor of personality. Despite being conceived as trait measures, parent reports for assessing infant temperament use short timescales, for example, the past 7 days, implying variability in temperament traits' expressions. In two daily diary studies, we use the perspective of whole-trait theory to investigate whether infant temperament is observable on a daily basis and to what degree infant temperament varies within-person across days. In Study 1, N = 137 mothers of infants aged 6-18 months reported on their infant’s daily (state) temperament (median number of days: 8, total observations: 984). The results suggest a substantial within-person variation in daily infant temperament (ICCs: .41 to .54). Study 2 (N = 199 mothers, median number of days: 7, total observations: 1375) replicated these results on the variability in infant state temperament (ICCs: .41 to .51). In addition, infant state temperament was related to infant trait temperament. However, certain temperament items – primarily those assessing surgency – were frequently rated as not applicable and did not seem suitable for daily assessments. Across both studies, results indicate substantial within-person variability in daily infant temperament and a strong trait component.

Background Detection of circulating tumour DNA (ctDNA) in biological fluids is a minimally invasive alternative to tissue biopsy for therapy monitoring. Cytokines are released in the tumour microenvironment to influence inflammation and tumorigenic mechanisms. Here, we investigated the potential biomarker utility of circulating cytokines vis-à-vis ctDNA in ALK-rearranged+ lung adenocarcinoma (ALK + NSCLC) and explored the optimal combination of molecular parameters that could indicate disease progression. Methods Longitudinal serum samples (n = 296) were collected from ALK + NSCLC patients (n = 38) under tyrosine kinase inhibitor (TKI) therapy and assayed to quantify eight cytokines: IFN-γ, IL-1β, IL-6, IL-8, IL-10, IL-12p70, MCP1 and TNF-α. Generalised linear mixed-effect modelling was performed to test the performance of different combinations of cytokines and previously determined ctDNA parameters in identifying progressive disease. Results Serum IL-6, IL-8 and IL-10 were elevated at progressive disease, with IL-8 having the most significant impact as a biomarker. Integrating changes in IL-8 with ctDNA parameters maximised the performance of the classifiers in identifying disease progression, but this did not significantly outperform the model based on ctDNA alone. Conclusions Serum cytokine levels are potential disease progression markers in ALK + NSCLC. Further validation in a larger and prospective cohort is necessary to determine whether the addition of cytokine evaluation could improve current tumour monitoring modalities in the clinical setting.

Normothermic machine perfusion (NMP) has emerged as an innovative organ preservation technique. Developing an understanding for the donor organ immune cell composition and its dynamic changes during NMP is essential. We aimed for a comprehensive characterization of immune cell (sub)populations, cell trafficking and cytokine release during liver NMP. Single-cell transcriptome profiling of human donor livers prior to, during NMP and after transplantation shows an abundance of CXC chemokine receptor 1+/2+ (CXCR1+/CXCR2+) neutrophils, which significantly decreased during NMP. This is paralleled by a large efflux of passenger leukocytes with neutrophil predominance in the perfusate. During NMP, neutrophils shift from a pro-inflammatory state towards an aged/chronically activated/exhausted phenotype, while anti-inflammatory/tolerogenic monocytes/macrophages are increased. We herein describe the dynamics of the immune cell repertoire, phenotypic immune cell shifts and a dominance of neutrophils during liver NMP, which potentially contribute to the inflammatory response. Our findings may serve as resource to initiate future immune-interventional studies.

Supplementary Data from A Costimulatory CAR Improves TCR-based Cancer Immunotherapy

T-cell receptors (TCR) recognize intracellular and extracellular cancer antigens, allowing T cells to target many tumor antigens. To sustain proliferation and persistence, T cells require not only signaling through the TCR (signal 1), but also costimulatory (signal 2) and cytokine (signal 3) signaling. Because most cancer cells lack costimulatory molecules, TCR engagement at the tumor site results in incomplete T-cell activation and transient antitumor effects. To overcome this lack of signal 2, we genetically modified tumor-specific T cells with a costimulatory chimeric antigen receptor (CoCAR). Like classical CARs, CoCARs combine the antigen-binding domain of an antibody with costimulatory endodomains to trigger T-cell proliferation, but CoCARs lack the cytotoxic CD3ζ chain to avoid toxicity to normal tissues. We first tested a CD19-targeting CoCAR in combination with an HLA-A*02:01-restricted, survivin-specific transgenic TCR (sTCR) in serial cocultures with leukemia cells coexpressing the cognate peptide–HLA complex (signal 1) and CD19 (signal 2). The CoCAR enabled sTCR+ T cells to kill tumors over a median of four additional tumor challenges. CoCAR activity depended on CD19 but was maintained in tumors with heterogeneous CD19 expression. In a murine tumor model, sTCR+CoCAR+ T cells improved tumor control and prolonged survival compared with sTCR+ T cells. We further evaluated the CoCAR in Epstein–Barr virus–specific T cells (EBVST). CoCAR-expressing EBVSTs expanded more rapidly than nontransduced EBVSTs and delayed tumor progression in an EBV+ murine lymphoma model. Overall, we demonstrated that the CoCAR can increase the activity of T cells expressing both native and transgenic TCRs and enhance antitumor responses.

