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Background Magnetic resonance imaging-based technologies are non-invasive diagnostic tests that can be used to assess non-alcoholic fatty liver disease. Objectives The study objectives were to assess the diagnostic test accuracy, clinical impact and cost-effectiveness of two magnetic resonance imaging-based technologies (LiverMultiScan and magnetic resonance elastography) for patients with non-alcoholic fatty liver disease for whom advanced fibrosis or cirrhosis had not been diagnosed and who had indeterminate results from fibrosis testing, or for whom transient elastography or acoustic radiation force impulse was unsuitable, or who had discordant results from fibrosis testing. Data sources The data sources searched were MEDLINE, MEDLINE Epub Ahead of Print, In-Process & Other Non-Indexed Citations, Embase, Cochrane Database of Systematic Reviews, Cochrane Central Database of Controlled Trials, Database of Abstracts of Reviews of Effects and the Health Technology Assessment. Methods A systematic review was conducted using established methods. Diagnostic test accuracy estimates were calculated using bivariate models and a summary receiver operating characteristic curve was calculated using a hierarchical model. A simple decision-tree model was developed to generate cost-effectiveness results. Results The diagnostic test accuracy review (13 studies) and the clinical impact review (11 studies) only included one study that provided evidence for patients who had indeterminate or discordant results from fibrosis testing. No studies of patients for whom transient elastography or acoustic radiation force impulse were unsuitable were identified. Depending on fibrosis level, relevant published LiverMultiScan diagnostic test accuracy results ranged from 50% to 88% (sensitivity) and from 42% to 75% (specificity). No magnetic resonance elastography diagnostic test accuracy data were available for the specific population of interest. Results from the clinical impact review suggested that acceptability of LiverMultiScan was generally positive. To explore how the decision to proceed to biopsy is influenced by magnetic resonance imaging-based technologies, the External Assessment Group presented cost-effectiveness analyses for LiverMultiScan plus biopsy versus biopsy only. Base-case incremental cost-effectiveness ratio per quality-adjusted life year gained results for seven of the eight diagnostic test strategies considered showed that LiverMultiScan plus biopsy was dominated by biopsy only; for the remaining strategy (Brunt grade ≥2), the incremental cost-effectiveness ratio per quality-adjusted life year gained was £1,266,511. Results from threshold and scenario analyses demonstrated that External Assessment Group base-case results were robust to plausible variations in the magnitude of key parameters. Limitations Diagnostic test accuracy, clinical impact and cost-effectiveness data for magnetic resonance imaging-based technologies for the population that is the focus of this assessment were limited. Conclusions Magnetic resonance imaging-based technologies may be useful to identify patients who may benefit from additional testing in the form of liver biopsy and those for whom this additional testing may not be necessary. However, there is a paucity of diagnostic test accuracy and clinical impact data for patients who have indeterminate results from fibrosis testing, for whom transient elastography or acoustic radiation force impulse are unsuitable or who had discordant results from fibrosis testing. Given the External Assessment Group cost-effectiveness analyses assumptions, the use of LiverMultiScan and magnetic resonance elastography for assessing non-alcoholic fatty liver disease for patients with inconclusive results from previous fibrosis testing is unlikely to be a cost-effective use of National Health Service resources compared with liver biopsy only. Study registration This study is registered as PROSPERO CRD42021286891. Funding Funding for this study was provided by the Evidence Synthesis Programme of the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 27, No. 10. See the NIHR Journals Library website for further project information. Plain language summary Non-alcoholic fatty liver disease includes a range of conditions that are caused by a build-up of fat in the liver, and not by alcohol consumption. This build-up of fat can cause inflammation. Persistent inflammation can cause scar tissue (fibrosis) to develop. It is important to identify patients with fibrosis because severe fibrosis can cause permanent liver damage (cirrhosis), which can lead to liver failure and liver cancer. In the National Health Service, patients with non-alcoholic fatty liver