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1.
Mol Cell ; 84(5): 854-866.e7, 2024 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-38402612

RESUMEN

Deaminases have important uses in modification detection and genome editing. However, the range of applications is limited by the small number of characterized enzymes. To expand the toolkit of deaminases, we developed an in vitro approach that bypasses a major hurdle with their toxicity in cells. We assayed 175 putative cytosine deaminases on a variety of substrates and found a broad range of activity on double- and single-stranded DNA in various sequence contexts, including CpG-specific deaminases and enzymes without sequence preference. We also characterized enzyme selectivity across six DNA modifications and reported enzymes that do not deaminate modified cytosines. The detailed analysis of diverse deaminases opens new avenues for biotechnological and medical applications. As a demonstration, we developed SEM-seq, a non-destructive single-enzyme methylation sequencing method using a modification-sensitive double-stranded DNA deaminase. The streamlined protocol enables accurate, base-resolution methylome mapping of scarce biological material, including cell-free DNA and 10 pg input DNA.


Asunto(s)
Citosina Desaminasa , Epigenoma , ADN/genética , Citosina , ADN de Cadena Simple/genética , Citidina Desaminasa/genética
2.
RNA ; 29(11): 1803-1817, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37625853

RESUMEN

The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here, we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity toward RNA containing secondary structures and a free 5' end, and a broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.


Asunto(s)
Nucleotidiltransferasas , ARN , Animales , Filogenia , ARN Mensajero/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/química , Metiltransferasas/genética , Guanina , Caperuzas de ARN/genética , Mamíferos/genética
3.
Genome Res ; 31(2): 291-300, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33468551

RESUMEN

The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA, leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite-treated DNA, we applied a new enzymatic deamination approach, termed enzymatic methyl-seq (EM-seq), to long-range sequencing technologies. Our methodology, named long-read enzymatic modification sequencing (LR-EM-seq), preserves the integrity of DNA, allowing long-range methylation profiling of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) over multikilobase length of genomic DNA. When applied to known differentially methylated regions (DMRs), LR-EM-seq achieves phasing of >5 kb, resulting in broader and better defined DMRs compared with that previously reported. This result showed the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long-range epigenetic analysis at single-molecule and single-nucleotide resolution.

4.
Genome Res ; 31(7): 1280-1289, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34140313

RESUMEN

Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.

5.
RNA ; 28(8): 1144-1155, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35680168

RESUMEN

Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5' cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5' cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of predefined short fragments from the 5' end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5' cleavage products, and LC-MS intact mass analysis. In this paper, we describe (1) a framework to design site-specific RNA cleavage using RNase H; (2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; (3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5' cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.


Asunto(s)
Liposomas , Ribonucleasa H , Nanopartículas , Caperuzas de ARN/genética , ARN Mensajero/metabolismo , Ribonucleasa H/genética , Ribonucleasa H/metabolismo
6.
Cancer Invest ; : 1-11, 2024 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-38965994

RESUMEN

Despite the emergence of various treatment strategies for rectal cancer based on neoadjuvant chemoradiotherapy, there is currently a lack of reliable biomarkers to determine which patients will respond well to neoadjuvant chemoradiotherapy. Through collecting hematological and biochemical parameters data of patients prior to receiving neoadjuvant chemoradiotherapy, we evaluated the predictive value of systemic inflammatory indices for pathological response and prognosis in rectal cancer patients. We found that baseline GRIm-Score was an independent predictor for MPR in rectal cancer patients. However, no association was observed between several commonly systemic inflammation indices and long-term outcome.

7.
Langmuir ; 40(16): 8427-8439, 2024 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-38607689

RESUMEN

This work aims to enhance the adsorption performance of Laponite @diatomite for organic pollutants by modifying it with cetyltrimethylammonium bromide (CTAB). The microstructure and morphology of the CTAB-modified Laponite @diatomite material were characterized using SEM, XRD, FTIR, BET, and TG. Furthermore, the influences of key parameters, containing pH, adsorbent dosage, reaction time, and reaction temperature, on the adsorption process were investigated. The kinetics, thermodynamics, and isotherm models of the adsorption process were analyzed. Finally, potential adsorption mechanisms were given based on the characterization. The research findings indicate that CTAB-La@D exhibits good adsorption performance toward Congo red (CR) over a broad pH range. The maximum adsorption capacity of CR was 451.1 mg/g under the optimum conditions (dosage = 10 mg, contact time = 240 min, initial CR concentration = 100 mg/L, temperature = 25 °C, and pH = 7). The adsorption process conformed to the pseudo-second-order kinetic model, and the adsorption isotherms indicated that the adsorption process of CR was more in line with the Langmuir model, and it was physical adsorption. Thermodynamic analysis illustrates that the adsorption process is exothermic and spontaneous. Additionally, the mechanisms of electrostatic adsorption and hydrophobic effect adsorption of CR were investigated through XPS and FTIR analysis. This work provides an effective pathway for designing high-performance adsorbents for the removal of organic dye, and the synthesized materials hold great capability for practical utilization in the treatment of wastewater.

