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Drugs that target immune checkpoint have become the most popular weapon in cancer immunotherapy, yet only have practical benefits for a small percentage of patients. Tumor cells constantly interact with their microenvironment, which is made up of a variety of immune cells as well as endothelial cells and fibroblasts. Immune checkpoint expression and blocked signaling of immune cells in the tumor microenvironment (TME) are key to tumor progression. In this study, we perform deliberation convolution on the TCGA database for human lung, breast, and colorectal cancer to infer crosstalk between immune checkpoint receptors (ICRs) and ligands (ICLs) in TME of pan-carcinogenic solid tumor types, validated by flow cytometry. Analysis of immune checkpoints showed that there was little variation between different tumor types. It showed that CD160, LAG3, TIGIT were found to be highly expressed in CD8+ T cells instead of CD4+ T cells, PD-L1, PD-L2, CD86, LGALS9, TNFRSF14, LILRB4 and other ligands were highly expressed on macrophages, FVR, NECTIN2, FGL1 were highly expressed on Epithelial cells, CD200 was highly expressed in Endothelial cells, and CD80 was highly expressed in CD8 High expression on T cells. Overall, our study provides a new resource for the expression of immune checkpoints in TME on various types of cells. Significance: This study provides immune checkpoint expression of immune cells of multiple cancer types to infer immune mechanisms in the tumor microenvironment and provide ideas for the development of new immune checkpoint-blocking drugs.
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Proteínas de Punto de Control Inmunitario , Microambiente Tumoral , Humanos , Microambiente Tumoral/inmunología , Proteínas de Punto de Control Inmunitario/metabolismo , Proteínas de Punto de Control Inmunitario/genética , Ligandos , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Antígenos CD/metabolismo , Antígenos CD/genética , Nectinas/metabolismo , Nectinas/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Proteína del Gen 3 de Activación de Linfocitos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , FemeninoRESUMEN
Although thousands of genes have been identified or cloned in rice (Oryza sativa) in the last two decades, the majority of them have only been separately characterized in specific varieties or single-gene modified backgrounds, thus limiting their practical application. We developed an optimized multiplex genome editing (MGE) toolbox that can efficiently assemble and stably express up to twelve sgRNA targets in a single plant expression vector. In this study, we established the MGE-based Rapid Directional Improvement (MRDI) strategy for directional improvement of complex agronomic traits in one small-scale rice transformation. This approach provides a rapid and practical procedure, encompassing sgRNA assembly, transgene-free screening and the creation of promising germplasm, by combining the precision of gene editing with phenotype-based field breeding. The MRDI strategy was used to generate the full diversity of twelve main agronomic genes in rice cultivar FXZ for the directional improvement of its growth duration and plant architecture. After applying the MRDI to FXZ, ideal plants with the desired traits of early heading date reduced plant height, and more effective panicles were generated without compromising yield, blast resistance and grain quality. Furthermore, the results of whole-genome sequencing (WGS), including the analysis of structural variations (SVs) and single nucleotide variations (SNVs) in the MGE plants, confirmed the high specificity and low frequency of unwanted mutations associated with this strategy. The MRDI breeding strategy would be a robust approach for exploring and applying crucial agronomic genes, as well as for generating novel elite germplasm in the future.
