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The main units of persimmon proanthocyanidins (PAs) are composed of flavan-3-ols including epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG). Precise quantification of GCG is challenging due to its trace amounts in persimmon. In this study, to establish the optimal UHPLC-Q-Exactive Orbitrap/MS technique for the determination of PAs monomer composition in persimmon fruit flesh of different astringency types, mass spectrometry and chromatographic conditions were optimized. The results showed that when operating in negative ion mode, using a T3 chromatographic column (a type of C18 column with high-strength silica), acetonitrile as the organic phase, a 0.1% mobile phase acid content, and a mobile phase flow rate of 0.2 mL/min, the chromatographic peak shape and resolution of the PAs monomer composition improved. Additionally, there was no tailing phenomenon observed in the chromatographic peaks. At the same time, the intra-day and inter-day precision, stability, and recovery of the procedure were good. The relative standard deviation (RSD) of stability was less than 5%. The intra-day precision was in the range of 1.14% to 2.36%, and the inter-day precision ranged from 1.03% to 2.92%, both of which were less than 5%. The recovery rate ranged from 94.43% to 98.59% with an RSD less than 5%. The results showed that the UHPLC-Q-Exactive Orbitrap/MS technique established in this study can not only be used for the quantification of EGCG and GCG in persimmon fruit flesh but also be suitable for analyzing other PAs monomer compositions, providing robust support for the related research on persimmon PAs.
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To evaluate the safety and efficacy of tubeless percutaneous nephrolithotomy (PCNL) in patients with Escherichia coli (E. coli) bacteriuria. We conducted a retrospective review of 84 patients with E. coli bacteriuria who underwent PCNL. Patients were divided into two groups according to whether a nephrostomy tube is placed at the end of the procedure. Preoperative clinical data, surgical outcomes, and postoperative complications were compared. Then, regression analysis of factors predicting success rate of PCNL in patients with E. coli bacteriuria was performed. After PCNL, residual fragments ≤ 4 mm were considered as success. At baseline, the two groups were similar with regard to age, gender, BMI, underlying disease, hydronephrosis, stone characteristics, and urinalysis. Postoperative fever occurred in 1 patient (3.8%) in the tubeless PCNL group, and in 5 patients (8.6%) in the conventional PCNL group (p > 0.05). There were no significant differences in terms of successful rate, decrease in hemoglobin, pain scores, blood transfusion, and hospitalization expenses. However, the tubeless PCNL group had significantly shorter operative time (60 vs. 70 min, p = 0.033), indwelling time of catheter (2 vs. 4 days, p < 0.001), and hospital stays (3 vs. 5 days, p < 0.001) than the conventional PCNL group. In the analysis of factors predicting success, the stone diameter, stone burden, and operative time were associated with success rate of PCNL. It is safe and effective to perform tubeless PCNL in patients with E. coli bacteriuria. Compared to conventional PCNL, tubeless PCNL accelerates patient recovery and shortens hospital stays.
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Bacteriuria , Nefrolitotomía Percutánea , Humanos , Nefrolitotomía Percutánea/efectos adversos , Escherichia coli , Catéteres , HospitalizaciónRESUMEN
To explore the preference for diagnosing and treating renal colic during pregnancy among Chinese urologists. A questionnaire was designed using the Sojump® platform. WeChat, the largest social networking platform in China, was used to distribute the questionnaire to urologists at hospitals of all levels in China. In total, 110 responses were included. Of the respondents, 100.0% used ultrasound to diagnose renal colic during pregnancy, followed by magnetic resonance imaging (17.3%) and low-dose CT (3.6%). Phloroglucinol (80.9%) and progesterone (72.7%) were the most commonly used antispasmodics and analgesics. Opioid analgesics were not commonly used (12.7%). Most of the respondents (63.6%) indicated that no more than 20% of the patients needed surgical intervention. If surgery was unavoidable, 95.5% preferred temporary renal drainage, including ureteral stenting (92.7%) and percutaneous nephrostomy (2.7%). However, some respondents still preferred definitive stone treatment, such as ureteroscopy lithotripsy (3.6%) and percutaneous nephrolithotomy (0.9%). Moreover, there were no differences in the choices of urologists with different professional titles regarding diagnostic tools, most therapeutic medications, or surgical methods (p > 0.05). Ultrasound is the preferred tool for diagnosing renal colic during pregnancy. Low-dose CT is still not widely accepted. Pregnant patients with renal colic are initially treated conservatively. Urologists prefer ureteral stenting when there are clinical indications for intervention.
