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Objective: In the past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. In this pathway, glycogen synthase kinase-3β (GSK3β) down-regulates transduction of the canonical Wnt signal by promoting degradation of β-catenin. In this study we wanted to further investigate the role of Gsk3β in cartilage maintenance. Design: Therefore, we have treated chondrocytes ex vivo and in vivo with GIN, a selective GSK3β inhibitor. Results: In E17.5 fetal mouse metatarsals, GIN treatment resulted in loss of expression of cartilage markers and decreased chondrocyte proliferation from day 1 onward. Late (3 days) effects of GIN included cartilage matrix degradation and increased apoptosis. Prolonged (7 days) GIN treatment resulted in resorption of the metatarsal. These changes were confirmed by microarray analysis showing a decrease in expression of typical chondrocyte markers and induction of expression of proteinases involved in cartilage matrix degradation. An intra-articular injection of GIN in rat knee joints induced nuclear accumulation of β-catenin in chondrocytes 72 h later. Three intra-articular GIN injections with a 2 days interval were associated with surface fibrillation, a decrease in glycosaminoglycan expression and chondrocyte hypocellularity 6 weeks later. Conclusions: These results suggest that, by down-regulating β-catenin, Gsk3β preserves the chondrocytic phenotype, and is involved in maintenance of the cartilage extracellular matrix. Short term β-catenin up-regulation in cartilage secondary to Gsk3β inhibition may be sufficient to induce osteoarthritis-like features in vivo. [ABSTRACT FROM AUTHOR]

Background: Regional lymph node metastasis is an important prognostic factor in head and neck squamous cell carcinoma (HNSCC) and plays a decisive role in the choice of treatment. Here, we present an independent gene expression validation study of metastasized versus non-metastasized HNSCC.Methods: We used a dataset recently published by Roepman et al. as reference dataset and an independent gene expression dataset of 11 metastasized and 11 non-metastasized HNSCC tumors as validation dataset. Reference and validation studies were performed on different microarray platforms with different probe sets and probe content. In addition to a supervised gene-based analysis, a supervised pathway-based analysis was performed, evaluating differences in gene expression for predefined tumorigenesis- and metastasis related gene sets.Results: The gene-based analysis showed 26 significant differentially expressed genes in the reference dataset, 21 of which were present on the microarray platform used in the validation study. 7 of these genes appeared to be significantly expressed in the validation dataset, but failed to pass the correction for multiple testing. The pathway-based analysis revealed 23 significant differentially expressed gene sets, 7 of which were statistically validated. These gene sets are involved in extracellular matrix remodeling (MMPs, MMP regulating pathways and the uPA system), hypoxia and angiogenesis (HIF1alpha regulated angiogenic factors and HIF1alpha regulated invasion).Conclusion: Pathways that are differentially expressed between metastasized and non-metastasized HNSCC are involved in the processes of extracellular matrix remodeling, hypoxia and angiogenesis. A supervised pathway-based analysis enhances the understanding of the biological context of the results, the comparability of results across different microarray studies, and reduces multiple testing problems by focusing on a limited number of pathways of interest instead of analyzing the large number of probes available on the microarray. [ABSTRACT FROM AUTHOR]

Purpose: To determine the independent effect of uteroplacental malperfusion on the development of retinopathy of prematurity (ROP)., Methods: This cohort study included 591 neonates with a gestational age (GA) ≤ 32 weeks or birthweight (BW) ≤ 1500 g. Clinical data was retrospectively collected and placentas were prospectively examined for maternal vascular malperfusion (e.g., abruption, infarct, distal villous hypoplasia, ischemia, and decidual necrosis) and fetal vascular malperfusion (e.g., thrombosis, fetal hypoxia, and hydrops parenchyma). The primary outcome was ROP. Secondary outcomes were GA, BW, small for gestational age (SGA), mechanical ventilation duration, postnatal corticosteroids, sepsis, and necrotizing enterocolitis., Results: Maternal vascular malperfusion was associated with higher GA, lower BW, and increased SGA rates, except placental abruption, which was associated with lower SGA rates. Fetal vascular malperfusion was associated with lower BW, increased SGA rates and lower duration of mechanical ventilation. Subgroup analysis of placentas without inflammation showed increased rates of distal villous hypoplasia (44% vs. 31%) and hydrops parenchyma (7% vs. 0%) in neonates with ROP. Multivariate regression analyses revealed three placenta factors to be independently associated with ROP: distal villous hypoplasia (OR = 1.7; 95% CI, 1.0-3.0), severe acute histological chorioamnionitis (OR = 2.1; 95% CI, 1.1-3.9) and funisitis (OR = 1.8; 95% CI, 1.0-3.1)., Conclusions: Placental evaluation of distal villous hypoplasia, severe acute chorioamnionitis and funisitis is a novel and valuable addition to the ROP risk profile. Evaluation of these placental risk factors shortly after birth can aid in identifying high-risk infants in an earlier stage than currently possible.

Objective:  Neonatal sepsis has been established as a risk factor for retinopathy of prematurity (ROP) but previous meta-analyses have predominately focused on late-onset sepsis (LOS). This meta-analysis aims to explore the association between early-onset sepsis (EOS) and the risk of ROP., Study Design:  Observational studies reporting (unadjusted) data on proven EOS in neonates with ROP were included. PubMed, Embase, and Cochrane Library were searched. Proven EOS was defined as a positive blood or cerebrospinal fluid culture. Effect sizes were calculated by using logistic random-effects models and meta-regression analyses. Primary outcomes were any stage ROP and severe ROP (≥stage 3, type I, aggressive [posterior] ROP, plus disease or requiring treatment). Potential confounders explored were gestational age at birth, birth weight, small for gestational age, maternal steroid use, necrotizing enterocolitis, LOS, and mechanical ventilation duration., Results:  Seventeen studies reporting the incidence of proven EOS in neonates with ROP were included. Proven EOS showed no significant association with any stage ROP (odds ratio [OR] = 1.90; 95% confidence interval [CI]: 0.96-3.79, p  = 0.067) but heterogeneity between studies was significantly high. Neonates with proven EOS had an increased risk for severe ROP (OR = 2.21; 95% CI: 1.68-2.90), and no significant confounders influencing this effect size were found in the meta-regression analysis., Conclusion:  Neonates with proven EOS are at increased risk of severe ROP. Neonatologists need to be aware that EOS is an early predictor of ROP and should adapt their policy and treatment decisions where possible to reduce ROP., Key Points: · This meta-analysis reveals a 2.2-fold increased risk of severe ROP in neonates with proven EOS.. · Future studies should distinguish between EOS and LOS when investigating risk factors of ROP.. · Treatment decisions should be adapted where possible in neonates with EOS before ROP screening begins.., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/).)

Closed testing has recently been shown to be optimal for simultaneous true discovery proportion control. It is, however, challenging to construct true discovery guarantee procedures in such a way that it focuses power on some feature sets chosen by users based on their specific interest or expertise. We propose a procedure that allows users to target power on prespecified feature sets, that is, "focus sets." Still, the method also allows inference for feature sets chosen post hoc, that is, "nonfocus sets," for which we deduce a true discovery lower confidence bound by interpolation. Our procedure is built from partial true discovery guarantee procedures combined with Holm's procedure and is a conservative shortcut to the closed testing procedure. A simulation study confirms that the statistical power of our method is relatively high for focus sets, at the cost of power for nonfocus sets, as desired. In addition, we investigate its power property for sets with specific structures, for example, trees and directed acyclic graphs. We also compare our method with AdaFilter in the context of replicability analysis. The application of our method is illustrated with a gene ontology analysis in gene expression data., (© 2024 The Author(s). Biometrical Journal published by Wiley‐VCH GmbH.)

