Rare Earth Element Variability in Italian Extra Virgin Olive Oils from Abruzzo Region.

Extra virgin olive oil is a food product from the Mediterranean area that is particularly and continuously experiencing to increasing instances of fraudulent geographical labeling. Therefore, origin protection must be improved, mainly based on its intrinsic chemical composition. This study aimed to perform a preliminary chemical characterization of Abruzzo extra virgin olive oils (EVOOs) using rare earth elements (REEs). REEs were evaluated in EVOO samples of different varieties produced in different geographical origins within the Abruzzo region (Italy) in three harvest years using ICP-MS chemometric techniques. Principal component, discriminant, and hierarchical cluster analyses were conducted to verify the influence of the variety, origin, and vintage of the REE composition. The results of a three-year study showed a uniform REE pattern and a strong correlation in most EVOOs, in particular for Y, La, Ce, and Nd. However, europium and erbium were also found in some oil samples. Compared with cultivar and origin, only the harvest year slightly influenced the REE composition, highlighting the interactions of the olive system with the climate and soil chemistry that could affect the multielement composition of EVOOs.

Breast cancer in the era of integrating "Omics" approaches.

Worldwide, breast cancer is the leading cause of cancer-related deaths in women. Breast cancer is a heterogeneous disease characterized by different clinical outcomes in terms of pathological features, response to therapies, and long-term patient survival. Thus, the heterogeneity found in this cancer led to the concept that breast cancer is not a single disease, being very heterogeneous both at the molecular and clinical level, and rather represents a group of distinct neoplastic diseases of the breast and its cells. Indubitably, in the past decades we witnessed a significant development of innovative therapeutic approaches, including targeted and immunotherapies, leading to impressive results in terms of increased survival for breast cancer patients. However, these multimodal treatments fail to prevent recurrence and metastasis. Therefore, it is urgent to improve our understanding of breast tumor and metastasis biology. Over the past few years, high-throughput "omics" technologies through the identification of novel biomarkers and molecular profiling have shown their great potential in generating new insights in the study of breast cancer, also improving diagnosis, prognosis and prediction of response to treatment. In this review, we discuss how the implementation of "omics" strategies and their integration may lead to a better comprehension of the mechanisms underlying breast cancer. In particular, with the aim to investigate the correlation between different "omics" datasets and to define the new important key pathway and upstream regulators in breast cancer, we applied a new integrative meta-analysis method to combine the results obtained from genomics, proteomics and metabolomics approaches in different revised studies.

Ceramic-on-metal coupling in THA: long term clinical and radiographic outcomes using two different short stems.

BACKGROUND: Hip prosthetic replacement surgery is the gold standard for patients affected by symptomatic osteoarthritis. The ceramic-on-metal hybrid hard-on-hard bearing was initially launched on the market with the purpose of reducing adhesive and corrosion wear, loss of metal debris and ions and risk of fracture and squeaking. However, this bearing was withdrawn from the market, in the apprehension of local and systemic toxicity. The aim of this study is to evaluate the reliability and safety of ceramic-on-metal bearing at long term follow-up. METHODS: From 2 cohorts of patients suffering of hip osteoarthritis who underwent total hip arthroplasty using ceramic-on-metal bearing with two different short stems, 19 of the GROUP A and 25 of the GROUP B were suitable for this study. All patients were compared clinically using the Harris Hip Score (HHS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), visual analogue scale (VAS), 12-item Short Form Health Survey (SF12P/M), and radiographically. Blood samples were collected in order to evaluate chromium and cobalt ions level. The two groups were compared in terms of metal ions blood levels, and finally all the implanted prostheses were compared with a healthy control group. RESULTS: All the implanted stems were well-positioned and osseointegrated at a mean follow-up of 114 months. Improvements were observed for all clinical scores comparing preoperative and postoperative values in both groups. Radiographic evaluation showed a good ability to restore proper articular geometry. Chromium and cobalt ion analysis revealed values below the safety threshold except for 1 case in GROUP A (cup malposition) and 2 cases in GROUP B (6.1%). No revision occurred. CONCLUSIONS: Ceramic-on-metal bearing is safe and reliable at long term follow-up in association to short stems arthroplasty, if the implant is correctly positioned. Chromium and cobalt metal ions blood levels evaluation should be performed annually.

Analytical Evaluation of the Ideal Strategy for High-Throughput Flow Injection Analysis by Tandem Mass Spectrometry in Routine Newborn Screening.

