RESUMEN
Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana actin-depolymerizing factor7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7-enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Citoesqueleto de Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Corriente Citoplasmática/genética , Genes Reporteros , Proteínas Fluorescentes Verdes , Modelos Moleculares , Datos de Secuencia Molecular , Tubo Polínico/genética , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Tiazolidinas/farmacologíaRESUMEN
Rapid actin turnover is essential for numerous actin-based processes. However, how it is precisely regulated remains poorly understood. Actin-interacting protein 1 (AIP1) has been shown to be an important factor by acting coordinately with actin-depolymerizing factor (ADF)/cofilin in promoting actin depolymerization, the rate-limiting factor in actin turnover. However, the molecular mechanism by which AIP1 promotes actin turnover remains largely unknown in plants. Here, we provide a demonstration that AIP1 promotes actin turnover, which is required for optimal growth of rice plants. Specific down-regulation of OsAIP1 increased the level of filamentous actin and reduced actin turnover, whereas over-expression of OsAIP1 induced fragmentation and depolymerization of actin filaments and enhanced actin turnover. In vitro biochemical characterization showed that, although OsAIP1 alone does not affect actin dynamics, it enhances ADF-mediated actin depolymerization. It also caps the filament barbed end in the presence of ADF, but the capping activity is not required for their coordinated action. Real-time visualization of single filament dynamics showed that OsAIP1 enhanced ADF-mediated severing and dissociation of pointed end subunits. Consistent with this, the filament severing frequency and subunit off-rate were enhanced in OsAIP1 over-expressors but decreased in RNAi protoplasts. Importantly, OsAIP1 acts coordinately with ADF and profilin to induce massive net actin depolymerization, indicating that AIP1 plays a major role in the turnover of actin, which is required to optimize F-actin levels in plants.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Microfilamentos/genética , Oryza/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Destrina/metabolismo , Regulación hacia Abajo , Expresión Génica , Proteínas de Microfilamentos/metabolismo , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Oryza/ultraestructura , Epidermis de la Planta/genética , Epidermis de la Planta/crecimiento & desarrollo , Epidermis de la Planta/metabolismo , Epidermis de la Planta/ultraestructura , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/ultraestructura , Plantas Modificadas Genéticamente , Profilinas/metabolismo , Protoplastos , ARN de Planta/genética , Proteínas Recombinantes , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Plantones/ultraestructura , Imagen de Lapso de Tiempo , Regulación hacia ArribaRESUMEN
The actin cytoskeleton is an important regulator of cell expansion and morphogenesis in plants. However, the molecular mechanisms linking the actin cytoskeleton to these processes remain largely unknown. Here, we report the functional analysis of rice (Oryza sativa) FH5/BENT UPPERMOST INTERNODE1 (BUI1), which encodes a formin-type actin nucleation factor and affects cell expansion and plant morphogenesis in rice. The bui1 mutant displayed pleiotropic phenotypes, including bent uppermost internode, dwarfism, wavy panicle rachis, and enhanced gravitropic response. Cytological observation indicated that the growth defects of bui1 were caused mainly by inhibition of cell expansion. Map-based cloning revealed that BUI1 encodes the class II formin FH5. FH5 contains a phosphatase tensin-like domain at its amino terminus and two highly conserved formin-homology domains, FH1 and FH2. In vitro biochemical analyses indicated that FH5 is capable of nucleating actin assembly from free or profilin-bound monomeric actin. FH5 also interacts with the barbed end of actin filaments and prevents the addition and loss of actin subunits from the same end. Interestingly, the FH2 domain of FH5 could bundle actin filaments directly and stabilize actin filaments in vitro. Consistent with these in vitro biochemical activities of FH5/BUI1, the amount of filamentous actin decreased, and the longitudinal actin cables almost disappeared in bui1 cells. The FH2 or FH1FH2 domains of FH5 could also bind to and bundle microtubules in vitro. Thus, our study identified a rice formin protein that regulates de novo actin nucleation and spatial organization of the actin filaments, which are important for proper cell expansion and rice morphogenesis.