The circulating methylome in childhood-onset inflammatory bowel disease.

The genetic contribution to inflammatory bowel disease (IBD) encompassing both Crohn's disease (CD) and ulcerative colitis (UC), accounts for around 20% of disease variance, highlighting the need to characterise environmental and epigenetic influences. Recently considerable progress has been made in characterising the adult methylome, in epigenome-wide association studies. We report detailed analysis of the circulating methylome in 86 patients with childhood-onset CD,UC and 30 controls using the Illumina Infinium Human MethylationEPIC platform. We derive and validate a 4-probe methylation biomarker (RPS6KA2, VMP1, CFI and ARHGEF3), with specificity and high diagnostic accuracy for paediatric IBD in UK and North American cohorts (AUC 0.90-0.94). Significant epigenetic age acceleration is present at diagnosis, with the greatest observed in CD patients. Cis-MeQTL analysis identifies genetic determinants underlying epigenetic alterations notably within the HLA 6p22.1-p21.33 region. Passive smoking exposure is associated with the development of UC rather than CD contrary to previous findings. These data provide new insights into epigenetic alterations in IBD and illustrate the reproducibility and translational potential of epigenome-wide association studies in complex diseases.

Macrophage-Stimulating 1 Polymorphism rs3197999 in Pediatric Patients with Inflammatory Bowel Disease.

Background and Objectives: Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), often necessitates long-term treatment and hospitalizations and also may require surgery. The macrophage-stimulating 1 (MST1) rs3197999 polymorphism is strongly associated with the risk of IBD but its exact clinical correlates remain under investigation. We aimed to characterize the relationships between the MST1 rs3197999 genotype and the clinical characteristics in children and adolescents with IBD within a multi-center cross-sectional study. Materials and Methods: Clinical data included serum C-reactive protein (CRP), albumin, activity indices (PUCAI, PCDAI), anthropometric data, pharmacotherapy details, surgery, and disease severity. Genotyping for rs3197999 was carried out using TaqMan hydrolysis probes. Results: The study included 367 pediatric patients, 197 with Crohn's disease (CD) (40.6% female; a median age of 15.2 years [interquartile range 13.2-17.0]) and 170 with ulcerative colitis (UC) (45.8% female; a median age of 15.1 years [11.6-16.8]). No significant relationships were found between MST1 genotypes and age upon first biologic use, time from diagnosis to biological therapy introduction, PUCAI, PCDAI, or hospitalizations for IBD flares. However, in IBD, the height Z-score at the worst flare was negatively associated with the CC genotype (p = 0.016; CC: -0.4 [-1.2-0.4], CT: -0.1 [-0.7-0.8], TT: 0.0 [-1.2-0.7)]). The TT genotype was associated with higher C-reactive protein upon diagnosis (p = 0.023; CC: 4.3 mg/dL [0.7-21.8], CT 5.3 mg/dL [1.3-17.9], TT 12.2 mg/dL [3.0-32.9]). Conclusions: This study identified links between MST1 rs3197999 and the clinical characteristics of pediatric IBD: height Z-score and CRP. Further studies of the associations between genetics and the course of IBD are still warranted, with a focus on more extensive phenotyping.

Reagent- and Ligand-Dependent Mechanistic Trichotomy in Fe-Catalyzed Borylative Cyclizations of Enynes.

Based on some very recent results on Fe-catalyzed boron-source-dependent regiodivergent hydroborations of internal alkynes, we report here a boron-source-dependent but also ligand-dependent mechanistic trichotomy in borylative cyclizations. The choice of ligand plus boron source allows the synthesis of three isomeric borylative cyclization products starting from a common substrate and using the same precatalyst ((Ph3P)2Fe(CO)(NO)H) and sets the stage for the development of a unifying concept in Fe-catalyzed borylative cyclizations.

Molecular properties and diagnostic potential of monoclonal antibodies targeting cytotoxic α-synuclein oligomers.

