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Purpose/objective(s): The translocator protein (TSPO) is overexpressed in high-grade glioma and can be visualized in vivo by TSPO PET even in areas without MRI contrast enhancement. Here, we assessed the value of TSPO PET using the high-affinity TSPO ligand 18F-GE-180 in primary irradiation (RT) and re-irradiation (reRT) treatment planning.Materials/methods: Radiation treatment plans of high-grade glioma patients, examined with the TSPO PET tracer 18F-GE-180 prior to RT and reRT between 08/2016 and 01/2019, were included. PET based biological tumor volumes (BTV) were built on basis of tumor-to-background activity thresholds of 1.6, 1.8, and 2.0. Consensus GTVs (cGTV) based on GTV volumes delineated on MRI by 4 independent observers were generated using the simultaneous truth and performance level estimation (STAPLE) algorithm. Conformity of cGTV with BTV1.6, BTV1.8 and BTV2.0 were assessed by Sørensen-Dice coefficient (SDC) and conformity index (CI). Minimal clinical target volume (CTV) margins including the whole BTV into the cGTV were calculated.Results: Target volumes of 35 RT and 16 reRT plans were assessed. Volumes of cGTVs were smaller than the corresponding volumes of BTV1.6, BTV1.8 and BTV2.0 in the primary RT plans (median volumes 22.6 vs. 67.4, 50.7, 39.1 cm³; P < 0.001, P < 0.001, P = 0.018; Wilcoxon-Test) and reRT plans (median volumes 22.7 vs. 80.5, 55.0, 41.6 cm³; P = 0.001, P = 0.007, P = 0.134; Wilcoxon-Test). Conformity between BTVs and cGTV calculated by SDC and CI for thresholds 1.6, 1.8 and 2.0 were low in median in the RT (SDC: 0.51, 0.55, 0.58; CI: 0.35, 0.38, 0.41) and reRT setting (SDC: 0.38, 0.40, 0.40; CI 0.24, 0.25, 0.25). Median minimal CTV margins to include the entire BTV in the expanded cGTV volume was smaller in the RT setting (16, 12, 10 mm) than in the reRT setting (> 20, 17.5, 11 mm) for thresholds 1.6, 1.8 and 2.0.Conclusion: TSPO PET based BTVs showed highest conformity and best comparability in terms of size with MRI based GTVs for the threshold 2.0 both in the primary and reRT setting. Recurrence pattern analyses are needed to assess the clinical relevance of TSPO PET in glioma RT and reRT treatment planning.Author Disclosure: D.F. Fleischmann: None. M. Buettner: None. M. Unterrainer: None. S. Corradini: None. B. Zollner: None. J. Hofmaier: None. R. Bodensohn: None. N. Thon: None. C. Belka: None. P. Bartenstein: None. N. Albert: None. M. Niyazi: None. [ABSTRACT FROM AUTHOR]

Spatially defined organoid damage enables the study of cellular repair processes. However, capturing dynamic events in living tissues is technically challenging. Here, we present a protocol for the application of single-cell damage in intestinal organoid models. We describe steps for isolating and cultivating murine colon organoids, lentivirus generation and transduction of organoids, single-cell ablation by a femtosecond laser, and follow-up imaging analysis. We provide examples for the image acquisition pipeline of dynamic processes in organoids using a confocal microscope. For complete details on the use and execution of this protocol, please refer to Donath et al. 1 , 2 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

EGFR-targeted therapies are efficacious, but toxicity is common and can be severe. Urokinase type plasminogen activator receptor (uPAR)-targeted drugs are only emerging, so neither their efficacy nor toxicity is fully established. Recombinant eBAT was created by combining cytokines EGF and uPA on the same single-chain molecule with truncated Pseudomonas toxin. Its purpose was to simultaneously target tumors and their vasculature in the tumor microenvironment. In prior studies on mice and dogs, the drug proved efficacious. Here, we report the safety of eBAT in normal wildtype, uPAR knockout, and immunoreplete and immunodeficient tumor-bearing mice, as well as in dogs with spontaneous sarcoma that more closely mirror human cancer onset. In immunocompetent mice, tumor-bearing mice, uPAR knockout mice, and mice receiving species-optimized eBAT, toxicities were mild and self-limiting. Likewise, in dogs with life-threatening sarcoma given dosages found to be biologically active, eBAT was well tolerated. In mice receiving higher doses, eBAT was associated with dose-dependent evidence of liver injury, including portal biliary hyperplasia, oval cell proliferation, lymphoplasmacytic inflammation, periportal hepatocellular microvesicular change, hemorrhage, necrosis, and apoptosis. The results support continuing the clinical development of eBAT as a therapeutic agent for individuals with sarcoma and other cancers.

