Asparagine transport through SLC1A5/ASCT2 and SLC38A5/SNAT5 is essential for BCP-ALL cell survival and a potential therapeutic target.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) blasts strictly depend on the transport of extra-cellular asparagine (Asn), yielding a rationale for L-asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP-ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V-9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase-sensitive RS4;11 cells and the relatively ASNase-insensitive NALM-6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP-ALL.

Colon-available mango (poly)phenols exhibit mitigating effects on the intestinal barrier function in human intestinal cell monolayers under inflammatory conditions.

This study investigated the impact of in vivo available colon-mango (poly)phenols on stress-induced impairment of intestinal barrier function. Caco-2/HT29-MTX cells were incubated with six extracts of ileal fluid collected pre- and 4-8 h post-mango consumption before being subjected to inflammatory stress. (Poly)phenols in ileal fluids were analysed by UHPLC-HR-MS. Epithelial barrier function was monitored by measurement of trans-epithelial electrical resistance (TEER) and the production of selected inflammatory markers (interleukin-8 (IL-8) and nitric oxide (NO)) and the major mucin of the mucosal layer (MUC2). Post-mango intake ileal fluids contained principally benzoic acids, hydroxybenzenes and galloyl derivatives. There was a high interindividual variability in the levels of these compounds, which was reflected by the degree of variability in the protective effects of individual ileal extracts on inflammatory changes in the treated cell cultures. The 24 h treatment with non-cytotoxic doses of extracts of 4-8 h post-mango intake ileal fluid significantly reduced the TEER decrease in monolayers treated with the inflammatory cytomix. This effect was not associated with changes in IL-8 expression and secretion or claudine-7 expression. The mango derived-ileal fluid extract (IFE) also mitigated cytomix-dependent nitrite secretion, as a proxy of NO production, and the MUC2 reduction observed upon the inflammatory challenge. These insights shed light on the potential protective effect of mango (poly)phenols on the intestinal barrier exposed to inflammatory conditions.

Taxonomic and metabolic development of the human gut microbiome across life stages: a worldwide metagenomic investigation.

The human gut microbiota is a dynamic community of microorganisms that undergo variable changes over the entire life span. To thoroughly investigate the possible fluctuations of the microbiota throughout human life, we performed a pooled analysis of healthy fecal samples across different age groups covering the entire human life span. Our study integrated data from 79 publicly available studies and new stool samples from an Italian cohort, i.e., the Parma Microbiota project, resulting in 6,653 samples processed through the shotgun metagenomic approach. This approach has allowed species-level taxonomic reconstruction of the gut microbiota and investigation of its metabolic potential across the human life span. From a taxonomic point of view, our findings confirmed and detailed at species-level accuracy that the microbial richness of the gut microbiota gradually increases in the first stage of life, becoming relatively stable during adolescence. Moreover, the analysis identified the potential core microbiota representative of distinct age groups, revealing age-related bacterial patterns and the continuous rearrangement of the microbiota in terms of relative abundances across the life span rather than the acquisition and loss of taxa. Furthermore, the shotgun approach provided insights into the functional contribution of the human gut microbiome. The metagenomic analysis revealed functional age-related differences, particularly in carbohydrate and fiber metabolism, suggesting a co-evolution of the microbiome assembly with diet. Additionally, we identified correlations between vitamin synthesis, such as thiamine and niacin, and early life, suggesting a potential role of the microbiome in human physiology, in particular in the functions of the host's nervous and immune systems. IMPORTANCE: In this study, we provided comprehensive insights into the dynamic nature of the human gut microbiota across the human life span. In detail, we analyzed a large data set based on a shotgun metagenomic approach, combining public data sets and new samples from the Parma Microbiota project and obtaining a detailed overview of the possible relationship between gut microbiota development and aging. Our findings confirmed the main stages in microbial richness development and revealed specific core microbiota associated with different age stages. Moreover, the shotgun metagenomic approach allowed the disentangling of the functional changes in the microbiome across the human life span, particularly in diet-related metabolism, which is probably correlated to bacterial co-evolution with dietary habits. Notably, our study also uncovered positive correlations with vitamin synthesis in early life, suggesting a possible impact of the microbiota on human physiology.

Genomic and ecological approaches to identify the Bifidobacterium breve prototype of the healthy human gut microbiota.