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Cólico Renal , Uréter , Cálculos Ureterales , Embarazo , Femenino , Humanos , Cólico Renal/diagnóstico , Cólico Renal/terapia , Urólogos , Ureteroscopía/métodos , Encuestas y Cuestionarios , Cálculos Ureterales/terapiaRESUMEN
OBJECTIVE: It is challenging to perform a tubeless percutaneous nephrolithotomy (PNL) in patients with tract bleeding. The present study was designed to study the safety and efficacy of the 1470 nm laser for hemostatic completion in tubeless PNL patients with tract bleeding. PATIENTS AND METHODS: Between January 2020 and October 2021, 120 patients were retrospectively included and divided into two groups. The hemostasis group included 60 patients receiving tubeless PNL, in which a 1470 nm laser was used to manage tract bleeding. The other group included 60 patients receiving tubeless PNL in which the hemostasis procedure was not performed, serving as the control group. The differences in the patients' demographic characteristics, procedural information, and posttreatment outcomes between the two groups were statistically compared. RESULTS: The differences associated with sex, age, weight, body mass index, urine culture, stone burden, calyx of puncture, degree of hydronephrosis and comorbidities between the two groups were not statistically significant. Compared with the control group, the hemostasis group showed greatly reduced blood loss (0.61 ± 0.31 vs. 0.85 ± 0.46 g/dL) and decreased postoperative hospitalization duration (2.83 ± 0.81 vs. 4.45 ± 0.91 days). The differences in operative time, stone-free rate, Visual Analogue Score and postoperative complications between the two groups were not statistically significant. In the subgroup analysis, the obese patients and patients with moderate to severe hydronephrosis in the hemostasis group also showed a significantly less blood loss (0.51 ± 0.22 vs. 0.83 ± 0.48 g/dL; 0.54 ± 0.27 vs. 0.85 ± 0.47 g/dL, respectively) and shorter length of postoperative hospitalization (2.62 ± 0.51 vs. 4.47 ± 1.19 days; 2.97 ± 0.63 vs. 4.41 ± 0.91 days, respectively) than those in the control group. CONCLUSIONS: Our results demonstrated that 1470 nm laser is a safe, feasible and effective method to obtain tract hemostasis in tubeless PNL.
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Hidronefrosis , Nefrolitotomía Percutánea , Nefrostomía Percutánea , Estudios Transversales , Hemostasis , Humanos , Rayos Láser , Nefrostomía Percutánea/métodos , Estudios RetrospectivosRESUMEN
Persimmon fruits accumulate a large amount of proanthocyanidins (PAs), which makes an astringent sensation. Proanthocyanidins (PAs) are the polymers of flavan-3-ols stored in plant vacuoles under laccase activation. A laccase gene, DkLAC2, is putatively involved in PAs biosynthesis and regulated by microRNA (DkmiR397) in persimmon. However, the polymerization of PAs in association with miRNA397 still needs to be explored in persimmon. Here, we identified pre-DkmiR397 and its target gene DkLAC2 in 'Eshi 1' persimmon. Histochemical staining with GUS and dual luciferase assay both confirmed DkmiR397-DkLAC2 binding after co-transformation in tobacco leaves. Diverse expression patterns of DkLAC2 and DkmiR397 were exhibited during persimmon fruit development stages. Moreover, a contrasting expression pattern was also observed after the combined DkLAC2-miR397 transformation in persimmon leaves, suggesting that DkmiR397 might be a negative regulator of DkLAC2. Similarly, the transient transformation of DkmiR397 in persimmon fruit discs in vitro also reduced PA accumulation by repressing DkLAC2, whereas the up-regulation of DkLAC2 increased the accumulation of PAs by short tandem target mimic STTM-miR397. A similar expression pattern was observed when overexpressing of DkLAC2 in Arabidopsis wild type (WT) and overexpression of DkLAC2, DkmiR397 in persimmon leaf callus. Our results revealed that the role of DkmiR397 repressed the expression of DkLAC2 concerning PA biosynthesis, providing a potential target for the manipulation of PAs metabolism in persimmon.
