EDI3 knockdown in ER-HER2+ breast cancer cells reduces tumor burden and improves survival in two mouse models of experimental metastasis.

BACKGROUND: Despite progress understanding the mechanisms underlying tumor spread, metastasis remains a clinical challenge. We identified the choline-producing glycerophosphodiesterase, EDI3 and reported its association with metastasis-free survival in endometrial cancer. We also observed that silencing EDI3 slowed cell migration and other cancer-relevant phenotypes in vitro. Recent work demonstrated high EDI3 expression in ER-HER2+ breast cancer compared to the other molecular subtypes. Silencing EDI3 in ER-HER2+ cells significantly reduced cell survival in vitro and decreased tumor growth in vivo. However, a role for EDI3 in tumor metastasis in this breast cancer subtype was not explored. Therefore, in the present work we investigate whether silencing EDI3 in ER-HER2+ breast cancer cell lines alters phenotypes linked to metastasis in vitro, and metastasis formation in vivo using mouse models of experimental metastasis. METHODS: To inducibly silence EDI3, luciferase-expressing HCC1954 cells were transduced with lentiviral particles containing shRNA oligos targeting EDI3 under the control of doxycycline. The effect on cell migration, adhesion, colony formation and anoikis was determined in vitro, and significant findings were confirmed in a second ER-HER2+ cell line, SUM190PT. Doxycycline-induced HCC1954-luc shEDI3 cells were injected into the tail vein or peritoneum of immunodeficient mice to generate lung and peritoneal metastases, respectively and monitored using non-invasive bioluminescence imaging. Metabolite levels in cells and tumor tissue were analyzed using targeted mass spectrometry and MALDI mass spectrometry imaging (MALDI-MSI), respectively. RESULTS: Inducibly silencing EDI3 reduced cell adhesion and colony formation, as well as increased susceptibility to anoikis in HCC1954-luc cells, which was confirmed in SUM190PT cells. No influence on cell migration was observed. Reduced luminescence was seen in lungs and peritoneum of mice injected with cells expressing less EDI3 after tail vein and intraperitoneal injection, respectively, indicative of reduced metastasis. Importantly, mice injected with EDI3-silenced cells survived longer. Closer analysis of the peritoneal organs revealed that silencing EDI3 had no effect on metastatic organotropism but instead reduced metastatic burden. Finally, metabolic analyses revealed significant changes in choline and glycerophospholipid metabolites in cells and in pancreatic metastases in vivo. CONCLUSIONS: Reduced metastasis upon silencing supports EDI3's potential as a treatment target in metastasizing ER-HER2+ breast cancer.

Activation of the nonneuronal cholinergic cardiac system by hypoxic preconditioning protects isolated adult cardiomyocytes from hypoxia/reoxygenation injury.

