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1.
Mol Cell ; 82(21): 4160-4175.e6, 2022 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-36272409

RESUMEN

CRISPR-Cas9-mediated genome editing depends on PAM recognition to initiate DNA unwinding. PAM mutations can abolish Cas9 binding and prohibit editing. Here, we identified a Cas9 from the thermophile Alicyclobacillus tengchongensis for which the PAM interaction can be robustly regulated by DNA topology. AtCas9 has a relaxed PAM of N4CNNN and N4RNNA (R = A/G) and is able to bind but not cleave targets with mutated PAMs. When PAM-mutated DNA was in underwound topology, AtCas9 exhibited enhanced binding affinity and high cleavage activity. Mechanistically, AtCas9 has a unique loop motif, which docked into the DNA major groove, and this interaction can be regulated by DNA topology. More importantly, AtCas9 showed near-PAMless editing of supercoiled plasmid in E. coli. In mammalian cells, AtCas9 exhibited broad PAM preference to edit plasmid with up to 72% efficiency and effective base editing at four endogenous loci, representing a potentially powerful tool for near-PAMless editing.


Asunto(s)
Sistemas CRISPR-Cas , Escherichia coli , Animales , Escherichia coli/genética , Escherichia coli/metabolismo , Edición Génica , ADN/genética , Plásmidos , Mamíferos/metabolismo
2.
BMC Neurol ; 18(1): 80, 2018 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-29866060

RESUMEN

BACKGROUNDS: Glioma is the most fatal primary brain glioma in central nervous system mainly attributed to its high invasion. Prucalopride, a Serotonin-4 (5-HT4) receptor agonist, has been reported to regulate neurodevelopment. This study aimed to investigate the influence of the Prucalopride on glioma cells and unveil underlying mechanism. METHODS: In this study, glioma cells proliferation was evaluated by Cell counting kit-8 (CCK8). Wound healing and transwell assay were used to test cellular migration and invasion. Flow cytometry was utilized to determine cellular apoptosis rate. Apoptosis related markers, autophagy markers, and protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway key molecules were detected using western blot assay. RESULTS: As a result, the proliferation, migration and invasiveness of glioma cells were impaired by Prucalopride treatment, the apoptosis rate of glioma cells was enhanced by Prucalopride stimulation, accompanied by the increased pro-apoptosis proteins Bax and Cleaved caspase-3 and decreased anti-apoptosis protein Bcl-2. Prucalopride significantly promoted autophagy by increased expression level of Beclin 1 and LC3-II, while decreased expression level of p62. Prucalopride administration resulted in obvious inhibitions of key molecules of AKT-mTOR pathway, including phosphorylated- (p-) AKT, p-mTOR and phosphorylated-ribosomal p70S6 kinase (p-P70S6K). CONCLUSIONS: Taking together, these results indicate that Prucalopride may be likely to play an anti-tumor role in glioma cells, which suggests potential implications for glioma promising therapy alternation in the further clinics.


Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Benzofuranos/farmacología , Glioma/patología , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Serina-Treonina Quinasas TOR/efectos de los fármacos
3.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 27(4): 374-6, 2011 Apr.
Artículo en Zh | MEDLINE | ID: mdl-21481309

RESUMEN

AIM: To explore the purification methods of wild-type and recombinant suilysin and to evaluate their biological activities. METHODS: Wild-type suilysin was purified by ammonium sulfate precipitation, anion-exchange chromatography and hydrophobic chromatography in turn, while recombinant suilysin was first refolded and purified by immobilized metal ion affinity chromatography, and further purified by Thiopropyl Sepharose 6B. The biological activities were evaluated by hemolysis test, cytotoxicity assay. RESULTS: Both prepared wild-type and recombinant suilysin, with purify over 90%, have hemolysis activity and could injure target cells at high concentration while cholesterol could completely inhibit their activities. CONCLUSION: Recombinant suilysin has similar biological activities with wild-type suilysin, and this work contributed to further study the functions of suilysin on pathogenesis of steptococcus suis.


Asunto(s)
Proteínas Hemolisinas/aislamiento & purificación , Proteínas Hemolisinas/farmacología , Humanos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Serotipificación , Streptococcus suis/química , Streptococcus suis/clasificación
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