RESUMO
The increase of CO2 emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO2 as a feedstock gains an extensive importance to establish CO2-neutral or CO2-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO2 as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. 13C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO2 as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L-1, while balancing the expression of key genes and modifying screening conditions led to 2 g L-1 itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO2 uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO2, to support sustainability of industrial bioprocesses.
Assuntos
Engenharia Metabólica , Pichia , Humanos , Pichia/metabolismo , Engenharia Metabólica/métodos , Dióxido de Carbono/metabolismo , Processos AutotróficosRESUMO
PURPOSE: To test a magnetic resonance (MR) scanning protocol as a noninvasive tool to determine hepatic hemodynamics and to assess the degree of liver fibrosis in an animal model of liver fibrosis and cirrhosis. MATERIALS AND METHODS: Fifty-four male Wistar rats were studied. Thirty-nine received thioacetamide (TAA) in their drinking water for either 12 or 16 weeks. MR measurements were performed using flow-sensitive 2D phase-contrast MRI and a 9.4T preclinical scanner. The following hemodynamic parameters were investigated: portal cross-sectional area, mean portal flow velocity, and portal and aortic flow volume rate. Therefore, rats (n = 46) were divided into three groups: CON (control, n = 13), FIB (fibrosis, n = 25), and CIR (cirrhosis, n = 8). Furthermore, the degree of liver fibrosis was assessed by a self-established MR score and verified by a standardized histological score (n = 48). RESULTS: Portal and aortic flow parameters could be reliably detected. A significant decrease in portal flow velocity was found in FIB (FIB vs. CON: -21%, P = 0.006 and CIR vs. CON: -17%, P = 0.105) and in portal flow volume rate in FIB and CIR (FIB vs. CON: -20%, P = 0.009 and CIR vs. CON: -25%, P = 0.024). If the histological score is taken as standard, the self-established MR score enabled discrimination between healthy and diseased livers (sensitivity to identify diseased livers: 89% and specificity to identify healthy livers: 100%). CONCLUSION: This MR scanning protocol presents a noninvasive tool to determine hepatic hemodynamics in healthy and diseased rats. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2017;46:1526-1534.
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Cirrose Hepática/diagnóstico por imagem , Fígado/diagnóstico por imagem , Imageamento por Ressonância Magnética , Animais , Hemodinâmica , Humanos , Hipertensão Portal/patologia , Processamento de Imagem Assistida por Computador , Fígado/irrigação sanguínea , Fígado/patologia , Masculino , Variações Dependentes do Observador , Veia Porta/patologia , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional , Tioacetamida/química , Água/químicaRESUMO
BACKGROUND: Conventional stainings (including H&E and special stains like Giemsa) are the most widely applied histopathologic detection methods of Helicobacter pylori (HP). MATERIALS AND METHODS: We aimed to compare the diagnostic performance of Giemsa staining with immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) on a monocentric cohort of 2896 gastric biopsies and relate results to histologic alterations in order to find such histopathologic subgroups in which these methods underperform. All cases were categorized regarding presence or absence of chronic gastritis, inflammatory activity, and mucosal structural alterations. RESULTS: Giemsa revealed 687 cases (23.7%), IHC 795 cases (27.5%), and FISH 788 cases (27.2%) as being HP positive. Giemsa showed significantly lower overall sensitivity (83.3%) compared to IHC (98.8%) and FISH (98.0%). Moreover, the sensitivity of Giemsa dramatically dropped to 33.6% in the nonactive cases. We found that sensitivity of Giemsa strongly depends on HP density and, accordingly, on the presence of activity. Structural alterations (intestinal metaplasia, atrophy, etc.) had only no or weak effect on sensitivity of the three stainings. Both IHC and FISH proved to be equally reliable HP detecting techniques whose diagnostic performance is minimally influenced by mucosal inflammatory and structural alterations contrary to conventional stainings. CONCLUSIONS: We highly recommend immunohistochemistry for clinically susceptible, nonactive chronic gastritis cases, if the conventional stain-based HP detection is negative. Moreover, we recommend to use IHC more widely as basic HP stain. Helicobacter pylori FISH technique is primarily recommended to determine bacterial clarithromycin resistance. Furthermore, it is another accurate diagnostic tool for HP.
