Cell Cytoskeleton Organization Research Articles

Biocompatibility, bioactivity and immunomodulatory properties of three calcium silicate-based sealers: an in vitro study on hPDLSCs

ObjectivesTo assess the biocompatibility, bioactivity, and immunomodulatory properties of three new calcium silicate cement-based sealers: Ceraseal (CS), Totalfill BC Sealer (TFbc) and WellRoot ST (WR-ST) on human periodontal ligament stem cells (hPDLSCs).Materials and methodsHPDLSCs were isolated from extracted third molars from healthy patients. Eluates (1:1, 1:2, and 1:4 ratio) and sample discs of CS, TFbc and WR-ST after setting were prepared. A series of assays were performed: cell characterization, cell metabolic activity (MTT assay) cell attachment and morphology (SEM assay), cell migration (wound-healing assay), cytoskeleton organization (phaloidin-based assay); IL-6 and IL-8 release (ELISA); differentiation marker expression (RT-qPCR assay), and cell mineralization (Alizarin Red S staining). HPDLSCs cultured in unconditioned (negative control) or osteogenic (positive control) culture media were used as a comparison. Statistical significance was established at p < 0.05.ResultsAll the tested sealers exhibited similar results in the cytocompatibility assays (cell metabolic activity, migration, attachment, morphology, and cytoskeleton organization) compared with a negative control group. CS and TFbc exhibited an upregulation of at least one osteo/cementogenic marker compared to the negative and positive control groups. CS and TFbc also showed a significantly higher calcified nodule formation than the negative and positive control groups. Both the marker expression and calcified nodule formation were significantly higher in CS-treated cells than TFbc treated cells. WR-ST exhibited similar results to the control group. CS and TFbc-treated cells exhibited a significant downregulation of IL-6 after 72 h of culture compared to the negative control group (p < 0.05).ConclusionAll the tested sealers exhibited an adequate cytocompatibility. CS significantly enhances cell differentiation by upregulating the expression of key genes associated with bone and cementum formation. Additionally, CS was observed to facilitate the mineralization of the extracellular matrix effectively. In contrast, the effects of TFbc and WR-ST on these processes were less pronounced compared to CS. Furthermore, both CS and TFbc exhibited an anti-inflammatory potential, contributing to their potential therapeutic benefits in regenerative endodontics.Clinical relevanceThis is the first study to compare the biological properties and immunomodulatory potential of Ceraseal, Totalfill BC Sealer, and WellRoot ST. The results act as supporting evidence for their use in root canal treatment.

Quantitative proteomics analysis reveals possible anticancer mechanisms of 5'-deoxy-5'-methylthioadenosine in cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is an aggressive cancer originating from bile duct epithelium, particularly prevalent in Asian countries with liver fluke infections. Current chemotherapy for CCA often fails due to drug resistance, necessitating novel anticancer agents. This study investigates the potential of 5'-deoxy-5'-methylthioadenosine (MTA), a naturally occurring nucleoside, against CCA. While MTA has shown promise against various cancers, its effects on CCA remain unexplored. We evaluated MTA's anticancer activity in CCA cell lines and drug-resistant sub-lines, assessing cell viability, migration, invasion, and apoptosis. The potential anticancer mechanisms of MTA were explored through proteomic analysis using LC-MS/MS and bioinformatic analysis. The results show a dose-dependent reduction in CCA cell viability, with enhanced effects on cancer cells compared to normal cells. Moreover, MTA inhibits growth, induces apoptosis, and suppresses cell migration and invasion. Additionally, MTA enhanced the anticancer effects of gemcitabine on drug-resistant CCA cells. Proteomics revealed the down-regulation of multiple proteins by MTA, affecting various molecular functions, biological processes, and cellular components. Network analysis highlighted MTA's role in inhibiting proteins related to mitochondrial function and energy derivation, crucial for cell growth and survival. Additionally, MTA suppressed proteins involved in cell morphology and cytoskeleton organization, important for cancer cell motility and metastasis. Six candidate genes, including ZNF860, KLC1, GRAMD1C, MAMSTR, TANC1, and TTC13, were selected from the top 10 most down-regulated proteins identified in the proteomics results and were subsequently verified through RT-qPCR. Further, KLC1 protein suppression by MTA treatment was confirmed through Western blotting. Additionally, based on TCGA data, KLC1 mRNA was found to be upregulated in the tissue of CCA patients compared to that of normal adjacent tissues. In summary, MTA shows promising anticancer potential against CCA by inhibiting growth, inducing apoptosis, and suppressing migration and invasion, while enhancing gemcitabine's effects. Proteomic analysis elucidates possible molecular mechanisms underlying MTA's anticancer activity, laying the groundwork for future research and development of MTA as a treatment for advanced CCA.

