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Energy metabolism is highly interdependent with adaptive cell migration in vivo. Mechanical confinement is a critical physical cue that induces switchable migration modes of the mesenchymal-to-amoeboid transition (MAT). However, the energy states in distinct migration modes, especially amoeboid-like stable bleb (A2) movement, remain unclear. In this report, we developed multivalent DNA framework-based nanomachines to explore strategical mitochondrial trafficking and differential ATP levels during cell migration in mechanically heterogeneous microenvironments. Through single-particle tracking and metabolomic analysis, we revealed that fast A2-moving cells driven by biomimetic confinement recruited back-end positioning of mitochondria for powering highly polarized cytoskeletal networks, preferentially adopting an energy-saving mode compared with a mesenchymal mode of cell migration. We present a versatile DNA nanotool for cellular energy exploration and highlight that adaptive energy strategies coordinately support switchable migration modes for facilitating efficient metastatic escape, offering a unique perspective for therapeutic interventions in cancer metastasis.
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Amoeba , Linhagem Celular Tumoral , Movimento Celular , Fenômenos FísicosRESUMO
The emerging outbreak of monkeypox is closely associated with the viral infection and spreading, threatening global public health. Virus-induced cell migration facilitates viral transmission. However, the mechanism underlying this type of cell migration remains unclear. Here we investigate the motility of cells infected by vaccinia virus (VACV), a close relative of monkeypox, through combining multi-omics analyses and high-resolution live-cell imaging. We find that, upon VACV infection, the epithelial cells undergo epithelial-mesenchymal transition-like transformation, during which they lose intercellular junctions and acquire the migratory capacity to promote viral spreading. After transformation, VACV-hijacked RhoA signaling significantly alters cellular morphology and rearranges the actin cytoskeleton involving the depolymerization of robust actin stress fibers, leading-edge protrusion formation, and the rear-edge recontraction, which coordinates VACV-induced cell migration. Our study reveals how poxviruses alter the epithelial phenotype and regulate RhoA signaling to induce fast migration, providing a unique perspective to understand the pathogenesis of poxviruses.
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Mpox , Vaccinia virus , Humanos , Movimento Celular , Surtos de Doenças , Células EpiteliaisRESUMO
Interleukin-8 (IL-8 or chemokine (C-X-C motif) ligand 8, CXCL8) is a chemokine produced by multiple cell types. It promotes chemotaxis and phagocytosis via interaction with chemokine receptors CXCR1 and CXCR2. Using published data, IL-8 gene (LcIL-8) of the large yellow croaker (Larimichthys crocea) was cloned into the pcDNA3.1 plasmid, and an interleukin-8 receptor (LcCXCR2) was cloned into the pEGFP-N1 plasmid. Secratory expression of LcIL-8 in HEK293T cells was carried out, and product in culture medium was collected for LcCXCR2 stimulation in HEK293â¯cells. Following receptor internalization observation and intracellular signaling detection, the functional interaction of LcIL-8 and LcCXCR2 was further determined and the ERK phosphorylation signal activation mediated by LcCXCR2 was demonstrated. Quantitative real-time PCR analysis was used to analyze transcription level regulation of LcIL-8 and LcCXCR2 in various tissues of large yellow croaker. Expression of LcIL-8 and LcCXCR2 was elevated in the spleen, head kidney, and liver after Vibrio parahemolyticus challenge. Results illustrated the functional interaction between LcIL-8 and LcCXCR2 in mediating intracellular ERK1/2 phosphorylation signaling and suggested that the LcIL-8 and LcCXCR2 system is part of the immune response induced by V. Parahemolyticus in L. crocea.
