RESUMEN
We report the synthesis, characterization, and magnetic properties of eight neutral functionalized trigonal lanthanide coordination complexes LnL with Ln = Gd (1), Tb (2), Dy (3), Ho (4), Er (5), Tm (6), Yb (7), Lu (8). These were prepared through a one-pot synthesis where, first, the ligand H3L was synthesized in situ through a Schiff base reaction of tris(2-aminoethyl)amine with 2,6-diformyl-p-cresol. Following addition of Ln(OTf)3·xH2O and base, LnL was obtained. Powder X-ray diffraction confirms that all complexes are isostructural. LnL contain pendant, noncoordinating carbonyl functions that are reactive and represent direct anchoring points to appropriately functionalized surfaces. Furthermore, these reactive carbonyl functions can be used to postfunctionalize LnL: for example, with aromatic π systems. We present herein the Schiff base condensation of 7 with benzylamine to yield 9 as well as the characterization and magnetic properties of 9. Our study establishes LnL as a truly versatile module for the surface deposition of Ln-based single-ion magnets.
RESUMEN
BACKGROUND & AIMS: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1-Prkaca fusion and monitored the mice for liver tumor development. METHODS: We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1-Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8-week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1-Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing. RESULTS: Livers from 12 of the 15 mice given the vectors to induce the Dnajb1-Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1-Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1-Prkaca fusion revealed a lack of mutations in genes commonly associated with liver cancers, as observed in human FL-HCC. CONCLUSIONS: Using CRISPR/Cas9 technology, we found generation of the Dnajb1-Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1-PRKACA might be developed as therapeutics for this form of liver cancer.
Asunto(s)
Biomarcadores de Tumor/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Carcinoma Hepatocelular/genética , Transformación Celular Neoplásica/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Edición Génica/métodos , Fusión Génica , Proteínas del Choque Térmico HSP40/genética , Neoplasias Hepáticas/genética , Animales , Biomarcadores de Tumor/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Proteínas del Choque Térmico HSP40/metabolismo , Neoplasias Hepáticas/metabolismo , Ratones , Fenotipo , Factores de TiempoRESUMEN
Selective serotonin re-uptake inhibitors are pharmaceuticals used to treat a range of psychological disorders. They are frequently found in surface waters in populated areas. In recent years, they have been shown to affect the behaviour of various aquatic organisms in a way that can have ecological effects. In this study, we exposed zebrafish of both sexes to nominally 0.00, 0.15 and 1.50 µg L-1 Escitalopram in flow-through tanks for three weeks. Subsequently, ten swimming behaviour parameters were quantified using high-resolution video tracking. There were noticeable gender differences in the behaviour responses to Escitalopram. Female fish exposed to 1.50 µg L-1 Escitalopram had a lower maximum swimming velocity, stopped less often and exhibited increased boldness (reduced thigmotaxis) compared to controls. Male fish exposed to 1.50 µg L-1 had a lower maximum swimming velocity compared to control fish. At the end of exposures, both length and weight of the females exposed to 1.50 µg L-1 Escitalopram were significantly less than the group of control fish. In addition, males exposed to 1.50 µg L-1 Escitalopram were significantly shorter than control fish. The behaviour, weight and body length of the fish exposed to nominally 0.15 µg L-1 was not significantly different from control fish in either sex. The results of this study demonstrate that Escitalopram can affect subtle but ecologically important aspects of fish behaviour and lends further credibility to the assumption that Escitalopram is an environmentally active pharmaceutical.
Asunto(s)
Citalopram/efectos adversos , Conducta Exploratoria/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/efectos adversos , Natación/fisiología , Contaminantes Químicos del Agua/efectos adversos , Pez Cebra/fisiología , Animales , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Distribución Aleatoria , Factores SexualesRESUMEN
The theoretical framework of cosmology is mainly defined by gravity, of which general relativity is the current model. Recent tests of general relativity within the Lambda Cold Dark Matter (ΛCDM) model have found a concordance between predictions and the observations of the growth rate and clustering of the cosmic web. General relativity has not hitherto been tested on cosmological scales independently of the assumptions of the ΛCDM model. Here we report an observation of the gravitational redshift of light coming from galaxies in clusters at the 99 per cent confidence level, based on archival data. Our measurement agrees with the predictions of general relativity and its modification created to explain cosmic acceleration without the need for dark energy (the f(R) theory), but is inconsistent with alternative models designed to avoid the presence of dark matter.
