NaCl enhances CD8+ T cell effector functions in cancer immunotherapy.

CD8+ T cells control tumors but inevitably become dysfunctional in the tumor microenvironment. Here, we show that sodium chloride (NaCl) counteracts T cell dysfunction to promote cancer regression. NaCl supplementation during CD8+ T cell culture induced effector differentiation, IFN-γ production and cytotoxicity while maintaining the gene networks responsible for stem-like plasticity. Accordingly, adoptive transfer of tumor-specific T cells resulted in superior anti-tumor immunity in a humanized mouse model. In mice, a high-salt diet reduced the growth of experimental tumors in a CD8+ T cell-dependent manner by inhibiting terminal differentiation and enhancing the effector potency of CD8+ T cells. Mechanistically, NaCl enhanced glutamine consumption, which was critical for transcriptional, epigenetic and functional reprogramming. In humans, CD8+ T cells undergoing antigen recognition in tumors and predicting favorable responses to checkpoint blockade immunotherapy resembled those induced by NaCl. Thus, NaCl metabolism is a regulator of CD8+ T cell effector function, with potential implications for cancer immunotherapy.

MSIpixel: a fully automated pipeline for compound annotation and quantitation in mass spectrometry imaging experiments.

Mass spectrometry imaging (MSI) is commonly used to map the spatial distribution of small molecules within complex biological matrices. One of the major challenges in imaging MS-based spatial metabolomics is molecular identification and metabolite annotation, to address this limitation, annotation is often complemented with parallel bulk LC-MS2-based metabolomics to confirm and validate identifications. Here we applied MSI method, utilizing data-dependent acquisition, to visualize and identify unknown molecules in a single instrument run. To reach this aim we developed MSIpixel, a fully automated pipeline for compound annotation and quantitation in MSI experiments. It overcomes challenges in molecular identification, and improving reliability and comprehensiveness in MSI-based spatial metabolomics.

Therapeutic induction of energy metabolism reduces neural tissue damage and increases microglia activation in severe spinal cord injury.

Neural tissue has high metabolic requirements. Following spinal cord injury (SCI), the damaged tissue suffers from a severe metabolic impairment, which aggravates axonal degeneration and neuronal loss. Impaired cellular energetic, tricarboxylic acid (TCA) cycle and oxidative phosphorylation metabolism in neuronal cells has been demonstrated to be a major cause of neural tissue death and regeneration failure following SCI. Therefore, rewiring the spinal cord cell metabolism may be an innovative therapeutic strategy for the treatment of SCI. In this study, we evaluated the therapeutic effect of the recovery of oxidative metabolism in a mouse model of severe contusive SCI. Oral administration of TCA cycle intermediates, co-factors, essential amino acids, and branched-chain amino acids was started 3 days post-injury and continued until the end of the experimental procedures. Metabolomic, immunohistological, and biochemical analyses were performed on the injured spinal cord sections. Administration of metabolic precursors enhanced spinal cord oxidative metabolism. In line with this metabolic shift, we observed the activation of the mTORC1 anabolic pathway, the increase in mitochondrial mass, and ROS defense which effectively prevented the injury-induced neural cell apoptosis in treated animals. Consistently, we found more choline acetyltransferase (ChAT)-expressing motor neurons and increased neurofilament-positive corticospinal axons in the spinal cord parenchyma of the treated mice. Interestingly, oral administration of the metabolic precursors increased the number of activated microglia expressing the CD206 marker suggestive of a pro-resolutive, M2-like phenotype. These molecular and histological modifications observed in treated animals ultimately led to a significant, although partial, improvement of the motor functions. Our data demonstrate that rewiring the cellular metabolism can represent an effective strategy to treat SCI.

Amyloid aggregates accumulate in melanoma metastasis modulating YAP activity.