The introduction of tandem mass spectrometry (MS/MS) to clinical laboratories and the advent of expanded newborn screening (NBS) were crucial changes to public health programs worldwide. Speed, robustness, accuracy, selectivity, and specificity of analysis are all requirements of expanded NBS and are needed to minimize false positive results risks, to possibly eliminate false negatives, and to improve the positive predictive value of NBS. In this study, we firstly evaluated the analytical performances of the RenataDX Screening System, a fully integrated flow-injection MS/MS (FIA-MS/MS) IVD system for high-throughput dried blood spot (DBS) analysis in a routine NBS laboratory. Since a choice of several commercial NBS kits is available, we sought to compare NeoBaseTM 2 (PerkinElmer®) and MassChrom® (Chromsystems) non-derivatized kits on the RenataDX platform by evaluating their analytical performances. Moreover, we verified the degree of correlation between data obtained by the two different NBS MS/MS kits by FIA-MS/MS of over 500 samples. Our data suggest that both methods correlate well with clinically insignificant differences that do not impact the NBS result. Finally, while NeoBase™ 2 offers an easier and faster sample preparation, MassChrom® provides a cleaner sample extract which empirically should improve instrument reliability.

Effects of low-dose methylcyclopentadienyl manganese tricarbonyl-derived manganese on the development of diencephalic dopaminergic neurons in zebrafish.

Fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT) is counted as an organic manganese (Mn)-derived compound. The toxic effects of Mn (alone and complexed) on dopaminergic (DA) neurotransmission have been investigated in both cellular and animal models. However, the impact of environmentally relevant Mn exposure on DA neurodevelopment is rather poorly understood. In the present study, the MMT dose of 100 µM (about 5 mg Mn/L) caused up-regulation of DA-related genes in association with cell body swelling and increase in the number of DA neurons of the ventral diencephalon subpopulation DC2. Furthermore, our analysis identified significant brain Mn bioaccumulation and enhancement of total dopamine levels in association with locomotor hyperactivity. Although DA levels were restored at adulthood, we observed a deficit in the acquisition and consolidation of memory. Collectively, these findings suggest that developmental exposure to low-level MMT-derived Mn is responsible for the selective alteration of diencephalic DA neurons and with long-lasting effects on fish explorative behaviour in adulthood.

Cobalt can fully recover the phenotypes related to zinc deficiency in Salmonella Typhimurium.

Cobalt is an essential element for living systems, which, however, make very limited use of this metal, using it mainly in cobalamin-containing enzymes. The reduced use of cobalt compared to other transition metals is generally attributed to the potential toxicity of this element. In this work, we demonstrate that cobalt not only does not have an obvious toxic effect on Salmonella Typhimurium, but that it can efficiently compensate for zinc deficiency in a znuABC deleted strain. In fact, cobalt, but not cobalamin supplementation, rescued all major phenotypic defects of the znuABC strain, including the reduced ability to grow and swim in zinc-deficient media and the high susceptibility to hydrogen peroxide stress. Growth in a cobalt-supplemented defined medium led to the accumulation of large amounts of cobalt both in the wild type and in the znuABC strain. These data suggest that atoms of cobalt may be incorporated in bacterial proteins in place of zinc, ensuring their functionality. In support of this hypothesis we have shown that, in vivo, cobalt can accumulate in ribosomes and replace zinc in a periplasmic Cu,Zn superoxide dismutase (SodCII). Finally, we provide evidence of the ability of cobalt to modulate the intracellular concentration of zinc-regulated proteins (ZnuA, ZinT, and SodCII). Although some observations suggest that in some proteins the replacement of zinc with cobalt can lead to subtle structural changes, the data reported in this study indicate that Salmonella has the ability to use cobalt instead of zinc, without evident harmful effects for cell physiology.

A False-Positive Case of Methylmalonic Aciduria by Tandem Mass Spectrometry Newborn Screening Dependent on Maternal Malnutrition in Pregnancy.

