Ubiquinone Metabolism and Transcription HIF-1 Targets Pathway Are Toxicity Signature Pathways Present in Extracellular Vesicles of Paraquat-Exposed Human Brain Microvascular Endothelial Cells.

Over the last decade, the knowledge in extracellular vesicles (EVs) biogenesis and modulation has increasingly grown. As their content reflects the physiological state of their donor cells, these "intercellular messengers" progressively became a potential source of biomarker reflecting the host cell state. However, little is known about EVs released from the human brain microvascular endothelial cells (HBMECs). The current study aimed to isolate and characterize EVs from HBMECs and to analyze their EVs proteome modulation after paraquat (PQ) stimulation, a widely used herbicide known for its neurotoxic effect. Size distribution, concentration and presence of well-known EV markers were assessed. Identification and quantification of PQ-exposed EV proteins was conducted by data-independent acquisition mass spectrometry (DIA-MS). Signature pathways of PQ-treated EVs were analyzed by gene ontology terms and pathway enrichment. Results highlighted that EVs exposed to PQ have modulated pathways, namely the ubiquinone metabolism and the transcription HIF-1 targets. These pathways may be potential molecular signatures of the PQ-induced toxicity carried by EVs that are reflecting their cell of origin by transporting with them irreversible functional changes.

Intracellular and Extracellular Markers of Lethality in Osteogenesis Imperfecta: A Quantitative Proteomic Approach.

Osteogenesis imperfecta (OI) is a heritable disorder that mainly affects the skeleton. The inheritance is mostly autosomal dominant and associated to mutations in one of the two genes, COL1A1 and COL1A2, encoding for the type I collagen α chains. According to more than 1500 described mutation sites and to outcome spanning from very mild cases to perinatal-lethality, OI is characterized by a wide genotype/phenotype heterogeneity. In order to identify common affected molecular-pathways and disease biomarkers in OI probands with different mutations and lethal or surviving phenotypes, primary fibroblasts from dominant OI patients, carrying COL1A1 or COL1A2 defects, were investigated by applying a Tandem Mass Tag labeling-Liquid Chromatography-Tandem Mass Spectrometry (TMT LC-MS/MS) proteomics approach and bioinformatic tools for comparative protein-abundance profiling. While no difference in α1 or α2 abundance was detected among lethal (type II) and not-lethal (type III) OI patients, 17 proteins, with key effects on matrix structure and organization, cell signaling, and cell and tissue development and differentiation, were significantly different between type II and type III OI patients. Among them, some non-collagenous extracellular matrix (ECM) proteins (e.g., decorin and fibrillin-1) and proteins modulating cytoskeleton (e.g., nestin and palladin) directly correlate to the severity of the disease. Their defective presence may define proband-failure in balancing aberrances related to mutant collagen.

Neuroinflammatory Response to TNFα and IL1ß Cytokines Is Accompanied by an Increase in Glycolysis in Human Astrocytes In Vitro.

Astrogliosis has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism, and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes as a function of age have been reported, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected in supporting a functional switch of astrocytes from neurotrophic to neurotoxic. This study aimed to explore the metabolic changes occurring in astrocytes during their activation. Astrocytes were derived from human ReN cell neural progenitors and characterized. They were activated by exposure to tumor necrosis factor alpha (TNFα) or interleukin 1ß (IL1ß) for 24 h. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics and extracellular flux analyses. ReN-derived astrocytes reactivity was observed by the modifications of genes and proteins linked to inflammation (cytokines, nuclear factor-kappa B (NFκB), signal transducers and activators of transcription (STATs)) and immune pathways (major histocompatibility complex (MHC) class I). Increased NFκB1, NFκB2 and STAT1 expression, together with decreased STAT3 expression, suggest an activation towards the detrimental pathway. Strong modifications of astrocyte cytoskeleton were observed, including a glial fibrillary acidic protein (GFAP) decrease. Astrogliosis was accompanied by changes in energy metabolism characterized by increased glycolysis and lactate release. Increased glycolysis is reported for the first time during human astrocyte activation. Astrocyte activation is strongly tied to energy metabolism, and a possible association between NFκB signaling and/or MHC class I pathway and glycolysis is suggested.