Melanoma progression is generally associated with increased transcriptional activity mediated by the Yes-associated protein (YAP). Mechanical signals from the extracellular matrix are sensed by YAP, which then activates the expression of proliferative genes, promoting melanoma progression and drug resistance. Which extracellular signals induce mechanotransduction, and how this is mediated, is not completely understood. Here, using secretome analyses, we reveal the extracellular accumulation of amyloidogenic proteins, i.e. premelanosome protein (PMEL), in metastatic melanoma, together with proteins that assist amyloid maturation into fibrils. We also confirm the accumulation of amyloid-like aggregates, similar to those detected in Alzheimer disease, in metastatic cell lines, as well as in human melanoma biopsies. Mechanistically, beta-secretase 2 (BACE2) regulates the maturation of these aggregates, which in turn induce YAP activity. We also demonstrate that recombinant PMEL fibrils are sufficient to induce mechanotransduction, triggering YAP signaling. Finally, we demonstrate that BACE inhibition affects cell proliferation and increases drug sensitivity, highlighting the importance of amyloids for melanoma survival, and the use of beta-secretase inhibitors as potential therapeutic approach for metastatic melanoma.

SMfinder: Small Molecules Finder for Metabolomics and Lipidomics Analysis.

Metabolomics and lipidomics studies are becoming increasingly popular but available tools for automated data analysis are still limited. The major issue in untargeted metabolomics is linked to the lack of efficient ranking methods allowing accurate identification of metabolites. Herein, we provide a user-friendly open-source software, named SMfinder, for the robust identification and quantification of small molecules. The software introduces an MS2 false discovery rate approach, which is based on single spectral permutation and increases identification accuracy. SMfinder can be efficiently applied to shotgun and targeted analysis in metabolomics and lipidomics without requiring extensive in-house acquisition of standards as it provides accurate identification by using available MS2 libraries in instrument independent manner. The software, downloadable at www.ifom.eu/SMfinder, is suitable for untargeted, targeted, and flux analysis.

Tetraspanins shape the synapse.

Tetraspanins are a family of proteins largely expressed in mammals. These proteins share very similar structures and are involved in several biological processes spanning from the immune system to cancer growth regulation. Moreover, tetraspanins are scaffold proteins that are able to interact with each other and with a subset of proteins involved in the regulation of the central nervous system, including synapse formation, function and plasticity. In this review, we will focus on the analysis of the literature on tetraspanins, highlighting their involvement in synapse formation and function through direct or indirect modulation of synaptic proteins.

Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth.

Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.

Correction: Rubiola et al. Risk Factors for Bovine Cysticercosis in North-West Italy: A Multi-Year Case-Control Study. Animals 2021, 11, 3049.

Error in Institutional Review Board Statement [...].

Opti-nQL: An Optimized, Versatile and Sensitive Nano-LC Method for MS-Based Lipidomics Analysis.

Lipidomics is the comprehensive analysis of lipids in a given biological system. This investigation is often limited by the low amount and high complexity of biological samples, therefore highly sensitive lipidomics methods are required. Nanoflow-LC/MS offers extremely high sensitivity; however, it is challenging as a more demanding maintenance is often needed compared to conventional microflow-LC approaches. Here, we developed a sensitive and reproducible lipidomics LC method, termed Opti-nQL, which can be applied to any biological system. Opti-nQL has been validated with cellular lipid extracts of human and mouse origin and with different lipid extraction methods. Among the resulting 4000 detected features, 700 and even more unique lipid molecular species have been identified covering 16 lipid sub-classes, while 400 lipids were uniquely structure defined by MS/MS. These results were obtained by analyzing an amount of lipids extract equivalent to 40 ng of proteins, being highly suitable for low abundant samples. MS analysis showed that theOpti-nQL method increases the number of identified lipids, which is evidenced by injecting 20 times less material than in microflow based chromatography, being more reproducible and accurate thus enhancing robustness of lipidomics analysis.

Risk Factors for Bovine Cysticercosis in North-West Italy: A Multi-Year Case-Control Study.

Taenia saginata is the causative agent of bovine cysticercosis, a zoonotic parasitic disease with a worldwide distribution. Bovine cysticercosis is considered to be an important food safety issue responsible for human taeniasis and a major economic concern since infected carcasses undergo condemnation, freezing and downgrading. The aim of the current investigation was to assess the presence of farm-level risk factors for bovine cysticercosis in an endemic area in North-West Italy. A questionnaire was designed to collect information relating to several farm structural features, as well as management practices, environmental characteristics and attitudes of farmers. The questionnaire was administered in two separate time intervals by direct interview to previously selected case and control farms. Overall, 32 case farms and 131 control farms were included between 2005 and 2011 and 50 case farms and 192 control farms were included between 2014 and 2020. The present survey showed a significant association between the detection of bovine cysticercosis cases at slaughter and farm proximity to picnic spots, closeness of wastewater treatment plant effluents, loose-housing systems and presence of employees along with the family members, highlighting the need for targeted awareness raising policies.

