RESUMO
CircRNAs are a class of endogenous non-coding RNAs implicated in the pathogenesis of many pregnancy related diseases, one of which is pre-eclampsia (PE). This study aims to investigate the role of CircPAPPA2 (circbase ID: hsa_circ_0015382) in regulating the migration and invasion of trophoblast cells. RNA sequencing was used to identify the differentially expressed circRNAs in placenta of PE and normal pregnant women. Quantitative polymerase chain reaction (qRT-PCR) was used to verify the expression of circPAPPA2 and two miRNAs (miR-942-5p, 5006-3p) in placenta of PE and normal pregnant women. CCK8 and transwell experiments were performed to assess the function of circPAPPA2 in PE development.The interaction between circPAPPA2 and miR-942-5p/miR-5006-3p was verified by dual-luciferase reporter assay. Finally, bioinformatics analyzed with gene ontology, Kyoto Encyclopedia of the target genes. The results showed that the expression of circPAPPA2 was increased in placenta of PE pregnant women. Also, circPAPPA2 impedes trophoblasts cell proliferation and invasion. Moreover, the expression of circPAPPA2 was positively correlated with systolic blood pressure and urine protein. In addition, circPAPPA2 serves as a sponge of miR-942-5p and miR-5006-3p. In conclusion, CircPAPPA2 regulates trophoblasts cell proliferation and invasion by mediating the miR-942/miR-5006-3p.
Assuntos
MicroRNAs , Placenta , Pré-Eclâmpsia , RNA Circular , Trofoblastos , Humanos , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Feminino , MicroRNAs/genética , MicroRNAs/metabolismo , Gravidez , RNA Circular/genética , Trofoblastos/metabolismo , Placenta/metabolismo , Adulto , Movimento Celular/genética , Proliferação de Células/genética , Estudos de Casos e ControlesRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea involucrata Kar.et Kir. (S.I.) has long been used as a precious national medicine and clinically proven to be an effective treatment for rheumatoid arthritis (RA) and cardiovascular diseases. In clinical practice, two extraction methods of S.I., including water decoction and alcohol extraction, are prescribed to treat the same conditions. Nevertheless, no study has been performed on the exposure differences of the pharmacodynamic material basis in vivo caused by different extraction methods. AIM OF THE STUDY: Based on the integrated strategy of metabolism, network pharmacology, and pharmacokinetics, we aimed to reveal exposure differences in pharmacodynamic substances caused by different extraction methods. MATERIALS AND METHODS: Ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) was employed to identify the chemical constituents of S.I. extracts and the metabolites in vivo after administration. Based on the analysis of prototype components in vivo, the major exposure active constituents, potential therapeutic targets and possible pharmacological mechanisms in RA treatment were investigated using network pharmacological analysis. Seven critical active components, including quercetin, hispidulin, apigenin, chlorogenic acid, arctigenin, syringin, and umbelliferone, were quantitatively compared between the alcohol, and aqueous extraction methods, which had been confirmed by the reference substance. RESULTS: The chemical comparison demonstrated that the types of chemicals in the two extracts were identical, mainly flavonoids, phenylpropanoids, coumarins, lignins, sesquiterpene lactones, and others, but the contents of the primary constituents in the aqueous extract were lower than those of the alcohol extract. A total of 30 prototype components and 174 metabolites were analyzed and identified in rat plasma, urine, fecal, and bile samples. Twenty-three prototype components were analyzed by network pharmacology, and seven critical active components were selected as representative markers for the pharmacokinetic study. Pharmacokinetic studies had shown that the Tmax values of apigenin, hispidulin, chlorogenic acid, arctigenin, and syringin after the oral administration of the alcohol extract were lower than those after the oral administration of the aqueous extract, and the above components in the alcohol extract could increase the absorption. Compared with the aqueous extract group, the Tmax and T1/2 of quercetin and umbelliferone were longer; it was suggested that alcohol extraction might have a slow-release and long-term effect on these two components. The relative bioavailability of apigenin, hispidulin, quercetin, chlorogenic acid, and umbelliferone in the alcohol extract group were higher than those in the aqueous extract group, which was consistent with the traditional clinical experience that alcohol extract could improve the efficacy of S.I. CONCLUSIONS: The major exposure active constituents in vivo were screened. The representative components that could be used in pharmacokinetics were determined by integrating network pharmacology and metabolism studies. The critical active compounds were quantitatively compared between the alcohol and aqueous extraction methods. This study clarified that flavonoids, coumarin, and phenylpropanoids might be the primary material basis that caused the exposure differences between aqueous and alcoholic extracts from S.I.. This research aimed to provide the basis of metabolism in vivo for further studying these pharmacodynamic differences.
