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Summary: The NLRP3 inflammasome is a key multi-protein complex controlling inflammation, particularly interleukin-1β (IL-1β) production. Here, we present a protocol to profile spatially resolved NLRP3 inflammasome complexes using ascorbic peroxidase 2 (APEX2)-based proximity labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). We describe steps for design and generation of the fusion construct, characterization of the stable FLAG-NLRP3-APEX2 expression cell line by western blotting/imaging, biotinylated proteome enrichment, and mass spectrometry analysis.For complete details on the use and execution of this protocol, please refer to Liang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Background Neoadjuvant cisplatin‐based chemotherapy (NAC) followed by cystectomy is the standard of care for patients with muscle‐invasive bladder cancer (MIBC). Pathologic complete response (pCR) is associated with favorable outcomes, but only 30%–40% of patients achieve that response. The aim of this study is to investigate the role played by the Tumor and Immune Microenvironment (TIME) in association with the clinical outcome of patients with MIBC undergoing NAC. Methods Nineteen patients received NAC and were classified as pCR (n = 10) or non‐pCR (n = 9). Bulk RNA‐seq and immune protein evaluations using Digital Spatial Profiling (DSP) were performed on formalin‐fixed paraffin‐embedded (FFPE) tumor biopsies collected before NAC (baseline). Immunohistochemistry (IHC) evaluation focused on CD3 and CD20 expression was performed on baseline and end‐of‐treatment (EOT) FFPEs. Baseline peripheral blood was assessed for lymphocyte and neutrophil counts. Kaplan–Meier analyses and Cox PH regression models were used for survival analyses (OS). Results In the periphery, pCR patients showed lower neutrophil counts, and neutrophil/ lymphocyte ratio (NLR) when compared to non‐pCR patients. In the tumor microenvironment (TME), gene expression analysis and protein evaluations highlighted an abundance of B cells and CD3+ T cells in pCR versus non‐pCR patients. On the contrary, increased protein expression of ARG1+ cells, and cells expressing immune checkpoints such as LAG3, ICOS, and STING were observed in the TME of patients with non‐pCR. Conclusions In the current study, we demonstrated that lower NLR levels and increased CD3+ T cells and B cell infiltration are associated with improved response and long‐term outcomes in patients with MIBC receiving NAC. These findings suggest that the impact of immune environment should be considered in determining the clinical outcome of MIBC patients treated with NAC.

ABSTRACT The interferon (IFN) system protects mammals from diseases caused by virus infections. IFN synthesis is induced by pattern recognition receptor signaling pathways activated by virus infection. IFN is secreted from the infected cells and acts upon neighboring cells by binding cell surface receptors and triggering induction of hundreds of IFN-stimulated genes and proteins, many of which block different steps of virus replication. The IFN-induced tetratricopeptide repeat proteins (IFIT) are a family of RNA-binding proteins. We and others have previously reported that IFIT2 protects mice from many neurotropic RNA viruses; indeed, Ifit2−/− mice are very susceptible to intranasal or subcutaneous infections with vesicular stomatitis virus (VSV). Here, using a newly generated conditional knockout mouse, we report that ablation of Ifit2 expression only in neuronal cells was sufficient to render mice susceptible to neuropathogenesis caused by intranasal, but not subcutaneous, infection of VSV. Another genetically modified mouse line, expressing a mutant IFIT2 that cannot bind RNA, was as susceptible to VSV infection as Ifit2−/− mice. These results demonstrated that IFIT2 RNA-binding activity is essential for protecting mice against neurological diseases caused by intranasal infection of VSV.IMPORTANCEInterferon’s (IFN’s) antiviral effects are mediated by the proteins encoded by the interferon-stimulated genes. IFN-stimulated genes (IFIT2) is one such protein, which inhibits replication of many RNA viruses in the mouse brain and the resultant neuropathology. Our study sheds light on how IFIT2 works. By ablating Ifit2 expression only in neuronal cells, using a newly generated conditional knockout mouse line, we showed that Ifit2 induction in the neurons of the infected mouse was necessary for antiviral function of interferon. IFIT2 has no known enzyme activity; instead, it functions by binding to cellular or viral proteins or RNAs. We engineered a new mouse line that expressed a mutant IFIT2 that cannot bind RNA. These mice were very susceptible to infection with vesicular stomatitis virus indicating that the RNA-binding property of IFIT2 was essential for its antiviral function in vivo.

Summary: Activation of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome complex is an essential innate immune signaling mechanism. To reveal how human NLRP3 inflammasome assembly and activation are controlled, in particular by components of the ubiquitin system, proximity labeling, affinity purification, and RNAi screening approaches were performed. Our study provides an intricate time-resolved molecular map of different phases of NLRP3 inflammasome activation. Also, we show that ubiquitin C-terminal hydrolase 1 (UCH-L1) interacts with the NACHT domain of NLRP3. Downregulation of UCH-L1 decreases pro-interleukin-1β (IL-1β) levels. UCH-L1 chemical inhibition with small molecules interfered with NLRP3 puncta formation and ASC oligomerization, leading to altered IL-1β cleavage and secretion, particularly in microglia cells, which exhibited elevated UCH-L1 expression as compared to monocytes/macrophages. Altogether, we profiled NLRP3 inflammasome activation dynamics and highlight UCH-L1 as an important modulator of NLRP3-mediated IL-1β production, suggesting that a pharmacological inhibitor of UCH-L1 may decrease inflammation-associated pathologies.

