Species Identification and Mycotoxigenic Potential of Aspergillus Section Flavi Isolated from Maize Marketed in the Metropolitan Region of Asunción, Paraguay.

Zea mays var. amylacea and Zea mays var. indurata are maize ecotypes from Paraguay. Aspergillus section Flavi is the main spoilage fungus of maize under storage conditions. Due to its large intraspecific genetic variability, the accurate identification of this fungal taxonomic group is difficult. In the present study, potential mycotoxigenic strains of Aspergillus section Flavi isolated from Z. mays var. indurata and Z. mays var. amylacea that are marketed in the metropolitan region of Asunción were identified by a polyphasic approach. Based on morphological characters, 211 isolates were confirmed to belong to Aspergillus section Flavi. A subset of 92 strains was identified as Aspergillus flavus by mass spectrometry MALDI-TOF and the strains were classified by MALDI-TOF MS into chemotypes based on their aflatoxins and cyclopiazonic acid production. According to the partial sequencing of ITS and CaM genes, a representative subset of 38 A. flavus strains was confirmed. Overall, 75 A. flavus strains (86%) were characterized as producers of aflatoxins. The co-occurrence of at least two mycotoxins (AF/ZEA, FUM/ZEA, and AF/ZEA/FUM) was detected for five of the Z. mays samples (63%). Considering the high mycological bioburden and mycotoxin contamination, maize marketed in the metropolitan region of Asunción constitutes a potential risk to food safety and public health and requires control measures.

Chemical Analysis and In Vitro Bioactivity of Essential Oil of Laurelia sempervirens and Safrole Derivatives against Oomycete Fish Pathogens.

In this study, the essential oil (EO) from Laurelia sempervirens was analyzed by GC/MS and safrole (1) was identified as the major metabolite 1, was subjected to direct reactions on the oxygenated groups in the aromatic ring and in the side chain, and eight compounds (4 to 12) were obtained by the process. EO and compounds 4-12 were subjected to biological assays on 24 strains of the genus Saprolegnia, specifically of the species 12 S. parasitica and 12 S. australis. EO showed a significant effect against Saprolegnia strains. Compound 6 presents the highest activity against two resistant strains, with minimum inhibitory concentration (MIC) and minimum oomyceticidal concentration (MOC) values of 25 to 100 and 75 to 125 µg/mL, respectively. The results show that compound 6 exhibited superior activities compared to the commercial controls bronopol and azoxystrobin used to combat these pathogens.

Key Characteristics of Human Hepatotoxicants as a Basis for Identification and Characterization of the Causes of Liver Toxicity.

Hazard identification regarding adverse effects on the liver is a critical step in safety evaluations of drugs and other chemicals. Current testing paradigms for hepatotoxicity rely heavily on preclinical studies in animals and human data (epidemiology and clinical trials). Mechanistic understanding of the molecular and cellular pathways that may cause or exacerbate hepatotoxicity is well advanced and holds promise for identification of hepatotoxicants. One of the challenges in translating mechanistic evidence into robust decisions about potential hepatotoxicity is the lack of a systematic approach to integrate these data to help identify liver toxicity hazards. Recently, marked improvements were achieved in the practice of hazard identification of carcinogens, female and male reproductive toxicants, and endocrine disrupting chemicals using the key characteristics approach. Here, we describe the methods by which key characteristics of human hepatotoxicants were identified and provide examples for how they could be used to systematically identify, organize, and use mechanistic data when identifying hepatotoxicants.

Absceso frío en una mano por un hongo dematiáceo. Primera comunicación en Chile de Pleurostomophora richardsiae / Hand cold abscess by a dematiaceous fungus. First report in Chile of Pleurostomophora richardsiae

Resumen Los hongos dematiáceos son un grupo heterogéneo de microorganismos capaces de sintetizar melanina. Las infecciones de este grupo que producen hifas en tejidos se denominan feohifomicosis y generalmente afectan la piel y tejidos vecinos. Presentamos el caso de un varón de 86 años con un tumor quístico blando progresivo en su mano y muñeca derecha, no asociado a dolor o signos inflamatorios. Se demostró una tenosinovitis de los flexores con pseudocapsula y sinovitis adherida a los tendones. El cultivo demostró un hongo dematiáceo compatible con Pleurostomophora richardsiae que se confirmó por secuenciación de la región ITS. La biopsia mostró una inflamación crónica granulomatosa e hifas. Después del drenaje quirúrgico, el paciente fue dado de alta sin terapia antifúngica, pero falleció por causas no relacionadas, tres meses después. Esta es la primera descripción de P. richardsiae como causa de feohifomicosis en Chile. Esta patología se puede sospechar cuando una lesión quística cutánea crónica involucra extremidades sin signos inflamatorios. Puede afectar a pacientes inmunocompetentes o inmunocomprometidos. El tratamiento contempla la escisión quirúrgica con o sin terapia antifúngica.

