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Development of novel medical devices for the treatment of musculoskeletal pain associated with neuro-muscular trigger points requires a model for relating the mechanical responses of in vivo biological tissues to applied palliative physical pressures and a method to design treatments for optimal effects. It is reasonable to hypothesize that the efficacy of therapeutic treatment is proportional to the maximum tensile strain at trigger point locations. This work presents modeling of the mechanical behavior of biological tissue structures and treatment simulations, supported by indentation experiments and finite element (FE) modeling. The steady-state indentation responses of the tissue structure of the posterior neck were measured with a testing device, and an FE model was constructed using a first-order Ogden hyperelastic material model and calibrated with the experimental data. The error between experimental and FE-generated displacement-load curves was minimized via a two-stage optimization process comprised of an Optimal Latin Hypercube design-of-experiments analysis and a Bayesian optimization loop. The optimized Ogden model had an initial shear modulus (µ) of 5.16 kPa and a deviatoric exponent (α) of 11.90. Another FE model was developed to simulate the deformation of the tissue structures in the posterior neck adjacent to the C3 vertebrae in response to indentation loading, in order to determine the optimal location and angle to apply an indentation force for maximum therapeutic benefit. The optimal location of indentation was determined to be 28° lateral from the sagittal plane along the surface of the skin, measured from the centerline of the spine, at an angle of 8° counterclockwise from the surface normal vector. The optimized spatial orientation of the indentation corresponded to the average of the maximum principal strain across the deep muscle region of the model.
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Análisis de Elementos Finitos , Ensayo de Materiales , Músculo Esquelético , Músculo Esquelético/fisiología , Ensayo de Materiales/instrumentación , Pruebas Mecánicas , Humanos , Fenómenos Mecánicos , Fenómenos Biomecánicos , Modelos Biológicos , Estrés Mecánico , Equipos y SuministrosRESUMEN
Ischemic stroke, constituting 80-90% of all strokes, is a leading cause of death and long-term disability in adults. There is an urgent need to discover new targets and therapies for this devastating condition. Protein kinase D (PKD), as a key target of diacylglycerol involved in ischemic responses, has not been well studied in ischemic stroke, particularly PKD2. In this study, we found that PKD2 expression and activity were significantly upregulated in the ipsilateral side of the brain after transient focal cerebral ischemia, which coincides with the upregulation of PKD2 in primary neurons in response to in vitro ischemia, implying a potential role of PKD2 in neuronal survival in ischemic stroke. Using kinase-dead PKD2 knock-in (PKD2-KI) mice, we examined whether loss of PKD2 activity affected stroke outcomes in mice subjected to 1 h of transient middle cerebral artery occlusion (tMCAO) and 24 h of reperfusion. Our data demonstrated that PKD2-KI mice exhibited larger infarction volumes and worsened neurological scores, indicative of increased brain injury, as compared to the wild-type (WT) mice, confirming a neuroprotective role of PKD2 in ischemia/reperfusion (I/R) injury. Mouse primary neurons obtained from PKD2-KI mice also exhibited increased cell death as compared to the WT neurons when subjected to in vitro ischemia. We have further identified AKT and CREB as two main signaling nodes through which PKD2 regulates neuronal survival during I/R injury. In summary, PKD2 confers neuroprotection in ischemic stroke by promoting AKT and CREB activation and targeted activation of PKD2 may benefit neuronal survival in ischemic stroke.
