Keratin 8/18 Regulation of Cell Stiffness-Extracellular Matrix Interplay through Modulation of Rho-Mediated Actin Cytoskeleton Dynamics

Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells

The malignant phenotype in breast cancer is driven by eIF4A1-mediated changes in the translational landscape

Human mRNA DeXD/H-box helicases are ubiquitous molecular motors that are required for the majority of cellular processes that involve RNA metabolism. One of the most abundant is eIF4A, which is required during the initiation phase of protein synthesis to unwind regions of highly structured mRNA that would otherwise impede the scanning ribosome. Dysregulation of protein synthesis is associated with tumorigenesis, but little is known about the detailed relationships between RNA helicase function and the malignant phenotype in solid malignancies. Therefore, immunohistochemical analysis was performed on over 3000 breast tumors to investigate the relationship among expression of eIF4A1, the helicase-modulating proteins eIF4B, eIF4E and PDCD4, and clinical outcome. We found eIF4A1, eIF4B and eIF4E to be independent predictors of poor outcome in ER-negative disease, while in contrast, the eIF4A1 inhibitor PDCD4 was related to improved outcome in ER-positive breast cancer. Consistent with these data, modulation of eIF4A1, eIF4B and PCDC4 expression in cultured MCF7 cells all restricted breast cancer cell growth and cycling. The eIF4A1-dependent translatome of MCF7 cells was defined by polysome profiling, and was shown to be highly enriched for several classes of oncogenic genes, including G-protein constituents, cyclins and protein kinases, and for mRNAs with G/C-rich 5′UTRs with potential to form G-quadruplexes and with 3′UTRs containing microRNA target sites. Overall, our data show that dysregulation of mRNA unwinding contributes to the malignant phenotype in breast cancer via preferential translation of a class of genes involved in pro-oncogenic signaling at numerous levels. Furthermore, immunohistochemical tests are promising biomarkers for tumors sensitive to anti-helicase therapies

c-di-GMP Turn-Over in Clostridium difficile Is Controlled by a Plethora of Diguanylate Cyclases and Phosphodiesterases

Clostridium difficile infections have become a major healthcare concern in the last decade during which the emergence of new strains has underscored this bacterium's capacity to cause persistent epidemics. c-di-GMP is a bacterial second messenger regulating diverse bacterial phenotypes, notably motility and biofilm formation, in proteobacteria such as Vibrio cholerae, Pseudomonas aeruginosa, and Salmonella. c-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a conserved GGDEF domain. It is degraded by phosphodiesterases (PDEs) that contain either an EAL or an HD-GYP conserved domain. Very little is known about the role of c-di-GMP in the regulation of phenotypes of Gram-positive or fastidious bacteria. Herein, we exposed the main components of c-di-GMP signalling in 20 genomes of C. difficile, revealed their prevalence, and predicted their enzymatic activity. Ectopic expression of 31 of these conserved genes was carried out in V. cholerae to evaluate their effect on motility and biofilm formation, two well-characterized phenotype alterations associated with intracellular c-di-GMP variation in this bacterium. Most of the predicted DGCs and PDEs were found to be active in the V. cholerae model. Expression of truncated versions of CD0522, a protein with two GGDEF domains and one EAL domain, suggests that it can act alternatively as a DGC or a PDE. The activity of one purified DGC (CD1420) and one purified PDE (CD0757) was confirmed by in vitro enzymatic assays. GTP was shown to be important for the PDE activity of CD0757. Our results indicate that, in contrast to most Gram-positive bacteria including its closest relatives, C. difficile encodes a large assortment of functional DGCs and PDEs, revealing that c-di-GMP signalling is an important and well-conserved signal transduction system in this human pathogen

An internal ribosome entry site in the 5′ untranslated region of epidermal growth factor receptor allows hypoxic expression

The expression of epidermal growth factor receptor (EGFR/ERBB1/HER1) is implicated in the progress of numerous cancers, a feature that has been exploited in the development of EGFR antibodies and EGFR tyrosine kinase inhibitors as anti-cancer drugs. However, EGFR also has important normal cellular functions, leading to serious side effects when EGFR is inhibited. One damaging characteristic of many oncogenes is the ability to be expressed in the hypoxic conditions associated with the tumour interior. It has previously been demonstrated that expression of EGFR is maintained in hypoxic conditions via an unknown mechanism of translational control, despite global translation rates generally being attenuated under hypoxic conditions. In this report, we demonstrate that the human EGFR 5′ untranslated region (UTR) sequence can initiate the expression of a downstream open reading frame via an internal ribosome entry site (IRES). We show that this effect is not due to either cryptic promoter activity or splicing events. We have investigated the requirement of the EGFR IRES for eukaryotic initiation factor 4A (eIF4A), which is an RNA helicase responsible for processing RNA secondary structure as part of translation initiation. Treatment with hippuristanol (a potent inhibitor of eIF4A) caused a decrease in EGFR 5′ UTR-driven reporter activity and also a reduction in EGFR protein level. Importantly, we show that expression of a reporter gene under the control of the EGFR IRES is maintained under hypoxic conditions despite a fall in global translation rates