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Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Interleucina-8/genética , Perciformes/genética , Perciformes/imunologia , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8B/genética , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Células HEK293 , Humanos , Interleucina-8/imunologia , Receptores de Interleucina-8A/imunologia , Receptores de Interleucina-8B/imunologiaRESUMO
Yohimbine is a novel compound for the treatment of erectile dysfunction derived from natural products, and pharmacokinetic study is important for its further development as a new medicine. In this work, we developed a novel PEEK tube-based solid-phase microextraction (SPME)-HPLC method for analysis of yohimbine in plasma and further for pharmacokinetic study. Poly (AA-EGDMA) was synthesized inside a PEEK tube as the sorbent for microextraction of yohimbine, and parameters that could influence extraction efficiency were systematically investigated. Under optimum conditions, the PEEK tube-based SPME method exhibits excellent enrichment efficiency towards yohimbine. By using berberine as internal standard, an online SPME-HPLC method was developed for analysis of yohimbine in human plasma sample. The method has wide linear range (2-1000 ng/mL) with an R2 of 0.9962; the limit of detection was determined and was as low as 0.1 ng/mL using UV detection. Finally, a pharmacokinetic study of yohimbine was carried out by the online SPME-HPLC method and the results have been compared with those of reported methods.
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Cromatografia Líquida de Alta Pressão/métodos , Microextração em Fase Sólida/métodos , Ioimbina/sangue , Ioimbina/farmacocinética , Administração Oral , Animais , Berberina/sangue , Cromatografia Líquida de Alta Pressão/normas , Estabilidade de Medicamentos , Desenho de Equipamento , Limite de Detecção , Masculino , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Microextração em Fase Sólida/instrumentação , Ioimbina/administração & dosagemRESUMO
Cathepsin L, a lysosomal endopeptidase, has been noted for its involvement in the innate immune response in invertebrates. Here, the cathepsin L cDNA of the sea cucumber Apostichopus japonicus (AjCatL) is identified from an EST library and then cloned by the rapid amplification of the cDNA ends (RACE) PCR. The full-length cDNA is 1678 bp long containing an open reading frame (ORF) of 1002 bp, an 80 bp 5' UTR and a 599 bp 3' UTR. The cDNA encodes 333 amino acid residues with a predicted molecular mass of 37.07 kDa and a theoretical isoelectric point (pI) of 5.01. The full-length AjCatL contains three active sites of eukaryotic thiol (cysteine) protease at positions 133-144, 278-288 and 295-314. Analysis of the predicted tertiary structure of prepro-CatL (17-333 aa) and mature-CatL (116-333 aa) reveals that the propeptide region (17-115 aa) blocks access to the substrate-binding cleft. Phylogenetic analysis shows that the AjCatL is clustered together with two other CatLs from Strongylocentrotus purpuratus. The enzymatic activity of AjCatL was verified using a substrate hydrolyzing assay with recombinant mAjCatL. Further analysis of real time-PCR demonstrates that the expression of AjCatL mRNA is significantly up-regulated in the coelomocytes in cases of infection with the common bacterial pathogen, Vibrio splendidus. This suggests that the AjCatL is likely to be involved in the immune response.
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Catepsina L/genética , Catepsina L/imunologia , Imunidade Inata , Stichopus/genética , Stichopus/microbiologia , Vibrio/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , Alinhamento de Sequência , Stichopus/classificação , Stichopus/imunologia , Regulação para CimaRESUMO
Electroporation techniques have emerged as attractive tools for intracellular delivery, rendering promising prospects towards clinical therapies. Transient disruption of membrane permeability is the critical process for efficient electroporation-based cargo delivery. However, smart nanotools for precise characterization of transient membrane changes induced by strong electric pulses are extremely limited. Herein, multivalent membrane-anchored fluorescent nanoprobes (MMFNPs) that take advantages of flexible functionalization and spatial arrangement of DNA frameworks are developed for in situ evaluation of electric field-induced membrane permeability during reversible electroporation . Single-molecule fluorescence imaging techniques are adopted to precisely verify the excellent analytical performance of the engineered MMFNPs. Benefited from tight membrane anchoring and sensitive adenosine triphosphate (ATP) profiling, varying degrees of membrane disturbances are visually exhibited under different intensities of the microsecond pulse electric field (µsPEF). Significantly, the dynamic process of membrane repair during reversible electroporation is well demonstrated via ATP fluctuations monitored by the designed MMFNPs. Furthermore, molecular dynamics (MD) simulations are performed for accurate verification of electroporation-driven dynamic cargo entry via membrane nanopores. This work provides an avenue for effectively capturing transient fluctuations of membrane permeability under external stimuli, offering valuable guidance for developing efficient and safe electroporation-driven delivery strategies for clinical diagnosis and therapeutics.