Objective: The role of placental inflammation in neonatal morbidities is underestimated due to lack of placental examination. This meta-analysis aims to assess the association between histological chorioamnionitis (HCA) with and without funisitis (FUN) and risk of retinopathy of prematurity (ROP)., Study Design: Forty-five studies reporting (unadjusted) data on HCA without FUN and HCA with FUN in neonates with ROP were included. Primary outcomes were any stage ROP and severe ROP. Potential confounders explored were gestational age (GA) at birth, birthweight, maternal steroid use, necrotizing enterocolitis, sepsis (suspected/proven) and mechanical ventilation duration., Results: Neonates with HCA had increased risk for any stage ROP (odds ratio [OR] 1.8; 95% confidence interval [CI] 1.3-2.4) and severe ROP (OR 1.5; 95% CI 1.2-1.8) compared with neonates without HCA. The rates of any stage ROP (OR 1.8; 95% CI 1.4-2.2) and severe ROP (OR 1.4; 95% CI 1.1-1.6) were higher in neonates with FUN compared with neonates without FUN. Multivariate meta-regression analysis suggests that lower GA increases the effect size between FUN and severe ROP., Conclusion: This meta-analysis confirms that presence of HCA and FUN are risk factors for any stage ROP and severe ROP. Structured histological placental examination of HCA and FUN may be a tool to further refine the ROP risk profile., Key Points: · This systematic review confirms that HCA is a risk factor for ROP.. · This meta-analysis reveals that FUN results in an even higher risk for developing ROP.. · Placental examination of HCA/FUN may be a tool to further refine the ROP risk profile.., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/).)

Background: Knowledge of the psychosocial impact of facial skin surgery on patients can help improve counselling strategies., Objectives: The objective was to measure the psychological impact of facial skin cancer surgery on patients over a 1-year period. Secondary objective was to measure the difference between Mohs micrographic surgery (MMS) and conventional excision (CE) on these parameters., Methods: This observational survey study was conducted between March 2019 and July 2020. Patients who had facial skin surgery using MMS or CE were selected. Five surveys were conducted on four timepoints (preoperative, 1 week, 3 months and 1 year post-operative) measuring the quality of life, perceived stigmatization, body image, satisfaction with facial appearance and psychosocial distress., Results: A total of 228 patients (MMS 154 patients, CE 74 patients) were included for the analysis. Scores for quality of life did not significantly change, in the year after surgery (PCS-12 mean 50.5, SD 9.3 and MCS-12 50.6, SD 9.4); however, stigmatization (F (3, 235,39) 7,26, p < 0.01, d = -0.07), body image concerns (F (3, 198,28) = 3.75, p < 0.01, d = -0.14), satisfaction with facial appearance (F (3, 205,18) = 10.74, p < 0.01, d = 0.43) and psychosocial distress (F (3, 208,69) = 9.26, p < 0.01, d = -0.15) did change over time. The use of MMS or CE did not significantly affect outcome scores after 1 year., Conclusion: Patients receiving facial skin cancer surgery exhibited low scores for perceived stigmatization and body image concerns. Their quality of life was not statistically influenced by facial surgery, and their satisfaction with their facial appearance and psychosocial distress even improved after 1 year. The results suggest that the surgical treatment type (MMS or CE) does not influence the outcome. The overall results can help in counselling strategies to improve expectations for patients receiving facial surgery., (© 2024 The Authors. Journal of the European Academy of Dermatology and Venereology published by John Wiley & Sons Ltd on behalf of European Academy of Dermatology and Venereology.)

Background: There is divergence in the rate at which people age. The concept of biological age is postulated to capture this variability, and hence to better represent an individual's true global physiological state than chronological age. Biological age predictors are often generated based on cross-sectional data, using biochemical or molecular markers as predictor variables. It is assumed that the difference between chronological and predicted biological age is informative of one's chronological age-independent aging divergence ∆., Methods: We investigated the statistical assumptions underlying the most popular cross-sectional biological age predictors, based on multiple linear regression, the Klemera-Doubal method or principal component analysis. We used synthetic and real data to illustrate the consequences if this assumption does not hold., Results: The most popular cross-sectional biological age predictors all use the same strong underlying assumption, namely that a candidate marker of aging's association with chronological age is directly informative of its association with the aging rate ∆. We called this the identical-association assumption and proved that it is untestable in a cross-sectional setting. If this assumption does not hold, weights assigned to candidate markers of aging are uninformative, and no more signal may be captured than if markers would have been assigned weights at random., Conclusions: Cross-sectional methods for predicting biological age commonly use the untestable identical-association assumption, which previous literature in the field had never explicitly acknowledged. These methods have inherent limitations and may provide uninformative results, highlighting the importance of researchers exercising caution in the development and interpretation of cross-sectional biological age predictors., (© 2024. The Author(s).)

Background: Glioblastoma patients are at high risk of developing venous thromboembolism (VTE). Tumor-intrinsic features are considered to play a role, but the underlying pathophysiological mechanisms remain incompletely understood., Objectives: To identify tumor-expressed genes and signaling pathways that associate with glioblastoma-related VTE by using next generation RNA-sequencing (RNA-Seq)., Methods: The tumor gene expression profile of 23 glioblastoma patients with VTE and 23 glioblastoma patients without VTE was compared using an unpaired analysis. Ingenuity Pathway Analysis (IPA) core analysis was performed on the top 50 differentially expressed genes to explore associated functions and pathways. Based on full RNA-Seq data, molecular glioblastoma subtypes were determined by performing cluster analysis., Results: Of the 19,327 genes, 1246 (6.4 %) were differentially expressed between glioblastoma patients with and without VTE (unadjusted P < 0.05). The most highly overexpressed gene was GLI1, a classical target gene in the Sonic Hedgehog (Shh) signaling pathway (log2 fold change: 3.7; unadjusted P < 0.0001, adjusted P = 0.219). In line, Shh signaling was among the top canonical pathways and processes associated with VTE. The proportion of patients with the proneural/neural glioblastoma subtype was higher among those with VTE than controls., Conclusion: Shh signaling may be involved in the development of glioblastoma-related VTE., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: F.A.K. has received research support from Bayer, BMS, BSCI, MSD, Leo Pharma, Actelion, Farm-X, The Netherlands Organization for Health Research and Development, The Dutch Thrombosis Foundation, The Dutch Heart Foundation and the Horizon Europe Program, all paid to his institution and outside the submitted work. The other authors have no conflicts of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Globaltest is a powerful test for the global null hypothesis that there is no association between a group of features and a response of interest, which is popular in pathway testing in metabolomics. Evaluating multiple feature sets, however, requires multiple testing correction. In this paper, we propose a multiple testing method, based on closed testing, specifically designed for the Globaltest. The proposed method controls the familywise error rate simultaneously over all possible feature sets, and therefore allows post hoc inference, that is, the researcher may choose feature sets of interest after seeing the data without jeopardizing error control. To circumvent the exponential computation time of closed testing, we derive a novel shortcut that allows exact closed testing to be performed on the scale of metabolomics data. An R package ctgt is available on comprehensive R archive network for the implementation of the shortcut procedure, with applications on several real metabolomics data examples., (© 2022 The International Biometric Society.)

