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In recent years, the toxicity of graphene-related materials (GRMs) has been evaluated in diverse models to guarantee their safety. In most applications, sublethal doses of GRMs contact human barriers such as skin in a subchronic way. Herein, the subchronic effect (30 day exposure) of three GRMs (GO 1, GO 2, and FLG) with different oxidation degrees and sizes was studied. The effects of these materials on human skin cells, HaCaTs, were assayed through high-throughput metabolic-based readout and other cell-based assays. A differential effect was found between the different GRMs. GO 2 induced a metabolic remodeling in epithelial cells, increasing the level of tricarboxylic acid components, mirrored by increased cell proliferation and changes in cell phenotype. The oxidation degree, size, and method of manufacture of GRMs dictated harmful effects on cell metabolism and behavior generated by nontoxic exposures. Therefore, a "safe by design" procedure is necessary when working with these nanomaterials.

Colistin resistance is caused by different lipopolysaccharide (LPS) modifications, and we propose to evaluate the effect on the innate immune response ofin vivoandin vitrocolistin resistance acquisition. We used 2 pairs of isogenic strains: (1)Escherichia coliATCC25922, susceptible to colistin and its isogenic transconjugant-carryingmcr-1 gene; and (2) OXA-48, CTX-M-15K. pneumoniaesusceptible to colistin (CS-Kp) isolated from a urinary infection and its colistin-resistant variant (CR-Kp) from the same patient after prolonged treatment with colistin. No mutation of described genes for colistin resistance (pmrA, pmrB, mgrB. phoP/QandcrrAB) were found in the CR-Kp genome; however, LPS modifications were characterized by negative-ion MALDI-TOF. The strains were co-cultured with human monocytes to determine their survival after phagocytosis and induction to apoptosis. Also, monocytes were stimulated with bacterial LPS to study cytokine and immunecheckpoint production. The addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A of CR-Kp accounted for the colistin resistance. CR-Kp survived significantly longer inside human monocytes after being phagocytosed compared with the CS-Kp strain, whereas no significant differences were observed for theE. coliisogenic strains. In addition, LPS from CR-Kp induced both higher apoptosis in monocytes and higher levels of cytokine and immune checkpoint production than LPS from CS-Kp. This effect was strictly the opposite forE. coli. Our data reveal a variable impact of colistin resistance on the innate immune system, depending on the responsible mechanism. Adding Ara4N to LPS increases bacterial survival after phagocytosis and elicits a higher inflammatory response than its colistin-susceptible counterpart.

[Purpose] Most monotherapies available against glioblastoma multiforme (GBM) target individual hallmarks of this aggressive brain tumor with minimal success. In this article, we propose a therapeutic strategy using coenzyme Q10 (CoQ10) as a pleiotropic factor that crosses the blood–brain barrier and accumulates in cell membranes acting as an antioxidant, and in mitochondrial membranes as a regulator of cell bioenergetics and gene expression., [Methods] Xenografts of U251 cells in nu/nu mice were used to assay tumor growth, hypoxia, angiogenesis, and inflammation. An orthotopic model was used to explore microglial infiltration, tumor growth, and invasion into the brain parenchyma. Cell proliferation, migration, invasion, proteome remodeling, and secretome were assayed in vitro. Conditioned media were used to assay angiogenesis, monocyte chemoattraction, and differentiation into macrophages in vitro., [Results] CoQ10 treatment decreased tumor volume in xenografts and orthotopic models, although its effect on tumor cell proliferation was not direct. Tumors from mice treated with CoQ10 were less hypoxic and vascularized, having less infiltration from inflammatory cells. Treatment-induced downregulation of HIF-1α and NF-kB led to a complete remodeling of the tumor cells proteome and secretome, impacting angiogenesis, monocyte infiltration, and their differentiation into macrophages. Besides, tumor cell migration and invasion were drastically restricted by mechanisms involving modulation of the actin cytoskeleton and downregulation of matrix metalloproteases (MMPs)., [Conclusions] CoQ10 has a pleiotropic effect on GBM growth, targeting several hallmarks simultaneously. Thus, its integration into current treatments of this fatal disease should be considered., This work has been supported by Grants 220020351 (James S. MacDonnell Foundation), GE20112221 (Universidad de Castilla-La Mancha), PPII-2014–010-P (Junta de Comunidades de Castilla-La Mancha), MTM2015-71200-R (MINECO-FEDER), and SAF2016-79311-R (MINECO-FEDER).