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Trifosfato de Adenosina , Permeabilidade da Membrana Celular , DNA , Eletroporação , Simulação de Dinâmica Molecular , Eletroporação/métodos , DNA/química , DNA/administração & dosagem , Trifosfato de Adenosina/metabolismo , Humanos , Corantes Fluorescentes/química , Membrana Celular/metabolismo , Membrana Celular/química , NanoporosRESUMO
The cell membrane separates the intracellular compartment from the extracellular environment, constraining exogenous molecules to enter the cell. Conventional electroporation typically employs high-voltage and short-duration pulses to facilitate the transmembrane transport of molecules impermeable to the membrane under natural conditions by creating temporary hydrophilic pores on the membrane. Electroporation not only enables the entry of exogenous molecules but also directs the intracellular distribution of the electric field. Recent advancements have markedly enhanced the efficiency of intracellular molecule delivery, achieved through the utilization of microstructures, microelectrodes, and surface modifications. However, little attention is paid to regulating the motion of molecules during and after passing through the membrane to improve delivery efficiency, resulting in an unsatisfactory delivery efficiency and high dose demand. Here, we proposed the strategy of regulating the motion of charged molecules during the delivery process by progressive electroporation (PEP), utilizing modulated electric fields. Efficient delivery of charged molecules with an expanded distribution and increased accumulation by PEP was demonstrated through numerical simulations and experimental results. The dose demand can be reduced by 10-40% depending on the size and charge of the molecules. We confirmed the safety of PEP for intracellular delivery in both short and long terms through cytotoxicity assays and transcriptome analysis. Overall, this work not only reveals the mechanism and effectiveness of PEP-enhanced intracellular delivery of charged molecules but also suggests the potential integration of field manipulation of molecular motion with surface modification techniques for biomedical applications such as cell engineering and sensitive cellular monitoring.
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Eletroporação , Eletroporação/métodos , Humanos , Membrana Celular/metabolismoRESUMO
Background: Tissue welding is an electrosurgical technique that can fuse tissue for small intestine anastomosis. However, limited knowledge exists on its application in mucosa-mucosa end-to-end anastomosis. This study investigates the effects of initial compression pressure, out-put power, and duration time on anastomosis strength ex vivo in mucosa-mucosa end-to-end anastomosis. Methods: Ex vivo porcine bowel segments were used to create 140 mucosa-mucosa end-to-end fusions. Different experimental parameters were employed for fusion, including initial com-pression pressure (50kPa-400 kPa), output power (90W, 110W, and 140W), and fusion time (5, 10, 15, 20 s). The fusion quality was measured by burst pressure and optical microscopes. Results: The best fusion quality was achieved with an initial compressive pressure between 200 and 250 kPa, an output power of 140W, and a fusion time of 15 s. However, an increase in output power and duration time resulted in a wider range of thermal damage. There was no significant difference between the burst pressure at 15 and 20 s (p > 0.05). However, a substantial increase in thermal damage was observed with longer fusion times of 15 and 20 s (p < 0.05). Conclusion: The best fusion quality for mucosa-mucosa end-to-end anastomosis ex vivo is achieved when the initial compressive pressure is between 200 and 250 kPa, the output power is approximately 140W, and the fusion time is approximately 15 s. These findings can serve as a valuable theoretical foundation and technical guidance for conducting animal experiments in vivo and subsequent tissue regeneration.