Aim: To build and externally validate an [ 18 F]FDG PET radiomic model to predict overall survival in patients with head and neck squamous cell carcinoma (HNSCC)., Methods: Two multicentre datasets of patients with operable HNSCC treated with preoperative afatinib who underwent a baseline and evaluation [ 18 F]FDG PET/CT scan were included (EORTC: n = 20, Unicancer: n = 34). Tumours were delineated, and radiomic features were extracted. Each cohort served once as a training and once as an external validation set for the prediction of overall survival. Supervised feature selection was performed using variable hunting with variable importance, selecting the top two features. A Cox proportional hazards regression model using selected radiomic features and clinical characteristics was fitted on the training dataset and validated in the external validation set. Model performances are expressed by the concordance index (C-index)., Results: In both models, the radiomic model surpassed the clinical model with validation C-indices of 0.69 and 0.79 vs. 0.60 and 0.67, respectively. The model that combined the radiomic features and clinical variables performed best, with validation C-indices of 0.71 and 0.82., Conclusion: Although assessed in two small but independent cohorts, an [ 18 F]FDG-PET radiomic signature based on the evaluation scan seems promising for the prediction of overall survival for HNSSC treated with preoperative afatinib. The robustness and clinical applicability of this radiomic signature should be assessed in a larger cohort.

Objective: Impaired amine metabolism has been associated with the etiology of migraine, that is, why patients continue to get migraine attacks. However, evidence from cerebrospinal fluid (CSF) is lacking. Here, we evaluated individual amine levels, global amine profiles, and amine pathways in CSF and plasma of interictal migraine patients and healthy controls., Methods: CSF and plasma were sampled between 8:30 am and 1:00 pm, randomly and interchangeably over the time span to avoid any diurnal and seasonal influences, from healthy volunteers and interictal migraine patients, matched for age, sex, and sampling time. The study was approved by the local medical ethics committee. Individual amines (n = 31), global amine profiles, and specific amine pathways were analyzed using a validated ultraperformance liquid chromatography mass spectrometry platform., Results: We analyzed n = 99 participants with migraine with aura, n = 98 with migraine without aura, and n = 96 healthy volunteers. Univariate analysis with Bonferroni correction indicated that CSF L-arginine was reduced in migraine with aura (10.4%, p < 0.001) and without aura (5.0%, p = 0.03). False discovery rate-corrected CSF L-phenylalanine was also lower in migraine with aura (6.9%, p = 0.011) and without aura (8.1%, p = 0.001), p = 0.088 after Bonferroni correction. Multivariate analysis revealed that CSF global amine profiles were similar for both types of migraine (p = 0.64), but distinct from controls (p = 0.009). Global profile analyses were similar in plasma. The strongest associated pathways with migraine were related to L-arginine metabolism., Interpretation: L-Arginine was decreased in the CSF (but not in plasma) of interictal patients with migraine with or without aura, and associated pathways were altered. This suggests that dysfunction of nitric oxide signaling is involved in susceptibility to getting migraine attacks. ANN NEUROL 2023;93:715-728., (© 2022 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Pelvic organ prolapse (POP) represents a major health care burden in women, but its underlying pathophysiological mechanisms have not been elucidated. We first used a case-control design to perform an exome chip study in 526 women with POP and 960 control women to identify single nucleotide variants (SNVs) associated with the disease. We then integrated the functional interactions between the POP candidate proteins derived from the exome chip study and other POP candidate molecules into a molecular landscape. We found significant associations between POP and SNVs in 54 genes. The proteins encoded by 26 of these genes fit into the molecular landscape, together with 43 other POP candidate molecules. The POP landscape is located in and around epithelial cells and fibroblasts of the urogenital tract and harbors four interacting biological processes-epithelial-mesenchymal transition, immune response, modulation of the extracellular matrix, and fibroblast function-that are regulated by sex hormones and TGFB1. Our findings were corroborated by enrichment analyses of differential gene expression data from an independent POP cohort. Lastly, based on the landscape and using vaginal fibroblasts from women with POP, we predicted and showed that metformin alters gene expression in these fibroblasts in a beneficial direction. In conclusion, our integrated molecular landscape of POP provides insights into the biological processes underlying the disease and clues towards novel treatments.

Background: Solid organ-transplant recipients (SOTR) have an increased risk of cutaneous squamous-cell carcinoma (cSCC), metastasis and death from cSCC. In immunocompetent patients with mucosal SCC, downregulation of HLA class I is associated with poor prognosis. Since the degree of HLA expression on tumor cells could play a role in immunogenicity and pathophysiology of cSCC metastasis, we hypothesized that decreased HLA expression is associated with an increased risk of metastasis., Methods: We compared HLA expression between primary metastasized cSCCs, their metastases, and non-metastasized cSCCs from the same patients. Samples were stained for HLA-A, HLA-B/-C and quantified by calculating the difference in immunoreactivity score (IRS) of the primary cSCC compared with all non-metastasized cSCCs., Results: The mean IRS score for HLA-B/C expression was 2.07 point higher in metastasized compared to non-metastasized cSCCs (p = 0.065, 95 % CI -0.18-4.32). 83.3 % of the primary metastasized cSCCs had an IRS score of 4 or higher, compared to 42.9 % in non-metastasized cSCCs. Moderately to poorly differentiated cSCCs had more HLA class I expression compared to well-differentiated cSCCs., Conclusion: Contrary to immunocompetent patients, HLA-B/C expression tends to be upregulated in metastasized cSCC compared to non-metastasized cSCC in SOTR, suggesting that different tumor escape mechanisms play a role in SOTR compared to immunocompetent patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mechanical stress influences scarring of a surgical wound. Several lines have been proposed for the best excision direction. It is unknown if these lines still apply when the body posture changes. The objective is to measure the skin reaction force in four directions and determine the direction of least force. Secondary objective is to determine if the reaction force varies in a different body posture. Skin reaction force was measured with the compressiometer in 30 participants on four different locations (forearm/upper arm/shoulder blade/lower back) in four directions (0°-45°-90°-135°) and two body postures. The direction of least skin reaction force changed with a different body posture and was significant for the forearm (p < 0.01) and shoulder blade (p = 0.05) The skin reaction force in all four direction changed significantly in a different body posture, except the 45° line in the upper arm and shoulder blade. Our results demonstrate that the skin reaction force in four directions in four locations varies with change in body posture. Focus should therefore not only lay on choosing the right direction, but also on managing skin tension postoperatively., (© 2023. The Author(s).)