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Microfilamentos/metabolismo , Oryza/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Gravitropismo , Proteínas de Microfilamentos/genética , Mutación , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Plantones/crecimiento & desarrolloRESUMEN
A dynamic actin cytoskeleton is essential for pollen germination and tube growth. However, the molecular mechanisms underlying the organization and turnover of the actin cytoskeleton in pollen remain poorly understood. Villin plays a key role in the formation of higher-order structures from actin filaments and in the regulation of actin dynamics in eukaryotic cells. It belongs to the villin/gelsolin/fragmin superfamily of actin binding proteins and is composed of six gelsolin-homology domains at its core and a villin headpiece domain at its C terminus. Recently, several villin family members from plants have been shown to sever, cap, and bundle actin filaments in vitro. Here, we characterized a villin isovariant, Arabidopsis thaliana VILLIN5 (VLN5), that is highly and preferentially expressed in pollen. VLN5 loss-of-function retarded pollen tube growth and sensitized actin filaments in pollen grains and tubes to latrunculin B. In vitro biochemical analyses revealed that VLN5 is a typical member of the villin family and retains a full suite of activities, including barbed-end capping, filament bundling, and calcium-dependent severing. The severing activity was confirmed with time-lapse evanescent wave microscopy of individual actin filaments in vitro. We propose that VLN5 is a major regulator of actin filament stability and turnover that functions in concert with oscillatory calcium gradients in pollen and therefore plays an integral role in pollen germination and tube growth.
Asunto(s)
Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Microfilamentos/metabolismo , Tubo Polínico/crecimiento & desarrollo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Calcio/metabolismo , Citocalasina D/farmacología , Proteínas de Microfilamentos/genética , Mutación , ARN de Planta/genética , Tiazolidinas/farmacologíaRESUMEN
Patients with ovarian cancer are at increased risk of venous thromboembolism (VTE), and the cumulative incidence is high, particularly at advanced stages of this disease. Nevertheless, it is challenging to investigate the molecular mechanisms of ovarian cancer-associated VTE (OC-VTE), mainly due to the lack of a well-developed animal model for this disease. We generated a mouse model for developing OC-VTE using ovarian cancer cell injection in combination with the inferior vena cava stenosis method. The rate of thrombosis in the OC-VTE group was 50%, compared with 0 in the control group. Moreover, we conducted a proteomic analysis using platelets from these models and revealed differentially expressed proteins between the OC-VTE and control groups, including upregulated and downregulated proteins. Gene Ontology analysis revealed that these differentially expressed proteins were mostly enriched in the biological process of negative regulation of fibrinolysis and the cellular component of the fibrinogen complex, both of which play key roles in thrombosis. In conclusion, this study lays the foundation for further investigation of the underlying mechanisms of how ovarian cancer promotes VTE formation.
Asunto(s)
Neoplasias Ováricas , Trombosis , Tromboembolia Venosa , Humanos , Femenino , Animales , Ratones , Tromboembolia Venosa/epidemiología , Proteómica , Incidencia , Factores de RiesgoRESUMEN
Despite significant strides promoting axon regeneration after spinal cord injury (SCI), meaningful functional recovery remains elusive. Using a combination of approaches, Squair et al.1 elegantly demonstrate that axons damaged after SCI must be reconnected with their natural targets to recover lost neurological functions.
