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OBJECTIVE: Endometrial carcinoma (EC) is a prevalent gynecological malignancy characterized by increasing incidence and mortality rates. This underscores the critical need for novel therapeutic targets. One such potential target is cell division cycle 20 (CDC20), which has been implicated in oncogenesis. This study investigated the effect of the CDC20 inhibitor Apcin on EC and elucidated the underlying mechanism involved. METHODS: The effects of Apcin on EC cell proliferation, apoptosis, and the cell cycle were evaluated using CCK8 assays and flow cytometry. RNA sequencing (RNA-seq) was subsequently conducted to explore the underlying molecular mechanism, and Western blotting and coimmunoprecipitation were subsequently performed to validate the results. Animal studies were performed to evaluate the antitumor effects in vivo. Bioinformatics analysis was also conducted to identify CDC20 as a potential therapeutic target in EC. RESULTS: Treatment with Apcin inhibited proliferation and induced apoptosis in EC cells, resulting in cell cycle arrest. Pathways associated with apoptosis and the cell cycle were activated following treatment with Apcin. Notably, Apcin treatment led to the upregulation of the cell cycle regulator p21, which was verified to interact with CDC20 and consequently decrease the expression of downstream cyclins in EC cells. In vivo experiments confirmed that Apcin treatment significantly impeded tumor growth. Higher CDC20 expression was observed in EC tissue than in nonmalignant tissue, and increased CDC20 expression in EC patients was associated with shorter overall survival and progress free interval. CONCLUSION: CDC20 is a novel molecular target in EC, and Apcin could be developed as a candidate antitumor drug for EC treatment.
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Apoptose , Proteínas Cdc20 , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21 , Neoplasias do Endométrio , Feminino , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/patologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Humanos , Apoptose/efeitos dos fármacos , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Camundongos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Proliferação de Células/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antineoplásicos/farmacologia , Camundongos NusRESUMO
Directed self-assembly (DSA) lithography has demonstrated significant potential in fabricating integrated circuits. However, DSA encounters limited processing windows due to the requirement for precise matching between the period of block copolymers (BCPs) and graphoepitaxy templates. We propose a binary BCP/homopolymer blending strategy to manipulate the self-assembly behavior and the processing window of graphoepitaxy DSA in contact hole shrinking. By carefully tailoring the blending rates of poly(methyl methacrylate) (PMMA) with different molecular weights in cylindrical polystyrene-b-poly(methyl methacrylate) (PS-b-PMMA), we manipulate the period and morphology of BCP/homopolymer self-assembly. Specifically, we employ BCP/homopolymer blending to fine-tune the critical dimension (CD) of contact holes with PS-affined topographical templates. Subsequent pattern transferring is achieved by selectively etching defect-free shrinkable cylinders as hard masks. Furthermore, self-consistent field theory (SCFT) simulation was employed to explore the self-assembly of BCP/homopolymer blending in confined cylindrical space and the results were in good consistency with the experimental results.
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The H9N2 avian influenza virus causes reduced production performance and immunosuppression in chickens. The chicken yolk sac immunoglobulins (IgY) receptor (FcRY) transports from the yolk into the embryo, providing offspring with passive immunity to infection against common poultry pathogens. FcRY is expressed in many tissues/organs of the chicken; however, there are no reports investigating FcRY expression in chicken macrophage cells, and how H9N2-infected HD11 cells (a chicken macrophage-like cell line) regulate FcRY expression remains uninvestigated. This study used the H9N2 virus as a model pathogen to explore the regulation of FcRY expression in avian macrophages. FcRY was highly expressed in HD11 cells, as shown by reverse transcription polymerase chain reactions, and indirect immunofluorescence indicated that FcRY was widely expressed in HD11 cells. HD11 cells infected with live H9N2 virus exhibited downregulated FcRY expression. Transfection of eukaryotic expression plasmids encoding each viral protein of H9N2 into HD11 cells revealed that nonstructural protein (NS1) and matrix protein (M1) downregulated FcRY expression. In addition, the use of a c-jun N-terminal kinase (JNK) activator inhibited the expression of FcRY, while a JNK inhibitor antagonized the downregulation of FcRY expression by live H9N2 virus, NS1 and M1 proteins. Finally, a dual luciferase reporter system showed that both the M1 protein and the transcription factor c-jun inhibited FcRY expression at the transcriptional level. Taken together, the transcription factor c-jun was a negative regulator of FcRY, while the live H9N2 virus, NS1, and M1 proteins downregulated the FcRY expression through activating the JNK signaling pathway. This provides an experimental basis for a novel mechanism of immunosuppression in the H9N2 avian influenza virus.
