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In this work, an analytical approach was developed for Pb, Sr, and Fe isotopic analysis of archaeological samples recovered from an iron work site by using multi-collector inductively coupled plasma - mass spectrometry (MC-ICP-MS). The sample types include slag, coal, clay and hammer scales, all obtained from an archaeological site at Hoeke (Belgium). Despite the wide concentration range of the target elements present in the samples and some sample manipulations necessarily performed outside of a clean laboratory facility, the analytical procedure yielded accurate and precise results for QA/QC standards while blank levels were negligible. Preliminary results concerning Pb, Sr and Fe isotope ratio variations in archaeological materials associated with iron working processes are provided. The samples revealed high variability in metal isotopic compositions, with the 208 Pb/ 207 Pb ratio ranging from 2.4261 to 2.4824, the 87 Sr/ 86 Sr ratio from 0.7100 to 0.7220, and δ 56 Fe values from -0.34 to +0.08‰, which was tentatively attributed to the mixing of materials during the iron production process or variability within the source material. Also, contamination introduced by coal and furnace/hearth lining material could have contributed to the wide range of isotopic compositions observed. Because of the absence of information and data for primary ore samples to compare with, the provenance of the materials could not be established. The present study highlights the challenges in interpreting archaeological data, particularly in terms of the isotopic variability observed. It underscores the necessity of integrating analysis data with historical and archaeological knowledge. Further research, involving detailed analysis of these source materials combined with robust historical evidence, is essential to validate hypotheses concerning the origin of iron., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

CRISPR-mediated simultaneous targeting of candidate tumor suppressor genes in Xenopus tropicalis allows fast functional assessment of co-driver genes for various solid tumors. Genotyping of tumors that emerge in the mosaic mutant animals rapidly exposes the gene mutations under positive selection for tumor establishment. However, applying this simple approach to the blood lineage has not been attempted. Multiple hematologic malignancies have mutations in EZH2, encoding the catalytic subunit of the Polycomb Repressive Complex 2. Interestingly, EZH2 can act as an oncogene or a tumor suppressor, depending on cellular context and disease stage. We show here that mosaic CRISPR/Cas9 mediated ezh2 disruption in the blood lineage resulted in early and penetrant acute myeloid leukemia (AML) induction. While animals were co-targeted with an sgRNA that induces notch1 gain-of-function mutations, sequencing of leukemias revealed positive selection towards biallelic ezh2 mutations regardless of notch1 mutational status. Co-targeting dnm2, recurrently mutated in T/ETP-ALL, induced a switch from myeloid towards acute T-cell leukemia. Both myeloid and T-cell leukemias engrafted in immunocompromised hosts. These data underline the potential of Xenopus tropicalis for modeling human leukemia, where mosaic gene disruption, combined with deep amplicon sequencing of the targeted genomic regions, can rapidly and efficiently expose co-operating driver gene mutations., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Owing to a lack of effective treatments, patients with metastatic disease have a median survival time of 6-12 months. We recently demonstrated that the Survival Associated Mitochondrial Melanoma Specific Oncogenic Non-coding RNA (SAMMSON) is essential for UM cell survival and that antisense oligonucleotide (ASO)-mediated silencing of SAMMSON impaired cell viability and tumor growth in vitro and in vivo . By screening a library of 2911 clinical stage compounds, we identified the mammalian target of rapamycin (mTOR) inhibitor GDC-0349 to synergize with SAMMSON inhibition in UM. Mechanistic studies revealed that mTOR inhibition enhanced uptake and reduced lysosomal accumulation of lipid complexed SAMMSON ASOs, improving SAMMSON knockdown and further decreasing UM cell viability. We found mTOR inhibition to also enhance target knockdown in other cancer cell lines as well as normal cells when combined with lipid nanoparticle complexed or encapsulated ASOs or small interfering RNAs (siRNAs). Our results are relevant to nucleic acid treatment in general and highlight the potential of mTOR inhibition to enhance ASO and siRNA-mediated target knockdown.