disease undergo tests to determine whether they have fibrosis. The test results are not always accurate and multiple tests can give conflicting results. Some of the tests may not be suitable for patients who have a very high body mass index. In the National Health Service, a liver biopsy may be offered to patients with inconclusive or conflicting test results or to those patients for whom other tests are unsuitable. However, liver biopsy is expensive, and is associated with side-effects such as pain and bleeding. Magnetic resonance imaging-based testing could be used as an extra test to help clinicians assess non-alcoholic fatty liver disease and identify patients who may need a liver biopsy. We assessed two magnetic resonance imaging-based diagnostic tests, LiverMultiScan and magnetic resonance elastography. LiverMultiScan is imaging software that is used alongside magnetic resonance imaging to measure markers of liver disease. Magnetic resonance elastography is used in some National Health Service centres to assess liver fibrosis; however, magnetic resonance elastography requires more equipment than just an magnetic resonance imaging scanner. We reviewed all studies examining how well LiverMultiScan and magnetic resonance elastography assess patients with non-alcoholic fatty liver disease. We also built an economic model to estimate the costs and benefits of using LiverMultiScan to identify patients who should be sent for a biopsy. Results from the model showed that LiverMultiScan may not provide good value for money to the National Health Service. Scientific summary Background Non-alcoholic fatty liver disease (NAFLD) is an umbrella term for a range of conditions caused by a build-up of fat in the liver that has not been caused by alcohol consumption. NAFLD covers a spectrum of histological lesions ranging from steatosis (simple fatty liver) to complex patterns of hepatocyte injury, inflammation and fibrosis. In the current National Health Service diagnostic pathway for staging fibrosis (based on guidelines and expert advice to NICE), patients with NAFLD (confirmed by ultrasound and liver aetiology screen) are referred for the fibrosis-4 (FIB-4), NAFLD fibrosis score (NFS) or enhanced liver fibrosis (ELF) test as first-line testing. Patients with an indeterminate result from first-line testing are referred for second-line testing using transient elastography (TE), acoustic radiation force impulse (ARFI) or the ELF test, if it had not already been used as a first-line test. Patients with indeterminate or discordant results from fibrosis testing and patients with high risk of advanced fibrosis are considered for liver biopsy. Magnetic resonance imaging (MRI)-based testing could be used as an additional, non-invasive, diagnostic test to help clinicians stage NAFLD and potentially identify which patients should be referred for liver biopsy. Liver biopsy is expensive and is an invasive procedure that is associated with complications. Objectives The objectives of this study were to assess the diagnostic test accuracy (DTA), the clinical impact and the cost-effectiveness of two non-invasive MRI-based technologies, namely LiverMultiScan and magnetic resonance elastography (MRE), for patients with NAFLD for whom advanced fibrosis or cirrhosis had not yet been diagnosed and who had indeterminate results from fibrosis testing, for whom TE or ARFI was unsuitable, or who had discordant results from fibrosis testing. To achieve the study objectives, the External Assessment Group (EAG): conducted a systematic literature review to evaluate the (1) DTA of MRI-based technologies for the assessment of fibrosis, inflammation, and steatosis for a patients with NAFLD for whom advanced fibrosis or cirrhosis had not yet been diagnosed, using liver biopsy as the reference standard, and (2) the clinical impact of MRI-based technologies conducted a systematic literature review to explore the cost-effectiveness of MRI-based technologies as diagnostic tools and built a de novo economic model to assess the cost-effectiveness of two diagnostic pathways, namely MRI-based technologies plus biopsy and liver biopsy. Methods: assessment of diagnostic test accuracy and clinical impact Electronic databases (MEDLINE, MEDLINE Epub Ahead of Print In-Process & Other Non-Indexed Citations, Embase, Cochrane Databases of Systematic Reviews, Cochrane Central Database of Controlled Trials, Database of Abstracts of Reviews of Effects, Health Technology Assessment Database) were searched from inception to 4 October 2021. Eligible studies assessed the DTA or clinical impact of LiverMultiScan or MRE for patients with NAFLD for whom advanced fibrosis or cirrhosis had not yet been diagnosed (who have indeterminate results from fibrosis testing, for whom