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1.
iScience ; 19: 492-503, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31437752

RESUMO

Cas12a is an RNA-guided endonuclease, which displays great potentials and several advantages over the well-known Cas9 in genome editing and engineering. Here, we established a quantitative kinetic scheme to describe the conformational dynamics of Cas12a/crRNA/dsDNA ternary complexes. The highly dynamic nature of Cas12a complexes, including their reversible formation, disassembly, and transition between different conformational states, is likely to be one of the key aspects contributing to their high specificity. The non-target strand is cleaved when its cleavage sites are released from DNA duplex after DNase activation of Cas12a. Cleaved non-target strand stabilizes target strand pre-cleavage states to permit subsequent cleavage and to ensure two DNA strands cleaved in a well-defined order. The extent of complementarity between crRNA and DNA modulates the relative stabilities of target strand pre-cleavage states targeting different cleavage sites. Our discoveries provide insights to fully elucidate the working mechanisms of Cas12a and to optimize it for genome engineering.

2.
Biomaterials ; 178: 147-157, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29933101

RESUMO

Lipid derivatives of nucleoside analogs have been highlighted for their potential for effective gene delivery. A novel class of nucleobase-lipids are rationally designed and readily synthesized, comprising thymine/cytosine, an ester/amide linker and an oleyl lipid. The diversity of four nucleobase-lipids termed DXBAs (DOTA, DNTA, DOCA and DNCA) is investigated. Besides, DNCA is demonstrated to be an effective neutral transfection material for nucleic acid delivery, which enbles to bind to oligonucleotides via H-bonding and π-π stacking with reduced toxicity in vitro and in vivo. Several kinds of nucleic acid drugs including aptamer, ssRNA, antisense oligonucleotide, and plasmid DNAs can be delivered by DXBAs, especially DNCA. In particular, G4-aptamer AS1411 encapsulated by DNCA exhibits cellular uptake enhancement, lysosome degradation reduction, cell apoptosis promotion, cell cycle phase alteration in vitro and duration prolongation in vivo, resulting in significant anti-proliferative activity. Our results demonstrate that DNCA is a promising transfection agent for G4-aptamers and exhibites bright application prospects in the permeation improvement of single-stranded oligonucleotides or plasmid DNAs.

3.
Cell Rep ; 22(2): 372-382, 2018 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-29320734

RESUMO

Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.

5.
J Obstet Gynaecol ; 37(8): 1076-1081, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28760055

RESUMO

Caesarean scar defect (CSD) can cause postmenstrual bleeding. Defect repair is an effective technique to improve this symptom, but there are still a few patients getting little improvement. This retrospective study evaluates the efficacy of scar repair and explores the factors associated with poor effect. In total, 123 patients were involved in the final analysis. All of them complained about menstruation period >7 days due to postmenstrual bleeding. Before surgery, 87.8% of patients had a menstruation period more than 10 days and 20.3% had a period more than 15 days. After surgery, a normal menstruation period (< =7 days) was achieved in 46.3% (95%CI 37.3%-55.6%) of patients and a menstruation period lasting no more than 10 days was achieved in 74.8% (95%CI 66.2%-82.2%). Through multivariate logistic analysis, four factors were found dependently associated with poor effect (defined as menstruation period >10 days after surgery): repeated caesarean section (OR 9.75, 95%CI 2.30-41.36, 0.002) was a risk factor, while defect volume >600 mm3 (OR 0.14, 95%CI 0.03-0.56, 0.006), interval from caesarean section to symptom emerging >3 months (OR 0.25, 95%CI 0.07-0.94, 0.041) and straight or retroflexed uterus (OR 0.19, 95%CI 0.05-0.79, 0.022) were protective factors. Impact statement What is already known on this subject? Caesarean scar defect can cause postmenstrual bleeding. Defect repair can improve this symptom, but there are still a few patients getting little improvement after surgery. What do the results of this study add? Defect volume >600 mm3, interval from caesarean section to symptom emerging >3 months and straight or retroflexed uterus are protective factors of poor effect (defined as menstruation period >10 days after surgery), and repeated caesarean section is a risk factor. What are the implications of these findings for clinical practice and/or further research? These findings may help in counselling the patients and in medical decision. Further researches are needed to explore other factors associated with surgical effect and build prediction models.