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Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Animais , Galinhas/metabolismo , Vírus da Influenza A Subtipo H9N2/fisiologia , Sistema de Sinalização das MAP Quinases , Linhagem Celular , Macrófagos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
INTRODUCTION: This study aimed to assess state-trait anxiety level changes in Chinese patients with dentofacial discrepancies before and after orthognathic surgery and to explore the feasibility of developing a reference index for the preoperative screening of postoperative patients with high anxiety. METHODS: A total of 96 Chinese patients with dentofacial discrepancies who underwent orthognathic surgery were included in this study. Data were collected before orthognathic surgery and at 2 weeks (T2), 3 months, and 6 months (T4) after surgery using the State-Trait Anxiety Inventory. Receiver operating characteristic and linear regression analyses were performed to screen for preoperative indicators of postoperative high-state anxiety. RESULTS: State-trait anxiety levels in patients with dentofacial discrepancies decreased after surgery (F = 18.95, P <0.01; F = 6.90, P <0.01). Trait Anxiety Inventory can be used to screen patients with high-state anxiety from T2 to T4 (area under cover 95% confidence interval: T2, 0.74 [0.62-0.86]; 3 months, 0.79 [0.69-0.90]; T4, 0.77 [0.66-0.87], P <0.01), corresponding to cutoff values of 48.5, 46.5, and 45.5, respectively. CONCLUSIONS: All participants' state-trait anxiety levels improved after surgery compared with their preoperative levels. Preoperative trait anxiety levels can be used as a reference indicator to screen patients who may have high-state anxiety levels after orthognathic surgery. The creation of a screening scale will assist health care professionals to more pertinently help patients with high anxiety.
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Ansiedade , Procedimentos Cirúrgicos Ortognáticos , Humanos , Feminino , Masculino , Estudos Longitudinais , Procedimentos Cirúrgicos Ortognáticos/psicologia , Adulto , Ansiedade/psicologia , Adulto Jovem , AdolescenteRESUMO
OBJECTIVE: To establish and assess the precision of pre-surgical condyle position planning using mandibular movement trajectory data for orthognathic surgery. METHODS: Skull data from large-field cone beam computed tomography (CBCT) and dental oral scan data were imported into IVSPlan 1.0.25 software for 3D reconstruction and fusion, creating 3D models of the maxilla and mandible. Trajectory data of mandibular movement were collected using a mandibular motion recorder, and the data were integrated with the jaw models within the software. Subsequently, three-dimensional trajectories of the condyle were obtained through matrix transformations, rendering them visually accessible. A senior oral and maxillofacial surgeon with experience in both diagnosis and treatment of temporomandibular joint disease and orthognathic surgery selected the appropriate condyle position using the condyle movement trajectory interface. During surgical design, the mobile mandibular proximal segment was positioned accordingly. Routine orthognathic surgical planning was completed by determining the location of the mandibular distal segment, which was based on occlusal relationships with maxilla and facial aesthetics. A virtual mandible model was created by integrating data from the proximal and distal segment bone. Subsequently, a solid model was generated through rapid prototyping. The titanium plate was pre-shaped on the mandibular model, and the screw hole positions were determined to design a condylar positioning guide device. In accordance with the surgical plan, orthognathic surgery was performed, involving mandibular bilateral sagittal split ramus osteotomy (SSRO). The distal segment of the mandible was correctly aligned intermaxillary, while the proximal bone segment was positioned using the condylar positioning guide device and the pre-shaped titanium plate. The accuracy of this procedure was assessed in a study involving 10 patients with skeletal class â ¡ malocclusion. Preoperative condyle location planning and intraoperative positioning were executed using the aforementioned techniques. CBCT data were collected both before the surgery and 2 weeks after the procedure, and the root mean square (RMS) distance between the preope-rative design position and the actual postoperative condyle position was analyzed. RESULTS: The RMS of the condyle surface distance measured was (1.59±0.36) mm (95%CI: 1.35-1.70 mm). This value was found to be significantly less than 2 mm threshold recommended by the expert consensus (P < 0.05). CONCLUSION: The mandibular trajectory may play a guiding role in determining the position of the mandibular proximal segment including the condyle in the orthognathic surgery. Through the use of a condylar positioning guide device and pre-shaped titanium plates, the condyle positioning can be personalized and customized with clinically acceptable accuracy.
