Regulation of Akt(ser473) phosphorylation by Choline kinase in breast carcinoma cells

<p>Abstract</p> <p>Background</p> <p>The serine/threonine kinase PKB/Akt plays essential role in various cellular processes including cell growth and proliferation, metabolism and cell survival. The importance of the Akt pathway is highlighted by the mutation of various components of the pathway such as the PTEN and PI3-kinase (P110α) in human cancers. In this paper, we employed an RNA interference library targeting all human kinases to screen for kinases involved in the regulation of Akt activation, in particular serine 473 phosphorylation. Here, we transfected the MDA-MB 468 breast cell line with the human kinome siRNA library and measured Akt activation using an antibody specific for phosphoserine 473 of Akt.</p> <p>Results</p> <p>The screen revealed that phosphorylation of Akt(ser473) can be regulated by more than 90 kinases. Interestingly, phosphorylation of Akt(ser473), but not thr308, can be severely reduced by inhibition of Choline kinase activity <it>via </it>siRNA or small molecule inhibitors. We show here that the regulation of Akt phosphorylation by Choline kinase is PI3K-independent. In addition, xenograft tumors treated with Choline kinase inhibitors demonstrated a statistically significant decrease in Akt(ser473) phosphorylation. Importantly, the reduction in phosphorylation correlates with regression of these xenograft tumors in the mouse model.</p> <p>Conclusion</p> <p>High Choline kinase expression and activity has previously been implicated in tumor development and metastasis. The mechanism by which Choline kinase is involved in tumor formation is still not fully resolved. From our data, we proposed that Choline kinase plays a key role in regulating Akt(ser473) phosphorylation, thereby promoting cell survival and proliferation.</p

Intracellular changes in Ca2 +, K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results

P. 155-158Although it is widely accepted that osmolality and ion fluxes are the main factors triggering sperm motility in fish, a complex universal mechanism for sperm motility activation does not exist in fish, and studies of marine fish species are even more scarce. Therefore, the main goal of this study was to estimate the intracellular variations in the main ions involved in sperm activation for the first time in European eel, in order to provide additional new data about this little-known process. It was observed that levels of intracellular Ca2 + and K+ sperm ions increased significantly 30 s after the hyperosmotic shock compared to baseline levels, and remained at this level until 120 s post-activation. In contrast, the intracellular pH remained constant during the first 30 s, and decreased gradually at 60 and 120 s post-activation. Our data agree with the current main theory for explaining motility activation in marine fish, in which internal fluctuations of Ca2 + and K+ seem to participate in sperm activation. In addition, fluorescent images showed that both Ca2 + and K+ were concentrated in the apical area of the sperm head, which corresponds to the location of the eel sperm mitochondria, suggesting this organelle plays an important role in sperm motility activation.S

Effects of global postural reeducation on postural control, dynamic balance, and ankle range of motion in patients with hallux abducto valgus. A randomized controlled trial.

AbstractHallux abducto valgus (HAV) is a common musculoskeletal disorder that has been addressed surgically. Nevertheless, the manual therapy approach may play an important role in the management of this condition. The present study aimed to determine the effectiveness of global postural reeducation (GPR) in subjects with symptomatic mild to moderate HAV in static postural control, dynamic stability, and ankle dorsiflexion range of motion (DFROM). A total of 80 patients with mild to moderate symptomatic HAV were allocated to the intervention group (GPR) or control group (CG) (no treatment) for 8 weeks. Outcome measures were assessed at baseline at 4 and 8 weeks including static postural control (Romberg test), dynamic balance (Star Excursion Balance Test [SEBT]), and ankle DFROM (Weight‐Bearing Lunge Test [WBLT]). No improvements were observed at 4 weeks, but there were improvements at 8 weeks in: static postural control mediolateral displacement (X) of center of pressure (CoP) in both eyes open (EO) and eyes closed (EC): XEO (t(36) = 2.892, p = .006, d = 0.67); XEC (t(68) = 2.280, p = .026, d = 054); and velocity (V) of CoP displacement: VEO (t(68) = 2.380, p = .020, d = 0.57); VEC (t(36) = 2.057, p = .047, d = 0.37). It were also improvements in: WBLT (t(36) = −2.869, p = .007, d = 0.54) and SEBT at three directions (anterior, ANT; posteromedial, PM; and posterolateral, PL): SEBT.ANT (t(36) = −2.292, p = .028, d = 0.23); SEBT.PM (t(36) = −4.075, p < .001, d = 0.43); SEBT.PL (t(62) = −3.506, p = .001, d = 0.34). The present study showed that GPR compared to the CG might be effective in enhancing ankle function including postural control, dynamic balance, and DFROM