Members of the genus Bifidobacterium are among the first microorganisms colonizing the human gut. Among these species, strains of Bifidobacterium breve are known to be commonly transmitted from mother to her newborn, while this species has also been linked with activities supporting human wellbeing. In the current study, an in silico approach, guided by ecology- and phylogenome-based analyses, was employed to identify a representative strain of B. breve to be exploited as a novel health-promoting candidate. The selected strain, i.e., B. breve PRL2012, was found to well represent the genetic content and functional genomic features of the B. breve taxon. We evaluated the ability of PRL2012 to survive in the gastrointestinal tract and to interact with other human gut commensal microbes. When co-cultivated with various human gut commensals, B. breve PRL2012 revealed an enhancement of its metabolic activity coupled with the activation of cellular defense mechanisms to apparently improve its survivability in a simulated ecosystem resembling the human microbiome.

GH136-encoding gene (perB) is involved in gut colonization and persistence by Bifidobacterium bifidum PRL2010.

Bifidobacteria are commensal microorganisms that typically inhabit the mammalian gut, including that of humans. As they may be vertically transmitted, they commonly colonize the human intestine from the very first day following birth and may persist until adulthood and old age, although generally at a reduced relative abundance and prevalence compared to infancy. The ability of bifidobacteria to persist in the human intestinal environment has been attributed to genes involved in adhesion to epithelial cells and the encoding of complex carbohydrate-degrading enzymes. Recently, a putative mucin-degrading glycosyl hydrolase belonging to the GH136 family and encoded by the perB gene has been implicated in gut persistence of certain bifidobacterial strains. In the current study, to better characterize the function of this gene, a comparative genomic analysis was performed, revealing the presence of perB homologues in just eight bifidobacterial species known to colonize the human gut, including Bifidobacterium bifidum and Bifidobacterium longum subsp. longum strains, or in non-human primates. Mucin-mediated growth and adhesion to human intestinal cells, in addition to a rodent model colonization assay, were performed using B. bifidum PRL2010 as a perB prototype and its isogenic perB-insertion mutant. These results demonstrate that perB inactivation reduces the ability of B. bifidum PRL2010 to grow on and adhere to mucin, as well as to persist in the rodent gut niche. These results corroborate the notion that the perB gene is one of the genetic determinants involved in the persistence of B. bifidum PRL2010 in the human gut.

Ecology- and genome-based identification of the Bifidobacterium adolescentis prototype of the healthy human gut microbiota.

Bifidobacteria are among the first microbial colonizers of the human gut, being frequently associated with human health-promoting activities. In the current study, an in silico methodology based on an ecological and phylogenomic-driven approach allowed the selection of a Bifidobacterium adolescentis prototype strain, i.e., B. adolescentis PRL2023, which best represents the overall genetic content and functional features of the B. adolescentis taxon. Such features were confirmed by in vitro experiments aimed at evaluating the ability of this strain to survive in the gastrointestinal tract of the host and its ability to interact with human intestinal cells and other microbial gut commensals. In this context, co-cultivation of B. adolescentis PRL2023 and several gut commensals revealed various microbe-microbe interactions and indicated co-metabolism of particular plant-derived glycans, such as xylan.IMPORTANCEThe use of appropriate bacterial strains in experimental research becomes imperative in order to investigate bacterial behavior while mimicking the natural environment. In the current study, through in silico and in vitro methodologies, we were able to identify the most representative strain of the Bifidobacterium adolescentis species. The ability of this strain, B. adolescentis PRL2023, to cope with the environmental challenges imposed by the gastrointestinal tract, together with its ability to switch its carbohydrate metabolism to compete with other gut microorganisms, makes it an ideal choice as a B. adolescentis prototype and a member of the healthy microbiota of adults. This strain possesses a genetic blueprint appropriate for its exploitation as a candidate for next-generation probiotics.

Amorphous silica nanoparticles and the human gut microbiota: a relationship with multiple implications.

Amorphous silica nanoparticles (ASNP) are among the nanomaterials that are produced in large quantities. ASNP have been present for a long time in several fast-moving consumer products, several of which imply exposure of the gastrointestinal tract, such as toothpastes, food additives, drug excipients, and carriers. Consolidated use and experimental evidence have consistently pointed to the very low acute toxicity and limited absorption of ASNP. However, slow absorption implies prolonged exposure of the intestinal epithelium to ASNP, with documented effects on intestinal permeability and immune gut homeostasis. These effects could explain the hepatic toxicity observed after oral administration of ASNP in animals. More recently, the role of microbiota in these and other ASNP effects has attracted increasing interest in parallel with the recognition of the role of microbiota in a variety of conditions. Although evidence for nanomaterial effects on microbiota is particularly abundant for materials endowed with bactericidal activities, a growing body of recent experimental data indicates that ASNPs also modify microbiota. The implications of these effects are recounted in this contribution, along with a discussion of the more important open issues and recommendations for future research.

