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One of the key events during spermiogenesis is the hypercondensation of chromatin by substitution of the majority of histones by protamines. In humans and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/Prm2) are expressed in a species-specific ratio. Using CRISPR-Cas9-mediated gene editing, we generated Prm1-deficient mice and demonstrated that Prm1+/- mice were subfertile, whereas Prm1-/- mice were infertile. Prm1-/- and Prm2-/- sperm showed high levels of reactive oxygen species-mediated DNA damage and increased histone retention. In contrast, Prm1+/- sperm displayed only moderate DNA damage. The majority of Prm1+/- sperm were CMA3 positive, indicating protamine-deficient chromatin, although this was not the result of increased histone retention in Prm1+/- sperm. However, sperm from Prm1+/- and Prm1-/- mice contained high levels of incompletely processed PRM2. Furthermore, the PRM1:PRM2 ratio was skewed from 1:2 in wild type to 1:5 in Prm1+/- animals. Our results reveal that PRM1 is required for proper PRM2 processing to produce mature PRM2, which, together with PRM1, is able to hypercondense DNA. Thus, the species-specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility.
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Astenozoospermia , Infertilidade Masculina , Protaminas/metabolismo , Animais , Cromatina , Histonas/genética , Infertilidade Masculina/genética , Masculino , Camundongos , Protaminas/genética , Motilidade dos Espermatozoides/genética , Espermatozoides/metabolismoRESUMO
INTRODUCTION: Hypercholesterolemia is a risk factor for premature arteriosclerosis. Inflammation and oxidative stress are thought to contribute to endothelial dysfunction preceding vasculopathy. We investigated the association between inflammation, glycocalyx biomarkers, endothelial function and vascular parameters in children with hypercholesterolemia. METHODS: In 22 patients (LDL-cholesterol > 130 mg/dl; median age [IQR]: 13 [2.3] years) and 22 controls (13 [2.5] years) tumor necrosis factor-alpha (TNF-α), oxidized cholesterol (oxLDL), and glycocalyx biomarkers (Syndecan-1, Hyaluronan) were measured using immunoassays. Endothelial function was assessed by peripheral arterial tonometry, sublingual glycocalyx and microcirculation by videomicroscopy and carotid intima media thickness by ultrasound. RESULTS: OxLDL was significantly higher in patients (78.9 [38.2] vs. 50.3 [16.6] U/l, p=0.002), whereas all other experimental parameters were comparable between groups. Multivariate analysis revealed a significant association of Syndecan-1 with TNF-α (ß=0.75, p<0.001) and with hypercholesterolemia (ß=0.31, p=0.030). The interaction term combining TNF-α and hypercholesterolemia showed a significant effect (p=0.034). Sex was an independent predictor of endothelial function. CONCLUSION: The combined effect of hypercholesterolemia and inflammation on glycocalyx perturbation and the impact of sex in the premature development of arteriosclerosis deserve further evaluation. Therapeutic approaches tackling low grade systemic inflammation-may offer potential to prevent or delay progression of CVD and cardiovascular complications. .
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A novel interdomain consortium composed of a methanogenic Archaeon and a sulfate-reducing bacterium was isolated from a microbial biofilm in an oil well in Cahuita National Park, Costa Rica. Both organisms can be grown in pure culture or as stable co-culture. The methanogenic cells were non-motile rods producing CH4 exclusively from H2/CO2. Cells of the sulfate-reducing partner were motile rods forming cell aggregates. They utilized hydrogen, lactate, formate, and pyruvate as electron donors. Electron acceptors were sulfate, thiosulfate, and sulfite. 16S rRNA sequencing revealed 99% gene sequence similarity of strain CaP3V-M-L2AT to Methanobacterium subterraneum and 98.5% of strain CaP3V-S-L1AT to Desulfomicrobium baculatum. Both strains grew from 20 to 42 °C, pH 5.0-7.5, and 0-4% NaCl. Based on our data, type strains CaP3V-M-L2AT (= DSM 113354 T = JCM 39174 T) and CaP3V-S-L1AT (= DSM 113299 T = JCM 39179 T) represent novel species which we name Methanobacterium cahuitense sp. nov. and Desulfomicrobium aggregans sp. nov.