Most monotherapies available against glioblastoma multiforme (GBM) target individual hallmarks of this aggressive brain tumor with minimal success. In this article, we propose a therapeutic strategy using coenzyme QXenografts of U251 cells in nu/nu mice were used to assay tumor growth, hypoxia, angiogenesis, and inflammation. An orthotopic model was used to explore microglial infiltration, tumor growth, and invasion into the brain parenchyma. Cell proliferation, migration, invasion, proteome remodeling, and secretome were assayed in vitro. Conditioned media were used to assay angiogenesis, monocyte chemoattraction, and differentiation into macrophages in vitro.CoQCoQ

The use of conventional and advanced biofuels is part of the efforts to reduce greenhouse gases and harmful exhaust gaseous emissions. This study investigates the unregulated emissions in gas and particles from a Euro 6b diesel engine, operated with four unconventional and advanced biofuels (two hydrogenated terpenic biofuels, a polyoxymethylene dimethyl ether, and a glycerol-derived biofuel), blended with diesel fuel and pure hydrotreated vegetable oil as base biofuel. The engine was operated following WLTC starting from cold-engine conditions. Gas phase samples were collected at each phase of the driving cycle and particulate matter (PM) samples were collected from a dilution tunnel at the end of the driving cycle. A total of 16 PAH and 13 carbonyls were analyzed. In addition, the apoptotic index induced by gas and particle emissions was determined. In the gaseous phase, the total PAH and carbonyl emission factors were higher at the low-speed phase for all fuels. Gas-phase PAH emission factors exceeded particle-bound PAH. Carbonyl emission factors ranged from 0.12 ± 0.012 to 25.3 ± 4.2 mg/km, markedly exceeding gaseous PAH emissions, which ranged from 20.7 ± 1.5 to 51.7 ± 8.9 μg/km. Diesel fuel exhibited the highest carbonyl emissions and its blend with 20% of hydrogenated turpentine exhibited the highest PAH emissions at the end of the WLTC, both due to high emissions at the low-speed phase. Although particle-bound PAH comprise only a small fraction of total PAH emissions, both phases (gas and particles) contributed approximately equal to the toxicity associated with carcinogenic PAH. The apoptotic cells percentage increased in a dose-dependent manner and was significantly higher in cells exposed to gas phase-derived samples. The apoptotic index induced by particulate matter samples did not show a concentration-response effect for any of the fuels.

Background Cystic fibrosis (CF) is an endotoxin tolerance (ET)-related disease. Given that increased PD-L1 has been reported in ET, its expression and physiological effects on cystic fibrosis monocytes should be studied. Methods We analyzed the phenotype and ex vivo response of immune system cells in 32 patients with CF, 19 of them colonized by Pseudomonas aeruginosa. An in vitro model was developed of Pseudomonas aeruginosa colonization using purified lipopolysaccharides (LPS) from one of the most prevalent strains in patients with CF (a CF-adapted Pseudomonas aeruginosa ST395 clone). Changes in the immune response, including cytokine production and T-lymphocyte proliferation, as well as expression of PD-L1, were evaluated. Results PD-L1 was overexpressed in the monocytes of patients with CF compared with healthy volunteers, and levels of this immune checkpoint were associated with Pseudomonas aeruginosa colonization. In addition, patients with Pseudomonas aeruginosa colonization showed a patent ET status, including poor inflammatory response, reduced HLA-DR expression and T-lymphocyte proliferation impairment. PD-L1/PD-1 blocking assays reverted the impaired adaptive response. Ultimately, monocytes from healthy volunteers cultured in the presence of the clinically relevant strain of Pseudomonas aeruginosa or serum collected from patients with CF colonized by Pseudomonas aeruginosa reproduced the previous observed features. Conclusions Pseudomonas aeruginosa colonization in patients with CF was associated with PD-L1 overexpression and impaired T cell response, and LPS from this pathogen induced the observed phenotype. Our findings open new avenues for the use of anti-PD-1/PD-L1 immunotherapy in patients with CF who are colonized by Pseudomonas aeruginosa.

The ‘cancer cell fusion’ theory is controversial due to the lack of methods available to identify hybrid cells and to follow the phenomenon in patients. However, it seems to be one of the best explanations for both the origin and metastasis of primary tumors. Herein, we co-cultured lung cancer stem cells with human monocytes and analyzed the dynamics and properties of tumor-hybrid cells (THC), as well as the molecular mechanisms beneath this fusion process by several techniques: electron-microscopy, karyotyping, CRISPR-Cas9, RNA-seq, immunostaining, signaling blockage, among others. Moreover, mice models were assessed for in vivo characterization of hybrids colonization and invasiveness. Then, the presence of THCs in bloodstream and samples from primary and metastatic lesions were detected by FACS and immunofluorescence protocols, and their correlations with TNM stages established. Our data indicate that the generation of THCs depends on the expression of CD36 on tumor stem cells and the oxidative state and polarization of monocytes, the latter being strongly influenced by microenvironmental fluctuations. Highly oxidized M2-like monocytes show the strongest affinity to fuse with tumor stem cells. THCs are able to proliferate, colonize and invade organs. THC-specific cell surface signature CD36+CD14+PANK+ allows identifying them in matched primary tumor tissues and metastases as well as in bloodstream from patients with lung cancer, thus functioning as a biomarker. THCs levels in circulation correlate with TNM classification. Our results suggest that THCs are involved in both origin and spread of metastatic cells. Furthermore, they might set the bases for future therapies to avoid or eradicate lung cancer metastasis., Research in the laboratory of EL-C was supported by “Fundación para la Investigación Biomédica La Paz Hospital” ((FIBHULP) and “Fundación Familia Alonso”.