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Edición Génica , Genoma de Planta , Oryza , Oryza/genética , Oryza/crecimiento & desarrollo , Edición Génica/métodos , Genoma de Planta/genética , Fitomejoramiento/métodos , Fenotipo , Sistemas CRISPR-Cas/genética , Plantas Modificadas Genéticamente/genéticaRESUMEN
BACKGROUND: Chronic stress promotes most hallmarks of cancer through impacting the malignant tissues, their microenvironment, immunity, lymphatic flow, etc. Existing studies mainly focused on the roles of stress-induced activation of systemic sympathetic nervous system and other stress-induced hormones, the organ specificity of chronic stress in shaping the pre-metastatic niche remains largely unknown. This study investigated the role of chronic stress in remodeling lung pre-metastatic niche of breast cancer. METHODS: Breast cancer mouse models with chronic stress were constructed by restraint or unpredictable stress. Expressions of tyrosine hydroxylase, vesicular acetylcholine transporter (VAChT), EpCAM and NETosis were examined by immunofluorescence and confocal microscopy. mRNA and protein levels of choline acetyltransferase (ChAT), VAChT, and peptidylarginine deiminase 4 were detected by qRT-PCR and Western blotting, respectively. Immune cell subsets were analyzed by flow cytometry. Acetylcholine (ACh) and chemokines were detected by ELISA and multi chemokine array, respectively. ChAT in lung tissues from patients was examined by immunohistochemistry. RESULTS: Breast cancer-bearing mice suffered chronic stress metastasized earlier and showed more severe lung metastasis than did mice in control group. VAChT, ChAT and ChAT+ epithelial cells were increased significantly in lung of model mice undergone chronic stress. ACh and chemokines especially CXCL2 in lung culture supernatants from model mice with chronic stress were profoundly increased. Chronic stress remodeled lung immune cell subsets with striking increase of neutrophils, enhanced NETosis in lung and promoted NETotic neutrophils to capture cancer cells. ACh treatment resulted in enhanced NETosis of neutrophils. The expression of ChAT in lung tissues from breast cancer patients with lung metastasis was significantly higher than that in patients with non-tumor pulmonary diseases. CONCLUSIONS: Chronic stress promotes production of CXCL2 that recruits neutrophils into lung, and induces pulmonary epithelial cells to produce ACh that enhances NETosis of neutrophils. Our findings demonstrate for the first time that chronic stress induced epithelial cell derived ACh plays a key role in remodeling lung pre-metastatic niche of breast cancer.
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Neoplasias de la Mama , Neoplasias Pulmonares , Humanos , Ratones , Animales , Femenino , Acetilcolina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Pulmón , Células Epiteliales/metabolismo , Quimiocinas , Microambiente TumoralRESUMEN
BACKGROUND: Dendritic cells (DCs) mediate divergent immune effects by activating T cells or negatively regulating the immune response to promote immune tolerance. They perform specific functions determined by their tissue distribution and maturation state. Traditionally, immature and semimature DCs were described to have immunosuppressive effects, leading to immune tolerance. Nonetheless, recent research has demonstrated that mature DCs can also suppress the immune response under certain circumstances. MAIN BODY: Mature DCs enriched in immunoregulatory molecules (mregDCs) have emerged as a regulatory module across species and tumour types. Indeed, the distinct roles of mregDCs in tumour immunotherapy have sparked the interest of researchers in the field of single-cell omics. In particular, these regulatory cells were found to be associated with a positive response to immunotherapy and a favourable prognosis. CONCLUSION: Here, we provide a general overview of the latest and most notable advances and recent findings regarding the basic features and complex roles of mregDCs in nonmalignant diseases and the tumour microenvironment. We also emphasise the important clinical implications of mregDCs in tumours.
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Células Dendríticas , Neoplasias , Humanos , Microambiente Tumoral , Inmunoterapia , Neoplasias/patología , InmunidadRESUMEN
T lymphocytes are the key protective contributors in chronic infection and tumor, but experience exhaustion by persistent antigen stimulation. As an unconventional lineage of T cells, γδ T cells can rapidly response to varied infectious and tumor challenges in a non-MHC-restricted manner and play key roles in immune surveillance via pleiotropic effector functions, showing promising as candidates for cellular tumor immunotherapy. Activated γδ T cells can also acquire exhaustion signature with elevated expression of immune checkpoints, such as PD-1, decreased cytokine production, and functional impairment. However, the exhaustion features of γδ T cells are distinct from conventional αß T cells. Here, we review the researches regarding the characteristics, heterogeneity, and mechanisms of γδ T cell exhaustion. These studies provide insights into the combined strategies to overcome the exhaustion of γδ T cells and enhance antitumor immunity. Summary sentence: Review of the characteristics, heterogeneity, and mechanisms of γδ T cell exhaustion provides insights into the combined strategies to enhance γδ T cell-based antitumor immunotherapy.