Metabolism of Stem and Progenitor Cells: Proper Methods to Answer Specific Questions.

Stem cells can stay quiescent for a long period of time or proliferate and differentiate into multiple lineages. The activity of stage-specific metabolic programs allows stem cells to best adapt their functions in different microenvironments. Specific cellular phenotypes can be, therefore, defined by precise metabolic signatures. Notably, not only cellular metabolism describes a defined cellular phenotype, but experimental evidence now clearly indicate that also rewiring cells towards a particular cellular metabolism can drive their cellular phenotype and function accordingly. Cellular metabolism can be studied by both targeted and untargeted approaches. Targeted analyses focus on a subset of identified metabolites and on their metabolic fluxes. In addition, the overall assessment of the oxygen consumption rate (OCR) gives a measure of the overall cellular oxidative metabolism and mitochondrial function. Untargeted approach provides a large-scale identification and quantification of the whole metabolome with the aim to describe a metabolic fingerprinting. In this review article, we overview the methodologies currently available for the study of in vitro stem cell metabolism, including metabolic fluxes, fingerprint analyses, and single-cell metabolomics. Moreover, we summarize available approaches for the study of in vivo stem cell metabolism. For all of the described methods, we highlight their specificities and limitations. In addition, we discuss practical concerns about the most threatening steps, including metabolic quenching, sample preparation and extraction. A better knowledge of the precise metabolic signature defining specific cell population is instrumental to the design of novel therapeutic strategies able to drive undifferentiated stem cells towards a selective and valuable cellular phenotype.

Glutamatergic synapses in neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) are a group of diseases whose symptoms arise during childhood or adolescence and that impact several higher cognitive functions such as learning, sociability and mood. Accruing evidence suggests that a shared pathogenic mechanism underlying these diseases is the dysfunction of glutamatergic synapses. We summarize present knowledge on autism spectrum disorders (ASD), intellectual disability (ID), Down syndrome (DS), Rett syndrome (RS) and attention-deficit hyperactivity disorder (ADHD), highlighting the involvement of glutamatergic synapses and receptors in these disorders. The most commonly shared defects involve α-amino-3-hydroxy-5-methyl- 4-isoxazole propionic acid receptors (AMPARs), N-methyl-d-aspartate receptors (NMDARs) and metabotropic glutamate receptors (mGluRs), whose functions are strongly linked to synaptic plasticity, affecting both cell-autonomous features as well as circuit formation. Moreover, the major scaffolding proteins and, thus, the general structure of the synapse are often deregulated in neurodevelopmental disorders, which is not surprising considering their crucial role in the regulation of glutamate receptor positioning and functioning. This convergence of defects supports the definition of neurodevelopmental disorders as a continuum of pathological manifestations, suggesting that glutamatergic synapses could be a therapeutic target to ameliorate patient symptomatology.

Trehalose reduces retinal degeneration, neuroinflammation and storage burden caused by a lysosomal hydrolase deficiency.

The accumulation of undegraded molecular material leads to progressive neurodegeneration in a number of lysosomal storage disorders (LSDs) that are caused by functional deficiencies of lysosomal hydrolases. To determine whether inducing macroautophagy/autophagy via small-molecule therapy would be effective for neuropathic LSDs due to enzyme deficiency, we treated a mouse model of mucopolysaccharidosis IIIB (MPS IIIB), a storage disorder caused by deficiency of the enzyme NAGLU (alpha-N-acetylglucosaminidase [Sanfilippo disease IIIB]), with the autophagy-inducing compound trehalose. Treated naglu-/ - mice lived longer, displayed less hyperactivity and anxiety, retained their vision (and retinal photoreceptors), and showed reduced inflammation in the brain and retina. Treated mice also showed improved clearance of autophagic vacuoles in neuronal and glial cells, accompanied by activation of the TFEB transcriptional network that controls lysosomal biogenesis and autophagic flux. Therefore, small-molecule-induced autophagy enhancement can improve the neurological symptoms associated with a lysosomal enzyme deficiency and could provide a viable therapeutic approach to neuropathic LSDs. ABBREVIATIONS: ANOVA: analysis of variance; Atg7: autophagy related 7; AV: autophagic vacuoles; CD68: cd68 antigen; ERG: electroretinogram; ERT: enzyme replacement therapy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GNAT2: guanine nucleotide binding protein, alpha transducing 2; HSCT: hematopoietic stem cell transplantation; INL: inner nuclear layer; LC3: microtubule-associated protein 1 light chain 3 alpha; MPS: mucopolysaccharidoses; NAGLU: alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); ONL: outer nuclear layer; PBS: phosphate-buffered saline; PRKCA/PKCα: protein kinase C, alpha; S1BF: somatosensory cortex; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; VMP/VPL: ventral posterior nuclei of the thalamus.