TE or ARFI is unsuitable, or who have discordant results from fibrosis testing). Two reviewers independently screened the titles and abstracts of all reports identified through electronic database searches and of all full-text articles subsequently obtained for assessment. Data extraction and quality assessment were conducted by one reviewer and checked for agreement by a second reviewer. The methodological quality of the included DTA studies was assessed using the QUality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. The methodological quality of randomised controlled trials (RCTs) evaluating the clinical impact of MRI-based technologies was assessed using the Cochrane Risk of Bias 2.0 tool. The National Institute of Health study quality-assessment tools for cohort studies, case-control studies and before–after (pre-post) studies with no control group were used to assess risk of bias of included non-randomised studies. Qualitative studies were assessed using the Critical Appraisal Skills Programme (CASP) qualitative studies checklist. The sensitivity and specificity of each index test were summarised in forest plots. Where at least three studies provided both sensitivity and specificity data for a specific combination of index test, diagnosis of interest, and cut-off value, a bivariate random-effects meta-analysis to provide pooled estimates of sensitivity and specificity was considered. We did not perform bivariate meta-analyses where statistical heterogeneity between the studies (assessed by visually examining forest plots) was so great that pooled estimates of sensitivity and specificity would have been meaningless. Where at least three studies provided both sensitivity and specificity data for a specific combination of index test and diagnosis of interest, but used different cut-off values for the index test, we used a hierarchical model to estimate a summary receiver operating characteristic (ROC) curve. Methods: assessment of cost-effectiveness The EAG appended an economic evaluation-specific search filter to the clinical search strategies to identify published cost-effectiveness studies. In addition, two databases of economic publications [EconLit (EBSCO) and the cost-effectiveness analysis (CEA) registry] were searched from inception until 4 October 2021. The EAG developed a simple, flexible de novo model to estimate the cost-effectiveness of an MRI-based technologies plus biopsy pathway versus liver biopsy only pathway. Results The EAG searches of the electronic databases and reference lists of relevant studies and systematic reviews identified 4489 records (3331 unique records). Although all the identified studies for inclusion in the DTA, clinical impact and cost-effectiveness reviews included patients with NAFLD for whom advanced fibrosis or cirrhosis had not yet been diagnosed, only one study provided results for patients with NAFLD who had indeterminate or discordant results from fibrosis testing. No studies were identified that considered patients for whom TE or ARFI was unsuitable. Diagnostic test accuracy The EAG identified 13 studies (15 publications). Two studies (four publications) were evaluations of LiverMultiScan, 10 studies (10 publications) were evaluations of MRE, and one study (one publication) was an evaluation of LiverMultiScan and MRE. MRI-based technology: LiverMultiScan For the LiverMultiScan proton density fat fraction (PDFF) and LiverMultiScan iron-corrected T1 (cT1) outputs, 2 × 2 data were available from three studies. The EAG considers that the Eddowes 2018 study is the most relevant study to this assessment. Eddowes 2018 recruited patients who were scheduled for non-targeted liver biopsy to stage fibrosis after inconclusive non-invasive assessment of fibrosis or to make a diagnosis after a range of non-invasive tests had not confirmed a diagnosis. For diagnosis of fibrosis, estimates from Eddowes 2018 ranged from 50% to 88% for sensitivity and from 42% to 75% for specificity. Sensitivity and specificity values for fibrosis testing in Eddowes 2018 were consistently higher for LiverMultiScan cT1 than for LiverMultiScan PDFF. Data from three studies were included in the meta-analyses for LiverMultiScan. For advanced fibrosis (≥F3), the pooled sensitivity and specificity values were higher for LiverMultiScan cT1 [sensitivity = 60.2%, 95% confidence interval (CI): 50.9% to 68.8%; specificity = 65.4%, 95% CI 55.8% to 73.9%] than for LiverMultiScan PDFF (sensitivity = 38.6%, 95% CI 23.8% to 56.0%; specificity = 43.6%, 95% CI 30.7% to 57.5%). MRI-based technology: magnetic resonance elastography For the MRE test, 2 × 2 data were available from four studies. Estimates of sensitivity and specificity for advanced fibrosis (≥F3) were high and ranged from 71% to 100% and 