8.
Surg Endosc ; 38(6): 3329-3336, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38689159

RESUMEN

OBJECTIVE: This study aims to investigate the efficacy and safety of snare traction-assisted endoscopic submucosal dissection (ESD) for the management of circumferential superficial esophageal cancer. METHODS: A total of 68 patients who underwent ESD for circumferential superficial esophageal cancer were included in this study. All the patients were divided into two groups based on whether the snare traction was used or not; the snare traction group (S-ESD, group n = 35) and the control group (C-ESD, group n = 33). RESULTS: There was no significant difference in the size of the resected area between the groups [21.98 (18.30, 27.00) cm2 vs 24.00 (15.28, 30.72) cm2, P = 0.976]. The snare traction group had a shorter dissection time [92.00 (74.00, 121.00) min vs 110.00 (92.50, 137.00) min, P = 0.017] and a faster resection speed [0.28 ± 0.13 cm2/min vs 0.22 ± 0.11cm2/min, P = 0.040] compared to the control group. There were no statistically significant differences between the two groups in terms of hospital stay, cost, en bloc resection rate, R0 resection rate, curative resection rate, bleeding rate, perforation rate, stricture rate, and recurrence rate (P > 0.05). CONCLUSION: Snare traction-assisted ESD is a safe and efficient approach for the treatment of circumferential superficial esophageal cancer. Its advantages includes shorter procedure so the anesthesia requirement, clear operative filed view, improved mucosal dissection efficiency, simple, and easily accessible equipment.


Asunto(s)
Resección Endoscópica de la Mucosa , Neoplasias Esofágicas , Humanos , Resección Endoscópica de la Mucosa/métodos , Resección Endoscópica de la Mucosa/instrumentación , Resección Endoscópica de la Mucosa/efectos adversos , Neoplasias Esofágicas/cirugía , Neoplasias Esofágicas/patología , Masculino , Femenino , Persona de Mediana Edad , Anciano , Resultado del Tratamiento , Tracción/métodos , Estudios Retrospectivos , Tempo Operativo , Esofagoscopía/métodos
9.
Dig Dis Sci ; 69(5): 1755-1761, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38483780

RESUMEN

OBJECTIVE: To investigate the safety and prognosis of enbloc or piecemeal removal after enbloc resection of a gastric GIST by comparing the clinical data of endoscopic en block resection and piecemeal removal (EP) and en block resection and complete removal (EC) of gastric GISTs. METHODS: A total of 111 (43 endoscopic piecemeal, and 68 complete removal) patients with gastric GIST's ≥ 2 cm in diameter who underwent endoscopic therapy from January 2016 to June 2020 at the First Affiliated Hospital of Zhengzhou University were retrospectively analyzed. In all cases, it was ensured that the tumor was intact during the resection, however, it was divided into EP group and EC group based on whether the tumor was completely removed or was cut into pieces which were then removed. The patients' recurrence-free survival rate and recurrence-free survival (RFS) were recorded. RESULTS: There was no statistically significant difference in RFS rates between the two groups (P = 0.197). The EP group had relatively high patient age, tumor diameter, risk classification, and operation time. However, there was no statistically significant difference in the number of nuclear fission images, postoperative hospitalization time, postoperative fasting time, complication rate and complication grading between the two groups (P > 0.05). CONCLUSION: Endoscopic piecemeal removal after en block resection of gastric GIST is safe and effective and achieves similar clinical outcomes as complete removal after en block resection.