Activation of the vagus nerve mediates cardioprotection and attenuates myocardial ischemia/reperfusion (I/R) injury. In response to vagal activation, acetylcholine (ACh) is released from the intracardiac nervous system (ICNS) and activates intracellular cardioprotective signaling cascades. Recently, however, a nonneuronal cholinergic cardiac system (NNCCS) in cardiomyocytes has been described as an additional source of ACh. To investigate whether the NNCCS mediates cardioprotection in the absence of vagal and ICNS activation, we used a reductionist approach of isolated adult rat ventricular cardiomyocytes without neuronal cells, using hypoxic preconditioning (HPC) as a protective stimulus. Adult rat ventricular cardiomyocytes were isolated, the absence of neuronal cells was confirmed, and HPC was induced by 10/20 min hypoxia/reoxygenation (H/R) before subjection to 30/5 min H/R to simulate I/R injury. Cardiomyocyte viability was assessed by trypan blue staining at baseline and after HPC+H/R or H/R. Intra- and extracellular ACh was quantified using liquid chromatography-coupled mass spectrometry at baseline, after HPC, after hypoxia, and after reoxygenation, respectively. In a subset of experiments, muscarinic and nicotinic ACh receptor (m- and nAChR) antagonists were added during HPC or during H/R. Cardiomyocyte viability at baseline (69 ± 4%) was reduced by H/R (10 ± 3%). With HPC, cardiomyocyte viability was preserved after H/R (25 ± 6%). Intra- and extracellular ACh increased during hypoxia; HPC further increased both intra- and extracellular ACh (from 0.9 ± 0.7 to 1.5 ± 1.0 nmol/mg; from 0.7 ± 0.6 to 1.1 ± 0.7 nmol/mg, respectively). The addition of mAChR and nAChR antagonists during HPC had no impact on HPC's protection; however, protection was abrogated when antagonists were added during H/R (cardiomyocyte viability after H/R: 23 ± 5%; 13 ± 4%). In conclusion, activation of the NNCCS is involved in cardiomyocyte protection; HPC increases intra- and extracellular ACh during H/R, and m- and nAChRs are causally involved in HPC's cardiomyocyte protection during H/R. The interplay between upstream ICNS activation and NNCCS activation in myocardial cholinergic metabolism and cardioprotection needs to be investigated in future studies.NEW & NOTEWORTHY The intracardiac nervous system is considered to be involved in ischemic conditioning's cardioprotection through the release of acetylcholine (ACh). However, we demonstrate that hypoxic preconditioning (HPC) protects from hypoxia/reoxygenation injury and increases intra- and extracellular ACh during hypoxia in isolated adult ventricular rat cardiomyocytes. HPC's protection involves cardiomyocyte muscarinic and nicotinic ACh receptor activation. Thus, besides the intracardiac nervous system, a nonneuronal cholinergic cardiac system may also be causally involved in cardiomyocyte protection by ischemic conditioning.

Immediate and Delayed Salivary Cytokine Responses during Repeated Exposures to Cold Pressor Stress.

INTRODUCTION: Excessive stress is increasingly recognized as an important trigger of many diseases prevalent in modern societies, and monitoring such stress-related effects could aid prevention. The measurement of salivary markers of inflammation is emerging as a promising tool to non-invasively quantify stress' effects on immune processes in everyday life and thereby detect early aberrations before the manifestation of serious health problems. However, more laboratory-controlled research is needed in order to establish the timescale and determinants of salivary cytokine responses to acute stress. METHODS: We repeatedly exposed participants to Cold Pressor Stress Test (CPT) or a control procedure and measured a wide array of salivary cytokines as well as subjective, cardiovascular, and cortisol stress reactions. CPT exposure was repeated every 15 min, 3 times in total, with a duration of 3 min each. Saliva was sampled immediately after the first two exposures as well as in 15-min intervals until 60 min after the onset of the first intervention. RESULTS: We found that many cytokines were detectable in saliva. Specific stress effects were limited to IL-8 and IL-6, however, which decreased immediately or 15 min after stress onset, respectively. Moreover, IL-8 was negatively correlated to cortisol output in the stress but not in the control group. Significant increases were also observed in salivary TNFα and IFNγ; however, these effects were similar under both stress and control conditions. DISCUSSION: Our results show that particular salivary cytokines may be sensitive to immediate effects of acute CPT-induced stress and also highlight the importance of employing control procedures to discern stress effects from unrelated variations in salivary cytokines.

A serum microRNA sequence reveals fragile X protein pathology in amyotrophic lateral sclerosis.