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Methanobacterium , Campos de Petróleo e Gás , Methanobacterium/genética , Costa Rica , RNA Ribossômico 16S/genética , Sulfatos/metabolismo , Filogenia , DNA Bacteriano/genética , Análise de Sequência de DNA , Ácidos GraxosRESUMO
A novel methanogenic strain, CaP3V-MF-L2AT, was isolated from an exploratory oil well from Cahuita National Park, Costa Rica. The cells were irregular cocci, 0.8-1.8 µm in diameter, stained Gram-negative and were motile. The strain utilized H2/CO2, formate and the primary and secondary alcohols 1-propanol and 2-propanol for methanogenesis, but not acetate, methanol, ethanol, 1-butanol or 2-butanol. Acetate was required as carbon source. The novel isolate grew at 25-40 °C, pH 6.0-7.5 and 0-2.5% (w/v) NaCl. 16S rRNA gene sequence analysis revealed that the strain is affiliated to the genus Methanofollis. It shows 98.8% sequence similarity to its closest relative Methanofollis ethanolicus. The G + C content is 60.1 mol%. Based on the data presented here type strain CaP3V-MF-L2AT (= DSM 113321T = JCM 39176T) represents a novel species, Methanofollis propanolicus sp. nov.
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Archaea , Methanomicrobiaceae , 1-Propanol , Archaea/genética , Costa Rica , DNA Arqueal/genética , Metano , Methanomicrobiaceae/genética , Campos de Petróleo e Gás , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
BACKGROUND: We aimed at clarifying the role of lipocalin-2 (LCN-2) in clear-cell renal cell carcinoma (ccRCC). Since LCN-2 was recently identified as a novel iron transporter, we explored its iron load as a decisive factor in conferring its biological function. METHODS: LCN-2 expression was analysed at the mRNA and protein level by using immunohistochemistry, RNAscope® and qRT-PCR in patients diagnosed with clear-cell renal cell carcinoma compared with adjacent healthy tissue. We measured LCN-2-bound iron by atomic absorption spectrometry from patient-derived samples and applied functional assays by using ccRCC cell lines, primary cells, and 3D tumour spheroids to verify the role of the LCN-2 iron load in tumour progression. RESULTS: LCN-2 was associated with poor patient survival and LCN-2 mRNA clustered in high- and low-expressing ccRCC patients. LCN-2 protein was found overexpressed in tumour compared with adjacent healthy tissue, whereby LCN-2 was iron loaded. In vitro, the iron load determines the biological function of LCN-2. Iron-loaded LCN-2 showed pro-tumour functions, whereas iron-free LCN-2 produced adverse effects. CONCLUSIONS: We provide new insights into the pro-tumour function of LCN-2. LCN-2 donates iron to cells to promote migration and matrix adhesion. Since the iron load of LCN-2 determines its pro-tumour characteristics, targeting either its iron load or its receptor interaction might represent new therapeutic options.