Summary: Precise regulation of Type I interferon signaling is crucial for combating infection and cancer while avoiding autoimmunity. Type I interferon signaling is negatively regulated by USP18. USP18 cleaves ISG15, an interferon-induced ubiquitin-like modification, via its canonical catalytic function, and inhibits Type I interferon receptor activity through its scaffold role. USP18 loss-of-function dramatically impacts immune regulation, pathogen susceptibility, and tumor growth. However, prior studies have reached conflicting conclusions regarding the relative importance of catalytic versus scaffold function. Here, we develop biochemical and cellular methods to systematically define the physiological role of USP18. By comparing a patient-derived mutation impairing scaffold function (I60N) to a mutation disrupting catalytic activity (C64S), we demonstrate that scaffold function is critical for cancer cell vulnerability to Type I interferon. Surprisingly, we discovered that human USP18 exhibits minimal catalytic activity, in stark contrast to mouse USP18. These findings resolve human USP18's mechanism-of-action and enable USP18-targeted therapeutics.

Abstract Actin beta-like 2 (ACTBL2) was recently identified as a new mediator of migration in ovarian cancer cells. Yet, its impact on tumor-infiltrating and thus migrating leukocytes (TILs) remains to date unknown. This study characterizes the subset of ACTBL2-expressing TILs in epithelial ovarian cancer (EOC) and elucidates their prognostic influence on the overall survival of EOC patients with special regard to different histological subtypes. Comprehensive immunohistochemical analyses of Tissue-Microarrays of 156 ovarian cancer patients revealed, that a tumor infiltration by ACTBL2-positive leukocytes was significantly associated with an improved overall survival (OS) (61.2 vs. 34.4 months; p = 0.006) and was identified as an independent prognostic factor (HR = 0.556; p = 0.038). This significant survival benefit was particularly evident in patients with low-grade serous carcinoma (OS: median not reached vs. 15.6 months, p

Abstract The IGF2BP family of RNA binding proteins consists of three paralogs that regulate intracellular RNA localization, RNA stability, and translational control. Although IGF2BP1 and 3 are oncofetal proteins, IGF2BP2 expression is maintained in many tissues, including the heart, into adulthood. IGF2BP2 is upregulated in cardiomyocytes during cardiac stress and remodeling and returns to normal levels in recovering hearts. We wondered whether IGF2BP2 might play an adaptive role during cardiac stress and recovery. Enhanced expression of an IGF2BP2 transgene in a conditional, inducible mouse line leads to dilated cardiomyopathy (DCM) and death within 3-4 weeks in newborn or adult hearts. Downregulation of the transgene after 2 weeks, however, rescues these mice, with complete recovery by 12 weeks. Hearts overexpressing IGF2BP2 downregulate sarcomeric and mitochondrial proteins and have fragmented mitochondria and elongated, thinner sarcomeres. IGF2BP2 is also upregulated in DCM or myocardial infarction patients. These results suggest that IGF2BP2 may be an attractive target for therapeutic intervention in cardiomyopathies.

Abstract The spatial organisation of cellular protein expression profiles within tissue determines cellular function and is key to understanding disease pathology. To define molecular phenotypes in the spatial context of tissue, there is a need for unbiased, quantitative technology capable of mapping proteomes within tissue structures. Here, we present a workflow for spatially-resolved, quantitative proteomics of tissue that generates maps of protein abundance across tissue slices derived from a human atypical teratoid-rhabdoid tumour at three spatial resolutions, the highest being 40 µm, to reveal distinct abundance patterns of thousands of proteins. We employ spatially-aware algorithms that do not require prior knowledge of the fine tissue structure to detect proteins and pathways with spatial abundance patterns and correlate proteins in the context of tissue heterogeneity and cellular features such as extracellular matrix or proximity to blood vessels. We identify PYGL, ASPH and CD45 as spatial markers for tumour boundary and reveal immune response-driven, spatially-organised protein networks of the extracellular tumour matrix. Overall, we demonstrate spatially-aware deep proteo-phenotyping of tissue heterogeneity, to re-define understanding tissue biology and pathology at the molecular level.

Abstract Background Patients with neurogenic lower urinary tract dysfunction (NLUTD) often rely on some type of catheterization for bladder emptying. Intermittent catheterization (IC) is considered the gold standard and is preferred over continuous catheterization, since it is considered to cause fewer urinary tract infections (UTIs) than indwelling catheterization. The main objective of our study was to describe UTI prevalence (at visit) and incidence (within the last 12 months) and urine culture characteristics between patients using an indwelling catheter versus (vs) those performing IC. Methods In this cross-sectional study, we prospectively evaluated from 02/2020 to 01/2021 patients with NLUTD undergoing urine cultures for prophylactic reasons or due to UTI symptoms. At visit, all patients underwent a standardized interview on current UTI symptoms as well as UTI history and antibiotic consumption within the past year. Patients using an indwelling catheter (n = 206) or IC (n = 299) were included in the analysis. The main outcome was between-group differences regarding UTI characteristics. Results Patients using an indwelling catheter were older (indwelling catheter vs IC: median 66 (Q1-Q3: 55—77) vs 55 (42—67) years of age) and showed a higher Charlson comorbidity index (indwelling catheter vs IC: median 4 (Q1-Q3: 2–6) vs 2 (1–4) (both p

Abstract Bacteriophages operate via pathogen-specific mechanisms of action distinct from conventional, broad-spectrum antibiotics and are emerging as promising alternative antimicrobials. However, phage-mediated killing is often limited by bacterial resistance development. Here, we engineer phages for target-specific effector gene delivery and host-dependent production of colicin-like bacteriocins and cell wall hydrolases. Using urinary tract infection (UTI) as a model, we show how heterologous effector phage therapeutics (HEPTs) suppress resistance and improve uropathogen killing by dual phage- and effector-mediated targeting. Moreover, we designed HEPTs to control polymicrobial uropathogen communities through production of effectors with cross-genus activity. Using phage-based companion diagnostics, we identified potential HEPT responder patients and treated their urine ex vivo. Compared to wildtype phage, a colicin E7-producing HEPT demonstrated superior control of patient E. coli bacteriuria. Arming phages with heterologous effectors paves the way for successful UTI treatment and represents a versatile tool to enhance and adapt phage-based precision antimicrobials.

