The underlying molecular mechanisms of the vasculoprotective effects of physical exercise are incompletely understood. Telomere erosion is a central component of aging, and telomere-associated ...proteins regulate cellular senescence and survival. This study examines the effects of exercising on vascular telomere biology and endothelial apoptosis in mice and the effects of long-term endurance training on telomere biology in humans.
C57/Bl6 mice were randomized to voluntary running or no running wheel conditions for 3 weeks. Exercise upregulated telomerase activity in the thoracic aorta and in circulating mononuclear cells compared with sedentary controls, increased vascular expression of telomere repeat-binding factor 2 and Ku70, and reduced the expression of vascular apoptosis regulators such as cell-cycle-checkpoint kinase 2, p16, and p53. Mice preconditioned by voluntary running exhibited a marked reduction in lipopolysaccharide-induced aortic endothelial apoptosis. Transgenic mouse studies showed that endothelial nitric oxide synthase and telomerase reverse transcriptase synergize to confer endothelial stress resistance after physical activity. To test the significance of these data in humans, telomere biology in circulating leukocytes of young and middle-aged track and field athletes was analyzed. Peripheral blood leukocytes isolated from endurance athletes showed increased telomerase activity, expression of telomere-stabilizing proteins, and downregulation of cell-cycle inhibitors compared with untrained individuals. Long-term endurance training was associated with reduced leukocyte telomere erosion compared with untrained controls.
Physical activity regulates telomere-stabilizing proteins in mice and in humans and thereby protects from stress-induced vascular apoptosis.
It has been well-established that mutations in BRCA1 and BRCA2, compromising functions in DNA double-strand break repair (DSBR), confer hereditary breast and ovarian cancer risk. Importantly, ...mutations in these genes explain only a minor fraction of the hereditary risk and of the subset of DSBR deficient tumors. Our screening efforts identified two truncating germline mutations in the gene encoding the BRCA1 complex partner ABRAXAS1 in German early-onset breast cancer patients. To unravel the molecular mechanisms triggering carcinogenesis in these carriers of heterozygous mutations, we examined DSBR functions in patient-derived lymphoblastoid cells (LCLs) and in genetically manipulated mammary epithelial cells. By use of these strategies we were able to demonstrate that these truncating ABRAXAS1 mutations exerted dominant effects on BRCA1 functions. Interestingly, we did not observe haploinsufficiency regarding homologous recombination (HR) proficiency (reporter assay, RAD51-foci, PARP-inhibitor sensitivity) in mutation carriers. However, the balance was shifted to use of mutagenic DSBR-pathways. The dominant effect of truncated ABRAXAS1 devoid of the C-terminal BRCA1 binding site can be explained by retention of the N-terminal interaction sites for other BRCA1-A complex partners like RAP80. In this case BRCA1 was channeled from the BRCA1-A to the BRCA1-C complex, which induced single-strand annealing (SSA). Further truncation, additionally deleting the coiled-coil region of ABRAXAS1, unleashed excessive DNA damage responses (DDRs) de-repressing multiple DSBR-pathways including SSA and non-homologous end-joining (NHEJ). Our data reveal de-repression of low-fidelity repair activities as a common feature of cells from patients with heterozygous mutations in genes encoding BRCA1 and its complex partners.
Postmenopausal bone loss stems from the inability of osteoblastic activity to match the increase in osteoclastic bone resorption induced by estrogen deficiency. However, the mechanism that uncouples ...osteoblast from osteoclast activities remains unexplained. We show that ovariectomy enhances the production of the osteoclastogenic cytokine IL-7, and that its neutralization in vivo prevents ovariectomy-induced bone loss. Surprisingly, serum osteocalcin levels, a biochemical marker of bone formation, suggested that the bone-sparing effects of IL-7 neutralization were due not only to inhibition of bone resorption, but also to stimulation of bone formation. Consistent with these data, addition of IL-7 to neonatal calvarial organ cultures blocked new bone formation, and injection of IL-7 into mice in vivo inhibited bone formation as measured by calcein incorporation into long bones. The antianabolic effects of IL-7 were consistent with an observed downregulation of the osteoblast-specific transcription factor core-binding factor alpha1/Runx2. Thus, because it targets both the osteoclast and the osteoblast pathways, IL-7 is central to the altered bone turnover characteristic of estrogen deficiency.