Assuntos
Medicamentos de Ervas Chinesas , Saussurea , Animais , Apigenina , Ácido Clorogênico , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/farmacologia , Flavonoides , Farmacologia em Rede , Extratos Vegetais/uso terapêutico , Quercetina , Ratos , Saussurea/química , UmbeliferonasRESUMO
Xanthine oxidase (XO) is a flavoprotein that exists in various organisms and can catalyze the uric acid formation in the human body. Based on the amide framework of N-(4-((3-cyanobenzyl)oxy)-3-(1H-tetrazol-1-yl)phenyl)isonicotinamide (compound 1) reported in our previous work, a series of N-(4-alkoxy-3-(1H-tetrazol-1-yl)phenyl) heterocyclic aromatic amide derivatives were designed, synthesized and evaluated as novel amide-based XO inhibitors. Structure-activity relationship campaign identified the most promising compound g25 (IC50 = 0.022 µM), which possesses a special 1H-imidazole-5-carboxamide scaffold and presented comparable XO inhibitory potency to topiroxostat (IC50 = 0.017 µM). Enzyme kinetic studies revealed that compound g25 acted as a mixed-type XO inhibitor. Molecular docking and molecular dynamics indicated that imidazole NH of g25 formed two stable hydrogen bonds with Glu1261 residue of XO that provided a vital contribution for the binding affinity. In addition, in vivo activity evaluation demonstrated that compound g25 exhibited obviously hypouricemic effect on a potassium oxonate induced hyperuricemic rat model.
Assuntos
Amidas , Xantina Oxidase , Álcoois , Amidas/farmacologia , Animais , Desenho de Fármacos , Inibidores Enzimáticos/química , Humanos , Imidazóis/farmacologia , Cinética , Simulação de Acoplamento Molecular , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
Our previous work identified a promising isonicotinamide based xanthine oxidase (XO) inhibitor, N-(3-cyano-4-((2-cyanobenzyl)oxy)phenyl)isonicotinamide (1), and concluded that amide is an effective linker in exploring the XO inhibitor chemical space that is completely different from the five-membered ring framework of febuxostat and topiroxostat. Indole, an endogenous bioactive substance and a popular drug construction fragment, was involved in the structural optimization campaign of the present effort. After the installation of some functional groups, N-(1-alkyl-3-cyano-1H-indol-5-yl) was generated and employed to mend the missing H-bond interaction between the 3'-cyano of 1 and Asn768 residue of XO by shortening their distance. In this context, eight kinds of heterocyclic aromatic amide chemotypes were rationally designed and synthesized to investigate the structure-activity relationship (SAR) of amide-based XO inhibitors. The optimized compound a6 (IC50 = 0.018 µM) exhibits 17.2-fold improved potency than the initial compound 1 (IC50 = 0.31 µM). Its potency is comparable to that of topiroxostat (IC50 = 0.013 µM). Molecular docking and molecular dynamics studies proved the existence of the stable H-bond between the cyano group and the Asn768 residue. Moreover, oral administration of a6 (11.8 mg/kg) could effectively reduce serum uric acid levels in an acute hyperuricemia rat model. Liver microsomal stability assay illustrated that compound a6 possesses well metabolic stability in rat liver microsomes. However, the in vivo potency of a6 was much lower than that of topiroxostat, which may be explained by the poor absorption found in the parallel artificial membrane permeability assay (PAMPA). In addition, 6a has non-cytotoxicity against normal cell lines MCF10A and 16HBE. Taken together, this work culminated in the identification of compound 6a as an excellent lead for further exploration of amide-based XO inhibitors.