Knowledge about converging disease mechanisms in the heterogeneous syndrome amyotrophic lateral sclerosis (ALS) is rare, but may lead to therapies effective in most ALS cases. Previously, we identified serum microRNAs downregulated in familial ALS, the majority of sporadic ALS patients, but also in presymptomatic mutation carriers. A 5-nucleotide sequence motif (GDCGG; D = G, A or U) was strongly enriched in these ALS-related microRNAs. We hypothesized that deregulation of protein(s) binding predominantly to this consensus motif was responsible for the ALS-linked microRNA fingerprint. Using microRNA pull-down assays combined with mass spectrometry followed by extensive biochemical validation, all members of the fragile X protein family, FMR1, FXR1 and FXR2, were identified to directly and predominantly interact with GDCGG microRNAs through their structurally disordered RGG/RG domains. Preferential association of this protein family with ALS-related microRNAs was confirmed by in vitro binding studies on a transcriptome-wide scale. Immunohistochemistry of lumbar spinal cord revealed aberrant expression level and aggregation of FXR1 and FXR2 in C9orf72- and FUS-linked familial ALS, but also patients with sporadic ALS. Further analysis of ALS autopsies and induced pluripotent stem cell-derived motor neurons with FUS mutations showed co-aggregation of FXR1 with FUS. Hence, our translational approach was able to take advantage of blood microRNAs to reveal CNS pathology, and suggests an involvement of the fragile X-related proteins in familial and sporadic ALS already at a presymptomatic stage. The findings may uncover disease mechanisms relevant to many patients with ALS. They furthermore underscore the systemic, extra-CNS aspect of ALS.

CHCHD10 mutations p.R15L and p.G66V cause motoneuron disease by haploinsufficiency.

Mutations in the mitochondrially located protein CHCHD10 cause motoneuron disease by an unknown mechanism. In this study, we investigate the mutations p.R15L and p.G66V in comparison to wild-type CHCHD10 and the non-pathogenic variant p.P34S in vitro, in patient cells as well as in the vertebrate in vivo model zebrafish. We demonstrate a reduction of CHCHD10 protein levels in p.R15L and p.G66V mutant patient cells to approximately 50%. Quantitative real-time PCR revealed that expression of CHCHD10 p.R15L, but not of CHCHD10 p.G66V, is already abrogated at the mRNA level. Altered secondary structure and rapid protein degradation are observed with regard to the CHCHD10 p.G66V mutant. In contrast, no significant differences in expression, degradation rate or secondary structure of non-pathogenic CHCHD10 p.P34S are detected when compared with wild-type protein. Knockdown of CHCHD10 expression in zebrafish to about 50% causes motoneuron pathology, abnormal myofibrillar structure and motility deficits in vivo. Thus, our data show that the CHCHD10 mutations p.R15L and p.G66V cause motoneuron disease primarily based on haploinsufficiency of CHCHD10.

Double genetic disruption of lactate dehydrogenases A and B is required to ablate the "Warburg effect" restricting tumor growth to oxidative metabolism.

Increased glucose consumption distinguishes cancer cells from normal cells and is known as the "Warburg effect" because of increased glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and believed to be the major enzyme responsible for pyruvate-to-lactate conversion. To elucidate its role in tumor growth, we disrupted both the LDHA and LDHB genes in two cancer cell lines (human colon adenocarcinoma and murine melanoma cells). Surprisingly, neither LDHA nor LDHB knockout strongly reduced lactate secretion. In contrast, double knockout (LDHA/B-DKO) fully suppressed LDH activity and lactate secretion. Furthermore, under normoxia, LDHA/B-DKO cells survived the genetic block by shifting their metabolism to oxidative phosphorylation (OXPHOS), entailing a 2-fold reduction in proliferation rates in vitro and in vivo compared with their WT counterparts. Under hypoxia (1% oxygen), however, LDHA/B suppression completely abolished in vitro growth, consistent with the reliance on OXPHOS. Interestingly, activation of the respiratory capacity operated by the LDHA/B-DKO genetic block as well as the resilient growth were not consequences of long-term adaptation. They could be reproduced pharmacologically by treating WT cells with an LDHA/B-specific inhibitor (GNE-140). These findings demonstrate that the Warburg effect is not only based on high LDHA expression, as both LDHA and LDHB need to be deleted to suppress fermentative glycolysis. Finally, we demonstrate that the Warburg effect is dispensable even in aggressive tumors and that the metabolic shift to OXPHOS caused by LDHA/B genetic disruptions is responsible for the tumors' escape and growth.

Identification of ADGRE5 as discriminating MYC target between Burkitt lymphoma and diffuse large B-cell lymphoma.