α-Synuclein (α-syn) accumulates as insoluble amyloid but also forms soluble α-syn oligomers (αSOs), thought to be even more cytotoxic than fibrils. To detect and block the unwanted activities of these αSOs, we have raised 30 monoclonal antibodies (mAbs) against different forms of αSOs, ranging from unmodified αSOs to species stabilized by lipid peroxidation products and polyphenols, αSOs formed by C-terminally truncated α-syn, and multivalent display of α-syn on capsid virus-like particles (cVLPs). While the mAbs generally show a preference for αSOs, they also bind fibrils, but to variable extents. Overall, we observe great diversity in the mAbs' relative affinities for monomers and αSOs, varied requirements for the C-terminal extension of α-syn, and only a modest effect on α-syn fibrillation. Several mAbs show several orders of magnitude preference for αSOs over monomers in in-solution studies, while the commercial antibody MJF14 only bound 10-fold more strongly to αSOs than monomeric α-syn. Gratifyingly, seven mAbs almost completely block αSO permeabilization of membrane vesicles. Five selected mAbs identified α-syn-related pathologies like Lewy bodies (LBs) and Lewy Neurites, as well as Glial Cytoplasmic Inclusions in postmortem brains from people diagnosed for PD, dementia with LBs or multiple system atrophy, although to different extents. Three mAbs were particularly useful for pathological evaluation of postmortem brain human tissue, including early stages of PD. Although there was no straightforward connection between the mAbs' biophysical and immunohistochemical properties, it is encouraging that this comprehensive collection of mAbs able to recognize different aggregated α-syn species in vitro also holds diagnostic potential.

Specific inhibition of α-synuclein oligomer generation and toxicity by the chaperone domain Bri2 BRICHOS.

Protein misfolding and aggregation are involved in several neurodegenerative disorders, such as α-synuclein (αSyn) implicated in Parkinson's disease, where new therapeutic approaches remain essential to combat these devastating diseases. Elucidating the microscopic nucleation mechanisms has opened new opportunities to develop therapeutics against toxic mechanisms and species. Here, we show that naturally occurring molecular chaperones, represented by the anti-amyloid Bri2 BRICHOS domain, can be used to target αSyn-associated nucleation processes and structural species related to neurotoxicity. Our findings revealed that BRICHOS predominantly suppresses the formation of new nucleation units on the fibrils surface (secondary nucleation), decreasing the oligomer generation rate. Further, BRICHOS directly binds to oligomeric αSyn species and effectively diminishes αSyn fibril-related toxicity. Hence, our studies show that molecular chaperones can be utilized as tools to target molecular processes and structural species related to αSyn neurotoxicity and have the potential as protein-based treatments against neurodegenerative disorders.

Vitamin K Status Based on K1, MK-4, MK-7, and Undercarboxylated Prothrombin Levels in Adolescent and Adult Patients with Cystic Fibrosis: A Cross-Sectional Study.

The available evidence on vitamin K status in cystic fibrosis (CF) is scarce, lacking data on vitamin K2 (menaquinones-MK). Therefore, we assessed vitamin K1, MK-4 and MK-7 concentrations (LC-MS/MS) in 63 pancreatic insufficient and modulator naïve CF patients, and compared to 61 healthy subjects (HS). Vitamin K1 levels did not differ between studied groups. MK-4 concentrations were higher (median <1st-3rd quartile>: 0.778 <0.589-1.086> vs. 0.349 <0.256-0.469>, p < 0.0001) and MK-7 levels lower (0.150 <0.094-0.259> vs. 0.231 <0.191-0.315>, p = 0.0007) in CF patients than in HS. MK-7 concentrations were higher in CF patients receiving K1 and MK-7 supplementation than in those receiving vitamin K1 alone or no supplementation. Moreover, vitamin K1 concentrations depended on the supplementation regime. Based on multivariate logistic regression analysis, we have found that MK-7 supplementation dose has been the only predictive factor for MK-7 levels. In conclusion, vitamin K1 levels in CF are low if not currently supplemented. MK-4 concentrations in CF patients supplemented with large doses of vitamin K1 are higher than in HS. MK-7 levels in CF subjects not receiving MK-7 supplementation, with no regard to vitamin K1 supplementation, are low. There do not seem to be any good clinical predictive factors for vitamin K status.

Altered DNA methylation within DNMT3A, AHRR, LTA/TNF loci mediates the effect of smoking on inflammatory bowel disease.