Abstract Dematiaceous fungi are a heterogeneous group of microorganisms able to synthesize melanin. Infections by this group that provoke tissular hyphae are called phaeohyphomycosis and usually involve skin and neighbor tissues. We present the case of a 86 years old men with a progressive soft cystic tumor in his right hand and wrist not associated to pain or inflammatory signs. A surgical intervention demonstrated flexor tenosynovitis with serous secretion, pseudocapsule and synovitis. Fungal culture demonstrated a dematiaceous fungi compatible with Pleurostomophora richardsiae that was confirmed by sequencing of the ITS region. Biopsy showed chronic inflammation with granuloma and hyphae. After surgical drainage, the patient was discharged without antifungal therapy but died of unrelated causes three month later. This is the first description of P. richardsiae as a cause of phaeohyphomycosis in Chile, a country with a template climate. Phaeohyphomycosis can be suspected when a chronic skin cystic lesion involves extremities without inflammatory signs, sometimes with an associated fistula. It may affect immunocompetent or immunosuppressed patients. Treatment involves surgical excision with or without antifungal therapy and prognosis is favorable.

Genotoxic stress triggers the activation of IRE1α-dependent RNA decay to modulate the DNA damage response.

The molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis.

Interference with ERK-dimerization at the nucleocytosolic interface targets pathological ERK1/2 signaling without cardiotoxic side-effects.

Dysregulation of extracellular signal-regulated kinases (ERK1/2) is linked to several diseases including heart failure, genetic syndromes and cancer. Inhibition of ERK1/2, however, can cause severe cardiac side-effects, precluding its wide therapeutic application. ERKT188-autophosphorylation was identified to cause pathological cardiac hypertrophy. Here we report that interference with ERK-dimerization, a prerequisite for ERKT188-phosphorylation, minimizes cardiac hypertrophy without inducing cardiac adverse effects: an ERK-dimerization inhibitory peptide (EDI) prevents ERKT188-phosphorylation, nuclear ERK1/2-signaling and cardiomyocyte hypertrophy, protecting from pressure-overload-induced heart failure in mice whilst preserving ERK1/2-activity and cytosolic survival signaling. We also examine this alternative ERK1/2-targeting strategy in cancer: indeed, ERKT188-phosphorylation is strongly upregulated in cancer and EDI efficiently suppresses cancer cell proliferation without causing cardiotoxicity. This powerful cardio-safe strategy of interfering with ERK-dimerization thus combats pathological ERK1/2-signaling in heart and cancer, and may potentially expand therapeutic options for ERK1/2-related diseases, such as heart failure and genetic syndromes.

Inflammation-associated suppression of metabolic gene networks in acute and chronic liver disease.

Inflammation has been recognized as essential for restorative regeneration. Here, we analyzed the sequential processes during onset of liver injury and subsequent regeneration based on time-resolved transcriptional regulatory networks (TRNs) to understand the relationship between inflammation, mature organ function, and regeneration. Genome-wide expression and TRN analysis were performed time dependently in mouse liver after acute injury by CCl4 (2 h, 8 h, 1, 2, 4, 6, 8, 16 days), as well as lipopolysaccharide (LPS, 24 h) and compared to publicly available data after tunicamycin exposure (mouse, 6 h), hepatocellular carcinoma (HCC, mouse), and human chronic liver disease (non-alcoholic fatty liver, HBV infection and HCC). Spatiotemporal investigation differentiated lobular zones for signaling and transcription factor expression. Acute CCl4 intoxication induced expression of gene clusters enriched for inflammation and stress signaling that peaked between 2 and 24 h, accompanied by a decrease of mature liver functions, particularly metabolic genes. Metabolism decreased not only in pericentral hepatocytes that underwent CCl4-induced necrosis, but extended to the surviving periportal hepatocytes. Proliferation and tissue restorative TRNs occurred only later reaching a maximum at 48 h. The same upstream regulators (e.g. inhibited RXR function) were implicated in increased inflammation and suppressed metabolism. The concomitant inflammation/metabolism TRN occurred similarly after acute LPS and tunicamycin challenges, in chronic mouse models and also in human liver diseases. Downregulation of metabolic genes occurs concomitantly to induce inflammation-associated genes as an early response and appears to be initiated by similar upstream regulators in acute and chronic liver diseases in humans and mice. In the acute setting, proliferation and restorative regeneration associated TRNs peak only later when metabolism is already suppressed.

