RESUMO
BACKGROUND: To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1. METHODS: The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vectors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay. RESULTS: The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) staining, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations. CONCLUSIONS: This study developed a radiation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.
Assuntos
Sistemas CRISPR-Cas , Neoplasias Hepáticas , Camundongos , Animais , Sistemas CRISPR-Cas/genética , Camundongos Endogâmicos C57BL , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/metabolismo , Mutação , Organoides/metabolismo , Organoides/patologia , Trifosfato de AdenosinaRESUMO
This research demonstrates a method to reduce the resistance of amorphous indium-gallium-zinc-oxide (a-IGZO) using a "vacuum-free solution-based metallization" (VSM) process, which revolutionizes the metallization process thanks to its simplicity, by simply dipping the a-IGZO into trimethyl aluminium (TMA, (CH3)3Al) solution. From the XPS results, it was found that oxygen vacancies were generated after the VSM process, resulting in the enhanced conductivity. Various metallization time and solution temperature conditions were investigated, and the measured conductivity of the a-IGZO could be enhanced up to 20.32 S cm-1, which is over 105 times larger compared to that of the untreated a-IGZO. By utilizing the VSM process, self-aligned top-gate (SATG) a-IGZO thin-film-transistors (TFTs) were successfully fabricated, and to provide an explanation for the mechanism, X-ray photoelectron spectroscopy (XPS) was employed.
RESUMO
The effect of Ag decoration on the gas sensing characteristics of SnO(2) nanowire (NW) networks was investigated. The Ag layers with thicknesses of 5-50 nm were uniformly coated on the surface of SnO(2) NWs via e-beam evaporation, which were converted into isolated or continuous configurations of Ag islands by heat treatment at 450 °C for 2 h. The SnO(2) NWs decorated by isolated Ag nano-islands displayed a 3.7-fold enhancement in gas response to 100 ppm C(2)H(5)OH at 450 °C compared to pristine SnO(2) NWs. In contrast, as the Ag decoration layers became continuous, the response to C(2)H(5)OH decreased significantly. The enhancement and deterioration of the C(2)H(5)OH sensing characteristics by the introduction of the Ag decoration layer were strongly governed by the morphological configurations of the Ag catalysts on SnO(2) NWs and their sensitization mechanism.
Assuntos
Técnicas Eletroquímicas/métodos , Etanol/análise , Nanopartículas Metálicas/química , Nanofios/química , Prata/química , Compostos de Estanho/química , Catálise , Gases/química , Nanopartículas Metálicas/ultraestrutura , Nanofios/ultraestrutura , TemperaturaRESUMO
Ginseng saponins, major active components of ginseng root used by folk medicine in the treatment of various diseases, produce multiple pharmacological responses having many effects on the central and peripheral nervous system. Specifically, ginsenoside Rg(2) has been shown to block the nicotinic acetylcholine receptors in bovine chromaffin cells. We have studied the effect of Rg(2) on different types of human neuronal nicotinic acetylcholine receptors (nAChRs), both homomeric and heteromeric, expressed in Xenopus oocytes. Rg(2) did not affect the acetylcholine (ACh)-induced currents in alpha(7) human receptors, however Rg(2) affected the peak currents, and mainly the desensitization of heteromeric receptors alpha(3)beta(4), alpha(3)beta(2), alpha(4)beta(4), and alpha(4)beta(2). Both effects, a diminution of peak current and an increase of desensitization, are dose-dependent and are very similar for all the receptors. The mechanism of action has been studied in more detail in alpha(3)beta(4) and alpha(4)beta(2) receptors where we found a negligible shift in the ACh dose-response curves and a persistence of the Rg(2) effects at high ACh concentrations, indicative of a noncompetitive antagonism. A lack of voltage dependence on the reduction of the peak currents induced by ACh also suggests that Rg(2) does not act as an open channel blocker of human nAChR. The results indicate that Rg(2) acts specifically on heteromeric human nAChRs modulating their desensitization and suggest a possible mechanism by which this saponin contributes to the multiple therapeutic effects of ginseng.