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Linfocitos Intraepiteliales , Neoplasias , Humanos , Receptores de Antígenos de Linfocitos T gamma-delta , Inmunoterapia , Neoplasias/terapiaRESUMEN
Breast cancer lung metastasis has a high mortality rate and lacks effective treatments, for the factors that determine breast cancer lung metastasis are not yet well understood. In this study, data from 1067 primary tumors in four public datasets revealed the distinct microenvironments and immune composition among patients with or without lung metastasis. We used multi-omics data of the TCGA cohort to emphasize the following characteristics that may lead to lung metastasis: more aggressive tumor malignant behaviors, severer genomic instability, higher immunogenicity but showed generalized inhibition of effector functions of immune cells. Furthermore, we found that mast cell fraction can be used as an index for individual lung metastasis status prediction and verified in the 20 human breast cancer samples. The lower mast cell infiltrations correlated with tumors that were more malignant and prone to have lung metastasis. This study is the first comprehensive analysis of the molecular and cellular characteristics and mutation profiles of breast cancer lung metastasis, which may be applicable for prognostic prediction and aid in choosing appropriate medical examinations and therapeutic regimens.
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BACKGROUND: Rice ratooning has traditionally been an important component of the rice cropping system in China. However, compared with the rice of the first harvest, few studies on factors effecting ratoon rice yield have been conducted. Because ratoon rice is a one-season rice cultivated using axillary buds that germinate on rice stakes and generate panicles after the first crop's harvest, its production is mainly affected by the growth of axillary buds. The objectives of this study were to evaluate the sprouting mechanism of axillary buds to improve the ratoon rice yield. RESULTS: First, we observed the differentiation and growth dynamics of axillary buds at different nodes of Shanyou 63, and found that they differentiated from bottom to top before the heading of the mother stem, and that they developed very slowly. After heading they differentiated from top to bottom, and the ones on the top, especially the top 2nd node, developed much faster than those at the other nodes. The average length and dry weight of the axillary buds were significantly greater than those at other nodes by the yellow ripe stage, and they differentiated into pistils and stamens by 6 d after the yellow ripe stage. The morphology of vegetative organs from regenerated tillers of Shanyou 63 also suggested the superior growth of the upper buds, which was regulated by hormones, in ratoon rice. Furthermore, a comprehensive proteome map of the rice axillary buds at the top 2nd node before and after the yellow ripe stage was established, and some proteins involved in steroid biosynthesis were significantly increased. Of these, four took part in brassinosteroid (BR) biosynthesis. Thus, BR signaling may play a role in the germination of axillary buds of ratoon rice. CONCLUSIONS: The data provide insights into the molecular mechanisms underlying BR signaling, and may allow researchers to explore further the biological functions of endogenous BRs in the germination of axillary buds of ratoon rice.
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Brasinoesteroides/metabolismo , Flores/fisiología , Germinación , Oryza/fisiología , Transducción de SeñalRESUMEN
This paper describes a highly controllable method to generate hollow calcium alginate microfibers using a double co-axial flow microdevice. The microdevice was fabricated by concentric assembly of two modules; each module consisted of a shortened cone-pulled glass capillary embedded in a polymethylmethacrylate fluidic block. Using this microdevice, cylindrical hollow calcium alginate microfibers with either straight or helical inner walls were stably and continuously generated. The radii of the hollow microfibers were not arbitrary, and in fact, the ratio of the outer to inner diameter was inversely correlated with the combination of core flow rate and the first sheath flow rate. The relationships between the geometrical features of the helix and the flow rates were also analyzed. The helical pitch and the spiral radius of the helical hollow microfibers were strongly influenced by the second sheath flow rate. Finally, guidelines for generating highly controllable straight and helical hollow microfibers and fabricating a seamless flow connector using this microfluidic device are suggested.
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Aptamers are promising alternatives to antibodies and can be used as high affinity agents for the cancer detection and the targeted drug transportation. In this manuscript, we highlight the advantages of aptamers, such as high affinities, specificity and excellent chemical stabilities, which are likely to benefit for the diagnosis of cancer in its early stages and then achieve molecular-level treatment. Also, we discuss the challenges and problems in the current application of aptamers.