Purpose: The purpose of this study was to investigate the impact of the type of data capture on the time and help needed for collecting patient-reported outcomes as well as on the proportion of missing scores., Methods: In a multinational prospective study, thyroid cancer patients from 17 countries completed a validated questionnaire measuring quality of life. Electronic data capture was compared to the paper-based approach using multivariate logistic regression., Results: A total of 437 patients were included, of whom 13% used electronic data capture. The relation between data capture and time needed was modified by the emotional functioning of the patients. Those with clinical impairments in that respect needed more time to complete the questionnaire when they used electronic data capture compared to paper and pencil (OR adj 24.0; p = 0.006). This was not the case when patients had sub-threshold emotional problems (OR adj 1.9; p = 0.48). The odds of having the researcher reading the questions out (instead of the patient doing this themselves) (OR adj 0.1; p = 0.01) and of needing any help (OR adj 0.1; p = 0.01) were lower when electronic data capture was used. The proportion of missing scores was equivalent in both groups (OR adj 0.4, p = 0.42)., Conclusions: The advantages of electronic data capture, such as real-time assessment and fewer data entry errors, may come at the price of more time required for data collection when the patients have mental health problems. As this is not uncommon in thyroid cancer, researchers need to choose the type of data capture wisely for their particular research question., (© 2023. The Author(s).)

Fasting has been practiced with different time span in different areas of the world and for various reasons. One of the types of fasting regimens is Ramadan intermittent fasting (RIF), which is described as intermittent dry fasting and known as the most commonly practiced form of religious fasting. Different studies have shown its effects on body composition parameters and mental health, fatigue and quality of life (QoL). Elucidating the relationship of RIF on biological parameters would also be of importance to show its mechanism. Therefore, we evaluated several biological mediators related to mental health, such as ß-nerve growth factor (ß-NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and insulin-like growth factor-1 (IGF-1), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and matrix-metalloproteinase-9 (MMP-9). This study consisted of fasting (FG; n  = 25) and non-fasting group (NFG; n  = 25). Four different time points were assessed for FG: one week before (T1), mid (T2), last days (T3), and one week after (T4) RIF. T1 and T3 were the assessment time points for NFG. Biological mediators were determined from serum samples by using Human Magnetic Luminex and enzyme-linked immunosorbent assay. Furthermore, we then performed correlation analyses between biological mediators and our previously published clinical parameters including body composition and mental health parameters at all time points. Significant alterations were shown in FG for ß-NGF (T2vsT3, p  < 0.05; T2vsT4, p  < 0.05), GDNF (T1vsT4, p  < 0.05; T2vsT4, p  < 0.05), IL-8 (T2vsT3, p  < 0.05; T3vsT4, p  < 0.05), TNF-α (T1vsT3, p  < 0.05; T1vsT4, p  < 0.001; T2vsT4, p  < 0.001), and MMP-9 (T1vsT4, p  < 0.01). There were no statistically significant differences between FG and NFG in all biological mediators at T1 and T3. Correlation analysis showed that MMP-9 levels had negative correlation with body mass index (BMI) at T3. At T3 BDNF levels had negative correlation with Epworth Sleepiness Scale (ESS) as one of measured QoL parameters. ß-NGF, GDNF, TNF-α, and MMP-9 had positive correlation with some of body composition and mental health parameters. Findings demonstrate that RIF altered different biological mediators could give benefit to health. Its benefit is mediated by the alteration of biological mediators., Competing Interests: BN and CG are employed by Hannover Rehabilitation Services and Science Consulting. The remaining 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 © 2024 Ghashang, Suwandi, Buettner, Hamdan, Grassl, Gutenbrunner and Nugraha.)

Omega-6 fatty acids are the primary polyunsaturated fatty acids in most Western diets, while their role in diabetes remains controversial. Exposure of omega-6 fatty acids to an oxidative environment results in the generation of a highly reactive carbonyl species known as trans, trans-2,4-decadienal (tt-DDE). The timely and efficient detoxification of this metabolite, which has actions comparable to other reactive carbonyl species, such as 4-hydroxynonenal, acrolein, acetaldehyde, and methylglyoxal, is essential for disease prevention. However, the detoxification mechanism for tt-DDE remains elusive. In this study, the enzyme Aldh9a1b is identified as having a key role in the detoxification of tt-DDE. Loss of Aldh9a1b increased tt-DDE levels and resulted in an abnormal retinal vasculature and glucose intolerance in aldh9a1b -/- zebrafish. Transcriptomic and metabolomic analyses revealed that tt-DDE and aldh9a1b deficiency in larval and adult zebrafish induced insulin resistance and impaired glucose homeostasis. Moreover, alterations in hyaloid vasculature is induced by aldh9a1b knockout or by tt-DDE treatment can be rescued by the insulin receptor sensitizers metformin and rosiglitazone. Collectively, these results demonstrated that tt-DDE is the substrate of Aldh9a1b which causes microvascular damage and impaired glucose metabolism through insulin resistance., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

The malate shuttle is traditionally understood to maintain NAD + /NADH balance between the cytosol and mitochondria. Whether the malate shuttle has additional functions is unclear. Here we show that chronic viral infections induce CD8 + T cell expression of GOT1, a central enzyme in the malate shuttle. Got1 deficiency decreased the NAD + /NADH ratio and limited antiviral CD8 + T cell responses to chronic infection; however, increasing the NAD + /NADH ratio did not restore T cell responses. Got1 deficiency reduced the production of the ammonia scavenger 2-ketoglutarate (2-KG) from glutaminolysis and led to a toxic accumulation of ammonia in CD8 + T cells. Supplementation with 2-KG assimilated and detoxified ammonia in Got1-deficient T cells and restored antiviral responses. These data indicate that the major function of the malate shuttle in CD8 + T cells is not to maintain the NAD + /NADH balance but rather to detoxify ammonia and enable sustainable ammonia-neutral glutamine catabolism in CD8 + T cells during chronic infection., (© 2023. The Author(s).)