Assuntos
Amidas/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Xantina Oxidase/antagonistas & inibidores , Amidas/química , Amidas/metabolismo , Animais , Bovinos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Feminino , Indóis/química , Masculino , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Leite/enzimologia , Modelos Moleculares , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Xantina Oxidase/metabolismoRESUMO
Our previous work demonstrated that amide is an efficient linker to explore chemical space of xanthine oxidase (XO) inhibitors that are entirely different from febuxostat and topiroxostat. In this effort, with 3-cyano-1H-indol-5-yl as a key moiety, two series of amide-based XO inhibitors, N-(3-cyano-1H-indol-5-yl)isonicotinamides (2a-w) and N-(3-cyano-1H-indol-5-yl)-1H-benzo[d]imidazole-5-carboxamides (3a-i), were designed and synthesized. The structure-activity relationship investigation identified N-(3-cyano-1-cyclopentyl-1H-indol-5-yl)-1H-benzo[d]imidazole-5-carboxamide (3i, IC50 = 0.62 µM) as the most promising compound, with 14.4-fold higher in vitro inhibitory potency than allopurinol (IC50 = 8.91 µM). Molecular simulations provided reasonable interaction modes for the representative compounds. Furthermore, in vivo activity evaluation demonstrated that compound 3i (oral dose of 12.8 mg/kg) has obviously hypouricemic effect on a potassium oxonate induced hyperuricemic rat model. Cytotoxicity assay and ADME prediction also supported that 3i is an excellent lead for further exploration of amide-based XO inhibitors.
Assuntos
Imidazóis/química , Imidazóis/farmacologia , Niacinamida/química , Niacinamida/farmacologia , Xantina Oxidase/antagonistas & inibidores , Amidas/química , Amidas/farmacologia , Animais , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Hiperuricemia/tratamento farmacológico , Hiperuricemia/metabolismo , Imidazóis/uso terapêutico , Masculino , Simulação de Acoplamento Molecular , Niacinamida/uso terapêutico , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Xantina Oxidase/metabolismoRESUMO
The molecular chaperone heat shock protein 90 (Hsp90) is a promising target for cancer therapy. Natural product aconitine is a potential Hsp90 inhibitor reported in our previous work. In this study, we designed and synthesized a series of 2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-one derivatives as potent Hsp90 inhibitors by simplifying and modifying aconitine scaffold. Among these compounds, 14t exhibited an excellent antiproliferative activity against LoVo cells with an IC50 value of 0.02 µM and a significant Hsp90α inhibitory activity with an IC50 value of 0.71 nM. Molecular docking studies provided a rational binding model of 14t in complex with Hsp90α. The following cell cycle and apoptosis assays revealed that compound 14t could arrest cell cycle at G1/S phase and induce cell apoptosis via up-regulation of bax and cleaved-caspase 3 protein expressions while inhibiting the expressions of bcl-2. Moreover, 14t could inhibit cell migration in LoVo and SW620 cell lines. Consistent with in vitro results, 14t significantly repressed tumor growth in the SW620 xenograft mouse model.
Assuntos
Aconitina/farmacologia , Antineoplásicos/farmacologia , Descoberta de Drogas , Aconitina/síntese química , Aconitina/química , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Compostos Aza/síntese química , Compostos Aza/química , Compostos Aza/farmacologia , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Octanos/síntese química , Octanos/química , Octanos/farmacologia , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química , Triazóis/farmacologiaRESUMO
Src plays a crucial role in many signaling pathways and contributes to a variety of cancers. Therefore, Src has long been considered an attractive drug target in oncology. However, the development of Src inhibitors with selectivity and novelty has been challenging. In the present study, pharmacophore-based virtual screening and molecular docking were carried out to identify potential Src inhibitors. A total of 891 molecules were obtained after pharmacophore-based virtual screening, and 10 molecules with high docking scores and strong interactions were selected as potential active molecules for further study. Absorption, distribution, metabolism, elimination and toxicity (ADMET) property evaluation was used to ascertain the drug-like properties of the obtained molecules. The proposed inhibitor-protein complexes were further subjected to molecular dynamics (MD) simulations involving root-mean-square deviation and root-mean-square fluctuation to explore the binding mode stability inside active pockets. Finally, two molecules (ZINC3214460 and ZINC1380384) were obtained as potential lead compounds against Src kinase. All these analyses provide a reference for the further development of novel Src inhibitors.