Preparation of human primary macrophages to study the polarization from monocyte-derived macrophages to pro- or anti-inflammatory macrophages at biomaterial interface in vitro.

Background/purpose: Testing of dental materials when in contact with innate immune cells has been so far hindered by the lack of proper in vitro models. Human primary monocyte-derived macrophages (MDMs) would be an excellent option to this aim. However, the inability to detach them from the tissue culture plates contrast the possibility to culture them on biomaterials. The goal of the present work is to present and validate an innovative protocol to obtain MDMs from peripheral blood monocytes, and to reseed them in contact with biomaterials without altering their viability and phenotype. Materials and methods: We differentiated MDMs on ultra-low attachment tissue culture plastics and recovered them with specific detachment solution in order to be reseeded on a secondary substrate. Therefore, using biological assays (RT-PCR, Western blot, and immunofluorescence) we compared their phenotype to MDMs differentiated on standard culture plates. Results: Transferred MDMs keep their differentiated M0 resting state, as well as the ability to be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages. Conclusion: These data provide the dental material research community the unprecedented possibility to investigate the immunomodulatory properties of biomaterials for dental application.

Disentangling the interactions between nasopharyngeal and gut microbiome and their involvement in the modulation of COVID-19 infection.

The human organism is inhabited by trillions of microorganisms, known as microbiota, which are considered to exploit a pivotal role in the regulation of host health and immunity. Recent investigations have suggested a relationship between the composition of the human microbiota and COVID-19 infection, highlighting a possible role of bacterial communities in the modulation of the disease severity. In this study, we performed a shotgun metagenomics analysis to explore and compare the nasopharyngeal microbiota of 38 hospitalized Italian patients with and without COVID-19 infection during the third and fourth pandemic waves. In detail, the metagenomic analysis combined with specific correlation analyses suggested a positive association of several microbial species, such as S. parasanguinis and P. melaninogenica, with the severity of COVID-19 infection. Furthermore, the comparison of the microbiota composition between the nasopharyngeal and their respective fecal samples highlighted an association between these different compartments represented by a sharing of several bacterial species. Additionally, lipidomic and deep-shotgun functional analyses of the fecal samples suggested a metabolic impact of the microbiome on the host's immune response, indicating the presence of key metabolic compounds in COVID-19 patients, such as lipid oxidation end products, potentially related to the inflammatory state. Conversely, the patients without COVID-19 displayed enzymatic patterns associated with the biosynthesis and degradation of specific compounds like lysine (synthesis) and phenylalanine (degradation) that could positively impact disease severity and contribute to modulating COVID-19 infection. IMPORTANCE The human microbiota is reported to play a major role in the regulation of host health and immunity, suggesting a possible impact on the severity of COVID-19 disease. This preliminary study investigated the possible correlation between nasopharyngeal microbiota and COVID-19 infection. In detail, the analysis of the nasopharyngeal microbiota of hospitalized Italian patients with and without COVID-19 infection suggested a positive association of several microbial species with the severity of the disease and highlighted a sharing of several bacteria species with the respective fecal samples. Moreover, the metabolic analyses suggested a possible impact of the microbiome on the host's immune response and the disease severity.

Genetic strategies for sex-biased persistence of gut microbes across human life.

Although compositional variation in the gut microbiome during human development has been extensively investigated, strain-resolved dynamic changes remain to be fully uncovered. In the current study, shotgun metagenomic sequencing data of 12,415 fecal microbiomes from healthy individuals are employed for strain-level tracking of gut microbiota members to elucidate its evolving biodiversity across the human life span. This detailed longitudinal meta-analysis reveals host sex-related persistence of strains belonging to common, maternally-inherited species, such as Bifidobacterium bifidum and Bifidobacterium longum subsp. longum. Comparative genome analyses, coupled with experiments including intimate interaction between microbes and human intestinal cells, show that specific bacterial glycosyl hydrolases related to host-glycan metabolism may contribute to more efficient colonization in females compared to males. These findings point to an intriguing ancient sex-specific host-microbe coevolution driving the selective persistence in women of key microbial taxa that may be vertically passed on to the next generation.

The SLC38A5/SNAT5 amino acid transporter: from pathophysiology to pro-cancer roles in the tumor microenvironment.

