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Exposure to atmospheric fne particulate matter (PM2.5) is currently recognized as a leading cause and/or a serious aggravating factor of several respiratory diseases, such as chronic obstructive pulmonary disease (COPD), pulmonary fbrosis, asthma, and lung cancer. Notably, pulmonary-altered metabolism has emerged as an important hallmark of those diseases, while a growing body of evidence shows that exposure to PM2.5 deeply modulates the bioenergetic profles and metabolic pathways of lung cells. Hence, there is great interest in understanding how PM-induced metabolic dysregulations may contribute to pulmonary pathobiological events. Herein, in vitro and in vivo studies assessing the impact of PM2.5 on lung cells metabolism are reviewed, with emphasis on lung bioenergetics and lipid composition. Overall, there is compelling evidence that the metabolism of lung cells is an early target of PM2.5 exposure, with changes in metabolic pathways being observed at PM doses that do not cause extensive cell death or tissue injury. Hence, characterizing metabolic dysregulations associated with specifc PM features and exposure conditions represents a valuable approach to further enlighten the toxicity mechanisms of PM2.5 and to identify novel biomarker signatures of air pollution exposure and health hazard. published

The widespread ethnomedical practice of dromedary urinotherapy as a remedy against various illnesses is well recognized in traditional dromedary countries, and multiple researchers tried to unravel its bioactive potential and provide scientific evidence through in vivo and in vitro experiments. None of these studies (i) measured urine osmolarity prior to bioactivity testing, which could deeply influence the results of in vitro tests, nor (ii) addressed issues related to cells’ morphological changes after exposure to camel urines. Taken together, the above aspects point to the need for a “good practice” to be shared by researchers in this field, in order to reduce the variability of in vitro testing of camel urine bioactivity. In this work, using a set of biological samples from animals differing in sex, age, and physiological status, we investigated, the antiproliferative activity of camel urine towards human non-tumoral (HK2) and tumoral renal cells (Caki-1), through cell viability and microscopy analysis, and taking the possible influence of osmolarity into account. We employed cell lines commonly used in toxicological research which, to the best of our knowledge, have not been previously exposed to camel urine. HK2 and Caki-1 cells tolerated well mannitol-induced hyperosmolarity up to 500 mOsm/L. Significant antiproliferative effects were observed only in Caki-1 cells, when exposed to urine solutions (diluted to

This study reports the formulation and delivery of hyaluronic acid-Zein (HA-Zein) nanogels loaded with Shikonin (SK) to selectively attenuate macrophage inflammasome. The self-assembled nanogels, produced by nanoprecipitation, exhibited high encapsulation efficiency, and were selectively internalized by human THP-1-derived macrophages without eliciting cytotoxic responses. Cell treatment with HA-Zein-SK nanogels before stimulation with LPS and Nigericin significantly suppressed caspase-1 activation and IL-1β production, indicating inflammasome inhibition. Importantly, HA-Zein-SK nanogels bioinstructed inflammasome activated macrophages towards an anti-inflammatory CD163highHLA-DRlow phenotype and led to a marked reduction in the release of pro-inflammatory mediators (TNF-α, IL-6 and IP-10). Extracellular metabolic profiling additionally revealed SK-mediated downregulation of cellular glycolytic activity, which was corroborated by a significant decrease of glycolytic genes transcription. All in all, our findings demonstrate the potential of bioactive SK-containing, self-assembled nanogels to modulate exacerbated responses in innate immune cells and, prospectively, in human tissues where NRLP3 inflammasome is abnormally activated upon injury or disease. published

In this work, untargeted metabolomics was used to unveil the impact of a Eucalyptus (Eucalyptus nitens) outer bark lipophilic extract on the metabolism of triple negative breast cancer (TNBC) and nontumor breast cells. Integrative analysis of culture medium, intracellular polar metabolites, and cellular lipids provided a comprehensive picture of cell metabolic adaptations, which enabled several hypotheses about the metabolic targets and pathways affected to be proposed. One of the most marked effects in MDA-MB-231 breast cancer cells, upon 48 h incubation with the E. nitens extract (15 μg/mL), was the enhancement of the NAD+/NADH ratio, likely reflecting a shift to mitochondrial respiration, which appeared to be fueled by amino acids and fatty acids resulting from hydrolysis of neutral lipids (triglycerides and cholesteryl esters). Contrastingly, in MCF-10A breast epithelial cells, the E. nitens extract appeared to intensify glycolysis and the tricarboxylic acid cycle (resulting in a decreased NAD+/NADH ratio), while having no effect on the cell lipid composition. This knowledge improves the current understanding of the biological activity of E. nitens bark extracts and is potentially useful to promote their development in the field of TNBC anticancer therapy.

