Abstract Background While genetics has been shown to direct wound healing responses toward healing or progressive fibrosis in liver and lung disease, attempts to identify genes involved in fibrostenotic Crohn’s disease (CD) have not been successful. This knowledge gap stems partly from the challenge to uniformly define fibrostenosis, for which the golden standard currently is surgery. We aimed to investigate the genetic basis of fibrostenotic CD by conducting a case-control genetic association study in a well-defined CD cohort, and assessing the presence of protein-coding (missense) mutations in patients carrying associated single nucleotide polymorphisms (SNPs) in interesting candidate genes through whole exome sequencing. Methods A retrospective case-control association study was performed by applying strict inclusion criteria to CD patients from three Belgian centres; early ileal fibrostenotic CD was primarily defined based on computed tomography or magnetic resonance enterography and occurring within five years following diagnosis (N = 56). The control cohort consisted of ileal CD patients without fibrostenotic or penetrating complications for a minimum of ten years (N = 252). Previously collected Immunochip data were subjected to logistic regression analysis correcting for disease location and the first five principal components. Exome sequencing of patients homozygous for at least one of the three top associated SNPs was performed. Genes in the proximity of the top SNPs, along with other candidate genes based on literature, were screened for the presence of missense mutations via Integrative Genomics Viewer. Mutations were evaluated for functional impact (PolyPhen-2) and evolutionary conservation (PhyloP), and frequencies were compared with European population data (ALFA dataset). Results Eight SNPs were associated with fibrostenosis (P<5e-05, Table 1), and three SNPs (rs6061883, rs9372122, and rs13176097) reached suggestive significance (P<10-6). The two top SNPs were located in cadherin 4 and autophagy related 5, two genes known to contribute to fibrosis.1, 2 We observed a significant shorter time to fibrosis between patients with a high amount (>3) of risk alleles (of the top eight associated SNPs), compared to patients with a low amount (<3). (P<0.001, Figure 1). Exome sequencing data of patients carrying associated SNPs revealed high risk variants (Table 2) in several candidate genes, including autophagy related 9B, epidermal growth factor receptor, and mitogen-activated protein kinase kinase kinase 1. Conclusion This carefully phenotyped association study reveals interesting SNPs and candidate genes to further study the genetic basis of early development of fibrostenosis in ileal CD. References 1.Li H, Peng X, Wang Y, et al. Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis. Autophagy. Sep 2016;12(9):1472-86. doi:10.1080/15548627.2016.1190071 2.Ding JH, Xiao Y, Zhao S, et al. Integrated analysis reveals the molecular features of fibrosis in triple-negative breast cancer. Mol Ther Oncolytics. Mar 17 2022;24:624-635. doi:10.1016/j.omto.2022.02.003

Abstract Background Inflammatory bowel diseases (IBD) are predominantly diagnosed during the second to third life decade. However, the disease may manifest itself at any age and current understanding of differences in clinical presentation and therapy use among age groups is still limited. We aimed to analyse age-related patterns in biological and surgical treatment among patients with IBD. Methods This study utilized data from the PANTHER cohort, a prospective Belgian inception cohort including 473 adult patients with IBD from 3 Belgian referral centres. Patient inclusion took place from 2015 to 2023. Patients were categorized into groups based on the age at diagnosis: 'young adult-onset' (18-39 years), 'adult-onset' (40-59 years), and ‘elderly-onset’ (³60 years). Baseline characteristics and treatments were analysed using Chi square, Mann-Whitney U tests, log-rank tests and/or Cox regression in SPSS. Results Baseline characteristics are shown in Table 1. No significant differences were found between age groups in terms of IBD diagnosis, gender, median follow-up duration, and smoking status. Use of biologics differed significantly across age groups with highest uptake in young-adult onset patients (65.8%, P<0.001). Significant differences were found in selection of first biologic with vedolizumab as most frequently selected option in the elderly compared to anti-TNF in the youngest age group (P=0.002). Time to initiation of a biological was earlier in the youngest cohort (P=0.002, Figure 1). Cox regression analysis revealed that older age at diagnosis (HR 0.987, 95%CI [0.98;0.996], P=0.006), UC (HR 0.495, 95%CI [0.50;0.39], P<0.001) and centre of follow-up (Brussels vs Leuven: HR 0.663, 95%CI [0.4;1], P=0.048) were associated with a lower risk of biological initiation. When analysing CD separately, independent risk factors associated with biological use were perianal disease (HR 2.26, 95%CI [1.6;3.2], P<0.001), L3 (HR 1.85, 95%CI [1.3;2.6], P<0.001,) and L4 (HR 12.7, 95%CI [1.6;98.7], P=0.015,) location (compared to L1 location). In UC patients, E2 (HR 7.9, 95%CI [2.8;22.4], P<0.001) and E3 (HR 6.9, 95%CI [2.4;19.3], P<0.001) were related to earlier use of biologics. During follow-up, 19% of CD and 5.1% of UC patients required IBD-related surgery. Univariate analysis showed a higher need for surgery in younger patients (P=0.042), however, this difference was no longer significant when analysing CD (P=0.272) and UC (P=0.09) patients as separate groups. Conclusion Analysis of the PANTHER Biobank reveals significant age-related variation in the administration of biological therapies among IBD patients.