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Background: Steroid-induced Osteonecrosis of the Femoral Head (SIONFH) is a skeletal disease with a high incidence and a poor prognosis. Whole body vibration therapy (WBVT), a new type of physical training, is known to promote bone formation. However, it remains unclear whether WBVT has a therapeutic effect on SIONFH. Materials and methods: Thirty adult male and female Sprague-Dawley (SD) rats were selected and randomly assigned to three experimental groups: the control group, the model group, and the mechanical vibration group, respectively. SIONFH induction was achieved through the combined administration of lipopolysaccharides (LPS) and methylprednisolone sodium succinate for injection (MPS). The femoral head samples underwent hematoxylin and eosin (H&E) staining to visualize tissue structures. Structural parameters of the region of interest (ROI) were compared using Micro-CT analysis. Immunohistochemistry was employed to assess the expression levels of Piezo1, BMP2, RUNX2, HIF-1, VEGF, CD31, while immunofluorescence was used to examine CD31 and Emcn expression levels. Results: The H&E staining results revealed a notable improvement in the ratio of empty lacuna in various groups following WBVT intervention. Immunohistochemical analysis showed that the expression levels of Piezo1, BMP2, RUNX2, HIF-1, VEGF, and CD31 in the WBVT group exhibited significant differences when compared to the Model group (p < 0.05). Additionally, immunofluorescence analysis demonstrated statistically significant differences in CD31 and Emcn expression levels between the WBVT group and the Model group (p < 0.05). Conclusion: WBVT upregulates Piezo1 to promote osteogenic differentiation, potentially by enhancing the HIF-1α/VEGF axis and regulating H-vessel angiogenesis through the activation of the Piezo1 ion channel. This mechanism may lead to improved blood flow supply and enhanced osteogenic differentiation within the femoral head.
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The tumor-treating fields (TTFields) technology has revolutionized the management of recurrent and newly diagnosed glioblastoma (GBM) cases. To ameliorate this treatment modality for GBM and other oncological conditions, it is necessary to understand the biophysical principles of TTFields better. In this study, we further analyzed the mechanism of the electromagnetic exposure with varying frequencies and electric field strengths on cells in mitosis, specifically in telophase. In reference to previous studies, an intuitive finite element model of the mitotic cell was built for electromagnetic simulations, predicting a local increase in the cleavage furrow region, which may help explain TTFields' anti-proliferative effects. Cell experiments confirmed that the reduction in proliferation and migration of glioma cell by TTFields was in a frequency- and field-strength-dependent manner. This work provides unique insights into the selection of frequencies in the anti-proliferative effect of TTFields on tumors, which could improve the application of TTFields.
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High-frequency electric field welding-induced tissue fusion has been explored as an advanced surgical method for intestinal anastomoses; however, intrinsic mechanisms remain unclear. The aim of this study was to investigate microcosmic changes of collagen within the fusion area, with various parameters. Ex vivo small intestine was fused with mucosa-mucosa. Four levels of compressive pressure (100 kPa, 150 kPa, 200 kPa, 250 kPa) were applied for 10 s in order to fuse the colons under a power level of 140 W. Then, collagen fibers of the fusion area were examined by fibrillar collagen alignment and TEM. Three levels of power (90 W, 110 W, 140 W) and three levels of time (5 s, 10 s, 20 s) were applied in order to fuse colons at 250 kPa, and then collagen within the fusion area was examined by Raman spectroscopy. Fibrillar collagen alignment analysis showed that with the increase in compression pressure, alignment of the collagen in the fusion area gradually increased, and the arrangement of collagen fibers tended to be consistent, which was conducive to the adhesion of collagen fibers. TEM showed that pressure changed the distribution and morphology of collagen fibers. Raman spectroscopy showed that increased power and time within a certain range contributed to collagen cross linking. Peak positions of amide I band and amide III band changed. These results suggested that higher power and a longer amount of time resulted in a decrease in non-reducible cross links and an increase in reducible cross links. Compression pressure, power, and time can affect the state of collagen, but the mechanisms are different. Compressive pressure affected the state of collagen by changing its orientation; power and time denatured collagen by increasing temperature and improved the reducible cross linking of collagen to promote tissue fusion.