Biosynthesis of Astrocytic Trehalose Regulates Neuronal Arborization in Hippocampal Neurons.

Trehalose is a nonreducing disaccharide that has recently attracted much attention because of its ability to inhibit protein aggregation, induce autophagy, and protect against dissections and strokes. In vertebrates, the biosynthesis of trehalose was long considered absent due to the lack of annotated genes involved in this process. In contrast, trehalase (TreH), which is an enzyme required for the cleavage of trehalose, is known to be conserved and expressed. Here, we show that trehalose is present as an endogenous metabolite in the rodent hippocampus. We found that primary astrocytes were able to synthesize trehalose and release it into the extracellular space. Notably, the TreH enzyme was observed only in the soma of neurons, which are the exclusive users of this substrate. A statistical analysis of the metabolome during different stages of maturation indicated that this metabolite is implicated in neuronal maturation. A morphological analysis of primary neurons confirmed that trehalose is required for neuronal arborization.

Sex-specific eNOS activity and function in human endothelial cells.

Clinical and epidemiological data show that biological sex is one of the major determinants for the development and progression of cardiovascular disease (CVD). Impaired endothelial function, characterized by an imbalance in endothelial Nitric Oxide Synthase (eNOS) activity, precedes and accelerates the development of CVD. However, whether there is any sexual dimorphism in eNOS activity and function in endothelial cells (ECs) is still unknown. Here, by independently studying human male and female ECs, we found that female ECs expressed higher eNOS mRNA and protein levels both in vitro and ex vivo. The increased eNOS expression was associated to higher enzymatic activity and nitric oxide production. Pharmacological and genetic inhibition of eNOS affected migratory properties only in female ECs. In vitro angiogenesis experiments confirmed that sprouting mostly relied on eNOS-dependent migration in female ECs. At variance, capillary outgrowth from male ECs was independent of eNOS activity but required cell proliferation. In this study, we found sex-specific differences in the EC expression, activity, and function of eNOS. This intrinsic sexual dimorphism of ECs should be further evaluated to achieve more effective and precise strategies for the prevention and therapy of diseases associated to an impaired endothelial function such as CVD and pathological angiogenesis.

Effects of ß-Carotene and Its Cleavage Products in Primary Pneumocyte Type II Cells.

ß-Carotene has been shown to increase the risk of developing lung cancer in smokers and asbestos workers in two large scale trails, the Beta-Carotene and Retinol Efficacy Trial (CARET) and the Alpha-Tocopherol Beta-carotene Cancer Prevention Trial (ATBC). Based on this observation, it was proposed that genotoxic oxidative breakdown products may cause this effect. In support of this assumption, increased levels of sister chromatid exchanges, micronuclei, and chromosomal aberrations were found in primary hepatocyte cultures treated with a mixture of cleavage products (CPs) and the major product apo-8'carotenal. However, because these findings cannot directly be transferred to the lung due to the exceptional biotransformation capacity of the liver, potential genotoxic and cytotoxic effects of ß-carotene under oxidative stress and its CPs were investigated in primary pneumocyte type II cells. The results indicate that increased concentrations of ß-carotene in the presence of the redox cycling quinone dimethoxynaphthoquinone (DMNQ) exhibit a cytotoxic potential, as evidenced by an increase of apoptotic cells and loss of cell density at concentrations > 10 µM. On the other hand, the analysis of micronucleated cells gave no clear picture due to the cytotoxicity related reduction of mitotic cells. Last, although CPs induced significant levels of DNA strand breaks even at concentrations ≥ 1 µM and 5 µM, respectively, ß-carotene in the presence of DMNQ did not cause DNA damage. Instead, ß-carotene appeared to act as an antioxidant. These findings are in contrast with what was demonstrated for primary hepatocytes and may reflect different sensitivities to and different metabolism of ß-carotene in the two cell types.