Transcriptional changes during crown-root development and emergence in barley (Hordeum vulgare L.)

BackgroundRoots play an important role during plant growth and development, ensuring water and nutrient uptake. Understanding the mechanisms regulating their initiation and development opens doors towards root system architecture engineering.ResultsHere, we investigated by RNA-seq analysis the changes in gene expression in the barley stem base of 1 day-after-germination (DAG) and 10DAG seedlings when crown roots are formed. We identified 2,333 genes whose expression was lower in the stem base of 10DAG seedlings compared to 1DAG seedlings. Those genes were mostly related to basal cellular activity such as cell cycle organization, protein biosynthesis, chromatin organization, cytoskeleton organization or nucleotide metabolism. In opposite, 2,932 genes showed up-regulation in the stem base of 10DAG seedlings compared to 1DAG seedlings, and their function was related to phytohormone action, solute transport, redox homeostasis, protein modification, secondary metabolism. Our results highlighted genes that are likely involved in the different steps of crown root formation from initiation to primordia differentiation and emergence, and revealed the activation of different hormonal pathways during this process.ConclusionsThis whole transcriptomic study is the first study aiming at understanding the molecular mechanisms controlling crown root development in barley. The results shed light on crown root emergence that is likely associated with a strong cell wall modification, death of the cells covering the crown root primordium, and the production of defense molecules that might prevent pathogen infection at the site of root emergence.

3D bioartificial stretchable scaffolds mimicking the mechanical hallmarks of human cardiac fibrotic tissue

Human cardiac fibrotic tissues are characterized by a higher stiffness relative to healthy cardiac tissues. These tissues are unable to spontaneously contract and are subjected to passive mechanical stimulation during heart functionality. Moreover, scaffolds that can recapitulate the in vivo mechanical properties of the cardiac fibrotic tissues are lacking. Herein, this study aimed to design and fabricate mechanically stretchable bioartificial scaffolds with biomimetic composition and stiffness comparable to human cardiac fibrotic tissues. Poly(&amp;epsilon;-caprolactone) (PCL) scaffolds with a stretchable mesh architecture were initially designed through structural and finite element method (FEM) analyses and subsequently fabricated by melt extrusion additive manufacturing (MEX). Scaffolds were surface functionalized by 3,4-dihydroxy-DL-phenylalanine (DOPA) polymerization (polyDOPA) to improve their interaction with natural polymers. Scaffold pores were then filled with different concentrations (5%, 7%, and 10% w/v) of gelatin methacryloyl (GelMA) hydrogels to support in vitro human cardiac fibroblasts (HCFs) 3D culture, thereby producing bioartificial PCL/GelMA scaffolds. Uniaxial tensile mechanical tests in static and dynamic conditions (1 Hz; 22% maximum strain) demonstrated that the bioartificial scaffolds had in vivo-like stretchability and similar stiffness to that of pathological cardiac tissue (tailored as a function of the number of PCL scaffold layers and GelMA hydrogel concentration). In vitro cell tests on bioartificial scaffolds using HCF-embedded GelMA hydrogels under static conditions displayed increasing cell viability, spreading, and cytoskeleton organization with decreasing GelMA hydrogel concentration. Moreover, &amp;alpha;-smooth muscle actin (&amp;alpha;-SMA)-positive cells were detected after 7 days of culture in static conditions followed by another 7 days of culture in dynamic conditions and not in HCF-loaded scaffolds cultured in static conditions for 14 days. These results suggested that in vitro culture under cyclic mechanical stimulations could induce an HCF phenotypic switch into myofibroblasts.

