Biomarkers to Be Used for Decision of Treatment of Hypogonadal Men with or without Insulin Resistance.

Male hypogonadism is a result of low testosterone levels, but patients could be insulin-sensitive (IS) or insulin-resistant (IR), showing different impaired metabolic pathways. Thus, testosterone coadministration, which is commonly used to reestablish testosterone levels in hypogonadism, must take into account whether or not insulin is still active. By comparing metabolic cycles recorded in IS and IR plasma before and after testosterone therapy (TRT), it is possible to know what metabolic pathways can be reactivated in the two different groups upon testosterone recovery, and it is possible to understand if antagonism or synergy exists between these two hormones. IS hypogonadism uses glycolysis, while IR hypogonadism activates gluconeogenesis through the degradation of branched-chain amino acids (BCAAs). Upon administration of testosterone, acceptable improvements are observed in IS patients, wherein many metabolic pathways are restored, while in IR patients, a reprogramming of metabolic cycles is observed. However, in both subgroups, lactate and acetyl-CoA increases significantly. In IS patients, lactate is used through the glucose-lactate cycle to produce energy, while in IR patients, both lactate and acetyl-CoA are metabolized into ketone bodies, which are used to produce energy. Thus, in IR patients, an ancestral molecular mechanism is activated to produce energy, mimicking insulin effects. Regarding lipids, in both groups, the utilization of fatty acids for energy (ß-oxidation) is blocked, even after TRT; free fatty acids (FFAs) increase in the blood in IS patients, while they are incorporated into triglycerides in those with IR. In both subgroups of hypogonadism, supplementation of useful chemicals is recommended during and after TRT when metabolites are not restored; they are listed in this review.

Extracellular serine empowers epidermal proliferation and psoriasis-like symptoms.

The contribution of nutrient availability to control epidermal cell proliferation, inflammation, and hyperproliferative diseases remains unknown. Here, we studied extracellular serine and serine/glycine metabolism using human keratinocytes, human skin biopsies, and a mouse model of psoriasis-like disease. We focused on a metabolic enzyme, serine hydroxymethyltransferase (SHMT), that converts serine into glycine and tetrahydrofolate-bound one­carbon units to support cell growth. We found that keratinocytes are both serine and glycine auxotrophs. Metabolomic profiling and hypoxanthine supplementation indicated that SHMT silencing/inhibition reduced cell growth through purine depletion, leading to nucleotide loss. In addition, topical application of an SHMT inhibitor suppressed both keratinocyte proliferation and inflammation in the imiquimod model and resulted in a decrease in psoriasis-associated gene expression. In conclusion, our study highlights SHMT2 activity and serine/glycine availability as an important metabolic hub controlling both keratinocyte proliferation and inflammatory cell expansion in psoriasis and holds promise for additional approaches to treat skin diseases.

On the Need to Distinguish between Insulin-Normal and Insulin-Resistant Patients in Testosterone Therapy.

Male hypogonadism is a disorder characterized by low levels of the hormone testosterone and patients may also have insulin sensitivity (IS) or insulin resistance (IR), such that they show different clinical complications and different metabolic pathways. In this review, we compare metabonomic differences observed between these two groups before and after testosterone therapy (TRT) in order to obtain information on whether the two hormones testosterone and insulin are synergistic or antagonistic. IS hypogonadism uses glucose as the main biofuel, while IR activates gluconeogenesis by the degradation of branched-chain amino acids. The Krebs (TCA) cycle is active in IS but connected with glutaminolysis, while in IR the TCA cycle stops at citrate, which is used for lipogenesis. In both cases, the utilization of fatty acids for energy (ß-oxidation) is hampered by lower amounts of acetylcarnitine, although it is favored by the absence of insulin in IR. Increased free fatty acids (FFAs) are free in the blood in IS, while they are partially incorporated in triglycerides in IR. Thus, upon TRT, the utilization of glucose is increased more in IS than in IR, revealing that in IR there is a switch from preferential glucose oxidation to lipid oxidation. However, in both cases, a high production of lactate and acetyl-CoA is the final result, with these levels being much higher in IR. Lactate is used in IS in the glucose-lactate cycle between the liver and muscle to produce energy, while in IR lactate and acetyl-CoA are biotransformed into ketone bodies, resulting in ketonuria. In conclusion, the restoration of testosterone values in hypogonadism gives better results in IS than in IR patients: in IS, TRT restores most of the metabolic pathways, while in IR TRT impairs insulin, and when insulin is inactive TRT activates an ancestral molecular mechanism to produce energy. This evidence supports the hypothesis that, over time, hypogonadism switches from IS to IR, and in the latter case most of the insulin-related metabolisms are not reactivated, at least within 60 days of TRT. However, testosterone therapy in both IS and IR might be of benefit given supplementation with metabolites that are not completely restored upon TRT, in order to help restore physiological metabolisms. This review underlines the importance of using a systems biology approach to shed light on the molecular mechanisms of related biochemical pathways involving insulin and testosterone.