Proteomic Characterization of the Olfactory Molecular Imbalance in Dementia with Lewy Bodies.

Olfactory dysfunction is one of the prodromal symptoms in dementia with Lewy bodies (DLB). However, the molecular pathogenesis associated with decreased smell function remains largely undeciphered. We generated quantitative proteome maps to detect molecular alterations in olfactory bulbs (OB) derived from DLB subjects compared to neurologically intact controls. A total of 3214 olfactory proteins were quantified, and 99 proteins showed significant alterations in DLB cases. Protein interaction networks disrupted in DLB indicated an imbalance in translation and the synaptic vesicle cycle. These alterations were accompanied by alterations in AKT/MAPK/SEK1/p38 MAPK signaling pathways that showed a distinct expression profile across the OB-olfactory tract (OT) axis. Taken together, our data partially reflect the missing links in the biochemical understanding of olfactory dysfunction in DLB.

Palmitate-Induced Insulin Hypersecretion and Later Secretory Decline Associated with Changes in Protein Expression Patterns in Human Pancreatic Islets.

In obese children with high circulating concentrations of free fatty acid palmitate, we have observed that insulin levels at fasting and in response to a glucose challenge were several times higher than in obese children with low concentrations of the fatty acid as well as in lean controls. Declining and even insufficient insulin levels were observed in obese adolescents with high levels of the fatty acid. In isolated human islets exposed to palmitate we have observed insulin hypersecretion after 2 days exposure. In contrast, insulin secretion from the islets was reduced after 7 days culture in the presence of the fatty acid. This study aims at identifying islet-related biological events potentially linked with the observed insulin hypersecretion and later secretory decline in these obese children and adolescents using the islet model. We analyzed protein expression data obtained from human islets exposed to elevated palmitate levels for 2 and 7 days by an improved methodology for statistical analysis of differentially expressed proteins. Protein profiling of islet samples by liquid chromatography-tandem mass spectrometry identified 115 differentially expressed proteins (DEPs). Several DEPs including sorcin were associated with increased glucose-stimulated insulin secretion in islets after 2 days of exposure to palmitate. Similarly, several metabolic pathways including altered protein degradation, increased autophagy, altered redox condition, and hampered insulin processing were coupled to the functional impairment of islets after 7 days of culture in the presence of palmitate. Such biological events, once validated in the islets, may give rise to novel treatment strategies aiming at normalizing insulin levels in obese children with high palmitate levels, which may reduce or even prevent obesity-related type 2 diabetes mellitus.

New molecular insights into modulation of platelet reactivity in aspirin-treated patients using a network-based approach.

Platelet reactivity (PR) is variable between individuals and modulates clinical outcome in cardiovascular (CV) patients treated with antiplatelet drugs. Although several data point to a genetic control of platelet reactivity, the genes contributing to the modulation of this phenotype are not clearly identified. Integration of data derived from high-throughput technologies may yield novel insights into the molecular mechanisms that govern platelet reactivity. The aim of this study is to identify candidate genes modulating platelet reactivity in aspirin-treated CV patients using an integrative network-based approach. Patients with extreme high (n = 6) or low PR (n = 6) were selected and data derived from quantitative proteomic of platelets and platelet sub-cellular fractions, as well as from transcriptomic analysis were integrated with a network biology approach. Two modules within the network containing 123 and 182 genes were identified. We then specifically assessed the level of miRNAs in these two groups of patients. Among the 12 miRNAs differentially expressed, 2 (miR-135a-5p and miR-204-5p) correlated with PR. The predicted targets of these miRNAs were mapped onto the network, allowing the identification of seven overlapping genes (THBS1, CDC42, CORO1C, SPTBN1, TPM3, GTPBP2, and MAPRE2), suggesting a synergistic effect of these two miRNAs on these predicted targets. Integration of several omics data sets allowed the identification of 2 candidate miRNAs and 7 candidate genes regulating platelet reactivity in aspirin-treated CV patients.

