RESUMO
Mycoplasma gallisepticum (MG) can induce persistent inflammatory damage to the tracheal mucosa of poultry and cause chronic respiratory diseases in chickens. To further investigate the mechanism of MG-induced injury to the tracheal mucosa, we used chick embryo tracheal organ culture (TOC) as a model to study the invasion and reproduction of MG, the effect of MG on tracheal morphology, and the potential factors that promote MG tissue invasion. The results showed that MG infection significantly damaged the tracheal epithelial structure and weakened tracheal epithelial barrier function; MG also increased the occurrence of bacterial displacement, with a significant (p < 0.05) increase in the bacterial load of the infected TOCs at 5 and 7 days post-infection. In addition, MG significantly (p < 0.05) increased the expression levels of inflammatory cytokines, such as TNF-α, interleukin-1ß (IL-1ß), and IL-6, and activated the NF-κB signalling pathway, leading to increased nuclear translocation of NF-κB p65. Simultaneously, the map kinase pathway (MAPK) was activated. This activation might be associated with increased myosin light chain (MLC) phosphorylation, which could lead to actin-myosin contraction and disruption of tight junction (TJ) protein function, potentially compromising epithelial barrier integrity and further catalysing MG migration into tissues. Overall, our results contribute to a better understanding of the interaction between MG and the host, provide insight into the mechanisms of damage to the tracheal mucosa induced by MG infection, and provide new insights into the possible pathways involved in Mycoplasma gallisepticum infection in vivo.
Assuntos
Infecções por Mycoplasma , NF-kappa B , Traqueia , Fator de Necrose Tumoral alfa , Animais , Embrião de Galinha , Mycoplasma gallisepticum , NF-kappa B/metabolismo , Traqueia/microbiologia , Fator de Necrose Tumoral alfa/metabolismo , Infecções por Mycoplasma/metabolismo , Infecções por Mycoplasma/patologiaRESUMO
Public-private partnerships (PPP), as an important model for collaboration between the public and private sectors, is an urgent and critical topic due to the serious financial losses of governments involved in transportation PPP projects in recent years. Current research focuses on the government subsidy model, in which the effective implementation of government subsidies relies on the design of incentives for stakeholder behavior. Although the positive externalities are strong, they are prone to the problem of "free riding," which leads to low project performance and challenges in compensating for the government's financial losses. Therefore, this study proposes a novel dynamic subsidy mechanism that can be adjusted based on actual changes in transportation demand and that is linked to project performance. We use evolutionary game theory to construct a two-party evolutionary game model of the government and social capital, focusing on the stability and influencing factors of these interactions. Our research unveils that reaching specific thresholds in both the incentive coefficient and benefit distribution ratio induces an "positive management-negative management" shift in the behavior of involved parties, leading to enhanced project outcomes. Notably, fluctuations in operational quality substantially enhance the efficiency of the active management of private sector, with no discernible impact on the subsidy efficiency of the government. Therefore, our study provides a theoretical framework for improving the revenue allocation and government subsidy mechanism, which has theoretical and practical implications for enhancing the effect of government incentives and improving the quality of operational social capital.
Assuntos
Meios de Transporte , Meios de Transporte/economia , Teoria dos Jogos , Parcerias Público-Privadas , Financiamento Governamental , Setor Privado , GovernoRESUMO
Helicity-resolved Raman spectroscopy (HRRS) can effectively distinguish the Raman modes of two-dimensional (2D) layered materials by phonon symmetry. In this paper, we systematically investigated the phonon helicity selection of basal and edge planes of MoS2 bulk by HRRS. We find that the symmetry of the crystal structure changes the helicity selection of the E1g, E1 2g, and A1g modes in the edge plane. The theoretical calculation results confirm that the E1 2g and A1g modes of the basal plane exhibit a perfect helicity exchange, and the helicity selections of the E1 2g and A1g modes of the edge plane are eliminated or weakened. Our study provides references for phonon helicity selection of 2D layered materials represented by MoS2.