BACKGROUND: MYC is a heterogeneously expressed transcription factor that plays a multifunctional role in many biological processes such as cell proliferation and differentiation. It is also associated with many types of cancer including the malignant lymphomas. There are two types of aggressive B-cell lymphoma, namely Burkitt lymphoma (BL) and a subgroup of diffuse large cell lymphoma (DLBCL), which both carry MYC translocations and overexpress MYC but both differ significantly in their clinical outcome. In DLBCL, MYC translocations are associated with an aggressive behavior and poor outcome, whereas MYC-positive BL show a superior outcome. METHODS: To shed light on this phenomenon, we investigated the different modes of actions of MYC in aggressive B-cell lymphoma cell lines subdivided into three groups: (i) MYC-positive BL, (ii) DLBCL with MYC translocation (DLBCLpos) and (iii) DLBCL without MYC translocation (DLBCLneg) for control. In order to identify genome-wide MYC-DNA binding sites a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) was performed. In addition, ChIP-Seq for H3K4me3 was used for determination of genomic regions accessible for transcriptional activity. These data were supplemented with gene expression data derived from RNA-Seq. RESULTS: Bioinformatics integration of all data sets revealed different MYC-binding patterns and transcriptional profiles in MYC-positive BL and DLBCL cell lines indicating different functional roles of MYC for gene regulation in aggressive B-cell lymphomas. Based on this multi-omics analysis we identified ADGRE5 (alias CD97) - a member of the EGF-TM7 subfamily of adhesion G protein-coupled receptors - as a MYC target gene, which is specifically expressed in BL but not in DLBCL regardless of MYC translocation. CONCLUSION: Our study describes a diverse genome-wide MYC-DNA binding pattern in BL and DLBCL cell lines with and without MYC translocations. Furthermore, we identified ADREG5 as a MYC target gene able to discriminate between BL and DLBCL irrespectively of the presence of MYC breaks in DLBCL. Since ADGRE5 plays an important role in tumor cell formation, metastasis and invasion, it might also be instrumental to better understand the different pathobiology of BL and DLBCL and help to explain discrepant clinical characteristics of BL and DLBCL.

Genetic determinants of steatosis and fibrosis progression in paediatric non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children and adolescents today. In comparison with adult disease, paediatric NAFLD may show a periportal localization, which is associated with advanced fibrosis. This study aimed to assess the role of genetic risk variants for histological disease pattern and severity in childhood NAFLD. METHODS: We studied 14 single nucleotide polymorphisms (SNP) in a cohort of 70 adolescents with biopsy-proven NAFLD. Genotype was compared to an adult control cohort (n = 200) and analysed in relation to histological disease severity and liver tissue proteomics. RESULTS: Three of the 14 SNPs were significantly associated with paediatric NAFLD after FDR adjustment, rs738409 (PNPLA3, P = 2.80 × 10-06 ), rs1044498 (ENPP1, P = 0.0091) and rs780094 (GCKR, P = 0.0281). The severity of steatosis was critically associated with rs738409 (OR=3.25; 95% CI: 1.72-6.52, FDR-adjusted P = 0.0070). The strongest variants associated with severity of fibrosis were rs1260326, rs780094 (both GCKR) and rs659366 (UCP2). PNPLA3 was associated with a portal pattern of steatosis, inflammation and fibrosis. Proteome profiling revealed decreasing levels of GCKR protein with increasing carriage of the rs1260326/rs780094 minor alleles and downregulation of the retinol pathway in rs738409 G/G carriers. Computational metabolic modelling highlighted functional relevance of PNPLA3, GCKR and UCP2 for NAFLD development. CONCLUSIONS: This study provides evidence for the role of PNPLA3 as a determinant of portal NAFLD localization and severity of portal fibrosis in children and adolescents, the risk variant being associated with an impaired hepatic retinol metabolism.

Dysregulation of a novel miR-1825/TBCB/TUBA4A pathway in sporadic and familial ALS.