Plasma Metabonomics in Insulin-Resistant Hypogonadic Patients Induced by Testosterone Treatment.

Hypogonadic subjects with insulin resistance (IR) showed different metabonomic profiles compared to normo-insulinemic subjects (IS). Testosterone replacement therapy (TRT) may have a different impact on the metabolisms of those with the presence or absence of insulin resistance. We evaluated the changes in the metabolism of IR hypogonadic patients before and after 60 days of TRT. The metabonomic plasma profiles from 20 IR hypogonadal patients were recorded using ultra-high-performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (HRMS). Plasma metabolites, before and after 60 days of TRT, were compared. In hypogonadic patients, carnosine, which is important for improving performance during exercise, increased. Conversely, proline and lysine-amino acids involved in the synthesis of collagen-reduced. Triglycerides decreased and fatty acids (FFAs) increased in the blood as a consequence of reduced FFA ß-oxidation. Glycolysis slightly improved, while the Krebs cycle was not activated. Gluconeogenesis (which is the main energy source for hypogonadal IR before TRT) stopped after treatment. As a consequence, lactate and acetyl CoA increased significantly. Both lactate and acetyl CoA were metabolized into ketone bodies which increased greatly, also due to leucine/isoleucine degradation. Ketone bodies were derived predominantly from acetyl CoA because the reaction of acetyl CoA into ketone bodies is catalyzed by mtHMGCoA synthase. This enzyme is inhibited by insulin, which is absent in IR patients but overexpressed following testosterone administration. Ketosis is an alternative route for energy supply and provides the same metabolic effects as insulin but at the metabolic or primitive control level, which bypasses the complex signaling pathway of insulin. After treatment, the hypogonadic patients showed clinical symptoms related to ketonuria. They presented similarly to those following a ketogenic diet, the so-called 'keto flu'. This must be taken into account before the administration of TRT to hypogonadic patients.

Plasma Metabolomics Profile of "Insulin Sensitive" Male Hypogonadism after Testosterone Replacement Therapy.

Male hypogonadism is a disorder characterized by low levels of testosterone, but patients can either show normal insulin (insulin-sensitive (IS)) or over time they can become insulin-resistant (IR). Since the two groups showed different altered metabolisms, testosterone replacement therapy (TRT) could achieve different results. In this paper, we analyzed plasma from 20 IS patients with low testosterone (<8 nmol/L) and HOMAi < 2.5. The samples, pre- and post-treatment with testosterone for 60 days, were analyzed by UHPLC and mass spectrometry. Glycolysis was significantly upregulated, suggesting an improved glucose utilization. Conversely, the pentose phosphate pathway was reduced, while the Krebs cycle was not used. Branched amino acids and carnosine metabolism were positively influenced, while ß-oxidation of fatty acids (FFA) was not activated. Cholesterol, HDL, and lipid metabolism did not show any improvements at 60 days but did so later in the experimental period. Finally, both malate and glycerol shuttle were reduced. As a result, both NADH and ATP were significantly lower. Interestingly, a significant production of lactate was observed, which induced the activation of the Cori cycle between the liver and muscles, which became the main source of energy for these patients without involving alanine. Thus, the treatment must be integrated with chemicals which are not restored in order to reactivate energy production.