International audience; Aspergillus fumigatus is a major opportunistic fungal pathogen of immunocompromised and immunocompetent hosts. To successfully establish an infection, A. fumigatus needs to use host carbon sources, such as acetate, present in the body fluids and peripheral tissues. However, utilization of acetate as a carbon source by fungi in the context of infection has not been investigated. This work shows that acetate is metabolized via different pathways in A. fumigatus and that acetate utilization is under the regulatory control of a transcription factor (TF), FacB. A. fumigatus acetate utilization is subject to carbon catabolite repression (CCR), although this is only partially dependent on the TF and main regulator of CCR CreA. The available extracellular carbon source, in this case glucose and acetate, significantly affected A. fumigatus virulence traits such as secondary metabolite secretion and cell wall composition, with the latter having consequences for resistance to oxidative stress, antifungal drugs, and human neutrophil-mediated killing. Furthermore, deletion of facB significantly impaired the in vivo virulence of A. fumigatus in both insect and mammalian models of invasive aspergillosis. This is the first report on acetate utilization in A. fumigatus, and this work further highlights the importance of available host-specific carbon sources in shaping fungal virulence traits and subsequent disease outcome, and a potential target for the development of antifungal strategies.IMPORTANCE Aspergillus fumigatus is an opportunistic fungal pathogen in humans. During infection, A. fumigatus is predicted to use host carbon sources, such as acetate, present in body fluids and peripheral tissues, to sustain growth and promote colonization and invasion. This work shows that A. fumigatus metabolizes acetate via different pathways, a process that is dependent on the transcription factor FacB. Furthermore, the type and concentration of the extracellular available carbon source were determined to shape A. fumigatus virulence determinants such as secondary metabolite secretion and cell wall composition. Subsequently, interactions with immune cells are altered in a carbon source-specific manner. FacB is required for A. fumigatus in vivo virulence in both insect and mammalian models of invasive aspergillosis. This is the first report that characterizes acetate utilization in A. fumigatus and highlights the importance of available host-specific carbon sources in shaping virulence traits and potentially subsequent disease outcome.

Decellularized extracellular matrix (dECM) is emerging as a valuable tool for generating 3D in vitro tumor models that better recapitulate tumor-stroma interactions. However, the development of dECM-3D heterotypic microtumors exhibiting a controlled morphology is yet to be materialized. Precisely controlling microtumors morphologic features is key to avoid an inaccurate evaluation of therapeutics performance during preclinical screening. To address this, herein we employed ultra-low adhesion surfaces for bioengineering organotypic 3D metastatic breast cancer-fibroblast models enriched with dECM microfibrillar fragments, as a bottom-up strategy to include major matrix components and their associated biomolecular cues during the early stages of 3D microtissue spheroids assembly, simulating pre-existing ECM presence in the in vivo setting. This biomimetic approach enabled the self-assembly of dECM-3D tumor-stroma spheroids with tunable size and reproducible morphology. Along time, dECM enriched and stroma-rich microtumors exhibited necrotic core formation, secretion of key biomarkers and higher cancer-cell specific resistance to different chemotherapeutics in comparison to standard spheroids. Exometabolomics profiling of dECM-Spheroid in vitro models further identified important breast cancer metabolic features including glucose/pyruvate consumption and lactate excretion, which suggest an intense glycolytic activity, recapitulating major hallmarks of the native microenvironment. Such organotypic dECM-enriched microtumors overcome the morphologic variability generally associated with cell-laden dECM models, while providing a scalable testing platform that can be foreseeable leveraged for high-throughput screening of candidate therapeutics. published

Tissue-mimetic silk hydrogels are being explored for diverse healthcare applications, including stem cell delivery. However, the impact of stress relaxation of silk hydrogels on human mesenchymal stem cell (MSC) biology is poorly defined. The aim of this study was to fabricate silk hydrogels with tuned mechanical properties that allowed the regulation of MSC biology in two dimensions. The silk content and stiffness of both elastic and viscoelastic silk hydrogels were kept constant to permit direct comparisons. Gene expression of IL-1��, IL-6, LIF, BMP-6, BMP-7, and protein tyrosine phosphatase receptor type C were substantially higher in MSCs cultured on elastic hydrogels than those on viscoelastic hydrogels, whereas this pattern was reversed for insulin, HNF-1A, and SOX-2. Protein expression was also mechanosensitive and the elastic cultures showed strong activation of IL-1�� signaling in response to hydrogel mechanics. An elastic substrate also induced higher consumption of glucose and aspartate, coupled with a higher secretion of lactate, than was observed in MSCs grown on viscoelastic substrate. However, both silk hydrogels changed the magnitude of consumption of glucose, pyruvate, glutamine, and aspartate, and also metabolite secretion, resulting in an overall lower metabolic activity than that found in control cells. Together, these findings describe how stress relaxation impacts the overall biology of MSCs cultured on silk hydrogels. ��

Cancer-associated pancreatic stellate cells installed in periacinar/periductal regions are master players in generating the characteristic biophysical shield found in pancreatic ductal adenocarcinoma (PDAC). Recreating this unique PDAC stromal architecture and its desmoplastic microenvironment in vitro is key to discover innovative treatments. However, this still remains highly challenging to realize. Herein, organotypic 3D microtumors that recapitulate PDAC-stroma spatial bioarchitecture, as well as its biomolecular, metabolic, and desmoplastic signatures, are bioengineered. Such newly engineered platforms, termed stratified microenvironment spheroid models - STAMS - mimic the spatial stratification of cancer-stromal cells, exhibit a reproducible morphology and sub-millimeter size. In culture, 3D STAMS secrete the key molecular biomarkers found in human pancreatic cancer, namely TGF-β, FGF-2, IL-1β, and MMP-9, among others. This is accompanied by an extensive desmoplastic reaction where collagen and glycosaminoglycans (GAGs) de novo deposition is observed. These stratified models also recapitulate the resistance to various chemotherapeutics when compared to standard cancer-stroma random 3D models. Therapeutics resistance is further evidenced upon STAMS inclusion in a tumor extracellular matrix (ECM)-mimetic hydrogel matrix, reinforcing the importance of mimicking PDAC-stroma bioarchitectural features in vitro. The 3D STAMS technology represents a next generation of biomimetic testing platforms with improved potential for advancing high-throughput screening and preclinical validation of innovative pancreatic cancer therapies. published

