RESUMEN
Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.
Asunto(s)
Sistemas CRISPR-Cas , Células Madre Pluripotentes , Humanos , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Reparación del ADN por Unión de Extremidades , MutagénesisRESUMEN
Pregnancy invokes a doubling of intestinal calcium absorption whereas lactation programs skeletal resorption to provide calcium to milk. Postweaning bone formation restores the skeleton's bone mineral content (BMC), but the factors that regulate this are not established. We used Pth-null mice to test whether parathyroid hormone (PTH) is required for postweaning skeletal recovery. On a normal 1% calcium diet, wild-type (WT) and Pth-null mice each gained BMC during pregnancy, declined 15% to 18% below baseline during lactation, and restored the skeleton above baseline BMC within 14 days postweaning. A 2% calcium diet reduced the lactational decline in BMC without altering the gains achieved during pregnancy and postweaning. The hypocalcemia and hyperphosphatemia of Pth-null mice normalized during lactation and serum calcium remained normal during postweaning. Osteocalcin and propeptide of type 1 collagen (P1NP) each rose significantly after lactation to similar values in WT and Pth-null. Serum calcitriol increased fivefold during pregnancy in both genotypes whereas vitamin D binding protein levels were unchanged. Absence of PTH blocked a normal rise in fibroblast growth factor-23 (FGF23) during pregnancy despite high calcitriol. A 30-fold higher expression of Cyp27b1 in maternal kidneys versus placenta suggests that the pregnancy-related increase in calcitriol comes from the kidneys. Conversely, substantial placental expression of Cyp24a1 may contribute significantly to the metabolism of calcitriol. In conclusion, PTH is not required to upregulate renal expression of Cyp27b1 during pregnancy or to stimulate recovery from loss of BMC caused by lactation. A calcium-rich diet in rodents suppresses skeletal losses during lactation, unlike clinical trials that showed no effect of supplemental calcium on lactational decline in BMC.
Asunto(s)
Huesos/metabolismo , Calcitriol/metabolismo , Lactancia/metabolismo , Hormona Paratiroidea/metabolismo , Reproducción , Regulación hacia Arriba , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Animales , Biomarcadores/metabolismo , Densidad Ósea/efectos de los fármacos , Remodelación Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Calcio/sangre , Calcio/farmacología , Dieta , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/genética , Glucuronidasa/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Proteínas Klotho , Lactancia/sangre , Lactancia/efectos de los fármacos , Ratones , Hormona Paratiroidea/deficiencia , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducción/efectos de los fármacos , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Proteína de Unión a Vitamina D/metabolismo , Vitamina D3 24-HidroxilasaRESUMEN
The ubiquitous occurrence of antibiotics in aquatic environments raises concerns about potential adverse effects on aquatic ecology and human health, including the promotion of increased antibiotic resistance. This study examined the oxidation of three widely detected antibiotics (ciprofloxacin, lincomycin, and trimethoprim) by potassium permanganate [KMnO(4); Mn(VII)]. Reaction kinetics were described by second-order rate laws, with apparent second-order rate constants (k(2)) at pH 7 and 25 degrees C in the order of 0.61 +/- 0.02 M(-1) s(-1) (ciprofloxacin) < 1.6 +/- 0.1 M(-1) s(-1) (trimethoprim) < 3.6 +/- 0.1 M(-1) s(-1) (lincomycin). Arrhenius temperature dependence was observed with apparent activation energies (E(a)) ranging from 49 kJ mol(-1) (trimethoprim) to 68 kJ mol(-1) (lincomycin). Rates of lincomycin and trimethoprim oxidation exhibited marked pH dependences, whereas pH had only a small effect on rates of ciprofloxacin oxidation. The effects of pH were quantitatively described by considering parallel reactions between KMnO(4) and individual acid-base species of the target antibiotics. Predictions from a kinetic model that included temperature, KMnO(4) dosage, pH, and source water oxidant demand as input parameters agreed reasonably well with measurements of trimethoprim and lincomycin oxidation in six drinking water utility sources. Although Mn(VII) reactivity with the antibiotics was lower than that reported for ozone and free chlorine, its high selectivity and stability suggests a promising oxidant for treating sensitive micropollutants in organic-rich matrices (e.g., wastewater).
Asunto(s)
Antibacterianos/química , Permanganato de Potasio/química , Purificación del Agua/métodos , Agua/química , Ciprofloxacina/química , Concentración de Iones de Hidrógeno , Cinética , Lincomicina/química , Manganeso/química , Modelos Químicos , Oxidación-Reducción , Temperatura , Trimetoprim/química , Abastecimiento de AguaRESUMEN
Experimental studies were conducted to examine the oxidation of carbamazepine, an anticonvulsant drug widely detected in surface waters and sewage treatment effluent, by potassium salts of permanganate (Mn(VII); KMnO4) and ferrate (Fe(VI); K2FeO4). Results show that both Mn(VII) and Fe(VI) rapidly oxidize carbamazepine by electrophilic attack at an olefinic group in the central heterocyclic ring, leading to ring-opening and a series of organic oxidation products. Reaction kinetics follow a generalized second-order rate law, with apparent rate constants at pH 7.0 and 25 degrees C of 3.0 (+/-0.3) x 10(2) M(-1) s(-1) for Mn(VII) and 70(+/-3) M(-1) s(-1) for Fe(VI). Mn(VII) reaction rates exhibit no pH dependence, whereas Fe(VI) reaction rates increase dramatically with decreasing pH, due to changing acid-base speciation of Fe(VI). Further studies with Mn(VII) show that most common nontarget water constituents, including natural organic matter, have no significant effect on rates of carbamazepine oxidation; reduced metals and (bi)sulfide exert a stoichiometric Mn(VII) demand that can be incorporated into the kinetic model. The removal of carbamazepine in two utility source waters treated with KMnO4 agrees closely with predictions from the kinetic model that was parametrized using experiments conducted in deionized water at much higher reagent concentrations.