Anti-Inflammatory Potential of Cow, Donkey and Goat Milk Extracellular Vesicles as Revealed by Metabolomic Profile.

In recent years, extracellular vesicles (EVs), cell-derived micro and nano-sized structures enclosed in a double-layer membrane, have been in the spotlight for their high potential in diagnostic and therapeutic applications. Indeed, they act as signal mediators between cells and/or tissues through different mechanisms involving their complex cargo and exert a number of biological effects depending upon EVs subtype and cell source. Being produced by almost all cell types, they are found in every biological fluid including milk. Milk EVs (MEVs) can enter the intestinal cells by endocytosis and protect their labile cargos against harsh conditions in the intestinal tract. In this study, we performed a metabolomic analysis of MEVs, from three different species (i.e., bovine, goat and donkey) by mass spectroscopy (MS) coupled with Ultrahigh-performance liquid chromatography (UHPLC). Metabolites, both common or specific of a species, were identified and enriched metabolic pathways were investigated, with the final aim to evaluate their anti-inflammatory and immunomodulatory properties in view of prospective applications as a nutraceutical in inflammatory conditions. In particular, metabolites transported by MEVs are involved in common pathways among the three species. These metabolites, such as arginine, asparagine, glutathione and lysine, show immunomodulating effects. Moreover, MEVs in goat milk showed a greater number of enriched metabolic pathways as compared to the other kinds of milk.

Testosterone replacement therapy in insulin-sensitive hypogonadal men restores phosphatidylcholine levels by regulation of arachidonic acid metabolism.

Male hypogonadism is notoriously associated with altered lipid metabolism. In this study, we performed an untargeted mass spectrometry-based profiling of plasma lipids from twenty healthy and twenty hypogonadal men before and after testosterone replacement therapy (TRT) for 60 days. Results demonstrated that hypogonadism was associated with a significant increase in sphingomyelin (SM), whereas phosphatidylcholine (PC) was mainly cleaved by activated phospholipase-A2 into lysophosphatidylcholine (LPC). In hypogonadal patients, arachidonic acid (AA), also produced through the latter cleavage, was prevalently bio-transformed into leukotriene B4 (LTB4) and not into endoperoxides from which prostaglandins and thromboxanes are derived. Interestingly, upon testosterone treatment SM, PC and LPC returned to levels similar to controls. Also, AA was newly converted into prostaglandin-A2, thromboxane-A2 and 5(S)-hydroxyeicosatetraenoic acid (HETE), suggesting that testosterone probably plays a role in controlling hypogonadal alterations above reported.

A metabolomics approach to investigate urine levels of neurotransmitters and related metabolites in autistic children.

Since recently metabolic abnormalities in autistic children have been associated with ASD disturbs, the aim of this study is to determine the neurotransmitter levels in urine samples of autistic children and to analyse the altered metabolic pathway involved in their production. Thus, ASD-specific urinary metabolomic patterns were explored in 40 ASD children and 40 matched controls using untargeted metabolomics through UHPLC-mass spectrometry (Q-exactive analyser), and by using XCMS Metlin software for data interpretation. Through this new advanced technique, a more considerable number of urinary altered metabolites were recorded in autistic children, than in the previous investigations, which allowed us to collect metabolites involved in neurotransmitter production. In these subjects, a high amount of dopamine was revealed and an increased amount of homovanillic acid, to the detriment of noradrenaline and adrenaline production, as well as MHPG and vanillylmandelic acid, which were found lower. This indicates that the accumulation of dopamine is not due to its greater production, but its lesser biotransformation into noradrenaline, due to the blockage of the dopamine ß-hydroxylase enzyme by 4-cresol and vitamin C, both found in high quantities in autistic subjects. Finally, a decreased amount of the active form of vitamin B6, pyridoxal phosphate (P5P), implicated in biotransformation of glutamate into γ-aminobutyric acid (GABA), was also detected, justifying the lower levels of latter. All of these alterations are correlated with a peculiar intestinal microbiome in autistic subjects, supporting the idea of a microbiota-gut-brain axis, then altered levels of neurotransmitters and altered neuronal transmission exist.