Genetic and functional studies suggest diverse pathways being affected in the neurodegenerative disease amyotrophic lateral sclerosis (ALS), while knowledge about converging disease mechanisms is rare. We detected a downregulation of microRNA-1825 in CNS and extra-CNS system organs of both sporadic (sALS) and familial ALS (fALS) patients. Combined transcriptomic and proteomic analysis revealed that reduced levels of microRNA-1825 caused a translational upregulation of tubulin-folding cofactor b (TBCB). Moreover, we found that excess TBCB led to depolymerization and degradation of tubulin alpha-4A (TUBA4A), which is encoded by a known ALS gene. Importantly, the increase in TBCB and reduction of TUBA4A protein was confirmed in brain cortex tissue of fALS and sALS patients, and led to motor axon defects in an in vivo model. Our discovery of a microRNA-1825/TBCB/TUBA4A pathway reveals a putative pathogenic cascade in both fALS and sALS extending the relevance of TUBA4A to a large proportion of ALS cases.

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations.

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

Comprehensive Metaboproteomics of Burkitt's and Diffuse Large B-Cell Lymphoma Cell Lines and Primary Tumor Tissues Reveals Distinct Differences in Pyruvate Content and Metabolism.

Burkitt's lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are pathologically and clinically distinct subtypes of aggressive non-Hodgkin B-cell lymphoma. To learn more about their biology, we employed metabolomic and proteomic methods to study both established cell lines as well as cryopreserved and formalin-fixed paraffin-embedded (FFPE) tissue sections of BL and DLBCL. Strikingly, NMR analyses revealed DLBCL cell lines to produce and secrete significantly (padj = 1.72 × 10-22) more pyruvic acid than BL cell lines. This finding could be reproduced by targeted GC/MS analyses of cryopreserved tissue sections of BL and DLBCL cases. Enrichment analysis of an overlapping set of N = 2315 proteins, that had been quantified by nanoLC-SWATH-MS in BL and DLBCL cultured cells and cryosections, supported the observed difference in pyruvic acid content, as glycolysis and pyruvate metabolism were downregulated, while one-carbon metabolism was upregulated in BL compared to DLBCL. Furthermore, 92.1% of the overlapping significant proteins showed the same direction of regulation in cryopreserved and FFPE material. Proteome data are available via ProteomeXchange with identifier PXD004936.

Causal Modeling of Cancer-Stromal Communication Identifies PAPPA as a Novel Stroma-Secreted Factor Activating NFκB Signaling in Hepatocellular Carcinoma.

Inter-cellular communication with stromal cells is vital for cancer cells. Molecules involved in the communication are potential drug targets. To identify them systematically, we applied a systems level analysis that combined reverse network engineering with causal effect estimation. Using only observational transcriptome profiles we searched for paracrine factors sending messages from activated hepatic stellate cells (HSC) to hepatocellular carcinoma (HCC) cells. We condensed these messages to predict ten proteins that, acting in concert, cause the majority of the gene expression changes observed in HCC cells. Among the 10 paracrine factors were both known and unknown cancer promoting stromal factors, the former including Placental Growth Factor (PGF) and Periostin (POSTN), while Pregnancy-Associated Plasma Protein A (PAPPA) was among the latter. Further support for the predicted effect of PAPPA on HCC cells came from both in vitro studies that showed PAPPA to contribute to the activation of NFκB signaling, and clinical data, which linked higher expression levels of PAPPA to advanced stage HCC. In summary, this study demonstrates the potential of causal modeling in combination with a condensation step borrowed from gene set analysis [Model-based Gene Set Analysis (MGSA)] in the identification of stromal signaling molecules influencing the cancer phenotype.

Chronic Toxoplasma gondii Infection Modulates Hearing Ability across the Adult Life Span.