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Carcinoma de Células Renais/metabolismo , Ferro/metabolismo , Neoplasias Renais/metabolismo , Lipocalina-2/metabolismo , RNA Mensageiro/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/cirurgia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Feminino , Humanos , Técnicas In Vitro , Neoplasias Renais/genética , Neoplasias Renais/patologia , Neoplasias Renais/cirurgia , Lipocalina-2/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Espectrofotometria Atômica , Esferoides Celulares , Células Tumorais CultivadasRESUMO
Lipocalin-2 (Lcn-2) is rapidly upregulated in macrophages after renal tubular injury and acts as renoprotective and pro-regenerative agent. Lcn-2 possesses the ability to bind and transport iron with high affinity. Therefore, the present study focuses on the decisive role of the Lcn-2 iron-load for its pro-regenerative function. Primary mouse tubular epithelial cells were isolated from kidney tissue of wildtype mice and incubated with 5µM Cisplatin for 24h to induce injury. Bone marrow-derived macrophages of wildtype and Lcn-2-/- mice were isolated and polarized with IL-10 towards an anti-inflammatory, iron-release phenotype. Their supernatants as well as recombinant iron-loaded holo-Lcn-2 was used for stimulation of Cisplatin-injured tubular epithelial cells. Incubation of tubular epithelial cells with wildtype supernatants resulted in less damage and induced cellular proliferation, whereas in absence of Lcn-2 no protective effect was observed. Epithelial integrity as well as cellular proliferation showed a clear protection upon rescue experiments applying holo-Lcn-2. Notably, we detected a positive correlation between total iron amounts in tubular epithelial cells and cellular proliferation, which, in turn, reinforced the assumed link between availability of Lcn-2-bound iron and recovery. We hypothesize that macrophage-released Lcn-2-bound iron is provided to tubular epithelial cells during toxic cell damage, whereby injury is limited and recovery is favored.
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Células Epiteliais/metabolismo , Rim/metabolismo , Lipocalina-2/metabolismo , Macrófagos/metabolismo , Regeneração , Animais , Proliferação de Células , Cisplatino/efeitos adversos , Ferro/metabolismo , Rim/efeitos dos fármacos , Rim/lesões , Lipocalina-2/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes , Regulação para CimaRESUMO
BACKGROUND: To minimize the surgical damage, minimally invasive mitral valve surgery (MIMVS) has become the therapy of choice. However, this approach is technically more challenging, especially in endocarditis. The data on MIMVS in endocarditis are scarce, we therefore retrospectively analyzed the result at our institute. METHODS: From January 2011 and July 2017, 420 MIMVS were performed, out of which 44 (10%) were for endocarditis. Mean age was 55 ± 17 years and 41% (n = 18) were male. RESULTS: Euroscore II was 7.3 (range: 2-38). Operation times, cardiopulmonary bypass times, and clamp times were 230 (±77), 158 (±56), and 84 (±39) minutes, respectively. Seven cases (16%) were cardiac redo operations. Mitral valve repair and replacement was performed in 46 (n = 20) and 54% (n = 24) of patients, respectively. Overall in-hospital mortality, apoplexy, and reoperation rates (all for bleeding) were 7 (n = 3), 0 (n = 0), and 11% (n = 5), respectively. New onset of dialysis was required in three patients (7%). No patient developed superficial wound infection. Overall intensive care unit and hospital stay was 3 (±3) and 24 (±32) days, respectively. CONCLUSION: MIMVS can be performed with acceptable outcome and low perioperative morbidity in patients with mitral valve endocarditis. Especially absence of any postoperative wound infections and low rate of endocarditis recurrence; use of MIMVS must be encouraged as an eligible approach in most cases.
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Procedimentos Cirúrgicos Cardíacos , Endocardite Bacteriana/cirurgia , Insuficiência da Valva Mitral/cirurgia , Valva Mitral/cirurgia , Adulto , Idoso , Antibacterianos/uso terapêutico , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Procedimentos Cirúrgicos Cardíacos/mortalidade , Endocardite Bacteriana/diagnóstico , Endocardite Bacteriana/microbiologia , Endocardite Bacteriana/mortalidade , Feminino , Mortalidade Hospitalar , Humanos , Masculino , Pessoa de Meia-Idade , Procedimentos Cirúrgicos Minimamente Invasivos , Valva Mitral/microbiologia , Insuficiência da Valva Mitral/diagnóstico , Insuficiência da Valva Mitral/microbiologia , Insuficiência da Valva Mitral/mortalidade , Duração da Cirurgia , Complicações Pós-Operatórias/mortalidade , Complicações Pós-Operatórias/terapia , Recidiva , Reoperação , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVE: The zinc finger transcription factor KLF4 is known to control diverse EC functions. METHODS: The functional role of KLF4 for angiogenesis and its association with CAD was examined in HUVECs and human CECs. RESULTS: In two different angiogenesis assays, siRNA-mediated KLF4 downregulation impaired HUVEC sprouting and network formation. Conversely, KLF4 overexpression increased HUVEC sprouting and network formation. Similar findings were observed after incubation of HUVECs with CdM from KLF4 cDNA-transfected cells, suggesting a role of paracrine factors for mediating angiogenic KLF4 effects. In this regard, VEGF expression was increased in KLF4-overexpressing HUVECs, whereas its expression was reduced in HUVECs transfected with KLF4 siRNA. To examine the relevance of our in vitro findings for human endothelial dysfunction, we analyzed the expression of KLF4 in CECs of patients with stable CAD. Flow cytometry analyses revealed decreased numbers of KLF4-positive CECs in peripheral blood from CAD patients compared to healthy controls. CONCLUSIONS: Our findings suggest that KLF4 may represent a potential biomarker for EC dysfunction. In the future, (therapeutic) modulation of KLF4 may be useful in regulating EC function during vascular disease processes.