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Cirurgia Ortognática , Procedimentos Cirúrgicos Ortognáticos , Humanos , Côndilo Mandibular/diagnóstico por imagem , Côndilo Mandibular/cirurgia , Titânio , Mandíbula , Procedimentos Cirúrgicos Ortognáticos/métodos , Osteotomia Sagital do Ramo Mandibular/métodosRESUMO
The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m6A) plays an essential role in regulating various biological processes. However, the function of m6A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m6A methyltransferase METTL3 or the m6A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m6A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m6A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m6A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m6A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m6A modification in restricting L1 retrotransposition.
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Azoospermia , Retroelementos , Masculino , Animais , Humanos , Retroelementos/genética , RNA , Azoospermia/genética , Diferenciação Celular/genética , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Mensageiro/genética , Mamíferos/metabolismoRESUMO
BACKGROUND: Small extracellular vesicles (EVs), exemplified by exosomes, mediate intercellular communication by transporting proteins, mRNAs, and miRNAs. Post-translational modifications are involved in controlling small EV secretion process. However, whether palmitoylation regulates small EV secretion, remains largely unexplored. METHODS: Vacuole Membrane Protein 1 (VMP1) was testified to be S-palmitoylated by Palmitoylation assays. VMP1 mutant plasmids were constructed to screen out the exact palmitoylation sites. Small EVs were isolated, identified and compared between wild-type VMP1 or mutant VMP1 transfected cells. Electron microscope and immunofluorescence were used to detect multivesicular body (MVB) number and morphology change when VMP1 was mutated. Immunoprecipitation and Mass spectrum were adopted to identify the protein that interacted with palmitoylated VMP1, while knock down experiment was used to explore the function of targeted protein ALIX. Taking human Sertoli cells (SCs) and human spermatogonial stem cell like cells (SSCLCs) as a model of intercellular communication, SSCLC maintenance was detected by flow cytometry and qPCR at 12 days of differentiation. In vivo, mouse model was established by intraperitoneal injection with palmitoylation inhibitor, 2-bromopalmitate (2BP) for 3 months. RESULTS: VMP1 was identified to be palmitoylated at cysteine 263,278 by ZDHHC3. Specifically, palmitoylation of VMP1 regulated its subcellular location and enhanced the amount of small EV secretion. Mutation of VMP1 palmitoylation sites interfered with the morphology and biogenesis of MVBs through suppressing intraluminal vesicle formation. Furthermore, inhibition of VMP1 palmitoylation impeded small EV secretion by affecting the interaction of VMP1 with ALIX, an accessory protein of the ESCRT machinery. Taking SCs and SSCLCs as a model of intercellular communication, we discovered VMP1 palmitoylation in SCs was vital to the growth status of SSCLCs in a co-culture system. Inhibition of VMP1 palmitoylation caused low self-maintenance, increased apoptosis, and decreased proliferation rate of SSCLCs. In vivo, intraperitoneal injection of 2BP inhibited VMP1 palmitoylation and exosomal marker expression in mouse testes, which were closely associated with the level of spermatogenic cell apoptosis and proliferation. CONCLUSIONS: Our study revealed a novel mechanism for small EV secretion regulated by VMP1 palmitoylation in Sertoli cells, and demonstrated its pivotal role in intercellular communication and SSC niche.