RESUMO
OBJECTIVE: To investigate whether mouse epididymis-specific mRNAs Adam7 and Crisp1 can be delivered into N2a and TM4 cells, and to provide an experimental basis for exploring the function of epididymal mRNAs. METHODS: Using RT-PCR, we detected the presence of epididymis-specific genes (Adam7, Crisp1, Defb22, Wfdc2, and Wfdc9) in the testis, epididymis, epididymosome and sperm of adult male BALB/c mice as well as in the human testis, seminal vesicles and sperm. We isolated epididymosomes of BALB/c mice by low-speed centrifugation, filtration and ultracentrifugation, fluorescently labeled them by PKH26, co-incubated them for 1 hour with the N2a and TM4 cells after 24 hours of starvation culture, and observed whether they were fused with the N2a and TM4 cells and ingested using the epididymosomes without PKH26 labeling, PKH26 dye without epididymosomes, and non- epididymosome or -PKH26 dye as controls. Then we detected the epididymis-specific genes in the N2a and TM4 cells after 1-hour co-incubation by RT-PCR. RESULTS: Adam7 and Crisp1 were present in the mouse epididymis, epididymosomes and sperm, and in the human seminal vesicles and sperm as well, but not in the testes of either the mice or men. PKH26 and Hoechst33258 fluorescence double-labeling showed that the mouse epididymosomes were fused with the N2a and TM4 cells and ingested; RT-PCR revealed the mRNAs of Adam7 and Crisp1 in the N2a and TM4 cells after 1-hour co-incubation; and Western blot exhibited the CRISP1 protein in the N2a and TM4 cells incubated with epididymosomes. CONCLUSION: Epididymosomes can deliver epididymis-specific mRNAs Adam7 and Crisp1 into N2a and TM4 cells, where Crisp1 may be translated into proteins, though their function and significance need to be further studied.
Assuntos
Epididimo , Testículo , Masculino , Humanos , Camundongos , Animais , Testículo/metabolismo , Maturação do Esperma/genética , Sêmen , Espermatozoides/metabolismo , Proteína 2 do Domínio Central WAP de Quatro Dissulfetos/metabolismoRESUMO
RESEARCH HIGHLIGHTSMG infection causes a persistent inflammatory response by increasing the expression of immune response genes.The ERK-MLCK signalling pathway activated by MG infection regulates tight junction proteins in the tracheal mucosa.These data provide a basis for future prevention and treatment studies to control MG infection.
Assuntos
Infecções por Mycoplasma , Mycoplasma gallisepticum , Doenças das Aves Domésticas , Animais , Galinhas , Mucosa , Infecções por Mycoplasma/veterináriaRESUMO
BACKGROUND: Gynecologic cancers are among the most prevalent malignancies in China. Cervical and uterine cancer respectively account for the sixth and eighth highest incidence of cancer among Chinese women. Abdominal surgery is one of the important treatment methods for gynecological tumors. However, the tumor- and surgery-related symptom burden are not well studied owing to a lack of a standardized and validated assessment tool in the Chinese population. The study aimed to translate and validate the MD Anderson Symptom Inventory for measuring perioperative symptom burden in gynecologic cancer patients (MDASI-PeriOp-GYN) and examine the utility of the Chinese version of MDASI-PeriOp-GYN. METHODS: The MDASI-PeriOp-GYN was translated in a stepwise manner. First, two native speakers independently translated the 9 PeriOp-GYN symptom items. Then the nine items were translated back into English by two different bilingual translators. After discussion and revision, the four translators reached an agreement. Finally, the finalized Chinese version was administered to women with three common gynecologic cancer types (cervical, ovarian, and endometrial cancers) recruited from the gynecological oncology department of Sichuan Cancer Hospital & Institute between July and October 2019. The reliability and validity of the translated version were assessed. RESULTS: Overall, 324 women with gynecologic cancers were enrolled. Cronbach's α values were 0.826 and 0.735 for the symptom severity and interference scales, respectively. Test-retest reliability values were 0.885, 0.873, and 0.914 for symptom severity, PeriOp-GYN, and interference scales. Significant correlations were found between the MDASI-PeriOp-GYN-C and EORTC QLQ-C30 along with the QLQ-OV28 module (- 0.608-0.871, P < 0.001). Known-group validity was supported by significant differences in the scores of the four scales grouped by time intervals, surgery type, and functional status (all P < 0.01). CONCLUSIONS: The MDASI-PeriOp-GYN-C is a valid and reliable tool for measuring symptoms in Chinese patients undergoing surgery for gynecologic cancers. The tool could be used in clinical practice and clinical trials to instantly gather patients' health and quality of life data.