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Isquemia Encefálica , Ataque Isquémico Transitorio , Accidente Cerebrovascular Isquémico , Fármacos Neuroprotectores , Daño por Reperfusión , Accidente Cerebrovascular , Ratones , Animales , Neuroprotección , Proteínas Proto-Oncogénicas c-akt/metabolismo , Isquemia Encefálica/metabolismo , Proteína Quinasa D2 , Transducción de Señal , Fármacos Neuroprotectores/farmacología , Infarto de la Arteria Cerebral MediaRESUMEN
All-solid-state batteries (ASSB) can potentially achieve high gravimetric and volumetric energy densities (900 Wh/L) if paired with a lithium metal anode and solid electrolyte. However, there is a lack in critical understanding about how to operate lithium metal cells at high capacities and minimize unwanted degradation mechanisms such as dendrites and voids. Herein, we investigate how pressure and temperature influence the formation and annihilation of unrecoverable voids in lithium metal upon stripping. Stack pressure and temperature are effective means to initiate creep-induced void filling and decrease charge transfer resistances. Applying stack pressure enables lithium to deform and creep below the yield stress during stripping at high current densities. Lithium creep is not sufficient to prevent cell shorting during plating. Three-electrode experiments were employed to probe the kinetic and morphological limitations that occur at the anode-solid electrolyte during high-capacity stripping (5 mAh/cm2). The role of cathode-LLZO interface, which dictates cyclability and capacity retention in full cells, was also studied. This work elucidates the important role that temperature (external or in situ generated) has on reversible operation of solid-state batteries.
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Protein kinase D (PKD) has been linked to inflammatory responses in various pathologic conditions; however, its role in inflammation-induced dermal fibrosis has not been evaluated. In this study, we aimed to investigate the roles and mechanisms of protein kinase D2 (PKD2) in inflammation-induced dermal fibrosis and evaluate the therapeutic potential of PKD inhibitors in this disease. Using homozygous kinase-dead PKD2 knock-in (KI) mice, we examined whether genetic ablation or pharmacologic inhibition of PKD2 activity affected dermal inflammation and fibrosis in a bleomycin (BLM)-induced skin fibrosis model. Our data showed that dermal thickness and collagen fibers were significantly reduced in BLM-treated PKD2 KI mice compared with that in wild-type mice, and so was the expression of α-smooth muscle actin and collagens and the mRNA levels of transforming growth factor-ß1 and interleukin-6 in the KI mice. Corroboratively, pharmacologic inhibition of PKD by CRT0066101 also significantly blocked BLM-induced dermal fibrosis and reduced α-smooth muscle actin, collagen, and interleukin-6 expression. Further analyses indicated that loss of PKD2 activity significantly blocked BLM-induced infiltration of monocytes/macrophages and neutrophils in the dermis. Moreover, using bone marrow-derived macrophages, we demonstrated that PKD activity was required for cytokine production and migration of macrophages. We have further identified Akt as a major downstream target of PKD2 in the early inflammatory phase of the fibrotic process. Taken together, our findings indicate that PKD2 promotes dermal fibrosis via regulating immune cell infiltration, cytokine production, and downstream activation of Akt in lesional skin, and targeted inhibition of PKD2 may benefit the treatment of this condition.
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Bleomicina , Proteína Quinasa D2 , Esclerodermia Sistémica , Animales , Ratones , Actinas/genética , Actinas/metabolismo , Bleomicina/toxicidad , Colágeno/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Inflamación/metabolismo , Interleucina-6 , Proteína Quinasa D2/genética , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-aktRESUMEN
All-solid-state batteries (ASSBs) using an alkali metal anode and a solid-state electrolyte (SE) face several problems due to poor physical and electrical contact. Recent experiments have shown that applying a stack pressure can improve the interface contact and suppress void formation. The mechanical properties of Na metal are different from those of Li metal, leading to differences in the mechanisms of the pressure-dependent interface evolution. Herein, we report a three-dimensional time-dependent model for tracking the evolution of interfaces formed between Na metal and Na-ßâ³-alumina SE. Our results show that Na metal contacts more conformally with the SE, providing a lower interfacial resistance, compared with Li metal, assuming equal resistance due to contamination. The differences due to contact elastoplasticity are larger than the differences in metal creep effects. In fact, we show that increased stack pressure can lead to lower creep because the contact is more conformal at high pressures. Our excellent agreement with recent experiments determines an effective hardness of Na in the Na-SE batteries to be 15 MPa. The results further reveal that the pressure dependence of void suppression is dominated by contact elastoplasticity.