While several studies have shown associations between hearing disorders and congenital toxoplasmosis, the present study investigated the impact of chronic, latent Toxoplasma gondii (T. gondii) infection on hearing loss. We used a regression analysis to explore whether latent T. gondii infection modulates changes in hearing thresholds over an age range from 20 to 70 years. We analyzed audiometric data of 162 T. gondii IgG-positive and 430 T. gondii-negative participants, collected in the Dortmund Vital Study (DVS, ClinicalTrials.gov Identifier: NCT05155397), a prospective study on healthy cognitive aging. The regression analysis indicated that latent toxoplasmosis was associated with an accelerated development in hearing loss over the observed age range. Hearing loss was less frequent in IgG-positive than in IgG-negative participants up to the age of about 40 for a low (0.125-1 kHz)-frequency range. For high (2-8 kHz) frequencies, this pattern reversed for ages above 65 years. We discuss these findings on hearing function in the context of a recently proposed model, suggesting that latent toxoplasmosis can differentially affect brain functions across a lifespan.

Presenilin 1/γ-secretase modulates P-cadherin processing and influences cell adhesion in oral squamous cell carcinoma cell lines.

P-cadherin belongs to a family of Ca(2+)-dependent homophilic cell-cell adhesion proteins that are important for correct cellular localization and tissue integrity in the oral epithelium. P-cadherin is only expressed in the basal and suprabasal cell layers of the oral epithelium, but in advanced oral squamous cell carcinoma (OSCC), a reduced membranous and an enhanced cytoplasmic truncated P-cadherin level is observed. In this study, we investigated the impact of presenilin (PS) 1/γ-secretase on P-cadherin processing in OSCC. Western blot analyses showed an enhanced PS1 expression in OSCC cell lines and in primary oral keratinocytes (POK) isolated from primary OSCC tissue (OSCC POK) compared with POKs isolated from normal oral mucosa. Immunocytochemical stainings and co-immunoprecipitation experiments revealed a cytoplasmic colocalization and a direct interaction of P-cadherin and PS1 in OSCC POKs. Blocking of PS1/γ-secretase activity by the PS1/γ-secretase inhibitors and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, another specific γ-secretase inhibitor yielded a 100 kDa P-cadherin band on western blots of OSCC cell line extracts. Small interfering RNA knockdown of PS1 equally generated a 100 kDa P-cadherin form in OSCC POKs. Mass spectrometry analyses and experiments with the glycosylation inhibitor tunicamycin characterized the appearing 100 kDa P-cadherin band as the unglycosylated full-length form of P-cadherin. On the functional level, cell attachment assays demonstrated an enhanced cell adhesion after PS1/γ-secretase inhibition only in the transiently P-cadherin expressing OSCC cell line PCI52 but not in the PCI52 control cells. In summary, our results show that PS1/γ-secretase contributes to P-cadherin processing and to reduced cell adhesion in OSCC.

Correction: Degen et al. Citrinin Exposure in Germany: Urine Biomarker Analysis in Children and Adults. Toxins 2023, 15, 26.

In the original publication [...].

Bladder cancer course, four genetic high-risk variants, and histopathological findings.

Urinary bladder cancer, a smoking and occupation related disease, was subject of several genome-wide association studies (GWAS). However, studies on the course of the disease based on GWAS findings differentiating between muscle invasive bladder cancer (MIBC) and non-muscle invasive bladder cancer (NMIBC) are rare. Thus we investigated 4 single nucleotide polymorphisms (SNPs) detected in GWAS, related to the genes coding for TACC3 (transforming, acidic coiled-coil containing protein 3), for FGFR3 (fibroblast growth factor receptor 3), for PSCA (prostate stem cell antigen) and the genes coding for CBX6 (chromobox homolog 6) and APOBEC3A (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A). This study is based on 712 bladder cancer patients and 875 controls from 3 different case control studies in Germany. The 4 SNPs of interest (PSCA rs2294008 and rs2978974, FGFR3-TACC3 rs798766, and CBX6-APOBEC3A rs1014971) were determined by real-time polymerase chain reaction. The distribution of the 4 SNPs does not vary significantly between cases and controls in the entire study group and in the 3 local subgroups, including two former highly industrialized areas and a region without such history. Also, no significant differences in the bladder cancer subgroups of MIBC and NMIBC were observed. The 4 investigated SNPs do not noticeably contribute differently to the bladder cancer risk for the bladder cancer subgroups of MIBC and NMIBC.