Asunto(s)
Axones , Traumatismos de la Médula Espinal , Humanos , Axones/fisiología , Regeneración Nerviosa/fisiología , Neuronas , Traumatismos de la Médula Espinal/terapia , Parálisis , Caminata , Médula Espinal , Recuperación de la Función/fisiologíaRESUMEN
Promotion of remyelination is an important therapeutic strategy to facilitate functional recovery after traumatic spinal cord injury (SCI). Transplantation of neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) has been used to enhance remyelination after SCI. However, the microenvironment in the injured spinal cord is inhibitory for oligodendrocyte (OL) differentiation of NSCs or OPCs. Identifying the signaling pathways that inhibit OL differentiation in the injured spinal cord could lead to new therapeutic strategies to enhance remyelination and functional recovery after SCI. In the present study, we show that reactive astrocytes from the injured rat spinal cord or their conditioned media inhibit OL differentiation of adult OPCs with concurrent promotion of astrocyte differentiation. The expression of bone morphogenetic proteins (BMP) is dramatically increased in the reactive astrocytes and their conditioned media. Importantly, blocking BMP activity by BMP receptor antagonist, noggin, reverse the effects of active astrocytes on OPC differentiation by increasing the differentiation of OL from OPCs while decreasing the generation of astrocytes. These data indicate that the upregulated bone morphogenetic proteins in the reactive astrocytes are major factors to inhibit OL differentiation of OPCs and to promote its astrocyte differentiation. These data suggest that manipulation of BMP signaling in the endogenous or grafted NSCs or OPCs may be a useful therapeutic strategy to increase their OL differentiation and remyelination and enhance functional recovery after SCI.
Asunto(s)
Astrocitos/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/fisiología , Oligodendroglía/metabolismo , Médula Espinal/metabolismo , Células Madre/metabolismo , Animales , Astrocitos/citología , Western Blotting , Células Cultivadas , Técnicas de Cocultivo , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/metabolismo , Oligodendroglía/citología , Ratas , Transducción de Señal/fisiología , Médula Espinal/citología , Traumatismos de la Médula Espinal/metabolismo , Células Madre/citologíaRESUMEN
Cytoplasmic actin cables are the most prominent actin structures in plant cells, but the molecular mechanism underlying their formation is unknown. The function of these actin cables, which are proposed to modulate cytoplasmic streaming and intracellular movement of many organelles in plants, has not been studied by genetic means. Here, we show that Arabidopsis thaliana formin3 (AFH3) is an actin nucleation factor responsible for the formation of longitudinal actin cables in pollen tubes. The Arabidopsis AFH3 gene encodes a 785-amino acid polypeptide, which contains a formin homology 1 (FH1) and a FH2 domain. In vitro analysis revealed that the AFH3 FH1FH2 domains interact with the barbed end of actin filaments and have actin nucleation activity in the presence of G-actin or G actin-profilin. Overexpression of AFH3 in tobacco (Nicotiana tabacum) pollen tubes induced excessive actin cables, which extended into the tubes' apices. Specific downregulation of AFH3 eliminated actin cables in Arabidopsis pollen tubes and reduced the level of actin polymers in pollen grains. This led to the disruption of the reverse fountain streaming pattern in pollen tubes, confirming a role for actin cables in the regulation of cytoplasmic streaming. Furthermore, these tubes became wide and short and swelled at their tips, suggesting that actin cables may regulate growth polarity in pollen tubes. Thus, AFH3 regulates the formation of actin cables, which are important for cytoplasmic streaming and polarized growth in pollen tubes.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Proteínas de la Membrana/metabolismo , Tubo Polínico/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Corriente Citoplasmática , ADN Complementario/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de la Membrana/genética , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Tubo Polínico/metabolismo , ARN de Planta/genéticaRESUMEN