RESUMEN
The RAS-stimulated RAF-MEK-ERK pathway confers epithelial cells with critical motile and invasive capacities during development, tissue regeneration, and carcinoma progression, often via promoting the epithelial-mesenchymal transition (EMT). Many mechanisms by which ERK exerts this control remain elusive. We demonstrate that the ERK-activated kinase RSK is necessary to induce mesenchymal motility and invasive capacities in nontransformed epithelial and carcinoma cells. RSK is sufficient to induce certain motile responses. Expression profiling analysis revealed that a primary role of RSK is to induce transcription of a potent promotile/invasive gene program by FRA1-dependent and -independent mechanisms. The program enables RSK to coordinately modulate the extracellular environment, the intracellular motility apparatus, and receptors mediating communication between these compartments to stimulate motility and invasion. These findings uncover a mechanism whereby the RAS-ERK pathway controls epithelial cell motility by identifying RSK as a key effector, from which emanate multiple highly coordinate transcription-dependent mechanisms for stimulation of motility and invasive properties.
Asunto(s)
Carcinoma/enzimología , Movimiento Celular , Transdiferenciación Celular , Transformación Celular Neoplásica/metabolismo , Células Epiteliales/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteínas ras/metabolismo , Animales , Carcinoma/genética , Carcinoma/patología , Línea Celular , Movimiento Celular/genética , Transdiferenciación Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Perros , Células Epiteliales/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genotipo , Humanos , Mesodermo/enzimología , Mesodermo/patología , Invasividad Neoplásica , Fenotipo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Transducción GenéticaRESUMEN
This paper describes the physics case for a new fixed target facility at CERN SPS. The SHiP (search for hidden particles) experiment is intended to hunt for new physics in the largely unexplored domain of very weakly interacting particles with masses below the Fermi scale, inaccessible to the LHC experiments, and to study tau neutrino physics. The same proton beam setup can be used later to look for decays of tau-leptons with lepton flavour number non-conservation, [Formula: see text] and to search for weakly-interacting sub-GeV dark matter candidates. We discuss the evidence for physics beyond the standard model and describe interactions between new particles and four different portals-scalars, vectors, fermions or axion-like particles. We discuss motivations for different models, manifesting themselves via these interactions, and how they can be probed with the SHiP experiment and present several case studies. The prospects to search for relatively light SUSY and composite particles at SHiP are also discussed. We demonstrate that the SHiP experiment has a unique potential to discover new physics and can directly probe a number of solutions of beyond the standard model puzzles, such as neutrino masses, baryon asymmetry of the Universe, dark matter, and inflation.
RESUMEN
Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30-70% at high transfection efficiencies and â¼ 2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with â¼ 1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9.
Asunto(s)
Proteínas Asociadas a CRISPR/genética , Desoxirribonucleasas/genética , Técnicas de Sustitución del Gen , Proteínas Luminiscentes/genética , Proteínas Asociadas a CRISPR/metabolismo , Línea Celular Tumoral , Separación Celular , Desoxirribonucleasas/metabolismo , Citometría de Flujo , Colorantes Fluorescentes , Genoma , Humanos , Células K562 , Proteínas Luminiscentes/metabolismo , Péptidos/química , Plásmidos/genética , Dedos de ZincRESUMEN
The glutamatergic neurotransmitter system is involved in important neurophysiological processes and thus constitutes a promising target for the treatment of neurological diseases. The two ionotropic glutamate receptor agonists kainic acid (KA) and dihydrokainic acid (DHK) have been used as research tools in various in vivo central nervous system disease models in rodents, as well as being templates in the design of novel ligands affecting the glutamatergic system. Both molecules are highly polar but yet capable of crossing the blood-brain barrier (BBB). We used an in situ rat brain perfusion technique to determine the brain uptake mechanism and permeability across the BBB. To determine KA and DHK concentrations in the rat brain, simple and rapid sample preparation and liquid chromatography mass spectrometer methods were developed. According to our results the BBB permeability of KA and DHK is low, 0.25 × 10(-6) and 0.28 × 10(-6) cm/s for KA and DHK, respectively. In addition, the brain uptake is mediated by passive diffusion, and not by active transport. Furthermore, the non-specific plasma and brain protein binding of KA and DHK was determined to be low, which means that the unbound drug volume of distribution in brain is also low. Therefore, even though the total KA and DHK concentrations in the brain are low after systemic dosing, the concentrations in the vicinity of the glutamate receptors are sufficient for their activation and thus the observed efficacy.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Permeabilidad Capilar/fisiología , Ácido Kaínico/análogos & derivados , Ácido Kaínico/metabolismo , Animales , Transporte Iónico/fisiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