Assuntos
Neoplasias dos Genitais Femininos , Qualidade de Vida , China , Feminino , Neoplasias dos Genitais Femininos/diagnóstico , Neoplasias dos Genitais Femininos/cirurgia , Humanos , Psicometria , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Inquéritos e QuestionáriosRESUMO
Copper complexes of monodentate phosphoramidites efficiently promote asymmetric arylation of N-azaaryl aldimines with arylboroxines. DFT calculations and experiments support an elementary step of 1,4-insertion in the reaction pathway, a step in which an aryl-copper species adds directly across four atoms of C=N-C=N in the N-azaaryl aldimines.
RESUMO
BACKGROUND Recent studies have shown that increased mobilization of Long Interspersed Nuclear Elements-1 (L1) can promote the pathophysiology of multiple neurological diseases. However, its role in Huntington's disease (HD) remains unknown. MATERIAL AND METHODS R6/2 mice - a common mouse model of HD - were used to evaluate changes in L1 mobilization. Pyrosequencing was used to evaluate methylation content changes. L1-ORF1 and L1-ORF2 expression analysis were evaluated by RT-PCR and immunoblotting. Changes in pro-survival signaling were evaluated by L1-ORF overexpression studies and validated in the mouse model by immunohistochemistry and immunoblotting. RESULTS We found an increased mobilization of L1 elements in the caudate genome of R6/2 mice (p<0.05) - a common mouse model of HD - but not in wild-type mice. Subsequent pyrosequencing and expression analysis showed that the L1 elements were hypomethylated and their respective ORFs were overexpressed in the affected tissues. In addition, a significant decrease in the pro-survival proteins such as the phosphoproteins of AKT target proteins, mTORC1 activity, and AMPK alpha levels was observed with the increase in the expression L1-ORF2. CONCLUSIONS These findings indicate that hyperactive retrotransposition of L1 triggers a downstream signaling pathway affecting the neuronal survival pathways via downregulation of mTORC1 activity and AMPKalpha and increasing apoptosis in neurons.
Assuntos
Doença de Huntington/genética , Elementos Nucleotídeos Longos e Dispersos/fisiologia , Animais , Metilação de DNA , Modelos Animais de Doenças , Progressão da Doença , Endonucleases/análise , Endonucleases/genética , Feminino , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Fosfoproteínas/genética , DNA Polimerase Dirigida por RNA/análise , DNA Polimerase Dirigida por RNA/genética , Retroelementos/genética , Transdução de Sinais , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
Cobalt-diphosphine catalysts have been found to promote intramolecular reactions between a vinylcyclopropane and an alkyne to selectively afford either the [5+2] cycloaddition product or the homo-ene reaction product under solvent control. The former product is exclusively formed in noncoordinating 1,2-dichloroethane, whereas the latter is dominant in coordinating solvents, such as acetonitrile and dimethylacetamide. Furthermore, a highly enantioselective variant of the homo-ene reaction afforded chiral tetrahydrofuran, pyrrolidine, and cyclopentane derivatives bearing 1,3-diene and alkylidene substituents.
RESUMO
Copper complexes of phosphoramidites efficiently catalyzed asymmetric addition of arylboron reagents to acyclic enones. Importantly, rare 1,4-insertion of arylcopper(I) was identified which led directly to O-bound copper enolates. The new mechanism is fundamentally different from classical oxidative addition/reductive elimination of organocopper(I) on enones.
RESUMO
BACKGROUND: Photodynamic therapy (PDT) is becoming a promising therapeutic modality for hematological malignancies. Hypericin is a natural photosensitizer possessing anti-depressant, anti-virus and anti-cancer activities. The present study was designed to explore the effect and mechanism of hypericin-mediated PDT on the mouse multiple myeloma (MM) cells in vitro. METHODS: The mouse myeloma SP2/0 cells were incubed with different concentrations of hypericin and then illuminated with different light doses. The inhibitory effect of hypericin-mediated PDT on tumor cell proliferation was assayed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method. The apoptosis related morphological changes of SP2/0 cells were observed by microscopy. The biochemical hallmarks of apoptosis such as DNA fragments, mitochondrial membrane potential changes were assessed. The expression of apoptosis related proteins were investigated by western blotting. RESULTS: Hypericin-mediated PDT induced the proliferation inhibition and apoptosis of tumor cells in a dose dependent manner. Tumor cells showed obvious morphological changes of apoptosis and necrosis and DNA fragmentation after treated by hypericin mediated PDT (0.025 ~ 0.05 µM). The mitochondria membrane potential in SP2/0 cells was decreased significantly after incubated with the 0.025 µM and 0.5 µM hypericin (P < 0.05). The expression level of caspase-3 was decreased, while caspase activity was elevated with the increasing drug dosage. The apoptosis of SP2/0 cells was blocked by a pan-caspase inhibitor Z-VAD-FMK and caspase-3 inhibitor Ac-DEVD-CHO. CONCLUSION: Hypericin-mediated PDT induced apoptosis mainly dependent on caspase related pathways. Hypericin-mediated PDT may be a potential and alternative therapy for MM.