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Antineoplásicos , Aptámeros de Nucleótidos , Sistemas de Liberación de Medicamentos/métodos , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/uso terapéutico , Humanos , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Previous studies have demonstrated that inflammatory cytokines are associated with matrix metalloproteinases (MMPs) and/or lysyl oxidases (LOXs) produced by anterior cruciate ligament (ACL) fibroblasts, which may contribute to the poor healing ability of the ACL. To evaluate whether the synovium also participates in ACL healing, the inflammatory microenvironment of the knee joint cavity was mimicked following ACL injury, and the combined effects of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) on the expression of MMPs and LOXs in synovial fibroblasts were studied. Cell viability was evaluated using trypan blue staining in the presence of TNF-α and IL-1ß, and the expression of LOXs and MMPs was measured by reverse transcription-quantitative polymerase chain reaction. MMP-2 activity was also measured by zymography. The results indicated that the combined effects of TNF-α and IL-1ß inhibited LOX expression, while promoting MMP-1, -2 and -3 expression and MMP-2 activity in synovial fibroblasts. These changes may impede healing by altering the balance between the degradative and biosynthetic arms of the ligament tissue remodeling process. Collectively, the present results suggest that the poor healing ability of cruciate ligaments may be due to the sensitivity of the synovium to inflammatory factors. Therefore, the synovium potentially serves a key regulatory role in the joint cavity microenvironment and in the healing process of the ACL, and thus should be considered as a therapeutic target to aid in the treatment of patients with ACL trauma.
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Fabrication of small diameter vascular grafts (SDVGs) with appropriate responses for clinical application is still challenging. In the present work, the production and characterization of solid alginate based microfibers as potential SDVG candidates through the method of microfluidics were considered original. A simple glass microfluidic device with a 'L-shape' cylindrical-flow channel in the microfluidic platform was developed. The gelation of microfibers occurred when the alginate solution and a CaCl2 solution were introduced as a core flow and as a sheath flow, respectively. The diameters of the microfibers could be controlled by varying the flow rates and the glass capillary tubes diameters at their tips. The generated microfibers had somewhat rough and porous surfaces, their suture retention strengths were comparable to the strength of other tissue engineered grafts. The encapsulated mesenchymal stem cells proliferated well in the microfibers, and showed a stable endothelialization under the angiogenesis effects of vascular endothelial growth factor and fibroblastic growth factor. The in vivo implant into the mice abdomens indicated that cell composite microfibers caused a mild host reaction. These encouraging results suggest great promise of the application of microfluidics as a future alternative in SDVGs engineering.
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Alginatos , Materiales Biocompatibles , Prótesis Vascular , Técnicas Analíticas Microfluídicas , Alginatos/química , Alginatos/metabolismo , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Diseño de Equipo , Femenino , Factores de Crecimiento de Fibroblastos , Ácido Glucurónico/química , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/química , Ácidos Hexurónicos/metabolismo , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Factor A de Crecimiento Endotelial Vascular/farmacocinética , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Grain number is an important factor in determining grain production of rice (Oryza sativa L.). The molecular genetic basis for grain number is complex. Discovering new genes involved in regulating rice grain number increases our knowledge regarding its molecular mechanisms and aids breeding programs. Here, we identified GRAINS NUMBER 2 (GN2), a novel gene that is responsible for rice grain number, from "Yuanjiang" common wild rice (O. rufipogon Griff.). Transgenic plants overexpressing GN2 showed less grain number, reduced plant height, and later heading date than control plants. Interestingly, GN2 arose through the insertion of a 1094-bp sequence from LOC_Os02g45150 into the third exon of LOC_Os02g56630, and the inserted sequence recruited its nearby sequence to generate the chimeric GN2 The gene structure and expression pattern of GN2 were distinct from those of LOC_Os02g45150 and LOC_Os02g56630 Sequence analysis showed that GN2 may be generated in the natural population of Yuanjiang common wild rice. In this study, we identified a novel functional chimeric gene and also provided information regarding the molecular mechanisms regulating rice grain number.