Asunto(s)
Tumores del Estroma Gastrointestinal , Humanos , Tumores del Estroma Gastrointestinal/cirugía , Tumores del Estroma Gastrointestinal/patología , Femenino , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Anciano , Neoplasias Gástricas/cirugía , Neoplasias Gástricas/patología , Adulto , Resultado del Tratamiento , Gastroscopía/métodos
10.
Nucleic Acids Res ; 50(18): e106, 2022 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-35871301

RESUMEN

With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine-two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5' cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC-MS/MS.


Asunto(s)
Endorribonucleasas/química , ARN Mensajero/química , Análisis de Secuencia de ARN/métodos , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , ADN Complementario , Humanos , Oligonucleótidos/análisis , ARN Mensajero/genética , Ribonucleasa T1/metabolismo , Espectrometría de Masas en Tándem/métodos
11.
Nucleic Acids Res ; 50(1): e2, 2022 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-34581823

RESUMEN

Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5' cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5' cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA-a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit.


Asunto(s)
Caperuzas de ARN/metabolismo , ARN/análisis , Análisis de Secuencia de ARN/métodos , Humanos , Fijación del Tejido
12.
Nucleic Acids Res ; 50(6): 3001-3017, 2022 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-34522950

RESUMEN

The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their cellular hosts, but whose biosynthetic origins are enigmatic. Up to half of thymidines in the Pseudomonas phage M6, the Salmonella phage ViI, and others, contain exotic chemical moieties synthesized through the post-replicative modification of 5-hydroxymethyluridine (5-hmdU). We have determined that these thymidine hypermodifications are derived from free amino acids enzymatically installed on 5-hmdU. These appended amino acids are further sculpted by various enzyme classes such as radical SAM isomerases, PLP-dependent decarboxylases, flavin-dependent lyases and acetyltransferases. The combinatorial permutations of thymidine hypermodification genes found in viral metagenomes from geographically widespread sources suggests an untapped reservoir of chemical diversity in DNA hypermodifications.


Asunto(s)
Bacteriófagos , Liasas , Aminoácidos/metabolismo , Bacteriófagos/genética , ADN/metabolismo , Timidina/metabolismo
13.
Proc Natl Acad Sci U S A ; 118(26)2021 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-34155108

RESUMEN

TET/JBP (ten-eleven translocation/base J binding protein) enzymes are iron(II)- and 2-oxo-glutarate-dependent dioxygenases that are found in all kingdoms of life and oxidize 5-methylpyrimidines on the polynucleotide level. Despite their prevalence, few examples have been biochemically characterized. Among those studied are the metazoan TET enzymes that oxidize 5-methylcytosine in DNA to hydroxy, formyl, and carboxy forms and the euglenozoa JBP dioxygenases that oxidize thymine in the first step of base J biosynthesis. Both enzymes have roles in epigenetic regulation. It has been hypothesized that all TET/JBPs have their ancestral origins in bacteriophages, but only eukaryotic orthologs have been described. Here we demonstrate the 5mC-dioxygenase activity of several phage TETs encoded within viral metagenomes. The clustering of these TETs in a phylogenetic tree correlates with the sequence specificity of their genomically cooccurring cytosine C5-methyltransferases, which install the methyl groups upon which TETs operate. The phage TETs favor Gp5mC dinucleotides over the 5mCpG sites targeted by the eukaryotic TETs and are found within gene clusters specifying complex cytosine modifications that may be important for DNA packaging and evasion of host restriction.


Asunto(s)
5-Metilcitosina/metabolismo , Bacteriófagos/metabolismo , ADN/metabolismo , Secuencia de Aminoácidos , Metilación de ADN , Dioxigenasas , Hidroxilación , Metagenómica , Motivos de Nucleótidos/genética , Oxidación-Reducción , Filogenia
14.
Int J Mol Sci ; 25(8)2024 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-38674153

RESUMEN

Anti-inflammatory drugs have become the second-largest class of common drugs after anti-infective drugs in animal clinical care worldwide and are often combined with other drugs to treat fever and viral diseases caused by various factors. In our previous study, a novel serine protease inhibitor-encoding gene (MDSPI16) with improved anti-inflammatory activity was selected from a constructed suppressive subducted hybridization library of housefly larvae. This protein could easily induce an immune response in animals and had a short half-life, which limited its wide application in the clinic. Thus, in this study, mPEG-succinimidyl propionate (mPEG-SPA, Mw = 5 kDa) was used to molecularly modify the MDSPI16 protein, and the modified product mPEG-SPA-MDSPI16, which strongly inhibited elastase production, was purified. It had good stability and safety, low immunogenicity, and a long half-life, and the IC50 for elastase was 86 nM. mPEG-SPA-MDSPI16 effectively inhibited the expression of neutrophil elastase and decreased ROS levels. Moreover, mPEG-SPA-MDSPI16 exerted anti-inflammatory effects by inhibiting activation of the NF-κB signaling pathway and the MAPK signaling pathway in neutrophils. It also exerted therapeutic effects on a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. In summary, mPEG-SPA-MDSPI16 is a novel anti-inflammatory protein modified with PEG that has the advantages of safety, nontoxicity, improved stability, and strong anti-inflammatory activity in vivo and in vitro and is expected to become an effective anti-inflammatory drug.


