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Abstract The EFSA Panel on Food Contact Materials (FCM) assessed the safety of the substances ‘wax, rice bran, oxidised’ and ‘wax, rice bran, oxidised, calcium salt’, used as additives up to 0.3% in polyethylene terephthalate (PET), polyamide (PA), thermoplastic polyurethane (TPU), polylactic acid (PLA) and poly(vinyl chloride) (PVC) in contact with all food types for long‐term storage at room temperature and below, after hot‐fill and/or heating. The substances consist of the chemical classes wax esters, carboxylic acids, alcohols and calcium salts of acids, along with an unidentified organic fraction up to ■■■■■ w/w. Migration into 10% ethanol and 4% acetic acid was below 0.012 mg/kg for each chemical class, and about 0.001 mg/kg for the unidentified fraction. In isooctane, migration was up to 0.297 mg/kg food for wax esters, below 0.01 mg/kg food for the other chemical classes and about 0.02 mg/kg food for the unidentified fraction. The contact with dry food and food simulated by 20% ethanol were considered covered by the migration tests with aqueous simulants. Based on genotoxicity assays and compositional analyses, the constituents of the chemical classes did not raise a concern for genotoxicity. The potential migration of individual constituents or groups of chemically‐related compounds of the unidentified fraction would result in exposures below (for aqueous food) and above (for fatty food) the threshold of toxicological concern for genotoxic carcinogens. Therefore, the FCM Panel concluded that the substances are not of safety concern for the consumer, if used as additives up to 0.3% w/w in PET, PLA and rigid PVC materials and articles intended for contact with all food types except for fatty foods, for long‐term storage at room temperature and below, including hot‐fill and/or heating up to 100°C for up to 2 h.

Background This cross-sectional study investigated quality of life (QoL) and illness-related parental stress in children with kidney diseases by (1) comparing mean levels of these two variables between several kidney disease categories; (2) exploring correlations between QoL and parental stress; and (3) describing which disease category reports lowest QoL and highest parental stress. Methods We included 295 patients with a kidney disease (0-18 years) and their parents, followed at 6 reference centers for pediatric nephrology. Children's QoL was assessed by the PedsQL[TM] 4.0 Generic Core Scales, and illness-related stress by the Pediatric Inventory for Parents. All patients were divided into 5 kidney disease categories according to the multidisciplinary care program criteria prescribed by the Belgian authorities: (1) structural kidney diseases, (2) tubulopathies and metabolic diseases, (3) nephrotic syndrome, (4) acquired diseases with proteinuria and hypertension, and (5) kidney transplantation. Results Child self-reports showed no differences in QoL between kidney disease categories, in contrast to parent proxy reports. Parents of transplant patients reported lower QoL in their child and more parental stress compared with the 4 non-transplant categories. QoL and parental stress were negatively correlated. Lowest QoL and highest parental stress scores were mainly found in transplant patients. Conclusions This study showed lower QoL and higher parental stress in pediatric transplant patients compared with non-transplants, based on parent reports. Higher parental stress is associated with worse QoL in the child. These results highlight the importance of multidisciplinary care for children with kidney diseases, with special attention to transplant patients and their parents. Graphical abstract, Author(s): Elke De Bruyne [sup.1] , Lore Willem [sup.2] , Koen Van Hoeck [sup.3] , Sarah Reynaert [sup.3] , Sylvie Vankerckhove [sup.4] , Brigitte Adams [sup.4] , Stéphanie Leroi [sup.5] [...]