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Genes de Plantas , Oryza/genética , Plantas Modificadas Genéticamente , Semillas/genética , Mutagénesis Insercional , Oryza/crecimiento & desarrollo , Semillas/crecimiento & desarrolloRESUMEN
We provide a facile and low-cost method (F-L) to fabricate a two-dimensional positive master using a milling technique for polydimethylsiloxane (PDMS)-based microchannel molding. This method comprises the following steps: (1) a positive microscale master of the geometry is milled on to an acrylic block; (2) pre-cured PDMS is used to mold the microscale positive master; (3) the PDMS plate is peeled off from the master and punctured with a blunt needle; and (4) the PDMS plate is O2 plasma bonded to a glass slide. Using this technique, we can fabricate microchannels with very simple protocols quickly and inexpensively. This method also avoids breakage of the end mill (Ï = 0.4 mm) of the computerized numerical control (CNC) system when fabricating the narrow channels (width < 50 µm). The prominent surface roughness of the milled bottom-layer could be overcomed by pre-cured PDMS with size trade-off in design. Finally, emulsion formation successfully demonstrates the validity of the proposed fabrication protocol. This work represents an important step toward the use of a milling technique for PDMS-based microfabrication.
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PURPOSE: The adult human posterior cruciate ligament (PCL) has poor functional healing response. The synovial tissue, which surrounds the PCL ligament, might be the major regulator of the microenvironment in the joint cavity after PCL injury, thus affecting the healing process. Here we establish a novel co-culture system for PCL fibroblasts and synovial cells (SC) in vitro to explore the direct influence of paracrine on PCL cells by characterizing the different expressions of the lysyl oxidase family (LOXs) and matrix metalloproteinases (MMP-1, 2, 3), which respectively facilitate extracellular matrix (ECM) repair and degradation. METHODS: Total RNA was harvested, reverse transcribed and assessed by semi-quantitative PCR and real-time PCR for the expression of LOXs and MMP-1, 2, 3 messenger RNAs. MMP-2 activity was assayed from the collected culture media samples by using zymography. RESULTS: We found co-culture could promote gene expressions of the LOXs and MMP-1, 2, 3 in normal PCL fibroblasts. But in injured PCL, we found that matrix crosstalk induced an increase of the MMP-1, 2, 3 expressions and a down-regulation of the LOXs. CONCLUSION: Based on these results, the crosstalk between PCL and SC strongly modified homeostatic balance of ECM and appeared to have a significant impact on PCL wound healing; decreased expression of cross-linking enzymes (LOXs) and increased expression of ECM-degrading proteinases (MMP-1, 2, 3) might be of great contribution to poor healing ability of PCL ligament.
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Fibroblastos/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Ligamento Cruzado Posterior/citología , Proteína-Lisina 6-Oxidasa/metabolismo , Membrana Sinovial/metabolismo , Adulto , Células Cultivadas , Técnicas de Cocultivo , Matriz Extracelular/metabolismo , Fibroblastos/citología , Expresión Génica , Humanos , Ligamento Cruzado Posterior/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Membrana Sinovial/citología , Cicatrización de Heridas/fisiologíaRESUMEN
BACKGROUND: The lysyl oxidase (LOX) family has the capacity to catalyze the cross-linking of collagen and elastin, implicating its important fundamental role in injury healing. Tumor necrosis factor alpha (TNF-α) is considered to be an important chemical mediator in the acute inflammatory phase of the ligament injury. The role of the lysyl oxidase family induced by TNF-α in the knee ligaments' wound healing process is poorly understood. Our purpose was to determine the different expressions of the LOXs in poorly self-healing anterior cruciate ligament (ACL) and well functionally self-healing medial collateral ligament (MCL) induced by TNF-α. METHODS: Semi-quantitative PCR, quantitative real-time PCR and western blot were performed for original research. RESULTS: The results showed that all LOX family members were expressed at higher levels in MCL than those in ACL fibroblasts; the significant differences existed in the down-regulations of the LOXs induced by TNF-α; and the TNF-α-mediated down-regulations of the LOXs were more prominent in ACL than those in MCL fibroblasts. 1-20 ng/ml TNF-α down-regulated mRNA levels in ACL and MCL fibroblasts by up to 76% and 58% in LOX; 90% and 45% in LOXL-1; 97.5% and 90% in LOXL-2; 89% and 68% in LOXL-3; 52% and 25% in LOXL-4, respectively. Protein assay also showed LOXs had lower expressions in ACL than those in MCL. CLINICAL RELEVANCE: Based on these results, the differential expressions of the LOXs might help to explain the intrinsic differences between the poorly self-healing ACL and well functionally self-healing MCL.