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Cancer is mainly caused by somatic genome alterations (SGAs). Precision oncology involves identifying and targeting tumor-specific aberrations resulting from causative SGAs. We developed a novel tumor-specific computational framework that finds the likely causative SGAs in an individual tumor and estimates their impact on oncogenic processes, which suggests the disease mechanisms that are acting in that tumor. This information can be used to guide precision oncology. We report a tumor-specific causal inference (TCI) framework, which estimates causative SGAs by modeling causal relationships between SGAs and molecular phenotypes (e.g., transcriptomic, proteomic, or metabolomic changes) within an individual tumor. We applied the TCI algorithm to tumors from The Cancer Genome Atlas (TCGA) and estimated for each tumor the SGAs that causally regulate the differentially expressed genes (DEGs) in that tumor. Overall, TCI identified 634 SGAs that are predicted to cause cancer-related DEGs in a significant number of tumors, including most of the previously known drivers and many novel candidate cancer drivers. The inferred causal relationships are statistically robust and biologically sensible, and multiple lines of experimental evidence support the predicted functional impact of both the well-known and the novel candidate drivers that are predicted by TCI. TCI provides a unified framework that integrates multiple types of SGAs and molecular phenotypes to estimate which genome perturbations are causally influencing one or more molecular/cellular phenotypes in an individual tumor. By identifying major candidate drivers and revealing their functional impact in an individual tumor, TCI sheds light on the disease mechanisms of that tumor, which can serve to advance our basic knowledge of cancer biology and to support precision oncology that provides tailored treatment of individual tumors.
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Neoplasias/genética , Algoritmos , Teorema de Bayes , Biología Computacional , Genoma Humano , Humanos , Modelos Genéticos , Mutación , Neoplasias/etiología , Oncogenes , Fenotipo , Medicina de PrecisiónRESUMEN
Minimizing friction and wear at a rubbing interface continues to be a challenge and has resulted in the recent surge toward the use of coatings such as diamond-like carbon (DLC) on machine components. The problem with the coating approach is the limitation of coating wear life. Here, we report a lubrication approach in which lubricious, wear-protective carbon-containing tribofilms can be self-generated and replenishable, without any surface pretreatment. Such carbon-containing films were formed under modest sliding conditions in a lubricant consisting of cyclopropanecarboxylic acid as an additive dissolved in polyalphaolefin base oil. These tribofilms show the same Raman D and G signatures that have been interpreted to be due to the presence of graphite- or DLC films. Our experimental measurements and reactive molecular dynamics simulations demonstrate that these tribofilms are in fact high-molecular weight hydrocarbons acting as a solid lubricant.
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We recently reported a new molecular heterocyclic friction modifier (FM) that exhibits excellent friction and wear reduction in the boundary lubrication regime. This paper explores the mechanisms by which friction reduction occurs with heterocyclic alkyl-cyclen FM molecules. We find that these chelating molecules adsorb onto (oxidized) steel surfaces far more tenaciously than conventional FMs such as simple alkylamines. Molecular dynamics simulations argue that the surface coverage of our heterocyclic FM molecules remains close to 100% even at 200 °C. This thermal stability allows the FMs to firmly anchor to the surface, allowing the hydrocarbon chains of the molecules to interact and trap base oil lubricant molecules. This results in thicker boundary film thickness compared with conventional FMs, as shown by optical interferometry measurements.