79% to 93%, respectively. However, the cut-off values used to indicate a positive result from the index test varied between studies, therefore a summary ROC curve was estimated. The summary ROC curve indicates high DTA. However, observed study results do not all lie close to the summary ROC curve, which could be due to small sample sizes and/or clinical and methodological heterogeneity between the included studies. Clinical impact Eleven studies (14 publications) were included in the clinical impact review. Five studies (eight publications) were evaluations of LiverMultiScan and six studies (six publications) were evaluations of MRE. MRI-based technology: LiverMultiScan Two studies reported on the prognostic ability of LiverMultiScan cT1. However, neither study reported results specifically for the subpopulation of patients with NAFLD for whom advanced fibrosis or cirrhosis had not yet been diagnosed. One study reported that LiverMultiScan cT1 and LiverMultiScan PDFF could reduce the number of unnecessary biopsies for patients with non-NAFLD and NAFLD to diagnose non-alcoholic steatohepatitis (NASH) and fibrosis unrelated to NAFLD [EAG calculated odds ratio (OR) = 0.65, 95% CI 0.22 to 1.96] and for patients with no to mild fibrosis (F0 to F1) to diagnose significant fibrosis to cirrhosis (F2 to F4; EAG calculated OR = 0.59, 95% CI 0.18 to 1.89) when compared to standard of care. Three studies reported the test failure rate of LiverMultiScan for patients with all liver aetiologies. The test failure rate ranged from 5.3% to 7.6%. One study reported the test failure rate for LiverMultiScan for patients with NAFLD (5.6%). Acceptability of LiverMultiScan was reported in a qualitative study and was generally positive. MRI-based technology: magnetic resonance elastography Six studies reported the test failure rate of MRE for patients with all liver aetiologies. The test failure rate ranged from 0.0% to 7.6%. Three studies reported the test failure rate for MRE specifically for patients with NAFLD. The EAG performed a fixed-effects meta-analysis to obtain a pooled estimate of 4.2% (95% CI 2.5% to 6.2%) test failure rate for patients with NAFLD. Despite conducting additional targeted searches, the EAG did not identify any relevant studies that provided evidence of the clinical impact of MRI-based technologies for patients with NAFLD for whom advanced fibrosis or cirrhosis has not been diagnosed, for the remaining clinical impact outcomes listed in the final scope issued by NICE. Cost-effectiveness The EAG base-case incremental cost-effectiveness ratio (ICER) per quality-adjusted life year (QALY) gained results for seven of the eight diagnostic test strategies considered, and showed that the LiverMultiScan plus biopsy pathway was dominated by the biopsy only pathway. For Brunt grade ≥2, the ICER per QALY gained was £1,266,511. Results from the EAG threshold and scenario analyses demonstrated that these results were robust to plausible variations in the magnitude of key parameters. Conclusions The DTA, clinical impact and cost-effectiveness data for MRI-based technologies are limited for patients who have indeterminate results from fibrosis testing, for whom TE or ARFI is unsuitable or patients who have discordant results from fibrosis testing. Only one small LiverMultiScan study provided DTA and population prevalence data for patients described in the final scope issued by NICE. It is unclear whether sensitivity and specificity estimates reported by this small study will give clinicians sufficient confidence to use LiverMultiScan test results to triage patients with inconclusive results from previous fibrosis testing to biopsy. Cost-effectiveness results from the EAG model are only informative if clinicians have confidence in LiverMultiScan DTA data. Using the available DTA and population prevalence data, EAG cost-effectiveness results showed that LiverMultiScan is unlikely to be cost-effective at current prices when used to triage patients with inconclusive results from previous fibrosis testing to biopsy. LiverMultiScan data are not available for patients for whom TE or ARFI was unsuitable. Further, no MRE DTA data were available for the population described in the final scope issued by NICE. The EAG was unable to generate cost-effectiveness results for this technology; however, even if MRE was 100% accurate, due to high population prevalence estimates it is unlikely that MRE would be cost-effective at current prices. Study registration This study is registered as PROSPERO CRD42021286891. Funding Funding for this study was provided by the Evidence Synthesis Programme of the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 27, No. 10. See the NIHR Journals Library website for further project information.