[Hand cold abscess by a dematiaceous fungus. First report in Chile of Pleurostomophora richardsiae]. / Absceso frío en una mano por un hongo dematiáceo. Primera comunicación en Chile de Pleurostomophora richardsiae.

Dematiaceous fungi are a heterogeneous group of microorganisms able to synthesize melanin. Infections by this group that provoke tissular hyphae are called phaeohyphomycosis and usually involve skin and neighbor tissues. We present the case of a 86 years old men with a progressive soft cystic tumor in his right hand and wrist not associated to pain or inflammatory signs. A surgical intervention demonstrated flexor tenosynovitis with serous secretion, pseudocapsule and synovitis. Fungal culture demonstrated a dematiaceous fungi compatible with Pleurostomophora richardsiae that was confirmed by sequencing of the ITS region. Biopsy showed chronic inflammation with granuloma and hyphae. After surgical drainage, the patient was discharged without antifungal therapy but died of unrelated causes three month later. This is the first description of P. richardsiae as a cause of phaeohyphomycosis in Chile, a country with a template climate. Phaeohyphomycosis can be suspected when a chronic skin cystic lesion involves extremities without inflammatory signs, sometimes with an associated fistula. It may affect immunocompetent or immunosuppressed patients. Treatment involves surgical excision with or without antifungal therapy and prognosis is favorable.

In vitro antioxidant and antiproliferative effect of the extracts of Ephedra chilensis K Presl aerial parts.

BACKGROUND: Ephedra chilensis K Presl, known locally as pingo-pingo, is a Chilean endemic plant used in traditional medicine as an anti-inflammatory and used in other treatments. However, unlike for the other Ephedra species, there have been no reports on the antioxidant and cytotoxic effects of this plant. The present study aims to explore the potential applications of E. chilensis extract as a cytotoxic agent against in vitro cancer cell lines and to explore the relationship between this extract and antioxidant activity. METHODS: Total anthraquinone, flavonoid, and phenolic contents, as well as antioxidant activity (DPPH, FRAP, and TRAP assays) and cytotoxic effect on several cancer cell lines (MCF-7, PC-3, DU-145, and HT-29) were measured for the hexane, dichloromethane and ethanol extracts of E. chilensis. In addition, several correlations among the phytochemical content, antioxidant activity, and cytotoxic effect were evaluated. Finally, GC-MS analyses of the most active extracts were carried out to identify their major components and to relate these components to the cytotoxic effect. RESULTS: Antioxidant activity was found in the EtOH extracts of Ephedra, and the results were correlated with the phenolic content. For the cytotoxic activity, the non-polar extracts of E. chilensis had the highest antiproliferative effect for the MCF-7 and PC-3 cancer lines; the extract was shown to be up to three times more selective than doxorubicin. However, the cytotoxic effect was not correlated with the antioxidant activity. Lastly, the GC-MS analysis showed a high concentration of saturated fatty acids (mainly n-hexadecanoic acid) and terpenoids (mainly 4-(hydroxy-ethyl)-γ-butanolactone). CONCLUSION: The cytotoxic activity and selectivity of the non-polar extracts of E. chilensis for the MCF-7 and PC-3 cell lines could be related to the terpenic compounds and fatty acids of the extracts or to the synergistic effect of all of the compounds in the extracts. These non-polar extracts can be used for the development of new drugs against breast and prostate cancer.

Synthesis and Antiproliferative Activity of New Cyclodiprenyl Phenols against Select Cancer Cell Lines.