In comparison with the technology platforms developed to localize transcripts and proteins, imaging tools for visualization of metabolite distributions in plant tissues are less well developed and lack versatility. This hampers our understanding of plant metabolism and dynamics. In this study, we demonstrate that desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of tissue imprints on porous Teflon may be used to accurately image the distribution of even labile plant metabolites such as hydroxynitrile glucosides, which normally undergo enzymatic hydrolysis by specific ß-glucosidases upon cell disruption. This fast and simple sample preparation resulted in no substantial differences in the distribution and ratios of all hydroxynitrile glucosides between leaves from wild-type Lotus japonicus and a ß-glucosidase mutant plant that lacks the ability to hydrolyze certain hydroxynitrile glucosides. In wild-type, the enzymatic conversion of hydroxynitrile glucosides and the concomitant release of glucose were easily visualized when a restricted area of the leaf tissue was damaged prior to sample preparation. The gene encoding the first enzyme in hydroxynitrile glucoside biosynthesis in L. japonicus leaves, CYP79D3, was found to be highly expressed during the early stages of leaf development, and the hydroxynitrile glucoside distribution in mature leaves reflected this early expression pattern. The utility of direct DESI-MSI of plant tissue was demonstrated using cryo-sections of cassava (Manihot esculenta) tubers. The hydroxynitrile glucoside levels were highest in the outer cell layers, as verified by LC-MS analyses. The unexpected discovery of a hydroxynitrile-derived di-glycoside shows the potential of DESI-MSI to discover and guide investigations into new metabolic routes.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Glucósidos/metabolismo , Lotus/metabolismo , Manihot/metabolismo , Sorghum/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Cromatografía Liquida , Sistema Enzimático del Citocromo P-450/genética , Genes Reporteros , Glucósidos/química , Hidrólisis , Lotus/química , Lotus/citología , Lotus/genética , Manihot/química , Manihot/citología , Espectrometría de Masas , Mutación , Nitrilos/química , Nitrilos/metabolismo , Hojas de la Planta/química , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Tubérculos de la Planta/química , Tubérculos de la Planta/citología , Tubérculos de la Planta/metabolismo , Regiones Promotoras Genéticas/genética , Plantones/química , Plantones/citología , Plantones/metabolismo , Sorghum/química , Espectrometría de Masa por Ionización de Electrospray/instrumentación , beta-Glucosidasa/metabolismoRESUMEN
Desorption electrospray ionization (DESI) mass spectrometry imaging is demonstrated as a detection technique for penetration experiments of drugs in skin. Lidocaine ointment was used as the model compound in ex vivo experiments with whole pig ears as the skin model. Follicular transport of lidocaine into the deeper skin layers is demonstrated for the first time. Furthermore, metabolism of lidocaine to 3-OH-lidocaine was observed in subcutaneous tissue as well as in lobules of white adipose tissue surrounding the hair follicles. These results suggest that it is advantageous to use full thickness skin, including subcutaneous tissue, for skin metabolism studies.
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Oído , Lidocaína/análisis , Piel/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray , Administración Tópica , Animales , Transporte Biológico , Epidermis/metabolismo , Lidocaína/química , Solventes/química , PorcinosRESUMEN
Beyond the smallest organisms, animals rely on tubes to transport cells, oxygen, nutrients, waste products, and a great variety of secretions. The cardiovascular system, lungs, gastrointestinal and genitourinary tracts, as well as major exocrine glands, are all composed of tubes. Paradoxically, despite their ubiquitous importance, most existing devices designed to study tubes are relatively complex to manufacture and/or utilize. The present work describes a simple method for generating tubes in vitro using nothing more than a low-cost 3D printer along with general lab supplies. The technology is termed "TruD", an acronym for true dimensional. Using this technology, it is readily feasible to cast tubes embedded in ECM with easy access to the lumen. The design is modular to permit more complex tube arrangements and to sustain flow. Importantly, by virtue of its simplicity, TruD technology enables typical molecular cell biology experiments where multiple conditions are assayed in replicate.