Assuntos
Descoberta de Drogas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Quinases da Família src/química , Sítios de Ligação , Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos Testes , Quinases da Família src/antagonistas & inibidoresRESUMO
In our previous study, we reported a series of N-phenylisonicotinamide derivatives as novel xanthine oxidase (XO) inhibitors and identified N-(3-cyano-4-((2-cyanobenzyl)oxy)phenyl)isonicotinamide (compound 1) as the most potent one with an IC50 value of 0.312⯵M. To further optimize the structure and improve the potency, a structure-based drug design (SBDD) strategy was performed to construct the missing H-bond between the small molecule and the Asn768 residue of XO. We introduced a tetrazole moiety at the 3'-position of the phenyl to serve as an H-bond acceptor and obtained a series of N-(3-(1H-tetrazol-1-yl)phenyl)isonicotinamide derivatives (2a-t and 6-8). Besides, to investigate the influence of the amide-reversal, some N-(pyridin-4-yl)-3-(1H-tetrazol-1-yl)benzamide derivatives (3c, 3e, 3i, 3k and 3u) were also synthesized and evaluated. Biological evaluation and structure-activity relationship analysis demonstrated that the 3'-(1H-tetrazol-1-yl) moiety was an excellent fragment for the N-phenylisonicotinamide scaffold; a substituted benzyloxy, especially, an m-cyanobenzyloxy (e.g., 2s), linking at the 4'-position was welcome for the potency; and the amide-reversal could damage the potency, so maintenance of the N-phenylisonicotinamide scaffold was essential. In summary, starting from compound 1, the SBDD effort successfully identified a promising XO inhibitor 2s (IC50â¯=â¯0.031⯵M), with a 10-fold gain in potency. Its potency was very close to the positive control topiroxostat (IC50â¯=â¯0.021⯵M). A Lineweaver-Burk plot indicated that compound 2s acted as a mixed-type XO inhibitor. Molecular docking and molecular dynamics simulations revealed that the tetrazole moiety could occupy the Asn768-sub-pocket with N-4 atom accepting an H-bond from the Asn768 residue, as expected.
Assuntos
Niacinamida/análogos & derivados , Niacinamida/síntese química , Xantina Oxidase/antagonistas & inibidores , Animais , Benzamidas/química , Bovinos , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Niacinamida/metabolismo , Nitrilas/metabolismo , Ligação Proteica , Piridinas/metabolismo , Relação Estrutura-Atividade , Tetrazóis/química , Ácido Úrico/metabolismo , Xantina Oxidase/metabolismoRESUMO
Patient safety education is conducive to medical students' cognition on patient safety and to improvement of medical quality and safety. Developing patient safety education for medical students is more and more widely recognized by World Health Organization and countries all over the world. However, in China, patient safety courses aiming at medical students are relatively few, and there are few reports about the effect of patient safety courses. This paper explored the influence of patient safety curriculum on medical students' attitude to and knowledge of patient safety. The patient safety curriculum was carried out for 2011-grade undergraduates of Tongji Medical College, Huazhong University of Science and Technology. The students participated in the class according to free choice. After the curriculum, the information of gender, major, attended course, attitude toward patient safety, and knowledge of laws and regulations of the 2011-grade undergraduates were collected. After rejecting invalid questionnaires, the number of undergraduates that participated in the survey was 112 (61 students did not take part in the curriculum; 51 took part in). Chi-square test was applied to analyze patient safety education's influence on medical students' attitude to patient safety and their knowledge mastery situation. The influence of patient safety education on the attitude of medical students to patient safety was not significant, but that on their knowledge of patient safety was remarkable. No matter male or female, as compared with medical students who had not accepted patient safety education, they both had a better acquisition of knowledge after having this education (for male students: 95% CI, 4.556-106.238, P<0.001; for female students: 95% CI, 3.183-33.238, P<0.001). Students majoring in Western Medicine had a relatively better mastery of knowledge of patient safety after receiving patient safety education (95% CI, 6.267-76.271, P<0.001). Short-term patient safety education cannot change medical students' stereotyped cognition on matters related to patient safety, but it can effectively enhance their knowledge of laws and regulations of patient safety.