Background & Aims: Improving clinical management of early stage colorectal cancers (T1CRCs) requires a better understanding of their underlying biology. Accumulating evidence shows that cancer-associated fibroblasts (CAFs) are important determinants of tumor progression in advanced colorectal cancer (CRC), but their role in the initial stages of CRC tumorigenesis is unknown. Therefore, we investigated the contribution of T1CAFs to early CRC progression., Methods: Primary T1CAFs and patient-matched normal fibroblasts (NFs) were isolated from endoscopic biopsy specimens of histologically confirmed T1CRCs and normal mucosa, respectively. The impact of T1CAFs and NFs on tumor behavior was studied using 3-dimensional co-culture systems with primary T1CRC organoids and extracellular matrix (ECM) remodeling assays. Whole-transcriptome sequencing and gene silencing were used to pinpoint mediators of T1CAF functions., Results: In 3-dimensional multicellular cultures, matrix invasion of T1CRC organoids was induced by T1CAFs, but not by matched NFs. Enhanced T1CRC invasion was accompanied by T1CAF-induced ECM remodeling and up-regulation of CD44 in epithelial cells. RNA sequencing of 10 NF-T1CAF pairs revealed 404 differentially expressed genes, with significant enrichment for ECM-related pathways in T1CAFs. Cathepsin H, a cysteine-type protease that was specifically up-regulated in T1CAFs but not in fibroblasts from premalignant lesions or advanced CRCs, was identified as a key factor driving matrix remodeling by T1CAFs. Finally, we showed high abundance of cathepsin H-expressing T1CAFs at the invasive front of primary T1CRC sections., Conclusions: Already in the earliest stage of CRC, cancer cell invasion is promoted by CAFs via direct interactions with epithelial cancer cells and stage-specific, cathepsin H-dependent ECM remodeling. RNA sequencing data of the 10 NF-T1CAF pairs can be found under GEO accession number GSE200660., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Reflex mismatch repair immunohistochemistry (MMR IHC) testing for MLH1, PMS2, MSH2 and MSH6 is used to screen for Lynch syndrome. Recently MMR-deficiency (MMRd) has been approved as a pan-cancer predictive biomarker for checkpoint inhibitor therapy, leading to a vast increase in the use of MMR IHC in clinical practice. We explored whether immunohistochemical staining with PMS2 and MSH6 can be used as a reliable substitute. This two-antibody testing algorithm has the benefit of saving tissue, cutting costs and saving time. PubMed, Embase and Cochrane library were systematically searched for articles reporting on MMR IHC. The weighed percentage of cases with isolated MLH1 or MSH2 loss or combined MLH1/MSH2 loss alone was analyzed using a random effects model meta-analysis in R. The search yielded 1704 unique citations, of which 131 studies were included, describing 9014 patients. A weighed percentage of 1.1% (95% CI 0.53-18.87, I = 87%) of cases with isolated MLH1 or MSH2 loss or combined MLH1/MSH2 loss alone was observed. In the six articles with the main aim of investigating the two-antibody testing algorithm all MMRd cases were detected with the two-antibody testing algorithm, there were no cases with isolated MLH1 or MSH2 loss or combined MLH1/MSH2 loss alone. This high detection rate of MMRd of the two-antibody testing algorithm supports its use in clinical practice by specialized pathologists. Staining of all four antibodies should remain the standard in cases with equivocal results of the two-antibody testing algorithm. Finally, educational sessions in which staining pattern pitfalls are discussed will continue to be important., (© 2022. The Author(s).)

Existing methods for differential network analysis could only infer whether two networks of interest have differences between two groups of samples, but could not quantify and localize network differences. In this work, a novel method, permutation-based Network True Discovery Proportions (NetTDP), is proposed to quantify the number of edges (correlations) or nodes (genes) for which the co-expression networks are different. In the NetTDP method, we propose an edge-level statistic and a node-level statistic, and detect true discoveries of edges and nodes in the sense of differential co-expression network, respectively, by the permutation-based sumSome method. Furthermore, the NetTDP method could further localize the differences by inferring the TDPs for edge or gene subsets of interest, which can be selected post hoc. Our NetTDP method allows inference on data-driven modules or biology-driven gene sets, and remains valid even when these sub-networks are optimized using the same data. Experimental results on both simulation data sets and five real data sets show the effectiveness of the proposed method in inferring the quantification and localization of differential co-expression networks. The R code is available at https://github.com/LiminLi-xjtu/NetTDP., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Virus-specific cellular and humoral responses are major determinants for protection from critical illness after SARS-CoV-2 infection. However, the magnitude of the contribution of each of the components to viral clearance remains unclear. Here, we studied the timing of viral clearance in relation to 122 immune parameters in 102 hospitalised patients with moderate and severe COVID-19 in a longitudinal design. Delayed viral clearance was associated with more severe disease and was associated with higher levels of SARS-CoV-2-specific (neutralising) antibodies over time, increased numbers of neutrophils, monocytes, basophils, and a range of pro-inflammatory cyto-/chemokines illustrating ongoing, partially Th2 dominating, immune activation. In contrast, early viral clearance and less critical illness correlated with the peak of neutralising antibodies, higher levels of CD4 T cells, and in particular naïve CD4+ T cells, suggesting their role in early control of SARS-CoV-2 possibly by proving appropriate B cell help. Higher counts of naïve CD4+ T cells also correlated with lower levels of MIF, IL-9, and TNF-beta, suggesting an indirect role in averting prolonged virus-induced tissue damage. Collectively, our data show that naïve CD4+ T cell play a critical role in rapid viral T cell control, obviating aberrant antibody and cytokine profiles and disease deterioration. These data may help in guiding risk stratification for severe COVID-19.

Purpose: The aim of this study was to identify positive predictors for survival in uveal melanoma (UM) patients treated with percutaneous hepatic perfusion with melphalan (M-PHP), by retrospectively pooling data from three centers., Materials and Methods: Retrospective analysis including patients ([Formula: see text] 18 years) treated with M-PHP between February 2014 and December 2019 for unresectable liver-dominant or liver-only metastases from UM. Predictors for OS were assessed using uni- and multivariate analyses. Other study outcome measures were response rate, progression-free survival (PFS), liver progression-free survival (LPFS), overall survival (OS) and complications according to CTCAEv5.0., Results: In total, 101 patients (47.5% males; median age 59.0 years) completed a minimum of one M-PHP. At a median follow-up time of 15.0 months, complete response (CR), partial response (PR), stable disease (SD) and progressive disease were seen in five (5.0%), 55 (54.5%), 30 (29.7%) and 11 (10.9%) patients, respectively, leading to a 89.1% disease control rate. Median PFS, LPFS and OS were 9.0, 11.0 and 20.0 months, respectively. Survival analyses stratified for radiological response demonstrated significant improved survival in patients with CR or PR and SD category. Treatment of the primary tumor with radiotherapy, ≥ 2 M-PHP and lactate dehydrogenase (LDH) < 248 U/L were correlated with improved OS. Thirty-day mortality was 1.1% (n = 2). Most common complication was hematological toxicity (self-limiting in most cases)., Conclusion: M-PHP is safe and effective in patients with UM liver metastases. Achieving CR, PR or SD is associated with improved survival. Primary tumor treatment with radiotherapy, normal baseline LDH and > 1 M-PHP cycles are associated with improved OS., (© 2022. The Author(s).)

Background: The standard treatment for cutaneous squamous cell carcinoma (cSCC) is surgical excision. Failure to radically remove a cSCC is a risk for recurrence, progression and metastasis., Objectives: This study investigates several risk factors for incomplete excision of cSCC., Methods: All consecutive patients in a single institution treated with wide local excision for primary cSCC over a 10-year period were included in this study. Risk factors such as: gender, age, immunosuppression, tumour size, location, differentiation grade, tumour depth, perineural and lymphovascular invasion (PNI and LVI) were extracted from the database. Univariable and (if applicable) multivariable logistic regression analysis were used to identify risk factors (P < 0.05). Generalized estimating equations (GEEs) were used for multiple tumours within the same patients., Results: A total of 566 patients with 1159 cSCC were identified. Univariable and multivariable logistic regression analysis showed that depth beyond the dermis (OR: 5.7 95% CI: 3.1-10.5) was the only risk factor for incomplete excision of cSCC. Immunosuppression was only a risk factor in the deep plane (OR: 2.5, 95% CI: 1.3-4.6)., Conclusion: Tumour depth beyond the dermis is the most important risk factor for incomplete excision of cSCC. Immunosuppression is a risk factor in the deep plane but its relevance is uncertain. Immunosuppression is not consistently included in the current cSCC staging systems, but care should be taken when treating these patients., (© 2022 The Authors. Journal of the European Academy of Dermatology and Venereology published by John Wiley & Sons Ltd on behalf of European Academy of Dermatology and Venereology.)