The μ-opioid receptor (μOR), a prototypical G protein-coupled receptor (GPCR), is the target of opioid analgesics such as morphine and fentanyl. Due to the severe side effects of current opioid drugs, there is considerable interest in developing novel modulators of μOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, represent alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the μOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded μOR ligand and uncover the molecular basis for μOR antagonism by determining the cryo-EM structure of the NbE-μOR complex. NbE displays a unique ligand binding mode and achieves μOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a β-hairpin loop formed by NbE that deeply protrudes into the μOR, we design linear and cyclic peptide analogs that recapitulate NbEs antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes lower molecular weight μOR ligands that can serve as a basis for therapeutic developments.

Crohn's disease (CD) is a complex and heterogeneous condition with no perfect preclinical model or cure. To address this, we explore adult stem cell-derived organoids that retain their tissue identity and disease-driving traits. We prospectively create a biobank of CD patient-derived organoid cultures (PDOs) from colonic biopsies of 53 subjects across all clinical subtypes and healthy subjects. Gene expression analyses enabled benchmarking of PDOs as tools for modeling the colonic epithelium in active disease and identified two major molecular subtypes: immune-deficient infectious CD (IDICD) and stress and senescence-induced fibrostenotic CD (S2FCD). Each subtype shows internal consistency in the transcriptome, genome, and phenome. The spectrum of morphometric, phenotypic, and functional changes within the "living biobank" reveals distinct differences between the molecular subtypes. Drug screens reverse subtype-specific phenotypes, suggesting phenotyped-genotyped CD PDOs can bridge basic biology and patient trials by enabling preclinical phase "0" human trials for personalized therapeutics.

Introduction Association between Hydraulic Fracturing and Perinatal Outcomes Hydraulic fracturing (HF), more commonly known as fracking, is the injection of water, sand, and chemicals into the ground at high pressures [...], BACKGROUND: Risk of preterm birth (PTB) and low birth weight (LBW) due to hydraulic fracturing (HF) exposure is a growing concern. Regional studies have demonstrated links, but results are often contradictory among studies. OBJECTIVES: This is the first US national study to our knowledge linking fracturing fluid ingredients to the human hormone pathways targeted--estrogen, testosterone, or other hormones (e.g., thyroid hormone)--to assess the effect of HF ingredients on rates of PTB and LBW. METHODS: We constructed generalized linear regression models of the impact of HF well density and hormone targeting chemicals in HF fluids (2001-2018) on the county-level average period prevalence rates of PTB and LBW (2015-2018) with each outcome measured in separate models. Our data sources consisted of publicly available datasets, including the WellExplorer database, which uses data from FracFocus, the March of Dimes Peristats, the US Census Bureau, the US Department of Agriculture, and the Centers for Disease Control and Prevention. We conducted additional stratified analyses to address issues of confounding. We used stratification to address issues regarding outcomes in rural vs. urban communities by assessing whether our models achieved similar results in nonmetro counties, as well as farming and mining counties. We also stratified by the year of the HF data to include HF data that was closer to the time of the birth outcomes. We also added covariate adjustment to address other important factors linked to adverse birth outcomes, including the proportion of the population belonging to various racial and ethnic minority populations (each modeled as a separate variable); education (bachelor's degree and high school); use of fertilizers, herbicides, and insecticides, acres of agricultural land per square mile; poverty; insurance status; marital status; population per square mile; maternal care deserts; and drug deaths per 100,000 people. RESULTS: We found that the density of HF wells in a county was significantly associated with both PTB and LBW rates (percentage of live births) in our fully adjusted models. We report the results from our more restrictive stratified analysis with a subset including only the 2014-2018 data, because this resulted in the most meaningful time frame for comparison. Across all models, the magnitude of effect was highest for wells with ingredients that include estrogen targeting chemicals (ETCs), testosterone targeting chemicals (TTCs) and other hormone targeting chemicals (OHTCs), and, finally, all wells grouped regardless of chemical type. For every unit increase in well density per square mile of wells that use chemicals that include