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Complexos Endossomais de Distribuição Requeridos para Transporte , Vesículas Extracelulares , Lipoilação , Proteínas de Membrana , Animais , Humanos , Camundongos , Comunicação Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Vesículas Extracelulares/metabolismo , Proteínas de Membrana/metabolismo , Vacúolos/metabolismoRESUMO
OBJECTIVE: In this study, the uptake characteristics of [18F]fibroblast activation protein inhibitor (FAPI) molecular imaging probe were investigated in acute radiation pneumonia and lung cancer xenografted mice before and after radiation to assess the future applicability of [18F]FAPI positron emission tomography/computed tomography (PET/CT) imaging in early radiotherapy response. METHODS: Initially, the biodistribution of [18F]FAPI tracer in vivo were studied in healthy mice at each time-point. A comparison of [18F]FAPI and [18F]fluorodeoxyglucose (FDG) PET/CT imaging efficacy in normal ICR, LLC tumor-bearing mice was evaluated. A radiation pneumonia model was then investigated using a gamma counter, small animal PET/CT, and autoradiography. The uptake properties of [18F]FAPI in lung cancer and acute radiation pneumonia were investigated using autoradiography and PET/CT imaging in mice. RESULTS: The tumor area was visible in [18F]FAPI imaging and the tracer was swiftly eliminated from normal tissues and organs. There was a significant increase of [18F]FDG absorption in lung tissue after radiotherapy compared to before radiotherapy, but no significant difference of [18F]FAPI uptake under the same condition. Furthermore, both the LLC tumor volume and the expression of FAP-É decreased after thorax irradiation. Correspondingly, there was no notable [18F]FAPI uptake after irradiation, but there was an increase of [18F]FDG uptake in malignancies and lungs. CONCLUSIONS: The background uptake of [18F]FAPI is negligible. Moreover, the uptake of [18F]FAPI may not be affected by acute radiation pneumonitis compared to [18F]FDG, which may be used to more accurately evaluate early radiotherapy response of lung cancer with acute radiation pneumonia.
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Neoplasias Pulmonares , Quinolinas , Pneumonite por Radiação , Animais , Camundongos , Camundongos Endogâmicos ICR , Pneumonite por Radiação/diagnóstico por imagem , Fluordesoxiglucose F18 , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Distribuição Tecidual , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/radioterapia , Modelos Animais de Doenças , Radioisótopos de GálioRESUMO
BACKGROUND: The aim of this study was to evaluate the efficacy of fluorine 18 (18F) labeled fibroblast activation protein inhibitor (FAPI) in identifying mediastinal and hilar lymph node metastases and to develop a model to quantitatively and repeatedly identify lymph node status. METHODS: Twenty-seven patients with 137 lymph nodes were identified by two PET/CT images. The sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of lymph node status were analyzed, and the optimal cut-off value was identified by ROC analysis. RESULTS: The SUVmax of metastatic lymph nodes on 18F-FAPI was higher than that on 18F-FDG PET/CT (10.87 ± 7.29 vs 6.08 ± 5.37, p < 0.001). 18F-FAPI presented much greater lymph node detection sensitivity, specificity, accuracy, PPV and NPV than 18F-FDG PET/CT (84% vs. 71%; 92% vs. 67%; 90% vs. 69%, 84% vs. 52%, and 92% vs. 83%, respectively). Additionally, the diagnostic effectiveness of 18F-FAPI in small lymph nodes was greater than that of 18F-FDG PET/CT (specificity: 96% vs. 72%; accuracy: 93% vs. 73%; PPV: 77% vs. 33%, respectively). Notably, the optimal cut-off value for specificity and PPV of 18F-FAPI SUVmax was 5.3; the optimal cut-off value for sensitivity and NPV was 2.5. CONCLUSION: 18F-FAPI showed promising diagnostic efficacy in metastatic mediastinal and hilar lymph nodes from lung cancer patients, with a higher SUVmax, especially in small metastatic nodes, compared with 18F-FDG. In addition, this exploratory work recommended optimal SUVmax cutoff values to distinguish between nonmetastatic and metastatic lymph nodes, thereby advancing the development of image-guided radiation. Trial registration ClinicalTrials.gov identifier: ChiCTR2000036091.