Methylmalonic Acidurias (MMAs) are a group of inborn errors of metabolism (IEMs), specifically of propionate catabolism characterized by gastrointestinal and neurometabolic manifestations resulting from a deficiency in the function of methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, and cobalamin metabolism. In Expanded Newborn Screening (NBS), increased levels of propionylcarnitine (C3) and/or of its ratios by MS/MS analysis of dried blood spots (DBS) samples are suggestive for either Propionic Acidemia or MMAs. C3 elevation is not considered a specific marker for these disorders, resulting in high false-positive rates. The use of analyte ratios improves specificity, but it still cannot resolve the diagnostic issue. Second-tier testing are strongly recommended as confirmation of primary NBS results and for a differential diagnosis. LC-MS/MS analysis allows the quantification of more specific markers of the disorder. Here, we report the case of a newborn with a suspected MMA at Expanded NBS and at second-tier test. Given the urgent situation, in-depth diagnostic investigations were performed. Further investigations surprisingly revealed a Vitamin B12 deficiency due to a maternal malnutrition during pregnancy. This case emphasized that metabolic alterations at NBS may not only be influenced by genome and related to IEMs, but also to external factors and to maternal conditions.

Proteomic and metabolomic characterization of streptozotocin-induced diabetic nephropathy in TIMP3-deficient mice.

AIMS: The tissue inhibitor of metalloproteinase TIMP3 is a stromal protein that restrains the activity of both protease and receptor in the extracellular matrix and has been found to be down-regulated in diabetic nephropathy (DN), the leading cause of end-stage renal disease in developed countries. METHODS: In order to gain deeper insights on the association of loss of TIMP3 and DN, we performed differential proteomic analysis of kidney and blood metabolic profiling of wild-type and Timp3-knockout mice before and after streptozotocin (STZ) treatment, widely used to induce insulin deficiency and hyperglycemia. RESULTS: Kidney proteomic data and blood metabolic profiles suggest significant alterations of peroxisomal and mitochondrial fatty acids ß-oxidation in Timp3-knockout mice compared to wild-type mice under basal condition. These alterations were exacerbated in response to STZ treatment. CONCLUSIONS: Proteomic and metabolomic approaches showed that loss of TIMP3 alone or in combination with STZ treatment results in significant alterations of kidney lipid metabolism and peripheral acylcarnitine levels, supporting the idea that loss of TIMP3 may generate a phenotype more prone to DN.

Metabolic Alterations of Thyroid Cancer as Potential Therapeutic Targets.

Thyroid cancer (TC) is the most frequent endocrine tumor with a growing incidence worldwide. Besides the improvement of diagnosis, TC increasing incidence is probably due to environmental factors and lifestyle modifications. The actual diagnostic criteria for TC classification are based on fine needle biopsy (FNAB) and histological examination following thyroidectomy. Since in some cases it is not possible to make a proper diagnosis, classical approach needs to be supported by additional biomarkers. Recently, new emphasis has been given to the altered cellular metabolism of proliferating cancer cells which require high amount of glucose for energy production and macromolecules biosynthesis. Also TC displays alteration of energy metabolism orchestrated by oncogenes activation and tumor suppressors inactivation leading to abnormal proliferation. Furthermore, TC shows significant metabolic heterogeneity within the tumor microenvironment and metabolic coupling between cancer and stromal cells. In this review we focus on the current knowledge of metabolic alterations of TC and speculate that targeting TC metabolism may improve current therapeutic protocols for poorly differentiated TC. Future studies will further deepen the actual understandings of the metabolic phenotype of TC cells and will give the chance to provide novel prognostic biomarkers and therapeutic targets in tumors with a more aggressive behavior.

Growth of Pseudomonas aeruginosa in zinc poor environments is promoted by a nicotianamine-related metallophore.

Previous studies have suggested that P. aeruginosa possesses redundant zinc uptake systems. To identify uncharacterized zinc transporters, we analyzed the genome-wide transcriptional responses of P. aeruginosa PA14 to zinc restriction. This approach led to the identification of an operon (zrmABCD) regulated by the zinc uptake regulator Zur, that encodes for a metallophore-mediated zinc import system. This operon includes the genes for an uncharacterized TonB-dependent Outer Membrane Protein (ZrmA) and for a putative nicotianamine synthase (ZrmB). The simultaneous inactivation of the ZnuABC transporter and of one of these two genes markedly decreases the ability of P. aeruginosa to grow in zinc-poor media and compromises intracellular zinc accumulation. Our data demonstrate that ZrmB is involved in the synthesis of a metallophore which is released outside the cell and mediates zinc uptake through the ZrmA receptor. We also show that alterations in zinc homeostasis severely affect the ability of P. aeruginosa to cause acute lung and systemic infections in C57BL/6 mice, likely due to the involvement of zinc in the expression of several virulence traits. These findings disclose a hitherto unappreciated role of zinc in P. aeruginosa pathogenicity and reveal that this microorganism can obtain zinc through a strategy resembling siderophore-mediated iron uptake.