Despite its success in achieving the long-term survival of 10-30% of treated individuals, immune therapy is still ineffective for most patients with cancer

The exposure of humans to nano-and microplastic particles (NMPs) is an issue recognized as a potential health hazard by scientists, authorities, politics, non-governmental organizations and the general public. The concentration of NMPs in the environment is increasing concomitantly with global plastic production and the usage of plastic materials. NMPs are detectable in numerous aquatic organisms and also in human samples, therefore necessitating a risk assessment of NMPs for human health. So far, a comprehensive risk assessment of NMPs is hampered by limited availability of appropriate reference materials, analytical obstacles and a lack of definitions and standardized study designs. Most studies conducted so far used polystyrene (PS) spheres as a matter of availability, although this polymer type accounts for only about 7% of total plastic production. Differently sized particles, different concentration and incubation times, and various biological models have been used, yielding hardly comparable data sets. Crucial physico-chemical properties of NMPs such as surface (charge, polarity, chemical reactivity), supplemented additives and adsorbed chemicals have been widely excluded from studies, although in particular the surface of NMPs determines the interaction with cellular membranes. In this manuscript we give an overview about the critical parameters which should be considered when performing risk assessments of NMPs, including novel reference materials, taking into account surface modifications (e.g., reflecting weathering processes), and the possible role of NMPs as a substrate and/or carrier for (pathogenic) microbes. Moreover, we make suggestions for biological model systems to evaluate immediate toxicity, long-term effects and the potential of NMPs to cross biological barriers. We are convinced that standardized reference materials and experimental parameters along with technical innovations in (nano)-particle sampling and analytics are a prerequisite for the successful realization of conclusive human health risk assessments of NMPs. ispartof: INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH vol:17 issue:23 ispartof: location:Switzerland status: published

Purpose: Prostate cancer is a major cause of cancer-related death in males worldwide and, in addition to impairing prostate function, also causes testicular adaptations. In this study, we aim to investigate the preventive effect of exercise training on PCa-induced testicular dysfunction. Methods: As a model, we used fifty Wistar Unilever male rats, randomly divided in four experimental groups. Prostate cancer was chemically and hormonally induced in two groups of animals (PCa groups). One control group and one PCa group was submitted to moderate intensity treadmill exercise training. Fifty weeks after the start of the training the animals were sacrificed and sperm, prostate, testis and serum were collected and analyzed. Sperm concentration and morphology, and testosterone serum levels were determined. In addition, histological analyses of the testes were performed, and testis proteomes and metabolomes were characterized. Results: We found that prostate cancer negatively affected testicular function, manifested as an arrest of spermatogenesis. Oxidative stress-induced DNA damage, arising from reduced testis blood flow, may also contribute to apoptosis of germ cells and consequential spermatogenic impairment. Decreased utilization of the glycolytic pathway, increased metabolism of ketone bodies and the accumulation of branched chain amino acids were also evident in the PCa animals. Conversely, we found that the treadmill training regimen activated DNA repair mechanisms and counteracted several metabolic alterations caused by PCa without impact on oxidative stress. Conclusions: These findings confirm a negative impact of prostate cancer on testis function and suggest a beneficial role for exercise training in the prevention of prostate cancer-induced testis dysfunction. published

Background The field of cancer immunotherapy is growing at a fast pace, with new developments in this field leading to a change in cancer therapy. Dendritic cells (DCs) are one of the central tools in cellular anti-tumour immunotherapy and the production of clinical grade monocyte-derived DCs (Mo-DCs) is the most frequent approach for antitumor vaccines production. However, there is a large space for improvement of protocols and a clear need for the establishment of clinical standard operating procedures (CSOP). Cancer stem cells (CSCs) are a recently identified small cell population present in the tumour, resistant to radio/chemotherapy and known to be responsible for disease recurrence. Here, we aim to contribute to the standardization of CSOPs and to target and eradicate CSCs by developing a DC-based immunotherapy vaccine for pancreatic and non-small cells lung cancer (NSCLC), comparing DC loading with CSCs vs. classical tumour lysates. Materials and Methods CSCs from PANC-1 (pancreatic cancer) and A549 (NSCLC) cell lines were isolated and characterized by RT-PCR and flow citometry. CSCs resistance to chemotherapy was also assessed. In vitro anti-tumour cytotoxicity assays were performed. We also defined and compared the effect of 4 culture media during human Mo-DCs production. Three Good Manufacturing Practice (GMP) serum-free culture media for clinical use were tested - DendriMACS, AIM-V and X-VIVO 15. RPMI was used as a comparative term given that it is largely used in pre-clinical research. We characterized DC viability, differentiation, maturation, internalization of tumour lysates, cytokines production and autologous T cell stimulatory capacity, as well as metabolomic profiles by Nuclear Magnetic Resonance (NMR) spectroscopy. Results CSCs from PANC-1 and A549 cell lines were successfully isolated and overexpressed the stem-like markers NANOG, OCT4, SOX2 and CD133, with resistance to gemcitabine. In terms of differentiation, maturation, antigen uptake capacity and metabolic profiles, AIM-V and X-VIVO 15 present similar results. However, the use of X-VIVO 15 shows an enhanced DC production of IL-12. DCs cultured in X-VIVO 15 and AIM-V media are able to induce a superior stimulation of T cells (CTLs and Th1 responses) while DCs cultured in DendriMACS are more prone to induce Treg polarization. Our data show that X-VIVO 15 and AIM-V culture media are preferable to support the differentiation of DCs to be used in immunostimulatory approaches such as in cancer immunotherapy. Conclusions Overall, our results demonstrate that blood monocytic precursors present considerable plasticity allowing a tailored differentiation of DCs just by changing the nutritive support. This highlights the need of critically defining the culture medium to be used in DC cancer immunotherapy, attaining the desired cell characteristics and consequent robust clinical responses. We are now assessing in vitro anti-tumour cytotoxicity to evaluate if DC loading with CSC antigens can be an efficient immunotherapy strategy to target and eliminate this specific and resistant cancer cell population. Funding ImmunoDCs@CancerStemCells: Cellular Immunotherapy towards the elimination of cancer stem cells (Ref.: POCI-01-0247-FEDER-033532), co-funded by FEDER, COMPETE2020 and University of Coimbra. Disclosure Information J. Calmeiro: None. M. Carrascal: A. Employment (full or part-time); Significant; Tecnimede Group. L. Mendes: None. I.F. Duarte: None. C. Gomes: None. J. Serra: A. Employment (full or part-time); Significant; Tecnimede Group. A. Falcao: None. M.T. Cruz: None. B.M. Neves: None.