Modulation of neuronal pentraxin 1 expression in rat pancreatic ß-cells submitted to chronic glucotoxic stress.

Insulin secretory granules are ß-cell vesicles dedicated to insulin processing, storage, and release. The secretion of insulin secretory granule content in response to an acute increase of glucose concentration is a highly regulated process allowing normal glycemic homeostasis. Type 2 diabetes is a metabolic disease characterized by chronic hyperglycemia. The consequent prolonged glucose exposure is known to exert deleterious effects on the function of various organs, notably impairment of insulin secretion by pancreatic ß-cells and induction of apoptosis. It has also been described as modifying gene and protein expression in ß-cells. Therefore, we hypothesized that a modulation of insulin secretory granule protein expression induced by chronic hyperglycemia may partially explain ß-cell dysfunction. To identify the potential early molecular mechanisms underlying ß-cell dysfunction during chronic hyperglycemia, we performed SILAC and mass spectrometry experiments to monitor changes in the insulin secretory granule proteome from INS-1E rat insulinoma ß-cells cultivated either with 11 or 30 mm of glucose for 24 h. Fourteen proteins were found to be differentially expressed between these two conditions, and several of these proteins were not described before to be present in ß-cells. Among them, neuronal pentraxin 1 was only described in neurons so far. Here we investigated its expression and intracellular localization in INS-1E cells. Furthermore, its overexpression in glucotoxic conditions was confirmed at the mRNA and protein levels. According to its role in hypoxia-ischemia-induced apoptosis described in neurons, this suggests that neuronal pentraxin 1 might be a new ß-cell mediator in the AKT/GSK3 apoptotic pathway. In conclusion, the modification of specific ß-cell pathways such as apoptosis and oxidative stress may partially explain the impairment of insulin secretion and ß-cell failure, observed after prolonged exposure to high glucose concentrations.

A proteomic approach supports the clinical relevance of TAT-Cx43266-283 in glioblastoma.

Glioblastoma (GBM) is the most frequent and aggressive primary brain cancer. The Src inhibitor, TAT-Cx43266-283, exerts antitumor effects in in vitro and in vivo models of GBM. Because addressing the mechanism of action is essential to translate these results to a clinical setting, in this study we carried out an unbiased proteomic approach. Data-independent acquisition mass spectrometry proteomics allowed the identification of 190 proteins whose abundance was modified by TAT-Cx43266-283. Our results were consistent with the inhibition of Src as the mechanism of action of TAT-Cx43266-283 and unveiled antitumor effectors, such as p120 catenin. Changes in the abundance of several proteins suggested that TAT-Cx43266-283 may also impact the brain microenvironment. Importantly, the proteins whose abundance was reduced by TAT-Cx43266-283 correlated with an improved GBM patient survival in clinical datasets and none of the proteins whose abundance was increased by TAT-Cx43266-283 correlated with shorter survival, supporting its use in clinical trials.

The Skp1-Cullin1-FBXO1 complex is a pleiotropic regulator required for the formation of gametes and motile forms in Plasmodium berghei.

Malaria-causing parasites of the Plasmodium genus undergo multiple developmental phases in the human and the mosquito hosts, regulated by various post-translational modifications. While ubiquitination by multi-component E3 ligases is key to regulate a wide range of cellular processes in eukaryotes, little is known about its role in Plasmodium. Here we show that Plasmodium berghei expresses a conserved SKP1/Cullin1/FBXO1 (SCFFBXO1) complex showing tightly regulated expression and localisation across multiple developmental stages. It is key to cell division for nuclear segregation during schizogony and centrosome partitioning during microgametogenesis. It is additionally required for parasite-specific processes including gamete egress from the host erythrocyte, as well as integrity of the apical and the inner membrane complexes (IMC) in merozoite and ookinete, two structures essential for the dissemination of these motile stages. Ubiquitinomic surveys reveal a large set of proteins ubiquitinated in a FBXO1-dependent manner including proteins important for egress and IMC organisation. We additionally demonstrate an interplay between FBXO1-dependent ubiquitination and phosphorylation via calcium-dependent protein kinase 1. Altogether we show that Plasmodium SCFFBXO1 plays conserved roles in cell division and is also important for parasite-specific processes in the mammalian and mosquito hosts.