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BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer death in the US. The protein kinase D (PKD) family has emerged as a promising target for cancer therapy with PKD1 being most intensively studied; however, its role in HNSCC has not been investigated. METHODS: The expression of PKD was evaluated in human HNSCC by quantitative RT-PCR, Western blot and immunohistochemistry. Cell proliferation, wound healing, and matrigel invasion assays were performed upon siRNA-mediated knockdown of PKD1 in HNSCC cells, and subcutaneous xenograft mouse model was established by implantation of the stable doxycycline (Dox)-inducible PKD1 expression cell lines for analysis of tumorigenic activity in vivo. RESULTS: PKD1 was frequently downregulated in HNSCC cell lines at both transcript and protein levels. In human HNSCC tissues, PKD1 was significantly down-regulated in localized tumors and metastases, and in patient-paired tumor tissues as compared to their normal counterparts, which was in part due to epigenetic modification of the PRKD1 gene. The function of PKD1 in HNSCC was analyzed using stable doxycycline-inducible cell lines that express native or constitutive-active PKD1. Upon induction, the rate of proliferation, survival, migration and invasion of HNSCC cells did not differ significantly between the control and PKD1 overexpressing cells in the basal state, and depletion of endogenous PKD1 did not impact the proliferation of HNSCC cells. However, the median growth rate of the subcutaneous HNSCC tumor xenografts over time was elevated with PKD1 induction, and the final tumor weight was significantly increased in Dox-induced vs. the non-induced tumors. Moreover, induced expression of PKD1 promoted bombesin-induced cell proliferation of HNSCC and resulted in sustained ERK1/2 activation in response to gastrin-releasing peptide or bombesin stimulation, suggesting that PKD1 potentiates GRP/bombesin-induced mitogenic response through the activation of ERK1/2 in HSNCC cells. CONCLUSIONS: Our study has identified PKD1 as a frequently downregulated gene in HNSCC, and functionally, under certain cellular context, may play a role in GRP/bombesin-induced oncogenesis in HNSCC.
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Proteínas Oncogénicas/genética , Proteína Quinasa C/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Adulto , Anciano , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Metilación de ADN , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Xenoinjertos , Histonas/metabolismo , Humanos , Inmunohistoquímica , Ratones , Persona de Mediana Edad , Familia de Multigenes/genética , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Proteínas Oncogénicas/metabolismo , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
The shear thinning of a lubricant significantly affects lubrication film generation at high shear rates. The critical shear rate, defined at the onset of shear thinning, marks the transition of lubricant behaviors. It is challenging to capture the entire shear-thinning curve by means of molecular dynamics (MD) simulations owing to the low signal-to-noise ratio or long calculation time at comparatively low shear rates (104-106 s-1), which is likely coincident with the shear rates of interest for lubrication applications. This paper proposes an approach that correlates the shear-thinning phenomenon with the change in the molecular conformation characterized by the radius of gyration of the molecule. Such a correlation should be feasible to capture the major mechanism of shear thinning for small- to moderate-sized non-spherical molecules, which is shear-induced molecular alignment. The idea is demonstrated by analyzing the critical shear rate for squalane (C30H62) and 1-decene trimer (C30H62); it is then implemented to study the behaviors of different molecular weight poly-α-olefin (PAO) structures. Time-temperature-pressure superpositioning (TTPS) is demonstrated and it helps further extend the ranges of the temperature and pressure for shear-thinning behavior analyses. The research leads to a relationship between molecular weight and critical shear rate for PAO structures, and the results are compared with those from the Einstein-Debye equation.
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Modern automotive engines operate at higher power densities than ever before, driving a need for new lubricant additives capable of reducing friction and wear further than ever before while not poisoning the catalytic converter. Reported in this paper is a new class of molecular friction modifier (FM), represented by 1,4,7,10-tetradodecyl-1,4,7,10-tetraazacyclododecane (1a), designed to employ thermally stable, sulfur- and phosphorus-free alkyl-substituted nitrogen heterocycles with multiple nitrogen centers per molecule. The multiple nitrogen centers enable cooperative binding to a surface which provides strong surface adsorption and lubricant film durability in the boundary lubrication (BL) regime. A 1 wt % loading of the cyclen FM 1a in Group III base oil exhibits strong surface adsorption, leading to excellent reductions in friction (70%) and wear (95%) versus the pure Group III oil across a wide temperature range. The lubricant with the new FM additive also outperforms two commercially available noncyclic amine-based FMs and a fully formulated commercial 5W30 motor oil.