Abstract The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Enterococcus spp., and Klebsiella spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥103 CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application.

Abstract Fatty liver disease or the accumulation of fat in the liver, has been reported to affect the global population. This comes with an increased risk for the development of fibrosis, cirrhosis, and hepatocellular carcinoma. Yet, little is known about the effects of a diet containing high fat and alcohol towards epigenetic aging, with respect to changes in transcriptional and epigenomic profiles. In this study, we took up a multi-omics approach and integrated gene expression, methylation signals, and chromatin signals to study the epigenomic effects of a high-fat and alcohol-containing diet on mouse hepatocytes. We identified four relevant gene network clusters that were associated with relevant pathways that promote steatosis. Using a machine learning approach, we predict specific transcription factors that might be responsible to modulate the functionally relevant clusters. Finally, we discover four additional CpG loci and validate aging-related differential CpG methylation. Differential CpG methylation linked to aging showed minimal overlap with altered methylation in steatosis.

Abstract Aim Chemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches. Methods Gene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipitation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses. Results We identified IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was significantly associated with resistance to selumetinib, gefitinib, and regorafenib in PDOs and to 5-fluorouracil and oxaliplatin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabilization of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confirmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells. Conclusions IGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mitochondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance.

Indoleamine 2,3-deoxygenase (IDO) plays an important role in the catabolism of the amino acid tryptophan. Tryptophan and its metabolites are key immune modulators. Increased IDO activity has been observed in various diseases and is associated with worse clinical outcomes. However, comprehensive research regarding its role in cardiac surgery remains limited. Therefore, we aimed to investigate perioperative changes in IDO activity and pathway metabolites, along with their impact on clinical outcomes in adult patients undergoing cardiac surgery. As an observational cohort study conducted at the Inselspital in Bern from January to December 2019, we retrospectively analyzed the data of prospectively collected biobank samples of patients undergoing cardiac surgery with the use of cardiopulmonary bypass. IDO pathway metabolite analysis was conducted by mass spectrometry. Perioperative dynamics were descriptively assessed and associated with pre-defined clinical outcome measures (30-day mortality, 1-year mortality, incidence of stroke and myocardial infarction, and length of hospital stay) through a multi-step exploratory regression analysis. A cohort of 192 adult patients undergoing cardiac surgery with the use of cardiopulmonary bypass were included (median age 67.0, IQR 60.0–73.0, 75.5% male). A significant perioperative decrease in the kynurenine/tryptophan (Kyn/Trp) ratio (−2.298, 95% CI −4.028 to −596, p = 0.009) and significant perioperative dynamics in the associated metabolites was observed. No association of perioperative changes in IDO activity and pathway metabolites with clinical outcomes was found. A significant decrease in the Kyn/Trp ratio among adult patients undergoing cardiac surgery indicates a perioperative downregulation of IDO, which stands in contrast to other pro-inflammatory conditions. Further studies are needed to investigate IDO in the setting of perioperative immunomodulation, which is a key driver of postoperative complications in cardiac surgery patients.

BackgroundMolecular mechanisms underlying perioperative acute phase reactions in cardiac surgery are largely unknown. We aimed to characterise perioperative alterations of the acute phase plasma proteome in a cohort of adult patients undergoing on-pump cardiac surgery using high-throughput mass spectrometry and to identify candidate proteins potentially relevant to postoperative clinical outcome through a novel, multi-step approach.MethodsThis study is an analysis of the Bern Perioperative Biobank, a prospective cohort of adults who underwent cardiac surgery with the use of cardiopulmonary bypass (CPB) at Bern University Hospital between January and December 2019. Blood samples were taken before induction of anaesthesia and on postoperative day one. Proteomic analyses were performed by mass spectrometry. Through a multi-step, exploratory approach, hit-proteins were first identified according to their perioperative prevalence and dynamics. The set of hit-proteins were associated with predefined clinical outcome measures (all-cause one-year mortality, length of hospital stay, postoperative myocardial infarction and stroke until hospital discharge).Results192 patients [75.5% male, median age 67.0 (IQR 60.0–73.0)] undergoing cardiac surgery with the use of CPB were included in this analysis. In total, we identified and quantified 402 proteins across all samples, whereof 30/402 (7%) proteins were identified as hit-proteins. Three hit-proteins—LDHB, VCAM1 and IGFBP2—demonstrated the strongest associations with clinical outcomes. After adjustment both for age, sex, BMI and for multiple comparisons, the scaled preoperative levels of IGFBP2 were associated with 1-year all-cause mortality (OR 10.63; 95% CI: 2.93–64.00; p = 0.046). Additionally, scaled preoperative levels of LDHB (OR 5.58; 95% CI: 2.58–8.57; p = 0.009) and VCAM1 (OR 2.32; 95% CI: 0.88–3.77; p = 0.05) were found to be associated with length of hospital stay.ConclusionsWe identified a subset of promising candidate plasma proteins relevant to outcome after on-pump cardiac surgery. IGFBP2 showed a strong association with clinical outcome measures and a significant association of preoperative levels with 1-year all-cause mortality. Other proteins strongly associated with outcome were LDHB and VCAM1, reflecting the dynamics in the acute phase response, inflammation and myocardial injury. We recommend further investigation of these proteins as potential outcome markers after cardiac surgery. Clinical Trial RegistrationClinicalTrials.gov; NCT04767685, data are available via ProteomeXchange with identifier PXD046496.