In this work, untargeted metabolomics was used to unveil the impact of a

This study employed NMR metabolomics to characterize macrophage responses to subtoxic concentrations of silver nanoparticles (AgNPs ca. 30 nm) and ionic silver (Ag+), with a view to further elucidate their immunomodulatory activity at the cell metabolism level. Exposure to AgNPs caused RAW 264.7 macrophages to decrease intracellular glucose utilization, possibly due to interference with glycolytic enzymes, and to reprogram the TCA cycle towards anaplerotic fueling and production of anti-inflammatory metabolites (e.g. itaconate and creatine). Moreover, AgNPs-exposed cells were able to control the levels of reactive oxygen/nitrogen species (ROS/RNS), likely through upregulation of glutathione synthesis. On the other hand, macrophages exposed to Ag+ at equivalent subtoxic concentrations showed reduced metabolic activity, lower ability to counterbalance ROS/RNS and alterations in membrane-related lipids. Overall, the metabolomics approach hereby employed provided novel insights into the differential effects of AgNPs and Ag+, which help explain the lower toxic potential of nanosilver than silver ions. published

In this study, we have employed 1H NMR metabolomics to assess the metabolic responses of PC3 prostate tumour cells to hypoxia and to pharmacological HIF-1α inhibition by DES or its polyacetal conjugate tert-DES. Oxygen deprivation prompted a number of changes in intracellular composition and metabolic activity, mainly reflecting upregulated glycolysis, amino acid catabolism and other compensatory mechanisms used by hypoxic cells to deal with oxidative imbalance and energy deficit. Cell treatment with a non-cytotoxic concentration of DES, under hypoxia, triggered significant changes in 17 metabolites. Among these, lactate, phosphocreatine and reduced glutathione, whose levels showed opposite variations in hypoxic and drug-treated cells, emerged as possible markers of DES-induced HIF-1α inhibition. Furthermore, the free drug had a much higher impact on the cellular metabolome than tert-DES, particularly concerning polyamine and pyrimidine biosynthetic pathways, known to be tightly involved in cell proliferation and growth. This is likely due to the different cell pharmacokinetics observed between free and conjugated DES. Overall, this study has revealed a number of unanticipated metabolic changes that inform on DES and tert-DES direct cellular effects, providing further insight into their mode of action at the biochemical level.

The respiratory tract is the route of entry for accidentally inhaled AgNPs, which can reach the lungs and redistribute to other main organs through systemic circulation. In the present work, we aimed to evaluate silver biodistribution and biological effects after 1 or 2 intratracheal instillations (IT) of two differently sized PVP-coated AgNPs (5 and 50 nm-3 mg/kg) and ionic silver (AgNO

The ability of flavonoids to attenuate macrophage pro-inflammatory activity and to promote macrophage-mediated resolution of inflammation is still poorly understood at the biochemical level. In this study, we have employed NMR metabolomics to assess how therapeutically promising flavonoids (quercetin, naringenin and naringin) affect the metabolism of human macrophages, with a view to better understand their biological targets and activity. In vitro-cultured human macrophages were polarized to the pro-inflammatory M1 phenotype, through incubation with LPS + IFN-γ, and subsequently treated with each flavonoid. The metabolic signatures of pro-inflammatory polarization and of flavonoid incubations were then characterized and compared. The results showed that all flavonoids modulated the cells endometabolome with the strongest impact being observed for quercetin. Many of the flavonoid-induced metabolic variations were in the opposite sense to those elicited by pro-inflammatory stimulation. In particular, the metabolic processes proposed to reflect flavonoid-mediated immunomodulation of macrophages included the downregulation of glycolytic activity, observed for all flavonoids, anti-inflammatory reprogramming of the TCA cycle (mainly quercetin), increased antioxidant protection (quercetin), osmoregulation (naringin), and membrane modification (naringenin). This work revealed key metabolites and metabolic pathways involved in macrophage responses to quercetin, naringenin and naringin, providing novel insights into their immunomodulatory activity.