Asunto(s)
Lesión Pulmonar Aguda , Lipopolisacáridos , Inhibidores de Serina Proteinasa , Animales , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/inducido químicamente , Ratones , Inhibidores de Serina Proteinasa/farmacología , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/uso terapéutico , Antiinflamatorios/farmacología , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Polietilenglicoles/química , Polietilenglicoles/farmacología , FN-kappa B/metabolismo , Masculino , Elastasa de Leucocito/metabolismo , Humanos , Transducción de Señal/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Modelos Animales de Enfermedad
15.
J Biol Chem ; 298(4): 101751, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35189146

RESUMEN

The phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1) is a methyltransferase that adds a methyl group to the N6-position of 2'O-methyladenosine (Am), generating N6, 2'O-dimethyladenosine (m6Am) when Am is the cap-proximal nucleotide. In addition, PCIF1 has ancillary methylation activities on internal adenosines (both A and Am), although with much lower catalytic efficiency relative to that of its preferred cap substrate. The PCIF1 preference for 2'O-methylated Am over unmodified A nucleosides is due mainly to increased binding affinity for Am. Importantly, it was recently reported that PCIF1 can methylate viral RNA. Although some viral RNA can be translated in the absence of a cap, it is unclear what roles PCIF1 modifications may play in the functionality of viral RNAs. Here we show, using in vitro assays of binding and methyltransfer, that PCIF1 binds an uncapped 5'-Am oligonucleotide with approximately the same affinity as that of a cap analog (KM = 0.4 versus 0.3 µM). In addition, PCIF1 methylates the uncapped 5'-Am with activity decreased by only fivefold to sixfold compared with its preferred capped substrate. We finally discuss the relationship between PCIF1-catalyzed RNA methylation, shown here to have broader substrate specificity than previously appreciated, and that of the RNA demethylase fat mass and obesity-associated protein (FTO), which demonstrates PCIF1-opposing activities on capped RNAs.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Nucleares , Caperuzas de ARN , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina/metabolismo , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Viral/metabolismo
16.
BMC Psychiatry ; 23(1): 907, 2023 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-38053101

RESUMEN

BACKGROUND: Patients' attitudes toward medication have been shown to be a predictor of nonadherence to antipsychotic treatment. However, most previous studies that explored this relationship used a cross-sectional design. It is important to explore the association of attitudes toward drugs with discontinuation at different time points during antipsychotic treatment. In this study, we investigated the association of attitudes toward drugs (measured by the Drug Attitude Inventory (DAI-10)) with adherence at seven time points (baseline, 4 weeks, 8 weeks, 12 weeks, 26 weeks, 39 weeks, and 52 weeks) during 1 year of treatment. Factors that were potentially associated with attitudes toward drugs at the time point of interest were also studied. METHODS: Demographic characteristics, psychopathology, social functioning, and attitudes toward drugs (measured by the DAI-10) were collected at baseline, 4 weeks, 8 weeks, 12 weeks, 26 weeks, 39 weeks and 52 weeks. The association of attitudes toward drugs (measured by DAI-10) with adherence at the seven time points was calculated using the Mann‒Whitney U test. The optimal cutoff point for the DAI-10 was then determined using receiver operating characteristic (ROC) analysis. Cox regression analysis was conducted to further investigate the association of DAI-10 scores with discontinuation, controlling for potential confounding variables. We used multiple regression analysis to identify the factors associated with DAI-10 scores. RESULTS: Among the six time points, only baseline DAI-10 total scores were significantly different between the completed and discontinued groups (p = 0.004). Female sex and a baseline DAI-10 total score greater than - 1 were found to be independent protective factors against discontinuation of antipsychotic drug treatments during the 1-year follow-up. At baseline, the severity of the disease (CGI-s) and insight regarding the disease were shown to be associated with DAI-10 total scores. CONCLUSION: Attitudes toward antipsychotic drugs at baseline were shown to play a crucial role in predicting treatment discontinuation. TRIAL REGISTRATION: The data were collected from a clinical trial and the clinical trials.gov ID of the study is NCT01057849.