A Systematic Analysis of Biological, Sociodemographic, Psychosocial, and Lifestyle Factors Contributing to Work Ability Across the Working Life Span: Cross-sectional Study.

BACKGROUND: As employees age, their physical and mental abilities decline and work ability decreases, enhancing the risk for long-term sick leave or even premature retirement. However, the relative impact of biological and environmental determinants on work ability with increasing age is poorly understood in terms of their complexity. OBJECTIVE: Previous research has shown relationships between work ability and job and individual resources, as well as specific demographic and lifestyle-related variables. However, other potentially important predictors of work ability remain unexplored, such as personality traits and biological determinants, including cardiovascular, metabolic, immunological, and cognitive abilities or psychosocial factors. Our aim was to systematically evaluate a wide range of factors to extract the most crucial predictors of low and high work ability across the working life span. METHODS: As part of the Dortmund Vital Study, 494 participants from different occupational sectors, aged between 20 and 69 years, completed the Work Ability Index (WAI) assessing employee's mental and physical resources. A total of 30 sociodemographic variables were grouped into 4 categories (social relationships, nutrition and stimulants, education and lifestyle, and work related), and 80 biological and environmental variables were grouped into 8 domains (anthropometric, cardiovascular, metabolic, immunologic, personality, cognitive, stress related, and quality of life) and have been related to the WAI. RESULTS: Using the analyses, we extracted important sociodemographic factors influencing work ability, such as education, social activities, or sleep quality, and identified age-dependent and age-independent determinants of work ability. Regression models explained up to 52% of the WAI variance. Negative predictors of work ability were chronological and immunological age, immunological inefficiency, BMI, neuroticism, psychosocial stress, emotional exhaustion, demands from work, daily cognitive failures, subclinical depression, and burnout symptoms. Positive predictors were maximum heart rate during ergometry, normal blood pressure, hemoglobin and monocyte concentration, weekly physical activity, commitment to the company, pressure to succeed, and good quality of life. CONCLUSIONS: The identified biological and environmental risk factors allowed us to evaluate work ability in its complexity. Policy makers, employers, and occupational safety and health personnel should consider the modifiable risk factors we identified to promote healthy aging at work through focused physical, dietary, cognitive, and stress-reduced preventive programs, in addition to well-balanced working conditions. This may also increase the quality of life, commitment to the job, and motivation to succeed, which are important factors to maintain or even enhance work ability in the aging workforce and to prevent early retirement. TRIAL REGISTRATION: ClinicalTrials.gov NCT05155397; https://clinicaltrials.gov/ct2/show/NCT05155397. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/32352.

Cognitive Functions, Neurotransmitter Alterations, and Hippocampal Microstructural Changes in Mice Caused by Feeding on Western Diet.

Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) is the most common chronic liver disease in Western countries. It is becoming increasingly evident that peripheral organ-centered inflammatory diseases, including liver diseases, are linked with brain dysfunctions. Therefore, this study aims to unravel the effect of MASLD on brain histology, cognitive functions, and neurotransmitters. For this purpose, mice fed for 48 weeks on standard (SD) or Western diet (WD) were evaluated by behavioral tests, followed by sacrifice and analysis of the liver-brain axis including histopathology, immunohistochemistry, and biochemical analyses. Histological analysis of the liver showed features of Metabolic Dysfunction-Associated Steatohepatitis (MASH) in the WD-fed mice including lipid droplet accumulation, inflammation, and fibrosis. This was accompanied by an elevation of transaminase and alkaline phosphatase activities, increase in inflammatory cytokine and bile acid concentrations, as well as altered amino acid concentrations in the blood. Interestingly, compromised blood capillary morphology coupled with astrogliosis and microgliosis were observed in brain hippocampus of the WD mice, indicating neuroinflammation or a disrupted neurovascular unit. Moreover, attention was impaired in WD-fed mice along with the observations of impaired motor activity and balance, enhanced anxiety, and stereotyped head-twitch response (HTR) behaviors. Analysis of neurotransmitters and modulators including dopamine, serotonin, GABA, glutamate, and acetylcholine showed region-specific dysregulation in the brain of the WD-fed mice. In conclusion, the induction of MASH in mice is accompanied by the alteration of cellular morphology and neurotransmitter expression in the brain, associated with compromised cognitive functions.