Comparative bioactivity and immunomodulatory potential of the new Bioroot Flow and AH Plus Bioceramic sealer: An in vitro study on hPDLSCs

ObjectivesTo evaluate the cytocompatibility, bioactivity, and anti-inflammatory potential of the new pre-mixed calcium silicate cement-based sealers Bioroot Flow (BrF) and AH Plus Bioceramic Sealer (AHPbcs) on human periodontal ligament stem cells (hPDLSCs) compared to the epoxy resin-based sealer AH Plus (AHP).Materials and methodsStandardized discs and 1:1, 1:2, and 1:4 eluates of BrF, AHPbcs and AHP after setting were prepared. The following assays were performed: cell attachment and morphology via SEM, cell viability via a MTT assay, cell migration/proliferation via a wound-healing assay, cytoskeleton organization via immunofluorescence staining; cytokine release via ELISA; osteo/cemento/odontogenic marker expression via RT-qPCR, and cell mineralized nodule formation via Alizarin Red S staining. HPDLSCs were isolated from extracted third molars from healthy patients. Comparisons were made with hPDLSCs cultured in unconditioned (negative control) or osteogenic (positive control) culture media. Statistical significance was established at p < 0.05.ResultsBoth BrF and AHPbcs showed significantly positive results in the cytocompatibility assays (cell metabolic activity, migration, attachment, morphology, and cytoskeleton organization) compared with a negative control group, while AHP showed significant negative results. BrF exhibited an upregulation of at least one osteo/cementogenic marker compared to the negative and positive control groups. BrF showed a significantly higher calcified nodule formation than AHPbcs, the negative and positive control groups, while AHPbcs was higher than the negative control group. Both were also significantly higher than AHP group.ConclusionBrF and AHPbcs exhibit adequate and comparable cytocompatibility on hPDLSCs. BrF also promoted the osteo/cementogenic differentiation of hPDLSCs. Both calcium silicate-based sealers favored the downregulation of the inflammatory cytokine IL-6 and the calcified nodule formation from hPDLSCs. BrF exerted a significantly higher influence on cell mineralization than AHPbcs.Clinical relevanceThis is the first study to elucidate the biological properties and immunomodulatory potential of Bioroot Flow and AH Plus Bioceramic Sealer. The results act as supporting evidence for their use in root canal treatment.

Modeled microgravity unravels the roles of mechanical forces in renal progenitor cell physiology

BackgroundThe glomerulus is a highly complex system, composed of different interdependent cell types that are subjected to various mechanical stimuli. These stimuli regulate multiple cellular functions, and changes in these functions may contribute to tissue damage and disease progression. To date, our understanding of the mechanobiology of glomerular cells is limited, with most research focused on the adaptive response of podocytes. However, it is crucial to recognize the interdependence between podocytes and parietal epithelial cells, in particular with the progenitor subset, as it plays a critical role in various manifestations of glomerular diseases. This highlights the necessity to implement the analysis of the effects of mechanical stress on renal progenitor cells.MethodsMicrogravity, modeled by Rotary Cell Culture System, has been employed as a system to investigate how renal progenitor cells respond to alterations in the mechanical cues within their microenvironment. Changes in cell phenotype, cytoskeleton organization, cell proliferation, cell adhesion and cell capacity for differentiation into podocytes were analyzed.ResultsIn modeled microgravity conditions, renal progenitor cells showed altered cytoskeleton and focal adhesion organization associated with a reduction in cell proliferation, cell adhesion and spreading capacity. Moreover, mechanical forces appeared to be essential for renal progenitor differentiation into podocytes. Indeed, when renal progenitors were exposed to a differentiative agent in modeled microgravity conditions, it impaired the acquisition of a complex podocyte-like F-actin cytoskeleton and the expression of specific podocyte markers, such as nephrin and nestin. Importantly, the stabilization of the cytoskeleton with a calcineurin inhibitor, cyclosporine A, rescued the differentiation of renal progenitor cells into podocytes in modeled microgravity conditions.ConclusionsAlterations in the organization of the renal progenitor cytoskeleton due to unloading conditions negatively affect the regenerative capacity of these cells. These findings strengthen the concept that changes in mechanical cues can initiate a pathophysiological process in the glomerulus, not only altering podocyte actin cytoskeleton, but also extending the detrimental effect to the renal progenitor population. This underscores the significance of the cytoskeleton as a druggable target for kidney diseases.