Business analytics in industry 4.0: a systematic review

Recently, the term “Industry 4.0” has emerged to characterize several Information Technology and Communication (ICT) adoptions in production processes (e.g., Internet-of-Things, implementation of digital production support information technologies). Business Analytics is often used within the Industry 4.0, thus incorporating its data intelligence (e.g., statistical analysis, predictive modelling, optimization) expert system component. In this paper, we perform a Systematic Literature Review (SLR) on the usage of Business Analytics within the Industry 4.0 concept, covering a selection of 169 papers obtained from six major scientific publication sources from 2010 to March 2020. The selected papers were first classified in three major types, namely, Practical Application, Reviews and Framework Proposal. Then, we analysed with more detail the practical application studies which were further divided into three main categories of the Gartner analytical maturity model, Descriptive Analytics, Predictive Analytics and Prescriptive Analytics. In particular, we characterized the distinct analytics studies in terms of the industry application and data context used, impact (in terms of their Technology Readiness Level) and selected data modelling method. Our SLR analysis provides a mapping of how data-based Industry 4.0 expert systems are currently used, disclosing also research gaps and future research opportunities.The work of P. Cortez was supported by FCT - Fundação para a Ciência e Tecnologia within the R&D Units Project Scope: UIDB/00319/2020. We would like to thank to the three anonymous reviewers for their helpful suggestions

Comparative ICE Genomics: Insights into the Evolution of the SXT/R391 Family of ICEs

Integrating and conjugative elements (ICEs) are one of the three principal types of self-transmissible mobile genetic elements in bacteria. ICEs, like plasmids, transfer via conjugation; but unlike plasmids and similar to many phages, these elements integrate into and replicate along with the host chromosome. Members of the SXT/R391 family of ICEs have been isolated from several species of gram-negative bacteria, including Vibrio cholerae, the cause of cholera, where they have been important vectors for disseminating genes conferring resistance to antibiotics. Here we developed a plasmid-based system to capture and isolate SXT/R391 ICEs for sequencing. Comparative analyses of the genomes of 13 SXT/R391 ICEs derived from diverse hosts and locations revealed that they contain 52 perfectly syntenic and nearly identical core genes that serve as a scaffold capable of mobilizing an array of variable DNA. Furthermore, selection pressure to maintain ICE mobility appears to have restricted insertions of variable DNA into intergenic sites that do not interrupt core functions. The variable genes confer diverse element-specific phenotypes, such as resistance to antibiotics. Functional analysis of a set of deletion mutants revealed that less than half of the conserved core genes are required for ICE mobility; the functions of most of the dispensable core genes are unknown. Several lines of evidence suggest that there has been extensive recombination between SXT/R391 ICEs, resulting in re-assortment of their respective variable gene content. Furthermore, our analyses suggest that there may be a network of phylogenetic relationships among sequences found in all types of mobile genetic elements

Sensitivity of Global Translation to mTOR Inhibition in REN Cells Depends on the Equilibrium between eIF4E and 4E-BP1

Initiation is the rate-limiting phase of protein synthesis, controlled by signaling pathways regulating the phosphorylation of translation factors. Initiation has three steps, 43S, 48S and 80S formation. 43S formation is repressed by eIF2α phosphorylation. The subsequent steps, 48S and 80S formation are enabled by growth factors. 48S relies on eIF4E-mediated assembly of eIF4F complex; 4E-BPs competitively displace eIF4E from eIF4F. Two pathways control eIF4F: 1) mTORc1 phosphorylates and inactivates 4E-BPs, leading to eIF4F formation; 2) the Ras-Mnk cascade phosphorylates eIF4E. We show that REN and NCI-H28 mesothelioma cells have constitutive activation of both pathways and maximal translation rate, in the absence of exogenous growth factors. Translation is rapidly abrogated by phosphorylation of eIF2α. Surprisingly, pharmacological inhibition of mTORc1 leads to the complete dephosphorylation of downstream targets, without changes in methionine incorporation. In addition, the combined administration of mTORc1 and MAPK/Mnk inhibitors has no additive effect. The inhibition of both mTORc1 and mTORc2 does not affect the metabolic rate. In spite of this, mTORc1 inhibition reduces eIF4F complex formation, and depresses translocation of TOP mRNAs on polysomes. Downregulation of eIF4E and overexpression of 4E-BP1 induce rapamycin sensitivity, suggesting that disruption of eIF4F complex, due to eIF4E modulation, competes with its recycling to ribosomes. These data suggest the existence of a dynamic equilibrium in which eIF4F is not essential for all mRNAs and is not displaced from translated mRNAs, before recycling to the next