Digoxin Induces Human Astrocyte Reaction In Vitro.

Astrocyte reaction is a complex cellular process involving astrocytes in response to various types of CNS injury and a marker of neurotoxicity. It has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes have been reported with age, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected. However, the relationship between energy metabolism and astrocyte reactivity in the context of neurotoxicity is not known. We hypothesized that changes in energy metabolism of astrocytes will be coupled to their activation by xenobiotics. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics, and extracellular flux analyses after 24 h of exposure of human ReN-derived astrocytes to digoxin (1-10 µM) or TNFα (30 ng/ml) used as a positive control. Strong astrocytic reaction was observed, accompanied by increased glycolysis at low concentrations of digoxin (0.1 and 0.5 µM) and after TNFα exposure, suggesting that increased glycolysis may be a common feature of reactive astrocytes, independent of the triggering molecule. In conclusion, whether astrocyte activation is triggered by cytokines or a xenobiotic, it is strongly tied to energy metabolism in human ReN-derived astrocytes. Increased glycolysis might be considered as an endpoint to detect astrocyte activation by potentially neurotoxic compounds in vitro. Finally, ReN-derived astrocytes may help to decipher mechanisms of neurotoxicity in ascertaining the ability of chemicals to directly target astrocytes.

Laser microdissection, proteomics, and multiplex immunohistochemistry: a bumpy ride into the study of paraffin-embedded fetal and pediatric lung tissues.

Background: Knowledge about lung development or lung disease is mainly derived from data extrapolated from mouse models. This has obvious drawbacks in developmental diseases, particularly due to species differences. Our objective is to describe the development of complementary analysis methods that will allow a better understanding of the molecular mechanisms involved in the pathogenesis of rare congenital diseases. Methods: Paraffin-embedded human pediatric and fetal lung samples were laser microdissected to enrich different lung regions, namely, bronchioli or alveoli. These samples were analyzed by data-independent acquisition-based quantitative proteomics, and the lung structures were subsequently compared. To confirm the proteomic data, we employed an optimized Sequential ImmunoPeroxidase Labeling and Erasing (SIMPLE) staining for specific proteins of interest. Results: By quantitative proteomics, we identified typical pulmonary proteins from being differentially expressed in different regions. While the receptor for advanced glycation end products (RAGE) and the surfactant protein C (SFTPC) were downregulated, tubulin beta 4B (TUBB4B) was upregulated in bronchioli, compared to alveoli. In fetal tissues, CD31 was downregulated in fetal bronchioli compared to canaliculi. Moreover, we confirmed their presence using SIMPLE staining. Some expected proteins did not show up in the proteomic data, such as SOX-9, which was only detected by means of immunohistochemistry in the SIMPLE analysis. Conclusion: Our data underline the robustness and applicability of this type of experimental approach, especially for rare paraffin-embedded tissue samples. It also strengthens the importance of these methods for future studies, particularly when considering developmental lung diseases, such as congenital lung anomalies.

Morphine-induced modulation of Nrf2-antioxidant response element signaling pathway in primary human brain microvascular endothelial cells.