Intestinal organoids represent a three-dimensional cell culture system mimicking the mammalian intestine. The application of single-cell ablation for defined wounding via a femtosecond laser system within the crypt base allowed us to study cell dynamics during epithelial restitution. Neighboring cells formed a contractile actin ring encircling the damaged cell, changed the cellular aspect ratio, and immediately closed the barrier. Using traction force microscopy, we observed major forces at the ablation site and additional forces on the crypt sides. Inhibitors of the actomyosin-based mobility of the cells led to the failure of restoring the barrier. Close to the ablation site, high-frequency calcium flickering and propagation of calcium waves occured that synchronized with the contraction of the epithelial layer. We observed an increased signal and nuclear translocation of YAP-1. In conclusion, our approach enabled, for the first time, to unveil the intricacies of epithelial restitution beyond in vivo models by employing precise laser-induced damage in colonoids., Competing Interests: The authors declare no competing interest., (© 2023 The Authors.)

Airway organoids derived from adult murine epithelial cells represent a complex 3D in vitro system mimicking the airway epithelial tissue's native cell composition and physiological properties. In combination with a precise damage induction via femtosecond laser-based nanosurgery, this model might allow for the examination of intra- and intercellular dynamics in the course of repair processes with a high spatio-temporal resolution, which can hardly be reached using in vivo approaches. For characterization of the organoids' response to single or multiple-cell ablation, we first analyzed overall organoid survival and found that airway organoids were capable of efficiently repairing damage induced by femtosecond laser-based ablation of a single to ten cells within 24 h. An EdU staining assay further revealed a steady proliferative potential of airway organoid cells. Especially in the case of ablation of five cells, proliferation was enhanced within the first 4 h upon damage induction, whereas ablation of ten cells was followed by a slight decrease in proliferation within this time frame. Analyzing individual trajectories of single cells within airway organoids, we found an increased migratory behavior in cells within close proximity to the ablation site following the ablation of ten, but not five cells. Bulk RNA sequencing and subsequent enrichment analysis revealed the differential expression of sets of genes involved in the regulation of epithelial repair, distinct signaling pathway activities such as Notch signaling, as well as cell migration after laser-based ablation. Together, our findings demonstrate that organoid repair upon ablation of ten cells involves key processes by which native airway epithelial wound healing is regulated. This marks the herein presented in vitro damage model suitable to study repair processes following localized airway injury, thereby posing a novel approach to gain insights into the mechanisms driving epithelial repair on a single-cell level., 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 © 2023 Gentemann, Donath, Seidler, Patyk, Buettner, Heisterkamp and Kalies.)

Purpose: The aim of this study was to validate the new European Organisation for Research and Treatment of Cancer Quality of Life Thyroid Cancer Module (EORTC QLQ-THY34). Methods: We enrolled 437 thyroid cancer patients from 17 countries. One group ( n  = 303), undergoing treatment or best supportive care, completed the questionnaires at three time points (before therapy [t1], 6 weeks later [t2], and 6 months after t2 [t3]). A second group (survivors ≥2 years after diagnosis, n  = 134) completed it at a random baseline time point and a second time 1 week later. We determined internal consistency (using Cronbach's alpha), the scale structure (with confirmatory factor analysis), and discriminant validity (using known-group comparisons). Group 1 data were used to assess responsiveness and group 2 data to determine test-retest reliability using intra-class correlations (ICC). Results: All 34 items fulfilled the criteria to be kept in the questionnaire. Cronbach's alpha was >0.70 in 8 of the 9 multi-item scales. All standardized factor loadings exceeded 0.40, confirming the proposed scale structure. The ICC was >0.70 in all scales expressing good test-retest reliability. Differences in scale scores between patients with different histology were >5 points in all scales. In all but one of the pre-specified scales ( Dry Mouth ), changes over time were ≥|4| points between at least two time points. Conclusion: The EORTC QLQ-THY34 with its 9 multi-item and 8 single-item scales is a reliable and valid tool to measure quality of life in thyroid cancer patients and can be used in future trials and studies.

Introduction: Bacterial infections and chronic intestinal inflammations triggered by genetic susceptibility, environment or an imbalance in the intestinal microbiome are usually long-lasting and painful diseases in which the development and maintenance of these various intestinal inflammations is not yet fully understood, research is still needed. This still requires the use of animal models and is subject to the refinement principle of the 3Rs, to minimize suffering or pain perceived by the animals. With regard to this, the present study aimed at the recognition of pain using the mouse grimace scale (MGS) during chronic intestinal colitis due to dextran sodium sulfate (DSS) treatment or after infection with Citrobacter rodentium ., Methods: In this study 56 animals were included which were divided into 2 experimental groups: 1. chronic intestinal inflammation ( n = 9) and 2. acute intestinal inflammation (with ( n = 23) and without ( n = 24) C. rodentium infection). Before the induction of intestinal inflammation in one of the animal models, mice underwent an abdominal surgery and the live MGS from the cage side and a clinical score were assessed before (bsl) and after 2, 4, 6, 8, 24, and 48 hours., Results: The highest clinical score as well as the highest live MGS was detected 2 hours after surgery and almost no sign of pain or severity were detected after 24 and 48 hours. Eight weeks after abdominal surgery B6- Il4/Il10-/- mice were treated with DSS to trigger chronic intestinal colitis. During the acute phase as well as the chronic phase of the experiment, the live MGS and a clinical score were evaluated. The clinical score increased after DSS administration due to weight loss of the animals but no change of the live MGS was observed. In the second C57BL/6J mouse model, after infection with C. rodentium the clinical score increased but again, no increased score values in the live MGS was detectable., Discussion: In conclusion, the live MGS detected post-operative pain, but indicated no pain during DSS-induced colitis or C. rodentium infection. In contrast, clinical scoring and here especially the weight loss revealed a decreased wellbeing due to surgery and intestinal inflammation., 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 © 2023 Peppermüller, Gehring, Zentrich, Bleich, Häger and Buettner.)