Assuntos
Infecções por Helicobacter/diagnóstico , Helicobacter pylori/isolamento & purificação , Histocitoquímica/métodos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Adulto , Idoso , Feminino , Infecções por Helicobacter/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Coloração e Rotulagem/métodosRESUMO
Barrett's esophagus (BE) is defined as metaplasia of the esophageal squamous epithelium with multiple cell layers into a single layer of intestinal columnar epithelial cells - or, in other words, skyscrapers are turned into town houses. The underlying pathomechanism(s) and the cell of origin of BE lesions have not been defined yet. However, four potential hypotheses for BE development have been suggested. The morphological changes during BE development are associated with rather well-described aberrant gene/protein expression patterns. However, the potential key regulators of this conversion process are still unclear. The process of metaplastic conversion is difficult to monitor in a spatiotemporal manner in vitro, and robust models are lacking. There is therefore a need for novel experimental systems. This review focuses on potential key regulators, microenvironmental influences, epigenetic alterations and experimental research systems related to BE.
Assuntos
Esôfago de Barrett/patologia , Células Epiteliais/patologia , Esôfago/patologia , Humanos , Metaplasia/patologiaRESUMO
BACKGROUND: Targeted next generation sequencing (tNGS) has become part of molecular pathology diagnostics for determining RAS mutation status in colorectal cancer (CRC) patients as predictive tool for decision on EGFR-targeted therapy. Here, we investigated mutation profiles of case-matched tissue specimens throughout the disease course of CRC, to further specify RAS-status dynamics and to identify de novo mutations associated with distant metastases. METHODS: Case-matched formalin-fixed and paraffin-embedded (FFPE) resection specimens (n = 70; primary tumours, synchronous and/or metachronous liver and/or lung metastases) of 14 CRC cases were subjected to microdissection of normal colonic epithelial, primary and metastatic tumour cells, their DNA extraction and an adapted library protocol for limited DNA using the 48 gene TruSeq Amplicon Cancer PanelTM, MiSeq sequencing and data analyses (Illumina). RESULTS: By tNGS primary tumours were RAS wildtype in 5/14 and mutated in 9/14 (8/9 KRAS exon 2; 1/9 NRAS Exon 3) of cases. RAS mutation status was maintained in case-matched metastases throughout the disease course, albeit with altered allele frequencies. Case-matched analyses further identified a maximum of three sequence variants (mainly in APC, KRAS, NRAS, TP53) shared by all tumour specimens throughout the disease course per individual case. In addition, further case-matched de novo mutations were detected in synchronous and/or metachronous liver and/or lung metastases (e.g. in APC, ATM, FBXW7, FGFR3, GNAQ, KIT, PIK3CA, PTEN, SMAD4, SMO, STK11, TP53, VHL). Moreover, several de novo mutations were more frequent in synchronous (e.g. ATM, KIT, PIK3CA, SMAD4) or metachronous (e.g. FBXW7, SMO, STK11) lung metastases. Finally, some de novo mutations occurred only in metachronous lung metastases (CDKN2A, FGFR2, GNAS, JAK3, SRC). CONCLUSION: Together, this study employs an adapted FFPE-based tNGS approach to confirm conservation of RAS mutation status in primary and metastatic tissue specimens of CRC patients. Moreover, it identifies genes preferentially mutated de novo in late disease stages of metachronous CRC lung metastases, several of which might be actionable by targeted therapies.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Pulmonares/secundário , Mutação , Adulto , Idoso , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Biblioteca Gênica , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
INTRODUCTION: As digital mammography and micro-computed tomography (CT) have been used for evaluation of stents deployed in experimental animal models, we compared the two methods regarding their sensitivity to detect abnormalities in three prototypes of intracranial stents. METHODS: Three different prototypes of intracranial stents (n = 84) were implanted in various animal models. Explanted stents were examined using digital mammography and micro-CT. The images were compared with respect to maintenance of material and form and the stents were compared to one another. Histological analysis was performed as well. RESULTS: In the open-cell stents, expansion of the stent cells was detected in the majority of cases (57.1 %) using micro-CT and less frequently using mammography (42.3 %). The closed-cell stent revealed kink stenoses in mammography as well as in micro-CT (3/7, 42.9 %). Detailed reconstructions of micro-CT images showed high-grade kink stenoses of the flow-diverter stent in two extremely curved vessels. Strut breaks were observed more frequently using micro-CT (6/84, 7.1 %) than by mammography (4/84, 4.8 %). Histology confirmed all changes of stent architecture. CONCLUSION: Significant changes of stent architecture can be observed and assessed even in the two-dimensional mammographic images. The use of micro-CT is recommended to detect subtle changes like single strut breaks and for three-dimensional information.