Asunto(s)
Impresión Tridimensional , Humanos , Animales , Impresión Tridimensional/instrumentación , Células Endoteliales/citología , Células Endoteliales/metabolismoRESUMEN
Defense against intracellular acidification of breast cancer tissue depends on net acid extrusion via Na+,HCO3--cotransporter NBCn1/Slc4a7 and Na+/H+-exchanger NHE1/Slc9a1. NBCn1 is increasingly recognized as breast cancer susceptibility protein and promising therapeutic target, whereas evidence for targeting NHE1 is discordant. Currently, selective small molecule inhibitors exist against NHE1 but not NBCn1. Cellular assays-with some discrepancies-link NHE1 activity to proliferation, migration, and invasion; and disrupted NHE1 expression can reduce triple-negative breast cancer growth. Studies on human breast cancer tissue associate high NHE1 expression with reduced metastasis and-in some molecular subtypes-improved patient survival. Here, we evaluate Na+/H+-exchange and therapeutic potential of the NHE1 inhibitor cariporide/HOE-642 in murine ErbB2-driven breast cancer. Ex vivo, cariporide inhibits net acid extrusion in breast cancer tissue (IC50 = 0.18 µM) and causes small decreases in steady-state intracellular pH (pHi). In vivo, we deliver cariporide orally, by osmotic minipumps, and by intra- and peritumoral injections to address the low oral bioavailability and fast metabolism. Prolonged cariporide administration in vivo upregulates NBCn1 expression, shifts pHi regulation towards CO2/HCO3--dependent mechanisms, and shows no net effect on the growth rate of ErbB2-driven primary breast carcinomas. Cariporide also does not influence proliferation markers in breast cancer tissue. Oral, but not parenteral, cariporide elevates serum glucose by â¼1.5 mM. In conclusion, acute administration of cariporide ex vivo powerfully inhibits net acid extrusion from breast cancer tissue but lowers steady-state pHi minimally. Prolonged cariporide administration in vivo is compensated via NBCn1 and we observe no discernible effect on growth of ErbB2-driven breast carcinomas.
Asunto(s)
Neoplasias de la Mama , Proliferación Celular , Guanidinas , Receptor ErbB-2 , Intercambiador 1 de Sodio-Hidrógeno , Sulfonas , Guanidinas/farmacología , Femenino , Animales , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/antagonistas & inhibidores , Intercambiador 1 de Sodio-Hidrógeno/metabolismo , Intercambiador 1 de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiador 1 de Sodio-Hidrógeno/genética , Ratones , Humanos , Sulfonas/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Simportadores de Sodio-Bicarbonato/metabolismo , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/antagonistas & inhibidores , Línea Celular Tumoral , Concentración de Iones de HidrógenoRESUMEN
A simple data reconstruction technique in CE-UV-ESI-MS (where UV stands for ultraviolet) is presented to overcome the drift in mobilities caused by various factors compromising the reproducibility of such data, for example Joule heating effects and the variation in thermostatic control along the capillary, drift in EOF and the suction effect caused by the nebulizing gas in coaxial CE-MS interfaces. We present here a method to transform the traditional time-based electropherogram into the corresponding temperature-correlated mobility scale allowing tracking of analytes independent from capillary dimensions, electric field strengths, temperature control, and distance between the detectors. The main principle of this alignment is based on including the current in the mobility calculations and relating this to the initial electrical resistance of the buffer-filled capillary. The temperature-correlated mobility calculation eliminates the peak shifts due to the viscosity changes, improves the precision of peak identification using the observed temperature-correlated mobilities, and allows a direct comparison of signals from different detection combinations. The method allows peaks from normal CE-UV separations to be correlated with the corresponding peak obtained by MS detection in CE-MS even for differences in capillary dimensions and thermostatic control.
Asunto(s)
Electroforesis Capilar/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Modelos Químicos , Procesamiento de Señales Asistido por Computador , Espectrofotometría Ultravioleta , TemperaturaRESUMEN
ARHGAP35 , which encodes p190A RhoGAP (p190A), is a major cancer gene. p190A is a tumor suppressor that activates the Hippo pathway. p190A was originally cloned via direct binding to p120 RasGAP (RasGAP). Here, we determine that a novel interaction of p190A with the tight junction-associated protein ZO-2 is dependent on RasGAP. We establish that both RasGAP and ZO-2 are necessary for p190A to activate LATS kinases, elicit mesenchymal-to-epithelial transition, promote contact inhibition of cell proliferation and suppress tumorigenesis. Moreover, RasGAP and ZO-2 are required for transcriptional modulation by p190A. Finally, we demonstrate that low ARHGAP35 expression is associated with shorter survival in patients with high, but not low, transcript levels of TJP2 encoding ZO-2. Hence, we define a tumor suppressor interactome of p190A that includes ZO-2, an established constituent of the Hippo pathway, and RasGAP, which despite strong association with Ras signaling, is essential for p190A to activate LATS kinases.