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Arabidopsis , Diospyros , Proantocianidinas , Arabidopsis/metabolismo , China , Diospyros/genética , Diospyros/metabolismo , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Lacasa/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proantocianidinas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
Sugar transporter proteins (STPs) play vital roles in sugar transport and allocation of carbon sources in plants. However, the evolutionary dynamics of this important gene family and their functions are still largely unknown in citrus, which is the largest fruit crop in the world. In this study, fourteen non-redundant CgSTP family members were identified in pummelo (Citrus grandis). A comprehensive analysis based on the biochemical characteristics, the chromosomal location, the exon-intron structures and the evolutionary relationships demonstrated the conservation and the divergence of CgSTPs. Moreover, CgSTP4, 11, 13, 14 were proofed to be localized in plasma membrane and have glucose transport activity in yeast. The hexose content were significantly increased with the transient overexpression of CgSTP11 and CgSTP14. In addition, antisense repression of CgSTP4 induced the shorter pollen tube length in vitro, implying the potential role of CgSTP4 in pummelo pollen tube growth. Taken together, this work explored a framework for understanding the physiological role of CgSTPs and laid a foundation for future functional studies of these members in citrus species.
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BACKGROUND: Sucrose (Suc) hydrolysis is directly associated with plants tolerance to multiple abiotic stresses. Invertase (INV) enzymes irreversibly catalyze Suc degradation to produce glucose (Glc) and fructose (Frc). However, genome-wide identification and function of individual members of the INV gene family in Poncirus trifoliata or its Citrus relatives in response to abiotic stresses are not fully understood. RESULTS: In this report, fourteen non-redundant PtrINV family members were identified in P. trifoliata including seven alkaline/neutral INV genes (PtrA/NINV1-7), two vacuolar INV genes (PtrVINV1-2), and five cell wall INV isoforms (PtrCWINV1-5). A comprehensive analysis based on the biochemical characteristics, the chromosomal location, the exon-intron structures and the evolutionary relationships demonstrated the conservation and the divergence of PtrINVs. In addition, expression analysis of INV genes during several abiotic stresses in various tissues indicated the central role of A/NINV7 among INV family members in response to abiotic stresses. Furthermore, our data demonstrated that high accumulation of Suc, Glc, Frc and total sugar contents were directly correlated with the elevated activities of soluble INV enzymes in the cold-tolerant P. trifoliata, C. ichangensis and C. sinensis, demonstrating the potential role of soluble INV enzymes for the cold tolerance of Citrus. CONCLUSIONS: This work offered a framework for understanding the physiological role of INV genes and laid a foundation for future functional studies of these genes in response to abiotic stresses.
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Adaptación Fisiológica/genética , Citrus/genética , Citrus/metabolismo , Frío , Poncirus/genética , Poncirus/metabolismo , Sacarosa/metabolismo , Productos Agrícolas/genética , Productos Agrícolas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Variación Genética , Estudio de Asociación del Genoma Completo , Genotipo , Familia de Multigenes , FilogeniaRESUMEN
Podoplanin, PDPN, is a mucin-type transmembrane glycoprotein widely expressed in many tissues, including lung, kidney, lymph nodes, and mineralized tissues. Its function is critical for lymphatic formation, differentiation of type I alveolar epithelial lung cells, and for bone response to biomechanical loading. It has previously been shown that Pdpn null mice die at birth due to respiratory failure emphasizing the importance of Pdpn in alveolar lung development. During the course of generation of Pdpn mutant mice, we found that most Pdpn null mice in the 129S6 and C57BL6/J mixed genetic background die at the perinatal stage, similar to previously published studies with Pdpn null mice, while all Pdpn null mice bred with Swiss outbred mice survived. Surviving mutant mice in the 129S6 and C57BL6/J mixed genetic background showed alterations in the osteocyte lacunocanalicular network, especially reduced osteocyte canaliculi in the tibial cortex with increased tibial trabecular bone. However, adult Pdpn null mice in the Swiss outbred background showed no overt differences in their osteocyte lacunocnalicular network, bone density, and no overt differences when challenged with exercise. Together, these data suggest that genetic variations present in the Swiss outbred mice compensate for the loss of function of PDPN in lung, kidney, and bone.