Assuntos
Artéria Carótida Primitiva/diagnóstico por imagem , Artéria Carótida Primitiva/cirurgia , Intensificação de Imagem Radiográfica/métodos , Stents , Tomografia Computadorizada por Raios X/métodos , Tomografia Computadorizada por Raios X/veterinária , Animais , Análise de Falha de Equipamento/métodos , Projetos Piloto , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie , Suínos , Resultado do TratamentoRESUMO
In older populations, sensitive fall risk assessment tools are important to timely intervene and prevent falls. Instrumented assessments have shown to be superior to standardized fall risk assessments such as the Timed Up and Go Test (TUG) and should capture both motor and cognitive functions. Therefore, the aim was to test novel instrumented assessments with and without a cognitive component. One hundred thirty-seven older adults aged 73.1 ± 7.3 years, 38 categorized as fallers and 99 as non-fallers, conducted five instrumented assessments on the Dividat Senso, a pressure sensitive stepping platform, and three standardized geriatric assessments (TUG, TUG-dual task, 30-s Sit-to-Stand Test (STS)). T-tests were applied to compare the test performance of fallers versus non-fallers. Furthermore, logistic regression analyses and area under the curve (AUC) analyses were performed. Statistically significant differences between fallers and non-fallers were found in the Go/No-Go test (p = .001, d = .72), the TUG (p = .014, d = .48), and the STS (p = .008, d = .51). Only the Go/No-Go test contributed significantly to all regression models. Significant AUC values were found for the Reaction Time Test (RTT) (AUC = .628, p = .023), Go/No-Go (AUC = .673, p = .002), TUG (AUC = .642, p = .012), and STS (AUC = .690, p = .001). The Go/No-Go test measuring inhibition showed the best discriminative ability suggesting added value of instrumented assessments with a cognitive component for clinical fall risk assessment in relatively healthy older adults. The study should be extended with a frailer population, in which TUG and the other instrumented assessments are possibly good predictors as well.
RESUMO
The current climatic change is predominantly driven by excessive anthropogenic CO2 emissions. As industrial bioprocesses primarily depend on food-competing organic feedstocks or fossil raw materials, CO2 co-assimilation or the use of CO2-derived methanol or formate as carbon sources are considered pathbreaking contributions to solving this global problem. The number of industrially-relevant microorganisms that can use these two carbon sources is limited, and even fewer can concurrently co-assimilate CO2. Here, we search for alternative native methanol and formate assimilation pathways that co-assimilate CO2 in the industrially-relevant methylotrophic yeast Komagataella phaffii (Pichia pastoris). Using 13C-tracer-based metabolomic techniques and metabolic engineering approaches, we discover and confirm a growth supporting pathway based on native enzymes that can perform all three assimilations: namely, the oxygen-tolerant reductive glycine pathway. This finding paves the way towards metabolic engineering of formate and CO2 utilisation to produce proteins, biomass, or chemicals in yeast.