Effects on cell cycle progression and cytoskeleton organization of five Bothrops spp. venoms in cell culture-based assays

Snake envenomation is a neglected tropical disease. In Brazil, the Bothrops genus is responsible for about 86% of snakebite accidents. Despite extensive evidence of the cytotoxicity of snake venoms, the cellular and molecular mechanisms involved are not fully understood, especially regarding the effects on cell cycle progression and cytoskeleton organization. Traditionally, the effectiveness and quality control tests of venoms and antivenoms are assessed by in vivo assays. Despite this, there is a rising effort to develop surrogate in vitro models according to the 3R principle (Replacement, Reduction, and Refinement). In this study, we treated rat liver cells (BRL-3A) with venoms from five Bothrops species (B. jararaca, B. jararacussu, B. moojeni, B. alternatus, and B. neuwiedi) and analyzed cell viability and IC50 by MTT assay, cell cycle phases distribution by flow cytometry, and morphology and cytoskeleton alterations by immunofluorescence. In addition, we evaluated the correlation between IC50 and the enzymatic and biological activities of each venom. Our results indicated that Bothrops spp. venoms decreased the cell viability of rat liver BRL-3A cells. The rank order of potency was B. jararacussu > B. moojeni > B. alternatus > B. jararaca > B. neuwiedi. The mechanisms of cytotoxicity were related to microtubules and actin network disruption, but not to cell cycle arrest. No clear correlation was found between the IC50 and retrieved literature data of in vitro enzymatic and in vivo biological activities. This work contributed to understanding cellular and molecular mechanisms underlying the Bothrops spp. venom cytotoxicity, which can help to improve envenomation treatment, as well as disclose potential therapeutic properties of snake venoms.

Recreating the Trabecular Outflow Tissue on Implantable, Micropatterned, Ultrathin, Porous Polycaprolactone Scaffolds.

Glaucoma, where increased intraocular pressure (IOP) leads to damage to the optic nerve and loss of sight, is amongst the foremost causes of irreversible blindness worldwide. In primary open angle glaucoma, the increased IOP is a result of the malfunctioning human trabecular meshwork (HTM) cells' inability to properly regulate the outflow of aqueous humor from the eye. A potential future treatment for glaucoma is to replace damaged HTM cells with a tissue-engineered substitute, thus restoring proper fluid outflow. Polycaprolactone (PCL) is a versatile, biodegradable, and implantable material that is widely used for cell culture and tissue engineering. In this work, PCL scaffolds were lithographically fabricated using a sacrificial process to produce submicron-thick scaffolds with openings of specific sizes and shapes (e.g., grid, hexagonal pattern). The HTM cell growth on gelatin-coated PCL scaffolds was assessed by scanning electron microscopy, tetrazolium metabolic activity assay, and cytoskeletal organization of F-actin. Expression of HTM-specific markers and ECM deposition were assessed by immunocytochemistry and qPCR analysis. Gelatin-coated, micropatterned, ultrathin, porous PCL scaffolds with a grid pattern supported proper HTM cell growth, cytoskeleton organization, HTM-marker expression, and ECM deposition, demonstrating the feasibility of using these PCL scaffolds to tissue-engineer implantable, healthy ocular outflow tissue.

Abstract 3616: Investigating proto-oncogene PBF-induced cell migration and invasion

Abstract The proto-oncogene pituitary tumor transforming gene binding factor (PTTG1IP/PBF) is overexpressed in multiple tumors and associated with tumor progression. PBF mediates several tumorigenic processes, including cell motility, whereby it potently induces cancer cell migration and invasion. PBF is phosphorylated by Src kinase at Y174 and mutation of this residue (Y174A) renders PBF unable to induce cell invasion, suggesting that phosphorylation mediates the stimulation of cancer cell motility by PBF. This study aimed to further elucidate the mechanism of PBF-induced cell motility. PBF-Y174 is also the key residue in a YXXΦ endocytosis motif. Both PBF phosphorylation and endocytosis were found to be essential for PBF-induced cell motility as mutants which disrupted either process were unable to stimulate thyroid and breast cancer cell migration and invasion. To elucidate molecular events downstream of PBF overexpression, phosphoproteomic and RNA-Seq analyses of normal thyroid cells (Nthy-ori 3-1) with stable PBF overexpression were performed and showed an enrichment in molecules involved in cell adhesion and cytoskeleton organisation. These findings prompted further investigation into a physiological role for PBF in cell motility. We utilised a novel Pbf knockout (Pbf−/−) mouse model generated through CRISPR/Cas9-mediated deletion of Pbf exon 4 in C57BL/6N mice to determine the impact of Pbf deletion on cellular mechanisms. Mouse embryonic fibroblasts (MEFs) were isolated at embryonic day 13.5 and used as primary cultures. Pbf−/- MEFs showed a significant reduction in migration and invasion compared with wild-type (Pbf+/+) MEFs. Interestingly, the loss of one functional copy of Pbf in heterozygote MEFs (Pbf+/−) resulted in an intermediate decrease in motility suggesting a gene-dosage effect. Initial immunofluorescent studies of Pbf−/− MEFs suggest alterations in focal adhesions (FAs). In Pbf+/+ MEFs focal adhesion kinase (FAK) and paxillin staining highlighted FA structures that were normally elongated and aligned with actin stress fibers. In contrast, Pbf−/- MEFs demonstrated a significant reduction in FAK and paxillin staining with smaller, punctate and more radially distributed FAs. These studies demonstrate a physiological role for PBF in cell adhesion and migration and further elucidate the mechanism by which PBF induces cell motility in tumor progression. Citation Format: Merve Kocbiyik, Selvambigai Manivannan, Ling Zha, Katie Brookes, Martin L Read, Chris J McCabe, Vicki E Smith. Investigating proto-oncogene PBF-induced cell migration and invasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3616.