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Soldagem , Colágeno/química , Colágenos Fibrilares , Intestino Delgado , AmidasRESUMO
The radiofrequency-induced intestine fusion has been widely studied as an alternative for traditional suture in surgery, but fusion quality cannot be evaluated directly. Impedance measurement can evaluate fusion quality, but the relation between impedance and the fusion quality needs optimization for best results. The present study reports the optimum resistance of small intestine fusion. As the feedback signal, resistance was considered the indicator of the fusion completion for the device design of intestine fusion and an in-depth study of microstructure change. A self-design pulse source was used for the small intestine fusion with adjustable voltage, duty ratio, frequency and output time. A frequency of 440 kHz was set, whereas voltage, output time and compression pressure (CP) of the small intestine were independent variables. Different conditions of voltage, CP and time were investigated for achieving the highest burst pressure (BP) measured with a pressure gauge and a peristaltic pump. Each parameter of the equivalent circuit model was calculated by an experimental waveform. Hematoxylin-eosin staining of fusion samples was used for assessing the quality of fusion. The real-time current was measured and recorded during the fusion for the calculation of capacitance and resistance. The highest BP of 38.9 mmHg was achieved with a CP of 900 kPa, a voltage of 50 V and a time of 5 s. Finally, an optimum extracellular resistance range of 61.0-86.2 Ω was found as the optimum resistance for the end of fusion, thus indicating automatic fusion with the best fusion quality.
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The kisspeptin system is a central modulator of the hypothalamic-pituitary-gonadal axis in vertebrates. Its existence outside the vertebrate lineage remains largely unknown. Here, we report the identification and characterization of the kisspeptin system in the sea cucumber Apostichopus japonicus. The gene encoding the kisspeptin precursor generates two mature neuropeptides, AjKiss1a and AjKiss1b. The receptors for these neuropeptides, AjKissR1 and AjKissR2, are strongly activated by synthetic A. japonicus and vertebrate kisspeptins, triggering a rapid intracellular mobilization of Ca2+, followed by receptor internalization. AjKissR1 and AjKissR2 share similar intracellular signaling pathways via Gαq/PLC/PKC/MAPK cascade, when activated by C-terminal decapeptide. The A. japonicus kisspeptin system functions in multiple tissues that are closely related to seasonal reproduction and metabolism. Overall, our findings uncover for the first time the existence and function of the kisspeptin system in a non-chordate species and provide new evidence to support the ancient origin of intracellular signaling and physiological functions that are mediated by this molecular system.
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Kisspeptinas , Receptores de Kisspeptina-1 , Transdução de Sinais , Stichopus , Animais , Kisspeptinas/genética , Kisspeptinas/metabolismo , Kisspeptinas/fisiologia , Receptores de Kisspeptina-1/genética , Receptores de Kisspeptina-1/metabolismo , Receptores de Kisspeptina-1/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Stichopus/genética , Stichopus/fisiologiaRESUMO
Background: The data on long-term outcomes of patients infected by SARS-CoV-2 and treated with extracorporeal membrane oxygenation (ECMO) in China are merely available. Methods: A retrospective study included 73 patients infected by SARS-CoV-2 and treated with ECMO in 21 intensive care units in Hubei, China. Data on demographic information, clinical features, laboratory tests, ECMO durations, complications, and living status were collected. Results: The 73 ECMO-treated patients had a median age of 62 (range 33-78) years and 42 (63.6%) were males. Before ECMO initiation, patients had severe respiratory failure on mechanical ventilation with a median PO2/FiO2 of 71.9 [interquartile range (IQR), 58.6-87.0] mmHg and a median PCO2 of 62 [IQR, 43-84] mmHg on arterial blood analyses. The median duration from symptom onset to invasive mechanical ventilation, and to ECMO initiation was19 [IQR, 15-25] days, and 23 [IQR, 19-31] days. Before and after ECMO initiation, the proportions of patients receiving prone position ventilation were 58.9 and 69.9%, respectively. The median duration of ECMO support was 18.5 [IQR 12-30] days. During the treatments with ECMO, major hemorrhages occurred in 31 (42.5%) patients, and oxygenators were replaced in 21 (28.8%) patients. Since ECMO initiation, the 30-day mortality and 60-day mortality were 63.0 and 80.8%, respectively. Conclusions: In Hubei, China, the ECMO-treated patients infected by SARS-CoV-2 were of a broad age range and with severe hypoxemia. The durations of ECMO support, accompanied with increased complications, were relatively long. The long-term mortality in these patients was considerably high.