Background: During the COVID-19 pandemic, several factors, such as improved hand hygiene, social distancing, and restricted hospital referral, may have had an influence on the epidemiology of Clostridioides difficile infections (CDI)., Methods: The annual CDI incidence rate of nine hospitals participating in the Dutch sentinel CI surveillance with complete data was compared between 2020 and the previous five surveillance years. Trends in characteristics of hospitalised CDI patients in 21-24 participating hospitals were compared between the first (March 13-May 12, 2020) or second Dutch COVID-19 wave (September 17, 2020-January 1, 2021) and the same calendar periods in 2015 through 2019. All analyses were adjusted for trend changes over time., Findings: The annual CDI incidence rate in 2020 was lower compared to previous years. During the second wave, the percentage of CDI patients with severe CDI was higher compared to earlier (25·8% in 2020 vs 17·9% in 2015-2019 (RR 1·6; 95%CI 1·1-2·3)). After adjustment for delayed C. difficile diagnostics (≥8 days from start symptoms), the increase disappeared. Delayed C. difficile diagnostics was indeed more common during the second wave (RR 1·7; 95%CI 1·1-2·6), but only for community-onset CDI (CO-CDI)., Interpretation: This study shows that a higher percentage of severe CDI cases was observed during the second COVID-19 wave. This may partially be caused by delayed diagnostics, potentially due to decreased visits to a physician or restricted hospital referral for CO-CDI patients., Funding: Dutch ministry of Health., Competing Interests: We declare that we have no conflicts of interest., (© 2022 The Authors.)

Background: Colistin is a last-resort treatment option for infections with multidrug-resistant Gram-negative bacteria. However, colistin resistance is increasing., Methods: A six-month prospective matched case-control study was performed in which 22 Dutch laboratories with 32 associated hospitals participated. Laboratories were invited to send a maximum of five colistin-resistant Escherichia coli or Klebsiella pneumoniae (COLR-EK) isolates and five colistin-susceptible isolates (COLS-EK) to the reference laboratory, matched for patient location, material of origin and bacterial species. Epidemiological/clinical data were collected and included in the analysis. Characteristics of COLR-EK/COLS-EK isolates were compared using logistic regression with correction for variables used for matching. Forty-six ColR-EK/ColS-EK pairs were analysed by next-generation sequencing (NGS) for whole-genome multi-locus sequence typing and identification of resistance genes, including mcr genes. To identify chromosomal mutations potentially leading to colistin resistance, NGS reads were mapped against gene sequences of pmrAB, phoPQ, mgrB and crrB ., Results: In total, 72 COLR-EK/COLS-EK pairs (75% E. coli and 25% K. pneumoniae ) were included. Twenty-one percent of COLR-EK patients had received colistin, in contrast to 3% of COLS-EK patients (OR > 2.9). Of COLR-EK isolates, five contained mcr-1 and two mcr-9 . One isolate lost mcr-9 after repeated sub-culturing, but retained colistin resistance. Among 46 sequenced COLR-EK isolates, genetic diversity was large and 19 (41.3%) isolates had chromosomal mutations potentially associated with colistin resistance., Conclusions: Colistin resistance is present but uncommon in the Netherlands and caused by the mcr gene in a minority of COLR-EK isolates. There is a need for surveillance of colistin resistance using appropriate susceptibility testing methods., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2022.)

Purpose: To assess errors associated with EPI-accelerated intracardiac 4D flow MRI (4DEPI) with EPI factor 5, compared with non-EPI gradient echo (4DGRE)., Methods: Three 3T MRI experiments were performed comparing 4DEPI to 4DGRE: steady flow through straight tubes, pulsatile flow in a left-ventricle phantom, and intracardiac flow in 10 healthy volunteers. For each experiment, 4DEPI was repeated with readout and blip phase-encoding gradient in different orientations, parallel or perpendicular to the flow direction. In vitro flow rates were compared with timed volumetric collection. In the left-ventricle phantom and in vivo, voxel-based speed and spatio-temporal median speed were compared between sequences, as well as mitral and aortic transvalvular net forward volume., Results: In steady-flow phantoms, the flow rate error was largest (12%) for high velocity (>2 m/s) with 4DEPI readout gradient parallel to the flow. Voxel-based speed and median speed in the left-ventricle phantom were ≤5.5% different between sequences. In vivo, mean net forward volume inconsistency was largest (6.4 ± 8.5%) for 4DEPI with nonblip phase-encoding gradient parallel to the main flow. The difference in median speed for 4DEPI versus 4DGRE was largest (9%) when the 4DEPI readout gradient was parallel to the flow., Conclusions: Velocity and flow rate are inaccurate for 4DEPI with EPI factor 5 when flow is parallel to the readout or blip phase-encoding gradient. However, mean differences in flow rate, voxel-based speed, and spatio-temporal median speed were acceptable (≤10%) when comparing 4DEPI to 4DGRE for intracardiac flow in healthy volunteers., (© 2021 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Purpose: The primary objective is to determine the minimal ablation margin required to achieve a local recurrence rate of < 10% in patients with hepatocellular carcinoma undergoing thermal ablation. Secondary objectives are to analyze the correlation between ablation margins and local recurrence and to assess efficacy., Materials and Methods: This study is a prospective, multicenter, non-experimental, non-comparative, open-label study. Patients > 18 years with Barcelona Clinic Liver Cancer stage 0/A hepatocellular carcinoma (or B with a maximum of two lesions < 5 cm each) are eligible. Patients will undergo dual-phase contrast-enhanced computed tomography directly before and after ablation. Ablation margins will be quantitatively assessed using co-registration software, blinding assessors (i.e. two experienced radiologists) for outcome. Presence and location of recurrence are evaluated independently on follow-up scans by two other experienced radiologists, blinded for the quantitative margin analysis. A sample size of 189 tumors (~ 145 patients) is required to show with 80% power that the risk of local recurrence is confidently below 10%. A two-sided binomial z-test will be used to test the null hypothesis that the local recurrence rate is ≥ 10% for patients with a minimal ablation margin ≥ 2 mm. Logistic regression will be used to find the relationship between minimal ablation margins and local recurrence. Kaplan-Meier estimates are used to assess local and overall recurrence, disease-free and overall survival., Discussion: It is expected that this study will result in a clear understanding of the correlation between ablation margins and local recurrence. Using co-registration software in future patients undergoing ablation for hepatocellular carcinoma may improve intraprocedural evaluation of technical success. Trial registration The Netherlands Trial Register (NL9713), https://www.trialregister.nl/trial/9713 ., (© 2022. The Author(s).)

When a ranking of institutions such as medical centers or universities is based on a numerical measure of performance provided with a standard error, confidence intervals (CIs) should be calculated to assess the uncertainty of these ranks. We present a novel method based on Tukey's honest significant difference test to construct simultaneous CIs for the true ranks. When all the true performances are equal, the probability of coverage of our method attains the nominal level. In case the true performance measures have no exact ties, our method is conservative. For this situation, we propose a rescaling method to the nominal level that results in shorter CIs while keeping control of the simultaneous coverage. We also show that a similar rescaling can be applied to correct a recently proposed Monte-Carlo based method, which is anticonservative. After rescaling, the two methods perform very similarly. However, the rescaling of the Monte-Carlo based method is computationally much more demanding and becomes infeasible when the number of institutions is larger than 30-50. We discuss another recently proposed method similar to ours based on simultaneous CIs for the true performance. We show that our method provides uniformly shorter CIs for the same confidence level. We illustrate the superiority of our new methods with a data analysis for travel time to work in the United States and on rankings of 64 hospitals in the Netherlands., (© 2020 The International Biometric Society.)