Assuntos
Dióxido de Carbono , Metanol , Metanol/metabolismo , Dióxido de Carbono/metabolismo , Glicina/metabolismo , Carbono/metabolismo , Formiatos/metabolismo , Oxigênio/metabolismo , Pichia/metabolismoAssuntos
Aciclovir/administração & dosagem , Colite Ulcerativa/virologia , Herpes Zoster/diagnóstico , Herpesvirus Humano 3/isolamento & purificação , Viremia/diagnóstico , Dor Abdominal/diagnóstico , Dor Abdominal/etiologia , Idoso , Biópsia por Agulha , Colite Ulcerativa/patologia , Feminino , Seguimentos , Mucosa Gástrica/patologia , Mucosa Gástrica/virologia , Gastroscopia/métodos , Herpes Zoster/tratamento farmacológico , Herpesvirus Humano 3/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Infusões Intravenosas , Doenças Raras , Índice de Gravidade de Doença , Resultado do Tratamento , Viremia/tratamento farmacológicoRESUMO
The influence of rhodanine and haematoxylin and eosin (HE) staining on the copper distribution and concentration in liver needle biopsy samples originating from patients with Wilson's disease (WD), a rare autosomal recessive inherited disorder of the copper metabolism, is investigated. In contemporary diagnostic of WD, rhodanine staining is used for histopathology, since rhodanine and copper are forming a red to orange-red complex, which can be recognized in the liver tissue using a microscope. In this paper, a laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method is applied for the analysis of eight different WD liver samples. Apart from a spatially resolved elemental detection as qualitative information, this LA-ICP-MS method offers also quantitative information by external calibration with matrix-matched gelatine standards. The sample set of this work included an unstained and a rhodanine stained section of each WD liver sample. While unstained sections of WD liver samples showed very distinct structures of the copper distribution with high copper concentrations, rhodanine stained sections revealed a blurred copper distribution with significant decreased concentrations in a range from 20 to more than 90%. This implies a copper removal from the liver tissue by complexation during the rhodanine staining. In contrast to this, a further HE stained sample of one WD liver sample did not show a significant decrease in the copper concentration and influence on the copper distribution in comparison to the unstained section. Therefore, HE staining can be combined with the analysis by means of LA-ICP-MS in two successive steps from one thin section of a biopsy specimen. This allows further information to be gained on the elemental distribution by LA-ICP-MS additional to results obtained by histological staining.
Assuntos
Cobre/metabolismo , Degeneração Hepatolenticular/metabolismo , Lasers , Fígado/metabolismo , Espectrometria de Massas/métodos , Gases em Plasma/química , Coloração e Rotulagem , Calibragem , Gelatina/química , Humanos , Fígado/patologia , Padrões de Referência , Rodanina/químicaRESUMO
The lipolysis-stimulated lipoprotein receptor (LSR) is a lipoprotein receptor, serves as host receptor for clostridial iota-like toxins and is involved in the formation of tricellular contacts. Of particular interest is the role of LSR in progression of various cancers. Here we aimed to study the tumor growth of LSR-deficient colon carcinoma-derived cell lines HCT116 and CaCo-2 in a mouse xenograft model. Whereas knockout of LSR had no effect on tumor growth of HCT116 cells, we observed that CaCo-2 LSR knockout tumors grew to a smaller size than their wild-type counterparts. Histological analysis revealed increased apoptotic and necrotic cell death in a tumor originating from LSR-deficient CaCo-2 cells. LSR-deficient CaCo-2 cells exhibited increased cell proliferation in vitro and an altered epithelial morphology with impaired targeting of tricellulin to tricellular contacts. In addition, loss of LSR reduced the transepithelial electrical resistance of CaCo-2 cell monolayers and increased permeability for small molecules. Moreover, LSR-deficient CaCo-2 cells formed larger cysts in 3D culture than their wild-type counterparts. Our study provides evidence that LSR affects epithelial morphology and barrier formation in CaCo-2 cells and examines for the first time the effects of LSR deficiency on the tumor growth properties of colon carcinoma-derived cell lines.