ALS skin fibroblasts reveal oxidative stress and ERK1/2-mediated cytoplasmic localization of TDP-43.

The main hallmark of many forms of familiar and sporadic amyotrophic lateral sclerosis (ALS) is a reduction in nuclear TDP-43 protein and its inclusion in cytoplasmic aggregates in motor neurons. In order to understand which cellular and molecular mechanisms underlie the mislocalization of TDP-43, we examined human skin fibroblasts from two individuals with familial ALS, both with mutations in TDP-43, and two individuals with sporadic ALS, both without TDP-43 mutations or mutations in other ALS related genes. We found that all ALS fibroblasts had a partially cytoplasmic localization of TDP-43 and had reduced cell metabolism as compared to fibroblasts from apparently healthy individuals. ALS fibroblasts showed an increase in global protein synthesis and an increase in 4E-BP1 and rpS6 phosphorylation, which is indicative of mTORC1 activity. We also observed a decrease in glutathione (GSH), which suggests that oxidative stress is elevated in ALS. ERK1/2 activity regulated the extent of oxidative stress and the localization of TDP-43 in the cytoplasm in all ALS fibroblasts. Lastly, ALS fibroblasts showed reduced stress granule formation in response to H2O2 stress. In conclusion, these findings identify specific cellular and molecular defects in ALS fibroblasts, thus providing insight into potential mechanisms that may also occur in degenerating motor neurons.

Brain protein changes in Mecp2 mouse mutant models: Effects on disease progression of Mecp2 brain specific gene reactivation.

Rett syndrome (RTT) is a leading cause of severe intellectual disability in females, caused by de novo loss-of function mutations in the X-linked methyl-CpG binding protein 2 (MECP2). To better investigate RTT disease progression/pathogenesis animal models of Mecp2 deficiency have been developed. Here, Mecp2 mouse models are employed to investigate the role of protein patterns in RTT. A proteome analysis was carried out in brain tissue from i) Mecp2 deficient mice at the pre-symptomatic and symptomatic stages and, ii) mice in which the disease phenotype was reversed by Mecp2 reactivation. Several proteins were shown to be differentially expressed in the pre-symptomatic (n = 18) and symptomatic (n = 20) mice. Mecp2 brain reactivated mice showed wild-type comparable levels of expression for twelve proteins, mainly related to proteostasis (n = 4) and energy metabolic pathways (n = 4). The remaining ones were found to be involved in redox homeostasis (n = 2), nitric oxide regulation (n = 1), neurodevelopment (n = 1). Ten out of twelve proteins were newly linked to Mecp2 deficiency. Our study sheds light on the relevance of the protein-regulation of main physiological process in the complex mechanisms leading from Mecp2 mutation to the RTT clinical phenotype. SIGNIFICANCE: We performed a proteomic study of a Mecp2stop/y mouse model for Rett syndrome (RTT) at the pre-symptomatic and symptomatic Mecp2 deficient mice stage and for the brain specific reactivated Mecp2 model. Our results reveal major protein expression changes pointing out to defects in proteostasis or energy metabolic pathways other than, to a lesser extent, in redox homeostasis, nitric oxide regulation or neurodevelopment. The Mecp2 mouse rescued model provides the possibility to select target proteins more susceptible to the Mecp2 gene mutation, potential and promising therapeutical targets.

Lymphatic Cannulation for Lymph Sampling and Molecular Delivery.