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The protein kinase D family of serine/threonine kinases, particularly PKD1, has been implicated in the regulation of a complex array of fundamental biological processes. However, its function and mechanism underlying PKD1-mediated the bone development and osteoblast differentiation are not fully understood. Here we demonstrate that loss of PKD1 function led to impaired bone development and osteoblast differentiation through STAT3 and p38 MAPK signaling using in vitro and in vivo bone-specific conditional PKD1-knockout (PKD1-KO) mice models. These mice developed markedly craniofacial dysplasia, scapula dysplasia, long bone length shortage and body weight decrease compared with wild-type littermates. Moreover, deletion of PKD1 in vivo reduced trabecular development and activity of osteoblast development, confirmed by Micro-CT and histological staining as well as expression of osteoblastic marker (OPN, Runx2 and OSX). Mechanistically, loss of PKD1 mediated the downregulation of osteoblast markers and impaired osteoblast differentiation through STAT3 and p38 MAPK signaling pathways. Taken together, these results demonstrated that PKD1 contributes to the osteoblast differentiation and bone development via elevation of osteoblast markers through activation of STAT3 and p38 MAPK signaling pathways.
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Desarrollo Óseo , Diferenciación Celular , Osteoblastos/citología , Osteoblastos/metabolismo , Canales Catiónicos TRPP/deficiencia , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Línea Celular , Fémur/patología , Eliminación de Gen , Quinasas Janus/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Tamaño de los Órganos , Especificidad de Órganos , Factor de Transcripción STAT3/metabolismo , Canales Catiónicos TRPP/metabolismo , Microtomografía por Rayos X , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In prostate cancer, androgen/androgen receptor (AR) and their downstream targets play key roles in all stages of disease progression. The protein kinase D (PKD) family, particularly PKD1, has been implicated in prostate cancer biology. Here, we examined the cross-regulation of PKD1 by androgen signaling in prostate cancer cells. Our data showed that the transcription of PKD1 was repressed by androgen in androgen-sensitive prostate cancer cells. Steroid depletion caused up regulation of PKD1 transcript and protein, an effect that was reversed by the AR agonist R1881 in a time- and concentration-dependent manner, thus identifying PKD1 as a novel androgen-repressed gene. Kinetic analysis indicated that the repression of PKD1 by androgen required the induction of a repressor protein. Furthermore, inhibition or knockdown of AR reversed AR agonist-induced PKD1 repression, indicating that AR was required for the suppression of PKD1 expression by androgen. Downstream of AR, we identified fibroblast growth factor receptor substrate 2 (FRS2) and its downstream MEK/ERK pathway as mediators of androgen-induced PKD1 repression. In summary, PKD1 was identified as a novel androgen-suppressed gene and could be downregulated by androgen through a novel AR/FRS2/MEK/ERK pathway. The upregulation of prosurvival PKD1 by anti-androgens may contribute to therapeutic resistance in prostate cancer treatment.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Andrógenos/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas de la Membrana/metabolismo , Neoplasias de la Próstata/patología , Canales Catiónicos TRPP/biosíntesis , Western Blotting , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Androgénicos/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Protein kinase D (PKD) belongs to a family of serine/threonine kinases in the calcium/calmodulin-dependent kinase superfamily. It modulates a number of signal transduction pathways involved in regulation of cell proliferation, survival, migration, angiogenesis, regulation of gene expression, and protein/membrane trafficking, mediated by variety of stimuli such as growth factors, hormones, and cellular stresses. Although its role in cancer progression remains elusive, current literature supports a potential tumor promoting function of the selective PKD isoforms in prostate cancer, making them promising therapeutic targets for cancer treatment.