Introduction: Vascular anatomy might affect endovascular treatment success in acute ischemic stroke patients with large vessel occlusion. We investigated the prognostic value of extracranial vascular characteristics on procedural time and revascularization success in patients with large vessel occlusion in the anterior cerebral circulation., Patients and Methods: We included 828 patients endovascularly treated within 6.5 hours of symptom onset from the Dutch MR CLEAN-Registry. We evaluated aortic arch configuration, stenosis and tortuosity of supra-aortic arteries, and internal carotid arteries (ICAs) on pre-intervention CTA. We constructed logistic prediction models for outcome variables procedural duration (≥60 minutes) and non-successful revascularization (extended thrombolysis in cerebral infarction (eTICI) of 0-2A) using baseline characteristics and assessed the effect of extracranial vascular characteristics on model performance., Results: Cervical ICA tortuosity and stenosis ≥99% improved prediction of long procedural duration compared with baseline characteristics from area under the curve of 0.61 (95% CI: 0.57-0.65) to 0.66 (95% CI: 0.62-0.70) ( P < 0.001). Cervical ICA tortuosity was significantly associated with non-successful recanalization. Prediction of non-successful revascularization did not improve after including aortic arch elongation, acute take-off angle, aortic variant, origin stenosis of supra-aortic arteries, and cervical ICA tortuosity, with an area under the curve of 0.63 (95% CI: 0.59-0.67) compared with 0.59 (95% CI: 0.55-0.63) ( P = 0.11)., Conclusion: Extracranial vascular characteristics have additional prognostic value for procedural duration, but not for revascularization success, compared with baseline characteristics. Performance of both prediction models is limited in patients treated for large vessel occlusion., Competing Interests: Declaration of Conflicting Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: AL received grants from: Cerenovus, Penumbra, Stryker, Medtronic, Dutch Heart Foundation, The Netherlands Organisation for Health Research and Development, Health Holland Top Sector Life Sciences & Health and from Dutch Brain Foundation. CBLMM received grants during the conduct of the study (paid to institution) from TWIN Foundation and outside of the submitted work (paid to institution) from CVON/Dutch Heart Foundation, European Commission, Dutch Health Evaluation Program and from Stryker; and is shareholder of Nico.Lab. HAM is co-founder and shareholder of Nico.Lab. GH, MPMES, TD, NDK, JJG, WJS, GJLN, LSFY, FJAM, MJHW and MAAW declared no conflict interest., (© European Stroke Organisation 2022.)

Background: Common and plantar warts are caused by human papillomaviruses (HPV). Mode of transmission of wart HPVs within families is largely unknown., Objective: To demonstrate similarity of HPV type(s) among wart cases, family members and household linen., Methods: In a cross-sectional study, swabs taken from 123 warts and foreheads of 62 index patients and 157 family members and from 58 kitchen towels and 59 bathroom mats were tested for DNA of 23 cutaneous wart-associated HPV types. Generalized estimating equations (GEE) were used to estimate the chance of detecting the same HPV type as was found in the index patients on the family contacts and on the kitchen towels and bathroom mats., Results: HPV1, HPV2, HPV27 and HPV57 were the most prevalent types in the warts of the index patients. Altogether, 60 (42.3%) of the 142 family members without warts had HPV DNA on their foreheads. When HPV1 and HPV2 were found in the warts, these types were also frequently (>50%) found on the foreheads of index patients and their family members, as well as on the kitchen towels and the bathroom mats. HPV27 and HPV57 were less frequently found (<25%) on foreheads and linen. No associations were found for age, sex and site of HPV DNA presence., Conclusion: Dissemination of skin wart-causing HPV types, from wart cases to household contacts and linen, such as kitchen towels and bathroom mats, is more likely for HPV1 and HPV2 than for HPV27 and HPV57. The role of towels and bathroom mats in HPV transmission deserves further investigation., (© 2021 The Authors. Journal of the European Academy of Dermatology and Venereology published by John Wiley & Sons Ltd on behalf of European Academy of Dermatology and Venereology.)

Research Objective: To investigate the correlation between clinical features and MRI-confirmed endolymphatic hydrops (EH) and blood-labyrinth barrier (BLB) impairment. Study Design: Retrospective cross-sectional study. Setting: Vertigo referral center (Haga Teaching Hospital, The Hague, the Netherlands). Methods: We retrospectively analyzed all patients that underwent 4 h-delayed Gd-enhanced 3D FLAIR MRI at our institution from February 2017 to March 2019. Perilymphatic enhancement and the degree of cochlear and vestibular hydrops were assessed. The signal intensity ratio (SIR) was calculated by region of interest analysis. Correlations between MRI findings and clinical features were evaluated. Results: Two hundred and fifteen patients with MRI-proven endolymphatic hydrops (EH) were included (179 unilateral, 36 bilateral) with a mean age of 55.9 yrs and median disease duration of 4.3 yrs. Hydrops grade is significantly correlated with disease duration ( P < 0.001), the severity of low- and high-frequency hearing loss (both P < 0.001), and the incidence of drop attacks ( P = 0.001). Visually increased perilymphatic enhancement was present in 157 (87.7%) subjects with unilateral EH. SIR increases in correlation with hydrops grade ( P < 0.001), but is not significantly correlated with the low or high Fletcher index ( P = 0.344 and P = 0.178 respectively). No significant differences were found between the degree of EH or BLB impairment and vertigo, tinnitus or aural fullness. Conclusion: The degree of EH positively correlates with disease duration, hearing loss and the incidence of drop attacks. The BLB is impaired in association with EH grade, but without clear contribution to the severity of audiovestibular symptoms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 de Pont, van Steekelenburg, Verhagen, Houben, Goeman, Verbist, van Buchem, Bommeljé, Blom and Hammer.)

Introduction: Meningoencephalitis patients are often severely impaired and benefit from early etiological diagnosis, though many cases remain without identified cause. Metagenomics as pathogen agnostic approach can result in additional etiological findings; however, the exact diagnostic yield when used as a secondary test remains unknown., Areas Covered: This review aims to highlight recent advances with regard to wet and dry lab methodologies of metagenomic testing and technical milestones that have been achieved. A selection of procedures currently applied in accredited diagnostic laboratories is described in more detail to illustrate best practices. Furthermore, a meta-analysis was performed to assess the additional diagnostic yield utilizing metagenomic sequencing in meningoencephalitis patients. Finally, the remaining challenges for successful widespread implementation of metagenomic sequencing for the diagnosis of meningoencephalitis are addressed in a future perspective., Expert Opinion: The last decade has shown major advances in technical possibilities for using mNGS in diagnostic settings including cloud-based analysis. An additional advance may be the current established infrastructure of platforms for bioinformatic analysis of SARS-CoV-2, which may assist to pave the way for global use of clinical metagenomics.

Motivation: Volcano plots are used to select the most interesting discoveries when too many discoveries remain after application of Benjamini-Hochberg's procedure (BH). The volcano plot suggests a double filtering procedure that selects features with both small adjusted $P$-value and large estimated effect size. Despite its popularity, this type of selection overlooks the fact that BH does not guarantee error control over filtered subsets of discoveries. Therefore the selected subset of features may include an inflated number of false discoveries., Results: In this paper, we illustrate the substantially inflated type I error rate of volcano plot selection with simulation experiments and RNA-seq data. In particular, we show that the feature with the largest estimated effect is a very likely false positive result. Next, we investigate two alternative approaches for multiple testing with double filtering that do not inflate the false discovery rate. Our procedure is implemented in an interactive web application and is publicly available., (© The Author(s) 2021. Published by Oxford University Press.)