Morphine is one of the most potent opioid analgesic used for pain treatment. Morphine action in the central nervous system requires crossing the blood-brain barrier. Due to the controversial relationship between morphine and oxidative stress, the potential pro- or antioxidant effects of morphine in the blood-brain barrier is important to be understood, as oxidative stress could cause its disruption and predispose to neurodegenerative diseases. However, investigation is scarce in human brain endothelial cells. Therefore, the present study evaluated the impact of morphine exposure at three different concentrations (1, 10 and 100 µM) for 24 h and 48 h on primary human brain microvascular endothelial cells. A quantitative data-independent acquisition mass spectrometry strategy was used to analyze proteome modulations. Almost 3000 proteins were quantified of which 217 were reported to be significantly regulated in at least one condition versus untreated control. Pathway enrichment analysis unveiled dysregulation of the Nrf2 pathway involved in oxidative stress response. Seahorse assay underlined mitochondria dysfunctions, which were supported by significant expression modulations of relevant mitochondrial proteins. In conclusion, our study revealed the dysregulation of the Nrf2 pathway and mitochondria dysfunctions after morphine exposure, highlighting a potential redox imbalance in human brain endothelial cells.

Impact of peptide permeation enhancer on tight junctions opening cellular mechanisms.

The myristoylated pentapeptide, L-R5, contains an amino acid sequence of the zeta inhibitory peptide (ZIP) portion (pseudosubstrate) of protein kinase C zeta (PKC ζ). As PKC ζ is involved in the modulation of epithelial tight junctions (TJs) through the phosphorylation of TJ proteins, L-R5 was suggested to interact with the enzyme resulting in the enhancement of paracellular permeability. This study shows that L-R5 does not bind to the enzyme but interacts directly with TJ proteins. We show here that the binding of PKC ζ to occludin and its successive phosphorylation is prevented by L-R5, which leads to TJ disruption and enhanced epithelial permeability. Although L-R5 did not show any in vitro cytotoxicity, a proteomics study revealed that L-R5 interferes with other regulatory pathways, e.g., apoptosis and immune response. We suggest that structural modification of the peptide may increase the specificity TJ protein-peptide interaction.

Oxidative Stress and Extracellular Matrix Remodeling Are Signature Pathways of Extracellular Vesicles Released upon Morphine Exposure on Human Brain Microvascular Endothelial Cells.

Morphine, a commonly used antinociceptive drug in hospitals, is known to cross the blood-brain barrier (BBB) by first passing through brain endothelial cells. Despite its pain-relieving effect, morphine also has detrimental effects, such as the potential induction of redox imbalance in the brain. However, there is still insufficient evidence of these effects on the brain, particularly on the brain endothelial cells and the extracellular vesicles that they naturally release. Indeed, extracellular vesicles (EVs) are nanosized bioparticles produced by almost all cell types and are currently thought to reflect the physiological state of their parent cells. These vesicles have emerged as a promising source of biomarkers by indicating the functional or dysfunctional state of their parent cells and, thus, allowing a better understanding of the biological processes involved in an adverse state. However, there is very little information on the morphine effect on human brain microvascular endothelial cells (HBMECs), and even less on their released EVs. Therefore, the current study aimed at unraveling the detrimental mechanisms of morphine exposure (at 1, 10, 25, 50 and 100 µM) for 24 h on human brain microvascular endothelial cells as well as on their associated EVs. Isolation of EVs was carried out using an affinity-based method. Several orthogonal techniques (NTA, western blotting and proteomics analysis) were used to validate the EVs enrichment, quality and concentration. Data-independent mass spectrometry (DIA-MS)-based proteomics was applied in order to analyze the proteome modulations induced by morphine on HBMECs and EVs. We were able to quantify almost 5500 proteins in HBMECs and 1500 proteins in EVs, of which 256 and 148, respectively, were found to be differentially expressed in at least one condition. Pathway enrichment analysis revealed that the "cell adhesion and extracellular matrix remodeling" process and the "HIF1 pathway", a pathway related to oxidative stress responses, were significantly modulated upon morphine exposure in HBMECs and EVs. Altogether, the combination of proteomics and bioinformatics findings highlighted shared pathways between HBMECs exposed to morphine and their released EVs. These results put forward molecular signatures of morphine-induced toxicity in HBMECs that were also carried by EVs. Therefore, EVs could potentially be regarded as a useful tool to investigate brain endothelial cells dysfunction, and to a different extent, the BBB dysfunction in patient circulation using these "signature pathways".