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Receptor activation upon ligand binding induces activation of multiple signaling pathways. To fully understand how these signaling pathways coordinate, it is essential to determine the dynamic nature of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we outline a detailed methodology for visualizing and quantitatively measuring the spatiotemporal activation of Ras and PKD1 by applying advanced fluorescence imaging techniques, including multichannel, simultaneous imaging and Förster resonance energy transfer (FRET).
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Microscopía Confocal , Imagen Molecular , Canales Catiónicos TRPP/metabolismo , Proteínas ras/metabolismo , Línea Celular Tumoral , Factores Quimiotácticos/metabolismo , Factores Quimiotácticos/farmacología , Activación Enzimática , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica , Genes Reporteros , Células HL-60 , Humanos , Microscopía Confocal/métodos , Fosforilación , Canales Catiónicos TRPP/genética , Proteínas ras/genéticaRESUMEN
A major challenge in lubrication technology is to enhance lubricant performance at extreme temperatures that exceed conventional engine oil thermal degradation limits. Soft noble metals such as silver have low reactivity and shear strength, which make them ideal solid lubricants for wear protection and friction reduction between contacting surfaces at high temperatures. However, achieving adequate dispersion in engine lubricants and metallic silver deposition over predetermined temperatures ranges presents a significant chemical challenge. Here we report the synthesis, characterization, and tribological implementation of the trimeric silver pyrazolate complex, [Ag(3,5-dimethyl-4-n-hexyl-pyrazolate)]3 (1). This complex is oil-soluble and undergoes clean thermolysis at â¼310 °C to deposit lubricious, protective metallic silver particles on metal/metal oxide surfaces. Temperature-controlled tribometer tests show that greater than 1 wt % loading of 1 reduces wear by 60% in PAO4, a poly-α-olefin lubricant base fluid, and by 70% in a commercial fully formulated 15W40 motor oil (FF oil). This silver-organic complex also imparts sufficient friction reduction so that the tribological transition from oil as the primary lubricant through its thermal degradation, to 1 as the primary lubricant, is experimentally undetectable.
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Protein kinase D (PKD) has been implicated in many aspects of tumorigenesis and progression, and is an emerging molecular target for the development of anticancer therapy. Despite recent advancement in the development of potent and selective PKD small molecule inhibitors, the availability of in vivo active PKD inhibitors remains sparse. In this study, we describe the discovery of a novel PKD small molecule inhibitor, SD-208, from a targeted kinase inhibitor library screen, and the synthesis of a series of analogs to probe the structure-activity relationship (SAR) vs. PKD1. SD-208 displayed a narrow SAR profile, was an ATP-competitive pan-PKD inhibitor with low nanomolar potency and was cell active. Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3. SD-208 also blocked prostate cancer cell survival and invasion, and arrested cells in the G2/M phase of the cell cycle. Mechanistically, SD-208-induced G2/M arrest was accompanied by an increase in levels of p21 in DU145 and PC3 cells as well as elevated phosphorylation of Cdc2 and Cdc25C in DU145 cells. Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL. Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment.
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Proliferación Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Proteína Quinasa C/antagonistas & inhibidores , Pteridinas/farmacología , Animales , Humanos , Masculino , Ratones , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/enzimología , Neoplasias Experimentales/patología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Pteridinas/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signalling, cells that underwent ADM can progress to pancreatic intraepithelial neoplasia (PanIN) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signalling mechanisms are not well understood. Here, using a conditional knockout approach, we show that protein kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras, to mediate formation of ductal structures through activation of the Notch pathway.