Assuntos
Cesárea/efeitos adversos , Cicatriz/cirurgia , Menstruação , Hemorragia Uterina/terapia , Adulto , Recesariana/efeitos adversos , Cicatriz/diagnóstico por imagem , Cicatriz/etiologia , Feminino , Humanos , Imagem por Ressonância Magnética , Estudos Retrospectivos , Fatores de Tempo , Resultado do Tratamento , Ultrassonografia , Hemorragia Uterina/epidemiologia
6.
Nat Commun ; 8: 16064, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28714467

RESUMO

The cellular ESCRT-III (endosomal sorting complex required for transport-III) and Vps4 (vacuolar protein sorting 4) comprise a common machinery that mediates a variety of membrane remodelling events. Vps4 is essential for the machinery function by using the energy from ATP hydrolysis to disassemble the ESCRT-III polymer into individual proteins. Here, we report the structures of the ATP-bound Vps4E233Q hexamer and its complex with the cofactor Vta1 (vps twenty associated 1) at resolutions of 3.9 and 4.2 Å, respectively, determined by electron cryo-microscopy. Six Vps4E233Q subunits in both assemblies exhibit a spiral-shaped ring-like arrangement. Locating at the periphery of the hexameric ring, Vta1 dimer bridges two adjacent Vps4 subunits by two different interaction modes to promote the formation of the active Vps4 hexamer during ESCRT-III filament disassembly. The structural findings, together with the structure-guided biochemical and single-molecule analyses, provide important insights into the process of the ESCRT-III polymer disassembly by Vps4.

7.
Proc Natl Acad Sci U S A ; 114(18): 4709-4714, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28424249

RESUMO

Depolymerizing lignin, the complex phenolic polymer fortifying plant cell walls, is an essential but challenging starting point for the lignocellulosics industries. The variety of ether- and carbon-carbon interunit linkages produced via radical coupling during lignification limit chemical and biological depolymerization efficiency. In an ancient fungus-cultivating termite system, we reveal unprecedentedly rapid lignin depolymerization and degradation by combining laboratory feeding experiments, lignocellulosic compositional measurements, electron microscopy, 2D-NMR, and thermochemolysis. In a gut transit time of under 3.5 h, in young worker termites, poplar lignin sidechains are extensively cleaved and the polymer is significantly depleted, leaving a residue almost completely devoid of various condensed units that are traditionally recognized to be the most recalcitrant. Subsequently, the fungus-comb microbiome preferentially uses xylose and cleaves polysaccharides, thus facilitating final utilization of easily digestible oligosaccharides by old worker termites. This complementary symbiotic pretreatment process in the fungus-growing termite symbiosis reveals a previously unappreciated natural system for efficient lignocellulose degradation.


Assuntos
Proteínas Fúngicas/metabolismo , Isópteros , Lacase/metabolismo , Lignina/metabolismo , Termitomyces/enzimologia , Animais
8.
Int J Gynaecol Obstet ; 134(3): 336-9, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27473332

RESUMO

OBJECTIVE: To evaluate operative and nonoperative therapies for cesarean scar defects (CSDs). METHODS: A prospective study was conducted among patients who underwent treatment of CSDs at a hospital in Shanghai, China, between April 1, 2010, and December 31, 2014. Treatment included laparoscopy (group 1), vaginal surgery (group 2), hysteroscopy (group 3), combined oral contraceptives (group 4), and the levonorgestrel intrauterine system (group 5). RESULTS: Among 142 participants, 119 patients underwent surgical repair and 23 received nonsurgical (conservative) treatment. Operation time, blood loss, and length of hospital stay reported for patients in group 3 (n=19) were all lower than those reported for patients in group 1 (n=86) and group 2 (n=14; P<0.001 for all comparisons). Patients who underwent surgery and those in group 4 (n=18) experienced shortened menstrual periods after treatment (P<0.001 for all comparisons). For group 5 (n=5), the duration of menstruation was similar before and after therapy (P=0.89). All 32 women who desired fertility underwent laparoscopy; 12 (37.5%) became pregnant after this procedure. CONCLUSION: All treatments for CSDs other than the levonorgestrel intrauterine system shortened menstrual periods. Laparoscopic surgery was effective for patients with fertility requirements.