Medium-chain plasma acylcarnitines, ketone levels, cognition, and gray matter volumes in healthy elderly, mildly cognitively impaired, or Alzheimer's disease subjects.

Aging, amyloid deposition, and tau-related pathology are key contributors to the onset and progression of Alzheimer's disease (AD). However, AD is also associated with brain hypometabolism and deficits of mitochondrial bioenergetics. Plasma acylcarnitines (ACCs) are indirect indices of altered fatty acid beta-oxidation, and ketogenesis has been found to be decreased on aging. Furthermore, in elderly subjects, alterations in plasma levels of specific ACCs have been suggested to predict conversion to mild cognitive impairment (MCI) or AD. In this study, we assayed plasma profiles of ACCs in a cohort of healthy elderly control, MCI subjects, and AD patients. Compared with healthy controls or MCI subjects, AD patients showed significant lower plasma levels of several medium-chain ACCs. Furthermore, in AD patients, these lower concentrations were associated with lower prefrontal gray matter volumes and the presence of cognitive impairment. Interestingly, lower levels of medium-chain ACCs were also found to be associated with lower plasma levels of 2-hydroxybutyric acid. Overall, these findings suggest that altered metabolism of medium-chain ACCs and impaired ketogenesis can be metabolic features of AD.

The capability of Pseudomonas aeruginosa to recruit zinc under conditions of limited metal availability is affected by inactivation of the ZnuABC transporter.

The ability of a large number of bacterial pathogens to multiply in the infected host and cause disease is dependent on their ability to express high affinity zinc importers. In many bacteria, ZnuABC, a transporter of the ABC family, plays a central role in the process of zinc uptake in zinc poor environments, including the tissues of the infected host. To initiate an investigation into the relevance of the zinc uptake apparatus for Pseudomonas aeruginosa pathogenicity, we have generated a znuA mutant in the PA14 strain. We have found that this mutant strain displays a limited growth defect in zinc depleted media. The znuA mutant strain is more sensitive than the wild type strain to calprotectin-mediated growth inhibition, but both the strains are highly resistant to this zinc sequestering antimicrobial protein. Moreover, intracellular zinc content is not evidently affected by inactivation of the ZnuABC transporter. These findings suggest that P. aeruginosa is equipped with redundant mechanisms for the acquisition of zinc that might favor P. aeruginosa colonization of environments containing low levels of this metal. Nonetheless, deletion of znuA affects alginate production, reduces the activity of extracellular zinc-containing proteases, including LasA, LasB and protease IV, and decreases the ability of P. aeruginosa to disseminate during systemic infections. These results indicate that efficient zinc acquisition is critical for the expression of various virulence features typical of P. aeruginosa and that ZnuABC also plays an important role in zinc homeostasis in this microorganism.

Stenotrophomonas maltophilia virulence and specific variations in trace elements during acute lung infection: implications in cystic fibrosis.

Metal ions are necessary for the proper functioning of the immune system, and, therefore, they might have a significant influence on the interaction between bacteria and host. Ionic dyshomeostasis has been recently observed also in cystic fibrosis (CF) patients, whose respiratory tract is frequently colonized by Stenotrophomonas maltophilia. For the first time, here we used an inductively mass spectrometry method to perform a spatial and temporal analysis of the pattern of changes in a broad range of major trace elements in response to pulmonary infection by S. maltophilia. To this, DBA/2 mouse lungs were comparatively infected by a CF strain and by an environmental one. Our results showed that pulmonary ionomic profile was significantly affected during infection. Infected mice showed increased lung levels of Mg, P, S, K, Zn, Se, and Rb. To the contrary, Mn, Fe, Co, and Cu levels resulted significantly decreased. Changes of element concentrations were correlated with pulmonary bacterial load and markers of inflammation, and occurred mostly on day 3 post-exposure, when severity of infection culminated. Interestingly, CF strain - significantly more virulent than the environmental one in our murine model - provoked a more significant impact in perturbing pulmonary metal homeostasis. Particularly, exposure to CF strain exclusively increased P and K levels, while decreased Fe and Mn ones. Overall, our data clearly indicate that S. maltophilia modulates pulmonary metal balance in a concerted and virulence-dependent manner highlighting the potential role of the element dyshomeostasis during the progression of S. maltophilia infection, probably exacerbating the harmful effects of the loss of CF transmembrane conductance regulator function. Further investigations are required to understand the biological significance of these alterations and to confirm they are specifically caused by S. maltophilia.