Assuntos
Permeabilidade da Membrana Celular , Neoplasias do Colo/metabolismo , Células Epiteliais/metabolismo , Receptores de Lipoproteínas/metabolismo , Animais , Sequência de Bases , Células CACO-2 , Proliferação de Células/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Nus , Receptores de Lipoproteínas/genética , Transplante Heterólogo , Carga Tumoral/genéticaRESUMO
EGFR-targeted therapy is a key treatment approach in patients with RAS wildtype metastatic colorectal cancers (CRC). Still, also RAS wildtype CRC may be resistant to EGFR-targeted therapy, with few predictive markers available for improved stratification of patients. Here, we investigated response of 7 CRC cell lines (Caco-2, DLD1, HCT116, HT29, LS174T, RKO, SW480) to Cetuximab and correlated this to NGS-based mutation profiles, EGFR promoter methylation and EGFR expression status as well as to E-cadherin expression. Moreover, tissue specimens of primary and/or recurrent tumors as well as liver and/or lung metastases of 25 CRC patients having received Cetuximab and/or Panitumumab were examined for the same molecular markers. In vitro and in situ analyses showed that EGFR promoter methylation and EGFR expression as well as the MSI and or CIMP-type status did not guide treatment responses. In fact, EGFR-targeted treatment responses were also observed in RAS exon 2 p.G13 mutated CRC cell lines or CRC cases and were further linked to PIK3CA exon 9 mutations. In contrast, non-response to EGFR-targeted treatment was associated with ATM mutations and low E-cadherin expression. Moreover, down-regulation of E-cadherin by siRNA in otherwise Cetuximab responding E-cadherin positive cells abrogated their response. Hence, we here identify ATM and E-cadherin expression as potential novel supportive predictive markers for EGFR-targeted therapy.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Caderinas/genética , Neoplasias Colorretais/genética , Receptores ErbB/genética , Mutação , Adulto , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígenos CD , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Western Blotting , Células CACO-2 , Caderinas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Metilação de DNA , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Células HCT116 , Células HT29 , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Panitumumabe , Fosfatidilinositol 3-Quinases/genética , Regiões Promotoras Genéticas/genética , Interferência de RNARESUMO
A laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method is developed and applied for the analysis of paraffin-embedded liver needle biopsy specimens of patients with Wilson's disease (WD), a rare autosomal recessive disorder of the copper metabolism causing various hepatic, neurological and psychiatric symptoms due to a copper accumulation in the liver and the central nervous system. The sample set includes two WD liver samples and one negative control sample. The imaging analysis was performed with a spatial resolution of 10 µm. Besides copper, iron was monitored because an elevated iron concentration in the liver is known for WD. In addition to this, both elements were quantified using an external calibration based on matrix-matched gelatine standards. The presented method offers low limits of detection of 1 and 5 µg/g for copper and iron, respectively. The high detection power and good spatial resolution allow the analysis of small needle biopsy specimen using this method. The two analyzed WD samples can be well differentiated from the control sample due to their inhomogeneous copper distribution and high copper concentrations of up to 1200 µg/g. Interestingly, the WD samples show an inverse correlation of regions with elevated copper concentrations and regions with high iron concentrations.
Assuntos
Diagnóstico por Imagem/métodos , Elementos Químicos , Degeneração Hepatolenticular/diagnóstico , Degeneração Hepatolenticular/patologia , Fígado/patologia , Espectrometria de Massas/métodos , Biópsia por Agulha , Calibragem , Cobre/metabolismo , Humanos , Ferro/metabolismo , Terapia a Laser , Microscopia de Fluorescência , Padrões de ReferênciaRESUMO
Primary systemic amyloidosis is a rare disorder resulting in extracellular deposition of insoluble fibrils in different organs. Liver involvement has been reported. Since hepatic amyloidosis often presents clinically asymptomatic without specific laboratory or imaging hallmarks, diagnosis is challenging. However, cases of progressive hepatic failure due to liver amyloidosis have been reported. A 63 year old man presented with newly diagnosed ascites to our department. The patient reported occasional alcohol consumption. Viral hepatitis, genetic-metabolic causes as well as hepatic vascular disorders were excluded and ultrasound did not show any signs of liver cirrhosis or intraabdominal malignancy. Initially, alcoholic hepatitis was suspected. Due to the rapid deterioration of liver function, however, transjugular liver biopsy was performed showing light chain amyloidosis of kappa isotype. As diagnosis of hepatic amyloidosis is challenging, early liver biopsy is mandatory in patients with unexplained acute or chronic liver disease to exclude rare diseases with high mortality.
Assuntos
Amiloidose , Hepatopatias , Falência Hepática , Diagnóstico Diferencial , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
A liver biopsy specimen from a Wilson's disease (WD) patient was analyzed by means of micro-X-ray fluorescence (µXRF) spectroscopy to determine the elemental distribution. First, bench-top µXRF was utilized for a coarse scan of the sample under laboratory conditions. The resulting distribution maps of copper and iron enabled the determination of a region of interest (ROI) for further analysis. In order to obtain more detailed elemental information, this ROI was analyzed by synchrotron radiation (SR)-based µXRF with a beam size of 4 µm offering a resolution at the cellular level. Distribution maps of additional elements to copper and iron like zinc and manganese were obtained due to a higher sensitivity of SR-µXRF. In addition to this, X-ray absorption near edge structure spectroscopy (XANES) was performed to identify the oxidation states of copper in WD. This speciation analysis indicated a mixture of copper(i) and copper(ii) within the WD liver tissue.