RNAi screen in the Drosophila wing of genes encoding proteins related to cytoskeleton organization and cell division

Cell division and cytoskeleton organization are fundamental processes participating in the development of Drosophila imaginal discs. In this manuscript we describe the phenotypes in the adult fly wing generated by knockdowns of 85% of Drosophila genes encoding proteins likely related to the regulation of cell division and cytoskeleton organization. We also compile a molecular classification of these proteins into classes that describe their expected or known main biochemical characteristics, as well as mRNA expression in the wing disc and likely protein subcellular localization for a subset of these genes. Finally, we analyze in more detail one protein family of cytoskeleton genes (Arp2/3 complex), and define the consequences of interfering with cell division for wing growth and patterning.

A role for heritable transcriptomic variation in maize adaptation to temperate environments

BackgroundTranscription bridges genetic information and phenotypes. Here, we evaluated how changes in transcriptional regulation enable maize (Zea mays), a crop originally domesticated in the tropics, to adapt to temperate environments.ResultWe generated 572 unique RNA-seq datasets from the roots of 340 maize genotypes. Genes involved in core processes such as cell division, chromosome organization and cytoskeleton organization showed lower heritability of gene expression, while genes involved in anti-oxidation activity exhibited higher expression heritability. An expression genome-wide association study (eGWAS) identified 19,602 expression quantitative trait loci (eQTLs) associated with the expression of 11,444 genes. A GWAS for alternative splicing identified 49,897 splicing QTLs (sQTLs) for 7614 genes. Genes harboring both cis-eQTLs and cis-sQTLs in linkage disequilibrium were disproportionately likely to encode transcription factors or were annotated as responding to one or more stresses. Independent component analysis of gene expression data identified loci regulating co-expression modules involved in oxidation reduction, response to water deprivation, plastid biogenesis, protein biogenesis, and plant-pathogen interaction. Several genes involved in cell proliferation, flower development, DNA replication, and gene silencing showed lower gene expression variation explained by genetic factors between temperate and tropical maize lines. A GWAS of 27 previously published phenotypes identified several candidate genes overlapping with genomic intervals showing signatures of selection during adaptation to temperate environments.ConclusionOur results illustrate how maize transcriptional regulatory networks enable changes in transcriptional regulation to adapt to temperate regions.

Extracellular Cues Govern Shape and Cytoskeletal Organization in Giant Unilamellar Lipid Vesicles.

Spontaneous and induced front-rear polarization and a subsequent asymmetric actin cytoskeleton is a crucial event leading to cell migration, a key process involved in a variety of physiological and pathological conditions such as tissue development, wound healing, and cancer. Migration of adherent cells relies on the balance between adhesion to the underlying matrix and cytoskeleton-driven front protrusion and rear retraction. A current challenge is to uncouple the effect of adhesion and shape from the contribution of the cytoskeleton in regulating the onset of front-rear polarization. Here, we present a minimal model system that introduces an asymmetric actin cytoskeleton in synthetic cells, which are resembled by giant unilamellar lipid vesicles (GUVs) adhering onto symmetric and asymmetric micropatterned surfaces. Surface micropatterning of streptavidin-coated regions with varying adhesion shape and area was achieved by maskless UV photopatterning. To further study the effects of GUV shape on the cytoskeletal organization, actin filaments were polymerized together with bundling proteins inside the GUVs. The micropatterns induce synthetic cell deformation upon adhesion to the surface, with the cell shape adapting to the pattern shape and size. As expected, asymmetric patterns induce an asymmetric deformation in adherent synthetic cells. Actin filaments orient along the long axis of the deformed GUV, when having a length similar to the size of the major axis, whereas short filaments exhibit random orientation. With this bottom-up approach we have laid the first steps to identify the relationship between cell front-rear polarization and cytoskeleton organization in the future. Such a minimal system will allow us to further study the major components needed to create a polarized cytoskeleton at the onset of migration.