During tumorigenesis, breast tumour cells undergo metabolic reprogramming, which generally includes enhanced glycolysis, tricarboxylic acid cycle activity, glutaminolysis and fatty acid biosynthesis. However, the extension and functional importance of these metabolic alterations may diverge not only according to breast cancer subtypes, but also depending on the interaction of cancer cells with the complex surrounding microenvironment. This microenvironment comprises a variety of non-cancerous cells, such as immune cells (e.g. macrophages, lymphocytes, natural killer cells), fibroblasts, adipocytes and endothelial cells, together with extracellular matrix components and soluble factors, which influence cancer progression and are predictive of clinical outcome. The continuous interaction between cancer and stromal cells results in metabolic competition and symbiosis, with oncogenic-driven metabolic reprogramming of cancer cells shaping the metabolism of neighbouring cells and vice versa. This review addresses current knowledge on this metabolic crosstalk within the breast tumour microenvironment (TME). Improved understanding of how metabolism in the TME modulates cancer development and evasion of tumour-suppressive mechanisms may provide clues for novel anticancer therapeutics directed to metabolic targets.

Ionic-liquid-based aqueous biphasic systems (IL-based ABS) have been extensively investigated in the separation of high-value biomolecules. However, the understanding of the molecular-level mechanisms ruling phase separation and extraction performance of these systems is crucial to successfully design effective separation processes. In this work, IL-based ABS composed of K2HPO4 and cholinium carboxylate ILs ([Ch][CnCO2] with n = 1 to 7, comprising anions with odd and even alkyl chain length) were investigated. The respective ternary phase diagrams, including binodal curves, tie-lengths, tie-line lengths and critical points, as well as the Setschenow salting-out coefficients (ks) that is a quantitative measure of the two-phase formation ability, were determined at 298 K. The extraction performance of these systems was then evaluated for four amino acids (L-tryptophan, L-phenylalanine, L-tyrosine, L-3,4-dihydroxyphenylalanine/L-dopa). It was found that ILs composed of anions with even alkyl chains display slightly higher ks values, meaning that these ILs are more easily salted-out or more easily phase separate to form ABS. On the other hand, ABS formed by ILs with anions comprising odd alkyl chains lead to slightly higher partition coefficients of amino acids. Beyond the neat ILs odd-even effect resulting from their nanostructuration, being this a well-known phenomenon occurring in a series of their thermophysical properties, it is here shown the existence of an odd-even effect displayed by the IL anion aliphatic moiety in aqueous solution, visible both in the two-phase formation ability and extraction performance of ABS. These findings contribute to elucidate the molecular-level mechanisms governing ABS formation and partitioning of biomolecules, ultimately allowing the design of effective separation platforms. published

Silk fibroin nanoparticles are emerging as promising nanomedicines, but their full therapeutic potential is yet to be realized. These nanoparticles can be readily PEGylated to improve colloidal stability and to tune degradation and drug release profiles; however, the relationship between silk fibroin nanoparticle PEGylation and macrophage activation still requires elucidation. Here, we used in vitro assays and nuclear magnetic resonance based metabolomics to examine the inflammatory phenotype and metabolic profiles of macrophages following their exposure to unmodified or PEGylated silk fibroin nanoparticles. The macrophages internalized both types of nanoparticles, but they showed different phenotypic and metabolic responses to each nanoparticle type. Unmodified silk fibroin nanoparticles induced the upregulation of several processes, including production of proinflammatory mediators (e.g., cytokines), release of nitric oxide, and promotion of antioxidant activity. These responses were accompanied by changes in the macrophage metabolomic profiles that were consistent with a proinflammatory state and that indicated an increase in glycolysis and reprogramming of the tricarboxylic acid cycle and the creatine kinase/phosphocreatine pathway. By contrast, PEGylated silk fibroin nanoparticles induced milder changes to both inflammatory and metabolic profiles, suggesting that immunomodulation of macrophages with silk fibroin nanoparticles is PEGylation-dependent. Overall, PEGylation of silk fibroin nanoparticles reduced the inflammatory and metabolic responses initiated by macrophages, and this observation could be used to guide the therapeutic applications of these nanoparticles. published

The antibacterial potential of silver nanoparticles (AgNPs) resulted in their increasing incorporation into consumer, industrial and biomedical products. Therefore, human and environmental exposure to AgNPs (either as an engineered product or a contaminant) supports the emergent research on the features conferring them different toxicity profiles. In this study, 30nm AgNPs coated with citrate or poly(ethylene glycol) (PEG) were used to assess the influence of coating on the effects produced on a human hepatoma cell line (HepG2), namely in terms of viability, apoptosis, apoptotic related genes, cell cycle and cyclins gene expression. Both types of coated AgNPs decreased cell proliferation and viability with a similar toxicity profile. At the concentrations used (11 and 5μg/mL corresponding to IC50 and ~IC10 levels, respectively) the amount of cells undergoing apoptosis was not significant and the apoptotic related genes BCL2 (anti-apoptotic gene) and BAX (pro-apoptotic gene) were both downregulated. Moreover, both AgNPs affected HepG2 cell cycle progression at the higher concentration (11μg/mL) by increasing the percentage of cells in S (synthesis phase) and G2 (Gap 2 phase) phases. Considering the cell-cycle related genes, the expression of cyclin B1 and cyclin E1 genes were decreased. Thus, this work has shown that citrate- and PEG-coated AgNPs impact on HepG2 apoptotic gene expression, cell cycle dynamics and cyclin regulation in a similar way. More research is needed to determine the properties that confer AgNPs at lower toxicity, since their use has proved helpful in several industrial and biomedical contexts.