It is estimated that up to 60% of people living with dementia go missing at least once during the course of their disease. Databases on missing incidents involving people living with dementia are managed in silos with minimal or incomplete data. A national strategy for the collection of data on missing incidents of people living with dementia would optimize time and resources spent on police and search and rescue and enhance chances of saving lives of those who go missing. Such a strategy would be a first step toward developing strategies to prevent future missing person incidents among this population. The objectives of this manuscript are to: (1) describe the issues and challenges related to the lack of integrated data on people living with dementia at risk of going missing, and (2) propose directions to create a national database.

Perioperative chemotherapy is the standard of care for patients undergoing curative resection for gastroesophageal adenocarcinoma. However, less than 50% of patients complete postoperative chemotherapy, and the added benefit to preoperative chemotherapy remains unclear. The aim of this study was to compare disease-free and overall survival (DFS and OS) in patients with perioperative chemotherapy to those who received preoperative chemotherapy only. In addition, a current literature overview is included. This multicenter, retrospective case series included 124 patients with gastroesophageal adenocarcinoma undergoing potentially curative resection and receiving pre- or perioperative chemotherapy between 2006 and 2010. Histopathological, demographic, clinical, and survival data were used to identify the impact of perioperative vs. preoperative chemotherapy on DFS and OS. Patients with perioperative chemotherapy had significantly improved DFS and OS (median DFS 28.0 months; 95%CI 0–62.4 vs. 19.0 months; 95%CI 10.5–27.5; p = 0.008 and median OS 35.7 months; 95%CI 0–73.6 vs. 19.2 months; 95%CI 7.8–30.4; p = 0.002). However, in contrast to patients with tumor-free lymph nodes at the time of resection, patients with positive lymph node status did not significantly benefit from additional postoperative chemotherapy in subgroup analysis. Further studies are encouraged to investigate optimal adjuvant treatment strategies for primary chemotherapy-resistant patients.

ZUSAMMENFASSUNGHintergrund: Die Rettungs-BOA ist ein Hilfsmittel zur schnellen Rettung von kritisch Verletzten aus Fahrzeugen unter Gewährleistung einer Teilimmobilisation der oberen Brustwirbelsäule und wird im Rahmen der PHTLS-Ausbildung der Bundeswehr gelehrt. Auf den Flugplätzen der Bundeswehr findet dieses Rettungshilfsmittel bisher keine flächendeckende Anwendung. Bei Flugunfällen kann es zu schweren Verletzungen der Luftfahrzeuginsassen kommen, wo genau diese Anforderungen an ein Hilfsmittel gestellt werden.Methoden: In einer Studie mit der Feuerwehr am Flugplatz Manching im August 2020 wurde die Anwendung der Rettungs-BOA an 7 verschiedenen Luftfahrzeugen getestet und mit anderen Rettungsmethoden (Rautek-Griff und KED-System) im Hinblick auf Rettungszeiten und mit Fragebögen zu Bedienerfreundlichkeit, Rückenschmerzen der Bediener sowie Vor- und Nachteilen bewertet. Die statistische Auswertung erfolgte mit IBM SPSS Statistik 17.0. Die Teilnahme erfolgte freiwillig. Die Daten wurden anonymisiert gespeichert.Ergebnisse: Die Rettung mittels Rautek-Griff geht erwartungsgemäß am schnellsten. Unwesentlich länger dauert die Rettung mittels BOA, gefolgt von der zeitaufwendigen Rettung mittels KED-System. In den Luftfahrzeugen Tornado, Eurofighter und A-400M erwies sich die BOA zeitlich nicht unterlegen. Insgesamt zeigte sich die Rettungs-BOA als einfaches, unkompliziertes und schnelles Rettungshilfsmittel, das sich auch bei Unerfahrenheit in der Anwendung universell in allen getesteten Luftfahrzeugen anwenden ließ.