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Doença da Artéria Coronariana/sangue , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fatores de Transcrição Kruppel-Like/sangue , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Feminino , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , MasculinoRESUMO
AIMS: The aim of this TeleCheck-AF sub-analysis was to evaluate motivation and adherence to on-demand heart rate/rhythm monitoring app in patients with atrial fibrillation (AF). METHODS AND RESULTS: Patients were instructed to perform 60 s app-based heart rate/rhythm recordings 3 times daily and in case of symptoms for 7 consecutive days prior to teleconsultation. Motivation was defined as number of days in which the expected number of measurements (≥3/day) were performed per number of days over the entire prescription period. Adherence was defined as number of performed measurements per number of expected measurements over the entire prescription period.Data from 990 consecutive patients with diagnosed AF [median age 64 (57-71) years, 39% female] from 10 centres were analyzed. Patients with both optimal motivation (100%) and adherence (≥100%) constituted 28% of the study population and had a lower percentage of recordings in sinus rhythm [90 (53-100%) vs. 100 (64-100%), P < 0.001] compared with others. Older age and absence of diabetes were predictors of both optimal motivation and adherence [odds ratio (OR) 1.02, 95% coincidence interval (95% CI): 1.01-1.04, P < 0.001 and OR: 0.49, 95% CI: 0.28-0.86, P = 0.013, respectively]. Patients with 100% motivation also had ≥100% adherence. Independent predictors for optimal adherence alone were older age (OR: 1.02, 95% CI: 1.00-1.04, P = 0.014), female sex (OR: 1.70, 95% CI: 1.29-2.23, P < 0.001), previous AF ablation (OR: 1.35, 95% CI: 1.03-1.07, P = 0.028). CONCLUSION: In the TeleCheck-AF project, more than one-fourth of patients had optimal motivation and adherence to app-based heart rate/rhythm monitoring. Older age and absence of diabetes were predictors of optimal motivation/adherence.
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Fibrilação Atrial , Diabetes Mellitus , Aplicativos Móveis , Humanos , Feminino , Pessoa de Meia-Idade , Masculino , Fibrilação Atrial/diagnóstico , Frequência Cardíaca , MotivaçãoRESUMO
Based on the structure of the HIV-1 glycoprotein gp120 in complex with its cellular receptor CD4, we have designed and synthesized peptides that mimic the binding site of CD4 for gp120. The ability of these peptides to bind to gp120 can be strongly enhanced by increasing their conformational stability through cyclization, as evidenced by binding assays, as well as through molecular-dynamics simulations of peptide-gp120 complexes. The specificity of the peptide-gp120 interaction was demonstrated by using peptide variants, in which key residues for the interaction with gp120 were replaced by alanine or D-amino acids.