RESUMO
The asymmetric intermolecular Heck reaction has been limited to aryl and vinyl triflates. Herein, we extend the reaction to aryl and vinyl bromides. Various cyclic olefins coupled with high enantioselectivity. Only bisphosphine oxides on a spiro backbone formed highly stereoselective Pd catalysts. The use of alcoholic solvents and alkylammonium salts were essential to promote halide dissociation from neutral arylpalladium complexes.
RESUMO
The brain parenchymal Langerhans cell histiocytosis (LCH) without systemic disease or lytic skull lesions is extremely rare. We report a 23-year-old male presenting with new onset 1 hour seizure with loss of consciousness 20 days prior to admission, and recurrent seizure 2 weeks later. Brain magnetic resonance imaging (MRI) showed an irregularly mass with enhancement involving the right frontal lobe. Microscopically, the lesion was characterized by sheets of Langerhans cells in addition to reactive inflammatory elements. Immunohistochemically, Langerhans cells were positive for Langerin, CD1a and S-100 protein. The patient received no chemotherapy or radiotherapy after surgery. After 24 months of follow-up, no recurrence or other systemic lesions were observed. Although there is no standard treatment for solitary cerebral LCH, the prognosis generally appears to be good.
RESUMO
The influence of RG-I domains on high methoxyl pectin (HMP) sugar-acid gel properties has rarely been reported. In our work, HMP was modified by enzymatic de-esterification and degradation of RG-I domains to compare and analyze the relationship between the structure and final sugar-acid gel properties. The results showed that the degree of esterification (DE) of REP (pectin degraded by rhamnosidase) and GEP (pectin debranched by galactosidase) was the same as that of untreated HMP, whereas the DE of PMEP (pectin de-esterified by pectin methyl esterase) decreased from 59.63 % to 54.69 %. The monosaccharide composition suggested no significant changes in the HG and RG-I structural domains of PMEP. In contrast, the percentage of RG-I structural domains of REP and GEP dropped from 37 % to about 28 %, accompanied by a reduction in the proportion of the RG-I backbones and side chains. The rheological characterization of sugar-acid gels demonstrated an enhanced gel grade for PMEP and a weakened one for REP and GEP. Moreover, we constructed a correlation relationship between the fine structure of pectin and the properties of the sugar-acid gels, confirming the critical contribution of the RG-I region (especially the neutral sugar side chains) to the HMP sugar-acid gels.
Assuntos
Pectinas , Açúcares , Pectinas/química , Esterificação , Géis/químicaRESUMO
Co-solutes such as sucrose and sugar alcohol play a significant part in low methoxyl pectin (LMP) gelation. To explore their gelation mechanism, we investigated the gelation behavior of LMP in the presence of erythritol and sucrose with Ca2+. Results revealed that the introduction of erythritol and sucrose improved the hardness of the gels, fixed more free water, accelerated the rate of gel structuring, and enhanced the gel strength. FT-IR confirmed the reinforced hydrogen bonding and hydrophobic forces between the pectin chains after introducing co-solutes. And it could be observed clearly by SEM that the cross-linking density of gel network enhanced with co-solutes. Furthermore, gel disruption experiments suggested the presence of ionic interaction, hydrogen bonding, and hydrophobic forces in LMP gels. Finally, we concluded that the egg-box regions cross-linked only by LMP and Ca2+ were too weak to form a stable gel network structure. Adding co-solutes could increase the amount of cross-linking between pectin chains and enlarge the cross-linking zones, which favored the formation of a dense gel network by more hydrogen bonding and hydrophobic forces. Sucrose gels had superior physicochemical properties and microstructure than erythritol gels due to sucrose's excellent hydration capacity and chemical structure characteristics.