RESUMEN
The trigonal lanthanide complexes LnL (H3L = tris(((3-formyl-5-methylsalicylidene)amino)ethyl)amine) contain three pendant aldehyde groups and are known to react with primary amines. Reacting LnL (Ln = Yb, Lu) with 1-octadecylamine yields the novel aliphatic lanthanide complexes LnL18 (H3L18 = tris(((3-(1-octadecylimine)-5-methylsalicylidene)amino)ethyl)amine) where the three aldehyde groups are transformed to 1-octadecylimine groups. Herein the syntheses, structural characterisation and magnetic properties of LnL18 are presented. The crystal structure of YbL18 shows that the reaction of YbL with 1-octadecylamine leads to only very subtle perturbations in the first coordination sphere of Yb(III), with the Yb(III) ion retaining its heptacoordination and similar bond lengths and angles to the ligand. The three octadecyl chains in each complex were found to direct crystal packing into lipophilic arrays of van der Waals interaction-driven hydrocarbon stacking. The static magnetic properties of YbL18 were compared to those of the non-derivatised complex YbL. The energy level splitting of the 2F7/2 ground multiplet was found, by emission spectroscopy, to be very similar between the derivatised and non-derivatised complexes. A.c. magnetic susceptibility measurements on YbL18 and YbL diluted at 4.8% and 4.2% into the diamagnetic hosts LuL18 and LuL, respectively, revealed that the spin-lattice relaxation of both complexes is governed by a low temperature direct process and a high temperature Raman process. In the high temperature regime, the derivatised complex was also found to have faster spin-lattice relaxation, which is likely due to the increased number of phonons in the octadecyl chains.
RESUMEN
ARHGAP35, which encodes p190A RhoGAP (p190A), is a major cancer gene. p190A is a tumor suppressor that activates the Hippo pathway. p190A was originally cloned via direct binding to p120 RasGAP (RasGAP). Here, we determine that interaction of p190A with the tight-junction-associated protein ZO-2 is dependent on RasGAP. We establish that both RasGAP and ZO-2 are necessary for p190A to activate large tumor-suppressor (LATS) kinases, elicit mesenchymal-to-epithelial transition, promote contact inhibition of cell proliferation, and suppress tumorigenesis. Moreover, RasGAP and ZO-2 are required for transcriptional modulation by p190A. Finally, we demonstrate that low ARHGAP35 expression is associated with shorter survival in patients with high, but not low, transcript levels of TJP2 encoding ZO-2. Hence, we define a tumor-suppressor interactome of p190A that includes ZO-2, an established constituent of the Hippo pathway, and RasGAP, which, despite strong association with Ras signaling, is essential for p190A to activate LATS kinases.
Asunto(s)
Proteínas Activadoras de GTPasa , Vía de Señalización Hippo , Humanos , Proliferación Celular , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Transducción de SeñalRESUMEN
The traditional way of calculating mobility and peak areas in capillary electrophoresis does not take into account the changes in the buffer viscosity at different thermostatic control and that the analytes may accelerate during the individual runs due to Joule heating effects. We present a method for accounting for these changes based on the monitored changes in current during the separation. The calculation method requires measuring the initial resistance of the buffer filled capillary, performed using a 0.2 min voltage ramping at the start of a separation. The mobility calculation corrected for current drift allowed identification of the tested analytes independent from capillary dimensions, electric field strengths and temperature control. Furthermore, the peak areas become less influenced by the experimental conditions, since the velocities of the analytes passing the detector are corrected for the acceleration during the run. The short voltage ramping could be further used to evaluate the heat transfer of the capillary to the surroundings and to estimate the temperature changes during the separation. The temperature was shown to change the ionization of 2-phenylethylamine in accordance to a pKa dependency of primary amines reported in literature.