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Neoplasias Pulmonares , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Humanos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Fluordesoxiglucose F18 , Compostos Radiofarmacêuticos , Linfonodos/diagnóstico por imagem , Linfonodos/patologiaRESUMO
As a core transcriptional factor regulating pluripotency, Krüppel-like factor 4 (KLF4) has gained much attention in the field of stem cells during the past decades. However, few research have focused on the function of KLF4 during human primordial germ cell (PGC) specification. Here, we induced human PGC-like cells (hPGCLCs) from human embryonic stem cells (hESCs) and the derived hPGCLCs upregulated PGC-related genes, like SOX17, BLIMP1, TFAP2C, NANOS3, and the naïve pluripotency gene KLF4. The KLF4-knockout hESCs formed typical multicellular colonies with clear borders, expressed pluripotency genes, such as NANOG, OCT4, and SOX2, and exhibited no differences in proliferation capacity compared with wild type hESCs. Notably, KLF4 deletion in hESCs did not influence the induction of PGCLCs in vitro. In contrast, overexpression of KLF4 during PGC induction process inhibited the efficiency of PGCLC formation from hESCs in vitro. Overexpression of KLF4 may regenerate the naïve ground state in hESCs and results in repression for PGC specification. Thus, KLF4 could be a downstream target of human PGC program and the upregulation of KLF4 is prepared for late stage of germline development.
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Células-Tronco Embrionárias Humanas , Fator 4 Semelhante a Kruppel , Humanos , Diferenciação Celular , Genes Homeobox , Células Germinativas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Fator 4 Semelhante a Kruppel/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The effects of pomegranate peel on the growth performance, intestinal morphology, and the cecal microbial community were investigated in broilers challenged with avian pathogenic Escherichia coli (APEC) O78. A total of 240 one-day-old chicks (120 males and 120 females) were randomly and evenly allotted into 4 treatment groups (each with 6 biological replicates each of 10 chicks), i.e., negative control (NC), positive control (PC), and 2 experimental groups treated with 0.2% fermented pomegranate peel (FP) and 0.2% unfermented pomegranate peel (UFP), respectively, with PC, FP, and UFP groups challenged with APEC O78 (5 × 108 CFU) on day 14. Results showed that the challenge of APEC O78 decreased the body weight (BW) and average daily gain (ADG) of broilers from 1 to 28 d (P < 0.01). These broilers exhibited more pathological conditions in the heart and liver and higher mortality rates in 28 d compared to the NC group. Diet supplemented with pomegranate peel (either fermented or unfermented) significantly increased BW, ADG, and the villus height/crypt depth ratio (VCR) of small intestine in 28 d compared to the NC group (P < 0.05). Results of the taxonomic structure of the gut microbiota showed that compared to the NC group, the APEC challenge significantly decreased the relative abundance of Bacteroidetes and increased the relative abundance of Firmicutes (P < 0.01). Compared to the PC group, the relative abundance of Ruminococcus_torques_group in FP group was increased, while the relative abundance of Alistipes was decreased. In summary, our study showed that the dietary supplementation of pomegranate peel could maintain the intestinal microbiota at a state favorable to the host, effectively reduce the abnormal changes in the taxonomic structure of the intestinal microbiota, and improve the growth performance in broilers treated with APEC.
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Infecções por Escherichia coli , Microbioma Gastrointestinal , Punica granatum , Probióticos , Masculino , Animais , Escherichia coli , Galinhas , Probióticos/farmacologia , Infecções por Escherichia coli/veterinária , Dieta/veterinária , Ração Animal/análiseRESUMO
INTRODUCTION: This study aimed to compare postsurgical stability between conventional (CSA) and surgery-first (SFA) approaches and investigate its prognostic factors in patients with a skeletal Class â ¢ extraction. METHODS: Twenty and 19 patients treated with LeFort I osteotomy and bilateral sagittal split ramus osteotomy (BSSRO) with premolar extraction were enrolled in SFA and CSA groups, respectively. Serial cone-beam computed tomography images obtained before surgery, immediately after surgery (T1), 3 months after surgery, and 12 months after surgery were used for 3-dimensional quantitative analysis. The condyle was segmented for analyzing volumetric changes. Repeated measures analysis of variance, independent t test, and chi-square test were used to compare time-course and intergroup differences. Pearson and Kendall correlation and multivariate linear regression analyses were used to explore prognostic factors affecting skeletal stability. RESULTS: In both CSA and SFA, postsurgical relapse mainly occurred in the mandible sagittal and vertical dimensions and during the first 3 months after surgery. Stability in SFA was significantly less than that in CSA. Intraoperatively, inferolateral condylar displacement with proximal segment inwards, clockwise rotation, and return movements after surgery were observed regardless of the treatment approach. The condylar volume remained stable over time. Multivariate regression analysis showed that posterior vertical dimension (VD) at T1 (-1.63 mm), surgical amount of mandibular setback (-10.33 mm), surgical condylar downwards displacement (-1.28 mm), and anterior overjet at T1 (6.43 mm) were the most important predictors of early mandibular relapse (r2 = 0.593). CONCLUSIONS: The risk of early relapse could be reduced by controlling the anterior, middle, and posterior constraints provided by the prediction model.