Assuntos
Educação Médica , Conhecimentos, Atitudes e Prática em Saúde , Segurança do Paciente , Adulto , China/epidemiologia , Currículo , Feminino , Humanos , Masculino , Estudantes de Medicina , Inquéritos e QuestionáriosRESUMO
Reversible lysine acetylation is one of the most important protein posttranslational modifications that plays essential roles in both prokaryotes and eukaryotes. However, only a few lysine deacetylases (KDACs) have been identified in prokaryotes, perhaps in part due to their limited sequence homology. Herein, we developed a 'clip-chip' strategy to enable unbiased, activity-based discovery of novel KDACs in the Escherichia coli proteome. In-depth biochemical characterization confirmed that YcgC is a serine hydrolase involving Ser200 as the catalytic nucleophile for lysine deacetylation and does not use NAD(+) or Zn(2+) like other established KDACs. Further, in vivo characterization demonstrated that YcgC regulates transcription by catalyzing deacetylation of Lys52 and Lys62 of a transcriptional repressor RutR. Importantly, YcgC targets a distinct set of substrates from the only known E. coli KDAC CobB. Analysis of YcgC's bacterial homologs confirmed that they also exhibit KDAC activity. YcgC thus represents a novel family of prokaryotic KDACs.
Assuntos
Amidoidrolases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Lisina/metabolismo , Especificidade por SubstratoRESUMO
Protein microarray technology is one of the most powerful tools presently available for proteomic studies. Numerous types of protein microarrays have been widely and successfully applied for both basic biological studies and clinical researches, including those designed to characterize protein-protein, protein-nucleic acid, protein-drug/small molecule and antibody-antigen interactions. In the past decade, a variety of protein microarrays have been developed, including those spotted with whole proteomes, smaller peptides, antibodies, and lectins. Featured as high-throughput, miniaturized, and capable of parallel analysis, the power of protein microarrays has already been demonstrated many times in both basic research and clinical applications. In this review, we have summarized the latest developments in the production and application of protein microarrays. We discuss several of the most important applications of protein microarray, ranging from proteome microarrays for large scale identification of protein-protein interactions to lectin microarrays for live cell surface glycan profiling, with special emphasis on their use in studies of drug mechanisms and biomarker discovery. Already with tremendous success, we envision protein microarrays will become an indispensible tool for any systems-wide studies, fostering the integration of basic research observations to clinically useful applications.
Assuntos
Análise Serial de Proteínas , Biologia de Sistemas , Lectinas/química , Miniaturização , Polissacarídeos/química , ProteomaRESUMO
Bcl2-associated athanogene 3 (BAG3), a member of the BAG family of co-chaperones, plays a critical role in regulating apoptosis, development, cell motility, autophagy, and tumor metastasis and in mediating cell adaptive responses to stressful stimuli. BAG3 carries a BAG domain, a WW domain, and a proline-rich repeat (PXXP), all of which mediate binding to different partners. To elucidate BAG3's interaction network at the molecular level, we employed quantitative immunoprecipitation combined with knockdown and human proteome microarrays to comprehensively profile the BAG3 interactome in humans. We identified a total of 382 BAG3-interacting proteins with diverse functions, including transferase activity, nucleic acid binding, transcription factors, proteases, and chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. In addition, we characterized interactions between BAG3 and some of its newly identified partners in greater detail. In particular, bioinformatic analysis revealed that the BAG3 interactome is strongly enriched in proteins functioning within the proteasome-ubiquitination process and that compose the proteasome complex itself, suggesting that a critical biological function of BAG3 is associated with the proteasome. Functional studies demonstrated that BAG3 indeed interacts with the proteasome and modulates its activity, sustaining cell survival and underlying resistance to therapy through the down-modulation of apoptosis. Taken as a whole, this study expands our knowledge of the BAG3 interactome, provides a valuable resource for understanding how BAG3 affects different cellular functions, and demonstrates that biologically relevant data can be harvested using this kind of integrated approach.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular Tumoral , Humanos , Análise Serial de Proteínas , Mapeamento de Interação de Proteínas , ProteomaRESUMO