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The coarse-graining of a simple all-atom 2D microscopic model of graphene, in terms of "blobs" described by center of mass variables, is presented. The equations of motion of the coarse-grained variables take the form of dissipative particle dynamics (DPD). The coarse-grained conservative forces and the friction of the DPD model are obtained via a bottom-up procedure from molecular dynamics (MD) simulations. The separation of timescales for blobs of 24 and 96 carbon atoms is sufficiently pronounced for the Markovian assumption, inherent to the DPD model, to provide satisfactory results. In particular, the MD velocity autocorrelation function of the blobs is well reproduced by the DPD model, provided that the effect of friction and noise is taken into account. However, DPD cross-correlations between neighbor blobs show appreciable discrepancies with respect to the MD results. Possible extensions to mend these discrepancies are briefly outlined.
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Grafite/química , Modelos Químicos , Simulação de Dinâmica MolecularRESUMO
While the importance of the iron-load of lipocalin-2 (Lcn-2) in promoting tumor progression is widely appreciated, underlying molecular mechanisms largely remain elusive. Considering its role as an iron-transporter, we aimed at clarifying iron-loaded, holo-Lcn-2 (hLcn-2)-dependent signaling pathways in affecting renal cancer cell viability. Applying RNA sequencing analysis in renal CAKI1 tumor cells to explore highly upregulated molecular signatures in response to hLcn-2, we identified a cluster of genes (SLC7A11, GCLM, GLS), which are implicated in regulating ferroptosis. Indeed, hLcn-2-stimulated cells are protected from erastin-induced ferroptosis. We also noticed a rapid increase in reactive oxygen species (ROS) with subsequent activation of the antioxidant Nrf2 pathway. However, knocking down Nrf2 by siRNA was not sufficient to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. In contrast, preventing oxidative stress through N-acetyl-l-cysteine (NAC) supplementation was still able to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. Besides an oxidative stress response, we noticed activation of the integrated stress response (ISR), shown by enhanced phosphorylation of eIF-2α and induction of ATF4 after hLcn-2 addition. ATF4 knockdown as well as inhibition of the ISR sensitized hLcn-2-treated renal tumor cells to ferroptosis, thus linking the ISR to pro-tumor characteristics of hLcn-2. Our study provides mechanistic details to better understand tumor pro-survival pathways initiated by iron-loaded Lcn-2.
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The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent remodeling. Here, we show that basophil granulocytes infiltrated infarcted murine hearts, with a peak occurring between days 3 and 7. Antibody-mediated and genetic depletion of basophils deteriorated cardiac function and resulted in enhanced scar thinning after MI. Mechanistically, we found that basophil depletion was associated with a shift from reparative Ly6Clo macrophages toward increased numbers of inflammatory Ly6Chi monocytes in the infarcted myocardium. Restoration of basophils in basophil-deficient mice by adoptive transfer reversed this proinflammatory phenotype. Cellular alterations in the absence of basophils were accompanied by lower cardiac levels of IL-4 and IL-13, two major cytokines secreted by basophils. Mice with basophil-specific IL-4/IL-13 deficiency exhibited a similarly altered myeloid response with an increased fraction of Ly6Chi monocytes and aggravated cardiac function after MI. In contrast, IL-4 induction in basophils via administration of the glycoprotein IPSE/α-1 led to improved post-MI healing. These results in mice were corroborated by the finding that initially low counts of blood basophils in patients with acute MI were associated with a worse cardiac outcome after 1 year, characterized by a larger scar size. In conclusion, we show that basophils promoted tissue repair after MI by increasing cardiac IL-4 and IL-13 levels.