Assuntos
Eritritol , Géis , Pectinas , Sacarose , Pectinas/química , Eritritol/química , Sacarose/química , Géis/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cálcio/química , Água/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Objectives. Construction worker safety behavior (CWSB) may be affected by a confluence of multilevel and interrelated factors. Cultivating and maintaining CWSB is vital for improving construction safety. Current studies focus on organization-level or individual-level CWSB antecedents. However, few studies have examined the influence of psychological factors on CWSB, thereby reducing the joint effects of multilevel factors on CWSB. Methods. To determine effective strategies for strengthening CWSB, this study adopted the Bayesian network technique to explore the interrelationships between CWSB and its antecedent factors. A Bayesian belief network model was developed and trained with data collected from Chinese construction workers, which connected organizational, individual and psychological factors with CWSB. Results. According to the sensitivity analysis, safety knowledge, safety climate and psychological capital are the three most significant influencing factors for CWSB. A combined strategy that enhances safety knowledge, safety climate and communication competence simultaneously is the most effective option for strengthening CWSB. The validation and robustness of the network showed good accuracy for safety behavior judgment. Conclusion. This study proposes an alternative way to improve safety behavior by identifying its interactive causes and illustrates the importance of initiating systematic safety measures, which may help to mitigate the problem of safety plateau.
RESUMO
OBJECTIVE: The objective of this study was to investigate the impact of Doxorubicin, Epirubicin, and Liposomal Doxorubicin (Anthracycline) on cardiac function in osteosarcoma patients and analyze the factors influencing this effect. METHODS: A retrospective study was conducted on 165 osteosarcoma patients admitted to our hospital from January 2020 to December 2022. Based on the chemotherapy regimen, the patients were divided into two groups: the control group (n = 62) treated with Cisplatin and cyclophosphamide, and the observation group (n = 103) treated with Doxorubicin, Epirubicin, and Liposomal Doxorubicin (Anthracycline). The general records of both groups were analyzed, and left ventricular ejection fraction (LVEF) was evaluated through echocardiography before and after chemotherapy. Blood cTnT and CK-MB levels were measured using immunoluminescence. The incidence of adverse reactions during chemotherapy was also analyzed. Univariate analysis was performed to identify patients with cardiotoxic events, and multiple logistic regression analysis was done to study the effects of Doxorubicin, Epirubicin, Liposomal Doxorubicin, and their dosages on cardiotoxicity in patients. RESULTS: The general records between the two groups showed no significant differences (P > 0.05). However, at the fourth cycle of chemotherapy, the observation group exhibited a lower LVEF (P < 0.05), and a higher percentage of LVEF decrease compared to the control group (P < 0.05). Moreover, the observation group had higher levels of blood cTnT and CK-MB (P < 0.05). The incidence of cardiotoxicity in the observation group was also higher (P < 0.05), but no significant differences were seen in other adverse reaction rates (P > 0.05). The occurrence of cardiotoxicity was found to be related to the choice and dosage of chemotherapy drugs (P < 0.05), but not significantly correlated with age, sex, and mediastinal irradiation in patients (P > 0.05). Furthermore, the use of Doxorubicin, Epirubicin, and Liposomal Doxorubicin in chemotherapy, as well as an increase in their dosages, was found to elevate the risk of cardiotoxicity in osteosarcoma patients (P < 0.05). However, age, sex, and mediastinal radiation were not significantly associated with cardiotoxicity in osteosarcoma patients (P > 0.05). CONCLUSION: We demonstrated that Doxorubicin, Epirubicin, Liposomal Doxorubicin (Anthracycline), and other drugs adversely affected cardiac function in osteosarcoma patients, increasing the risk of cardiac toxicity. Therefore, close monitoring of cardiac function during chemotherapy is crucial, and timely adjustments to the chemotherapy regimen are necessary. In addition, rational control of drug selection and dosage is essential to minimize the occurrence of cardiac toxicity.