an ETC, we observed a 3.789-higher PTB rate (95% CI: 1.83,5.74) compared with counties with no ETC wells from 2014 to 2018 and likewise, we observed a 1.964-higher LBW rate (95% CI: 0.41,3.52). Similarly, for every unit increase in well density per square mile of wells that use TTC, we observed a 3.192-higher PTB rate (95% CI: 1.62,4.77) compared with counties with no TTC wells. Likewise, for LBW, we found a 1.619-higher LBW rate (95% CI: 0.37,2.87). We also found that an increase in well density per square mile among wells that use chemicals that include an OHTC resulting in a 2.276-higher PTB rate (95% CI: 1.25, 3.30) compared with counties with no OHTC wells, and for LBW, we found a 1.244-higher LBW rate (95% CI: 0.43,2.06). We also explored the role of HF well exposure in general (regardless of the chemicals used) and found that an increase in total well density (grouped regardless of hormonal targeting status of the chemicals used) resulted in a 1.228-higherPTB rate (95% CI: 0.66, 1.80) compared with counties with no wells, and for LBW, we found a 0.602-higher LBW rate (95% CI: 0.15,1.05) compared with counties with no wells. We found similar results in our primary analysis that used all data without any exclusions and the statistical significance did not change. DISCUSSION: Our findings reinforce previously identified regional associations between HF and PTB and LBW, but on a national scale. Our findings point to dysregulation of hormonal pathways underpinning HF exposure risk on birth outcomes, which warrants further exploration. Future research must consider the specific ingredients used in HF fluids to properly understand the differential effects of exposure. https://doi.org/10.1289/EHP12628

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning.

* Context.--Respiratory infections complicate lung transplantation and increase the risk of allograft dysfunction. Allograft lungs may have different susceptibilities to infection than native lungs, potentially leading to different disease severity in lungs of single lung transplant recipients (SLTRs). Objective.--To study whether infections affect allograft and native lungs differently in SLTRs but similarly in double LTRs (DLTRs). Design.--Using an institutional database of LTRs, medical records were searched, chest computed tomography studies were systematically reviewed, and histopathologic features were recorded per lung lobe and graded semiquantitatively. A multilobar-histopathology score (MLHS) including histopathologic data from each lung and a bilateral ratio ([MLHS.sub.ratio]) comparing histopathologies between both lungs were calculated in SLTRs and compared to DLTRs. Results.--Six SLTRs died of infection involving the lungs. All allografts showed multifocal histopathologic evidence of infection, but at least 1 lobe of the native lung was uninvolved. In 4 of 5 DLTRs, histopathologic evidence of infection was seen in all lung lobes. On computed tomography, multifocal ground-glass and/or nodular opacities were found in a bilateral distribution in all DLTRs but in only 2 of 6 SLTRs. In SLTRs, the [MLHS.sub.Allograft] was higher than [MLHS.sub.Native] (P = .02). The [MLHS.sub.ratio] values of SLTR and DLTR were significantly different (P < .001). Conclusions.--Allograft and native lungs appear to harbor different susceptibilities to infections. The results are important for the management of LTRs. doi: 10.5858/arpa.2023-0227-OA), The median overall survival of lung transplant recipients (LTRs) is only 6.7 years, which is shorter than for other solid organ transplantations. (1) Noncytomegalovirus (nonCMV) infections play a major role [...]

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.

Abstract APC/C is a multi-subunit complex that functions as a master regulator of cell division. It controls progression through the cell cycle by timely marking mitotic cyclins and other cell cycle regulatory proteins for degradation. The APC/C itself is regulated by the sequential action of its coactivator subunits CDC20 and CDH1, post-translational modifications, and its inhibitory binding partners EMI1 and the mitotic checkpoint complex. In this study, we took advantage of developments in cryo-electron microscopy to determine the structures of human APC/CCDH1:EMI1 and apo-APC/C at 2.9 Å and 3.2 Å resolution, respectively, providing insights into the regulation of APC/C activity. The high-resolution maps allow the unambiguous assignment of an α-helix to the N-terminus of CDH1 (CDH1α1) in the APC/CCDH1:EMI1 ternary complex. We also identify a zinc-binding module in APC2 that confers structural stability to the complex, and we confirm the presence of zinc ions experimentally. Finally, due to the higher resolution and well defined density of these maps, we are able to build, aided by AlphaFold predictions, several intrinsically disordered regions in different APC/C subunits that likely play a role in proper APC/C assembly and regulation of its activity.