Altered cellular metabolism is a hallmark of cancer pathogenesis and progression; for example, a near-universal feature of cancer is increased metabolic flux through the hexosamine biosynthetic pathway (HBP). This pathway produces uridine diphosphate N -acetylglucosamine (UDP-GlcNAc), a potent oncometabolite that drives multiple facets of cancer progression. In this study, we synthesized and evaluated peracetylated hexosamine analogs designed to reduce flux through the HBP. By screening a panel of analogs in pancreatic cancer and glioblastoma multiform (GBM) cells, we identified Ac 4 Glc2Bz─a benzyl-modified GlcNAc mimetic─as an antiproliferative cancer drug candidate that down-regulated oncogenic metabolites and reduced GBM cell motility at concentrations non-toxic to non-neoplastic cells. More specifically, the growth inhibitory effects of Ac 4 Glc2Bz were linked to reduced levels of UDP-GlcNAc and concomitant decreases in protein O-GlcNAc modification in both pancreatic cancer and GBM cells. Targeted metabolomics analysis in GBM cells showed that Ac 4 Glc2Bz disturbed glucose metabolism, amino acid pools, and nucleotide precursor biosynthesis, consistent with reduced proliferation and other anti-oncogenic properties of this analog. Furthermore, Ac 4 Glc2Bz reduced the invasion, migration, and stemness of GBM cells. Importantly, normal metabolic functions mediated by UDP-GlcNAc were not disrupted in non-neoplastic cells, including maintenance of endogenous levels of O-GlcNAcylation with no global disruption of N-glycan production. Finally, a pilot in vivo study showed that a potential therapeutic window exists where animals tolerated 5- to 10-fold higher levels of Ac 4 Glc2Bz than projected for in vivo efficacy. Together, these results establish GlcNAc analogs targeting the HBP through salvage mechanisms as a new therapeutic approach to safely normalize an important facet of aberrant glucose metabolism associated with cancer.

Introduction: Sheep are frequently used in translational surgical orthopedic studies. Naturally, a good pain management is mandatory for animal welfare, although it is also important with regard to data quality. However, methods for adequate severity assessment, especially considering pain, are rather rare regarding large animal models. Therefore, in the present study, accompanying a surgical pilot study, telemetry and the Sheep Grimace Scale (SGS) were used in addition to clinical scoring for severity assessment after surgical interventions in sheep., Methods: Telemetric devices were implanted in a first surgery subcutaneously into four German black-headed mutton ewes (4-5 years, 77-115 kg). After 3-4 weeks of recovery, sheep underwent tendon ablation of the left M. infraspinatus. Clinical scoring and video recordings for SGS analysis were performed after both surgeries, and the heart rate (HR) and general activity were monitored by telemetry., Results: Immediately after surgery, clinical score and HR were slightly increased, and activity was decreased in individual sheep after both surgeries. The SGS mildly elevated directly after transmitter implantation but increased to higher levels after tendon ablation immediately after surgery and on the following day., Conclusion: In summary, SGS- and telemetry-derived data were suitable to detect postoperative pain in sheep with the potential to improve individual pain recognition and postoperative management, which consequently contributes to refinement., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Introduction: Obesity increases the risk of several diseases, such as type 2 diabetes mellitus and cardiovascular disease. Obesity also affects the immune system. When dietary lipids are transported via the lymphatics, they pass the mesenteric lymph nodes (mLNs). In these secondary lymphoid organs, immune responses towards pathogens are generated, or tolerance against harmless antigens is induced., Methods: In this study, the effects of diet-induced obesity (DIO) on mLN induced oral tolerance induction were examined in C57BL/6NCrl mice. Therefore, mice were fed a high-fat or a low-fat diet for 14 weeks. After 10 weeks of feeding oral tolerance induction started, ending up in measuring the delayed-type hypersensitivity reaction, the cell subset composition and cytokine expression., Results: We detected an impaired oral tolerance induction during DIO, but changes were reversible after switching the feed to standard chow. Thus, the altered immunological function of mLNs depends on the intake of dietary lipids. Additionally, our results show an influence of the microenvironment on the development of oral tolerance during DIO as oral tolerance was induced in transplanted peripheral lymph nodes., Conclusion: This indicates a functional influence of dietary lipids on stromal cells involved in immune system induction in the mLNs., (© 2022 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA 's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between animal facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multicentre study, whereby inbred C57BL/6J mice from a single breeding cohort were allocated to and reared in 5 different animal facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of the rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that environmental variation among animal facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organisation. We detected changes in chromatin organisation in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity, and regulation of behaviour. Our findings demonstrate that common environmental differences between animal facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and thus argue that study designs should take environmental background into account to increase the robustness and replicability of findings., Competing Interests: The authors have declared that no competing interests exist.