Formin is a major protein responsible for regulating the nucleation of actin filaments, and as such, it permits the cell to control where and when to assemble actin arrays. It is encoded by a multigene family comprising 21 members in Arabidopsis thaliana. The Arabidopsis formins can be separated into two phylogenetically-distinct classes: there are 11 class I formins and 10 class II formins. Significant questions remain unanswered regarding the molecular mechanism of actin nucleation and elongation stimulated by each formin isovariant, and how the different isovariants coordinate to regulate actin dynamics in cells. Here, we characterize a class II formin, AtFH19, biochemically. We found that AtFH19 retains all general properties of the formin family, including nucleation and barbed end capping activity. It can also generate actin filaments from a pool of actin monomers bound to profilin. However, both the nucleation and barbed end capping activities of AtFH19 are less efficient compared to those of another well-characterized formin, AtFH1. Interestingly, AtFH19 FH1FH2 competes with AtFH1 FH1FH2 in binding actin filament barbed ends, and inhibits the effect of AtFH1 FH1FH2 on actin. We thus propose a mechanism in which two quantitatively different formins coordinate to regulate actin dynamics by competing for actin filament barbed ends.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Microfilamentos/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Microfilamentos/química , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
Multifaceted functions of the so-called "oncogene" Wip1 have been reported in a previous study, while its actual role remains to be explored in serous ovarian cancer (SOC). In this study, by performing bioinformatic analysis with a public database and immunohistochemical staining of Wip1 in tumour tissue from SOC, we concluded that decreased expression of Wip1 was associated with a higher rate of tumour metastasis and platinum-based therapy resistance and increased ascites volume, which led to poorer prognosis in SOC patients. We also found that overexpression of Wip1 in SKOV3 cells decreased the levels of several cytokines, including VEGF, by secretome profiling analysis, and Wip1 overexpression suppressed angiogenesis both in vitro and in vivo. Mechanistic studies indicated that overexpression of Wip1 decreased the expression of VEGF at both the protein and mRNA levels and that the inhibitory effect was mediated by dephosphorylation of STAT3 at Ser727. Our study uncovered the role of Wip1 in SOC and provides a novel therapeutic strategy for suppressing angiogenesis.
Asunto(s)
Cistadenocarcinoma Seroso , Neoplasias Ováricas , Proteína Fosfatasa 2C , Factor de Transcripción STAT3 , Factor A de Crecimiento Endotelial Vascular , Carcinoma Epitelial de Ovario/irrigación sanguínea , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Línea Celular Tumoral , Cistadenocarcinoma Seroso/irrigación sanguínea , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Femenino , Humanos , Neovascularización Patológica/genética , Neoplasias Ováricas/irrigación sanguínea , Neoplasias Ováricas/patología , Proteína Fosfatasa 2C/genética , Proteína Fosfatasa 2C/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Locomotor function after spinal cord injury (SCI) is critical for assessing recovery. Currently, available means to improve locomotor function include surgery, physical therapy rehabilitation and exoskeleton. Stem cell therapy with neural progenitor cells (NPCs) transplantation is a promising reparative strategy. Along this line, patient-specific induced pluripotent stem cells (iPSCs) are a remarkable autologous cell source, which offer many advantages including: great potential to generate isografts avoiding immunosuppression; the availability of a variety of somatic cells without ethical controversy related to embryo use; and vast differentiation. In this current work, to realize the therapeutic potential of iPSC-NPCs for the treatment of SCI, we transplanted purified iPSCs-derived NPCs into a cervical contusion SCI rat model. Our results showed that the iPSC-NPCs were able to survive and differentiate into both neurons and astrocytes and, importantly, improve forelimb locomotor function as assessed by the grooming task and horizontal ladder test. Purified iPSC-NPCs represent a promising cell type that could be further tested and developed into a clinically useful cell source for targeted cell therapy for cervical SCI patients.
Asunto(s)
Médula Cervical , Células Madre Pluripotentes Inducidas , Células-Madre Neurales , Traumatismos de la Médula Espinal , Animales , Miembro Anterior , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratas , Traumatismos de la Médula Espinal/metabolismoRESUMEN
Increasing evidence supports the notion that filamentous actin (F-actin) and globular actin exist in the nuclei of somatic cells, and are involved in chromatin remodeling, gene transcription regulation and DNA damage repair. However, the underlying mechanisms of how nuclear F-actin are polymerized in cells remain incompletely understood. Here, we identify potential kinase targets that participate in nuclear F-actin polymerization in ovarian cancer cells using small-molecule inhibitor library screening in combination with a deep learning approach. The analysis of the targets of the inhibitors used in this study suggest that the PI3K-AKT pathway are involved in regulating nuclear F-actin organization in ovarian cancer cells. Our work lays the foundation for uncovering the important roles of nuclear F-actin in the context of ovarian cancer, and for understanding how nuclear F-actin structures are organized.