This work aims to investigate how smoking exerts effect on the development of inflammatory bowel disease (IBD). A prospective cohort study and a Mendelian randomization study are first conducted to evaluate the association between smoking behaviors, smoking-related DNA methylation and the risks of Crohn's disease (CD) and ulcerative colitis (UC). We then perform both genome-wide methylation analysis and co-localization analysis to validate the observed associations. Compared to never smoking, current and previous smoking habits are associated with increased CD (P = 7.09 × 10-10) and UC (P < 2 × 10-16) risk, respectively. DNA methylation alteration at cg17742416 [DNMT3A] is linked to both CD (P = 7.30 × 10-8) and UC (P = 1.04 × 10-4) risk, while cg03599224 [LTA/TNF] is associated with CD risk (P = 1.91 × 10-6), and cg14647125 [AHRR] and cg23916896 [AHRR] are linked to UC risk (P = 0.001 and 0.002, respectively). Our study identifies biological mechanisms and pathways involved in the effects of smoking on the pathogenesis of IBD.

Helping proteins come in from the cold: 5 burning questions about cold-active enzymes.

Enzymes from psychrophilic (cold-loving) organisms have attracted considerable interest over the past decades for their potential in various low-temperature industrial processes. However, we still lack large-scale commercialization of their activities. Here, we review their properties, limitations and potential. Our review is structured around answers to 5 central questions: 1. How do cold-active enzymes achieve high catalytic rates at low temperatures? 2. How is protein flexibility connected to cold-activity? 3. What are the sequence-based and structural determinants for cold-activity? 4. How does the thermodynamic stability of psychrophilic enzymes reflect their cold-active capabilities? 5. How do we effectively identify novel cold-active enzymes, and can we apply them in an industrial context? We conclude that emerging screening technologies combined with big-data handling and analysis make it reasonable to expect a bright future for our understanding and exploitation of cold-active enzymes.

Current and emerging biomarkers for ulcerative colitis.

INTRODUCTION: Ulcerative colitis (UC) is a chronic illness requiring lifelong management that could be enhanced by personalizing care using biomarkers. AREAS COVERED: The main biomarker discovery modalities are reviewed, highlighting recent results across the spectrum of applications, including diagnostics (serum anti-αvß6 antibodies achieving an area under the curve [AUC] = 0.99; serum oncostatin M AUC = 0.94), disease activity assessment (fecal calprotectin and serum trefoil factor 3: AUC > 0.90), prognostication of the need for treatment escalation (whole blood transcriptomic panels and CLEC5A/CDH2 ratio: AUC > 0.90), prediction of treatment response, and early identification of patients with subclinical disease. The use of established biomarkers is discussed, along with new evidence regarding autoantibodies, proteins, proteomic panels, transcriptomic signatures, deoxyribonucleic acid methylation patterns, and UC-specific glycomic and metabolic disturbances. EXPERT OPINION: Novel biomarkers will pave the way for optimized UC care. However, validation, simplification, and direct clinical translation of complex models may prove challenging. Currently, few candidates exist to assess key characteristics, such as UC susceptibility, histological disease activity, drug response, and long-term disease behavior. Further research will likely not only reveal new tools to tackle these issues but also contribute to understanding UC pathogenesis mechanisms.

Periostin C-Terminal Is Intrinsically Disordered and Interacts with 143 Proteins in an In Vitro Epidermal Model of Atopic Dermatitis.

Human periostin is a 78-91 kDa matricellular protein implicated in extracellular matrix remodeling, tumor development, metastasis, and inflammatory diseases like atopic dermatitis, psoriasis, and asthma. The protein consists of six domains, including an N-terminal Cys-rich CROPT domain, four fasciclin-1 domains, and a C-terminal domain. The exons encoding the C-terminal domain may be alternatively spliced by shuffling four exons, generating ten variants of unknown function. Here, we investigate the structure and interactome of the full-length variant of the C-terminal domain with no exons spliced out. The structural analysis showed that the C-terminal domain lacked a tertiary structure and was intrinsically disordered. In addition, we show that the motif responsible for heparin-binding is in the conserved very C-terminal part of periostin. Pull-down confirmed three known interaction partners and identified an additional 140 proteins, among which nine previously have been implicated in atopic dermatitis. Based on our findings, we suggest that the C-terminal domain of periostin facilitates interactions between connective tissue components in concert with the four fasciclin domains.

Transcriptomic Context of RUNX3 Expression in Monocytes: A Cross-Sectional Analysis.