Introduction: Polymicrobial sepsis causes acute anorexia (loss of appetite), leading to lipolysis in white adipose tissue and proteolysis in muscle, and thus release of free fatty acids (FFAs), glycerol and gluconeogenic amino acids. Since hepatic peroxisome proliferator-activated receptor alpha (PPARα) and glucocorticoid receptor (GR) quickly lose function in sepsis, these metabolites accumulate (causing toxicity) and fail to yield energy-rich molecules such as ketone bodies (KBs) and glucose. The mechanism of PPARα and GR dysfunction is not known., Methods & Results: We investigated the hypothesis that hypoxia and/or activation of hypoxia inducible factors (HIFs) might play a role in these issues with PPARα and GR. After cecal ligation and puncture (CLP) in mice, leading to lethal polymicrobial sepsis, bulk liver RNA sequencing illustrated the induction of the genes encoding HIF1α and HIF2α, and an enrichment of HIF-dependent gene signatures. Therefore, we generated hepatocyte-specific knock-out mice for HIF1α, HIF2α or both, and a new HRE-luciferase reporter mouse line. After CLP, these HRE-luciferase reporter mice show signals in several tissues, including the liver. Hydrodynamic injection of an HRE-luciferase reporter plasmid also led to (liver-specific) signals in hypoxia and CLP. Despite these encouraging data, however, hepatocyte-specific HIF1α and/or HIF2α knock-out mice suggest that survival after CLP was not dependent on the hepatocyte-specific presence of HIF proteins, which was supported by measuring blood levels of glucose, FFAs, and KBs. The HIF proteins were also irrelevant in the CLP-induced glucocorticoid resistance, but we found indications that the absence of HIF1α in hepatocytes causes less inactivation of PPARα transcriptional function., Conclusion: We conclude that HIF1α and HIF2α are activated in hepatocytes in sepsis, but their contribution to the mechanisms leading to lethality are minimal., 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 Vanderhaeghen, Timmermans, Eggermont, Watts, Vandewalle, Wallaeys, Nuyttens, De Temmerman, Hochepied, Dewaele, Berghe, Sanders, Wielockx, Beyaert and Libert.)

High-quality care for patients with COPD is necessary. To achieve quality improvement in primary care, the general practitioner and the electronic health record (EHR) play an important role. The aim of this study was to develop a set of evidence-based and EHR extractable quality indicators (QIs) to measure and improve the quality of COPD primary care. We composed a multidisciplinary expert panel of 12 members, including patients, and used a RAND-modified Delphi method. The SMART principle was applied to select recommendations and QIs from international guidelines as well as existing sets of QIs, and these recommendations and QIs were added to an individual written questionnaire. Based on the median score, prioritization and degree of agreement, the recommendations and QIs were rated as having a high, uncertain or low potential to measure the quality of COPD primary care and were then discussed in an online consensus meeting for inclusion or exclusion. After a final validation, a core set of recommendations was translated into QIs. From 37 recommendations, obtained out of 10 international guidelines, and 5 existing indicators, a core set of 18 recommendations and 2 QIs was derived after the rating procedure. The expert panel added one new recommendation. Together, the recommendations and QIs were translated and merged into a final set of 21 QIs. Our study developed a set of 21 evidence-based and EHR-extractable QIs for COPD in primary care. These indicators can be used in an automated quality assessment to measure and improve the quality of COPD primary care., (© 2022. The Author(s).)

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GR dim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GR D/D ) have previously helped to define the functions of GR monomers and dimers. Since GR D/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GR D+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GR L/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and K d values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Here, we investigate the impact of hypoxia on the hepatic response of glucocorticoid receptor (GR) to dexamethasone (DEX) in mice via RNA-sequencing. Hypoxia causes three types of reprogramming of GR: (i) much weaker induction of classical GR-responsive genes by DEX in hypoxia, (ii) a number of genes is induced by DEX specifically in hypoxia, and (iii) hypoxia induces a group of genes via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Transcriptional profiles are reflected by changed GR DNA-binding as measured by ChIP sequencing. The HPA axis is induced by hypothalamic HIF1α and HIF2α activation and leads to GR-dependent lipolysis and ketogenesis. Acute inflammation, induced by lipopolysaccharide, is prevented by DEX in normoxia but not during hypoxia, and this is attributed to HPA axis activation by hypoxia. We unfold new physiological pathways that have consequences for patients suffering from GC resistance., (© 2021 The Authors.)

Long non-coding RNAs (lncRNAs) can exhibit cell-type and cancer-type specific expression profiles, making them highly attractive as therapeutic targets. Pan-cancer RNA sequencing data revealed broad expression of the SAMMSON lncRNA in uveal melanoma (UM), the most common primary intraocular malignancy in adults. Currently, there are no effective treatments for UM patients with metastatic disease, resulting in a median survival time of 6-12 months. We aimed to investigate the therapeutic potential of SAMMSON inhibition in UM. Antisense oligonucleotide (ASO)-mediated SAMMSON inhibition impaired the growth and viability of a genetically diverse panel of uveal melanoma cell lines. These effects were accompanied by an induction of apoptosis and were recapitulated in two uveal melanoma patient derived xenograft (PDX) models through subcutaneous ASO delivery. SAMMSON pulldown revealed several candidate interaction partners, including various proteins involved in mitochondrial translation. Consequently, inhibition of SAMMSON impaired global, mitochondrial and cytosolic protein translation levels and mitochondrial function in uveal melanoma cells. The present study demonstrates that SAMMSON expression is essential for uveal melanoma cell survival. ASO-mediated silencing of SAMMSON may provide an effective treatment strategy to treat primary and metastatic uveal melanoma patients., (© 2021. The Author(s).)