Asunto(s)
Antipsicóticos , Esquizofrenia , Humanos , Femenino , Esquizofrenia/tratamiento farmacológico , Antipsicóticos/uso terapéutico , Estudios Prospectivos , Estudios Transversales , Cumplimiento de la Medicación
17.
Mol Cell ; 57(4): 750-761, 2015 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-25639471

RESUMEN

Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biological functions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC chemical labeling enrichment method. The sensitive method enables detection of low-abundance 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated a genome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC in non-CpG context exhibit lower abundance, more dynamically, than those in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H = A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite conversion-free method can be applied to biological samples with limited starting material or low-abundance cytosine modifications.


Asunto(s)
Citosina/análogos & derivados , Mapeo Restrictivo/métodos , 5-Metilcitosina/análogos & derivados , Animales , Secuencia de Bases , Citosina/química , Enzimas de Restricción del ADN/química , Células Madre Embrionarias , Epigénesis Genética , Biblioteca de Genes , Histonas/metabolismo , Ratones
18.
Nucleic Acids Res ; 49(9): 5265-5277, 2021 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-33885787

RESUMEN

Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca2+)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca2+-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca2+ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca2+ in the modulation of RNase I activity. Mutation of a previously overlooked Ca2+ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca2+, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca2+-dependency of RNase I may be useful as a tool in applied molecular biology.


Asunto(s)
Calcio/metabolismo , Endorribonucleasas/metabolismo , ARN Bicatenario/metabolismo , Sustitución de Aminoácidos , ADN , Endorribonucleasas/química , Endorribonucleasas/genética , Metales/metabolismo , ARN/metabolismo , Ribonucleasas/metabolismo , Especificidad por Sustrato
19.
Wei Sheng Yan Jiu ; 52(4): 549-553, 2023 Jul.
Artículo en Zh | MEDLINE | ID: mdl-37679078

RESUMEN

OBJECTIVE: To explore whether there are differences in the levels of protein, glucose and blood lipids in umbilical vein and umbilical artery blood of newborns with different delivery modes, and to evaluate their value as indicators of fetal intrauterine nutrition and nutritional support. METHODS: A total of 89 pairs of mothers and infants who were delivered in Danyang People's Hospital of Jiangsu Province from June to September 2021 were selected as the study subjects, including 38 cases of spontaneous delivery and 51 cases of cesarean section. The basic information of pregnant women, pregnancy information, newborn delivery and physical examination information were extracted from the medical record information system of the hospital. According to the mode of delivery, HITACHI 7600 automatic biochemical analyzer was used to measure the levels of protein, glucose and blood lipids in umbilical vein and umbilical artery blood, including total protein(TP), albumin(ALB), glucose(GLU), total cholesterol(TC), triglyceride(TG), high density lipoprotein cholesterol(HDL-C), low density lipoprotein cholesterol(LDL-C). The data were statistically analyzed using IBM SPSS Statistics 26.0 statistical software. RESULTS: The levels of TP, ALB, GLU, TC, TG, HDL-C and LDL-C in the umbilical vein blood of the spontaneous delivery group were(56.40±5.83)g/L, (38.41±3.43)g/L, (4.55±1.53)mmol/L, (1.68±0.42)mmol/L, (0.25±0.11)mmol/L, (0.84±0.17)mmol/L and(0.69±0.23)mmol/L, respectively. The levels of TP, ALB, GLU, TC, TG, HDL-C and LDL-C in umbilical artery blood were(56.49±9.91)g/L, (37.72±4.77)g/L, (4.07±1.52)mmol/L, (1.60±0.42)mmol/L, (0.24±0.10)mmol/L, (0.80±0.18)mmol/L and(0.68±0.24)mmol/L, respectively. The levels of TP, ALB, GLU, TC, TG, HDL-C and LDL-C in umbilical vein blood of cesarean section group were(52.08±4.12)g/L, (36.12±2.13)g/L, (3.45±1.16)mmol/L, (1.61±0.39)mmol/L, (0.19±0.08)mmol/L, (0.82±0.18)mmol/L and(0.61±0.20)mmol/L, respectively. The levels of TP, ALB, GLU, TC, TG, HDL-C and LDL-C in umbilical artery blood were(51.49±7.59)g/L, (35.40±3.60)g/L, (3.09±1.15)mmol/L, (1.48±0.40)mmol/L, (0.19±0.08)mmol/L, (0.78±0.18)mmol/L and(0.60±0.20)mmol/L, respectively. The levels of TP, ALB, Glu and TG in cord vein blood and cord artery blood in spontaneous labor group were significantly higher than those in cesarean section group(P<0.05); The levels of Glu, TC, TG and HDL-C in cord vein blood were significantly higher in spontaneous labor group and cesarean section group than those in cord artery blood(P<0.05). CONCLUSION: The levels of protein, glucose and blood lipids in umbilical vein and umbilical artery blood were different among different delivery modes.