To promote improved trial design in upcoming randomized clinical trials in childhood chronic kidney disease (CKD), insight in the within- and inter-patient variability of uremic toxins with its nutritional, treatment- and patient-related confounding factors is of utmost importance. In this study, the within- and inter-patient variability of a selection of uremic toxins in a longitudinal cohort of children diagnosed with CKD was assessed, using the intraclass correlation coefficient (ICC) and the within-patient coefficient of variation (CV). Subsequently, the contribution of anthropometry, estimated glomerular filtration rate (eGFR), dietary fiber and protein, and use of (prophylactic) antibiotics to uremic toxin variability was evaluated. Based on 403 observations from 62 children (median seven visits per patient; 9.4 ± 5.3 years; 68% males; eGFR 38.5 [23.1; 64.0] mL/min/1.73 m2) collected over a maximum of 2 years, we found that the within-patient variability is high for especially protein-bound uremic toxins (PBUTs) (ICC < 0.7; within-patient CV 37–67%). Moreover, eGFR was identified as a predominant contributor to the within- and inter-patient variability for the majority of solutes, while the impact of the child’s anthropometry, fiber and protein intake, and antibiotics on the variability of uremic toxin concentrations was limited. Based on these findings, we would recommend future intervention studies that attempt to decrease uremic toxin levels to select a (non-dialysis) CKD study population with a narrow eGFR range. As the expected effect of the selected intervention should exceed the inter-patient variability of the selected uremic toxins, a narrow eGFR range might aid in improving the trial design.

Background The Flemish Collaborative Glomerulonephritis Group (FCGG) registry is a population-based kidney biopsy registry that has been including all native kidney biopsies performed in children in Flanders (Belgium), since 2017. Methods From 2017 to 2020, 148 pediatric ( 5 glomeruli. Results Kidney biopsy rate was high (29.0 biopsies per million children per year), median age was 10.0 years (IQR 5.8-14.7), and boys predominated (56.1% males). A total of 140 biopsies (94.6%) showed a representative pathology result. Glomerular disease was most prevalent, with IgA nephropathy/IgA vasculitis (43 biopsies, 29.1%) and minimal change disease (MCD) (29 biopsies, 19.6%) being the overall most frequent diagnoses. In general, diagnostic concordance was high (80.7%). In Alport syndrome and focal segmental glomerulosclerosis (FSGS), concordance was lower, as the nephrological diagnosis was often determined by results of genetic analysis. Nephrotic syndrome was the most frequent indication for kidney biopsy (31.8%) and was mainly caused by MCD and FSGS. The degree of disease chronicity on kidney biopsies was generally low, although 27.3% of biopsies with a diagnosis of FSGS showed moderate-to-severe chronic damage. Conclusions The presented epidemiological findings validate data from previous European registry studies and may inspire kidney biopsy registries worldwide to implement novel features such as clinicopathological concordance and chronicity grading. Graphical abstract, Author(s): Dries Deleersnijder [sup.1] [sup.2] , Noël Knops [sup.3] [sup.4] , Dominique Trouet [sup.5] [sup.6] , Koen Van Hoeck [sup.6] , Sevasti Karamaria [sup.7] [sup.8] , Johan Vande Walle [sup.7] [...]

Abstract DNA methylation is important for establishing and maintaining cell identity and for genomic stability. This is achieved by regulating the accessibility of regulatory and transcriptional elements and the compaction of subtelomeric, centromeric, and other inactive genomic regions. Carcinogenesis is accompanied by a global loss in DNA methylation, which facilitates the transformation of cells. Cancer hypomethylation may also cause genomic instability, for example through interference with the protective function of telomeres and centromeres. However, understanding the role(s) of hypomethylation in tumor evolution is incomplete because the precise mutational consequences of global hypomethylation have thus far not been systematically assessed. Here we made genome-wide inventories of all possible genetic variation that accumulates in single cells upon the long-term global hypomethylation by CRISPR interference-mediated conditional knockdown of DNMT1. Depletion of DNMT1 resulted in a genomewide reduction in DNA methylation. The degree of DNA methylation loss was similar to that observed in many cancer types. Hypomethylated cells showed reduced proliferation rates, increased transcription of genes, reactivation of the inactive X-chromosome and abnormal nuclear morphologies. Prolonged hypomethylation was accompanied by increased chromosomal instability. However, there was no increase in mutational burden, enrichment for certain mutational signatures or accumulation of structural variation to the genome. In conclusion, the primary consequence of hypomethylation is genomic instability, which in cancer leads to increased tumor heterogeneity and thereby fuels cancer evolution.