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Basófilos/imunologia , Interleucina-13/imunologia , Interleucina-4/imunologia , Infarto do Miocárdio/imunologia , Animais , Basófilos/patologia , Basófilos/fisiologia , Modelos Animais de Doenças , Humanos , Interleucina-13/deficiência , Interleucina-13/genética , Interleucina-4/deficiência , Interleucina-4/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio com Supradesnível do Segmento ST/imunologia , Infarto do Miocárdio com Supradesnível do Segmento ST/patologia , Infarto do Miocárdio com Supradesnível do Segmento ST/fisiopatologiaRESUMO
INTRODUCTION: The TeleCheck-AF approach is an on-demand mobile health (mHealth) infrastructure incorporating mobile app-based heart rate and rhythm monitoring through teleconsultation. We evaluated feasibility and accuracy of self-reported mHealth-based AF risk factors and CHA2DS2-VASc-score in atrial fibrillation (AF) patients managed within this approach. MATERIALS AND METHODS: Consecutive patients from eight international TeleCheck-AF centers were asked to complete an app-based 10-item questionnaire related to risk factors, associated conditions and CHA2DS2-VASc-score components. Patient's medical history was retrieved from electronic health records (EHR). RESULTS: Among 994 patients, 954 (96%) patients (38% female, median age 65 years) completed the questionnaire and were included in this analysis. The accuracy of self-reported assessment was highest for pacemaker and anticoagulation treatment and lowest for heart failure and arrhythmias. Patients who knew that AF increases the stroke risk, more often had a 100% or ≥80% correlation between EHR- and app-based results compared to those who did not know (27 vs. 14% or 84 vs. 77%, P = 0.001). Thromboembolic events were more often reported in app (vs. EHR) in all countries, whereas higher self-reported hypertension and anticoagulant treatment were observed in Germany and heart failure in the Netherlands. If the app-based questionnaire alone was used for clinical decision-making on anticoagulation initiation, 26% of patients would have been undertreated and 6.1%-overtreated. CONCLUSION: Self-reported mHealth-based assessment of AF risk factors is feasible. It shows high accuracy of pacemaker and anticoagulation treatment, nevertheless, displays limited accuracy for some of the CHA2DS2-VASc-score components. Direct health care professional assessment of risk factors remains indispensable to ensure high quality clinical-decision making.
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Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid- and chip-based three-dimensional cell cultures of approximately 300 µm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-to-noise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols - in particular, for screening purposes - clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.
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Accumulating evidence suggests that iron homeostasis is disturbed in tumors. We aimed at clarifying the distribution of iron in renal cell carcinoma (RCC). Considering the pivotal role of macrophages for iron homeostasis and their association with poor clinical outcome, we investigated the role of macrophage-secreted iron for tumor progression by applying a novel chelation approach. We applied flow cytometry and multiplex-immunohistochemistry to detect iron-dependent markers and analyzed iron distribution with atomic absorption spectrometry in patients diagnosed with RCC. We further analyzed the functional significance of iron by applying a novel extracellular chelator using RCC cell lines as well as patient-derived primary cells. The expression of iron-regulated genes was significantly elevated in tumors compared to adjacent healthy tissue. Iron retention was detected in tumor cells, whereas tumor-associated macrophages showed an iron-release phenotype accompanied by enhanced expression of ferroportin. We found increased iron amounts in extracellular fluids, which in turn stimulated tumor cell proliferation and migration. In vitro, macrophage-derived iron showed pro-tumor functions, whereas application of an extracellular chelator blocked these effects. Our study provides new insights in iron distribution and iron-handling in RCC. Chelators that specifically scavenge iron in the extracellular space confirmed the importance of macrophage-secreted iron in promoting tumor growth.