In hemophilia, the unmet needs regarding adherence to prophylaxis and lack of effective long-term prophylaxis regimens, especially in patients with inhibitors, led to the production of emicizumab, the first non-factor medicine for subcutaneous administration in patients with severe and moderate hemophilia A with or without factor VIII inhibitors. This review describes the research steps behind the development of this game-changing medication as well as its success in the prophylaxis of bleeding episodes, as witnessed by the results of pivotal clinical trials but also by real-life use in the frame of a still expanding global market. We also discuss potential and actual adverse events and the nuances related to clinical use, such as laboratory monitoring, development of neutralizing antidrug antibodies, risk of thrombosis/hypercoagulability and role in the management of surgical operations. The potential of emicizumab to prevent bleeding in other congenital and acquired coagulation disorders is also outlined.

Mutations within viral epitopes can result in escape from T cells, but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two SARS-CoV-2 nucleoprotein CD8+ epitopes, we investigated the contribution of these flanking mutations to proteasomal processing and T cell activation. We found decreased NP9-17-B*27:05 CD8+ T cell responses to the NP-Q7K mutation, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103 N/Y mutations flanking the NP9-17-B*27:05 and NP105-113-B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on proteasomal processing, either contributing to T cell escape or enhancement that may be exploited for future vaccine design.

Highlights USP18 knockdown results in transcriptome-wide changes that cause a switch from proliferation to muscle cell differentiation. Fully differentiated muscles cells (or simply 'myotubes') formed in absence of USP18 lack sarcomeric integrity (in both cell culture and engineered muscle models). An increase in USP18 expression during muscle cell differentiation is independent of its role in the interferon type 1 stimulated response. During normal muscle cell differentiation, USP18 accumulates in the cell nucleus regulating multiple cellular processes, including proliferation, differentiation initiation and muscle cell maturation. In the absence of USP18, muscle cells stop proliferating and undergo accelerated fusion as part of the differentiation process by altering the expression levels of myogenic regulatory (co-)transcription factors, whereas maturation is impaired due to reduced expression of calcium-flux genes and sarcomeric genes.

The ability for density functional theory with the B3LYP functional with the lanl2dz basis set to predict the 1st (Wave 1) and 2nd (Wave 2) reductions of the diazine ring in a series of thirty-seven (37) 1,4-di-N-oxide quinoxaline-2-carboxamide derivatives in dimethylformamide was examined. The B3LYP/lanl2dz method had a strong correlation and low correlation to the experimental potentials for Wave 1 and Wave 2, respectively. There are nine identifiable analogs based on similarities of structure. The predicted reduction potentials for the derivatives of each analog generally fit the modified Hammett equation. The B3LYP/lanl2dz method is shown to be useful in accurately predicting the Wave 1 potentials for quinoxaline-di-N-oxide derivatives. For derivatives with assessable anti-tuberculosis activity, the predicted Wave 1 potentials have a similar correlation with the bioactivity when compared to the experimental wave 1 potentials.

Context: The role of urodynamic studies (UDSs) in the diagnosis of lower urinary tract symptoms (LUTS) is crucial. Although expert statements and guidelines underline their value for clinical decision-making in various clinical settings, the academic debate as to their impact on patient outcomes continues. Objective: To summarise the evidence from all randomised controlled trials assessing the clinical usefulness of UDS in the management of LUTS. Evidence acquisition: For this systematic review, searches were performed without language restrictions in three electronic databases until November 18, 2020. The inclusion criteria were randomised controlled study design and allocation to receive UDS or not prior to any clinical management. Quality assessment was performed by two reviewers independently, using the Cochrane Collaboration’s tool for assessing the risk of bias. A random-effect meta-analysis was performed on the uniformly reported outcome parameters. Evidence synthesis: Eight trials were included, and all but two focused on women with pure or predominant stress urinary incontinence (SUI). A meta-analysis of six studies including 942 female patients was possible for treatment success, as defined by the authors (relative risk 1.00, 95% confidence interval: 0.93–1.07), indicating no difference in efficacy when managing women with UDS. Conclusions: Although UDSs are not replaceable in diagnostics, since there is no other equivalent method to find out exactly what the lower urinary tract problem is, there are little data supporting its impact on outcomes. Randomised controlled trials have focussed on a small group of women with uncomplicated SUI and showed no added value, but these findings cannot be extrapolated to the overall patient population with LUTS, warranting further well-designed trials. Patient summary: Despite urodynamics being the gold standard to assess lower urinary tract symptoms (LUTS), as it is the only method that can specify lower urinary tract dysfunction, more studies assessing the clinical usefulness of urodynamic studies (UDSs) in the management of LUTS are needed. UDS investigation is not increasing the probability of success in the treatment of stress urinary incontinence.

Background: The RNA-binding protein IGF2BP2/IMP2/VICKZ2/p62 is an oncofetal protein that is overexpressed in several cancer entities. Employing IMP2 knockout colorectal cancer cells, we could show the important role of IMP2 in several hallmarks of cancer. This study aimed to functionally characterize IMP2 in lung (A549, LLC1) and hepatocellular carcinoma (HepG2, Huh7) cell lines to assess its role as a potential target for these cancer entities. Methods: IMP2 knockouts were generated by CRISPR/Cas9 and its variant approach prime editing; the editing efficiency of two single guide RNAs (sgRNAs) was verified via next-generation sequencing. We studied the effect of IMP2 knockout on cell proliferation, colony formation, and migration and employed small-molecule inhibitors of IMP2. Results: Despite multiple attempts, it was not possible to generate IMP2 biallelic knockouts in A549 and Huh7 cells. Both sgRNAs showed good editing efficiency. However, edited cells lost their ability to proliferate. The attempt to generate an IMP2 biallelic knockout in LLC1 cells using CRISPR/Cas9 was successful. Monoallelic knockout cell lines of IMP2 showed a reduction in 2D cell proliferation and reduced migration. In 3D cultures, a change in morphology from compact spheroids to loose aggregates and a distinct reduction in the colony formation ability of the IMP2 knockouts was observed, an effect that was mimicked by previously identified IMP2 inhibitor compounds that also showed an inhibitory effect on colony formation. Conclusions: Our in vitro target validation supports that IMP2 is essential for tumor cell proliferation, migration, and colony formation in several cancer entities.