Single-Cell Transcriptomics in Cancer Immunobiology: The Future of Precision Oncology

Cancer is a heterogeneous and complex disease. Tumors are formed by cancer cells and a myriad of non-cancerous cell types that together with the extracellular matrix form the tumor microenvironment. These cancer-associated cells and components contribute to shape the progression of cancer and are deeply involved in patient outcome. The immune system is an essential part of the tumor microenvironment, and induction of cancer immunotolerance is a necessary step involved in tumor formation and growth. Immune mechanisms are intimately associated with cancer progression, invasion, and metastasis; as well as to tumor dormancy and modulation of sensitivity to drug therapy. Transcriptome analyses have been extensively used to understand the heterogeneity of tumors, classifying tumors into molecular subtypes and establishing signatures that predict response to therapy and patient outcomes. However, the classification of the tumor cell diversity and specially the identification of rare populations has been limited in these transcriptomic analyses of bulk tumor cell populations. Massively-parallel single-cell RNAseq analysis has emerged as a powerful method to unravel heterogeneity and to study rare cell populations in cancer, through unsupervised sampling and modeling of transcriptional states in single cells. In this context, the study of the role of the immune system in cancer would benefit from single cell approaches, as it will enable the characterization and/or discovery of the cell types and pathways involved in cancer immunotolerance otherwise missed in bulk transcriptomic information. Thus, the analysis of gene expression patterns at single cell resolution holds the potential to provide key information to develop precise and personalized cancer treatment including immunotherapy. This review is focused on the latest single-cell RNAseq methodologies able to agnostically study thousands of tumor cells as well as targeted single-cell RNAseq to study rare populations within tumors. In particular, we will discuss methods to study the immune system in cancer. We will also discuss the current challenges to the study of cancer at the single cell level and the potential solutions to the current approaches

Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen

P. 529-543There has been a marked reduction in natural stocks of eels (genus Anguilla) over the past 60 years, and the culture of eels is still based on the capture of very large quantities of juveniles. It is necessary to close the life cycle in captivity in order to ease the pressure on wild populations. The aims of the present study were to evaluate sperm subpopulations (through cluster analysis of computer-aided sperm analysis data) in the European eel (Anguilla anguilla) and to assess the effects of motility acquisition time after activation (i.e. at 30, 60 and 90 s), the thermal regimen (i.e. 10°C (T10) or 15°C (T15) and up to 20°C, or constant at 20°C (T20)) and hormonal treatments (i.e. human chorionic gonadotropin (hCG), recombinant (r) hCG or pregnant mare serum gonadotropin (PMSG)) on these subpopulations. In all cases, we obtained three subpopulations of spermatozoa: low velocity and linear (S1); high velocity with low linearity (S2); and high velocity and linear (S3; considered high quality). Total motility and S1 were affected by acquisition time; thus, 30 s is recommended as the standard time for motility acquisition. When eels were kept at 20°C (T20), motility data fitted quadratic models, with the highest motility and proportion of S3 between Weeks 8 and 12 after the first injection. Lower temperatures (T10, T15) delayed spermiation and the obtaining of high-quality spermatozoa (S3), but did not seem to alter the spermiation process (similar subpopulation pattern). Conversely, the hormonal treatments altered both the dynamics of the subpopulation pattern and the onset of spermiation (with PMSG delaying it). Total motility and the yield of S3 with the widely used hCG treatment varied throughout the spermiation period. However, using rhCG allowed us to obtain high-quality and constant motility for most of the study (Weeks 7–20), and the S3 yield was also higher overall (61.8 ± 1.3%; mean ± s.e.m.) and more stable over time than the other hormonal treatments (averaging 53.0 ± 1.4%). Using T20 and rhCG would be more economical and practical, allowing us to obtain a higher number of S3 spermatozoa over an extended time.S

Capillary microfluidic platform for sulfite determination in wines

A microfluidic paper-based analytical device integrating a chromoreactand – a formylazo dye– has been fabri- cated and used for a colorimetric assay of sulfites. The chromoreactand was covalently linked to paper by vinyl sulfone chemistry. This work presents two robust capillary microfluidic devices to determine sulfite in wine without any pretreatment. One of them based on thread (µTPAD) useful to determine it in white wine and another based on paper (µPAD) to specifically determine sulfite in red wine as well as in white wine. Both are based on the selective recognition of sulfite by means of a chromoreactand that turns from orange to yellow in the presence of sulfite. The colour information acquired (H coordinate) using a digital camera readout allows for a range of appli- cation of the µTPAD from 7.8⋅10−5 M (8.1 mg L−1) to 2.7⋅10−3 M (279.3 mg L−1) with a limit of detection (LOD) of 78 µM. The strong interference caused by the dyes present in red wine is eliminated by including a laminated paper channel in the µPAD structure that allows for the separation of colorants from red wine before the recognition of the sulfite. This makes it possible to adjust the µPAD procedure to the usual sulfite concentration in wine, with an LOD of 2.2⋅10−4 M (22.7 mg L−1) and a CV of 2.6%.This work was founded by Spanish “Ministerio de Economía y Competitividad” (Projects PID2019-103938RB-I00) and Junta de Andalucía (Projects B-FQM-243-UGR18 and P18-RT-2961). The projects were partially supported by European Regional Development Funds (ERDF)