Assuntos
Cobre/metabolismo , Degeneração Hepatolenticular/diagnóstico , Ferro/metabolismo , Fígado/patologia , Espectrometria por Raios X/métodos , Espectroscopia por Absorção de Raios X/métodos , Zinco/metabolismo , Degeneração Hepatolenticular/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/métodos , Fígado/metabolismoRESUMO
Little is known about histone modifiers and histone marks in colorectal cancers (CRC). The present study therefore addressed the role of histone acetylation and histone deacetylases (HDAC) in CRCs in situ and in vitro. Immunohistochemistry of primary CRCs (n=47) revealed that selected histone marks were frequently present (H3K4me3: 100%; H3K9me3: 77%; H3K9ac: 75%), partially displayed intratumoral heterogeneity (H3K9me3; H3K9ac) and were significantly linked to higher pT category (H3K9me3: p=0.023; H3K9ac: p=0.028). Furthermore, also HDAC1 (62%), HDAC2 (100%) and HDAC3 (72%) expression was frequent, revealing four CRC types: cases expressing 1) HDAC1, HDAC2 and HDAC3 (49%), 2) HDAC2 and HDAC3 (30%), 3) HDAC1 and HDAC2 (10.5%) and 4) exclusively HDAC2 (10.5%). Correlation to clinico-pathological parameters (pT, pN, G, MSI status) revealed that heterogeneous HDAC1 expression correlated with lymph node status (p=0.012). HDAC expression in situ was partially reflected by six CRC cell lines, with similar expression of all three HDACs (DLD1, LS174T), preferential HDAC2 and HDAC3 expression (SW480, Caco2) or lower HDAC2 and HDAC3 expression (HCT116, HT29). HDAC activity was variably higher in HCT116, HT29, DLD1 and SW480 compared to LS174T and Caco2 cells. Treatment with broad (SAHA) and specific (MS-275; FK228) HDAC inhibitors (HDACi) caused loss of cell viability in predominantly MSIpositive CRC cells (HCT116, LS174T, DLD1; SAHA, MS-275 and in part FK228). In contrast, MSI-negative CRC cells (Caco2, HT29, SW480) were resistant, except for high doses of FK228 (Caco2, HT29). Cell viability patterns were not linked to different efficacies of HDACi on reduction of HDAC activity or histone acetylation, p21 expression and/or induction of DNA damage (γH2A-X levels). In summary, this study reveals inter- and intra-tumoral heterogeneity of histone marks and HDAC expression in CRCs. This is reflected by diverse HDACi responses in vitro, which do not follow known modes of action. Together, this implies further exploitation of histone alterations in CRC for molecular classification and/or novel treatment options.
RESUMO
OBJECTIVES: The pressure along vascular clamp jaws may be unequally distributed, with greater pressure near the clamp hinge than at its top. Such unequal pressure distribution may cause aortic injury, especially in large aortas. We evaluated pressure distribution along different currently availably clamp jaws. METHODS: Seven descending thoracic aortas from pigs (diameter 2.0-3.0 cm) were plainly dissected and all side arteries closed. Aortas were filled up with water and cross-clamped. The pressure inside the aorta was raised to 100 mmHg and the aorta was clamped so tightly that no water exited from the distal aortic end. Each aorta was clamped seven times at different sites with the following clamps: DeBakey, Satinsky, femoral, iliac, Chitwood, angled handle Fogarty and straight handle Fogarty. The pressure along the clamp jaws was measured with a pressure-detecting film placed between the clamp jaws and aorta. The collagen-fibre disorganization was examined in haemotoxylin-eosin- and Elastica van Gieson-stained tissue samples. RESULTS: The DeBakey clamp revealed the lowest maximum pressure along the clamp jaws after complete aortic occlusion (1.43 ± 0.49 MPa), whereas the Chitwood clamp's pressure was the highest (3.26 ± 1.93 MPa, P < 0.001). The angled handle Fogarty clamp displayed the lowest difference between maximum pressures across the jaws (33%), with the greatest difference measured in the iliac (72%) and Chitwood (66%) clamps. The highest collagen-fibre disorganization score was observed in the proximal-to-the-clamp-hinge quartile after clamping with the angled handle Fogarty (2.8 ± 0.4), straight handle Fogarty (2.3 ± 0.8) and Chitwood (2.3 ± 0.5) clamps. CONCLUSIONS: The pressure along clamp jaws is unequally distributed in all the currently available vascular clamps. The Chitwood clamp is associated with the highest maximum pressure during complete aortic occlusion and with the most unequal pressure distribution along the jaws.