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OBJECTIVE: Drug retention enema is a common therapy for various illnesses. However, it is impossible to keep the drug in the colon for a long time, due to the limitation of the current equipment, and it is unable to achieve the purpose of retention enema. A retention enema device was designed by the department of intensive care unit (ICU) of Dongfeng Hospital Affiliated to Hubei University of Medicine. The retention enema device adds a spindle shaped inflatable air bag on the basis of the traditional enema device, which not only fix on the anus, but also prevent the leakage of enema fluid. It can achieve retention enema, play the enema drug effect fully, and significantly reduce the nursing workload, in addition, the silica gel material of the retention enema device ensures the comfort of the patients, the decompression air bag also avoids the damage of the high pressure of the spindle fixed air bag for the patients, which is worthy of clinical use.
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Enema/instrumentação , Colo , HumanosRESUMO
Neuropeptide Y (NPY) receptors are suggested to mediate the multi-physiological functions of NPY family peptides, such as food intake, in teleost fish. However, the structure and signaling of fish NPY receptors are yet to be fully elucidated. In this study, we report the cloning and characterization of two neuropeptide Y receptor subtypes, Y2 (NPY2R) and Y7 (NPY7R), in yellow croaker Larimichthys crocea (L. crocea) (LcNPY2R, LcNPY7R). The gene structure, pharmacological characterization, cell location, and tissue expression of these two receptors were explored. The phylogenetic results showed that LcNPY2R and LcNPY7R had typical G protein-coupled receptor profiles, associated with the Y2 subfamily, with coding sequences that are highly conserved in vertebrates. The expression of both LcNPY2R and LcNPY7R could be activated by LcNPY in HEK293 cells. However, truncated LcNPY18-36 was only able to activate LcNPY2R at the same level as full length LcNPY. Expression analysis revealed that LcNPY2R mRNA was predominantly expressed in the intestine and liver, whereas LcNPY7R was expressed in the stomach, which indicated that both receptors were related to the digestive system. Overall, our data establishes a molecular basis to determine the actions of LcNPY2R and LcNPY7R, which could be used to elucidate the conserved roles of these receptor-ligand pairs in vertebrates.
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Proteínas de Peixes , Regulação da Expressão Gênica/fisiologia , Perciformes , Receptores de Neuropeptídeo Y , Animais , Proteínas de Peixes/biossíntese , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Especificidade de Órgãos/fisiologia , Perciformes/genética , Perciformes/metabolismo , Receptores de Neuropeptídeo Y/biossíntese , Receptores de Neuropeptídeo Y/genéticaRESUMO
BACKGROUND: The aim of the study was to evaluate the diagnostic accuracy of cervical elastography in predicting preterm delivery (PTD). METHODS: We searched the PubMed, EMBASE, and Cochrane databases to identify relevant studies that applied ultrasound (US) elastography to assess cervical stiffness and predict PTD. All the studies were published before December 11, 2018, and only studies published in English were collected. The cervical length (CL) was considered a comparator, and the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool was applied to assess the quality of the included studies. Summary receiver operating characteristic (SROC) modeling was performed to evaluate the diagnostic performance of cervical elastography in predicting PTD. Subgroup analyses were also performed. RESULTS: Seven studies, including 1488 pregnant women, were included in this meta-analysis. Cervical elastography showed a summary sensitivity of 0.84 [95% confidence interval (CI): 0.68, 0.93], a specificity of 0.82 (95% CI: 0.63, 0.93), a diagnostic odds ratio of 25 (95% CI: 7, 93), and an area under the curve (AUC) of SROC of 0.90 (95% CI: 0.87-0.93). CL measurement showed that the AUC of SROC was 0.60 (95% CI: 0.56-0.64). The results of subgroup analysis showed that the summary sensitivity and specificity were different in the QUADAS-2 score subgroups. CONCLUSION: Cervical elastography is a promising and reliable method to predict PTD. Cervical elastography showed better diagnostic performance to predict PTD than CL measurement.
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Colo do Útero , Técnicas de Imagem por Elasticidade/métodos , Nascimento Prematuro/diagnóstico , Colo do Útero/diagnóstico por imagem , Colo do Útero/fisiopatologia , Precisão da Medição Dimensional , Feminino , Humanos , Valor Preditivo dos Testes , GravidezRESUMO
Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.