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The identification of cerebral microinfarctions with magnetic resonance imaging (MRI) and histological methods remains challenging in aging and dementia. Here, we matched pathological changes in the microvasculature of cortical cerebral microinfarcts to MRI signals using single 100 µm-thick histological sections scanned with ultra-high-resolution 11.7 T MRI. Histologically, microinfarcts were located in superficial or deep cortical layers or transcortically, compatible with the pattern of layer-specific arteriolar blood supply of the cerebral cortex. Contrary to acute microinfarcts, at chronic stages the core region of microinfarcts showed pallor with extracellular accumulation of lipofuscin and depletion of neurons, a dense meshwork of collagen 4-positive microvessels with numerous string vessels, CD68-positive macrophages and glial fibrillary acidic protein (GFAP)-positive astrocytes. In MRI scans, cortical microinfarcts at chronic stages, called chronic cortical microinfarcts here, gave hypointense signals in T1-weighted and hyperintense signals in T2-weighted images when thinning of the tissue and cavitation and/or prominent iron accumulation were present. Iron accumulation in chronic microinfarcts, histologically verified with Prussian blue staining, also produced strong hypointense T2*-weighted signals. In summary, the microinfarct core was occupied by a dense microvascular meshwork with string vessels, which was invaded by macrophages and astroglia and contained various degrees of iron accumulation. While postmortem ultra-high-resolution single-section imaging improved MRI-histological matching and the structural characterization of chronic cortical cerebral microinfarcts, miniscule microinfarcts without thinning or iron accumulation could not be detected with certainty in the MRI scans. Moreover, string vessels at the infarct margin indicate disturbances in the microcirculation in and around microinfarcts, which might be exploitable in the diagnostics of cortical cerebral microinfarcts with MRI in vivo.
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Astrócitos/patologia , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/patologia , Infarto Cerebral/diagnóstico por imagem , Infarto Cerebral/patologia , Macrófagos/patologia , Microvasos/patologia , Doença Aguda , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Astrócitos/metabolismo , Autopsia , Doenças de Pequenos Vasos Cerebrais/diagnóstico por imagem , Doenças de Pequenos Vasos Cerebrais/patologia , Doença Crônica , Colágeno Tipo IV/metabolismo , Demência/diagnóstico por imagem , Demência/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Macrófagos/metabolismo , Imageamento por Ressonância Magnética , Masculino , Microvasos/metabolismo , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/patologiaRESUMO
The inflammatory tumor microenvironment is an important regulator of carcinogenesis. Tumor-infiltrating immune cells promote each step of tumor development, exerting crucial functions from initiation, early neovascularization, to metastasis. During tumor outgrowth, tumor-associated immune cells, including myeloid cells and lymphocytes, acquire a tumor-supportive, anti-inflammatory phenotype due to their interaction with tumor cells. Microenvironmental cues such as inflammation and hypoxia are mainly responsible for creating a tumor-supportive niche. Moreover, it is becoming apparent that the availability of iron within the tumor not only affects tumor growth and survival, but also the polarization of infiltrating immune cells. The interaction of tumor cells and infiltrating immune cells is multifaceted and complex, finally leading to different activation phenotypes of infiltrating immune cells regarding their functional heterogeneity and plasticity. In recent years, it was discovered that these phenotypes are mainly implicated in defining tumor outcome. Here, we discuss the role of the metabolic activation of both tumor cells and infiltrating immune cells in order to adapt their metabolism during tumor growth. Additionally, we address the role of iron availability and the hypoxic conditioning of the tumor with regard to tumor growth and we describe the relevance of therapeutic strategies to target such metabolic characteristics.
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Imunidade Adaptativa , Carcinogênese/imunologia , Imunidade Inata , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/metabolismo , Microambiente Tumoral/imunologia , Animais , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Humanos , Hipóxia/metabolismo , Ferro/metabolismo , Camundongos , Neoplasias/imunologia , RatosRESUMO
The bromodomain and plant homeodomain finger-containing (BRPF) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Here, we describe NI-57 (16) as new pan-BRPF chemical probe of the bromodomain (BRD) of the BRPFs. Inhibitor 16 preferentially bound the BRD of BRPF1 and BRPF2 over BRPF3, whereas binding to BRD9 was weaker. Compound 16 has excellent selectivity over nonclass IV BRD proteins. Target engagement of BRPF1B and BRPF2 with 16 was demonstrated in nanoBRET and FRAP assays. The binding of 16 to BRPF1B was rationalized through an X-ray cocrystal structure determination, which showed a flipped binding orientation when compared to previous structures. We report studies that show 16 has functional activity in cellular assays by modulation of the phenotype at low micromolar concentrations in both cancer and inflammatory models. Pharmacokinetic data for 16 was generated in mouse with single dose administration showing favorable oral bioavailability.