Assuntos
Cesárea , Cicatriz/cirurgia , Doenças Uterinas/cirurgia , Adulto , China , Cicatriz/patologia , Feminino , Procedimentos Cirúrgicos em Ginecologia/métodos , Humanos , Histeroscopia , Tempo de Internação , Gravidez , Estudos Prospectivos , Resultado do Tratamento , Doenças Uterinas/patologia
9.
Environ Microbiol ; 18(5): 1440-51, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26346907

RESUMO

Fungus-cultivating termites (Macrotermitinae) possess an elaborate strategy of lignocellulose digestion. It involves a lignocellulose-degrading fungal symbiont (genus Termitomyces), a diverse gut microbiota and a characteristic labour division in food processing. In this study, using pyrotag sequencing and electron microscopy, we analysed the bacterial microbiota in the hindgut of Odontotermes formosanus and its fungus comb to investigate the spatial organization, establishment and temporal succession of the bacterial communities colonizing specific microhabitats. Our results document strong differences between the communities at the hindgut epithelium and the luminal fluid of newly moulted, young and old worker termites. The differences in community structure were consistent with the density, morphology and spatial distribution of bacterial cells and the pools of microbial metabolites in the hindgut compartment, underlining that both gut development and the age-specific changes in diet affect the composition and functional role of their gut microbiota. These findings provide strong support for the concept that changes in diet and gut environment are important determinants of community structure because they create new niches for microbial symbionts.


Assuntos
Envelhecimento/fisiologia , Bactérias/classificação , Sistema Digestório/microbiologia , Microbioma Gastrointestinal/fisiologia , Isópteros/microbiologia , Animais , Fungos/classificação , Isópteros/fisiologia
10.
J Econ Entomol ; 108(1): 266-73, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26470129

RESUMO

Laboratory rearing systems are useful models for studying Rhinotermitid behavior. Information on the biology of fungus-growing termites, however, is limited because of the difficulty of rearing colonies in the laboratory settings. The physical structure of termite nests makes it impossible to photograph or to observe colonies in the field. In this study, an artificial rearing system for field-collected colonies of the fungus-growing termite Odontotermes formosanus (Shiraki) was developed to facilitate observation in the laboratory. We recorded colony activity within the artificial rearing system and documented a variety of social behaviors that occurred throughout the food processing of the colony. This complex miniature ecosystem was cooperatively organized via division of labor in the foraging and processing of plant materials, and the observed patterns largely resembled the caste and age-based principles present in Macrotermes colonies. This work extends our insights into polyethism in the subfamily Macrotermitinae.


Assuntos
Criação de Animais Domésticos/instrumentação , Comportamento Animal , Isópteros , Fatores Etários , Animais , Fungos
11.
J Biomed Biotechnol ; 2011: 917232, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22203787

RESUMO

A procedure for simultaneous identification and quantification of canrenone and its biotransformed product 11-α-hydroxy-canrenone by high-performance liquid chromatography with ultraviolet detector (HPLC-UVD) and mass spectrometry (LC-MS) methods was proposed. The optimal determination variables on the HPLC-UVD or LC-MS coupled with a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 µm) were set as follows: detection wavelength of 280 nm, mobile phase of water and methanol gradient elution, temperature for the chromatographic column of 30°C, flow rate of mobile phase of 0.8 mL/min, sample injection volume of 5 µL, and elution time of 40 min. The MS conditions were set as follows: the flow rate of sheath gas, aux gas, and sweep gas were kept at 35 arb, 5 arb, and 0 arb, respectively. The temperature of capillary was held at 300°C, and capillary voltage was set at 30.00 V. Tube lens were performed at 100.00 V. The proposed method was validated by linearity (r² ≥ 0.9910), average recovery (94.93%, RSD1.21%), precision (RSD ≤ 1.31%), limit of detection, and limit of quantification (LOD 0.1~0.12 mg/L, LOQ 0.5~0.67 mg/L), which proved to be affordable for simultaneously determining canrenone and its bio-transformed product 11-α-hydroxy-canrenone.


Assuntos
Canrenona/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Biotransformação , Canrenona/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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