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Ligamento Cruzado Anterior/citología , Regulación hacia Abajo , Fibroblastos/metabolismo , Ligamento Colateral Medial de la Rodilla/citología , Proteína-Lisina 6-Oxidasa/metabolismo , Factor de Necrosis Tumoral alfa/administración & dosificación , Adulto , Análisis de Varianza , Western Blotting , Supervivencia Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Agar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Proteína-Lisina 6-Oxidasa/genética , ARN Mensajero/metabolismoRESUMEN
The progress of research on the the anterior cruciate ligament (ACL) wound healing demonstrates that the synovial tissue in the knee joint plays a very important role in the healing process of injured ACL. Therefore, the molecular response mechanisms of lysyl oxidase (LOX) and matrix metalloproteina (MMP) in normal/injured ACL fibroblast cells could be considered to perform the major analysis function of injured ACL healing mechanism. The mRNA expressions of LOXs and MMPs and the activity expressions of MMP-2 in ACL fibroblasts co-cultured with synovial cells were analyzed by quantitative real-time PCR and zymography. The results showed that co-culture could regulate the mRNA expressions of LOXs and MMPs in the ACL fibroblasts cells. These results suggest that the differential expressions of LOXs and MMP-1, 2, 3 in co-cultured ACL indicate that interaction crosstalk do exist between ACL cells and synovial cells and provide a theoretical basis for subsequent exploration of the mechanisms and treatment of ACL injury and repair.
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Ligamento Cruzado Anterior/citología , Fibroblastos/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Membrana Sinovial/citología , Lesiones del Ligamento Cruzado Anterior , Técnicas de Cocultivo , Fibroblastos/citología , Humanos , Traumatismos de la Rodilla/fisiopatología , Articulación de la Rodilla/citología , Metaloproteinasas de la Matriz/genética , Proteína-Lisina 6-Oxidasa/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Lysyl oxidase (LOX) family has the capacity to catalyse the cross-linking of collagen and elastin, implicating its important fundamental roles in tissue development and injury healing. However, the variations in expression of the LOX family in the normal and injured anterior cruciate ligament (ACL) are not fully known. To better understand the role of LOX family in the self-healing inability mechanism of injured ACL, this study is to measure the LOX family's differential expressions in ACL and medial collateral ligament (MCL) fibroblasts after mechanical injury induced by using an equi-biaxial stretching chamber. The cells received various degrees of mechanical stretch 0% (resting state), 6% (physiological state) and 12% (injurious state), respectively. The gene profile and protein expressions were analysed by semi-quantitative PCR, quantitative real-time PCR and Western blotting. At physiological state, gene expression showed LOX in ACL was 2.6-5.2 folds higher than that in MCL in all culture time periods, LOXL-4 1.2-3.6 folds, but LOXL-3 in MCL showed 1.1-4.8 folds higher than that in ACL. In injurious state, MCL gene expressions were 2.8-29.6 folds higher than ACL in LOX, LOXL-2, LOXL-3 and LOXL-4 at 2, 6 and 12h periods. These differential expression profiles of the LOX family in the two ligament tissues were further used to explain the intrinsic differences between ACL and MCL, and why injured ACL could not be amenable to repair itself, whereas MCL could.