Since the ban on single-use plastic articles in Europe, the food contact material (FCM) industry has been forced to move to more sustainable alternatives. Paper and board FCM are convenient alternatives but must be safe for consumers. This study aims to investigate potential migrations of various substances (e.g., plasticizers, photoinitiators, primary aromatic amines, mineral oil, and bisphenols) from straws and takeaway articles made of paper and board. Twenty straws and fifty-eight takeaway articles were carefully selected and investigated using liquid and gas chromatography coupled with tandem mass spectrometry or flame ionization detector. Fourteen substances of all the targeted categories were found in takeaway articles, including seven plasticizers, two photoinitiators, one primary aromatic amine, two bisphenols, and the saturated and aromatic fraction of mineral oil (MOSH and MOAH, respectively). In straws, fewer substances were detected, i.e., six substances, including three plasticizers, one photoinitiator, MOSH, and MOAH. At least one of the target substances was detected in 88% of the samples, demonstrating the importance of further evaluation of these materials. Finally, the associated risks were assessed, highlighting the potential risks for several types of articles regarding bisphenol A, one primary aromatic amine (3.3-DMB), and MOSH and MOAH.

Specific anti-cancer therapies are highly mutagenic to cancer cells but the mutational impact on healthy tissues remains elusive. Here, the authors use organoids and whole-genome sequencing to characterise somatic mutations in healthy colon and liver adult stem cells after chemo- or radiotherapy.

Background Fruit and vegetable intake is commonly discouraged in children with chronic kidney disease (CKD) to avoid hyperkalemia. However, direct evidence in support of this widespread practice is lacking. Furthermore, the resultant restricted fiber exposure may deprive CKD patients from potential health benefits associated with the latter. Therefore, we investigated associations between dietary potassium intake, fiber intake, and serum potassium levels in pediatric CKD. Methods This study is a longitudinal analysis of a 2-year, prospective, multi-institutional study, following children with CKD at 3-month intervals. At each visit, dietary potassium and fiber intake were assessed, using 24-h recalls and 3-day food records. On the same occasion, serum potassium concentrations were determined. Associations between dietary potassium intake, dietary fiber intake, and serum potassium concentrations were determined using linear mixed models. Results Fifty-two CKD patients (7 transplant recipients, none on dialysis) aged 9 [4;14] years with an estimated glomerular filtration rate (eGFR) of 49 [25;68] mL/min/1.73 m.sup.2 were included. For every g/day decrease in dietary potassium intake, the estimated mean daily fiber intake was 5.1 g lower (95% confidence interval (CI), 4.3-5.9 g/day; p < 0.001). Neither dietary potassium intake (p = 0.40) nor dietary fiber intake (p = 0.43) was associated with circulating potassium in a model adjusted for time point, eGFR, treatment with a renin-angiotensin-aldosterone system blocker, serum bicarbonate concentration, and body surface area. Conclusions Dietary potassium and fiber intake are closely related but were not associated with circulating potassium levels in pediatric CKD. Graphical abstract A higher-resolution version of the graphical abstract is available as Supplementary information., Author(s): Amina El Amouri [sup.1] , Kato Delva [sup.2] , Aurélie Foulon [sup.2] , Charlotte Vande Moortel [sup.2] , Koen Van Hoeck [sup.3] , Griet Glorieux [sup.2] , Wim Van [...]

The original tumor location can be unclear for metastatic tumors. Here, the authors show that DNA sequencing of whole genomes can be used to classify metastatic tumors using a machine learning model, Cancer of Unknown Primary Location Resolver, in order to improve diagnosis and inform treatment decisions.