CobB is a bacterial NAD(+)-dependent protein deacetylase. Although progress has been made in functional studies of this protein in recent years, its substrates and biological functions are still largely unclear. Using proteome microarray technology, potential substrates of Escherichia coli CobB were screened and nine proteins were identified, including N-hydroxyarylamine O-acetyltransferase (NhoA). In vitro acetylation/deacetylation of NhoA was verified by western blotting and mass spectrometry, and two acetylated lysine residues were identified. Site-specific mutagenesis experiments showed that mutation of each acetylated lysine decreased the acetylation level of NhoA in vitro. Further analysis showed that variant NhoA proteins carrying substitutions at the two acetylated lysine residues are involved in both the O-acetyltransferase and N-acetyltransferase activity of NhoA. Structural analyses were also performed to explore the effects of the acetylated lysine residues on the activity of NhoA. These results suggest that reversible acetylation may play a role in the activity of Escherichia coli NhoA.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Processamento de Proteína Pós-Traducional , Sirtuínas/metabolismo , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Escherichia coli/química , Cinética , Lisina/análogos & derivados , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Análise Serial de Proteínas , Proteoma/metabolismo , Sirtuínas/químicaRESUMO
Both basic research and clinical medicine have urgent demands for highly efficient strategies to simultaneously identify many different DNA sequences within a single tube. Effective and simultaneous amplification of multiple target sequences is a prerequisite for any successful multiple nucleic acid detection method. Multiplex PCR is one of the best choices for this purpose. However, due to the intrinsic interference and competition among primer pairs in the same tube, multiple rounds of highly empirical optimization procedures are usually required to establish a successful multiplex PCR reaction. To address this challenge, we report here a universal multiplex PCR strategy that is capable of over 100-plex amplification using a specially designed microarray in which hydrophilic microwells are patterned on a hydrophobic chip. On such an array, primer pairs tagged with a universal sequence are physically separated in individual hydrophilic microwells on an otherwise hydrophobic chip, enabling many unique PCR reactions to be proceeded simultaneously during the first step of the procedure. The PCR products are then isolated and further amplified from the universal sequences, producing a sufficient amount of material for analysis by conventional gel electrophoresis or DNA microarray technology. This strategy is abbreviated as "MPH&HPM" for "Multiplex PCR on a Hydrophobically and Hydrophilically Patterned Microarray". The feasibility of this method is first demonstrated by a multiplex PCR reaction for the simultaneous detection of eleven pneumonia-causing pathogens. Further, we demonstrate the power of this strategy with a highly successful 116-plex PCR reaction that required only little prior optimization. The effectiveness of the MPH&HPM strategy with clinical samples is then illustrated with the detection of deleted exons of the Duchenne Muscular Dystrophy (DMD) gene, the results are in excellent agreement with the clinical records. Because of its generality, simplicity, flexibility, specificity and capacity of more than 100-plex amplification, the MPH&HPM strategy should have broad applications in both laboratory research and clinical applications when multiplex nucleic acid analysis is required.
Assuntos
Reação em Cadeia da Polimerase Multiplex , Análise de Sequência com Séries de Oligonucleotídeos , DNA/análise , Primers do DNA/química , DNA Bacteriano/análise , Éxons , Humanos , Interações Hidrofóbicas e Hidrofílicas , Distrofia Muscular de Duchenne/genética , Mycobacterium tuberculosis/genética , Pneumonia/microbiologiaRESUMO
OBJECTIVE: To explore and evaluate the combined conservative managements in the treatment of cervical chylous leakage. METHODS: Thirty nine cases of cervical chylous leakage from June 1992 to June 2008 were retrospectively analyzed in this hospital. All of the 39 cases were cured by treating with conservative individualized therapy, including the applying of diet with high calorie, high protein and low fat and fatty food should only contains medium-chain triglycerides, total parenteral nutrition, keep the balance of hydrogen and electrolyte and correct hypoproteinemia, local pressure dressing, high persistent vacuum drainage (-50 approximately -80 kPa) and/or somatostatin analogue. RESULTS: All the cases of chylous leakage happened 2nd to 5th days after the operation. Among the 39 cases, 7 were high flow (drainage>or=500 ml/d) chylous leakage, the amount of drainage reached as high as 1440 ml per day. The time of chylous leakage closure was 3 approximately 12 days, and the mean time was 7 days. No one experienced re-operation, wound hydrops or wound infection. CONCLUSIONS: The conservative individualized therapy may play a key role in the treatment of cervical chylous leakage.