Biosynthesis of glycerol phosphate is associated with long-term potentiation in hippocampal neurons.

INTRODUCTION: Neurons have a very high energy requirement, and their metabolism is tightly regulated to ensure delivery of adequate substrate to sustain neuronal activity and neuroplastic changes. The mechanisms underlying the regulation of neuronal metabolism, however, are not completely clear. OBJECTIVE: The objective of this study was to investigate the central carbon metabolism in neurons, in order to identify the regulatory pathways governing neuronal anabolism and catabolism. METHODS: Here we first have applied MS-based endometabolomics to elucidate the metabolic dynamics in cultured hippocampal primary neurons. Using nanoLC-ESI-LTQ Orbitrap MS approach followed by statistical analysis, we measure the dynamics of uniformly labeled 13C-glucose entering neurons. We adapted the method by coupling offline patch-clamp setup with MS to confirm findings in vivo. RESULTS: According to non-parametric statistical analysis of metabolic dynamics, in cultured hippocampal neurons, the glycerol phosphate shuttle is active and correlates with the metabolic flux in the pentose phosphate pathway. In the hippocampus, glycerol-3-phosphate biosynthesis was activated in response to long-term potentiation together with the upregulation of glycolysis and the TCA cycle, but was inactive or silenced in basal conditions. CONCLUSIONS: We identified the biosynthesis of glycerol-3-phosphate as a key regulator in mechanisms implicated in learning and memory. Notably, defects in enzymes linked with the glycerol phosphate shuttle have been implicated in neurological disorders and intellectual disability. These results could improve our understanding of the general mechanisms of learning and memory and facilitate the development of novel therapies for metabolic disorders linked with intellectual disability.

Fast sampling method for mammalian cell metabolic analyses using liquid chromatography-mass spectrometry.

Metabolomics has emerged as a powerful tool for addressing biological questions. Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used for metabolic characterization, including targeted and untargeted approaches. Despite recent innovations, a crucial aspect of this technique is the sample preparation for accurate data analyses. In this protocol, we present a robust and adaptable workflow for metabolic analyses of mammalian cells from adherent cell cultures, which is particularly suited for qualitative and quantitative central metabolite characterization by LC-MS. Each sample consists of 600,000 mammalian cells grown on cover glasses, allowing for fast and complete transfer of the cells for metabolite extraction or medium exchange, e.g., for labeling experiments. The sampling procedure includes a fast and efficient washing step in liquid flow in water, which reduces cross-contamination and matrix effects while minimizing perturbation of the metabolic steady state of the cells; it is followed by quenching cell metabolism. The latter is achieved by using a -20 °C cold methanol acetonitrile mixture acidified with formic acid, followed by freeze drying, metabolite extraction and LC-MS. The protocol requires 2 s for cell sampling until quenching, and the entire protocol takes a total of 1.5 h per sample when the provided nanoscale LC-MS method is applied.

Analysis of 11 tetrameric STRs in wild boars for forensic purposes.

STR profiling of animal species has a wide range of applications, including forensic identification, wildlife preservation, veterinary public health protection and food safety. We tested the efficacy of a multiplex PCR-based assay including 11 porcine-specific tetrameric STRs in a population sample of wild boars (n=142) originating from Piedmont (North West Italy). Multiple deviations from Hardy-Weinberg expectations were observed, mostly due to a reduction in observed heterozygosity indicative of a high degree of inbreeding. A value of θ of 0.046 and an inbreeding coefficient of 0.089 were estimated. Combined power of discrimination and probability of exclusion values for the STR panel were 0.9999999999996 and 0.99989. In order to test the suitability of the method for meat traceability purposes, a domestic pig reference sample (n=412), consisting of commercial lines commonly used in the meat production process, was also typed. A Bayesian cluster analysis carried out using the observed genotypes, showed a percentage of correct subspecies assignment of individual samples of 0.974 for wild boars and 0.991 for pigs, thus demonstrating the usefulness of the multiplex STR-typing system for discrimination purposes.