Gaining Insights Into Metabolic Networks Using Chemometrics and Bioinformatics: Chronic Kidney Disease as a Clinical Model.

Because of its ability to generate biological hypotheses, metabolomics offers an innovative and promising approach in many fields, including clinical research. However, collecting specimens in this setting can be difficult to standardize, especially when groups of patients with different degrees of disease severity are considered. In addition, despite major technological advances, it remains challenging to measure all the compounds defining the metabolic network of a biological system. In this context, the characterization of samples based on several analytical setups is now recognized as an efficient strategy to improve the coverage of metabolic complexity. For this purpose, chemometrics proposes efficient methods to reduce the dimensionality of these complex datasets spread over several matrices, allowing the integration of different sources or structures of metabolic information. Bioinformatics databases and query tools designed to describe and explore metabolic network models offer extremely useful solutions for the contextualization of potential biomarker subsets, enabling mechanistic hypotheses to be considered rather than simple associations. In this study, network principal component analysis was used to investigate samples collected from three cohorts of patients including multiple stages of chronic kidney disease. Metabolic profiles were measured using a combination of four analytical setups involving different separation modes in liquid chromatography coupled to high resolution mass spectrometry. Based on the chemometric model, specific patterns of metabolites, such as N-acetyl amino acids, could be associated with the different subgroups of patients. Further investigation of the metabolic signatures carried out using genome-scale network modeling confirmed both tryptophan metabolism and nucleotide interconversion as relevant pathways potentially associated with disease severity. Metabolic modules composed of chemically adjacent or close compounds of biological relevance were further investigated using carbon transfer reaction paths. Overall, the proposed integrative data analysis strategy allowed deeper insights into the metabolic routes associated with different groups of patients to be gained. Because of their complementary role in the knowledge discovery process, the association of chemometrics and bioinformatics in a common workflow is therefore shown as an efficient methodology to gain meaningful insights in a clinical context.

Early activation of the fatty acid metabolism pathway by chronic high glucose exposure in rat insulin secretory beta-cells.

Pancreatic beta-cells are responsible for insulin secretion that regulates blood glucose homeostasis. In the development of type II diabetes, a progressive impairment of insulin secretion by the pancreatic beta-cells occurs called beta-cell dysfunction or beta-cell failure. Chronic hyperglycemia has been shown being involved in beta-cell dysfunction, a phenomenon known as glucotoxicity. The molecular mechanisms underlying the impairment of insulin secretion by beta-cells induced by glucotoxicity are still not fully understood. In this work, quantitative proteomics was employed to identify early key players involved in beta-cell dysfunction induced by glucotoxicity. For this, the stable isotope labeling by amino acids in cell culture strategy was used on the slowly-growing rat beta-cell line INS-1E. We showed that the stable isotope labeling by amino acids in cell culture approach did not induce any detectable biological effects on these beta-cells, as measured at both the transcriptomic and proteomic levels. Proteins differentially expressed between control cells and cells submitted to chronic high glucose concentrations were identified and verified. The results obtained reinforce the link between glucotoxicity and lipogenesis and suggest that the fatty acid metabolism pathway may rapidly be stimulated in beta-cells submitted to chronic high glucose concentrations.

Proteomics of regulated secretory organelles.

Regulated secretory organelles are important subcellular structures of living cells that allow the release in the extracellular space of crucial compounds, such as hormones and neurotransmitters. Therefore, the regulation of biogenesis, trafficking, and exocytosis of regulated secretory organelles has been intensively studied during the last 30 years. However, due to the large number of different regulated secretory organelles, only a few of them have been specifically characterized. New insights into regulated secretory organelles open crucial perspectives for a better comprehension of the mechanisms that govern cell secretion. The combination of subcellular fractionation, protein separation, and mass spectrometry is also possible to study regulated secretory organelles at the proteome level. In this review, we present different strategies used to isolate regulated secretory organelles, separate their protein content, and identify the proteins by mass spectrometry. The biological significance of regulated secretory organelles-proteomic analysis is discussed as well.