Objectives: This study determined: 1) the interobserver agreement; 2) valvular flow variation; and 3) which variables independently predicted the variation of valvular flow quantification from 4-dimensional (4D) flow cardiac magnetic resonance (CMR) with automated retrospective valve tracking at multiple sites., Background: Automated retrospective valve tracking in 4D flow CMR allows consistent assessment of valvular flow through all intracardiac valves. However, due to the variance of CMR scanners and protocols, it remains uncertain if the published consistency holds for other clinical centers., Methods: Seven sites each retrospectively or prospectively selected 20 subjects who underwent whole heart 4D flow CMR (64 patients and 76 healthy volunteers; aged 32 years [range 24 to 48 years], 47% men, from 2014 to 2020), which was acquired with locally used CMR scanners (scanners from 3 vendors; 2 1.5-T and 5 3-T scanners) and protocols. Automated retrospective valve tracking was locally performed at each site to quantify the valvular flow and repeated by 1 central site. Interobserver agreement was evaluated with intraclass correlation coefficients (ICCs). Net forward volume (NFV) consistency among the valves was evaluated by calculating the intervalvular variation. Multiple regression analysis was performed to assess the predicting effect of local CMR scanners and protocols on the intervalvular inconsistency., Results: The interobserver analysis demonstrated strong-to-excellent agreement for NFV (ICC: 0.85 to 0.96) and moderate-to-excellent agreement for regurgitation fraction (ICC: 0.53 to 0.97) for all sites and valves. In addition, all observers established a low intervalvular variation (≤10.5%) in their analysis. The availability of 2 cine images per valve for valve tracking compared with 1 cine image predicted a decreasing variation in NFV among the 4 valves (beta = -1.3; p = 0.01)., Conclusions: Independently of locally used CMR scanners and protocols, valvular flow quantification can be performed consistently with automated retrospective valve tracking in 4D flow CMR., Competing Interests: Funding Support And Author Disclosures Drs. Zhang and Zhong were supported by the National Medical Research Council (NMRC/OFIRG/0018/2016; NMRC/BnB/0017/2015; MOH-000358; MOH-000351) and SingHealth Duke-NUS Academic Medicine Research Grants (AM/TP015/2018 (SRDUKAMR1814); AM/TP032/2020 (SRDUKAMR2032). All other authors have reported that they have no relationships relevant to the content of this paper to disclose., (Copyright © 2021 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: To evaluate longitudinal variations in diffusion tensor imaging (DTI) metrics of different white matter (WM) tracts of newly diagnosed SLE patients, and to assess whether DTI changes relate to changes in clinical characteristics over time., Methods: A total of 17 newly diagnosed SLE patients (19-55 years) were assessed within 24 months from diagnosis with brain MRI (1.5 T Philips Achieva) at baseline, and after at least 12 months. Fractional anisotropy, mean diffusivity (MD), radial diffusivity (RD) and axial diffusivity values were calculated in several normal-appearing WM tracts. Longitudinal variations in DTI metrics were analysed by repeated measures analysis of variance. DTI changes were separately assessed for 21 WM tracts. Associations between longitudinal alterations of DTI metrics and clinical variables (SLEDAI-2K, complement levels, glucocorticoid dosage) were evaluated using adjusted Spearman correlation analysis., Results: Mean MD and RD values from the normal-appearing WM significantly increased over time (P = 0.019 and P = 0.021, respectively). A significant increase in RD (P = 0.005) and MD (P = 0.012) was found in the left posterior limb of the internal capsule; RD significantly increased in the left retro-lenticular part of the internal capsule (P = 0.013), and fractional anisotropy significantly decreased in the left corticospinal tract (P = 0.029). No significant correlation was found between the longitudinal change in DTI metrics and the change in clinical measures., Conclusion: Increase in diffusivity, reflecting a compromised WM tissue microstructure, starts in initial phases of the SLE disease course, even in the absence of overt neuropsychiatric (NP) symptoms. These results indicate the importance of monitoring NP involvement in SLE, even shortly after diagnosis., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

We propose a top-down approach for pathway analysis of longitudinal metabolite data. We apply a score test based on a shared latent process mixed model which can identify pathways with differentially progressing metabolites. The strength of our approach is that it can handle unbalanced designs, deals with potential missing values in the longitudinal markers, and gives valid results even with small sample sizes. Contrary to bottom-up approaches, correlations between metabolites are explicitly modeled leveraging power gains. For large pathway sizes, a computationally efficient solution is proposed based on pseudo-likelihood methodology. We demonstrate the advantages of the proposed method in identification of differentially expressed pathways through simulation studies. Finally, longitudinal metabolite data from a mice experiment is analyzed to demonstrate our methodology., (© 2021 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd.)

Controlled human infections provide opportunities to study the interaction between the immune system and malaria parasites, which is essential for vaccine development. Here, we compared immune signatures of malaria-naive Europeans and of Africans with lifelong malaria exposure using mass cytometry, RNA sequencing and data integration, before and 5 and 11 days after venous inoculation with Plasmodium falciparum sporozoites. We observed differences in immune cell populations, antigen-specific responses and gene expression profiles between Europeans and Africans and among Africans with differing degrees of immunity. Before inoculation, an activated/differentiated state of both innate and adaptive cells, including elevated CD161 + CD4 + T cells and interferon-γ production, predicted Africans capable of controlling parasitemia. After inoculation, the rapidity of the transcriptional response and clusters of CD4 + T cells, plasmacytoid dendritic cells and innate T cells were among the features distinguishing Africans capable of controlling parasitemia from susceptible individuals. These findings can guide the development of a vaccine effective in malaria-endemic regions.

The estimated accuracy of a classifier is a random quantity with variability. A common practice in supervised machine learning, is thus to test if the estimated accuracy is significantly better than chance level. This method of signal detection is particularly popular in neuroimaging and genetics. We provide evidence that using a classifier's accuracy as a test statistic can be an underpowered strategy for finding differences between populations, compared to a bona fide statistical test. It is also computationally more demanding than a statistical test. Via simulation, we compare test statistics that are based on classification accuracy, to others based on multivariate test statistics. We find that the probability of detecting differences between two distributions is lower for accuracy-based statistics. We examine several candidate causes for the low power of accuracy-tests. These causes include: the discrete nature of the accuracy-test statistic, the type of signal accuracy-tests are designed to detect, their inefficient use of the data, and their suboptimal regularization. When the purpose of the analysis is the evaluation of a particular classifier, not signal detection, we suggest several improvements to increase power. In particular, to replace V-fold cross-validation with the Leave-One-Out Bootstrap., (© The Author 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Long scan times prohibit a widespread clinical applicability of 4D flow MRI in Fontan patients. As pulsatility in the Fontan pathway is minimal during the cardiac cycle, acquiring non-ECG gated 3D flow MRI may result in a reduction of scan time while accurately obtaining time-averaged clinical parameters in comparison with 2D and 4D flow MRI. Thirty-two Fontan patients prospectively underwent 2D (reference), 3D and 4D flow MRI of the Fontan pathway. Multiple clinical parameters were assessed from time-averaged flow rates, including the right-to-left pulmonary flow distribution (main endpoint) and systemic-to-pulmonary collateral flow (SPCF). A ten-fold reduction in scan time was achieved [4D flow 15.9 min (SD 2.7 min) and 3D flow 1.6 min (SD 7.8 s), p < 0.001] with a superior signal-to-noise ratio [mean ratio of SNRs 1.7 (0.8), p < 0.001] and vessel sharpness [mean ratio 1.2 (0.4), p = 0.01] with 3D flow. Compared to 2D flow, good-excellent agreement was shown for mean flow rates (ICC 0.82-0.96) and right-to-left pulmonary flow distribution (ICC 0.97). SPCF derived from 3D flow showed good agreement with that from 4D flow (ICC 0.86). 3D flow MRI allows for obtaining time-averaged flow rates and derived clinical parameters in the Fontan pathway with good-excellent agreement with 2D and 4D flow, but with a tenfold reduction in scan time and significantly improved image quality compared to 4D flow.