RESUMEN
Müllerian tissue-specific oncogenes, prototyped by PAX8, underlie ovarian tumorigenesis and represent unique molecular vulnerabilities. Further delineating such lineage-dependency factors and associated therapeutic implications would provide valuable insights into ovarian cancer biology and treatment. In this study, we identified SOX17 as a new lineage-survival master transcription factor, which shared co-expression pattern with PAX8 in epithelial ovarian carcinoma. Genetic disruption of SOX17 or PAX8 analogously inhibited neoplastic cell viability and downregulated a spectrum of lineage-related transcripts. Mechanistically, we showed that SOX17 physically interacted with PAX8 in cultured cell lines and clinical tumor specimens. The two nuclear proteins bound to overlapping genomic regions and regulated a common set of downstream genes, including those involved in cell cycle and tissue morphogenesis. In addition, we revealed that small-molecule inhibitors of transcriptional cyclin-dependent kinases (CDKs) effectively reduced SOX17 and PAX8 expression. ZSQ1722, a novel orally bioavailable CDK12/13 covalent antagonist, exerted potent anti-tumor activity in xenograft models. These findings shed light on an actionable lineage-survival transcriptional complex in ovarian cancer, and facilitated drug discovery by generating a serial of candidate compounds to pharmacologically target this difficult-to-treat malignancy.
Asunto(s)
Neoplasias Ováricas , Factor de Transcripción PAX8 , Factores de Transcripción SOXF , Ciclo Celular , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Factor de Transcripción PAX8/genética , Factor de Transcripción PAX8/metabolismo , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismoRESUMEN
Spinal cord injury (SCI) is one of the most devastating neural injuries without effective therapeutic solutions. Astrocytes are the predominant component of the scar. Understanding the complex contributions of reactive astrocytes to SCI pathophysiologies is fundamentally important for developing therapeutic strategies. We have studied the molecular changes in the injury environment and the astrocyte-specific responses by astrocyte purification from injured spinal cords from acute to chronic stages. In addition to protein-coding genes, we have systematically analyzed the expression profiles of long non-coding RNAs (lncRNAs) (>200 bp), which are regulatory RNAs that play important roles in the CNS. We have identified a highly conserved lncRNA, Zeb2os, and demonstrated using functional assays that it plays an important role in reactive astrogliosis through the Zeb2os/Zeb2/Stat3 axis. These studies provide valuable insights into the molecular basis of reactive astrogliosis and fill the knowledge gap regarding the function(s) of lncRNAs in astrogliosis and SCI.
Asunto(s)
Astrocitos/metabolismo , Gliosis/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Humanos , Ratones , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Cotton fiber development at the stages of elongation and secondary wall synthesis determines the traits of fiber length and strength. To date, the mechanisms controlling the progression of these two phases remain elusive. In this work, the function of a fiber-preferential actin-binding protein (GhPFN2) was characterized by cytological and molecular studies on the fibers of transgenic green-colored cotton (Gossypium hirsutum) through three successive generations. Overexpression of GhPFN2 caused pre-terminated cell elongation, resulting in a marked decrease in the length of mature fibers. Cytoskeleton staining and quantitative assay revealed that thicker and more abundant F-actin bundles formed during the elongation stage in GhPFN2-overexpressing fibers. Accompanying this alteration, the developmental reorientation of transverse microtubules to the oblique direction was advanced by 2 d at the period of transition from elongation to secondary wall deposition. Birefringence and reverse transcription-PCR analyses showed that earlier onset of secondary wall synthesis occurred in parallel. These data demonstrate that formation of the higher actin structure plays a determinant role in the progression of developmental phases in cotton fibers, and that GhPFN2 acts as a critical modulator in this process. Such a function of the actin cytoskeleton in cell phase conversion may be common to other secondary wall-containing plant cells.