The runt-related transcription factor 3 (RUNX3) regulates the differentiation of monocytes and their response to inflammation. However, the transcriptomic context of RUNX3 expression in blood monocytes remains poorly understood. We aim to learn about RUNX3 from its relationships within transcriptomes of bulk CD14+ cells in adults. This study used immunomagnetically sorted CD14+ cell gene expression microarray data from the Multi-Ethnic Study of Atherosclerosis (MESA, n = 1202, GSE56047) and the Correlated Expression and Disease Association Research (CEDAR, n = 281, E-MTAB-6667) cohorts. The data were preprocessed, subjected to RUNX3-focused correlation analyses and random forest modeling, followed by the gene ontology analysis. Immunity-focused differential ratio analysis with intermediary inference (DRAIMI) was used to integrate the data with protein-protein interaction network. Correlation analysis of RUNX3 expression revealed the strongest positive association for EVL (rmean = 0.75, pFDR-MESA = 5.37 × 10-140, pFDR-CEDAR = 5.52 × 10-80), ARHGAP17 (rmean = 0.74, pFDR-MESA = 1.13 × 10-169, pFDR-CEDAR = 9.20 × 10-59), DNMT1 (rmean = 0.74, pFDR-MESA = 1.10 × 10-169, pFDR-CEDAR = 1.67 × 10-58), and CLEC16A (rmean = 0.72, pFDR-MESA = 3.51 × 10-154, pFDR-CEDAR = 2.27 × 10-55), while the top negative correlates were C2ORF76 (rmean = -0.57, pFDR-MESA = 8.70 × 10-94, pFDR-CEDAR = 1.31 × 10-25) and TBC1D7 (rmean = -0.55, pFDR-MESA = 1.36 × 10-69, pFDR-CEDAR = 7.81 × 10-30). The RUNX3-associated transcriptome signature was involved in mRNA metabolism, signal transduction, and the organization of cytoskeleton, chromosomes, and chromatin, which may all accompany mitosis. Transcriptomic context of RUNX3 expression in monocytes hints at its relationship with cell growth, shape maintenance, and aspects of the immune response, including tyrosine kinases.

Defining Interactions Between the Genome, Epigenome, and the Environment in Inflammatory Bowel Disease: Progress and Prospects.

Recent advances in our understanding of the pathogenesis of inflammatory bowel disease (IBD) have highlighted the complex interplay between the genome, the epigenome, and the environment. Despite the exciting advances in genomics that have enabled the identification of over 200 susceptibility loci, these only account for a small proportion of the disease variance and the estimated heritability in IBD. It is likely that gene-environment (GxE) interactions contribute to "missing heritability" and these may act through epigenetic mechanisms. Several environmental factors, such as the microbiome, nutrition, and tobacco smoking, induce alterations in the epigenome of children and adults, which may impact disease susceptibility. Other mechanisms for GxE interactions are also directly pertinent in early life. We discuss a model in which environmental factors imprint disease risk in a window of susceptibility during infancy that may contribute to later disease onset, whereas other elements of the exposome act later in life and contribute directly to the pathogenesis and course of the disease. Understanding the mechanisms underlying GxE interactions may provide the basis for new therapeutic targets or preventative strategies for IBD.

Autoantibodies and Clinical Correlations in Polish Systemic Sclerosis Patients: A Cross-Sectional Study.