Assuntos
Ginsenosídeos , Panax/química , Receptores Nicotínicos/metabolismo , Saponinas/farmacologia , Animais , Relação Dose-Resposta a Droga , Vetores Genéticos/antagonistas & inibidores , Vetores Genéticos/metabolismo , Humanos , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Receptores Nicotínicos/fisiologia , Saponinas/química , Transfecção , Xenopus/genéticaRESUMO
We investigated the effects of ginsenosides, the active ingredient of ginseng, on neuronal or muscle-type nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal alpha3beta4, alpha7 or human muscle alphabetadeltavarepsilon subunits. Treatment with acetylcholine elicited an inward peak current (I(ACh)) in oocytes expressing nicotinic acetylcholine receptor subtypes. Cotreatment with ginsenoside Rg2 and acetylcholine inhibited I(ACh) in oocytes expressing with alpha3beta4 or alphabetadeltavarepsilon but not in oocytes expressing alpha7 nicotinic acetylcholine receptors. The inhibition of I(ACh) by ginsenoside Rg2 was reversible and dose-dependent. The half-inhibitory concentrations (IC50) of ginsenoside Rg2 were 60.2+/-14.1 and 15.7+/-3.5 microM in oocytes expressing alpha3beta4 and alphabetadeltavarepsilon nicotinic acetylcholine receptors, respectively. The inhibition of I(ACh) by ginsenoside Rg2 was voltage-independent and noncompetitive. Other ginsenosides besides ginsenoside Rg2 also inhibited I(ACh) in oocytes expressing alpha3beta4 or alphabetadeltavarepsilon nicotinic acetylcholine receptors. The order of potency for the inhibition of I(ACh) was ginsenoside Rg2>Rf>Re>Rg1>Rc>Rb2>Rb1 in oocytes expressing alpha3beta4 nicotinic acetylcholine receptors and was ginsenoside Rg2>Rf>Rg1>Re>Rb1>Rc>Rb2 in oocytes expressing alphabetadeltavarepsilon nicotinic acetylcholine receptors. These results indicate that ginsenosides might regulate nicotinic acetylcholine receptors in a differential manner and this regulation might be one of the pharmacological actions of Panax ginseng.
Assuntos
Panax/química , Receptores Nicotínicos/fisiologia , Saponinas/farmacologia , Acetilcolina/farmacologia , Animais , Bovinos , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica , Ginsenosídeos , Humanos , Potenciais da Membrana/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Receptores Nicotínicos/genética , Saponinas/química , Xenopus laevisRESUMO
We have shown that ginsenoside Rf (Rf) regulates voltage-dependent Ca(2+) channels through pertussis toxin (PTX)-sensitive G proteins in rat sensory neurons. These results suggest that Rf can act through a novel G protein-linked receptor in the nervous system. In the present study, we further examined the effect of Rf on G protein-coupled inwardly rectifying K(+) (GIRK) channels after coexpression with size-fractionated rat brain mRNA and GIRK1 and GIRK4 (GIRK1/4) channel cRNAs in Xenopus laevis oocytes using two-electrode voltage-clamp techniques. We found that Rf activated GIRK channel in a dose-dependent and reversible manner after coexpression with subfractions of rat brain mRNA and GIRK1/4 channel cRNAs. This Rf-evoked current was blocked by Ba(2+), a potassium channel blocker. The size of rat brain mRNA responding to Rf was about 6 to 7 kilobases. However, Rf did not evoke GIRK current after injection with this subfraction of rat brain mRNA or GIRK1/4 channel cRNAs alone. Other ginsenosides, such as Rb(1) and Rg(1), evoked only slight induction of GIRK currents after coexpression with the subfraction of rat brain mRNA and GIRK1/4 channel cRNAs. Acetylcholine and serotonin almost did not induce GIRK currents after coexpression with the subfraction of rat brain mRNA and GIRK1/4 channel cRNAs. Rf-evoked GIRK currents were not altered by PTX pretreatment but were suppressed by intracellularly injected guanosine-5'-(2-O-thio) diphosphate, a nonhydrolyzable GDP analog. These results indicate that Rf activates GIRK channel through an unidentified G protein-coupled receptor in rat brain and that this receptor can be cloned by the expression method demonstrated here.