Clinical relevance of the transcriptional signature regulated by CDC42 in colorectal cancer

CDC42 is an oncogenic Rho GTPase overexpressed in colorectal cancer (CRC). Although CDC42 has been shown to regulate gene transcription, the specific molecular mechanisms regulating the oncogenic ability of CDC42 remain unknown. Here, we have characterized the transcriptional networks governed by CDC42 in the CRC SW620 cell line using gene expression analysis. Our results establish that several cancer-related signaling pathways, including cell migration and cell proliferation, are regulated by CDC42. This transcriptional signature was validated in two large cohorts of CRC patients and its clinical relevance was also studied. We demonstrate that three CDC42-regulated genes offered a better prognostic value when combined with CDC42 compared to CDC42 alone. In particular, the concordant overexpression of CDC42 and silencing of the putative tumor suppressor gene CACNA2D2 dramatically improved the prognostic value. The CACNA2D2/CDC42 prognostic classifier was further validated in a third CRC cohort as well as in vitro and in vivo CRC models. Altogether, we show that CDC42 has an active oncogenic role in CRC via the transcriptional regulation of multiple cancer-related pathways and that CDC42-mediated silencing of CACNA2D2 is clinically relevant. Our results further support the use of CDC42 specific inhibitors for the treatment of the most aggressive types of CRCThis work has been supported by grants to JCL from Ministerio de Ciencia e Innovación (SAF2008- 03750, RD06-0020-0016 and RD12/0036/0019) and to DGO Cancer Institute New South Wales (2017/CDF625). FVM is a National Breast Cancer Foundation/Cure Cancer Australia Foundation Postdoctoral Training Fellow

A Data Fusion System for Simulation of Critical Scenarios and Decision-Making

The decision-making (DM) process in critical environments is a complex process that can be simulated due to current telematic capabilities, which allow the real time interaction of large amounts of data. This document describes the proposed architecture from a research process, developed by the FAC Aerospace Technology Development Center (CETAD), where using computational and expert system tools, allowed to create a computational environment for decision maker evaluated his options to prepares for real events, simulating characteristics, resources and strategies in a real time environment. This document describes an investigation product resulted in a simulation system, based on a combination of fuzzy logic, genetic algorithms and decision trees which let modelled and simulated various entities and their automatic response according to simulated patterns and situations, in which, through operators, decision maker can modify entities behaviour, according to parameterized restrictions and physical conditions. Also based on business intelligence tools, reports are generated to evaluate the decisions made. This type of technologies improves planning capacity and facilitate the decision-making process. System allows simulating any media deployment in national security and critical events context. Thus, a case study was developed for implementation of a support in natural disaster scenario simulatio

Intravital FRAP imaging using an E-cadherin-GFP mouse reveals disease- and drug-dependent dynamic regulation of cell-cell junctions in live tissue

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments

ROBO2 is a stroma suppressor gene in the pancreas and acts via TGF-β signalling

Whereas genomic aberrations in the SLIT-ROBO pathway are frequent in pancreatic ductal adenocarcinoma (PDAC), their function in the pancreas is unclear. Here we report that in pancreatitis and PDAC mouse models, epithelial Robo2 expression is lost while Robo1 expression becomes most prominent in the stroma. Cell cultures of mice with loss of epithelial Robo2 (Pdx1 ;Robo2 ) show increased activation of Robo1 myofibroblasts and induction of TGF-β and Wnt pathways. During pancreatitis, Pdx1 ;Robo2 mice present enhanced myofibroblast activation, collagen crosslinking, T-cell infiltration and tumorigenic immune markers. The TGF-β inhibitor galunisertib suppresses these effects. In PDAC patients, ROBO2 expression is overall low while ROBO1 is variably expressed in epithelium and high in stroma. ROBO2 ;ROBO1 patients present the poorest survival. In conclusion, Robo2 acts non-autonomously as a stroma suppressor gene by restraining myofibroblast activation and T-cell infiltration. ROBO1/2 expression in PDAC patients may guide therapy with TGF-β inhibitors or other stroma /immune modulating agents