SLC38A5/SNAT5 is a system N transporter that can mediate net inward or outward transmembrane fluxes of neutral amino acids coupled with Na+ (symport) and H+ (antiport). Its preferential substrates are not only amino acids with side chains containing amide (glutamine and asparagine) or imidazole (histidine) groups, but also serine, glycine, and alanine are transported by the carrier. Expressed in the pancreas, intestinal tract, brain, liver, bone marrow, and placenta, it is regulated at mRNA and protein levels by mTORC1 and WNT/ß-catenin pathways, and it is sensitive to pH, nutritional stress, inflammation, and hypoxia. SNAT5 expression has been found to be altered in pathological conditions such as chronic inflammatory diseases, gestational complications, chronic metabolic acidosis, and malnutrition. Growing experimental evidence shows that SNAT5 is overexpressed in several types of cancer cells. Moreover, recently published results indicate that SNAT5 expression in stromal cells can support the metabolic exchanges occurring in the tumor microenvironment of asparagine-auxotroph tumors. We review the functional role of the SNAT5 transporter in pathophysiology and propose that, due to its peculiar operational and regulatory features, SNAT5 may play important pro-cancer roles when expressed either in neoplastic or in stromal cells of glutamine-auxotroph tumors.NEW & NOTEWORTHY The transporter SLC38A5/SNAT5 provides net influx or efflux of glutamine, asparagine, and serine. These amino acids are of particular metabolic relevance in several conditions. Changes in transporter expression or activity have been described in selected types of human cancers, where SNAT5 can mediate amino acid exchanges between tumor and stromal cells, thus providing a potential therapeutic target. This is the first review that recapitulates the characteristics and roles of the transporter in physiology and pathology.

Exploring Molecular Interactions between Human Milk Hormone Insulin and Bifidobacteria.

Multiple millennia of human evolution have shaped the chemical composition of breast milk toward an optimal human body fluid for nutrition and protection and for shaping the early gut microbiota of newborns. This biological fluid is composed of water, lipids, simple and complex carbohydrates, proteins, immunoglobulins, and hormones. Potential interactions between hormones present in mother's milk and the microbial community of the newborn are a very fascinating yet unexplored topic. In this context, insulin, in addition to being one of the most prevalent hormones in breast milk, is also involved in a metabolic disease that affects many pregnant women, i.e., gestational diabetes mellitus (GDM). Analysis of 3,620 publicly available metagenomic data sets revealed that the bifidobacterial community varies in relation to the different concentrations of this hormone in breast milk of healthy and diabetic mothers. Starting from this assumption, in this study, we explored possible molecular interactions between this hormone and bifidobacterial strains that represent bifidobacterial species commonly occurring in the infant gut using 'omics' approaches. Our findings revealed that insulin modulates the bifidobacterial community by apparently improving the persistence of the Bifidobacterium bifidum taxon in the infant gut environment compared to other typical infant-associated bifidobacterial species. IMPORTANCE Breast milk is a key factor in modulating the infant's intestinal microbiota composition. Even though the interaction between human milk sugars and bifidobacteria has been extensively studied, there are other bioactive compounds in human milk that may influence the gut microbiota, such as hormones. In this article, the molecular interaction of the human milk hormone insulin and the bifidobacterial communities colonizing the human gut in the early stages of life has been explored. This molecular cross talk was assessed using an in vitro gut microbiota model and then analyzed by various omics approaches, allowing the identification of genes associated with bacterial cell adaptation/colonization in the human intestine. Our findings provide insights into the manner by which assembly of the early gut microbiota may be regulated by host factors such as hormones carried by human milk.

Nanoemulsions Embedded in Alginate Beads as Bioadhesive Nanocomposites for Intestinal Delivery of the Anti-Inflammatory Drug Tofacitinib.

Oral administration of nanoparticles (NPs) is a promising strategy to overcome solubility and stability issues of many active compounds. However, this route faces major obstacles related to the hostile gastrointestinal (GI) environment, which impairs the efficacy of orally administered nanomedicines. Here, we propose nanocomposites as a promising approach to increase the retention time of NPs in the intestinal tract by using bio- and mucoadhesive matrixes able to protect the cargo until it reaches the targeted area. A microfluidic-based approach has been applied for the production of tailored nanoemulsions (NEs) of about 110 nm, used for the encapsulation of small hydrophobic drugs such as the anti-inflammatory JAK-inhibitor tofacitinib. These NEs proved to be efficiently internalized into a mucus-secreting human intestinal monolayer of Caco-2/HT29-MTX cells and to deliver tofacitinib to subepithelial human THP-1 macrophage-like cells, reducing their inflammatory response. NEs were then successfully encapsulated into alginate hydrogel microbeads of around 300 µm, which were characterized by rheological experiments and dried to create a long-term stable system for pharmaceutical applications. Finally, ex vivo experiments on excised segments of rats' intestine proved the bioadhesive ability of NEs embedded in alginate hydrogels compared to free NEs, showing the advantage that this hybrid system can offer for the treatment of intestinal pathologies.