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Fibroblastos/citología , Ligamentos/lesiones , Proteína-Lisina 6-Oxidasa/biosíntesis , Cicatrización de Heridas/fisiología , Adulto , Ligamento Cruzado Anterior/citología , Ligamento Cruzado Anterior/metabolismo , Lesiones del Ligamento Cruzado Anterior , Femenino , Fibroblastos/metabolismo , Humanos , Masculino , Ligamento Colateral Medial de la Rodilla/citología , Ligamento Colateral Medial de la Rodilla/lesiones , Ligamento Colateral Medial de la Rodilla/metabolismo , Persona de Mediana Edad , Proteína-Lisina 6-Oxidasa/metabolismoRESUMEN
Tiller angle is a key feature of the architecture of cultivated rice (Oryza sativa), since it determines planting density and influences rice yield. Our previous work identified Tiller Angle Control 1 (TAC1) as a major quantitative trait locus that controls rice tiller angle. To further clarify the evolutionary characterization of the TAC1 gene, we compared a TAC1-containing 3164-bp genomic region among 113 cultivated varieties and 48 accessions of wild rice, including 43 accessions of O. rufipogon and five accessions of O. nivara. Only one single nucleotide polymorphism (SNP), a synonymous substitution, was detected in TAC1 coding regions of the cultivated rice varieties, whereas one synonymous and one nonsynonymous SNP were detected among the TAC1 coding regions of wild rice accessions. These data indicate that little natural mutation and modification in the TAC1 coding region occurred within the cultivated rice and its progenitor during evolution. Nucleotide diversities in the TAC1 gene regions of O. sativa and O. rufipogon of 0.00116 and 0.00112, respectively, further indicate that TAC1 has been highly conserved during the course of rice domestication. A functional nucleotide polymorphism (FNP) of TAC1 was only found in the japonica rice group. A neutrality test revealed strong selection, especially in the 3'-flanking region of the TAC1 coding region containing the FNP in the japonica rice group. However, no selection occurred in the indica and wild-rice groups. A phylogenetic tree derived from TAC1 sequence analysis suggests that the indica and japonica subspecies arose independently during the domestication of wild rice.
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Evolución Molecular , Genes de Plantas/genética , Oryza/genética , Mutación/genética , Oryza/crecimiento & desarrollo , Filogenia , Polimorfismo Genético/genética , Selección Genética , Especificidad de la EspecieRESUMEN
PURPOSE: The lysyl oxidase (LOX) family plays a crucial role in the formation and stabilisation of extracellular matrix (ECM) by catalysing the cross-linking of collagen and elastin, implicating its important fundamental roles in injury healing. A high level of transforming growth factor-ß(1) (TGF-ß(1)) accompanies the inflammatory phase of an injury of the knee joint. Our purpose was to detect the expressions of the LOX family in anterior cruciate ligament (ACL) and medial collateral ligament (MCL) response to TGF-ß(1). METHODS: This study used reversed transcript PCR, real time quantitative PCR and Western blot for analyses. RESULTS: The results showed significant increases in mRNA levels of LOX family members. At 5 ng/ml concentration of TGF-ß(1,) the gene profiles of LOXs showed most active, and LOX and LOXL-3 showed increasing peaks at 12 hours after TGF-ß(1) treatment (LOX: 7.2, 8.8-fold and LOXL-3: 3.8, 5.4-fold compared with normal controls in ACL and MCL, respectively); LOXL-1, LOXL-2 and LOXL-4 reached their highest amounts at six hours (LOXL-1: 1.9, 2.4-fold; LOXL-2: 14.8, 16.2-fold; LOXL-4: 2.5, 4.4-fold in ACL and MCL, respectively). Protein assays revealed that LOXs in ACL cells had relatively lower response to TGF-ß(1) compared with those in MCL cells. CONCLUSIONS: The differential expression and activities of LOXs might help to explain the intrinsic difference between ACL and MCL, and LOXs could imply a potential capability for ACL healing.
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Ligamento Cruzado Anterior/citología , Fibroblastos/citología , Ligamento Colateral Medial de la Rodilla/citología , Proteína-Lisina 6-Oxidasa/genética , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacos , Adulto , Animales , Western Blotting , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Proteína-Lisina 6-Oxidasa/metabolismoRESUMEN
Microfluidics deals with the manipulation of fluidics in the structure with dimensions of micrometers or nanometers. As an emerging field, microfluidics has numerous advantages, such as controllable fluid flow and reduced reagents consumption. Recently, microfluidic has been applied into the area of cell transfection, providing opportunities to investigate cell transfection process on microscale. This review summarizes recent technical development of cell transfection based on microfluidics, including transfeceted microarray, transfecetion established in miniaturization flowing space, microdrops, microinjection and microfluidic electroporation. The factors that affect the transfection efficiency and improvement approaches are also discussed.