Sepsis is a potentially lethal syndrome resulting from a maladaptive response to infection. Upon infection, glucocorticoids are produced as a part of the compensatory response to tolerate sepsis. This tolerance is, however, mitigated in sepsis due to a quickly induced glucocorticoid resistance at the level of the glucocorticoid receptor. Here, we show that defects in the glucocorticoid receptor signaling pathway aggravate sepsis pathophysiology by lowering lactate clearance and sensitizing mice to lactate-induced toxicity. The latter is exerted via an uncontrolled production of vascular endothelial growth factor, resulting in vascular leakage and collapse with severe hypotension, organ damage, and death, all being typical features of a lethal form of sepsis. In conclusion, sepsis leads to glucocorticoid receptor failure and hyperlactatemia, which collectively leads to a lethal vascular collapse., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The hypothalamic-pituitary-adrenal (HPA) axis forms a complex neuroendocrine system that regulates the body's response to stress such as starvation. In contrast with the glucocorticoid receptor (GR), Zinc finger and BTB domain containing 32 (ZBTB32) is a transcription factor with poorly described functional relevance in physiology. This study shows that ZBTB32 is essential for the production of glucocorticoids (GCs) in response to starvation, since ZBTB32 -/- mice fail to increase their GC production in the absence of nutrients. In terms of mechanism, GR-mediated upregulation of adrenal Scarb1 gene expression was absent in ZBTB32 -/- mice, implicating defective cholesterol import as the cause of the poor GC synthesis. These lower GC levels are further associated with aberrations in the metabolic adaptation to starvation, which could explain the progressive weight gain of ZBTB32 -/- mice. In conclusion, ZBTB32 performs a crosstalk with the GR in the metabolic adaptation to starvation via regulation of adrenal GC production., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

The cytokine TNF drives inflammatory diseases, e.g., Crohn's disease. In a mouse model of TNF-induced systemic inflammatory response syndrome (SIRS), severe impact on intestinal epithelial cells (IECs) is observed. Zinc confers complete protection in this model. We found that zinc no longer protects in animals which lack glucocorticoids (GCs), or express mutant versions of their receptor GR in IECs, nor in mice which lack gut microbiota. RNA-seq studies in IECs showed that zinc caused reduction in expression of constitutive (STAT1-induced) interferon-stimulated response (ISRE) genes and interferon regulatory factor (IRF) genes. Since some of these genes are involved in TNF-induced cell death in intestinal crypt Paneth cells, and since zinc has direct effects on the composition of the gut microbiota (such as several Staphylococcus species) and on TNF-induced Paneth cell death, we postulate a new zinc-related anti-inflammatory mechanism. Zinc modulates the gut microbiota, causing less induction of ISRE/IRF genes in crypt cells, less TNF-induced necroptosis in Paneth cells, and less fatal evasion of gut bacteria into the system., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Despite a variety of mainly school-driven prevention strategies, the number of adolescent pregnancies in Rwanda is worryingly high and is even expected to increase. The aim of this study is to empower Kirehe secondary school students aged 15-19 years old in sexual and reproductive health (SRH) by developing a peer education program., Methods: A combination of quantitative and qualitative research will be used. A pre- and post-survey will examine adolescents' knowledge and attitudes regarding SRH. In addition, six focus group interviews will explore these knowledge, attitudes but also SRH needs more in depth. Based on the obtained information, and after retrieving experts' input, a peer education program is being developed in which Midwifery students obtain training in SRH and educational skills (= first train-the-trainer module). In turn, these students will educate and train a selected group of secondary school students (= second train the trainer module). Finally, these trained in-school students can act as reliable peers for other in-school students in the context of SRH., Discussion: The project will contribute to 1) more independent and thoughtful decisions in contraception and sexual behavior, and consequently less adolescent pregnancies, and 2) the reinforcement of the Rwandan Midwifery education., Trial Registration: University of Rwanda, College of Medicine and Health Sciences, Institutional Review Board, Approval No 158/CMHS IRB/2019.