Identification of a prototype human gut Bifidobacterium longum subsp. longum strain based on comparative and functional genomic approaches.

Bifidobacteria are extensively exploited for the formulation of probiotic food supplements due to their claimed ability to exert health-beneficial effects upon their host. However, most commercialized probiotics are tested and selected for their safety features rather than for their effective abilities to interact with the host and/or other intestinal microbial players. In this study, we applied an ecological and phylogenomic-driven selection to identify novel B. longum subsp. longum strains with a presumed high fitness in the human gut. Such analyses allowed the identification of a prototype microorganism to investigate the genetic traits encompassed by the autochthonous bifidobacterial human gut communities. B. longum subsp. longum PRL2022 was selected due to its close genomic relationship with the calculated model representative of the adult human-gut associated B. longum subsp. longum taxon. The interactomic features of PRL2022 with the human host as well as with key representative intestinal microbial members were assayed using in vitro models, revealing how this bifidobacterial gut strain is able to establish extensive cross-talk with both the host and other microbial residents of the human intestine.

The last battle of Alessandro Farnese (1545-1592).

BACKGROUND AND AIM: Alessandro Farnese (1545-1592), 3rd Duke of Parma and Piacenza, one of the most important generals and politicians of his age. He died after a rapid deterioration of his health. The available documents testify that the Duke suffered for a long time from various health problems, such as jaundice, intestinal disorders, gout, dropsy but very little is known about the cause(s) of his death. The aim of this article is to offer for the first time a complete clinical interpretation of Alessandro Farnese's last months of life Methods: A collection of descriptions of symptoms and signs described by his court physician and by the leading biographers of Farnese has been compiled. This collected medical evidence has been interpreted in the light of current medical knowledge, to obtain a final interpretation. RESULTS: The results led us to consider liver diseases, neoplastic diseases (especially pancres) and infectious diseases (including typhus and malaria) as causes or contributing causes of death. CONCLUSIONS: The accurate autopsy description, in association with the anamnestic information provided by the historical documents studied, suggests that Alessandro Farnese was a hepatopathic patient suffering from spontaneous bacterial peritonitis. In the pre-antibiotic era, the pathological organ alterations described certainly have at least contributed to making the infectious episode (that the autopsy describes of pulmonary origin) fatal.

Designation of optimal reference strains representing the infant gut bifidobacterial species through a comprehensive multi-omics approach.

The genomic era has resulted in the generation of a massive amount of genetic data concerning the genomic diversity of bacterial taxa. As a result, the microbiological community is increasingly looking for ways to define reference bacterial strains to perform experiments that are representative of the entire bacterial species. Despite this, there is currently no established approach allowing a reliable identification of reference strains based on a comprehensive genomic, ecological, and functional context. In the current study, we developed a comprehensive multi-omics approach that will allow the identification of the optimal reference strains using the Bifidobacterium genus as test case. Strain tracking analysis based on 1664 shotgun metagenomics datasets of healthy infant faecal samples were employed to identify bifidobacterial strains suitable for in silico and in vitro analyses. Subsequently, an ad hoc bioinformatic tool was developed to screen local strain collections for the most suitable species-representative strain alternative. The here presented approach was validated using in vitro trials followed by metagenomics and metatranscriptomics analyses. Altogether, these results demonstrated the validity of the proposed model for reference strain selection, thus allowing improved in silico and in vitro investigations both in terms of cross-laboratory reproducibility and relevance of research findings.

The TLR4/NFκB-Dependent Inflammatory Response Activated by LPS Is Inhibited in Human Macrophages Pre-Exposed to Amorphous Silica Nanoparticles.