Summary: Cancer is a leading cause of mortality worldwide. Over 50% of cancers are diagnosed late, rendering many treatments ineffective. Existing liquid biopsy studies demonstrate a minimally invasive and inexpensive approach for disease detection but lack parsimonious biomarker selection, exhibit poor cancer detection performance and lack appropriate validation and testing. We established a tailored machine learning pipeline, DEcancer, for liquid biopsy analysis that addresses these limitations and improved performance. In a test set from a published cohort of 1,005 patients including 8 cancer types and 812 cancer-free individuals, DEcancer increased stage 1 cancer detection sensitivity across cancer types from 48 to 90%. In addition, with a test set cohort of patients from a high dimensional proteomics dataset of 61 lung cancer patients and 80 cancer-free individuals, DEcancer’s performance using a 14-43 protein panel was comparable to 1,000 original proteins. DEcancer is a promising tool which may facilitate improved cancer detection and management.

Background: There is limited epidemiological evidence describing contemporary neuro-urological management of persons with acute spinal cord injury (SCI). Objective: To describe neurogenic lower urinary tract dysfunction (NLUTD) management at discharge from SCI rehabilitation. Design, setting, and participants: The population-based Swiss Spinal Cord Injury (SwiSCI) cohort study prospectively collected data from 602 adults undergoing specialized postacute SCI rehabilitation from 2013 to 2020. The management strategy was based on the European Association of Urology (EAU) Guidelines on Neuro-Urology. Outcome measurements and statistical analysis: Data were collected at discharge using the International SCI Lower Urinary Tract Function Basic Data Set. Multivariable logistic regression adjusting for demographics, SCI characteristics, and center, with inverse probability weighting accounting for sampling bias, was used to produce prevalence estimates and identify predictors of lower urinary tract symptoms (LUTS) and NLUTD management outcomes. Results and limitations: At discharge (median time after SCI: 5.0 mo [Q1-Q3: 3.0–7.2]), the prevalence of LUTS or managed NLUTD was 82% (95% confidence interval [CI]: 79–85%). SCI completeness was the main predictor of LUTS and managed NLUTD. The risk of urinary incontinence was elevated in females (odds ratio 1.98 [95% CI: 1.18–3.32]) and with complete lesions (odds ratio 4.71 [95% CI: 2.52–8.81]). Voiding dysfunction was most commonly managed with intermittent catheterization (prevalence 39% [95% CI: 35–42%]), followed by indwelling catheterization (prevalence 22% [95% CI: 18–25%]). The prevalence of antimuscarinic or mirabegron use was 29% (95% CI: 26–33%). Urodynamic and renal function data were not collected. Conclusions: Our population-based description of urological management in Swiss SCI centers utilizing the EAU Guidelines on Neuro-Urology may be used as a reference for evaluation in other settings. Data further indicate a need for sex-specific neuro-urological management research. Patient summary: At discharge from spinal cord injury (SCI) rehabilitation, a majority of patients have lower urinary tract problems, especially those with complete SCI. Women have a higher risk of urinary incontinence.

Despite the fact that a majority of patients with an injury to the spinal cord develop lower urinary tract dysfunction, only few treatment options are available currently once the dysfunction arises. Tibial nerve stimulation has been used in pilot clinical trials, with some promising results. Hence, we investigated whether the early application of transcutaneous tibial nerve stimulation in the animal model of spinal cord injured rats can prevent the development of detrusor overactivity and/or detrusor-sphincter-dyssynergia. Rats were implanted with a bladder catheter and external urethral sphincter electromyography electrodes. A dorsal over-hemisection, resulting in an incomplete spinal cord injury at the T8/9 spinal level, induced immediate bladder paralysis. One week later, the animals received daily tibial nerve or sham stimulation for 15 days. Effects of stimulation on the lower urinary tract function were assessed by urodynamic investigation. Measurements showed improvements of several key parameters of lower urinary tract function?in particular, non-voiding bladder contractions and intravesical pressure?immediately after the completion of the stimulation period in the stimulated animals. These differences extinguished one week later, however. In the dorsal horn of the lumbosacral spinal cord, a small significant increase of the density of C-fiber afferents layers I-II was found in the stimulated animals at four weeks after spinal cord injury. Tibial nerve stimulation applied acutely after spinal cord injury in rats had an immediate beneficial effect on lower urinary tract dysfunction; however, the effect was transitory and did not last over time. To achieve more sustainable, longer lasting effects, further studies are needed looking into different stimulation protocols using optimized stimulation parameters, timing, and treatment schedules.

Ischemia-reperfusion injury (IRI) is a hallmark for tissue injury in donation after circulatory death (DCD) kidneys. The implementation of hypothermic machine perfusion (HMP) provides a platform for improved preservation of DCD kidneys. Doxycycline administration has shown protective effects during IRI. Therefore, we explored the impact of doxycycline on proteolytic degradation mechanisms and the urinary proteome of perfused kidney grafts. Porcine kidneys underwent 30 min of warm ischemia, 24 h of oxygenated HMP (control/doxycycline) and 240 min of ex vivo reperfusion. A proteomic analysis revealed distinctive clustering profiles between urine samples collected at T15 min and T240 min. High-efficiency undecanal-based N-termini (HUNTER) kidney tissue degradomics revealed significantly more proteolytic activity in the control group at T-10. At T240, significantly more proteolytic activity was observed in the doxycycline group, indicating that doxycycline alters protein degradation during HMP. In conclusion, doxycycline administration during HMP led to significant proteomic and proteolytic differences and protective effects by attenuating urinary NGAL levels. Ultimately, we unraveled metabolic, and complement and coagulation pathways that undergo alterations during machine perfusion and that could be targeted to attenuate IRI induced injury.