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Má Oclusão Classe III de Angle , Côndilo Mandibular , Humanos , Côndilo Mandibular/cirurgia , Estudos Prospectivos , Estudos Retrospectivos , Má Oclusão Classe III de Angle/diagnóstico por imagem , Má Oclusão Classe III de Angle/cirurgia , Mandíbula/diagnóstico por imagem , Mandíbula/cirurgia , Osteotomia Sagital do Ramo Mandibular/métodos , Cefalometria/métodos , Recidiva , SeguimentosRESUMO
Riemerella anatipestifer is an important bacterial pathogen in poultry. Pathogenic bacteria recruit host complement factors to resist the bactericidal effect of serum complement. Vitronectin (Vn) is a complementary regulatory protein that inhibits the formation of the membrane attack complex (MAC). Microbes use outer membrane proteins (OMPs) to hijack Vn for complement evasion. However, the mechanism by which R. anatipestifer achieves evasion is unclear. This study aimed to characterise OMPs of R. anatipestifer which interact with duck Vn (dVn) during complement evasion. Far-western assays and comparison of wild-type and mutant strains that were treated with dVn and duck serum demonstrated particularly strong binding of OMP76 to dVn. These data were confirmed with Escherichia coli strains expressing and not expressing OMP76. Combining tertiary structure analysis and homology modelling, truncated and knocked-out fragments of OMP76 showed that a cluster of critical amino acids in an extracellular loop of OMP76 mediate the interaction with dVn. Moreover, binding of dVn to R. anatipestifer inhibited MAC deposition on the bacterial surface thereby enhancing survival in duck serum. Virulence of the mutant strain ΔOMP76 was attenuated significantly relative to the wild-type strain. Furthermore, adhesion and invasion abilities of ΔOMP76 decreased, and histopathological changes showed that ΔOMP76 was less virulent in ducklings. Thus, OMP76 is a key virulence factor of R. anatipestifer. The identification of OMP76-mediated evasion of complement by recruitment of dVn contributes significantly to the understanding of the molecular mechanism by which R. anatipestifer escapes host innate immunity and provides a new target for the development of subunit vaccines.
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Infecções por Flavobacteriaceae , Doenças das Aves Domésticas , Animais , Virulência , Patos , Proteínas de Membrana , Vitronectina , Proteínas de Bactérias/metabolismo , Infecções por Flavobacteriaceae/microbiologia , Infecções por Flavobacteriaceae/prevenção & controle , Fatores Imunológicos , Proteínas do Sistema Complemento , Doenças das Aves Domésticas/microbiologiaRESUMO
Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic porcine enteric coronavirus that causes severe watery diarrhea and even death in piglets. The neonatal Fc receptor (FcRn) is the only transport receptor for IgG. FcRn expressed by intestinal epithelial cells can transport IgG from breast milk to piglets to provide immune protection. Previous studies have shown that viral infection affects FcRn expression. In this study, we showed for the first time, to our knowledge, that FcRn expression can be influenced by methyltransferases. In addition, we found that PEDV inhibited FcRn protein synthesis in porcine small intestinal epithelial cells postinfection. Then, we found that PEDV interfered with the transcription of genes through aberrant methylation modification of the FcRn promoter. DNA methyltransferase 3b (DNMT3b) has been implicated in this process. Using a series of PEDV structural and nonstructural protein (nsp) expression plasmids, we showed that nsp13 plays an important role in this aberrant methylation modification. PEDV nsp13 can affect the NF-κB canonical pathway and promote DNMT3b protein expression by facilitating p65 protein binding to chromatin. PEDV caused aberrant methylation of the FcRn promoter via DNMT3b. The same phenomenon was found in animal experiments with large white piglets. IgG transcytosis demonstrated that PEDV nsp13 can inhibit bidirectional IgG transport by FcRn. In addition, the core region of nsp13 (230-597 aa) is critical for FcRn inhibition. Taken together, to our knowledge, our findings revealed a novel immune escape mechanism of PEDV and shed new light on the design and development of vaccines and drugs.