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Células Epiteliales Alveolares/metabolismo , Diferenciación Celular/genética , Linfangiogénesis/genética , Glicoproteínas de Membrana/genética , Animales , Calcificación Fisiológica/genética , Hueso Esponjoso/crecimiento & desarrollo , Hueso Esponjoso/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Riñón/crecimiento & desarrollo , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Ganglios Linfáticos/crecimiento & desarrollo , Ratones , Osteocitos/metabolismo , Tibia/crecimiento & desarrollo , Tibia/metabolismoRESUMEN
Biochar (BC) application to low fertility soils is a promising approach to increase crop yield, improve soil quality, and mitigate climate change simultaneously. Only few studies evaluated the combined effects of BC and nitrogen (N) fertilization rates on crop productivity and N losses under field conditions. The objectives were to investigate combined effects of BC (2 rates) and N (5 rates) fertilization on crop productivity and N losses in a long-term field experiment started in 2008 in a winter wheat/summer maize rotation system in the North China Plain. Linear-plateau models best described the responses of wheat and maize yields to N rates. N2O fluxes, NH3 volatilization, and soil mineral N contents increased exponentially with N fertilization rates. Despite the effect of BC on wheat or maize yields was negligible, BC retains of mineral N at 240â¯kgâ¯Nâ¯ha-1â¯yr-1. BC application increased NH3 volatilization by 31% in wheat season and 26% in maize season because of pH increase. BC reduced N2O emissions by 8-23% in the wheat season and by 24% at lower N rates (≤60â¯kgâ¯ha-1) in the maize season, due to BC induced complete denitrification to N2. BC stimulated N2O emissions by 18-26% compared to soils without BC in maize season at N rates higher than 60â¯kgâ¯ha-1. The combination of increased mineral N retention and C availability with BC addition increased nitrification and/or denitrification rates, leading to increased N2O emissions. For the wheat/maize rotation system, BC application decreased N2O emissions at lower N rates (≤120â¯kgâ¯ha-1â¯yr-1) but had no effects at higher N rates.
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Agricultura/métodos , Carbón Orgánico , Fertilizantes , Nitrógeno , China , Óxido Nitroso , Suelo/químicaRESUMEN
Tetraploids have been reported to exhibit increased stress tolerance, but the underlying molecular and physiological mechanisms remain poorly understood. In this study, autotetraploid plants were identified by screening natural seedlings of trifoliate orange (Poncirus trifoliata). The tetraploids exhibited different morphology and displayed significantly enhanced drought and dehydration tolerance in comparison with the diploid progenitor. Transcriptome analysis indicated that a number of stress-responsive genes and pathways were differentially influenced and enriched in the tetraploids, in particular those coding for enzymes related to antioxidant process and sugar metabolism. Transcript levels and activities of antioxidant enzymes (peroxidase and superoxide dismutase) and sucrose-hydrolysing enzyme (vacuolar invertase) were increased in the tetraploids upon exposure to the drought, concomitant with greater levels of glucose but lower level of reactive oxygen species (ROS). These data indicate that the tetraploids might undergo extensive transcriptome reprogramming of genes involved in ROS scavenging and sugar metabolism, which contributes, synergistically or independently, to the enhanced stress tolerance of the tetraploid. Our results reveal that the tetraploids take priority over the diploid for stress tolerance by maintaining a more robust system of ROS detoxification and osmotic adjustment via elevating antioxidant capacity and sugar accumulation in comparison with the diploid counterpart.