Memory CD8 + T cells mature after antigen clearance and ultimately express CD8 protein at levels higher than those detected in effector CD8 + T cells. However, it is not clear whether engagement of CD8 in the absence of antigenic stimulation will result in the functional activation of T cells. Here, we found that CD8 antibody-mediated activation of memory CD8 + T cells triggered T cell receptor (TCR) downstream signaling, enhanced T cell-mediated cytotoxicity and promoted effector cytokine production in a glucose- and glutamine-dependent manner. Furthermore, pretreatment of memory CD8 + T cells with an agonistic anti-CD8 antibody enhanced their tumoricidal activity in vitro and in vivo. From these studies, we conclude that CD8 agonism activates glucose and glutamine metabolism in memory T cells and enhances the efficacy of memory T cell-based cancer immunotherapy., (© 2022 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Basic processes of the fatty acid metabolism have an important impact on the function of intestinal epithelial cells (IEC). However, while the role of cellular fatty acid oxidation is well appreciated, it is not clear how de novo fatty acid synthesis (FAS) influences the biology of IECs. We report here that interfering with de novo FAS by deletion of the enzyme Acetyl-CoA-Carboxylase (ACC)1 in IECs results in the loss of epithelial crypt structures and a specific decline in Lgr5 + intestinal epithelial stem cells (ISC). Mechanistically, ACC1-mediated de novo FAS supports the formation of intestinal organoids and the differentiation of complex crypt structures by sustaining the nuclear accumulation of PPARδ/β-catenin in ISCs. The dependency of ISCs on cellular de novo FAS is tuned by the availability of environmental lipids, as an excess delivery of external fatty acids is sufficient to rescue the defect in crypt formation. Finally, inhibition of ACC1 reduces the formation of tumors in colitis-associated colon cancer, together highlighting the importance of cellular lipogenesis for sustaining ISC function and providing a potential perspective to colon cancer therapy., (© 2022. The Author(s).)

The pdx1-/- zebrafish mutant was recently established as a novel animal model of diabetic retinopathy. In this study, we investigate whether knockout of pdx1 also leads to diabetic kidney disease (DKD). pdx1-/- larvae exhibit several signs of early DKD, such as glomerular hypertrophy, impairments in the filtration barrier corresponding to microalbuminuria, and glomerular basement membrane (GBM) thickening. Adult pdx1-/- mutants show progressive GBM thickening in comparison with the larval state. Heterozygous pdx1 knockout also leads to glomerular hypertrophy as initial establishment of DKD similar to the pdx1-/- larvae. RNA sequencing of adult pdx1+/- kidneys uncovered regulations in multiple expected diabetic pathways related to podocyte disruption and hinting at early vascular dysregulation without obvious morphological alterations. Metabolome analysis and pharmacological intervention experiments revealed the contribution of phosphatidylethanolamine in the early establishment of kidney damage. In conclusion, this study identified the pdx1 mutant as a novel model for the study of DKD, showing signs of the early disease progression already in the larval stage and several selective features of later DKD in adult mutants., (© 2022 by the American Diabetes Association.)