Six new cyclodiprenyl phenols were synthesized by direct coupling of perillyl alcohol and the appropriate phenol. Their structures were established by IR, HRMS and mainly NMR. Three human cancer cell lines-breast (MCF-7), prostate (PC-3) and colon (HT-29)-were used in antiproliferative assays, with daunorubicin and dunnione as positive controls. Results described in the article suggest that dihydroxylated compounds 2⁻4 and monohydroxylated compound 5 display selectivity against cancer cell lines, cytotoxicity, apoptosis induction, and mitochondrial membrane impairment capacity. Compound 2 was identified as the most effective of the series by displaying against all cancer cell lines a cytotoxicity close to dunnione antineoplastic agent, suggesting that the cyclodiprenyl phenols from perillyl alcohol deserve more extensive investigation of their potential medicinal applications.

Autumn Royal and Ribier Grape Juice Extracts Reduced Viability and Metastatic Potential of Colon Cancer Cells.

Antioxidants are known to be beneficial to health. This paper evaluates the potential chemopreventive and anticancer properties of phenolic compounds present in grape juice extracts (GJE) from Autumn Royal and Ribier varieties. The effects of these GJE on viability (SRB day assay) and metastatic potential (migration and invasion parameters) of colon cancer cell lines HT-29 and SW-480 were evaluated. The effects of GJE on two matrix metalloproteinase gene expressions (MMP2 and MMP9) were also evaluated via qRT-PCR. In the former, GJE reduced cell viability in both cell lines in a dose-dependent manner. GJE treatment also reduced cell migration and invasion. Moreover, MMP-2 and MMP-9 gene expression diminished depending on extract and on cell type. Conclusions. These results provide novel information concerning anticancer properties of selected GJE by revealing selective cytotoxicity and the ability to reduce invasiveness of colon cancer cells.

Adverse outcome pathways: opportunities, limitations and open questions.

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

The chilean network of microbial culture collections: establishment and operation / Red chilena de colecciones de cultivos microbianos: establecimiento y operación

According to the data available at the World Data Center for Microorganism-WDCM from the World Federation for Culture Collection-WFCC, Chile has four registered culture collections that preserve 2777 microbial strains. At the global point of view, the culture collections in Chile are in different level of operation regarding its own infrastructure and compliancy with quality standards for preservation of strains and for services provide. The absence of funding to support the preservation of the Chilean microbial assets is a key issue for the development of the Chilean bioeconomy. Considering this, the Chilean culture collections started working together to establish the Chilean Network of Microbial Culture Collections (RCCCM, acronym in Spanish). In this note, the establishment and operation of the RCCCM is presented and discussed.

De acuerdo con los datos disponibles en el World Data Center for Microorganism-WDCM de la Federación Mundial para la Colección de Cultivos - WFCC, Chile tiene cuatro colecciones de cultivos registradas que preservan 2777 cepas microbianas. Desde el punto de vista global, las colecciones de cultivos en Chile se encuentran en diferentes niveles de operación con respecto a su propia infraestructura y cumplimiento con estándares de calidad para la preservación de las cepas y para los servicios que proporcionan. La ausencia de financiamiento para apoyar la preservación de los activos microbianos chilenos es un tema clave para el desarrollo de la bioeconomía chilena. Considerando esto, las colecciones chilenas de cultivos comenzaron a trabajar conjuntamente para establecer la Red Chilena de Colecciones de Cultivos Microbianos (RCCCM). En esta nota se presenta y discute el establecimiento y funcionamiento de la RCCCM.

Toxicogenomics directory of chemically exposed human hepatocytes.