Abstract Nucleocytoplasmatic transport plays an essential role in eukaryotic cell homeostasis and is mediated by karyopherins. Importin β1 (KPNB1) and its adaptor protein importin α1 (KPNA2) are the best-characterized karyopherins that effect nuclear import. Here, we identify a novel small-molecule inhibitor of the importin β1-mediated nuclear import. We design a reporter cell line by stably tagging endogenous importin α1 with a fluorescent protein to screen for compounds affecting its subcellular localization. We identify a series of compounds that trigger cytoplasmatic accumulation of importin α1. The lead compound, ibetazol, is further characterized in a broad sequence of cellular nuclear transport assays. Ibetazol is shown to inhibit all importin β1-mediated nuclear import quickly and specifically, without affecting transport mediated by other karyopherins. Detailed molecular mechanism of action studies demonstrate that ibetazol inhibits importin β1 by covalently targeting Cys585. In summary, ibetazol is a novel small molecule inhibitor of importin β1 enabling pharmacological inhibition of the importin β1-mediated nuclear import process with wide applicability in different fields.

BackgroundUlcerative colitis (UC) and Crohn's disease are 2 types of inflammatory bowel disease (IBD), a group of chronic digestive disorders caused by aberrant immune responses to intestinal microbes. Although changes in the composition of immune cell subsets in the context of IBD have been previously described, the interactions and communication among cells are less well understood. Moreover, the precise mechanisms of action underlying many biologic therapies, including the anti-α4β7 integrin antagonist vedolizumab, remain incompletely understood. Our study aimed to explore possible additional mechanisms through which vedolizumab acts.MethodsWe performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) on peripheral blood and colon immune cells derived from patients with ulcerative colitis treated with the anti-α4β7 integrin antagonist vedolizumab. We applied a previously published computational approach, NicheNet, to predict immune cell-cell interactions, revealing putative ligand-receptor pairs and key transcriptional changes downstream of these cell-cell communications (CCC).ResultsWe observed decreased proportions of T helper 17 (TH17) cells in UC patients who responded to vedolizumab and therefore focused the study on identifying cell-cell communications and signals of TH17 cells with other immune cells. For example, we observed that colon TH17 cells from vedolizumab nonresponders were predicted to have a greater degree of interactions with classical monocytes compared with responders, whereas colon TH17 cells from vedolizumab responders exhibited more interactions with myeloid dendritic cells compared with nonresponders.ConclusionsOverall, our results indicate that efforts to elucidate cell-cell communications among immune and nonimmune cell types may increase the mechanistic understanding of current and investigational therapies for IBD.

Tissue resident memory CD8+ T (TRM) cells offer rapid and long-term protection at sites of reinfection1. Tumour-infiltrating lymphocytes with characteristics of TRM cells maintain enhanced effector functions, predict responses to immunotherapy and accompany better prognoses2,3. Thus, an improved understanding of the metabolic strategies that enable tissue residency by T cells could inform new approaches to empower immune responses in tissues and solid tumours. Here, to systematically define the basis for the metabolic reprogramming supporting TRM cell differentiation, survival and function, we leveraged in vivo functional genomics, untargeted metabolomics and transcriptomics of virus-specific memory CD8+ T cell populations. We found that memory CD8+ T cells deployed a range of adaptations to tissue residency, including reliance on non-steroidal products of the mevalonate-cholesterol pathway, such as coenzyme Q, driven by increased activity of the transcription factor SREBP2. This metabolic adaptation was most pronounced in the small intestine, where TRM cells interface with dietary cholesterol and maintain a heightened state of activation4, and was shared by functional tumour-infiltrating lymphocytes in diverse tumour types in mice and humans. Enforcing synthesis of coenzyme Q through deletion of Fdft1 or overexpression of PDSS2 promoted mitochondrial respiration, memory T cell formation following viral infection and enhanced antitumour immunity. In sum, through a systematic exploration of TRM cell metabolism, we reveal how these programs can be leveraged to fuel memory CD8+ T cell formation in the context of acute infections and enhance antitumour immunity.