A Graphene Oxide-Angiogenin Theranostic Nanoplatform for the Therapeutic Targeting of Angiogenic Processes: The Effect of Copper-Supplemented Medium

Graphene oxide (GO) nanosheets with different content in the defective carbon species bound to oxygen sp3 were functionalized with the angiogenin (ANG) protein, to create a novel nanomedicine for modulating angiogenic processes in cancer therapies. The GO@ANG nanocomposite was scrutinized utilizing UV-visible and fluorescence spectroscopies. GO exhibits pro- or antiangiogenic effects, mostly attributed to the disturbance of ROS concentration, depending both on the total concentration (i.e., &gt;100 ng/mL) as well as on the number of carbon species oxidized, that is, the C/O ratio. ANG is considered one of the most effective angiogenic factors that plays a vital role in the angiogenic process, often in a synergic role with copper ions. Based on these starting hypotheses, the GO@ANG nanotoxicity was assessed with the MTT colorimetric assay, both in the absence and in the presence of copper ions, by in vitro cellular experiments on human prostatic cancer cells (PC-3 line). Laser confocal microscopy (LSM) cell imaging evidenced an enhanced internationalization of GO@ANG than bare GO nanosheets, as well as significant changes in cell cytoskeleton organization and mitochondrial staining compared to the cell treatments with free ANG.

Molecular analyses of metastatic collecting ducts renal cell carcinoma from the phase 2 prospective trial of cabozantinib as first-line treatment (BONSAI trial Meeturo 2).

e16507 Background: The phase II BONSAI trial ( n = 25 ; NCT 03354884) met the primary endpoint demonstrating activity of cabozantinib in untreated metastatic collecting ducts carcinoma (mCDC), a rare and biologically poorly characterized disease. Here we report on molecular analyses of baseline tissue samples. Methods: Formalin-fixed paraffin-embedded (FFPE) samples from 18 mCDC patients enrolled in BONSAI were sequenced by TruSeq RNA Exome kit (Illumina). The data were mapped and quantified using STAR and htseq, respectively. Globaltest and edgeR packages in R were used to assess the correlation between transcriptional profiles and survival data. Nineteen samples underwent DNA Sequencing with the Oncomine Comprehensive Assay Plus (ThermoFisher Scientific). The reads were aligned to the human genome reference (hg19) and analyzed with Opencravat and IonReporter software. Germline variants were excluded based on 1000 Genomes, GnomAd and Exac databases, and Clinical annotation of somatic variants was performed using ClinVar. Results: The global expression levels of thirty-one genes have been found significantly associated with overall survival (OS). The natural grouping of the 18 tumor samples based on the 31-gene signature identified a main group of 11 cases, showing global higher expression levels in 22 out of the 31 genes. This group displayed overall a significant higher OS rate in comparison to the remaining 7 patients, carrying opposite expression trend and mostly undergoing to disease progression. The identified signature was enriched in biological processes like cell junction, cytoskeleton organization and methylation. FOS oncogene was among the 9 genes negatively associated to OS, showing common higher expression in the poorer survival rate group. Furthermore, a 22-gene signature was found significantly associated with progression-free survival (PFS), involving mainly genes associated to cell cycle regulation or recognized as components of Golgi apparatus. Only three genes were globally up-regulated in the group of 9 patients characterized by shorter PFS. Finally, heterogeneous pattern of somatic mutations was identified in 19 tumor samples, with at least 8 genes recurrently affected in more than three patients. Notably, mutated genes were mostly involved in DNA repair and chromatin modification processes. Conclusions: Our findings for the first time define specific molecular signatures that differentiate therapy-specific outcomes in first-line mCDC, warranting further investigation of their involvement in the tumor biology. Clinical trial information: NCT 03354884.