Organoids represent the cellular composition of natural tissue. So called colonoids, organoids derived from colon tissue, are a good model for understanding regeneration. However, next to the cellular composition, the surrounding matrix, the cell-cell interactions, and environmental factors have to be considered. This requires new approaches for the manipulation of a colonoid. Of key interest is the precise application of localized damage and the following cellular reaction. We have established multiphoton imaging in combination with femtosecond laser-based cellular nanosurgery in colonoids to ablate single cells in the colonoids' crypts, the proliferative zones, and the differentiated zones. We observed that half of the colonoids recovered within six hours after manipulation. An invagination of the damaged cell and closing of the structure was observed. In about a third of the cases of targeted crypt damage, it caused a stop in crypt proliferation. In the majority of colonoids ablated in the crypt, the damage led to an increase in Wnt signalling, indicated via a fluorescent lentiviral biosensor. qRT-PCR analysis showed increased expression of various proliferation and Wnt -associated genes in response to damage. Our new model of probing colonoid regeneration paves the way to better understand organoid dynamics on a single cell level.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro , in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA 's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU . They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Objective: Little is known on optimal screening population for detecting new atrial fibrillation (AF) in the community. We describe characteristics and estimate cost-effectiveness for a single timepoint electrocardiographic screening., Methods: We performed a 12-lead ECG in the German population-based Gutenberg Health Study between 2007 and 2012 (n=15 010), mean age 55±11 years, 51% men and collected more than 120 clinical and biomarker variables, including N-terminal pro B-type natriuretic peptide (Nt-proBNP), risk factors, disease symptoms and echocardiographic variables., Results: Of 15 010 individuals, 466 (3.1%) had AF. New AF was found in 32 individuals, 0.2% of the total sample, 0.5% of individuals aged 65-74 years and predominantly men (86%). The classical risk factor burden was high in individuals with new AF. The median estimated stroke risk was 2.2%/year, while risk of developing heart failure was 21% over 10 years. In the 65-74 year age group, the cost per quality-adjusted life-year gained resulting from a single timepoint screening was €30 361. In simulations, the costs were highly sensitive to AF detection rates, proportion of treatment and type of oral anticoagulant. Prescreening by Nt-proBNP measurements was not cost-effective in the current setting., Conclusions: In our middle-aged population cohort, we identified 0.2% new AF by single timepoint screening. There was a significant estimated risk of stroke and heart failure in these individuals. Cost-effectiveness for screening may be reached in individuals aged 65 years and older. The simple age cut-off is not improved by using Nt-proBNP as a biomarker to guide a screening programme., Competing Interests: Competing interests: CW and CM and employees of the Institute for Epidemiology, Statistics and Informatics GmbH. The Institute has received grants from Bristol-Myers Squibb and Pfizer, and CSL Behring outside the submitted work. BF reports prior fees and advisory board honoraria from Bayer Pharma AG, Boehringer Ingelheim, Daiichi-Sankyo, Omron and Pfizer/BMS and grants to the institution for investigator-initiated studies from BMS/Pfizer. RBS has received speaker honoraria from Bristol-Myers Squibb/Pfizer. MB received lecture fees from Lilly and Takeda, all outside of the submitted work., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In recent years, stem cell-derived organoids have become a cell culture standard that is widely used for studying various scientific issues that were previously investigated through animal experiments and using common tumor cell lines. After their initial hype, concerns regarding their standardization have been raised. Here, we aim to provide some insights into our experience in standardizing murine colonic epithelial organoids, which we use as a replacement method for research on inflammatory bowel disease. Considering good scientific practice, we examined various factors that might challenge the design and outcome of experiments using these organoids. First, to analyze the impact of antibiotics/antimycotics, we performed kinetic experiments using ZellShield® and measured the gene expression levels of the tight junction markers Ocln , Zo-1 , and Cldn4 , the proliferation marker Ki67 , and the proinflammatory cytokine Tnfα . Because we found no differences between cultivations with and without ZellShield®, we then performed infection experiments using the probiotic Escherichia coli Nissle 1917 as an already established model setup to analyze the impact of technical, interexperimental, and biologic replicates. We demonstrate that interexperimental differences pose the greatest challenge for reproducibility and explain our strategies for addressing these differences. Additionally, we conducted infection experiments using freshly isolated and cryopreserved/thawed organoids and found that cryopreservation influenced the experimental outcome during early passages. Formerly cryopreserved colonoids exhibited a premature appearance and a higher proinflammatory response to bacterial stimulation. Therefore, we recommend analyzing the growth characteristics and reliability of cryopreserved organoids before to their use in experiments together with conducting several independent experiments under standardized conditions. Taken together, our findings demonstrate that organoid culture, if standardized, constitutes a good tool for reducing the need for animal experiments and might further improve our understanding of, for example, the role of epithelial cells in inflammatory bowel disease development., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Talke F. zur Bruegge et al.)

Background: Studies show that proallergic T H 2 cells decrease after successful allergen-specific immunotherapy (AIT). It is likely that iatrogenic administration of allergens drives these cells to exhaustion due to chronic T-cell receptor stimulation. This study aimed to investigate the exhaustion of T cells in connection with allergen exposure during AIT in mice and two independent patient cohorts., Methods: OVA-sensitized C57BL/6J mice were challenged and treated with OVA, and the development of exhaustion in local and systemic T H 2 cells was analyzed. In patients, the expression of exhaustion-associated surface markers on T H 2 cells was evaluated using flow cytometry in a cross-sectional grass pollen allergy cohort with and without AIT. The treatment effect was further studied in PBMC collected from a prospective long-term AIT cohort., Results: The exhaustion-associated surface markers CTLA-4 and PD-1 were significantly upregulated on T H 2 cells upon OVA aerosol exposure in OVA-allergic compared to non-allergic mice. CTLA-4 and PD-1 decreased after AIT, in particular on the surface of local lung T H 2 cells. Similarly, CTLA-4 and PD-1 expression was enhanced on T H 2 cells from patients with allergic rhinitis with an even stronger effect in those with concomitant asthma. Using an unbiased Louvain clustering analysis, we discovered a late-differentiated T H 2 population expressing both markers that decreased during up-dosing but persisted long term during the maintenance phase., Conclusions: This study shows that allergen exposure promotes CTLA-4 and PD-1 expression on T H 2 cells and that the dynamic change in frequencies of exhausted T H 2 cells exhibits a differential pattern during the up-dosing versus the maintenance phases of AIT., (© 2021 Zentrum Allergie und Umwelt. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Ramadan fasting (RF) is a type of diurnal intermittent fasting. Previous studies reported the benefits of RF in healthy subjects on mood and health related to quality of life (QoL). Cortisol and brain-derived neurotrophic factor (BDNF) have been shown to play a role in mood, body composition parameters, and health-related QoL. This study aimed at elucidating the mechanism of the benefit of RF, particularly cortisol and BNDF and their association with mood and QoL. Insulin growth factor-1 (IGF-1), interleukin (IL)-8, matrix metalloproteinase (MMP)-9, and myoglobin were determined. Thirty-four healthy men and women were recruited. Serum from peripheral venous blood samples was collected at five time points: 1 week before RF (T1); mid of RF (T2), last days of RF (T3), 1 week after RF (T4), and 1 month after RF (T5). The amounts of biological mediators in the serum samples were determined by enzyme-linked immunosorbent assay (ELISA) and Luminex assays. BDNF and cortisol significantly decreased at T3 ( p < 0.05) and T4 ( p < 0.001) compared to T1, respectively. It seems the benefits of RF for mood-related symptoms are mediated by different biological mediators, particularly cortisol and BDNF., 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 Riat, Suwandi, Ghashang, Buettner, Eljurnazi, Grassl, Gutenbrunner and Nugraha.)

