Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis.

BackgroundKey effector(s) of mutated KRAS in lung cancer progression and metastasis are unknown. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic KRAS signal in non-small cell lung cancer (NSCLC).MethodsWe used NSCLC clinical specimens to examine the correlation among KRAS mutations (codon 12, 13 and 61); PAK1/Crk axis activation [p-PAK1(Thr423), p-Crk(Ser41)]; and adhesion molecules expression by immunohistochemistry. For assessing the role of proto-oncogene c-Crk as a KRAS effector, we inhibited KRAS in NSCLC cells by a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and measured p-Crk-II(Ser41) by western blotting. Finally, we disrupted the signaling network downstream of KRAS by blocking KRAS/PAK1/Crk axis with PAK1 inhibitors (i.e., IPA-3, FRAX597 or FRAX1036) along with partial inhibition of all other KRAS effectors by prenylation inhibitors (FTI + GGTI) and examined the motility, morphology and proliferation of the NSCLC cells.ResultsImmunohistochemical analysis demonstrated an inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type. KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent. Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells.ConclusionsOur data provide evidence that proto-oncogene c-Crk is operative downstream of KRAS in NSCLC. Previously we demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of KRAS signal bring forward the importance of KRAS/PAK1/Crk axis as a prominent pathway in the oncogenesis of KRAS mutant lung cancer

Diseño de un sistema robótico para la clasificación automatizada de espárragos empleando visión artificial por imágenes

Se sabe que la clasificación de frutos en la industria agrícola peruana aún se realiza de manera muy rudimentaria, en especial, si se trata de productos de exportación que generan gran ingreso de divisas; por ello, el trabajo de investigación dio una solución eficaz para la clasificación automatizada de espárragos por medio de la integración de un sistema robótico, procesamiento de imágenes y redes neuronales artificiales. Para dicho propósito se adquirieron imágenes de dicha hortaliza que sirvieron de entrada a un algoritmo basado en redes neuronales previamente entrenado con características de tamaño y color que permitan la clasificación mediante el software Matlab. Posteriormente, se procedió al diseño del sistema robótico, cámara web, dispositivos electrónicos, etc. Por otro lado, un controlador fue la intermediaria en la comunicación del algoritmo y el actuador que realizó la clasificación de espárragos. Finalmente, se obtuvo un porcentaje de acierto del 97.5% de la red neuronal para la clasificación de espárragos con lo cual se concluyó que la visión artificial es una solución óptima para aumentar la productividad de la industria agroexportadora.Trabajo de investigaciónCampus Lima Centr

A2ML1 and otitis media : novel variants, differential expression, and relevant pathways

A genetic basis for otitis media is established, however, the role of rare variants in disease etiology is largely unknown. Previously a duplication variant within A2ML1 was identified as a significant risk factor for otitis media in an indigenous Filipino population and in US children. In this report exome and Sanger sequencing was performed using DNA samples from the indigenous Filipino population, Filipino cochlear implantees, US probands, Finnish, and Pakistani families with otitis media. Sixteen novel, damaging A2ML1 variants identified in otitis media patients were rare or low-frequency in population-matched controls. In the indigenous population, both gingivitis and A2ML1 variants including the known duplication variant and the novel splice variant c.4061 + 1 G>C were independently associated with otitis media. Sequencing of salivary RNA samples from indigenous Filipinos demonstrated lower A2ML1 expression according to the carriage of A2ML1 variants. Sequencing of additional salivary RNA samples from US patients with otitis media revealed differentially expressed genes that are highly correlated with A2ML1 expression levels. In particular, RND3 is upregulated in both A2ML1 variant carriers and high-A2ML1 expressors. These findings support a role for A2ML1 in keratinocyte differentiation within the middle ear as part of otitis media pathology and the potential application of ROCK inhibition in otitis media.Peer reviewe

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Diseño de un sistema robótico para la clasificación automatizada de espárragos empleando visión artificial por imágenes