The fire modeling community currently lacks full-scale experi- mental data from fires in residential-style structures with heat- ing, ventilation, and air conditioning (HVAC) systems. Further, there is an absence of data quantifying the generation of H2O due to combustion and the subsequent transport of those gases with a structure. Propane gas burner fire experiments were con- ducted in a purpose-built two-story structure instrumented to measure temperature, pressure, velocity, and gas concentrations. Experiments were conducted to assess heat and gas species trans- fer due to HVAC operating status (off vs. on), fire location and heat release rate, and bedroom door position (open vs. closed).

Abstract Objective: To investigate clinically relevant microbiological characteristics of uropathogens and to compare patients with catheter-associated urinary tract infections (CAUTIs) to those with non-CAUTIs. Methods: All urine cultures from the calendar year 2019 of the Swiss Centre for Antibiotic Resistance database were analyzed. Group differences in the proportions of bacterial species and antibiotic-resistant isolates from CAUTI and non-CAUTI samples were investigated. Results: Data from 27,158 urine cultures met the inclusion criteria. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus mirabilis together represented 70% and 85% of pathogens identified in CAUTI and non-CAUTI samples, respectively. Pseudomonas aeruginosa was significantly more often detected in CAUTI samples. The overall resistance rate for the empirically often-prescribed antibiotics ciprofloxacin (CIP), norfloxacin (NOR), and trimethoprim-sulfamethoxazole (TMP-SMX) was between 13% and 31%. Except for nitrofurantoin, E. coli from CAUTI samples were more often resistant (P ≤ .048) to all classes of antibiotics analyzed, including third-generation cephalosporines used as surrogate for extended-spectrum β-lactamase (ESBL). Significanty higher resistance proportions in CAUTI samples versus non-CAUTI samples were observed for CIP (P = .001) and NOR (P = .033) in K. pneumoniae, for NOR (P = .011) in P. mirabilis, and for cefepime (P = .015), and piperacillin-tazobactam (P = .043) in P. aeruginosa. Conclusion: CAUTI pathogens were more often resistant to recommended empirical antibiotics than non-CAUTI pathogens. This finding emphasizes the need for urine sampling for culturing before initiating therapy for CAUTI and the importance of considering therapeutic alternatives.

BackgroundEvidence regarding favorable treatment of patients with functional mitral regurgitation (FMR) using transcatheter edge-to-edge repair (TEER) is constantly growing. However, there is only few data directly comparing TEER and surgical mitral valve repair (SMVr).AimsTo compare baseline characteristics, short-term and 1-year outcomes in FMR patients undergoing mitral valve (MV) TEER or SMVr using a meta-analytic approach.MethodsSystematic database search identified 1,703 studies reporting on TEER or SMVr for treatment of FMR between January 2010 and December 2020. A meta-analytic approach was used to compare outcomes from single-arm and randomized studies based on measures by means of their corresponding 95% confidence intervals (CI). Statistical significance was assumed if CIs did not overlap. A total of 21 TEER and 37 SMVr studies comprising 4,304 and 3,983 patients were included.ResultsPatients in the TEER cohort presented with higher age (72.0 ± 1.7 vs. 64.7 ± 4.7 years, p < 0.001), greater burden of comorbidities like hypertension (p < 0.001), atrial fibrillation (p < 0.001), lung disease (p < 0.001) and chronic renal disease (p = 0.005) as well as poorer left ventricular ejection fraction (30.9 ± 5.7 vs. 36.6 ± 5.3%, p < 0.001). In-hospital mortality was significantly lower with TEER [3% (95%-CI 0.02–0.03) vs. 5% (95%-CI 0.04–0.07)] and 1-year mortality did not differ significantly [18% (95%-CI 0.15–0.21) vs. 11% (0.07–0.18)]. NYHA [1.06 (95%-CI 0.87–1.26) vs. 1.15 (0.74–1.56)] and MR reduction [1.74 (95%-CI 1.52–1.97) vs. 2.08 (1.57–2.59)] were comparable between both cohorts.ConclusionDespite considerably higher age and comorbidity burden, in-hospital mortality was significantly lower in FMR patients treated with TEER, whereas a tendency toward increased 1-year mortality was observed in this high-risk population. In terms of functional status and MR grade reduction, comparable 1-year results were achieved.

The transcription factor, IRF5, has been implicated in the regulation of inflammation, but how IRF5 protein is activated is still unclear. Here the authors use inhibitor library screening, biochemical analyses and in vivo/ex vivo data to show that a protein tyrosine kinase, Pyk2, may be key for the activation of IRF5 in macrophages and inflammatory responses in the gut.

I have had the pleasure of many visits to Brazil since 2001. During these visits, I have collaborated with ANVISA on training programs at the start of the Sentinel Hospital program. I have lectured on medication safety topics to students, practitioners, private companies, and healthcare administrators. I have worked closely with safety organizations such as ISMP- Brazil, a national leader in promoting safety awareness and safe practices. I have seen Brazil experience an awakening to the problems of safety in healthcare. Over these last 16 years, good things have happened because of the personal dedication of professionals, leaders, and organizations devoted to safety. There have been well-attended local, regional and national congresses on safety. In general, there is an appetite for learning how to be safer and how to make changes that improve quality.