Surgery faces a paradigm shift since it has developed rapidly in recent decades, becoming a high-tech discipline. Increasingly powerful technological developments such as modern operating rooms, featuring digital and interconnected equipment and novel imaging as well as robotic procedures, provide several data sources resulting in a huge potential to improve patient therapy and surgical outcome by means of Surgical Data Science. The emerging field of Surgical Data Science aims to improve the quality of surgery through acquisition, organization, analysis, and modeling of data, in particular using machine learning (ML). An integral part of surgical data science is to analyze the available data along the surgical treatment path and provide a context-aware autonomous action by means of ML methods. Autonomous actions related to surgical decision-making include preoperative decision support, intraoperative assistance functions, as well as robot-assisted actions. The goal is to democratize surgical skills and enhance the collaboration between surgeons and cyber-physical systems by quantifying surgical experience and making it accessible to machines, thereby improving patient therapy and outcome. The article introduces basic ML concepts as enablers for autonomous actions in surgery, highlighting examples for such actions along the surgical treatment path.

Ligation of programmed cell death-1 (PD-1) in the tumor microenvironment is known to inhibit effective adaptive antitumor immunity. Blockade of PD-1 in humans has resulted in impressive, durable regression responses in select tumor types. However, durable responses have been elusive in ovarian cancer patients. PD-1 was recently shown to be expressed on and thereby impair the functions of tumor-infiltrating murine and human myeloid dendritic cells (TIDC) in ovarian cancer. In the present work, we characterize the regulation of PD-1 expression and the effects of PD-1 blockade on TIDC. Treatment of TIDC and bone marrow–derived dendritic cells (DC) with IL10 led to increased PD-1 expression. Both groups of DCs also responded to PD-1 blockade by increasing production of IL10. Similarly, treatment of ovarian tumor–bearing mice with PD-1 blocking antibody resulted in an increase in IL10 levels in both serum and ascites. While PD-1 blockade or IL10 neutralization as monotherapies were inefficient, combination of these two led to improved survival and delayed tumor growth; this was accompanied by augmented antitumor T- and B-cell responses and decreased infiltration of immunosuppressive MDSC. Taken together, our findings implicate compensatory release of IL10 as one of the adaptive resistance mechanisms that undermine the efficacy of anti–PD-1 (or anti–PD-L1) monotherapies and prompt further studies aimed at identifying such resistance mechanisms. Cancer Res; 77(23); 6667–78. ©2017 AACR.

Expression of PD-1 on BMDCs and T cells after exposure to IL-6 and IL-10

In mitosis, the kinetochores of chromosomes that lack full microtubule attachments and/or mechanical tension activate a signaling pathway called the mitotic spindle checkpoint that blocks progression into anaphase and prevents premature segregation of the chromatids until chromosomes become aligned at the metaphase plate. The spindle checkpoint is responsible for arresting cells in mitosis in response to chemotherapeutic spindle poisons such as paclitaxel or vinblastine. Some cancer cells show a weakened checkpoint signaling system that may contribute to chromosome instability in tumors. Because complete absence of the spindle checkpoint leads to catastrophic cell division, we reasoned that drugs targeting the checkpoint might provide a therapeutic window in which the checkpoint would be eliminated in cancer cells but sufficiently preserved in normal cells. We developed an assay to identify lead compounds that inhibit the spindle checkpoint. Most cells respond to microtubule drugs by activating the spindle checkpoint and arresting in mitosis with a rounded morphology. Our assay depended on the ability of checkpoint inhibitor compounds to drive mitotic exit and cause cells to flatten onto the substrate in the continuous presence of microtubule drugs. In this study, we characterize one of the compounds, OM137, as an inhibitor of Aurora kinases. We find that this compound is growth inhibitory to cultured cells when applied at high concentration and potentiates the growth inhibitory effects of subnanomolar concentrations of paclitaxel. [Cancer Res 2009;69(4):1509–16]

Supplementary Video 4 from A High Throughput, Whole Cell Screen for Small Molecule Inhibitors of the Mitotic Spindle Checkpoint Identifies OM137, a Novel Aurora Kinase Inhibitor

Supplementary Legends 1-4 from A High Throughput, Whole Cell Screen for Small Molecule Inhibitors of the Mitotic Spindle Checkpoint Identifies OM137, a Novel Aurora Kinase Inhibitor

Supplementary Video 1 from A High Throughput, Whole Cell Screen for Small Molecule Inhibitors of the Mitotic Spindle Checkpoint Identifies OM137, a Novel Aurora Kinase Inhibitor

Supplementary Video 3 from A High Throughput, Whole Cell Screen for Small Molecule Inhibitors of the Mitotic Spindle Checkpoint Identifies OM137, a Novel Aurora Kinase Inhibitor

Supplementary Video 2 from A High Throughput, Whole Cell Screen for Small Molecule Inhibitors of the Mitotic Spindle Checkpoint Identifies OM137, a Novel Aurora Kinase Inhibitor