Urban areas are facing challenges for the provision of public services, with water scarcity arising as one of the main problems. A twin track approach of supply and demand management is essential, and water-loss management contributes to reducing water demand. However, small municipalities from developing countries have technical, information, and financial limitations to locate and monitor water losses. This paper presents the estimation of real and apparent losses in a small municipality from a developing country in a data-scarce situation. For this, several tools were used, allowing data integration that resulted in a water balance, from which water losses were estimated at 46%, and four alternatives for water-loss reduction were developed. A cost-benefit analysis and financial indicators were estimated for the proposed alternatives, resulting in a water savings of 19%, a payback period of 3 years and an internal rate of return of 39%. The proposed strategies have potential to improve water quantity and quality, the technical stability of the system, utility performance, and water security.

This chapter describes protocols for identifying Ca+2- and GTP-binding proteins using whole cellular protein extracts from noncultured human psoriatic keratinocytes and COS-1 cells. The protocol for blot overlay detection of Ca+2- and GTP-binding proteins is exemplified using whole cellular protein extracts from noncultured human psoriatic keratinocytes and COS-1 cells, but can be applied to a variety of other cultured cells, tissue samples, and biological fluids. Calcium ion is a universal intracellular signal that acts as an important second messenger for many cellular processes and whose effect is modulated by specific calcium-binding proteins. Proteins are separated by means of one (1D)- or two-dimensional (2D) gel electrophoresis, transferred to a nitrocellulose membrane, and overlaid with radioactive 45Ca. Calcium-binding proteins are detected by autoradiography or phosphorimaging. No significant differences in calcium binding are observed when using membranes that had been dried before probing or membranes that had been used directly after blotting.

This chapter describes a protocol for the effective transient transfection of COS-1 cells using LipofectAMINE as a vehicle agent. Transient and stable expression of exogenous cDNAs in eukaryotic cells provides a powerful approach for examining the corresponding gene products. Plasmid pMT21 is used as an expression vector utilizing the adenovirus major late promoter for transcription initiation. The full-length cDNA of interest is subcloned into the pMT21 plasmid according to standard protocols. COS-1 monkey kidney cells are grown in complete Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). In average, about 15–30% transfection efficiency can be achieved as estimated by immunofluorescence. Transcription of the construct starts approximately 10 h after the addition of the transfection mixture to the cells as determined by Northen hybridization. Given the high pMT21 promoter activity, it is possible to harvest the cells for gene activity assays about 24 h after starting transfection.

High-resolution two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE), often referred as gel-based proteomics, multidimensional chromatography, and protein biochips in combination with mass spectrometry are among the proteomic tools that are available for biomarker and drug target discovery. Proteomic technologies are expected to play a key role in the study and treatment of diseases as they provide invaluable resources to define and characterize regulatory and functional networks, investigate the precise molecular defect in diseased tissues and biological fluids, and for developing specific reagents to precisely pinpoint a particular disease or stage of a disease. Today, 2D PAGE can be carried out using two separation modes in the first dimension: conventional isoelectic focusing (IEF) gels and immobilized pH gradient (IPG) gels. At the end of the labeling period, one should remove the medium with the aid of a drawn-out Pasteur pipette. Keep the medium if one wants to analyze secreted or externalized proteins. Place the 96-well plate at an angle to facilitate removal of the liquid. Dispose of the radioactive medium according to the regulations enforced in the laboratory.

This chapter describes a procedure that facilitates protein detection in gels by silver staining. Silver staining is one of the procedures, in addition to Coomassie blue, R and G types, and fluorescent dyes. The basic mechanisms underlying silver staining of proteins in gels are relatively well understood. Basically, protein detection depends on the binding of silver ions to the amino acid side chains, primary the sulfhydril and carboxiyl groups of proteins. The protein bands are visualized as spots where the reduction occurs and, as a result, the image of protein distribution within the gel is based on the difference in oxidation reduction potential between the gel area occupied by the proteins and the free adjacent sites. The silver amine or alkaline methods usually have lower background and, as a result, are most sensitive but require extended procedures. Acidic protocols, however, are faster but slightly less sensitive. The gel fixation and washing procedure can be carried in a staining try, but make sure that these are only used for silver staining. The size of the container has to be big enough to perform free movement of the gel during the shaking.

Synchronised cells of Tetrahymena pyriformis GL were labelled with 3H thymidine at a stage in the cell cycle when only the mitochondrial and extrachromosomal nucleolar ribosomal DNAs were replicating. In this way it was possible to prepare nuclei labelled selectively in the DNA of the ribosomal RNA genes. Since the ribosomal RNA cistrons of these cells are also very active in serving as a template for transcription, experiments were performed to test whether these genes are organised upon a nucleoprotein subunit structure of the kind that has been found in the total chromatin of a wide range of eukaryotic cell types. Tetrahymena macronuclei were prepared labelled uniformly in their DNA with 32P and labelled only in their nucleolar ribosomal DNA with 3H. Both the ribosomal genes and the bulk chromatin were then degraded in situ using micrococcal nuclease. The DNA fragments resulting from mild digestion were analysed on gels to reveal an identical DNA degradation pattern within both the ribosomal and bulk chromatins. It is concluded that the nucleoprotein structure of nucleolar rRNA cistrons posesses a periodic repeat along the DNA which is identical to that found in the substructure of unfractionated chromatin.