Assuntos
Aorta Torácica/fisiopatologia , Procedimentos Cirúrgicos Cardiovasculares/instrumentação , Animais , Aorta Torácica/química , Aorta Torácica/lesões , Aorta Torácica/patologia , Colágeno/análise , Constrição , Desenho de Equipamento , Pressão , Sus scrofaRESUMO
BRAF mutations are associated with aggressive, less-differentiated and therapy-resistant colorectal carcinoma. However, the underlying mechanisms for these correlations remain unknown. To understand how oncogenic B-Raf contributes to carcinogenesis, in particular to aspects other than cellular proliferation and survival, we generated three isogenic human colorectal carcinoma cell line models in which we can dynamically modulate the expression of the B-Raf(V600E) oncoprotein. Doxycyclin-inducible knockdown of endogenous B-Raf(V600E) decreases cellular motility and invasion in conventional and three-dimensional (3D) culture, whereas it promotes cell-cell contacts and induces various hallmarks of differentiated epithelia. Importantly, all these effects are recapitulated by B-Raf (PLX4720, vemurafenib, and dabrafenib) or MEK inhibitors (trametinib). Surprisingly, loss of B-Raf(V600E) in HT29 xenografts does not only stall tumor growth, but also induces glandular structures with marked expression of CDX2, a tumor-suppressor and master transcription factor of intestinal differentiation. By performing the first transcriptome profiles of PLX4720-treated 3D cultures of HT29 and Colo-205 cells, we identify several upregulated genes linked to epithelial differentiation and effector functions, such as claudin-1, a Cdx-2 target gene encoding a critical tight junction component. Thereby, we provide a mechanism for the clinically observed correlation between mutant BRAF and the loss of Cdx-2 and claudin-1. PLX4720 also suppressed several metastasis-associated transcripts that have not been implicated as targets, effectors or potential biomarkers of oncogenic B-Raf signaling so far. Together, we identify a novel facet of clinically applied B-Raf or MEK inhibitors by showing that they promote cellular adhesion and differentiation of colorectal carcinoma cells.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Técnicas de Silenciamento de Genes , Células HT29 , Humanos , Camundongos , Camundongos SCID , Camundongos Transgênicos , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genética , Distribuição Aleatória , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Soy protein is used for meal replacement therapy in obesity, however the influence on renal function parameters is not adequately investigated. This study evaluates glomerular filtration rate (GFR) and renal plasma flow (RPF) in patients with the metabolic syndrome and healthy controls after ingestion of different amounts of soy protein. METHODS: 10 patients with the metabolic syndrome but no signs of kidney disease and 10 healthy controls ingested 1 g protein/kg body weight of a commercial soy-yoghurt-honeypreparation. The patient group was also given a protein challenge of 0.3 g/kg body weight. RESULTS: Baseline GFR and RPF both were significantly higher in the patient group (147 ± 34.8 vs. 116 ± 21.1 ml/min, p=0.01 and 848 ± 217 vs. 637 ± 121 ml/min, p=0.02) and were strongly correlated with body weight. Use of different algorithms to estimate GFR resulted in underestimation of GFR, particularly in the patients with the metabolic syndrome. The challenge with an acute protein load of 1g protein per kilogram body weight induced a significant increase in GFR and RPF in healthy controls (GFR: +12.6 ± 11.0 % (p=0.01), RPF: +13.6 ± 15.6 % (p=0.04)) and even more in patients with the metabolic syndrome (GFR: +31.5 ± 32.2 % (p=0.01); RPF: +19.4 ± 22.7 % (p=0.02)). The ingestion of 0.3 g protein/ kg body weight did not induce significant changes. CONCLUSIONS: Basic renal function is changed in patients with the metabolic syndrome, even without microalbuminuria. In addition, there is an elevated susceptibility for protein load. However, the protein amount recommended for use in soy-protein based meal replacement therapy induced no significant changes.