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Sequías , Poncirus/fisiología , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Azúcares/metabolismo , Tetraploidía , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas , Plantas Modificadas GenéticamenteRESUMEN
CYP2C11 is involved in the metabolism of many drugs in rats. To assess the roles of CYP2C11 in physiology and drug metabolism, a CYP2C11-null rat model was generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9method. A 2-base pair insertion was added to exon 6 of CYP2C11 in Sprague-Dawley rats. CYP2C11 was not detected by western blotting in liver microsomes of CYP2C11-null rats. No off-target effects were found at 11 predicted sites of the knockout model. The CYP2C11-null rats were viable and had no obvious abnormalities, with the exception of reduced fertility. Puberty in CYP2C11-null rats appeared to be delayed by â¼20 days, and the average litter size fell by 43%. Tolbutamide was used as a probe in this drug metabolism study. In the liver microsomes of CYP2C11-null rats, the Vmax and intrinsicclearance values decreased by 22% and 47%, respectively, compared with those of wild-type rats. The Km values increased by 47% compared with that of wild types. However, our pharmacokinetics study showed no major differences in any parameters between the two strains, in both males and females. In conclusion, a CYP2C11-null rat model was successfully generated and is a valuable tool to study the in vivo function of CYP2C11.
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Hidrocarburo de Aril Hidroxilasas/genética , Sistemas CRISPR-Cas/genética , Familia 2 del Citocromo P450/genética , Esteroide 16-alfa-Hidroxilasa/genética , Animales , Sistemas CRISPR-Cas/efectos de los fármacos , Femenino , Inactivación Metabólica/fisiología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Modelos Animales , Ratas , Ratas Sprague-Dawley , Tolbutamida/farmacologíaRESUMEN
AIM: To investigate the clinical relevance and functional role of HOXA13 in prostate cancer Methods: PCR, western blot and immunohistochemistry were performed to determine the expression. Kaplan-Meier and Cox regression survival analyses investigated the clinical relevance. Cell viability, flow cytometry and transwell assays were used to determine the functional roles. RESULTS: HOXA13 expression is sharply increased in carcinoma tissues and is significantly associated with poor prognosis of prostate cancer patients. Interestingly, nucleus not cytoplasm HOXA13 expression is associated with unfavorable survival of the patients. Furthermore, nucleus HOXA13 expression represents an unfavorable and independent prognosis factor of histological grade 2 or Gleason grade <8 patients. Functionally, forced expression of HOXA13 obviously promotes tumor cell proliferation, migration and invasion, whereas inhibits tumor cell apoptosis. CONCLUSION: HOXA13 is an unfavorable prognostic factor and a novel oncogene for prostate cancer.
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Carcinoma/genética , Proteínas de Homeodominio/genética , Oncogenes/genética , Neoplasias de la Próstata/genética , Anciano , Anciano de 80 o más Años , Apoptosis/genética , Biomarcadores de Tumor/genética , Carcinoma/epidemiología , Carcinoma/patología , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia sin Enfermedad , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Pronóstico , Neoplasias de la Próstata/epidemiología , Neoplasias de la Próstata/patologíaRESUMEN
Objective To investigate the effect of 1.94 μm thulium laser enucleation of benign prostatic hyperplasia (BPH) with volume >80 ml by morcellator. Methods From September 2014 to June 2016, there were 95 BPH patients with prostate volume over 80 ml treated by thulium laser were divided into two groups according to the surgical procedure: 45 cases in group A, prostate tissue were washed out of bladder after vapoenucleation by 1.94 μm thulium laser; 50 cases in group B, the enucleated prostate tissue were extracted by morcellator. The operation time, the decreasing level of hemoglobin on the first day after surgery, the hospitalization time, the gland tissue weight, catheterization duration, short-term incidence of complications, and the IPSS, PVP, Qmax, QOL in 3 months after surgeon of the two groups were observed and recorded. Results There was significant difference in operation time and gland tissue weight between the two groups. The group B have significantly short operation time compared with group A (P < 0.05), and obtained gland tissue remarkably exceed the group A (P < 0.05). No significant difference was found in hemoglobin level, hospitalization time, catheterization duration, and short-term complication between the two groups (P > 0.05). The IPSS, PVR, Qmax and QOL of 3 month, after operation were significantly improved but without any significant difference between the two groups (P < 0.05). Conclusion Vaporization cutting tissue or morcellating tissue after 1.94 μm thulium laser enucleation has high safety, good curative effect and low complication, while extraction prostate tissue by morcellator can shorten the operation time and get more tissues.