Asunto(s)
Electroforesis Capilar/métodos , Electroforesis Capilar/normas , Campos Electromagnéticos , Modelos Químicos , Fenetilaminas/química , Reproducibilidad de los Resultados , Temperatura , ViscosidadRESUMEN
Pain treatment in ancient Greece, and through the middle ages in Europe, was to a great extent based on the expertise of the Greek physician Galen (c. 129-200 A.D.). Galen makes particular reference to "Olympic Victor's Dark Ointment" (OVDO), which is listed with a number of collyria. Galen states that OVDO can be useful for treating extreme pain and swellings, forming one of the best eye salves. Olympic Victor's Dark Ointment, an opium-based treatment, forms a "patch" when applied externally as an ointment, because it quickly dries to cover a localized region but still retains its elastic properties. This study has recreated OVDO and applied the ointment to abdominal mouse skin, in vitro. To assess the efficacy of OVDO, the transdermal transfer of morphine was measured when given as OVDO and compared to morphine administered in the form of a solution of Opium + PBS (ringer). Olympic Victor's Dark Ointment showed a transdermal transfer of morphine over time comparable to 25% of the most efficient modern transdermal opioid patches, while hardly any morphine was able to penetrate the skin when applied mixed in PBS. We conclude that OVDO is very efficient in its composition and may carry some forgotten abilities in terms of drug delivery, which could be transferred to modern medicine. Indeed, this may lead to a better choice of morphine use and controlled management in individual patient cases, taking both pain relief and anti-inflammatory aspects into account.
Asunto(s)
Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/historia , Historia Antigua , Dolor/tratamiento farmacológico , Animales , Antigua Grecia , Humanos , Masculino , Ratones , Parche TransdérmicoRESUMEN
Transporters of the inner mitochondrial membrane are essential to metabolism. We demonstrate that metabolism as represented by expression of genes encoding SLC25 transporters differentiates human cancers. Tumor to normal tissue expression ratios for clear cell renal cell carcinoma, colon adenocarcinoma, lung adenocarcinoma and breast invasive carcinoma were found to be highly significant. Affinity propagation trained on SLC25 gene expression patterns from 19 human cancer types (6825 TCGA samples) and normal tissues (2322 GTEx samples) was used to generate clusters. They differentiate cancers from normal tissues. They also indicate cancer subtypes with survivals distinct from the total patient population of the cancer type. Probing the kidney, colon, lung, and breast cancer clusters, subtype pairs of cancers were identified with distinct prognoses and differing in expression of protein coding genes from among 2080 metabolic enzymes assayed. We demonstrate that SLC25 expression clusters facilitate the identification of the tissue-of-origin, essential to efficacy of most cancer therapies, of CUPs (cancer-unknown-primary) known to have poor prognoses. Different cancer types within a single cluster have similar metabolic patterns and this raises the possibility that such cancers may respond similarly to existing and new anti-cancer therapies.
Asunto(s)
Adenocarcinoma , Neoplasias de la Mama , Carcinoma de Células Renales , Neoplasias del Colon , Neoplasias Renales , Adenocarcinoma/genética , Neoplasias de la Mama/genética , Carcinoma de Células Renales/patología , Neoplasias del Colon/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Renales/patología , PronósticoRESUMEN
The Rac GTPase regulates Rho signaling in a broad range of physiological settings and in oncogenic transformation [1-3]. Here, we report a novel mechanism by which crosstalk between Rac and Rho GTPases is achieved. Activated Rac1 binds directly to p190B Rho GTPase-activating protein (RhoGAP), a major modulator of Rho signaling. p190B colocalizes with constitutively active Rac1 in membrane ruffles. Moreover, activated Rac1 is sufficient to recruit p190B into a detergent-insoluble membrane fraction, a process that is accompanied by a decrease in GTP-bound RhoA from membranes. p190B is recruited to the plasma membrane in response to integrin engagement [4]. We demonstrate that collagen type I, a potent inducer of Rac1-dependent cell motility in HeLa cells, counteracts cytoskeletal collapse resulting from overexpression of wild-type p190B, but not that resulting from overexpression of a p190B mutant specifically lacking the Rac1-binding sequence. Furthermore, this p190B mutant exhibits dramatically enhanced RhoGAP activity, consistent with a model whereby binding of Rac1 relieves autoinhibition of p190B RhoGAP function. Collectively, these observations establish that activated Rac1, through direct interaction with p190B, modulates subcellular RhoGAP localization and activity, thereby providing a novel mechanism for Rac control of Rho signaling in a broad range of physiological processes.