A long-term goal of numerous research projects is to identify biomarkers for in vitro systems predicting toxicity in vivo. Often, transcriptomics data are used to identify candidates for further evaluation. However, a systematic directory summarizing key features of chemically influenced genes in human hepatocytes is not yet available. To bridge this gap, we used the Open TG-GATES database with Affymetrix files of cultivated human hepatocytes incubated with chemicals, further sets of gene array data with hepatocytes from human donors generated in this study, and publicly available genome-wide datasets of human liver tissue from patients with non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular cancer (HCC). After a curation procedure, expression data of 143 chemicals were included into a comprehensive biostatistical analysis. The results are summarized in the publicly available toxicotranscriptomics directory ( http://wiki.toxbank.net/toxicogenomics-map/ ) which provides information for all genes whether they are up- or downregulated by chemicals and, if yes, by which compounds. The directory also informs about the following key features of chemically influenced genes: (1) Stereotypical stress response. When chemicals induce strong expression alterations, this usually includes a complex but highly reproducible pattern named 'stereotypical response.' On the other hand, more specific expression responses exist that are induced only by individual compounds or small numbers of compounds. The directory differentiates if the gene is part of the stereotypical stress response or if it represents a more specific reaction. (2) Liver disease-associated genes. Approximately 20 % of the genes influenced by chemicals are up- or downregulated, also in liver disease. Liver disease genes deregulated in cirrhosis, HCC, and NASH that overlap with genes of the aforementioned stereotypical chemical stress response include CYP3A7, normally expressed in fetal liver; the phase II metabolizing enzyme SULT1C2; ALDH8A1, known to generate the ligand of RXR, one of the master regulators of gene expression in the liver; and several genes involved in normal liver functions: CPS1, PCK1, SLC2A2, CYP8B1, CYP4A11, ABCA8, and ADH4. (3) Unstable baseline genes. The process of isolating and the cultivation of hepatocytes was sufficient to induce some stress leading to alterations in the expression of genes, the so-called unstable baseline genes. (4) Biological function. Although more than 2,000 genes are transcriptionally influenced by chemicals, they can be assigned to a relatively small group of biological functions, including energy and lipid metabolism, inflammation and immune response, protein modification, endogenous and xenobiotic metabolism, cytoskeletal organization, stress response, and DNA repair. In conclusion, the introduced toxicotranscriptomics directory offers a basis for a rationale choice of candidate genes for biomarker evaluation studies and represents an easy to use source of background information on chemically influenced genes.

Feohifomicosis causada por Colletotrichum gloeosporioides y Alternaria infectoria en un paciente trasplantado renal / Phaeohyphomycosis caused by Colletotrichum gloeosporioides and Alternaría infectoria in renal transplant recipient

Several species of black fungi have been reported as agents of subcutaneous phaeohyphomycosis. Although most of these fungi are considered opportunistic pathogens, they play an important role in phaeohyphomycosis, a disease considered an emergent mycosis among solid organ recipients. We report a case of phaeohyphomycosis caused by Alternaria infectoria of the left hand and the 4th finger of the right hand of a 68-year-old male who underwent a renal transplant 35 months before. The lesion was treated with surgical excision. One year later, the patient presented a new lesion on the 5th finger of the right hand, but this time caused by Colletotrichum gloeosporioides that was also removed surgically. Both lesions did not relapse after being removed. Antifungal susceptibility testing was performed against five antifungal drugs (amphotericin B, itraconazole, flucytosine, fluconazole and voriconazole). Alternaria infectoria was resistant to all five drugs and C. gloeosporioides was sensitive only to amphotericin B and voriconazole. We emphasize the need of histopathologic and microbiologic studies of new lesions of phaeohyphomycosis, since in this case the same patient was infected twice by two different fungi.

Diversas especies de hongos negros han sido descritos como agentes de feohifomicosis subcutáneas. A pesar que la gran mayoría de estos se consideran como oportunistas, juegan un papel importante en la feohifomicosis, enfermedad considerada una micosis emergente en pacientes trasplantados de órganos sólidos. Reportamos un caso de feohifomicosis subcutánea causada por Alternaria infectoria en el dorso de la mano izquierda y 4° falange de la mano derecha de un paciente de sexo masculino de 68 años de edad, que había sido sometido a un trasplante de riñón 35 meses antes. Se realizó escisión quirúrgica de la lesión. Después de un año, el paciente presentó una nueva lesión, esta vez en la 5° falange de la mano derecha causada por Colletotrichum gloeosporioides, lesión que también fue removida quirúrgicamente. El paciente no presentó nuevas lesiones después de las cirugías. Las pruebas de susceptibilidad antifúngica fueron realizados utilizando cinco antifúngicos (anfotericina B, itraconazol, 5-fluorcitosina, fluconazol y voriconazol). Alternaria infectoria presentó resistencia a los cinco antifíngicos y C. gloeosporioides fue susceptible solamente a anfotericina B y voriconazol. Enfatizamos la necesidad de realizar estudios histopatológicas y microbiológicos de nuevas lesiones de feohifomicosis, ya que el mismo paciente fue infectado por dos hongos diferentes.