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Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Animais , Suínos , NF-kappa B/metabolismo , Vírus da Diarreia Epidêmica Suína/genética , Transdução de Sinais , Imunoglobulina GRESUMO
Duck infectious serositis is an acute and infectious disease caused by Riemerella anatipestifer (R. anatipestifer) that leads to perihepatitis, pericarditis, meningitis, and airbag inflammation in ducks, which causes serious economic losses to the global duck industry. The phoP/phoR is a novel 2-component signal transduction system first reported in gram-negative bacteria, of which phoP acts as a global regulator and virulence factor. In this study, the phoP gene from the R. anatipestifer YM strain was knocked out using homologous recombination technology and replaced with the spectinomycin resistance gene (Spec). The virulence of the R. anatipestifer YMΔphoP strain was reduced by approximately 47,000 times compared to that of the wild-type R. anatipestifer YM strain. Ducks were immunized with live R. anatipestifer YMΔphoP strain by subcutaneous inoculation at a dose of 106 to 107 CFU (0.2 mL per duck) and challenged with the wild-type R. anatipestifer YM strain 14 days later. The protection rate in the immunized group was 100%. The growth characteristics of ducks in the immunized and negative control groups were normal, and the research demonstrated R. anatipestifer YMΔphoP strain have suitable immunogenicity and protective effects. Thus, the study findings suggest that the novel R. anatipestifer YMΔphoP strain may provide a candidate for the development of a gene deletion activated vaccine against duck infectious serositis.
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Infecções por Flavobacteriaceae , Doenças das Aves Domésticas , Riemerella , Serosite , Animais , Proteínas de Bactérias/genética , Patos/microbiologia , Infecções por Flavobacteriaceae/veterinária , Deleção de Genes , Doenças das Aves Domésticas/microbiologia , Riemerella/genética , Sorogrupo , Serosite/genética , Serosite/veterináriaRESUMO
OBJECTIVES: This study aimed to evaluate the soft tissue prediction accuracy of patients undergoing orthognathic surgery to correct skeletal class III malocclusion using maxillofacial regional aesthetic units. MATERIALS AND METHODS: Pre- and postoperative cone-beam computed tomography (CBCT) and 3D facial scans were taken for 58 patients who had undergone orthognathic surgery. The preoperative 3D facial scan was integrated with the preoperative CBCT using ProPlan CMF software. The software simulated the surgery and generated postoperative soft tissue prediction. The simulated 3D facial scan was then compared with the actual 3D facial scan obtained at least 6 months after the surgery by the maxillofacial regional aesthetic units and the facial soft tissue landmark points. RESULTS: The anatomical regions of the upper lip, lower lip, chin, right external buccal and left external buccal prediction were above 2.0 mm. As for the soft tissue landmarks, at chl, chr, ls, stm and li, the position of predicted scan was higher than that of the actual postoperative scan. CONCLUSIONS: The accuracy of 3D soft tissue predictions using ProPlan CMF software in Skeletal III patients was clinically satisfactory according to maxillofacial regional aesthetic units combined with facial soft tissue landmark points. However, the accuracy of prediction still needed improvement in some areas. CLINICAL RELEVANCE: The accuracy of soft tissue prediction can be analyzed more clearly through maxillofacial regional aesthetic units so that clinicians have a deeper understanding of the use of the software to predict soft tissue change after orthognathic surgery.
Assuntos
Má Oclusão Classe III de Angle , Cirurgia Ortognática , Procedimentos Cirúrgicos Ortognáticos , Humanos , Procedimentos Cirúrgicos Ortognáticos/métodos , Imageamento Tridimensional/métodos , Estética Dentária , Má Oclusão Classe III de Angle/diagnóstico por imagem , Má Oclusão Classe III de Angle/cirurgia , Cefalometria/métodos , Mandíbula/cirurgiaRESUMO
To investigate morphological changes of the total and inferior part of the maxillary sinus following Le Fort I osteotomy. 21 skeletal class II and 49 skeletal III patients who underwent orthognathic surgery were enrolled in this retrospective study. Cone-beam computed tomography taken before (T1) and 6 to 24 months after (T2) orthognathic surgery were imported into Mimics 20.0 software to analyze morphological changes of the total and inferior part of the maxillary sinus. Volume of the whole maxillary sinus was significantly reduced after surgery ( P ≤0.008), while the volume of the inferior part of the maxillary sinus was significantly greater than before surgery ( P ≤0.004). Maxillary sinus floor moved occlusally after Le Fort I osteotomy. Movement in the pitch direction of the posterior maxilla affected the state of the maxillary sinus mucosa after orthognathic surgery. Le Fort I osteotomy exerts a significant impact on the morphology of the total and inferior part of the maxillary sinus.