Glucocorticoid resistance (GCR) is defined as an unresponsiveness to the therapeutic effects, including the antiinflammatory ones of glucocorticoids (GCs) and their receptor, the glucocorticoid receptor (GR). It is a problem in the management of inflammatory diseases and can be congenital as well as acquired. The strong proinflammatory cytokine TNF-alpha (TNF) induces an acute form of GCR, not only in mice, but also in several cell lines: e.g., in the hepatoma cell line BWTG3, as evidenced by impaired Dexamethasone (Dex)-stimulated direct GR-dependent gene up- and down-regulation. We report that TNF has a significant and broad impact on this transcriptional performance of GR, but no impact on nuclear translocation, dimerization, or DNA binding capacity of GR. Proteome-wide proximity-mapping (BioID), however, revealed that the GR interactome was strongly modulated by TNF. One GR cofactor that interacted significantly less with the receptor under GCR conditions is p300. NFκB activation and p300 knockdown both reduced direct transcriptional output of GR whereas p300 overexpression and NFκB inhibition reverted TNF-induced GCR, which is in support of a cofactor reshuffle model. This hypothesis was supported by FRET studies. This mechanism of GCR opens avenues for therapeutic interventions in GCR diseases., Competing Interests: The authors declare no conflict of interest.

A detrimental role for matrix metalloproteinase 8 (MMP8) has been identified in several pathological conditions, e.g., lethal hepatitis and the systemic inflammatory response syndrome. Since matrix MMP8-deficient mice are protected in the above-mentioned diseases, specific MMP8 inhibitors could be of clinical value. However, targeting a specific matrix metalloproteinase remains challenging due to the strong structural homology of matrix metalloproteinases, which form a family of 25 members in mammals. Single-domain antibodies, called nanobodies, offer a range of possibilities toward therapy since they are easy to generate, express, produce, and modify, e.g., by linkage to nanobodies directed against other target molecules. Hence, we generated small MMP8-binding nanobodies, and established a proof-of-principle for developing nanobodies that inhibit matrix metalloproteinase activity. Also, we demonstrated for the first time the possibility of expressing nanobodies systemically by in vivo electroporation of the muscle and its relevance as a potential therapy in inflammatory diseases.

Background: Silent brain infarcts (SBIs) are highly prevalent in the aged population and relate to the occurrence of further stroke and dementia. Serum N-glycome levels have been previously associated with aging and they might be related as well to the presence of SBIs and age-related white matter hyperintensities., Methods and Results: We determined the serum N-glycome profile in a cohort study comprising 972 subjects and evaluated the relationship between N-glycome levels and the presence and number of SBIs and with age-related white matter hyperintensities grades, assessed by brain magnetic resonance imaging. Decreasing concentrations of bigalacto core-α-1,6-fucosylated biantennary glycan and increasing concentrations of branching α-1,3-fucosylated triantennary glycan remained as independent predictors of SBIs (odds ratio 0.4, 95% CI 0.3-0.7 and odds ratio 1.8, 95% CI 1-3.2, respectively), after controlling for the presence of age and classic vascular risk factors. A similar pattern was found to be related to an increasing number of SBIs and white matter hyperintensities grade., Conclusions: N-glycome levels might be potentially useful as biomarkers for the presence of silent cerebrovascular disease., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: Glycosylation, i.e the enzymatic addition of oligosaccharides (or glycans) to proteins and lipids, known as glycosylation, is one of the most common co-/posttranslational modifications of proteins. Many important biological roles of glycoproteins are modulated by N-linked oligosaccharides. As glucose levels can affect the pathways leading to glycosylation of proteins, we investigated whether metabolic syndrome (MS) and type 2 diabetes mellitus (T2DM), pathological conditions characterized by altered glucose levels, are associated with specific modifications in serum N-glycome., Methods: We enrolled in the study 562 patients with Type 2 Diabetes Mellitus (T2DM) (mean age 65.6±8.2 years) and 599 healthy control subjects (CTRs) (mean age, 58.5±12.4 years). N-glycome was evaluated in serum glycoproteins., Results: We found significant changes in N-glycan composition in the sera of T2DM patients. In particular, α(1,6)-linked arm monogalactosylated, core-fucosylated diantennary N-glycans (NG1(6)A2F) were significantly reduced in T2DM compared with CTR subjects. Importantly, they were equally reduced in diabetic patients with and without complications (P<0.001) compared with CTRs. Macro vascular-complications were found to be related with decreased levels of NG1(6)A2F. In addition, NG1(6)A2F and NG1(3)A2F, identifying, respectively, monogalactosylated N-glycans with α(1,6)- and α(1,3)-antennary galactosylation, resulted strongly correlated with most MS parameters. The plasmatic levels of these two glycans were lower in T2DM as compared to healthy controls, and even lower in patients with complications and MS, that is the extreme "unhealthy" phenotype (T2DM+ with MS)., Conclusions: Imbalance of glycosyltransferases, glycosidases and sugar nucleotide donor levels is able to cause the structural changes evidenced by our findings. Serum N-glycan profiles are thus sensitive to the presence of diabetes and MS. Serum N-glycan levels could therefore provide a non-invasive alternative marker for T2DM and MS.