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Parathyroid Hormone (PTH) can exert both anabolic and catabolic effects on the skeleton, potentially through expression of the PTH type1 receptor (PTH1R), which is highly expressed in osteocytes. To determine the cellular and molecular mechanisms responsible, we examined the effects of PTH on osteoblast to osteocyte differentiation using primary osteocytes and the IDG-SW3 murine cell line, which differentiate from osteoblast to osteocyte-like cells in vitro and express GFP under control of the dentin matrix 1 (Dmp1) promoter. PTH treatment resulted in an increase in some osteoblast and early osteocyte markers and a decrease in mature osteocyte marker expression. The gene expression profile of PTH-treated Day 28 IDG-SW3 cells was similar to PTH treated primary osteocytes. PTH treatment induced striking changes in the morphology of the Dmp1-GFP positive cells in IDG-SW3 cultures and primary cells from Dmp1-GFP transgenic mice. The cells changed from a more dendritic to an elongated morphology and showed increased cell motility. E11/gp38 has been shown to be important for cell migration, however, deletion of the E11/gp38/podoplanin gene had no effect on PTH-induced motility. The effects of PTH on motility were reproduced using cAMP, but not with protein kinase A (PKA), exchange proteins activated by cAMP (Epac), protein kinase C (PKC) or phosphatidylinositol-4,5-bisphosphonate 3-kinase (Pi3K) agonists nor were they blocked by their antagonists. However, the effects of PTH were mediated through calcium signaling, specifically through L-type channels normally expressed in osteoblasts but decreased in osteocytes. PTH was shown to increase expression of this channel, but decrease the T-type channel that is normally more highly expressed in osteocytes. Inhibition of L-type calcium channel activity attenuated the effects of PTH on cell morphology and motility but did not prevent the downregulation of mature osteocyte marker expression. Taken together, these results show that PTH induces loss of the mature osteocyte phenotype and promotes the motility of these cells. These two effects are mediated through different mechanisms. The loss of phenotype effect is independent and the cell motility effect is dependent on calcium signaling.
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Canales de Calcio Tipo L/metabolismo , Osteocitos/efectos de los fármacos , Osteocitos/metabolismo , Hormona Paratiroidea/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Análisis por Conglomerados , Colforsina/farmacología , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Perfilación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
CRISPR Cas9 and other sequence-specific endonucleases are fundamental genome editors supporting gene knockout and gene therapy. A speedy and accurate computational allele designer is required for a high through-put gene mutagenesis pipeline using these new techniques. An automatic system, Cas9 online designer (COD), was created to screen Cas9 targets and off-targets, as well as to provide gene knockout and genotyping strategies. A gene knockout rat model was successfully created and genotyped under the direction of this online system confirming its ability to predict real targets and off-targets. Gene knockout strategies to mutate 72 rat cytochrome P450 genes were designed instantly by the system to demonstrate its high-throughput efficiency. Also, the system used an off-target scoring matrix which can be applied to any sequence-specific genome editing tools besides Cas9. The COD system (http://cas9.wicp.net) has established a speedy, accurate, flexible and high through-put computational gene knockout pipeline supporting the sequence-specific endonuclease induced mutagenesis.
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We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKO(ob)) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKO(ob) mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKO(ob) osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA(+) MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells.