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Células Acinares/enzimología , Células Acinares/patología , Carcinoma in Situ/enzimología , Reprogramación Celular , Progresión de la Enfermedad , Neoplasias Pancreáticas/enzimología , Proteína Quinasa C/metabolismo , Células Acinares/efectos de los fármacos , Animales , Carcinoma in Situ/patología , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/patología , Reprogramación Celular/efectos de los fármacos , Ratones Endogámicos C57BL , Conductos Pancreáticos/efectos de los fármacos , Conductos Pancreáticos/patología , Neoplasias Pancreáticas/patología , Fenotipo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptores Notch/metabolismo , Factor de Crecimiento Transformador alfa/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Chemotaxis requires precisely coordinated polymerization and depolymerization of the actin cytoskeleton at leading fronts of migrating cells. However, GPCR activation-controlled F-actin depolymerization remains largely elusive. Here, we reveal a novel signaling pathway, including Gαi, PLC, PKCß, protein kinase D (PKD), and SSH2, in control of cofilin phosphorylation and actin cytoskeletal reorganization, which is essential for neutrophil chemotaxis. We show that PKD is essential for neutrophil chemotaxis and that GPCR-mediated PKD activation depends on PLC/PKC signaling. More importantly, we discover that GPCR activation recruits/activates PLCγ2 in a PI3K-dependent manner. We further verify that PKCß specifically interacts with PKD1 and is required for chemotaxis. Finally, we identify slingshot 2 (SSH2), a phosphatase of cofilin (actin depolymerization factor), as a target of PKD1 that regulates cofilin phosphorylation and remodeling of the actin cytoskeleton during neutrophil chemotaxis.
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Quimiotaxis de Leucocito/inmunología , Cofilina 1/metabolismo , Neutrófilos/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Transducción de Señal , Citoesqueleto de Actina/metabolismo , Animales , Femenino , Humanos , Masculino , Ratones , Neutrófilos/enzimología , Neutrófilos/inmunología , Fosfolipasa C beta/metabolismo , Fosfolipasa C gamma/metabolismo , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/inmunologíaRESUMEN
A series of light- and air-stable silver(I) pyrazolylmethylpyridine complexes [Ag(L(R))]n(BF4)n (L = pyrazolylmethylpyridine; R = H, 1; R = Me, 2; R = i-Pr, 3) and [Ag(L(R))(NO3)]2 (L = pyrazolylmethylpyridine; R = H, 4; R = Me, 5; R = i-Pr, 6) has been synthesized and structurally and spectroscopically characterized. In all of the molecular structures, the pyrazolylmethylpyridine ligands bridge two metal centers, thus giving rise to dinuclear (2, 4, 5, and 6) or polynuclear structures (1 and 3). The role played by the counteranions is also of relevance, because dimeric structures are invariably obtained with NO3(-) (4, 5, and 6), whereas the less-coordinating BF4(-) counteranion affords polymeric structures (1 and 3). Also, through atoms-in-molecules (AIM) analysis of the electron density, an argentophilic Ag···Ag interaction is found in complexes 2 and 4. Thermogravimetric analysis (TGA) shows that the thermolytic properties of the present complexes can be significantly modified by altering the ligand structure and counteranion. These complexes were further investigated as thin silver film precursors by spin-coating solutions, followed by annealing at 310 °C on 52100 steel substrates. The resulting polycrystalline cubic-phase Ag films of â¼55 nm thickness exhibit low levels of extraneous element contamination by X-ray photoelectron spectroscopy (XPS). Atomic force microscopy (AFM) and scanning electron microscopy (SEM) indicate that film growth proceeds primarily via an island growth (Volmer-Weber) mechanism. Complex 4 was also evaluated as a lubricant additive in ball-on-disk tribological tests. The results of the friction evaluation and wear measurements indicate a significant reduction in wear (â¼ 88%) at optimized Ag complex concentrations with little change in friction. The enhanced wear performance is attributed to facile shearing of Ag metal in the contact region, resulting from thermolysis of the silver complexes, and is confirmed by energy-dispersive X-ray analysis of the resulting wear scars.