Inhibiting the glycerophosphodiesterase EDI3 in ER-HER2+ breast cancer cells resistant to HER2-targeted therapy reduces viability and tumour growth.

BACKGROUND: Intrinsic or acquired resistance to HER2-targeted therapy is often a problem when small molecule tyrosine kinase inhibitors or antibodies are used to treat patients with HER2 positive breast cancer. Therefore, the identification of new targets and therapies for this patient group is warranted. Activated choline metabolism, characterized by elevated levels of choline-containing compounds, has been previously reported in breast cancer. The glycerophosphodiesterase EDI3 (GPCPD1), which hydrolyses glycerophosphocholine to choline and glycerol-3-phosphate, directly influences choline and phospholipid metabolism, and has been linked to cancer-relevant phenotypes in vitro. While the importance of choline metabolism has been addressed in breast cancer, the role of EDI3 in this cancer type has not been explored. METHODS: EDI3 mRNA and protein expression in human breast cancer tissue were investigated using publicly-available Affymetrix gene expression microarray datasets (n = 540) and with immunohistochemistry on a tissue microarray (n = 265), respectively. A panel of breast cancer cell lines of different molecular subtypes were used to investigate expression and activity of EDI3 in vitro. To determine whether EDI3 expression is regulated by HER2 signalling, the effect of pharmacological inhibition and siRNA silencing of HER2, as well as the influence of inhibiting key components of signalling cascades downstream of HER2 were studied. Finally, the influence of silencing and pharmacologically inhibiting EDI3 on viability was investigated in vitro and on tumour growth in vivo. RESULTS: In the present study, we show that EDI3 expression is highest in ER-HER2 + human breast tumours, and both expression and activity were also highest in ER-HER2 + breast cancer cell lines. Silencing HER2 using siRNA, as well as inhibiting HER2 signalling with lapatinib decreased EDI3 expression. Pathways downstream of PI3K/Akt/mTOR and GSK3ß, and transcription factors, including HIF1α, CREB and STAT3 were identified as relevant in regulating EDI3 expression. Silencing EDI3 preferentially decreased cell viability in the ER-HER2 + cells. Furthermore, silencing or pharmacologically inhibiting EDI3 using dipyridamole in ER-HER2 + cells resistant to HER2-targeted therapy decreased cell viability in vitro and tumour growth in vivo. CONCLUSIONS: Our results indicate that EDI3 may be a potential novel therapeutic target in patients with HER2-targeted therapy-resistant ER-HER2 + breast cancer that should be further explored.

Early changes in the liver-soluble proteome from mice fed a nonalcoholic steatohepatitis inducing diet.

Despite the increasing incidence of nonalcoholic steatohepatitis (NASH) with the rise in lifestyle-related diseases such as the metabolic syndrome, little is known about the changes in the liver proteome that precede the onset of inflammation and fibrosis. Here, we investigated early changes in the liver-soluble proteome of female C57BL/6N mice fed an NASH-inducing diet by 2D-DIGE and nano-HPLC-MS/MS. In parallel, histology and measurements of hepatic content of triglycerides, cholesterol and intermediates of the methionine cycle were performed. Hepatic steatosis manifested itself after 2 days of feeding, albeit significant changes in the liver-soluble proteome were not evident before day 10 in the absence of inflammatory or fibrotic signs. Proteomic alterations affected mainly energy and amino acid metabolism, detoxification processes, urea cycle, and the one-carbon/S-adenosylmethionine pathways. Additionally, intermediates of relevant affected pathways were quantified from liver tissue, confirming the findings from the proteomic analysis.