Protocols for staining of bile canalicular and sinusoidal networks of human, mouse and pig livers, three-dimensional reconstruction and quantification of tissue microarchitecture by image processing and analysis.

Histological alterations often constitute a fingerprint of toxicity and diseases. The extent to which these alterations are cause or consequence of compromised organ function, and the underlying mechanisms involved is a matter of intensive research. In particular, liver disease is often associated with altered tissue microarchitecture, which in turn may compromise perfusion and functionality. Research in this field requires the development and orchestration of new techniques into standardized processing pipelines that can be used to reproducibly quantify tissue architecture. Major bottlenecks include the lack of robust staining, and adequate reconstruction and quantification techniques. To bridge this gap, we established protocols employing specific antibody combinations for immunostaining, confocal imaging, three-dimensional reconstruction of approximately 100-µm-thick tissue blocks and quantification of key architectural features. We describe a standard procedure termed 'liver architectural staining' for the simultaneous visualization of bile canaliculi, sinusoidal endothelial cells, glutamine synthetase (GS) for the identification of central veins, and DAPI as a nuclear marker. Additionally, we present a second standard procedure entitled 'S-phase staining', where S-phase-positive and S-phase-negative nuclei (stained with BrdU and DAPI, respectively), sinusoidal endothelial cells and GS are stained. The techniques include three-dimensional reconstruction of the sinusoidal and bile canalicular networks from the same tissue block, and robust capture of position, size and shape of individual hepatocytes, as well as entire lobules from the same tissue specimen. In addition to the protocols, we have also established image analysis software that allows relational and hierarchical quantifications of different liver substructures (e.g. cells and vascular branches) and events (e.g. cell proliferation and death). Typical results acquired for routinely quantified parameters in adult mice (C57Bl6/N) include the hepatocyte volume (5,128.3 ± 837.8 µm(3)) and the fraction of the hepatocyte surface in contact with the neighbouring hepatocytes (67.4 ± 6.7 %), sinusoids (22.1 ± 4.8 %) and bile canaliculi (9.9 ± 3.8 %). Parameters of the sinusoidal network that we also routinely quantify include the radius of the sinusoids (4.8 ± 2.25 µm), the branching angle (32.5 ± 11.2°), the length of intersection branches (23.93 ± 5.9 µm), the number of intersection nodes per mm(3) (120.3 × 103 ± 42.1 × 10(3)), the average length of sinusoidal vessel per mm(3) (5.4 × 10(3) ± 1.4 × 10(3)mm) and the percentage of vessel volume in relation to the whole liver volume (15.3 ± 3.9) (mean ± standard deviation). Moreover, the provided parameters of the bile canalicular network are: length of the first-order branches (7.5 ± 0.6 µm), length of the second-order branches (10.9 ± 1.8 µm), length of the dead-end branches (5.9 ± 0.7 µm), the number of intersection nodes per mm(3) (819.1 × 10(3) ± 180.7 × 10(3)), the number of dead-end branches per mm(3) (409.9 × 10(3) ± 95.6 × 10(3)), the length of the bile canalicular network per mm(3) (9.4 × 10(3) ± 0.7 × 10(3) mm) and the percentage of the bile canalicular volume with respect to the total liver volume (3.4 ± 0.005). A particular strength of our technique is that quantitative parameters of hepatocytes and bile canalicular as well as sinusoidal networks can be extracted from the same tissue block. Reconstructions and quantifications performed as described in the current protocols can be used for quantitative mathematical modelling of the underlying mechanisms. Furthermore, protocols are presented for both human and pig livers. The technique is also applicable for both vibratome blocks and conventional paraffin slices.

Interferon-inducible guanylate binding protein (GBP2) is associated with better prognosis in breast cancer and indicates an efficient T cell response.