We evaluated the prevalence of systemic sclerosis (SSc)-related autoantibodies and their clinical significance and compared the sensitivity of two line immunoblot assays on a prospective study group of 96 Polish SSc patients (ACR-EULAR 2013 criteria) whose sera were assessed by indirect immunofluorescence (HEp-2 and monkey liver) and line immunoblot assays: ANA Profile 3 and Systemic Sclerosis Profile by EUROIMMUN (Lübeck, Germany). Organ involvement was evaluated according to the EUSTAR Minimal Essential Data Set. The following autoantibodies' prevalence was found: Scl-70 (36%), Ro-52 (28%), CENP-B (22%), CENP-A (20%), PM-Scl-75 (20%), PM-Scl-100 (14%), fibrillarin (7%), Th/To (7%), RNA polymerase III 11 kDa (5%), RNA polymerase III 155 kDa (3%), PDGFR (3%), NOR-90 (2%), and Ku (1%). Significant associations between the autoantibodies' presence and organ involvement were found: ATA (dcSSc > lcSSc, less prevalent muscle weakness), Ro-52 (gangrene, DLCO < 60), CENP-B and A (lcSSc > dcSSc, normal CK), CENP-B (rarer digital ulcers and joint contractures), PM-Scl-100 and 75 (PM/SSc overlap, CK increase, muscle weakness, muscle atrophy), PM-Scl-100 (dcSSc unlikely), PM-Scl-75 (lung fibrosis), fibrillarin (muscle atrophy, proteinuria, conduction blocks, palpitations), Th/To (proteinuria, arthritis, muscle weakness, and rarer esophageal symptoms), RNA Polymerase III 11 kDa (arterial hypertension, renal crisis), RNA polymerase III 155 kDa (renal crisis), and PDGFR (dcSSc, tendon friction rubs). Additionally, the Systemic Sclerosis Profile was significantly more sensitive in detecting SSc-related autoantibodies than ANA Profile 3 (p = 0.002). In conclusion, individual autoantibodies associated with specific characteristics of SSc.

Analysis of Systemic Epigenetic Alterations in Inflammatory Bowel Disease: Defining Geographical, Genetic and Immune-Inflammatory influences on the Circulating Methylome.

BACKGROUND: Epigenetic alterations may provide valuable insights into gene-environment interactions in the pathogenesis of inflammatory bowel disease [IBD]. METHODS: Genome-wide methylation was measured from peripheral blood using the Illumina 450k platform in a case-control study in an inception cohort (295 controls, 154 Crohn's disease [CD], 161 ulcerative colitis [UC], 28 IBD unclassified [IBD-U)] with covariates of age, sex and cell counts, deconvoluted by the Houseman method. Genotyping was performed using Illumina HumanOmniExpressExome-8 BeadChips and gene expression using the Ion AmpliSeq Human Gene Expression Core Panel. Treatment escalation was characterized by the need for biological agents or surgery after initial disease remission. RESULTS: A total of 137 differentially methylated positions [DMPs] were identified in IBD, including VMP1/MIR21 [p = 9.11 × 10-15] and RPS6KA2 [6.43 × 10-13], with consistency seen across Scandinavia and the UK. Dysregulated loci demonstrate strong genetic influence, notably VMP1 [p = 1.53 × 10-15]. Age acceleration is seen in IBD [coefficient 0.94, p < 2.2 × 10-16]. Several immuno-active genes demonstrated highly significant correlations between methylation and gene expression in IBD, in particular OSM: IBD r = -0.32, p = 3.64 × 10-7 vs non-IBD r = -0.14, p = 0.77]. Multi-omic integration of the methylome, genome and transcriptome also implicated specific pathways that associate with immune activation, response and regulation at disease inception. At follow-up, a signature of three DMPs [TAP1, TESPA1, RPTOR] were associated with treatment escalation to biological agents or surgery (hazard ratio of 5.19 [CI: 2.14-12.56], logrank p = 9.70 × 10-4). CONCLUSION: These data demonstrate consistent epigenetic alterations at diagnosis in European patients with IBD, providing insights into the pathogenetic importance and translational potential of epigenetic mapping in complex disease.

Methylation of RUNX3 Promoter 2 in the Whole Blood of Children with Ulcerative Colitis.

Ulcerative colitis (UC) results from a complex interplay between the environment, gut microbiota, host genetics, and immunity. Runt-related transcription factor 3 (RUNX3) regulates Th1/Th2 balance and, thus, the synthesis of cytokines and inflammation. We aimed to analyze the dependence of RUNX3 promoter 2 (P2) methylation level on: age, sex, body mass index (BMI), C-reactive protein (CRP), serum albumin, disease duration, Pediatric Ulcerative Colitis Activity Index (PUCAI), the Paris classification, and exposure to medications. This multicenter, cross-sectional study recruited hospitalized children with UC. Methylation of RUNX3 P2 was measured with methylation-sensitive restriction enzymes in the whole blood DNA. Sixty-four children were enrolled, with a mean age of 14.5 ± 2.8 years. Half of them were female (51.6%), and the average BMI Z-score was -0.44 ± 1.14. The mean methylation of RUNX3 P2 was 54.1 ± 13.3%. The methylation level of RUNX3 P2 did not correlate with age, sex, nutritional status, CRP, albumin, PUCAI, or the extent of colitis (Paris E1-E4). RUNX3 P2 methylation did not differ between patients recruited within two and a half months of diagnosis and children who had UC for at least a year. Current or past exposure to biologics, immunosuppressants, or steroids was not associated with RUNX3 P2 methylation. Methylation of RUNX3 promoter 2 in whole blood DNA does not seem to be associated with the characteristics of UC in children.