Assuntos
Neoplasias Ósseas , Cardiotoxicidade , Doxorrubicina , Epirubicina , Osteossarcoma , Humanos , Osteossarcoma/tratamento farmacológico , Epirubicina/efeitos adversos , Epirubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Doxorrubicina/análogos & derivados , Feminino , Masculino , Estudos Retrospectivos , Adulto , Adulto Jovem , Neoplasias Ósseas/tratamento farmacológico , Cardiotoxicidade/etiologia , Adolescente , Volume Sistólico/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Função Ventricular Esquerda/efeitos dos fármacos , Antibióticos Antineoplásicos/efeitos adversos , Antibióticos Antineoplásicos/uso terapêutico , Ecocardiografia , Troponina T/sangue , Creatina Quinase Forma MB/sangue , Ciclofosfamida/efeitos adversos , Ciclofosfamida/administração & dosagem , Criança , Cisplatino/efeitos adversos , Cisplatino/administração & dosagem , PolietilenoglicóisRESUMO
BACKGROUND: DNA methylation analysis is useful for investigation of male fertility in mammals, whereas the reliance on tissues limits the research on human. We have previously found the presence of high concentration of cell-free seminal DNA (cfsDNA) in human semen. We proposed that some testis and epididymis-specific methylated promoters could be detected in human cfsDNA, and thus hold promise as noninvasive epigenetic biomarkers for male infertility, of which most cases are caused by defects in testicular sperm production or epididymal sperm maturation. RESULTS: The ejaculate of successfully vasectomized men does not contain any secretion from testis and epididymis. Here we compared genome-wide promoter methylation profiles in cfsDNA between health donors and post-vasectomy men. Promoters of 367 testis and epididymis-specific hypomethylated genes and 134 hypermethylated genes were identified. Subsequent validation by Methyl-DNA immunoprecipitation and MethyLight analysis confirmed the result of promoter microarray. Gene Ontology analysis revealed many genes involved in male reproduction. CONCLUSION: We detected the testis and epididymis-specific methylated promoters in human cfsDNA, which may be used for noninvasive epigenetic biomarkers for the study and diagnosis of male infertility.
Assuntos
Metilação de DNA , Epididimo/metabolismo , Infertilidade Masculina/diagnóstico , Regiões Promotoras Genéticas/fisiologia , Sêmen/química , Testículo/metabolismo , Biomarcadores/análise , DNA/metabolismo , Epigênese Genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Anotação de Sequência Molecular , Regiões Promotoras Genéticas/genética , Maturação do Esperma , VasectomiaRESUMO
Cell-free DNA, also referred to as extracellular DNA, has been detected in many kinds of human body fluids, including blood plasma, urine, cerebrospinal fluid, bronchoalveolar lavage fluid, amniotic fluid, and seminal plasma. At present, cell-free DNA has been reported widely as promising noninvasive biomarkers for disease diagnosis and research. Recent years have witnessed some progress in the studies of the general characteristics of cell-free DNA, such as its concentration, extent of molecular weight, origin and existing forms, as well as in its clinical application. Cell-free seminal DNA has been proposed as promising noninvasive biomarkers for the studies and diagnosis of male idiopathic infertility, and the early diagnosis, treatment evaluation and outcome prediction of testicular germ cell tumors and prostatic cancer. This review summarizes the general characteristics and biological functions of cell-free DNA, and outlines the research status and application perspective of cell-free seminal DNA.
Assuntos
DNA , Infertilidade Masculina/diagnóstico , Sêmen/química , Biomarcadores/análise , Humanos , Infertilidade Masculina/genética , MasculinoRESUMO
OBJECTIVE: To establish a method of methyl-DNA immunoprecipitation (MeDIP)-real time quantitative PCR (qPCR) for detecting the promoter methylation level in cell-free seminal DNA (cfsDNA). METHODS: We obtained cfsDNA samples from 6 normozoospermia men (the NZ group) and 6 post-vasectomy patients (the PV group), and mixed the samples from different individuals of each group, respectively. Then we made DNA fragments by ultrasonication, separated the methylated DNA fragments by MeDIP, and determined the methylation level of the promoters in cfsDNA by qPCR. RESULTS: The methylation levels of the promoters PRAME, PEG10, MORC1, GML, HOXA5, DNMT3L, SNURF, MSH4, DAZ1 and CLPB were 14.93, 2.64, 0.69, 2.66, 17.50, 21.10, 5.98, 2.28, 13.50 and 3.86%, respectively, in the NZ group, obviously lower than 121.25, 73.62, 16.25, 42.90, 76.74, 112.40, 59.79, 25.85, 91.90 and 64.53% in the PV group. The results of MeDIP-qPCR for the methylation of PRAME, MORC1, GML, HOXA5, DNMT3L, SNURF, MSH4 and DAZ1 were coincident with the results of genome-wide promoter methylation microarray. CONCLUSION: MeDIP-qPCR can quantitatively measure the promoter methylation level in cfsDNA, and effectively determine the testis- and epididymis-specific methylated promoters in human semen.