Asunto(s)
Fibra de Algodón , Gossypium/genética , Proteínas de Plantas/metabolismo , Profilinas/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Pared Celular/metabolismo , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Gossypium/crecimiento & desarrollo , Gossypium/metabolismo , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Profilinas/genética , ARN de Planta/genéticaRESUMEN
Injection of viral vectors into peripheral nerves may transfer specific genes into their dorsal root ganglion (DRG) neurons and motoneurons. However, myelin sheaths of peripheral axons block the entry of viral particles into nerves. We studied whether mild, transient peripheral nerve demyelination prior to intraneural viral vector injection would enhance gene transfer to target DRG neurons and motoneurons. The right sciatic nerve of C57BL/6 mice was focally demyelinated with 1% lysolecithin, and the left sciatic nerve was similarly injected with saline (control). Five days after demyelination, 0.5 microl of Ad5-GFP was injected into both sciatic nerves at the site of previous injection. The effectiveness of gene transfer was evaluated by counting GFP(+) neurons in the DRGs and ventral horns. After peripheral nerve demyelination, there was a fivefold increase in the number of infected DRG neurons and almost a 15-fold increase in the number of infected motoneurons compared with the control, nondemyelinated side. Focal demyelination reduced the myelin sheath barrier, allowing greater virus-axon contact. Increased CXADR expression on the demyelinated axons facilitated axoplasmic viral entry. No animals sustained any prolonged neurological deficits. Increased gene delivery into DRG neurons and motoneurons may provide effective treatment for amyotrophic lateral sclerosis, pain, and spinal cord injury.
Asunto(s)
Adenoviridae/genética , Enfermedades Desmielinizantes/patología , Ganglios Espinales/fisiología , Neuronas Motoras/fisiología , Neuronas/fisiología , Nervios Periféricos/fisiología , Animales , Western Blotting , Enfermedades Desmielinizantes/inducido químicamente , Enterovirus , Femenino , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Inmunohistoquímica , Locomoción/fisiología , Lisofosfatidilcolinas , Ratones , Ratones Endogámicos C57BL , Neuritis/inducido químicamente , Neuritis/patología , Receptores Virales/genética , Nervio Ciático/metabolismo , Nervio Ciático/patología , Sensación/fisiologíaRESUMEN
Previous studies suggest locomotion training could be an effective non-invasive therapy after spinal cord injury (SCI) using primarily acute thoracic injuries. However, the majority of SCI patients have chronic cervical injuries. Regaining hand function could significantly increase their quality of life. In this study, we used a clinically relevant chronic cervical contusion to study the therapeutic efficacy of rehabilitation in forelimb functional recovery. Nude rats received a moderate C5 unilateral contusive injury and were then divided into two groups with or without Modified Montoya Staircase (MMS) rehabilitation. For the rehabilitation group, rats were trained 5 days a week starting at 8 weeks post-injury (PI) for 6 weeks. All rats were assessed for skilled forelimb functions with MMS test weekly and for untrained gross forelimb locomotion with grooming and horizontal ladder (HL) tests biweekly. Our results showed that MMS rehabilitation significantly increased the number of pellets taken at 13 and 14 weeks PI and the accuracy rates at 12 to 14 weeks PI. However, there were no significant differences in the grooming scores or the percentage of HL missteps at any time point. Histological analyses revealed that MMS rehabilitation significantly increased the number of serotonergic fibers and the amount of presynaptic terminals around motor neurons in the cervical ventral horns caudal to the injury and reduced glial fibrillary acidic protein (GFAP)-immunoreactive astrogliosis in spinal cords caudal to the lesion. This study shows that MMS rehabilitation can modify the injury environment, promote axonal sprouting and synaptic plasticity, and importantly, improve reaching and grasping functions in the forelimb, supporting the therapeutic potential of task-specific rehabilitation for functional recovery after chronic SCI.
RESUMEN
The inter-differentiation between cell states promotes cancer cell survival under stress and fosters non-genetic heterogeneity (NGH). NGH is, therefore, a surrogate of tumor resilience but its quantification is confounded by genetic heterogeneity. Here we show that NGH in serous ovarian cancer (SOC) can be accurately measured when informed by the molecular signatures of the normal fallopian tube epithelium (FTE) cells, the cells of origin of SOC. Surveying the transcriptomes of â¼6,000 FTE cells, predominantly from non-ovarian cancer patients, identified 6 FTE subtypes. We used subtype signatures to deconvolute SOC expression data and found substantial intra-tumor NGH. Importantly, NGH-based stratification of â¼1,700 tumors robustly correlated with survival. Our findings lay the foundation for accurate prognostic and therapeutic stratification of SOC.