Asunto(s)
Cesárea , Glucosa , Recién Nacido , Embarazo , Lactante , Femenino , Humanos , LDL-Colesterol , Arterias , Lípidos
20.
Wei Sheng Yan Jiu ; 52(3): 424-428, 2023 May.
Artículo en Zh | MEDLINE | ID: mdl-37500522

RESUMEN

OBJECTIVE: To investigate the difference of cortical hormones in cord artery and vein blood of newborns with different delivery modes. METHODS: A total of 65 pregnant women who delivered in the People's Hospital of Danyang City, Jiangsu Province from June to September 2021 were selected as the study subjects, including 26 cases of spontaneous delivery and 39 cases of cesarean section. The basic information of 65 pregnant women and newborns was collected by questionnaire survey. According to the mode of delivery, the levels of corticosteroids in umbilical vein and umbilical artery blood were determined by liquid chromatography-tandem mass spectrometry(LC-MS/MS), including corticosterone, 11-desoxycorticosterone, aldosterone, cortisol, 11-deoxycortisol and cortisone. The data were statistically analyzed using IBM SPSS Statistics 26.0 statistical software. RESULTS: The levels of cortisol, 11-deoxycortisol, aldosterone, cortisol, 11-deoxycortisol and cortisone in the umbilical vein blood of the spontaneous delivery group were(2.44±1.87), (0.64±0.29), (0.49±0.35), (54.95±40.80), (3.20±1.23) and(142.27±57.42)ng/mL, respectively. The levels of corticosterone, 11-deoxycortisol, aldosterone, cortisol, 11-deoxycortisol and cortisone in umbilical artery blood were(4.51±4.47), (0.57±0.28), (0.42±0.29), (60.79±45.53), (2.69±1.25) and(123.10±46.32)ng/mL, respectively. The levels of corticosterone, 11-deoxycortisone, aldosterone, cortisol, 11-deoxycortisone and cortisone in umbilical vein blood of cesarean section group were(0.94±1.09), (0.47±0.14), (0.26±0.14), (22.63±19.82), (2.30±0.90) and(84.51±29.49)ng/mL, respectively. The levels of corticosterone, 11-deoxycortisol, aldosterone, cortisol, 11-deoxycortisol and cortisone in umbilical artery blood were(2.22±2.24), (0.43±0.17), (0.27±0.14), (30.09±25.93), (1.87±0.76) and(75.03±24.90)ng/mL, respectively. The levels of corticosterone, 11-desoxycorticosterone, aldosterone, cortisol, 11-deoxycortisol and cortisone in cord vein blood and cord artery blood in spontaneous labor group were significantly higher than those in cesarean section group(P<0.05). The levels of corticosterone and cortisol in cord vein blood were significantly lower in spontaneous labor group and cesarean section group than those in cord artery blood(P<0.05), the levels of 11-desoxycorticosterone, 11-deoxycortisol and cortisone in cord vein blood were significantly higher in spontaneous labor group and cesarean section group than those in cord artery blood(P<0.05). CONCLUSION: There are differences in the level of cortical hormones in cord artery and vein blood in different delivery modes.


Asunto(s)
Corticosterona , Cortisona , Femenino , Recién Nacido , Embarazo , Humanos , Hidrocortisona , Aldosterona , Cortodoxona , Cesárea , Cromatografía Liquida , Espectrometría de Masas en Tándem , Desoxicorticosterona , Sangre Fetal , Arterias
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