Unexpected Actors in Inflammatory Bowel Disease Revealed by Machine Learning from Whole-Blood Transcriptomic Data.

Although big data from transcriptomic analyses have helped transform our understanding of inflammatory bowel disease (IBD), they remain underexploited. We hypothesized that the application of machine learning using lasso regression to transcriptomic data from IBD patients and controls can help identify previously overlooked genes. Transcriptomic data provided by Ostrowski et al. (ENA PRJEB28822) were subjected to a two-stage process of feature selection to discriminate between IBD and controls. First, a principal component analysis was used for dimensionality reduction. Second, the least absolute shrinkage and selection operator (lasso) regression was employed to identify genes potentially involved in the pathobiology of IBD. The study included data from 294 participants: 100 with ulcerative colitis (48 adults and 52 children), 99 with Crohn's disease (45 adults and 54 children), and 95 controls (46 adults and 49 children). IBD patients presented a wide range of disease severity. Lasso regression preceded by principal component analysis successfully selected interesting features in the IBD transcriptomic data and yielded 12 models. The models achieved high discriminatory value (range of the area under the receiver operating characteristic curve 0.61-0.95) and identified over 100 genes as potentially associated with IBD. PURA, GALNT14, and FCGR1A were the most consistently selected, highlighting the role of the cell cycle, glycosylation, and immunoglobulin binding. Several known IBD-related genes were among the results. The results included genes involved in the TGF-beta pathway, expressed in NK cells, and they were enriched in ontology terms related to immunity. Future IBD research should emphasize the TGF-beta pathway, immunoglobulins, NK cells, and the role of glycosylation.

Characterisation of the Circulating Transcriptomic Landscape in Inflammatory Bowel Disease Provides Evidence for Dysregulation of Multiple Transcription Factors Including NFE2, SPI1, CEBPB, and IRF2.

AIM: To assess the pathobiological and translational importance of whole-blood transcriptomic analysis in inflammatory bowel disease [IBD]. METHODS: We analysed whole-blood expression profiles from paired-end sequencing in a discovery cohort of 590 Europeans recruited across six countries in the IBD Character initiative (newly diagnosed patients with Crohn's disease [CD; n = 156], ulcerative colitis [UC; n = 167], and controls [n = 267]), exploring differential expression [DESeq2], co-expression networks [WGCNA], and transcription factor involvement [EPEE, ChEA, DoRothEA]. Findings were validated by analysis of an independent replication cohort [99 CD, 100 UC, 95 controls]. In the discovery cohort, we also defined baseline expression correlates of future treatment escalation using cross-validated elastic-net and random forest modelling, along with a pragmatic ratio detection procedure. RESULTS: Disease-specific transcriptomes were defined in IBD [8697 transcripts], CD [7152], and UC [8521], with the most highly significant changes in single genes, including CD177 (log2-fold change [LFC] = 4.63, p = 4.05 × 10-118), MCEMP1 [LFC = 2.45, p = 7.37 × 10-109], and S100A12 [LFC = 2.31, p = 2.15 × 10-93]. Significantly over-represented pathways included IL-1 [p = 1.58 × 10-11], IL-4, and IL-13 [p = 8.96 × 10-9]. Highly concordant results were obtained using multiple regulatory activity inference tools applied to the discovery and replication cohorts. These analyses demonstrated central roles in IBD for the transcription factors NFE2, SPI1 [PU.1], CEBPB, and IRF2, all regulators of cytokine signalling, based on a consistent signal across cohorts and transcription factor ranking methods. A number of simple transcriptome-based models were associated with the need for treatment escalation, including the binary CLEC5A/CDH2 expression ratio in UC (hazard ratio = 23.4, 95% confidence interval [CI] 5.3-102.0). CONCLUSIONS: Transcriptomic analysis has allowed for a detailed characterisation of IBD pathobiology, with important potential translational implications.