Trabado de investigacionSe sabe que la clasificación de frutos en la industria agrícola peruana aún se realiza de manera muy rudimentaria, en especial, si se trata de productos de exportación que generan gran ingreso de divisas; por ello, el trabajo de investigación dio una solución eficaz para la clasificación automatizada de espárragos por medio de la integración de un sistema robótico, procesamiento de imágenes y redes neuronales artificiales. Para dicho propósito se adquirieron imágenes de dicha hortaliza que sirvieron de entrada a un algoritmo basado en redes neuronales previamente entrenado con características de tamaño y color que permitan la clasificación mediante el software Matlab. Posteriormente, se procedió al diseño del sistema robótico, cámara web, dispositivos electrónicos, etc. Por otro lado, un controlador fue la intermediaria en la comunicación del algoritmo y el actuador que realizó la clasificación de espárragos. Finalmente, se obtuvo un porcentaje de acierto del 97.5% de la red neuronal para la clasificación de espárragos con lo cual se concluyó que la visión artificial es una solución óptima para aumentar la productividad de la industria agroexportadora

PAK1 kinase promotes cell motility and invasiveness through CRK-II serine phosphorylation in non-small cell lung cancer cells.

The role of c-Crk (CRK) in promoting metastasis is well described however the role of CRK phosphorylation and the corresponding signaling events are not well explained. We have observed CRK-II serine 41 phosphorylation is inversely correlated with p120-catenin and E-cadherin expressions in non-small cell lung cancer (NSCLC) cells. Therefore, we investigated the role of CRK-II serine 41 phosphorylation in the down-regulation of p120-catenin, cell motility and cell invasiveness in NSCLC cells. For this purpose, we expressed phosphomimetic and phosphodeficient CRK-II serine 41 mutants in NSCLC cells. NSCLC cells expressing phosphomimetic CRK-II seine 41 mutant showed lower p120-catenin level while CRK-II seine 41 phosphodeficient mutant expression resulted in higher p120-catenin. In addition, A549 cells expressing CRK-II serine 41 phosphomimetic mutant demonstrated more aggressive behavior in wound healing and invasion assays and, on the contrary, expression of phosphodeficient CRK-II serine 41 mutant in A549 cells resulted in reduced cell motility and invasiveness. We also provide evidence that PAK1 mediates CRK-II serine 41 phosphorylation. RNAi mediated silencing of PAK1 increased p120-catenin level in A549 and H157 cells. Furthermore, PAK1 silencing decreased cell motility and invasiveness in A549 cells. These effects were abrogated in A549 cells expressing phosphomimetic CRK-II serine 41. In summary, these data provide evidence for the role of PAK1 in the promotion of cell motility, cell invasiveness and the down regulation of p120-catenin through CRK serine 41 phosphorylation in NSCLC cells

Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis.

BackgroundKey effector(s) of mutated KRAS in lung cancer progression and metastasis are unknown. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic KRAS signal in non-small cell lung cancer (NSCLC).MethodsWe used NSCLC clinical specimens to examine the correlation among KRAS mutations (codon 12, 13 and 61); PAK1/Crk axis activation [p-PAK1(Thr423), p-Crk(Ser41)]; and adhesion molecules expression by immunohistochemistry. For assessing the role of proto-oncogene c-Crk as a KRAS effector, we inhibited KRAS in NSCLC cells by a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and measured p-Crk-II(Ser41) by western blotting. Finally, we disrupted the signaling network downstream of KRAS by blocking KRAS/PAK1/Crk axis with PAK1 inhibitors (i.e., IPA-3, FRAX597 or FRAX1036) along with partial inhibition of all other KRAS effectors by prenylation inhibitors (FTI + GGTI) and examined the motility, morphology and proliferation of the NSCLC cells.ResultsImmunohistochemical analysis demonstrated an inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type. KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent. Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells.ConclusionsOur data provide evidence that proto-oncogene c-Crk is operative downstream of KRAS in NSCLC. Previously we demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of KRAS signal bring forward the importance of KRAS/PAK1/Crk axis as a prominent pathway in the oncogenesis of KRAS mutant lung cancer

A-Wound healing assays in A549 cells stably expressing pCMV vector, wild type CRK-II (WT), CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants.

<p>Endogenous CRK is silenced in all conditions by siRNA. B- Measurement of wound surface area 24 hours after establishment of the wound among the above mentioned groups. The average is calculated following measurement of three separate experiments. (2 tailed student’s t-test: ** P<0.01; *** P<0.001; error bars represent ± standard deviation).C- Invasion assays following incubation of stably transfected A549 cells expressing either pCMV vector; wild type CRK-II (WT); CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants. 1×10⁵ cells were incubated over night as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042012#s4" target="_blank">Methods</a> section and stained at 24 hours after incubation. D- Quantitative measurement of the invaded cells. Five separate sections of the invaded cells were counted. (2 tailed student’s t-test: ** P<0.01; *** P<0.001; error bars represent ± standard deviation).</p