Objective: To explore the usefulness of the 'differential renal length index' (iDRL) before and after pyeloplasty, as the anteroposterior diameter is commonly used to quantify hydronephrosis but inaccuracies arise due to interobserver variability, hydration status and pure intra‐renal dilatation. Patients and Methods: Prospectively collected data, from two centres, of all children undergoing pyeloplasty for isolated unilateral pelvi‐ureteric junction obstruction (PUJO) (2015–2021) were analysed. Subgroup analysis was undertaken: Group A – differential renal function (DRF) ≥40%, Group B – subnormal DRF (20–39%), and Group C – symptomatic. Children with structural anomalies of upper and lower urinary tract, bilateral involvement, and subnormal DRF (<20%) were excluded. All the children had a pre‐ and postoperative ultrasound scan and Tc99m mercapto‐acetyltriglycine (MAG3) renograms. The iDRL was calculated as follows: iDRL = ([a – b]/b) × 100, where 'a' is the length of hydronephrotic kidney (cm) and 'b' is the length of contralateral normal kidney (cm). The mean difference and standard error of mean (SEM) between the pre‐ and postoperative iDRL was evaluated using the paired Student's t‐test, with P < 0.05 considered statistically significant. Results: A total of 119 children with 1‐year follow‐up were included. For the entire cohort, the mean (SEM) preoperative iDRL was 27.7 (1.4) and postoperatively was 12.5 (1.1), with a mean (range) DRF improvement of 54% (44–66%) (P < 0.001). In Group A (n = 97), the mean (SEM) preoperative iDRL was 26.6 (1.5) and postoperatively was 13.1 (1.2), with a mean (range) DRF improvement of 50% (38–63%) (P < 0.001). In Group B (n = 22), the mean (SEM) preoperative iDRL was 32.6 (3.5) and postoperatively was 10.0 (2.8), with a mean (range) DRF improvement of 69% (49–89%) (P < 0.001). In Group C (n = 28), the mean (SEM) preoperative iDRL was 19.9 (2.3) and postoperatively was 7.7 (1.9), with a mean (range) DRF improvement of 61% (38–85%) (P < 0.001). Conclusion: Our study identifies the iDRL as a useful measure of improvement following successful pyeloplasty. In the subgroup with DRF of >39% minimum improvement was >37%. Similar minimum DRF improvement was also noted (>37%) in hypo‐functioning kidneys and symptomatic PUJO. [ABSTRACT FROM AUTHOR]

Cereulide is an emetic toxin produced by some strains of Bacillus cereus. This bacterial toxin, a cyclic 1.2 kDa dodecadepsipeptide, is stable to heat and acids and causes nausea and vomiting when ingested via contaminated food. This work aimed to develop and validate a targeted analytical method applying liquid chromatography-tandem mass spectrometry (LC–MS/MS) to quantify this toxin in food and human faeces. Samples were extracted with acetonitrile in the presence of 13C6-cereulide, a labelled internal standard, and purified by centrifugation and filtration. The limits of quantification were 0.5 and 0.3 µg kg−1 for food and faeces, respectively. The linearity of the method was very good, with calculated R2 values above 0.995. The mean recovery of the method was within the acceptable range of 70.0%–120.0%, the repeatability was not higher than 7.3%, and the highest intra-laboratory reproducibility was 8.9%. The estimated range for the expanded measurement uncertainty was between 5.1% and 18.0%. The LC–MS/MS method was used to analyse one food sample (rice) from a Belgian foodborne outbreak and five faecal samples from patients with clinical symptoms after consumption of the contaminated rice. The levels of cereulide were 12.22 µg g−1 for food and between 6.32 and 773.37 ng g−1 for faecal samples.

Abstract Background The collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data. Results Here, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights. Conclusions This novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns .

The objective of the present study was to assess the dietary intake of steviol glycosides in the Belgian population and to conduct a risk assessment by comparing the estimated intakes to the acceptable daily intake (ADI). A tiered approach was adopted in this study. First, a Tier 2 assessment was performed using maximum permitted levels. Next, the calculations were refined because market share data were used (refined Tier 2). Finally, the actual concentration data of 198 samples purchased from the Belgian market were used for Tier 3 exposure assessment. Based on a Tier 2 assessment, the ADI was exceeded for the high-consumer children population. However, the results of a more refined exposure assessment (Tier 3) of high consumers (P95) within the child, adolescent and adult populations were 13·75, 10 and 6·25 % of the ADI, respectively, using mean analytical results. Even with more conservative refined approaches, the estimated daily intake remained below 20 % of ADI. Flavoured drinks, flavoured fermented milk products and jams, jellies, and marmalades were the top three contributing food groups to steviol intake at 26–49 %, 12–27 % and 5–13 %, respectively. Despite the high concentrations (up to 94 000 mg/kg) of steviol glycosides in tabletop sweeteners, their contribution to the total intake remains low. The impact of the use of food supplements on the total intake was also considered to be limited. It was concluded that there was no risk for the Belgian population related to dietary exposure to steviol glycoside.