Transcutaneous tibial nerve stimulation (TTNS) is a promising treatment for neurogenic lower urinary tract symptoms. However, the evidence is limited due to a general lack of randomised controlled trials (RCTs) and, also, inconsistency in the sham and blinding conditions. In the context of much-needed RCTs, we aimed to develop a suitable sham-control protocol for a clinical setting to maintain blinding but avoid meaningful stimulation of the tibial nerve. Three potential electrode positions (lateral malleolus/5th metatarsal/plantar calcaneus) and two electrode sizes (diameter: 2.5 cm/3.2 cm) were tested to determine which combination provided the optimal sham configuration for a TTNS approach, based on a visible motor response. Sixteen healthy volunteers underwent sensory and motor assessments for each sham configuration. Eight out of them came back for an extra TTNS visit. Sensory thresholds were present for all sham configurations, with linear regression models revealing a significant effect regarding electrode position (highest at plantar calcaneus) but not size. In addition, motor thresholds varied with the position—lowest for the 5th metatarsal. Only using this position and 3.2 cm electrodes attained a 100% response rate. Compared to TTNS, sensory and motor thresholds were generally higher for the sham configurations; meanwhile, perceived pain was only higher at the lateral malleolus. In conclusion, using the 5th metatarsal position and 3.2 cm electrodes proved to be the most suitable sham configuration. Implemented as a four-electrode setup with standardized procedures, this appears to be a suitable RCT protocol for maintaining blinding and controlling for nonspecific TTNS effects in a clinical setting.

Abstract Background Bleeding in people with hemophilia A can be life threatening, and intra‐articular bleeds can result in joint damage. Most clinical studies focus on treated bleeds, while bleeds not treated with coagulation factor(s) (untreated bleeds) are underreported. Objectives We assessed the incidence of untreated bleeds during a noninterventional study (NIS) wherein people with hemophilia A, with or without factor VIII (FVIII) inhibitors, were managed according to standard practice. Patients/Methods Using the Bleed and Medication Questionnaire, we prospectively collected data from three cohorts: Cohort A, adults/adolescents (age ≥12 years) with FVIII inhibitors; Cohort B, children (aged

Oftedal et al. generate mice lacking the chaperonin subunit CCT8 in T cells. They find that loss of CCT8 leads to reduced formation of nuclear actin filaments, changes in proteostasis, defective Th2 cell polarization and T cell metabolism and a failed antigenic response to intestinal helminths.

Overactive bladder (OAB) is a global problem reducing the quality of life of patients and increasing the costs of any healthcare system. The etiology of OAB is understudied but likely involves supraspinal network alterations. Here, we characterized supraspinal resting-state functional connectivity in 12 OAB patients and 12 healthy controls (HC) who were younger than 60 years. Independent component analysis showed that OAB patients had a weaker presence of the salience (Cohen’s d = 0.9) and default mode network (Cohen’s d = 1.1) and weaker directed connectivity between the fronto-parietal network and salience network with a longer lag time compared to HC. A region of interest analysis demonstrated weaker connectivity in OAB compared to HC (Cohen’s d > 1.6 or < −1.6), particularly within the frontal and prefrontal cortices. In addition, weaker seed (insula, ventrolateral prefrontal cortex) to voxel (anterior cingulate cortex, frontal gyrus, superior parietal lobe, cerebellum) connectivity was found in OAB compared to HC (Cohen’s d > 1.9). The degree of deviation in supraspinal connectivity in OAB patients (relative to HC) appears to be an indicator of the severity of the lower urinary tract symptoms and an indication that such symptoms are directly related to functional supraspinal alterations. Thus, future OAB therapy options should also consider supraspinal targets, while neuroimaging techniques should be given more consideration in the quest for better phenotyping of OAB.

Spinal cord injury (SCI) produces damage to the somatic and autonomic pathways that regulate lower urinary tract, sexual, and bowel function, and increases the risk of autonomic dysreflexia. The recovery of these functions has a high impact on health, functioning, and quality of life and is set as the utmost priority by patients. The application of reliable models to predict lower urinary tract, sexual, and bowel function, and autonomic dysreflexia is important for guiding counseling, rehabilitation, and social reintegration. Moreover, a reliable prediction is essential for designing future clinical trials to optimize patients’ allocation to different treatment groups. To date, reliable and simple algorithms are available to predict lower urinary tract and bowel outcomes after traumatic and ischemic SCI. Previous studies identified a few risk factors to develop autonomic dysreflexia, albeit a model for prediction still lacks. On the other hand, there is an urgent need for a model to predict the evolution of sexual function. The aim of this review is to examine the available knowledge and models for the prediction of lower urinary tract, sexual, and bowel function, and autonomic dysreflexia after SCI, and critically discuss the research priorities in these fields.

We used clinical parameters to develop a prediction model for the occurrence of urodynamic risk factors for upper urinary tract (UUT) damage during the first year after acute spinal cord injury (SCI). A total of 97 patients underwent urodynamic investigation at 1, 3, 6, and 12 months after acute SCI, within the framework of a population-based longitudinal study at a single university SCI center. Candidate predictors included demographic characteristics and neurological and functional statuses 1 month after SCI. Outcomes included urodynamic risk factors for UUT damage: detrusor overactivity combined with detrusor sphincter dyssynergia, maximum storage detrusor pressure (pDetmax) ≥ 40 cmH2O, bladder compliance < 20 mL/cmH2O, and vesicoureteral reflux. Multivariable logistic regression was used for the prediction model development and internal validation, using the area under the receiver operating curve (aROC) to assess model discrimination. Two models showed fair discrimination for pDetmax ≥ 40 cmH2O: (i) upper extremity motor score and sex, aROC 0.79 (95% CI: 0.69–0.89), C-statistic 0.78 (95% CI: 0.69–0.87), and (ii) neurological level, American Spinal Injury Association Impairment Scale grade, and sex, aROC 0.78 (95% CI: 0.68–0.89), C-statistic 0.76 (95% CI: 0.68–0.85). We identified two models that provided fair predictive values for urodynamic risk factors of UUT damage during the first year after SCI. Pending external validation, these models may be useful for clinical trial planning, although less so for individual-level patient management. Therefore, urodynamics remains essential for reliably identifying patients at risk of UUT damage.