Proteomic profiling of exosomes in a mouse model of Candida albicans endophthalmitis

Exosomes play pivotal roles in intercellular communication, and pathophysiological functions. In this study, we aimed to understand the role of exosomal proteome derived from C. albicans infected mice (C57BL/6) eyeball. Exosomes were characterized by Dynamic Light Scattering and Western blot, quantified and subjected to LC-MS/MS and cytokine quantification by ELISA. The average size of exosomes was 170–200 nm with number of exosomes amounted to 1.42 × 1010 in infected set compared to control (1.24 × 109). Western blot was positive for CD9, CD63 and CD81 confirming the presence of exosomes. IL-6, IL1β, TNF-α, and IFN-γ levels were significantly elevated in infected eye at 72 h.p.i. Proteomic analysis identified 42 differentially expressed proteins, of these 37 were upregulated and 5 were downregulated. Gene Ontology (GO) revealed enrichment of cell adhesion, cytoskeleton organisation, and cellular response proteins such as aquaporin-5, gasdermin-A, CD5 antigen-like, Catenin, V-ATPase, and vesicle associated protein. Additionally, KEGG pathway analysis indicated the association of metabolic and carbon signalling pathways with exosomes from C. albicans infected eye. The protein cargo in exosomes released during endophthalmitis with C. albicans seems to play a unique role in the pathogenesis of the disease and further validations with larger cohort of patients is required to confirm them as biomarkers.

DNA sequence features underlying large-scale duplications and deletions in human

Copy number variants (CNVs) may cover up to 12% of the whole genome and have substantial impact on phenotypes. We used 5867 duplications and 33,181 deletions available from the 1000 Genomes Project to characterise genomic regions vulnerable to CNV formation and to identify sequence features characteristic for those regions. The GC content for deletions was lower and for duplications was higher than for randomly selected regions. In regions flanking deletions and downstream of duplications, content was higher than in the random sequences, but upstream of duplication content was lower. In duplications and downstream of deletion regions, the percentage of low-complexity sequences was not different from the randomised data. In deletions and upstream of CNVs, it was higher, while for downstream of duplications, it was lower as compared to random sequences. The majority of CNVs intersected with genic regions — mainly with introns. GC content may be associated with CNV formation and CNVs, especially duplications are initiated in low-complexity regions. Moreover, CNVs located or overlapped with introns indicate their role in shaping intron variability. Genic CNV regions were enriched in many essential biological processes such as cell adhesion, synaptic transmission, transport, cytoskeleton organization, immune response and metabolic mechanisms, which indicates that these large-scaled variants play important biological roles.

Endothelial-immune crosstalk contributes to vasculopathy in nonalcoholic fatty liver disease.

The top cause of mortality in patients with nonalcoholic fatty liver disease (NAFLD) is cardiovascular complications. However, mechanisms of NAFLD‐associated vasculopathy remain understudied. Here, we show that blood outgrowth endothelial cells (BOECs) from NAFLD subjects exhibit global transcriptional upregulation of chemokines and human leukocyte antigens. In mouse models of diet‐induced NAFLD, we confirm heightened endothelial expressions of CXCL12 in the aortas and the liver vasculatures, and increased retention of infiltrated leukocytes within the vessel walls. To elucidate endothelial‐immune crosstalk, we performed immunoprofiling by single‐cell analysis, uncovering T cell intensification in NAFLD patients. Functionally, treatment with a CXCL12‐neutralizing antibody is effective at moderating the enhanced chemotactic effect of NAFLD BOECs in recruiting CD8+ T lymphocytes. Interference with the CXCL12‐CXCR4 axis using a CXCR4 antagonist also averts the impact of immune cell transendothelial migration and restores endothelial barrier integrity. Clinically, we detect threefold more circulating damaged endothelial cells in NAFLD patients than in healthy controls. Our work provides insight into the modulation of interactions with effector immune cells to mitigate endothelial injury in NAFLD.

Fucoidan (Undaria pinnatifida)/Polydopamine Composite-Modified Surface Promotes Osteogenic Potential of Periodontal Ligament Stem Cells.