Amorphous silica nanoparticles (ASNP) are present in a variety of products and their biological effects are actively investigated. Although several studies have documented pro-inflammatory effects of ASNP, the possibility that they also modify the response of innate immunity cells to natural activators has not been thoroughly investigated. Here, we study the effects of pyrogenic ASNP on the LPS-dependent activation of human macrophages differentiated from peripheral blood monocytes. In macrophages, 24 h of pre-exposure to non-cytotoxic doses of ASNP markedly inhibited the LPS-dependent induction of pro-inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10). The inhibitory effect was associated with the suppression of NFκB activation and the increased intracellular sequestration of the TLR4 receptor. The late induction of glutamine synthetase (GS) by LPS was also prevented by pre-exposure to ASNP, while GS silencing did not interfere with cytokine secretion. It is concluded that (i) macrophages exposed to ASNP are less sensitive to LPS-dependent activation and (ii) GS induction by LPS is likely secondary to the stimulation of cytokine secretion. The observed interference with LPS effects may point to a dampening of the acute inflammatory response after exposure to ASNP in humans.

Mesenchymal stromal cells cultured in physiological conditions sustain citrate secretion with glutamate anaplerosis.

Bone marrow mesenchymal stromal cells (MSCs) have immunomodulatory and regenerative potential. However, culture conditions govern their metabolic processes and therapeutic efficacy. Here we show that culturing donor-derived MSCs in Plasmax™, a physiological medium with the concentrations of nutrients found in human plasma, supports their proliferation and stemness, and prevents the nutritional stress induced by the conventional medium DMEM. The quantification of the exchange rates of metabolites between cells and medium, untargeted metabolomics, stable isotope tracing and transcriptomic analysis, performed at physiologically relevant oxygen concentrations (1%O2), reveal that MSCs rely on a high rate of glucose to lactate conversion, coupled with parallel anaplerotic fluxes from glutamine and glutamate to support citrate synthesis and secretion. These distinctive traits of MSCs shape the metabolic microenvironment of the bone marrow niche and can influence nutrient cross-talks under physiological and pathological conditions.

Oral status of a noble European couple from the 16th century: A morphologic analysis of the teeth of Alessandro Farnese and his wife Maria D'Aviz.

Objective. To report the results of dental morphological analysis performed on the remains of two European nobles (Duke Alessandro Farnese and his wife Maria D'Aviz) who lived in the 16th century, together with hypotheses on their nutrition and oral hygiene habits. Design and results. The remains of Alessandro Farnese (1545-1592) and Princess Maria D'Aviz (1538-1577) were exhumed in Parma, Italy, in the context of an historical investigation into their possible causes of death. The skulls were examined and analysed through direct inspection, high-detailed photographs and radiographs. Ante mortem tooth loss (AMTL), postmortem tooth loss (PMTL), Scott and Smith dental wear indexes and the Kerr periodontal index were used to assess the dental and periodontal status of the couple. Alessandro Farnese suffered from severe dental wear while it was presumed that Maria D'Aviz was affected by periodontal disease and tooth decay. Conclusions. Based on the findings of the present analysis, we hypothesise that Alessandro Farnese's diet was mainly based on hard and unrefined foods, also suggested by limited historical reports. It is likely that Maria D'Aviz's nutrition was based on sugar-enriched foods.

ALL blasts drive primary mesenchymal stromal cells to increase asparagine availability during asparaginase treatment.

Mechanisms underlying the resistance of acute lymphoblastic leukemia (ALL) blasts to l-asparaginase are still incompletely known. Here we demonstrate that human primary bone marrow mesenchymal stromal cells (MSCs) successfully adapt to l-asparaginase and markedly protect leukemic blasts from the enzyme-dependent cytotoxicity through an amino acid trade-off. ALL blasts synthesize and secrete glutamine, thus increasing extracellular glutamine availability for stromal cells. In turn, MSCs use glutamine, either synthesized through glutamine synthetase (GS) or imported, to produce asparagine, which is then extruded to sustain asparagine-auxotroph leukemic cells. GS inhibition prevents mesenchymal cells adaptation to l-asparaginase, lowers glutamine secretion by ALL blasts, and markedly hinders the protection exerted by MSCs on leukemic cells. The pro-survival amino acid exchange is hindered by the inhibition or silencing of the asparagine efflux transporter SNAT5, which is induced in mesenchymal cells by ALL blasts. Consistently, primary MSCs from ALL patients express higher levels of SNAT5 (P < .05), secrete more asparagine (P < .05), and protect leukemic blasts (P < .05) better than MSCs isolated from healthy donors. In conclusion, ALL blasts arrange a pro-leukemic amino acid trade-off with bone marrow mesenchymal cells, which depends on GS and SNAT5 and promotes leukemic cell survival during l-asparaginase treatment.