In vivo studies have shown T cells to be central to the mechanism by which estrogen deficiency induces bone loss, but the mechanism involved remains, in part, undefined. In vitro, T cells from ...ovariectomized mice produce increased amounts of tumor necrosis factor (TNF), which augments receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. However, both the mechanism and the relevance of this phenomenon in vivo remain to be established. In this study, we found that ovariectomy increased the number of bone marrow T cell-producing TNF without altering production of TNF per T cell. Attesting to the essential contribution of TNF, ovariectomy induced rapid bone loss in wild type (wt) mice but failed to do so in TNF-deficient TNF-/-mice. Furthermore, ovariectomy induced bone loss, which was absent in T cell-deficient nude mice, was restored by adoptive transfer of wt T cells, but not by reconstitution with T cells from TNF-/-mice. These findings demonstrate the key causal role of T cell-produced TNF in the bone loss after estrogen withdrawal. Finally, ovariectomy caused bone loss in wt mice and in mice lacking p75 TNF receptor but failed to do so in mice lacking the p55 TNF receptor. These findings demonstrate that enhanced T cell production of TNF resulting from increased bone marrow T cell number is a key mechanism by which estrogen deficiency induces bone loss in vivo. The data also demonstrate that the bone-wasting effect of TNF in vivo is mediated by the p55 TNF receptor.
IL-7, a powerful lymphopoietic cytokine, is elevated in rheumatoid arthritis (RA) and known to induce bone loss when administered in vivo. IL-7 has been suggested to induce bone loss, in part, by ...stimulating the proliferation of B220+ cells, a population capable of acting as early osteoclast (OC) precursors. However, the mechanism by which IL-7 leads to differentiation of precursors into mature OCs remains unknown. We previously reported that, in vitro, IL-7 up-regulated T cell cytokines including receptor activator of nuclear factor κB ligand (RANKL). To demonstrate the importance of T cells to the bone-wasting effect of IL-7 in vivo, we have now examined IL-7-induced bone loss in T cell-deficient nude mice. We show that T cell-replete mice undergo significant osteoclastic bone loss after IL-7 administration, concurrent with induction of RANKL and tumor necrosis factor α (TNF-α) secretion by splenic T cells. In contrast, nude mice were resistant to IL-7-induced bone loss and showed no detectable increase in either RANKL or TNF-α, despite an up-regulation of B220+ cells. Importantly, T cell adoptive transfer into nude mice restored IL-7-induced bone loss, and RANKL and TNF-α secretion, demonstrating that T cells are essential mediators of IL-7-induced bone loss in vivo.
Expansion of the pool of tumor necrosis factor (TNF)-α-producing T cells is instrumental for the bone loss induced by estrogen deficiency, but the responsible mechanism is unknown. Here we show that ...ovariectomy up-regulates IFN-γ-induced class II transactivator, a multitarget immune modulator, resulting in increased antigen presentation by macrophages, enhanced T cell activation, and prolonged lifespan of active T cells. Up-regulation of class II transactivator derives from increased production of IFN-γ by T helper 1 cells, resulting from enhanced secretion of IL-12 and IL-18 by macrophages. The resulting T cell expansion and bone loss are prevented in vivo by both blockade of antigen presenting cell-induced T cell activation, and silencing of IFN-γ receptor signaling. Thus, increased IFN-γ-induced class II transactivator expression and the resulting enhanced T cell proliferation and lifespan are critical to the bone wasting effect of estrogen deficiency.
There are only limited treatment options for metastatic
mutant melanoma patients with resistance to immune checkpoint inhibitors. Besides activation of the mitogen-activated protein (MAP) kinase ...pathway, they often have additional disturbances in cell cycle regulation. However, unlike
mutant melanoma, no targeted therapy has yet been approved for
mutant melanoma so far. Here we present a
mutant melanoma patient with response to combined binimetinib and ribociclib therapy following characterization of the molecular defects of the tumor by panel sequencing. Next generation sequencing (708 cancer genes) of a soft tissue metastasis revealed a homozygous deletion of
in addition to the previously known
mutation, as well as amplification of
and
Immunohistochemical staining of the altered cell cycle genes confirmed loss of p16, reduced expression of p21 and high expression of CDK6 and cyclin D1. As the patient had been progressive on combined immunotherapy, targeted therapy with combined MEK and CDK4/6 inhibition was initiated as recommended by the molecular tumor board. Response to treatment was monitored with PET/CT and liquid biopsy, serum LDH, and S100. In addition, a patient-derived xenograft (PDX) was used to prove the efficacy of the two drugs in combination. Furthermore, senescence-associated beta-galactosidase staining showed that more cells were senescent under the combination treatment of binimetinib and ribociclib. Our case demonstrates how an individualized, molecular-based therapeutic approach could be found based on next-generation sequencing results. Furthermore our report highlights the fruitful and efficient collaboration of dermatooncologists, human geneticists, molecular pathologists, biochemists, radiologists, and nuclear physicians. Further studies are urgently needed to expand the very limited therapeutic landscape of
mutated melanoma.