Fucoidan, a marine-sulfated polysaccharide derived from brown algae, has been recently spotlighted as a natural biomaterial for use in bone formation and regeneration. Current research explores the osteoinductive and osteoconductive properties of fucoidan-based composites for bone tissue engineering applications. The utility of fucoidan in a bone tissue regeneration environment necessitates a better understanding of how fucoidan regulates osteogenic processes at the molecular level. Therefore, this study designed a fucoidan and polydopamine (PDA) composite-based film for use in a culture platform for periodontal ligament stem cells (PDLSCs) and explored the prominent molecular pathways induced during osteogenic differentiation of PDLSCs through transcriptome profiling. Characterization of the fucoidan/PDA-coated culture polystyrene surface was assessed by scanning electron microscopy and X-ray photoelectron spectroscopy. The osteogenic differentiation of the PDLSCs cultured on the fucoidan/PDA composite was examined through alkaline phosphatase activity, intracellular calcium levels, matrix mineralization assay, and analysis of the mRNA and protein expression of osteogenic markers. RNA sequencing was performed to identify significantly enriched and associated molecular networks. The culture of PDLSCs on the fucoidan/PDA composite demonstrated higher osteogenic potency than that on the control surface. Differentially expressed genes (DEGs) (n = 348) were identified during fucoidan/PDA-induced osteogenic differentiation by RNA sequencing. The signaling pathways enriched in the DEGs include regulation of the actin cytoskeleton and Ras-related protein 1 and phosphatidylinositol signaling. These pathways represent cell adhesion and cytoskeleton organization functions that are significantly involved in the osteogenic process. These results suggest that a fucoidan/PDA composite promotes the osteogenic potential of PDLSCs by activation of critical molecular pathways.

A new function for the serine protease HtrA2 in controlling radiation-induced senescence in cancer cells.

Radiation therapy can induce cellular senescence in cancer cells, leading to short‐term tumor growth arrest but increased long‐term recurrence. To better understand the molecular mechanisms involved, we developed a model of radiation‐induced senescence in cultured cancer cells. The irradiated cells exhibited a typical senescent phenotype, including upregulation of p53 and its main target, p21, followed by a sustained reduction in cellular proliferation, changes in cell size and cytoskeleton organization, and senescence‐associated beta‐galactosidase activity. Mass spectrometry‐based proteomic profiling of the senescent cells indicated downregulation of proteins involved in cell cycle progression and DNA repair, and upregulation of proteins associated with malignancy. A functional siRNA screen using a cell death‐related library identified mitochondrial serine protease HtrA2 as being necessary for sustained growth arrest of the senescent cells. In search of direct HtrA2 substrates following radiation, we determined that HtrA2 cleaves the intermediate filament protein vimentin, affecting its cytoplasmic organization. Ectopic expression of active cytosolic HtrA2 resulted in similar changes to vimentin filament assembly. Thus, HtrA2 is involved in the cytoskeletal reorganization that accompanies radiation‐induced senescence and the continuous maintenance of proliferation arrest.

Temporal Proteomic Profiling During Differentiation of Normal and Dystrophin-Deficient Human Muscle Cells.

Myogenesis is a dynamic process involving temporal changes in the expression of many genes. Lack of dystrophin protein such as in Duchenne muscular dystrophy might alter the natural course of gene expression dynamics during myogenesis. To gain insight into the dynamic temporal changes in protein expression during differentiation of normal and dystrophin deficient myoblasts to myotubes. A super SILAC spike-in strategy in combination and LC-MS/MS was used for temporal proteome profiling of normal and dystrophin deficient myoblasts during differentiation. The acquired data was analyzed using Proteome Discoverer 2.2. and data clustering using R to define significant temporal changes in protein expression. sFour major temporal protein clusters that showed sequential dynamic expression profiles during myogenesis of normal myoblasts were identified. Clusters 1 and 2, consisting mainly of proteins involved mRNA splicing and processing expression, were elevated at days 0 and 0.5 of differentiation then gradually decreased by day 7 of differentiation, then remained lower thereafter. Cluster 3 consisted of proteins involved contractile muscle and actomyosin organization. They increased in their expression reaching maximum at day 7 of differentiation then stabilized thereafter. Cluster 4 consisting of proteins involved in skeletal muscle development glucogenesis and extracellular remodeling had a lower expression during myoblast stage then gradually increased in their expression to reach a maximum at days 11-15 of differentiation. Lack of dystrophin expression in DMD muscle myoblast caused major alteration in temporal expression of proteins involved in cell adhesion, cytoskeleton, and organelle organization as well as the ubiquitination machinery. Time series proteome profiling using super SILAC strategy is a powerful method to assess temporal changes in protein expression during myogenesis and to define the downstream consequences of lack of dystrophin on these temporal protein expressions. Key alterations were identified in dystrophin deficient myoblast differentiation compared to normal myoblasts. These alterations could be an attractive therapeutic target.