Silver nanoparticles (AgNPs) are widely used in industrial, cosmetic, and biomedical products, and humans are frequently exposed to these products through the skin. It is widely recognized that the characteristics of AgNPs (e.g., size, coating) may influence their cytotoxic effects, but their correlation with DNA damage and mitotic disorders remains poorly explored. In this study, human keratinocytes (HaCaT cell line) were exposed to well-characterized 30 nm AgNPs coated with citrate, and their effects on viability, DNA fragmentation (assessed by the comet assay), and micronuclei (MNi) induction (assessed by the cytokinesis-block micronucleus cytome assays, CBMN) were investigated. The results showed that 10 and 40 μg/mL AgNPs decreased cell proliferation and viability, and induced a significant genetic damage. This was observed by an increase of DNA amount in comet tail, which linearly correlated with dose and time of exposure. Also, cytostaticity (increase of mononucleated cells) and MNi rates increased in treated cells. In contrast, no significant changes were observed in nucleoplasmatic bridges (NPBs) or nuclear buds (NBUDs), although NBUDs tended to increase in all conditions and periods. The cytostatic effects on HaCaT cells were also shown by the decrease of their nuclear division index. Thus, both comet and CBMN assays supported the observation that citrate-AgNPs induced genotoxic effects on HaCaT cells. Considering that AgNPs are present in a vast number of consumer products and also in multiple nanomedicine skin applications and formulations, more research is needed to determine the properties that confer less toxicity of AgNPs to different cell lines.

Spectroscopic non-targeted methods are gaining ever-growing importance in quality control and authenticity assessment of food products because of their strong potential for identification of specific features of the products by data-driven classifiers. One of the factors hampering the diffusion of spectroscopic non-targeted methods and data-driven classifiers is the lack of harmonized guidelines for their development and validation. In particular, to date, neither conditions to directly compare spectra recorded by different spectrometers nor studies demonstrating the statistical equivalence of the spectra are available. Among the spectroscopic analytical techniques suitable for the development of non-targeted methods, nuclear magnetic resonance (NMR) offers the unique opportunity to generate statistically equivalent signals. In this paper, the feasibility of NMR spectroscopy to generate statistically equivalent NMR signals from a number of different spectrometers was demonstrated for complex mixtures (aqueous extracts of wheat and flour) by organizing an inter-laboratory comparison involving 36 NMR spectrometers. Univariate statistics along with multivariate analysis were exploited to establish unbiased criteria for assessing the statistical equivalence of the NMR signals. The aspects affecting the signal equivalence were investigated, and possible solutions to reduce the extent of the human error were proposed and applied with satisfactory results. This study furnishes the scientific community with an appropriate and easy procedure to validate non-targeted NMR methods and provides error values to be used as a reference for future studies.

In an era of “omic” sciences and big data analysis, many research areas still constitute new ground for the application of such nontraditional strategies, which may potentially provide significant and rapid advances in scientific knowledge. Biomaterial research is an example of this, in particular regarding the potential of metabolomics in providing further insight into various steps of biomaterial development, from its preparation to the assessment and fine-tuning of its performance in a biological context. This review firstly presents the basic concepts of metabolomics and the analytic strategies entailed, while briefly stating the major advances it has fostered in health research. The recent translation of metabolomics to biomaterial research is here illustrated in three major aspects: (1) stem cell (SC) research, based on the link between metabolic traits and SC function or differentiation, amenable to biomaterial guidance; (2) measurement and monitoring of biomaterials' biological performance; and (3) follow-up and optimization of the synthesis of biopolymers used as biomaterials. Furthermore, the present text attempts to provide the reader with a view of possible future developments in the use of metabolomics to enhance biomaterial research, from improving biomaterial formulations to fostering new strategies for the clinical follow-up of biomaterial interventions.

Recognition of neoplastic metabolic reprogramming as one of cancer's hallmarks has paved the way for developing novel metabolism-targeted therapeutic approaches. The use of plant-derived natural bioactive compounds for this endeavor is especially promising, due to their diverse structures and multiple targets. Hence, over the past decade, a growing number of studies have assessed the impact of phytochemicals on tumor cell metabolism, aiming at improving current knowledge on their mechanisms of action and, at the same time, evaluating their potential as anti-cancer metabolic modulators. In this Review, we focus on three classes of plant-derived compounds with promising anti-cancer activity-phenolic compounds, isoprenoids, and alkaloids-to describe their effects on major energetic and biosynthetic pathways of human tumor cells. Such a comprehensive and integrated account of the ability of these compounds to hit different metabolic targets is expected to contribute to the rational design and critical assessment of novel anti-cancer therapies based on natural-product-mediated metabolic reprogramming.

Bioseparation processes are a relevant part of modern biotechnology, particularly regarding the development of efficient and biocompatible methods for the separation and purification of added-value biologically active compounds. In this field, ionic liquids (ILs) have been proposed, either in liquid-liquid extractions, in which non-water miscible ILs or aqueous biphasic systems (ABS) formed by ILs can be used, or in solid-liquid extractions, in which they are covalently attached to create supported IL phases (SILPs). Aprotic ILs possess unique properties, such as non-volatility and designability, which are valuable in their use in bioseparation processes. In this chapter, we summarize and discuss bioseparation processes based on ILs, including both liquid-liquid and solid-liquid extractions, applied to amino acids and proteins. The most recent and remarkable advances in this area are emphasized, and improvements brought by the use of ILs properly discussed. New insights and envisaged directions with IL-based bioseparation processes are suggested. Graphical Abstract.