Abstract Sex differences in jealousy responses to sexual and emotional infidelity are robust in samples of heterosexual adults, especially in more gender egalitarian nations. However, investigations of when and how these differences develop have been scant. We applied two forced choice infidelity scenarios in a large community sample of high school students (age 16–19, N = 1266). In line with previous findings on adults using the forced choice paradigm, adolescent males found the sexual aspect of imagined infidelity more distressing than adolescent females did. Nevertheless, there was no effect of age on the jealousy responses, and age did not moderate the sex difference. There were neither any effects of three covariates (having had first sexual intercourse, being in a committed romantic relationship, and sociosexuality), neither as markers of pubertal maturation nor as psychosocial environmental stimuli. Future research needs to investigate even younger samples in order to specify at what age the sex difference in jealousy responses emerges.

Abstract Controlling the activation of the NLRP3 inflammasome by post-translational modifications (PTMs) of critical protein subunits has emerged as a key determinant in inflammatory processes as well as in pathophysiology. In this review, we put into context the kinases, ubiquitin processing and other PTM enzymes that modify NLRP3, ASC/PYCARD and caspase-1, leading to inflammasome regulation, activation and signal termination. Potential target therapeutic entry points for a number of inflammatory diseases focussed on PTM enzyme readers, writers and erasers, leading to the regulation of inflammasome function, are discussed. Video Abstract

The BLM helicase interacts with the topoisomerase TOP3A and RMI1 to form the BTR complex. Here, the authors reveal that this complex contains multiple binding sites for the single-stranded DNA-binding complex RPA, and that RPA-binding stimulates BLM recruitment to stalled replication forks to promote their restart after replication stress.

Abstract Middle- and inner-ear surgery is a vital treatment option in hearing loss, infections, and tumors of the lateral skull base. Segmentation of otologic structures from computed tomography (CT) has many potential applications for improving surgical planning but can be an arduous and time-consuming task. We propose an end-to-end solution for the automated segmentation of temporal bone CT using convolutional neural networks (CNN). Using 150 manually segmented CT scans, a comparison of 3 CNN models (AH-Net, U-Net, ResNet) was conducted to compare Dice coefficient, Hausdorff distance, and speed of segmentation of the inner ear, ossicles, facial nerve and sigmoid sinus. Using AH-Net, the Dice coefficient was 0.91 for the inner ear; 0.85 for the ossicles; 0.75 for the facial nerve; and 0.86 for the sigmoid sinus. The average Hausdorff distance was 0.25, 0.21, 0.24 and 0.45 mm, respectively. Blinded experts assessed the accuracy of both techniques, and there was no statistical difference between the ratings for the two methods (p = 0.93). Objective and subjective assessment confirm good correlation between automated segmentation of otologic structures and manual segmentation performed by a specialist. This end-to-end automated segmentation pipeline can help to advance the systematic application of augmented reality, simulation, and automation in otologic procedures.

Purpose This study aimed to assess the feasibility of a rapid diffusion tensor imaging (DTI) for evaluation of the female urinary sphincter function based on differentiation between rest and muscle contraction. Methods Magnetic resonance imaging (MRI) of the lower pelvis was performed at 3 Tesla in 10 healthy female volunteers (21–36 years; body mass index, 20.8±3.6 kg/m2) between June and July 2019. High-resolution T1- and T2-weighted images were acquired for anatomical reference, and following DTI performed in 4 experiment phases: twice during rest (denoted rest-1, rest-2) and contraction (contraction-1, contraction-2). Manual segmentation of the urinary sphincter and the levator ani muscles were performed by 2 independent readers. Mean diffusivity (MD) and fractional anisotropy (FA) values derived from DTI volumes were compared in search for significant differences between the experiment phases. Interreader agreement was assessed by intraclass correlation coefficient (ICC). Results Kruskal-Wallis test showed significant differences between MD values among all the experiment phases, by both independent readers (1st: X2 [3,76]=17.16, P

Abstract Muscle wasting and atrophy are regulated by multiple molecular processes, including mRNA processing. Reduced levels of the polyadenylation binding protein nucleus 1 (PABPN1), a multifactorial regulator of mRNA processing, cause muscle atrophy. A proteomic study in muscles with reduced PABPN1 levels suggested dysregulation of sarcomeric and cytoskeletal proteins. Here we investigated the hypothesis that reduced PABPN1 levels lead to an aberrant organization of the cytoskeleton. MURC, a plasma membrane-associated protein, was found to be more abundant in muscles with reduced PABPN1 levels, and it was found to be expressed at regions showing regeneration. A polarized cytoskeletal organization is typical for muscle cells, but muscle cells with reduced PABPN1 levels (named as shPAB) were characterized by a disorganized cytoskeleton that lacked polarization. Moreover, cell mechanical features and myogenic differentiation were significantly reduced in shPAB cells. Importantly, restoring cytoskeletal stability, by actin overexpression, was beneficial for myogenesis, expression of sarcomeric proteins and proper localization of MURC in shPAB cell cultures and in shPAB muscle bundle. We suggest that poor cytoskeletal mechanical features are caused by altered expression levels of cytoskeletal proteins and contribute to muscle wasting and atrophy.