Assuntos
Seio Maxilar , Levantamento do Assoalho do Seio Maxilar , Humanos , Seio Maxilar/cirurgia , Estudos Retrospectivos , Osteotomia de Le Fort/métodos , Maxila/cirurgia , Tomografia Computadorizada de Feixe Cônico/métodos , Osteotomia MaxilarRESUMO
African swine fever (ASF) and postweaning multisystemic wasting syndrome (PMWS) are acute infectious diseases caused by the African swine fever virus (ASFV) and porcine circovirus type 2 (PCV2). At present, there are no effective vaccines for the prevention of ASFV. PMWS, which is harmful to the domestic and even the world pig industry, is difficult to cure and has a high mortality. So, developing simple, inexpensive, and accurate analytical methods to detect and effectively diagnose ASFV and PCV2 can be conducive to avoid ASFV and PCV2 infection. CRISPR has become a potentially rapid diagnostic tool due to recent discoveries of the trans-cleavage properties of CRISPR type V effectors. Herein, we report the visual detection based on CRISPR-Cas12a (cpf1), which is more convenient than fluorescence detection. Through in vitro cleavage target DNA activation, Cas12a can trans-cleavage ssDNA G-quadruplex. TMB/H2O2 and Hemin cannot be catalyzed by cleavaged G-DNA to produce green color products. This protocol is useful for the detection of ASFV and PCV2 with high sensitivity. This method can enable the development of visual and label-free ASFV and PCV2 detection and can be carried out in the field without relying on instruments or power. This method can complete nucleic acid detection at 37 °C without using other instruments or energy. Our research has expanded the application of Cas12a and laid the foundation for the field's rapid detection of viral nucleic acid in future.
RESUMO
With the development of frontier technology in emerging semiconductor processes, self-assembling (SA) and directed self-assembly (DSA) of block copolymers (BCPs) have attracted great attention from scientific researchers and become promising candidates for advanced photolithography. Using an optimal coating and baking process, highly ordered assembly morphologies (e.g., cylinder and lamella) of two BCPs in thin films were obtained without an additional topcoat material layer. Moreover, the whole experimental study also provides an optimal process for integrating the two BCPs into the same topographic guiding pattern substrate fabricated by electron beam lithography (EBL) to achieve specific self-assembly. This topographic guiding substrate achieves not only lamellar micro-domains aligned perpendicular to the sidewalls of trench edges but also cylindrical micro-domains (PMMA phase in a PS matrix) aligned parallel to trench edges respectively, which provides insights and valuable information for further applications in lithography and electronic devices.
RESUMO
It has become a significant problem to develop and improve carbonized polymer dot (CPD)-based fluorescence sensors with environmental detection features. In this study, fluorescent "turn on" sensors of CPDs were prepared by a one-step hydrothermal method using o-phenylenediamine (o-PD) and ferric chloride (FeCl3) as raw materials. Fe-doped CPDs exhibited excellent fluorescence properties, stability, and the sensitive and selective "turn on" detection of hydrazine hydrate (N2H4·H2O). In this detection system, Fe3+ acts as an effective fluorescence inhibitor that inhibits the yellow fluorescence emission from CPDs, while Fe3+ is reduced upon the addition of the N2H4·H2O reducing agent. The fluorescence intensity of CPDs gradually increased with the increasing concentration of N2H4·H2O, while the limit of detection (LOD) could reach 0.168 µM. In addition, CPD-based polyvinyl alcohol (CPDs@PVA) films were also prepared, which still maintained excellent sensitivity to N2H4·H2O. The results show that CPDs and their composite films can detect N2H4·H2O with good detection performance.