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Proteína Morfogenética Ósea 2/metabolismo , Células Madre Mesenquimatosas/metabolismo , Neovascularización Patológica/metabolismo , Osteoblastos/metabolismo , Animales , Diferenciación Celular , Células Madre Mesenquimatosas/fisiología , Ratones , Periostio , Transducción de SeñalRESUMEN
With the support of free-air carbon dioxide enrichment (FACE) system and by using isotope 13C technique, and through planting wheat (Triticum aestivum L., C3 crop) on a soil having been planted with maize (Zea mays L., C4 crop) for many years, this paper studied the effects of elevated atmospheric CO2 and nitrogen application on the delta 13C value of soil emitted CO2 and the wheat rhizosphere respiration. With the growth of wheat, the delta 13C value of soil emitted CO2 had a gradual decrease. Elevated atmospheric CO2 concentration (200 micromol mol(-1)) decreased the delta 13C value of emitted CO2 at booting and heading stages significantly when the nitrogen application rate was 250 kg hm(-2) (HN), and at jointing and booting stages significantly when the nitrogen application rate was 150 kg hm(-2) (LN). Nevertheless, the elevated atmospheric CO2 promoted the proportions of wheat rhizosphere respiration to soil respiration at booting and heading stages significantly. From jointing stage to maturing stage, the proportions of wheat rhizosphere respiration to soil respiration were 24%-48% (HN) and 21%-48% (LN) under elevated atmospheric CO2, and 20%-36% (HN) and 19%-32% (LN) under ambient atmospheric CO2. Under both elevated and ambient atmospheric CO2 concentrations, the delta 13C value of emitted CO2 and the rhizosphere respiration had different responses to the increased nitrogen application rate, and there was a significant interactive effect of atmospheric CO2 concentration and nitrogen application rate on the wheat rhizosphere respiration at jointing stage.
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Atmósfera/análisis , Dióxido de Carbono/análisis , Nitrógeno/química , Rizosfera , Triticum/metabolismo , Fertilizantes , Suelo/química , Triticum/fisiologíaRESUMEN
Since little is known regarding osteocytes, cells embedded within the mineralized bone matrix, a proteomics approach was used to discover proteins more highly expressed in osteocytes than in osteoblasts to determine osteocyte-specific function. Two proteomic profiles obtained by two different proteomic approaches using total cell lysates from the osteocyte cell line MLO-Y4 and the osteoblast cell line MC3T3 revealed unique differences. Three protein clusters, one related to glycolysis (Phosphoglycerate kinase 1, fructose-bisphosphate aldolase A, hypoxia up-regulated 1 [ORP150], triosephosphate isomerase), one to protein folding (Mitochondrial Stress-70 protein, ORP150, Endoplasmin), and one to actin cytoskeleton regulation (Macrophage-capping protein [CapG], destrin, forms of lamin A and vimentin) were identified. Higher protein expression of ORP-150, Cap G, and destrin in MLO-Y4 cells compared with MC3T3 cells was validated by gene expression, Western blotting, and in vivo expression. These proteins were shown to be selective in osteocytes in vivo using immuno-staining of mouse ulnae. Destrin was most highly expressed in embedding osteoid osteocytes, GapG in embedded osteocytes, and ORP150 in deeply embedded osteocytes. In summary, the proteomic approach has yielded important information regarding molecular mechanisms used by osteocytes for embedding in matrix, the formation of dendritic processes, and protection within a hypoxic environment.
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Huesos/química , Huesos/citología , Osteocitos/química , Proteínas/análisis , Proteoma/análisis , Animales , Huesos/fisiología , Línea Celular , Expresión Génica , Ratones , Osteoblastos/química , Osteoblastos/fisiología , Osteocitos/fisiología , Proteínas/genética , Proteómica/métodosRESUMEN
The mammalian organ of Corti of the inner ear is a highly sophisticated sensory end organ responsible for detecting sound. Noggin is a secreted glycoprotein, which antagonizes bone morphogenetic proteins 2 and 4 (Bmp2 and Bmp4). The lack of this antagonist causes increased rows of inner and outer hair cells in the organ of Corti. In mice, Bmp2 is expressed transiently in nascent cochlear hair cells. To investigate whether Noggin normally modulates the levels of Bmp2 for hair cell formation, we deleted Bmp2 in the cochlear hair cells using two cre strains, Foxg1(cre/+) and Gfi1(cre/+). Bmp2 conditional knockout cochleae generated using these two cre strains show normal hair cells. Furthermore, Gfi1(cre/+);Bmp2(lox/-) mice are viable and have largely normal hearing. The combined results of Noggin and Bmp2 mutants suggest that Noggin is likely to regulate other Bmps in the cochlea such as Bmp4.