The expansion of biomedical and therapeutic applications of silver nanoparticles (AgNPs) raises the need to further understand their biological effects on human cells. In this work, NMR metabolomics has been applied to reveal the metabolic effects of AgNPs toward human hepatoma (HepG2) cells, which are relevant with respect to nanoparticle accumulation and detoxification. Cellular responses to widely disseminated citrate-coated AgNPs (Cit30) and to emergent biogenic AgNPs prepared using an aqueous plant extract as reducing and stabilizing agent (GS30) have been compared with a view to assess the influence of nanoparticle coating on the metabolic effects produced. Subtoxic concentrations (IC5 and IC20) of both nanoparticle types caused profound changes in the cellular metabolome, suggesting adaptations in energy production processes (glucose metabolism and the phosphocreatine system), antioxidant defenses, protein degradation and lipid metabolism. These signatures were proposed to reflect mainly metabolism...

Bioseparation processes are a relevant part of modern biotechnology, particularly regarding the development of efficient and biocompatible methods for the separation and purification of added-value biologically active compounds. In this field, ionic liquids (ILs) have been proposed, either in liquid–liquid extractions, in which non-water miscible ILs or aqueous biphasic systems (ABS) formed by ILs can be used, or in solid–liquid extractions, in which they are covalently attached to create supported IL phases (SILPs). Aprotic ILs possess unique properties, such as non-volatility and designability, which are valuable in their use in bioseparation processes. In this chapter, we summarize and discuss bioseparation processes based on ILs, including both liquid–liquid and solid–liquid extractions, applied to amino acids and proteins. The most recent and remarkable advances in this area are emphasized, and improvements brought by the use of ILs properly discussed. New insights and envisaged directions with IL-based bioseparation processes are suggested.

Although silver nanoparticles (AgNPs) are widely disseminated and show great potential in the biomedical field, there is a recognized need to better understand their action at the metabolic and functional levels. In this work, we have used NMR metabolomics, together with conventional clinical chemistry and histological examination, to characterize multi-organ and systemic metabolic responses to AgNPs intravenously administered to mice at 8 mg/kg body weight (a dose not eliciting overt toxicity). The major target organs of AgNPs accumulation, liver and spleen, showed the greatest metabolic changes, in a clear 2-stage response. In particular, the liver of dosed mice was found to switch from glycogenolysis and lipid storage, at 6 h postinjection, to glycogenesis and lipolysis, at subsequent times up to 48 h. Moreover, metabolites related to antioxidative defense, immunoregulation and detoxification seemed to play a crucial role in avoiding major hepatic damage. The spleen showed several early changes, including depletion of several amino acids, possibly reflecting impairment of hemoglobin recycling, while only a few differences remained at 48 h postinjection. In the heart, the metabolic shift towards TCA cycle intensification and increased ATP production possibly reflected a beneficial adaptation to the presence of AgNPs. On the other hand, the TCA cycle appeared to be down regulated in the lungs of injected mice, which showed signs of inflammation. Thekidneys showed the mildest metabolic response to AgNPs. Overall, this study has shown that NMR metabolomics is a powerful tool to monitor invivo metabolic responses to nanoparticles, revealing unforeseen effects.

Citrate-coated silver nanoparticles (citrate-AgNPs) are among the most commonly used nanomaterials, widely present in industrial and biomedical products. In this study, the cytotoxicity of 30-nm citrate-AgNPs on the macrophage cell line RAW 264.7 was evaluated, using a battery of cytotoxicity endpoints (viability, oxidative stress, and cytostaticity/clastogenicity), at 24 and 48 h of exposure. Citrate-AgNPs decreased cell proliferation and viability only at 75 μg/mL, suggesting a low sensitivity of RAW cells to lower doses of these AgNPs. After 24 h of exposure, ROS content decreased in cells exposed to 60 μg/mL AgNPs (IC20 value), corroborating the high tolerance of these cells to citrate-AgNPs. However, these cells suffered an impairment of the cell cycle, shown by an increase at the sub-G1 phase. This increase of the sub-G1 population was correlated with an increase of DNA fragmentation, suggesting an increase of apoptosis. Thus, our data are important to understand the effects of low concentrations (IC20) of citrate-AgNPs on in vitro vital macrophage functions.

A metabolomics study of Pd

Ionic-liquid-based aqueous biphasic systems (IL-based ABS) have been described as promising platforms for the extraction and separation of proteins. However, imidazolium-based ILs have been the preferred choice, which may raise some biocompatibility and biodegradability concerns. In this work, novel ABS composed of tetraalk- ylammonium-based ILs and potassium phosphate solutions at different pH values (pH = 7, 8, 9 and 13, using K2HPO4/KH2PO4 or K3PO4) were investigated in terms of extraction efficiency and recovery yield for two proteins, namely ovalbumin and lysozyme. These proteins were selected due to their wide application in several sectors, being present in egg white. At pH 7, the complete extraction and recovery of lysozyme to the IL-rich phase are achieved in all systems; however, low recovery yields of ovalbumin are obtained with ABS formed by ILs with longer alkyl side chains. Furthermore, an increase in the pH above the proteins isoelectric point is deleterious for their recovery in the IL-rich phase. In order to characterize the molecular-level mechanisms that could maximize the proteins recovery, molecular docking studies were carried out, showing that ILs that preferentially establish hydrophobic interactions with these proteins are those that lead to their aggregation and lower recovery yields. Finally, it is shown the proteins recovery from the IL-rich phase by ice cold ethanol precipitation, where up to 99% of lysozyme can be recovered. These results support the viability of adequate IL-based ABS to extract ovalbumin and lysozyme and the possibility of recovering stable proteins from the IL-rich phase into an adequate buffered aqueous solution, thus contributing to the design of effective separation processes. published