Background
Targeted sequencing approaches such as gene panel or exome sequencing have become standard of care for the diagnosis of rare and common genetic disease. The detection and interpretation of ...point mutations, small insertions and deletions, and even exon‐level copy number variants are well established in clinical genetic testing. Other types of genetic variation such as mobile elements insertions (MEIs) are technically difficult to detect. In addition, their downstream clinical interpretation is more complex compared to point mutations due to a larger genomic footprint that can not only predict a clear loss of protein function but might disturb gene regulation and splicing even when located within the non‐coding regions. As a consequence, the contribution of MEIs to disease and tumor development remains largely unexplored in routine diagnostics.
Methods
In this study, we investigated the occurrence of MEIs in 7,693 exome datasets from individuals with rare diseases and healthy relatives as well as 788 cancer patients analyzed by panel sequencing.
Results
We present several exemplary cases highlighting the diagnostic value of MEIs and propose a strategy for the detection, prioritization, and clinical interpretation of MEIs in routine clinical diagnostics.
Conclusion
In this paper, we state that detection and interpretation of MEIs in clinical practice in targeted NGS data can be performed relatively easy despite the fact that MEIs very rarely occur in coding parts of the human genome. Large scale reanalysis of MEIs in existing cohorts may solve otherwise unsolvable cases.
Mobile elements insertions (MEIs) are technically difficult to detect in targeted NGS data. In this study, we investigated the occurrence of MEIs in 7,693 exome datasets from individuals with rare diseases and healthy relatives as well as 788 cancer patients analyzed by panel sequencing. We present several exemplary cases highlighting the diagnostic value of MEIs and propose a strategy for the detection, prioritization, and clinical interpretation of MEIs in routine clinical diagnostics.
Hepatocyte growth factor (HGF) is a pleiotropic cytokine promoting proliferation, migration and survival in several cell types. HGF and its cognate receptor c-Met are expressed in cardiac cells ...during early cardiogenesis, but data concerning its role in cardiac differentiation of embryonic stem cells (ESCs) and the underlying molecular mechanisms involved are limited. In the present study we show that HGF significantly increases the number of beating embryoid bodies of differentiating ESCs without affecting beating frequency. Furthermore, HGF up-regulates the expression of the cardiac-specific transcription factors Nkx 2.5 and GATA-4 and of markers of differentiated cardiomyocytes, i.e. α-MHC, β-MHC, ANF, MLC2v and Troponin T. The HGF-induced increase in Nkx 2.5 expression was inhibited by co-treatment with the PI3 kinase inhibitors Wortmannin and LY294002, but not by its inactive homolog LY303511, suggesting an involvement of the PI3 kinase/Akt pathway in this effect. We conclude that HGF is an important growth factor involved in cardiac differentiation and/or proliferation of ESCs and may therefore be critical for the in vitro generation of pre- or fully differentiated cardiomyocytes as required for clinical use of embryonic stem cells in cardiac diseases.
Objectives
Germline mutations in the
CDH1
-gene are identified as the cause of 30–40% of cases of hereditary diffuse gastric cancer, an autosomal-dominant inherited cancer predisposition syndrome. ...Given this high risk of developing diffuse gastric cancer, carriers of a pathogenic
CDH1
germline mutation are advised to undergo prophylactic gastrectomy. For patients preferring conservative management, endoscopic surveillance is recommended. The detection of diffuse gastric cancer using white light endoscopy, however, remains challenging.
Methods
Patients with pathogenic
CDH1
mutation underwent (chromo)endoscopic surveillance or endoscopy prior to surgery. Biopsies were taken at suspicious sites identified by chromoendoscopy. In addition, endoscopically normal areas were assessed with mapping biopsies. Detection rates from endoscopic biopsies (mapping vs. targeted) and gastrectomy specimen were then compared.
Result
Between 11/2015 and 12/2020, ten patients from four families with a known
CDH1
germline mutation had a total of
n
= 24 endoscopies with
n
= 518 total biopsies being examined. Three patients were diagnosed with GC during the study period. These patients all had suspicious chromoendoscopic lesions (= detection rate 100%). In two of three patients who had suspicious chromoendoscopic lesions, signet cell carcinoma was also detected in mapping biopsies and multiple additional cancer
foci
were identified in the gastrectomy specimen.
Conclusion
Chromoendoscopy facilitated detection of gastric carcinoma
foci
in
CDH1
mutation carriers. Chromoendoscopy identified all patients with gastric cancer, but not all cancer
foci
present in these patients. We conclude that for patients opting against prophylactic total gastrectomy, the addition of chromoendoscopy to white light could be used to enhance diagnostic reliability of endoscopic surveillance.
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