Asunto(s)
Células Epiteliales/patología , Neoplasias de las Trompas Uterinas/metabolismo , Trompas Uterinas/patología , Neoplasias Ováricas/patología , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Epitelio/metabolismo , Epitelio/patología , Neoplasias de las Trompas Uterinas/genética , Neoplasias de las Trompas Uterinas/patología , Trompas Uterinas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Heterogeneidad Genética , Humanos , Neoplasias Ováricas/metabolismoRESUMEN
Metastasis is a complex multistep process that involves critical interactions between cancer cells and a variety of stromal components in the tumor microenvironment, which profoundly influence the different aspects of the metastatic cascade and organ tropism of disseminating cancer cells. Ovarian cancer is the most lethal gynecological malignancy and is characterized by peritoneal disseminated metastasis. Evidence has demonstrated that ovarian cancer possesses specific metastatic tropism for the adipose-rich omentum, which has a pivotal role in the creation of the metastatic tumor microenvironment in the intraperitoneal cavity. Considering the distinct biology of ovarian cancer metastasis, the elucidation of the cellular and molecular mechanisms underlying the reciprocal interplay between ovarian cancer cells and surrounding stromal cell types in the adipose-rich metastatic microenvironment will provide further insights into the development of novel therapeutic approaches for patients with advanced ovarian cancer. Herein, we review the biological mechanisms that regulate the highly orchestrated crosstalk between ovarian cancer cells and various cancer-associated stromal cells in the metastatic tumor microenvironment with regard to the omentum by illustrating how different stromal cells concertedly contribute to the development of ovarian cancer metastasis and metastatic tropism for the omentum.
Asunto(s)
Tejido Adiposo/patología , Neoplasias Ováricas/patología , Microambiente Tumoral/fisiología , Femenino , Humanos , Epiplón/patología , Tropismo/fisiologíaRESUMEN
Contusive spinal cord injury (SCI) is the most common type of spinal injury seen clinically. Several rat contusion SCI models have been described, and all have strengths and weaknesses with respect to sensitivity, reproducibility, and clinical relevance. We developed the Louisville Injury System Apparatus (LISA), which contains a novel spine-stabilizing device that enables precise and stable spine fixation, and is based on tissue displacement to determine the severity of injury. Injuries graded from mild to moderately severe were produced using 0.2-, 0.4-, 0.6-, 0.8-, 1.0-, and 1.2-mm spinal cord displacement in rats. Basso, Beattie, and Bresnahan (BBB) and Louisville Swim Score (LSS) could not significantly distinguish between 0.2-mm lesion severities, except those of 0.6- and 0.8-mm BBB scores, but could between 0.4-mm injury differences or if the data were grouped (0.2-0.4, 0.6-0.8, and 1.0-1.2). Transcranial magnetic motor evoked potential (tcMMEP) response amplitudes were decreased 10-fold at 0.2-mm displacement, barely detected at 0.4-mm displacement, and absent with greater displacement injuries. In contrast, somatosensory evoked potentials (SSEPs) were recorded at 0.2- and 0.4-mm displacements with normal amplitudes and latencies but were detected at lower amplitudes at 0.6-mm displacement and absent with more severe injuries. Analyzing combined BBB, tcMMEP, and SSEP results enabled statistically significant discrimination between 0.2-, 0.4-, 0.6-, and 0.8-mm displacement injuries but not the more severe injuries. Present data document that the LISA produces reliable and reproducible SCI whose parameters of injury can be adjusted to more accurately reflect clinical SCI. Moreover, multiple outcome measures are necessary to accurately detect small differences in functional deficits and/or recovery. This is of crucial importance when trying to detect functional improvement after therapeutic intervention to treat SCI.