Old problem, new solutions: biomarker discovery for acetaminophen liver toxicity.

Introduction: Although the hepatotoxicity of acetaminophen is a well-known problem, the search for reliable biomarker of toxicity is still a current issue as clinical tools are missing to assess patients intoxicated following chronic use, sequential ingestion, use of modified release formulations or in case of delayed arrival to hospital. The need for new specific and robust biomarkers for acetaminophen toxicity has prompted many studies exploring the use of blood levels of acetaminophen derivatives, mitochondrial damage markers, liver cell apoptosis and/or necrosis markers and circulating microRNAs. Areas covered: In this review, we present a concise overview of the most promising biomarkers currently under evaluation including descriptions of their properties with respect to exposure type, APAP specificity, and potential clinical application. In addition, we illustrate the power of new technologies for biomarker research and describe their current application to the field of acetaminophen-induced hepatotoxicity. Expert opinion: Recently the use of extracellular vesicles isolation in combination with omics techniques has opened a new perspective to the field of biomarker research. However, the potential of those new technologies for the prediction and monitoring of hepatic diseases and acetaminophen toxicity has not yet been fully taken into consideration.

3D Cellular Architecture Affects MicroRNA and Protein Cargo of Extracellular Vesicles.

The success of malignant tumors is conditioned by the intercellular communication between tumor cells and their microenvironment, with extracellular vesicles (EVs) acting as main mediators. While the value of 3D conditions to study tumor cells is well established, the impact of cellular architecture on EV content and function is not investigated yet. Here, a recently developed 3D cell culture microwell array is adapted for EV production and a comprehensive comparative analysis of biochemical features, RNA and proteomic profiles of EVs secreted by 2D vs 3D cultures of gastric cancer cells, is performed. 3D cultures are significantly more efficient in producing EVs than 2D cultures. Global upregulation of microRNAs and downregulation of proteins in 3D are observed, indicating their dynamic coregulation in response to cellular architecture, with the ADP-ribosylation factor 6 signaling pathway significantly downregulated in 3D EVs. The data strengthen the biological relevance of cellular architecture for production and cargo of EVs.

The olfactory bulb proteotype differs across frontotemporal dementia spectrum.

Mild olfactory dysfunction has been observed in frontotemporal dementias (FTD). However, the underlying molecular mechanisms associated to this deficit are poorly understood. We applied quantitative proteomics to analyze pathological effects on the olfactory bulb (OB) from progressive supranuclear palsy (PSP) and frontotemporal lobar degeneration (FTLD-TDP43) subjects respect to elderly non-FTD group. Our data revealed: i) a mitochondrial and calcium homeostasis impairment in PSP and ii) a disruption of protein synthesis and vesicle trafficking in FTLD-TDP43. Although differential OB proteomes clearly differ between both FTD phenotypes, functional analyses pointed out an imbalance in survival signaling in both pathologies. A common alteration of olfactory mitogen-activated protein kinases (MAPKs), calcium/calmodulin dependent protein kinase II (CAMKII), and protein kinase C (PKC) signaling pathways was observed in PSP and FTLD subjects. In contrast, a specific shut off in mitogen-activated protein kinase kinase 4 (SEK1/MKK4)/stress-activated protein kinase (SAPK) axis was exclusively observed in PSP, whereas a specific phosphoinositide-dependent protein kinase 1 (PDK1) inactivation was observed in FTLD-TDP43. In summary, our data contribute to a better understanding of the molecular mechanisms that are modulated in PSP and FTLD-TDP43 at olfactory level, highlighting cross-disease similarities and differences in the regulation of survival pathways across FTD spectrum. SIGNIFICANCE: This work reflects differential olfactory molecular disarrangements in PSP and FTLD-TDP43, two clinically similar FTD disorders, but with different neuropathological signature. Besides FTDs present mild olfactory dysfunction, our data provide basic information for understanding the implication of the OB in the pathophysiology of FTDs.