Background: Steviol glycosides, constituents of Stevia rebaudiana Bertoni, are high-intensity sweeteners that enhance glucose-induced insulin secretion and decrease hyperglycemia in mice and rats. Given the extreme sweetness and bitter aftertaste of these compounds, the non-tasting aglycon steviol might be a promising alternative food additive that also harbors anti-hyperglycemic effects. Furthermore, it has been suggested that steviol glucuronide is the main bioactive metabolite of steviol and steviol glycosides that appears in blood. Aim: To investigate whether steviol and/or steviol glucuronide reduce hyperglycemia in a high-fat diet (HFD)-induced diabetic mouse model. Methods: Steviol was administered acute per os (p.o.), chronic via drinking water and intravenous (i.v.) to HFD-induced diabetic mice. The effect on glucose homeostasis was assessed using glucose tolerance tests (GTTs). Following chronic and i.v. steviol administration, steviol and steviol glucuronide plasma levels were determined. Steviol glucuronide was administered i.v. and the effect on glycemia was assessed with GTTs. Results: Acute and chronic oral steviol administration did not lower glycemia. This is due to the very limited and short-lived bioavailability of steviol in plasma. Only after high dosage i.v. steviol, glycemia was transiently reduced shortly after administration (Δ 15 min: 76.4 ± 30.9 mg dL−1, p = 0.035). In contrast, i.v. steviol glucuronide resulted in a 16.4 ± 5.9% lower AUCGlucose(0–120 min) compared to mice that received vehicle (p = 0.040). Moreover, steviol glucuronide enhanced the glucose-induced calcium oscillation frequency in insulin-secreting pancreatic islets. Conclusion: Our findings demonstrate that steviol is not a useful derivative of steviol glycosides to improve hyperglycemia in type 2 diabetes. On the other hand, steviol glucuronide has significant anti-hyperglycemic effects in living mice with type 2 diabetes.

The food contact materials (FCMs) industry is forced to develop substitute materials due to constant pressure from consumers and authorities to reduce fossil-based plastic. Several alternatives are available on the market. However, market share, trends, and consumer preferences are still unclear. Therefore, this study aims to provide an overview of the Belgian FCMs market, the available substitute materials, and their uses. The market analysis was performed with an integrated web-based approach. Fifty-two sources were investigated, covering e-shops selling materials intended to replace disposable plastic materials or being advertised as environmentally friendly and websites describing homemade FCMs. The first screening identified 10,523 articles. The following data cleaning process resulted in a homogeneous dataset containing 2688 unique entries, systematically categorised into fifteen material categories and seven utilisation classes. Paper and board was the most popular material category (i.e., 37% of the entries), followed by bagasse, accounting for 9% of the entries. Takeaway and food serving (44.4% and 22.8% of the entries) were the most common usage categories. The study pursued to provide insights into current trends and consumer preferences, highlighting priorities for safety assessment and future policy making.

The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.

Abstract GRIDSS2 is the first structural variant caller to explicitly report single breakends—breakpoints in which only one side can be unambiguously determined. By treating single breakends as a fundamental genomic rearrangement signal on par with breakpoints, GRIDSS2 can explain 47% of somatic centromere copy number changes using single breakends to non-centromere sequence. On a cohort of 3782 deeply sequenced metastatic cancers, GRIDSS2 achieves an unprecedented 3.1% false negative rate and 3.3% false discovery rate and identifies a novel 32–100 bp duplication signature. GRIDSS2 simplifies complex rearrangement interpretation through phasing of structural variants with 16% of somatic calls phasable using paired-end sequencing.