Uncontrolled proliferation and altered metabolic reprogramming are hallmarks of cancer. Active glycolysis and glutaminolysis are characteristic features of these hallmarks and required for tumorigenesis. A fine balance between cancer metabolism and autophagy is a prerequisite of homeostasis within cancer cells. Here we show that glutamate pyruvate transaminase 2 (GPT2), which serves as a pivot between glycolysis and glutaminolysis, is highly upregulated in aggressive breast cancers, particularly the triple-negative breast cancer subtype. Abrogation of this enzyme results in decreased tricarboxylic acid cycle intermediates, which promotes the rewiring of glucose carbon atoms and alterations in nutrient levels. Concordantly, loss of GPT2 results in an impairment of mechanistic target of rapamycin complex 1 activity as well as the induction of autophagy. Furthermore, in vivo xenograft studies have shown that autophagy induction correlates with decreased tumor growth and that markers of induced autophagy correlate with low GPT2 levels in patient samples. Taken together, these findings indicate that cancer cells have a close network between metabolic and nutrient sensing pathways necessary to sustain tumorigenesis and that aminotransferase reactions play an important role in maintaining this balance., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

The balance between the responsiveness of the intestinal immune system and the gut environment is fundamental for the maintenance of intestinal homeostasis, which is required for an adequate recognition of entering antigens. The disruption of this homeostasis by exaggerated immune response to harmless antigens can lead to the development of intestinal disorders such as inflammatory bowel disease. Stromal cells are sessile non-hematopoietic cells that build the backbone of the lymph node, an important site for the immune response induction, but also contribute to immune response and tolerance induction. However, the knowledge about the role of stromal cells in the regulation of inflammatory responses is still limited. Therefore, in this study we analyzed the influence of stromal cells on the development of chronic intestinal inflammation. Here, we show that intestinal inflammation alters the immune activation of the mesenteric lymph node-derived stromal cells. Podoplanin + and CD21/35 + stromal cells showed increased expression of MHC class II molecules, but CD106 expression on CD21/35 + cells was reduced. Stromal cells secreted cytokines and chemokines such as CCL7 and CXCL16 influenced the gut-homing phenotype and proliferation of CD4 + and CD8 + T cells. Furthermore, stromal cells of peripheral lymph nodes transplanted into the mesentery attenuated colitis severity in B6- Il10 -/- mice. The reduced colitis severity in these mice was associated with increased expression of IL4 and distinct activation pattern of stromal cells derived from transplanted peripheral lymph nodes. Altogether, our results demonstrate that lymph node stromal cells impact development of chronic colitis via T cell induction. Moreover, lymph node stromal cells from different draining area due to neonatally imprinted processes distinctly regulate the induction of immune responses., 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 Basic, Peppermüller, Bolsega, Bleich, Bornemann, Bode and Buettner.)

Bisphenol (BP) A, a known food contaminant, is a possible risk factor in the epidemic of non-communicable diseases (NCD) including food intolerance and inflammatory bowel diseases (IBD). Regulatory restrictions regarding BPA usage led to BPA removal and replacement by poorly described substitutes, like BPS or BPF (few data on occurrence in food and human samples and biological effect). Oral tolerance protocol to ovalbumin (OVA) in WT mice and Il10 -/- mice prone to IBD were used respectively to address immune responses towards food and microbial luminal antigens following BP oral exposure. Both mice models were orally exposed for five weeks to BPA, BPS or BPF at 0.5, 5 and 50 μg/kg of body weight (bw)/day (d). Oral exposure to BPs at low doses (0.5 and 5 μg/kg bw/d) impaired oral tolerance as indicated by higher humoral and pro-inflammatory cellular responses in OVA-tolerized mice. However, only BPF exacerbate colitis in Il10 -/- prone mice associated with a defect of fecal IgA and increased secretion of TNF-α in colon. These findings provide a unique comparative study on effects of adult oral exposure to BPs on immune responses and its consequences on NCD related to intestinal luminal antigen development., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The liver is known to possess extensive regenerative capabilities, the processes and pathways of which are not fully understood. A necessary step towards a better understanding involves the analysis of regeneration on the microscopic level in the in vivo environment. We developed an evaluation method combining longitudinal imaging analysis in vivo with simultaneous manipulation on single cell level. An abdominal imaging window was implanted in vivo in Balb/C mice for recurrent imaging after implantation. Intravenous injection of Fluorescein Isothiocyanate (FITC)-Dextran was used for labelling of vessels and Rhodamine 6G for hepatocytes. Minimal cell injury was induced via ablation with a femtosecond laser system during simultaneous visualisation of targeted cells using multiphoton microscopy. High-resolution imaging in vivo on single cell level including re-localisation of ablated regions in follow-up measurements after 2-7 days was feasible. Targeted single cell manipulation using femtosecond laser pulses at peak intensities of 3-6.6 μJ led to enhancement of FITC-Dextran in the surrounding tissue. These reactions reached their maxima 5-15 minutes after ablation and were no longer detectable after 24 hours. The procedures were well tolerated by all animals. Multiphoton microscopy in vivo, combined with a femtosecond laser system for single cell manipulation provides a refined procedure for longitudinal evaluation of liver micro-regeneration in the same region of interest. Immediate reactions after cell ablation and tissue regeneration can be analysed., Competing Interests: The authors have declared that no competing interests exist.