Background During the last two decades, over 100 proteomics studies have identified a variety of potential biomarkers in CSF of Alzheimer’s (AD) patients. Although several reviews have proposed specific biomarkers, to date, the statistical relevance of these proteins has not been investigated and no peptidomic analyses have been generated on the basis of specific up- or down- regulation. Herein, we perform an analysis of all unbiased explorative proteomics studies of CSF biomarkers in AD to critically evaluate whether proteins and peptides identified in each study are consistent in distribution; direction change; and significance, which would strengthen their potential use in studies of AD pathology and progression. Methods We generated a database containing all CSF proteins whose levels are known to be significantly altered in human AD from 47 independent, validated, proteomics studies. Using this database, which contains 2022 AD and 2562 control human samples, we examined whether each protein is consistently present on the basis of reliable statistical studies; and if so, whether it is over- or under-represented in AD. Additionally, we performed a direct analysis of available mass spectrometric data of these proteins to generate an AD CSF peptide database with 3221 peptides for further analysis. Results Of the 162 proteins that were identified in 2 or more studies, we investigated their enrichment or depletion in AD CSF. This allowed us to identify 23 proteins which were increased and 50 proteins which were decreased in AD, some of which have never been revealed as consistent AD biomarkers (i.e. SPRC or MUC18). Regarding the analysis of the tryptic peptide database, we identified 87 peptides corresponding to 13 proteins as the most highly consistently altered peptides in AD. Analysis of tryptic peptide fingerprinting revealed specific peptides encoded by CH3L1, VGF, SCG2, PCSK1N, FBLN3 and APOC2 with the highest probability of detection in AD. Conclusions Our study reveals a panel of 27 proteins and 21 peptides highly altered in AD with consistent statistical significance; this panel constitutes a potent tool for the classification and diagnosis of AD.

CoQ10 is an endogenous antioxidant produced in all cells that plays an essential role in energy metabolism and antioxidant protection. CoQ10 distribution is not uniform among different organs, and the highest concentration is observed in the heart, though its levels decrease with age. Advanced age is the major risk factor for cardiovascular disease and endothelial dysfunction triggered by oxidative stress that impairs mitochondrial bioenergetic and reduces NO bioavailability, thus affecting vasodilatation. The rationale of the use of CoQ10 in cardiovascular diseases is that the loss of contractile function due to an energy depletion status in the mitochondria and reduced levels of NO for vasodilatation has been associated with low endogenous CoQ10 levels. Clinical evidence shows that CoQ10 supplementation for prolonged periods is safe, well-tolerated and significantly increases the concentration of CoQ10 in plasma up to 3–5 µg/mL. CoQ10 supplementation reduces oxidative stress and mortality from cardiovascular causes and improves clinical outcome in patients undergoing coronary artery bypass graft surgery, prevents the accumulation of oxLDL in arteries, decreases vascular stiffness and hypertension, improves endothelial dysfunction by reducing the source of ROS in the vascular system and increases the NO levels for vasodilation.

Sepsis, among other pathologies, is an endotoxin tolerance (ET)-related disease. On admission, we classified 48 patients with sepsis into 3 subgroups according to the ex vivo response to lipopolysaccharide. This response correlates with the Acute Physiology and Chronic Health Evaluation (APACHE) II score and the ET degree. Moreover, the ET-related classification determines the outcome of these patients. Programmed cell death-ligand 1 (PD-L1) expression on septic monocytes is also linked with ET status. In addition to the regulation of cytokine production, one of the hallmarks of ET that significantly affects patients with sepsis is T-cell proliferation impairment or a poor switch to the adaptive response. PD-L1/programmed cell death-1 (PD-1) blocking and knockdown assays on tolerant monocytes from both patients with sepsis and the in vitro model reverted the impaired adaptive response. Mechanistically, the transcription factor hypoxia-inducible factor-1α (HIF1α) has been translocated into the nucleus and drives PD-L1 expression during ET in human monocytes. This fact, together with patient classification according to the ex vivo lipopolysaccharide response, opens an interesting field of study and potential personalized clinical applications, not only for sepsis but also for all ET-associated pathologies.