Non-inferiority in the anamnestic antibody response is conventionally determined by comparing seroconversion rates after revaccination. However, this approach is inadequate in the case of high pre-booster antibody titers. Therefore, we propose an alternative method to determine non-inferiority of booster responses. We used anonymized data from a randomized controlled trial (NCT01388985; EudraCT 2011-001612-62) in 500 adults, comparing a two-visit primary vaccination schedule (two intradermal 0.1 mL rabies vaccine doses on day 0 and 7) with a three-visit schedule (single intradermal 0.1 mL dose on day 0, 7, and 28). Participants were revaccinated intradermally (single dose) 1 to 3 years later. Rabies virus neutralizing antibody titers were measured on day 0 and 7 after revaccination. After log 3 -transformation of antibody titers, the mean increase in titers after revaccination was compared between schedules. Non-inferiority was defined as the lower bound of the two-sided 95% confidence interval not exceeding -0.369. Four hundred and ten participants fulfilled the inclusion criteria. The mean increase in log 3 titer was 2.21 and 2.31 for the two-visit and three-visit schedule, respectively. The difference between these increases was -0.10 [-0.28, 0.08], meeting the non-inferiority criterion. In conclusion, comparing mean increases in log-transformed titers after revaccination appears to be a feasible and more informative method of studying non-inferiority regarding the anamnestic antibody response.

Hookworms are soil-transmitted helminths that use immune-evasive strategies to persist in the human duodenum where they are responsible for anemia and protein loss. Given their location and immune regulatory effects, hookworms likely impact the bacterial microbiota. However, microbiota studies struggle to deconvolute the effect of hookworms from confounders such as coinfections and malnutrition. We thus used an experimental human hookworm infection model to explore temporal changes in the gut microbiota before and during hookworm infection. Volunteers were dermally exposed to cumulative dosages of 50, 100 or 150 L3 Necator americanus larvae. Fecal samples were collected for microbiota profiling through 16S rRNA gene amplicon sequencing at weeks zero, four, eight, fourteen and twenty. During the acute infection phase (trial week zero to eight) no changes in bacterial diversity were detected. During the established infection phase (trial week eight to twenty), bacterial richness (Chao1, p = .0174) increased significantly over all volunteers. No relation was found between larval dosage and diversity, stability or relative abundance of individual bacterial taxa. GI symptoms were associated with an unstable microbiota during the first eight weeks and rapid recovery at week twenty. Barnesiella , amongst other taxa, was more abundant in volunteers with more GI symptoms throughout the study. In conclusion, this study showed that clinical GI symptoms following N. americanus infection are associated with temporary microbiota instability and relative abundance of specific bacterial taxa. These results suggest a possible role of hookworm-induced enteritis on microbiota stability.

Data analysis workflows in many scientific domains have become increasingly complex and flexible. Here we assess the effect of this flexibility on the results of functional magnetic resonance imaging by asking 70 independent teams to analyse the same dataset, testing the same 9 ex-ante hypotheses 1 . The flexibility of analytical approaches is exemplified by the fact that no two teams chose identical workflows to analyse the data. This flexibility resulted in sizeable variation in the results of hypothesis tests, even for teams whose statistical maps were highly correlated at intermediate stages of the analysis pipeline. Variation in reported results was related to several aspects of analysis methodology. Notably, a meta-analytical approach that aggregated information across teams yielded a significant consensus in activated regions. Furthermore, prediction markets of researchers in the field revealed an overestimation of the likelihood of significant findings, even by researchers with direct knowledge of the dataset 2-5 . Our findings show that analytical flexibility can have substantial effects on scientific conclusions, and identify factors that may be related to variability in the analysis of functional magnetic resonance imaging. The results emphasize the importance of validating and sharing complex analysis workflows, and demonstrate the need for performing and reporting multiple analyses of the same data. Potential approaches that could be used to mitigate issues related to analytical variability are discussed.

In this article, we introduce a novel procedure for improving power of multiple testing procedures (MTPs) of interval hypotheses. When testing interval hypotheses the null hypothesis $P$-values tend to be stochastically larger than standard uniform if the true parameter is in the interior of the null hypothesis. The new procedure starts with a set of $P$-values and discards those with values above a certain pre-selected threshold, while the rest are corrected (scaled-up) by the value of the threshold. Subsequently, a chosen family-wise error rate (FWER) or false discovery rate MTP is applied to the set of corrected $P$-values only. We prove the general validity of this procedure under independence of $P$-values, and for the special case of the Bonferroni method, we formulate several sufficient conditions for the control of the FWER. It is demonstrated that this "filtering" of $P$-values can yield considerable gains of power., (© The Author 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The treatment of choice for cutaneous squamous cell carcinoma is complete surgical excision. Incomplete excision of cutaneous squamous cell carcinoma has an increased risk of local recurrence, deep subclinical progression, and metastasis. This study aimed to investigate the proportion and risk factors of incomplete excised cutaneous squamous cell carcinoma. A systematic review of the literature was performed. Incomplete excision rates for cutaneous squamous cell carcinoma ranged from 0.4% to 35.7%. The pooled incomplete excision risk estimate was 13% (95% confidence interval 9-17%). Risk factors noted in more than one study for incomplete excision included tumor depth and size, type of operator, head and neck localization, and former incomplete excision. We found an overall incomplete excision rate of 13% for cutaneous squamous cell carcinoma. Risk factors should be taken into account in the management of cutaneous squamous cell carcinoma surgical treatment.

Early diagnosis of leprosy is challenging, particularly its inflammatory reactions, the major cause of irreversible neuropathy in leprosy. Current diagnostics cannot identify which patients are at risk of developing reactions. This study assessed blood RNA expression levels as potential biomarkers for leprosy. Prospective cohorts of newly diagnosed leprosy patients, including reactions, and healthy controls were recruited in Bangladesh, Brazil, Ethiopia and Nepal. RNA expression in 1,090 whole blood samples was determined for 103 target genes for innate and adaptive immune profiling by dual color Reverse-Transcription Multiplex Ligation-dependent Probe Amplification (dcRT-MLPA) followed by cluster analysis. We identified transcriptomic biomarkers associated with leprosy disease, different leprosy phenotypes as well as high exposure to Mycobacterium leprae which respectively allow improved diagnosis and classification of leprosy patients and detection of infection. Importantly, a transcriptomic signature of risk for reversal reactions consisting of five genes (CCL2, CD8A, IL2, IL15 and MARCO) was identified based on cross-sectional comparison of RNA expression. In addition, intra-individual longitudinal analyses of leprosy patients before, during and after treatment of reversal reactions, indicated that several IFN-induced genes increased significantly at onset of reaction whereas IL15 decreased. This multi-site study, situated in four leprosy endemic areas, demonstrates the potential of host transcriptomic biomarkers as correlates of risk for leprosy. Importantly, a prospective five-gene signature for reversal reactions could predict reversal reactions at least 2 weeks before onset. Thus, transcriptomic biomarkers provide promise for early detection of these acute inflammatory episodes and thereby help prevent permanent neuropathy and disability in leprosy patients.