Novel tools in humane animal research should benefit the animal as well as the experimentally obtained data. Imaging technologies have proven to be versatile and also in accordance with the demands of the 3 R principle. However, most imaging technologies are either limited by the target organs, number of repetitive imaging sessions, or the maximal resolution. We present a technique-, which enables multicolor abdominal imaging on a tissue level. It is based on a small imaging fiber endoscope, which is guided by a second commercial endoscope. The imaging fiber endoscope allows the distinction of four different fluorescence channels. It has a size of less than 1 mm and can approximately resolve single cells. The imaging fiber was successfully tested on cells in vitro, excised organ tissue, and in mice in vivo. Combined with neural networks for image restauration, high quality images from various abdominal organs of interest were realized. The second endoscope ensured a precise placement of the imaging fiber in vivo. Our approach of guided tissue imaging in vivo, combined with neuronal networks for image restauration, permits the acquisition of fluorescence-microscope like images with minimal invasive surgery in vivo. Therefore, it is possible to extend our approach to repetitive imaging sessions. The cost below 30 thousand euros allows an establishment of this approach in various scenarios.

In obesity, increased dietary lipids are taken up and transported by the lymphatic systems into the circulatory system. Increased fat accumulation results in impairments in the lymph fluid and lymph node (LN) atrophy. LNs filter the lymph fluid for foreign antigens to induce and control immune responses, and the alteration of this function during obesity remains underexplored. Here, the changes within the microarchitecture of mesenteric LNs (mLNs) during high levels of lipid transport were investigated, and the role of stromal cells in mice fed a high-fat diet for 10 weeks was assessed. Microarray experiments revealed that gene probes involved in lipid metabolism are expressed by mLN stromal cells. Transmission electron microscopy enabled the identification of lipid droplets in lymphatic endothelial cells, different reticulum cells, and macrophages, and the lipid droplet sizes as well as their numbers and intercellular distances increased after 10 weeks of high-fat diet feeding. The results indicate that changes in the microarchitecture and increased accumulation of lipid droplets in stromal cells and macrophages influence the immunological function of mLNs., Competing Interests: 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., (© 2020 THE AUTHORS. Published by Elsevier BV on behalf of Cairo University.)

Obesity has emerged as a major global health problem and is associated with various diseases, such as metabolic syndrome, type 2 diabetes mellitus, and cardiovascular diseases. The inbred C57BL/6 mouse strain is often used for various experimental investigations, such as metabolic research. However, over time, genetically distinguishable C57BL/6 substrains have evolved. The manifestation of genetic alterations has resulted in behavioral and metabolic differences. In this study, a comparison of diet-induced obesity in C57BL/6JHanZtm, C57BL/6NCrl and C57BL/6 J mice revealed several metabolic and immunological differences such as blood glucose level and cytokine expression, respectively, among these C57BL/6 substrains. For example, C57BL/6NCrl mice developed the most pronounced adiposity, whereas C57BL/6 J mice showed the highest impairment in glucose tolerance. Moreover, our results indicated that the immunological phenotype depends on the intestinal microbiota, as the cell subset composition of the colon was similar in obese ex-GF B6NRj B6JHanZtm and obese B6JHanZtm mice. Phenotypic differences between C57BL/6 substrains are caused by a complex combination of genetic and microbial alterations. Therefore, in performing metabolic research, considering substrain-specific characteristics, which can influence the course of study, is important. Moreover, for unbiased comparison of data, the entire strain name should be shared with the scientific community.

Cd14 and Alpk1 both encode pathogen recognition receptors and are known candidate genes for affecting severity in inflammatory bowel diseases. CD14 acts as a coreceptor for bacterial lipopolysaccharide (LPS), while ALPK1 senses ADP-D-glycero-beta-D-manno-heptose, a metabolic intermediate of LPS biosynthesis. Intestinal barrier integrity can be influenced by CD14, whereas to date, the role of ALPK1 in maintaining barrier function remains unknown. We used colon-derived 3D organoids, first characterised for growth, proliferation, stem cell markers, and expression of tight junction (TJ) components using qPCR and immunohistochemistry. They showed characteristic crypt stem cells, apical shedding of dead cells, and TJ formation. Afterwards, organoids of different genotypes (WT, Il10 -/- , Cd14 -/- , and Alpk1 -/- ) were then stimulated with either LPS or Escherichia coli Nissle 1917 ( Ec N). Gene expression and protein levels of cytokines and TJ components were analysed. WT organoids increased expression of Tnfα and tight junction components. Cd14 -/- organoids expressed significantly less Tnfα and Ocln after LPS stimulation than WT organoids but reacted similarly to WT organoids after Ec N stimulation. In contrast, compared to WT, Alpk1 -/- organoids showed decreased expression of different TJ and cytokine genes in response to Ec N but not LPS. However, Western blotting revealed an effect of ALPK1 on TJ protein levels. These findings demonstrate that Cd14 , but not Alpk1 , alters the response to LPS stimulation in colonic epithelial cells, whereas Alpk1 is involved in the response upon bacterial challenge., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Pascal Brooks et al.)