The preparation of graphene-based nanomaterials (GBNs) with appropriate stability and biocompatibility is crucial for their use in biomedical applications. In this work, three GBNs differing in size and/or functionalization have been synthetized and characterized, and their in vitro biological effects were compared. Pegylated graphene oxide (GO-PEG, 200–500 nm) and flavin mononucleotide-stabilized pristine graphene with two different sizes (PG-FMN, 200–400 nm and 100–200 nm) were administered to macrophages, chosen as cellular model due to their key role in the processing of foreign materials and the regulation of inflammatory responses. The results showed that cellular uptake of GBNs was mainly influenced by their lateral size, while the inflammatory potential depended also on the type of functionalization. PG-FMN nanomaterials (both sizes) triggered significantly higher nitric oxide (NO) release, together with some intracellular metabolic changes, similar to those induced by the prototypical inflammatory stimulus LPS. NMR metabolomics revealed that macrophages incubated with smaller PG-FMN displayed increased levels of succinate, itaconate, phosphocholine and phosphocreatine, together with decreased creatine content. The latter two variations were also detected in cells incubated with larger PG-FMN nanosheets. On the other hand, GO-PEG induced a decrease in the inflammatory metabolite succinate and a few other changes distinct from those seen in LPS-stimulated macrophages. Assessment of TNF-α secretion and macrophage surface markers (CD80 and CD206) further corroborated the low inflammatory potential of GO-PEG. Overall, these findings revealed distinct phenotypic and metabolic responses of macrophages to different GBNs, which inform on their immunomodulatory activity and may contribute to guide their therapeutic applications., This work was developed in the scope of the projects CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, and CESAM, FCT Ref. UID/AMB/50017/2019, financed by national funds through FCT/MCTES, Portugal. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). Further acknowledgments are due to Bruker BioSpinGmbH and the European Union Framework Programme for Research and Innovation HORIZON 2020, under the TEAMING Grant agreement No 739572 - The Discoveries CTR. I.F.D. and H.O. acknowledge FCT/MCTES for research contracts under the Programs ‘Investigador FCT’ and Stimulus to Scientific Employment (CEECIND/04050/2017), respectively, and M.C. acknowledges the FCT-awarded research grant SFRH/BPD/101468/2014. This research was also supported by the Spanish Ministerio de Economía y Competitividad (MAT2016-75611-R AEI/FEDER, UE). M.A.-V., S.V.-R. and J.I.P. acknowledge financial support from the Spanish Ministerio de Economía y Competitividad (MINECO) and the European Regional Development Fund (ERDF) through project MAT2015-69844-R, and from the Spanish Ministerio de Ciencia, Innovación y Universidades, the Spanish Agencia Estatal de Investigación and ERDF, through project RTI2018-100832-B-I00. Partial funding by Plan de Ciencia, Tecnología e Innovación (PCTI) 2013-2017 del Principado de Asturias and ERDF (project IDI/2018/000233) is also acknowledged.

e14009 Background: Dendritic cells (DCs) are one of the central tools in cellular anti-tumor immunotherapy, being characterized by their capacity for acquiring and processing antigens and ability to produce strong antitumor immune responses. The production of clinical grade ex-vivo monocyte-derived DCs (Mo-DCs) is the most frequent approach for antitumor vaccines production. Recently, therapeutic resistance to radio/chemotherapy and disease recurrence was shown to be in part due to a small cancer stem cell (CSCs) population present in tumors. Methods: Here, we aim to target and eradicate CSCs by developing a novel DC-based immunotherapy vaccine for pancreatic and non-small cells lung cancer (NSCLC), comparing the loading of CSCs vs. classical tumor lysates. Results: CSCs from PANC-1 (pancreatic cancer) and A549 (NSCLC) cell lines were successfully isolated and characterized, overexpressing stem-like markers: NANOG, OCT4, SOX2 and CD133. CSCs resistance to Gemcitabine was also assessed. Before comparing the 2 types of vaccine loading, we also analyzed the impact of 3 GMP free-serum culture media on the phenotype and functional abilities of Mo-DCs. DCs cultured in X-VIVO 15 and AIM-V media show enhanced production of IL-12 and are able to induce a superior stimulation of T cells, mainly CTLs and Th1 subsets. By contrast, DCs cultured in DendriMACS are more prone to induce Treg polarization. Conclusions: Overall, our data demonstrate that blood monocytic precursors present considerable plasticity allowing a tailored differentiation of DCs just by changing the nutritive support. We also highlight the need of critically defining the culture medium to be used in DC cancer immunotherapy in order to attain desired cell characteristics and by consequent robust responses. Finally, our preliminary results indicate that loading DCs with CSCs antigens may be an effective strategy to target and destroy this resilient cancer cell population.

The knowledge that the organism's metabolome is a potentially informative mirror of the impact of disease and its dynamics has led to promising developments in cancer research, strongly geared toward the discovery of new biomarkers of disease onset and progression. The present text reviews the advances made in the last 10 years in lung cancer research making use of the metabolomics strategies, particularly concerning metabolite profiling of human biofluids (blood serum and plasma, urine and others), expected to reflect the deviant metabolic behavior of lung tumors. The main goal of this article is to provide the reader with an up-to-date summary of the main metabolic variations taking place in biofluids, in relation to lung cancer, as well as of the analytical strategies employed to unveil them. Furthermore, particular needs and challenges are identified and possible developments envisaged.