Proteomic and ionomic profiling reveals significant alterations of protein expression and calcium homeostasis in cystic fibrosis cells.

Cystic fibrosis (CF) is an autosomal recessive disorder associated with mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and defective chloride transport across the epithelial cell membranes. Abnormal epithelial ion transport is the primary cause of persistent airway infections and chronic inflammation in CF patients. In order to gain further insight into the mechanisms of epithelial dysfunctions linked to CFTR mutations, we performed and integrated proteomic and ionomic analysis of human bronchial epithelial IB3-1 cells and compared them with a CFTR-complemented isogenic cell line (C38). Aside from changes that were consistent with known effects related to CFTR mutations, such as differences in glycolytic and gluconeogenic pathways and unfolded protein responses, differential proteomics highlighted significant alteration of protein expression and, in particular, of the 14-3-3 signalling pathway that is known to be involved in cellular calcium (Ca) homeostasis. Of note, restoring chloride efflux by acting on Ca cellular homeostasis has been shown to be a promising therapeutic intervention for CF. Ionomic analysis showed significant changes in the IB3-1 element profile compared with C38 cells and in particular we observed an increase of intracellular Ca that significantly correlates with intracellular zinc (Zn) levels, suggesting a synergistic role of Ca and Zn influx. This finding is particularly intriguing because Zn has been reported to be effective in CF treatment increasing Ca influx. Taken together, our proteomic and ionomic data reveal that CFTR mutation sets in motion endogenous mechanisms counteracting impaired chloride transport mainly acting on epithelial ion transport and increasing intracellular Ca, suggesting potential links between protein expression and this response.

Characterisation of element profile changes induced by long-term dietary supplementation of zinc in the brain and cerebellum of 3xTg-AD mice by alternated cool and normal plasma ICP-MS.

Metal dyshomeostasis plays a crucial role in promoting several neurodegenerative diseases including Alzheimer's disease (AD), a condition that has been linked to deregulation of brain levels of Al, Fe, Mn, Cu, and Zn. Thus, quantitative multi-element profiling of brain tissues from AD models can be of great value in assessing the pathogenic role of metals as well as the value of therapeutic interventions aimed at restoring metal homeostasis in the brain. In this study, we employed low resolution inductively coupled plasma mass spectrometry (ICP-MS) to evaluate levels of ultra-trace, trace, and major elements in brains and cerebella of 3xTg-AD mice, a well characterized transgenic (Tg) AD model. This method is based on alternated cool and hot plasma ICP-MS. The essay fulfilled analytical requirements for the quantification of 14 elements in the Central Nervous System (CNS) of our Tg model. Quantification of Li, Al, Cr, and Co, a procedure that requires a pre-concentration step, was validated by high resolution ICP-MS. Changes in element profiles occurring in 3xTg-AD mice were compared to the ones observed in wild type (WT) mice. We also investigated variations in element profiles in 3xTg-AD mice receiving a long-term (17 months) dietary supplementation of Zn. Our data indicate that, compared to WT animals, 3xTg-AD mice displayed signs of altered brain metal homeostasis. We also found that long-term Zn administration promoted decreased brain levels of some metals (K, Ca, and Fe) and restored levels of Al, Cr, and Co to values found in WT mice.

Phenotypic profile linked to inhibition of the major Zn influx system in Salmonella enterica: proteomics and ionomics investigations.

Zinc is required for a wide variety of cellular functions and plays a key role in bacterial metabolism and virulence. However, Zn can also be toxic and, therefore, its influx is tightly regulated. The high affinity zinc uptake transporter ZnuABC is the main Zn influx system in Salmonella enterica under conditions of Zn starvation. It has been shown that deletion of the gene encoding for its periplasmic subunit ZnuA significantly affects S. Typhimurium growth rate and virulence, highlighting the importance of this system in the host-pathogen interaction. To gain further insight into the mechanisms involved in Zn influx regulation, we characterized the main alterations in the ionome and proteome of S. Typhimurium wild type and znuA mutant strains grown either under Zn starvation or under Zn-replete conditions. We found significant differences in the element profile and protein expression that were reversed by Zn supplementation. In particular, several of the differentially regulated proteins are predicted to be metal-binding proteins. Interestingly, their over-expression in the znuA mutant strain strictly depends on Zn starvation and correlates with the differences found at the ionome level. In conclusion, our data demonstrate that inhibition of Zn influx has relevant effects either on the bacterial ionome or proteome and shed new light on the role of the ZnuABC system and Zn influx in S. Typhimurium pathogenicity.