BACKGROUND: Recently, interferon-inducible guanylate binding protein (GBP2) has been discussed as a possible control factor in tumor development, which is controlled by p53, and inhibits NF-Kappa B and Rac protein as well as expression of matrix metalloproteinase 9. However, the potential role that GBP2 plays in tumor development and prognosis has not yet been studied. METHODS: We analyzed whether GBP2 mRNA levels are associated with metastasis-free interval in 766 patients with node negative breast carcinomas who did not receive systemic chemotherapy. Furthermore, response to anthracycline-based chemotherapy was studied in 768 breast cancer patients. RESULTS: High expression of GBP2 in breast carcinomas was associated with better prognosis in the univariate (P < 0.001, hazard ratio 0.763, 95 % CI 0.650-0.896) as well as in the multivariate Cox analysis (P = 0.008, hazard ratio 0.731, 95 % CI 0.580-0.920) adjusted to the established clinical factors age, pT stage, grading, hormone and ERBB2 receptor status. The association was particularly strong in subgroups with high proliferation and positive estrogen receptor status but did not reach significance in carcinomas with low expression of proliferation associated genes. Besides its prognostic capacity, GBP2 also predicted pathologically complete response to anthracycline-based chemotherapy (P = 0.0037, odds ratio 1.39, 95 % CI 1.11-1.74). Interestingly, GBP2 correlated with a recently established T cell signature, indicating tumor infiltration with T cells (R = 0.607, P < 0.001). CONCLUSION: GBP2 is associated with better prognosis in fast proliferating tumors and probably represents a marker of an efficient T cell response.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Pharmacokinetics explain in vivo/in vitro discrepancies of carcinogen-induced gene expression alterations in rat liver and cultivated hepatocytes.

Cultivated hepatocytes represent a well-established in vitro system. However, the applicability of hepatocytes in toxicogenomics is still controversially discussed. Recently, an in vivo/in vitro discrepancy has been described, whereby the non-genotoxic rat liver carcinogen methapyrilene alters the expression of the metabolizing genes SULT1A1 and ABAT, as well as the DNA damage response gene GADD34 in vitro, but not in vivo. If the collagen sandwich cultures of hepatocytes really produce false-positive data, this would compromise its application in toxicogenomics. To revisit the putative in vivo/in vitro discrepancy, we first analyzed and modeled methapyrilene concentrations in the portal vein of rats. The relatively short half-life of 2.8 h implies a rapid decrease in orally administered methapyrilene in vivo below concentrations that can cause gene expression alterations. This corresponded to the time-dependent alteration levels of GADD34, ABAT and SULT1A1 RNA in the liver: RNA levels are altered 1, 6 and 12 h after methapyrilene administration, but return to control levels after 24 and 72 h. In contrast, methapyrilene concentrations in the culture medium supernatant of primary rat hepatocyte cultures decreased slowly. This explains why GADD34, ABAT and SULT1A1 were still deregulated after 24 h exposure in vitro, but not in vivo. It should also be considered that the earliest analyzed time point in the previous in vivo studies was 24 h after methapyrilene administration. In conclusion, previously observed in vitro/in vivo discrepancy can be explained by different pharmacokinetics present in vitro and in vivo. When the in vivo half-life is short, levels of some initially altered genes may have returned to control levels already 24 h after administration.

Identification of RARRES1 as a core regulator in liver fibrosis.

Genetic factors contribute to progression and modulation of hepatic fibrosis. High throughput genomics/transcriptomics approaches aiming at identifying key regulators of fibrosis development are tainted with the difficulty of separating essential biological "driver" from modifier genes. We applied a comparative transcriptomics approach and investigated fibrosis development in different organs to identify overlapping expression changes, since these genes may be part of core pathways in fibrosis development. Gene expression was analysed on publicly available microarray data from liver, lung and kidney fibrosis. RARRES1, AGER and S100A2 were differentially regulated in all fibrosis experiments. RARRES1 was extensively analysed by means of advanced bioinformatics analyses and functional studies. Microarray and Western Blot analysis of a standard liver fibrosis model (CCl(4)) demonstrated an early induction of RARRES1 mRNA and protein expression. In addition, quantitative RT-PCR in tissue samples from patients with advanced liver fibrosis showed higher expression as compared to non-fibrotic biopsies. Microarray analysis of RARRES1 overexpressing cells identified an enrichment of a major signature associated with fibrosis. Furthermore, RARRES1 expression increased during in vitro activation of hepatic stellate cells. To further verify the pro-fibrogenic role across organs, we demonstrated an increase in RARRES1 expression in a rat lung fibrosis model induced by adenoviral TGF-ß1 induction. We have performed a comparative transcriptomics analysis in order to identify core pathways of liver fibrogenesis, confirmed a candidate gene and enlightened the up- and downstream mechanisms of its action leading to fibrosis across organs and species.