Unlike the blood, the interstitial fluid and the deriving lymph are directly bathing the cellular layer of each organ. As such, composition analysis of the lymphatic fluid can provide more precise biochemical and cellular information on an organ's health and be a valuable resource for biomarker discovery. In this study, we describe a protocol for cannulation of mouse and rat lymphatic collectors that is suitable for the following: the "omic" sampling of pre- and postnodal lymph, collected from different anatomical districts; the phenotyping of immune cells circulating between parenchymal organs and draining lymph nodes; injection of known amounts of molecules for quantitative immunological studies of nodal trafficking and/or clearance; and monitoring an organ's biochemical omic changes in pathological conditions. Our data indicate that probing the lymphatic fluid can provide an accurate snapshot of an organ's physiology/pathology, making it an ideal target for liquid biopsy.

A novel oral micellar fenretinide formulation with enhanced bioavailability and antitumour activity against multiple tumours from cancer stem cells.

BACKGROUND: An increasing number of anticancer agents has been proposed in recent years with the attempt to overcome treatment-resistant cancer cells and particularly cancer stem cells (CSC), the major culprits for tumour resistance and recurrence. However, a huge obstacle to treatment success is the ineffective delivery of drugs within the tumour environment due to limited solubility, short circulation time or inconsistent stability of compounds that, together with concomitant dose-limiting systemic toxicity, contribute to hamper the achievement of therapeutic drug concentrations. The synthetic retinoid Fenretinide (4-hydroxy (phenyl)retinamide; 4-HPR) formerly emerged as a promising anticancer agent based on pre-clinical and clinical studies. However, a major limitation of fenretinide is traditionally represented by its poor aqueous solubility/bioavailability due to its hydrophobic nature, that undermined the clinical success of previous clinical trials. METHODS: Here, we developed a novel nano-micellar fenretinide formulation called bionanofenretinide (Bio-nFeR), based on drug encapsulation in an ion-pair stabilized lipid matrix, with the aim to raise fenretinide bioavailability and antitumour efficacy. RESULTS: Bio-nFeR displayed marked antitumour activity against lung, colon and melanoma CSC both in vitro and in tumour xenografts, in absence of mice toxicity. Bio-nFeR is suitable for oral administration, reaching therapeutic concentrations within tumours and an unprecedented therapeutic activity in vivo as single agent. CONCLUSION: Altogether, our results indicate Bio-nFeR as a novel anticancer agent with low toxicity and high activity against tumourigenic cells, potentially useful for the treatment of solid tumours of multiple origin.

A new bioavailable fenretinide formulation with antiproliferative, antimetabolic, and cytotoxic effects on solid tumors.

Fenretinide is a synthetic retinoid characterized by anticancer activity in preclinical models and favorable toxicological profile, but also by a low bioavailability that hindered its clinical efficacy in former clinical trials. We developed a new formulation of fenretinide complexed with 2-hydroxypropyl-beta-cyclodextrin (nanofenretinide) characterized by an increased bioavailability and therapeutic efficacy. Nanofenretinide was active in cell lines derived from multiple solid tumors, in primary spheroid cultures and in xenografts of lung and colorectal cancer, where it inhibited tumor growth independently from the mutational status of tumor cells. A global profiling of pathways activated by nanofenretinide was performed by reverse-phase proteomic arrays and lipid analysis, revealing widespread repression of the mTOR pathway, activation of apoptotic, autophagic and DNA damage signals and massive production of dihydroceramide, a bioactive lipid with pleiotropic effects on several biological processes. In cells that survived nanofenretinide treatment there was a decrease of factors involved in cell cycle progression and an increase in the levels of p16 and phosphorylated p38 MAPK with consequent block in G0 and early G1. The capacity of nanofenretinide to induce cancer cell death and quiescence, together with its elevated bioavailability and broad antitumor activity indicate its potential use in cancer treatment and chemoprevention.

Multi-omics profiling of calcium-induced human keratinocytes differentiation reveals modulation of unfolded protein response signaling pathways.

By proteomic, metabolomic and transcriptomic approaches we shed light on the molecular mechanism by which human keratinocytes undergo to terminal differentiation upon in vitro calcium treatment. Proteomic analysis revealed a selective induction of the ribosomal proteins RSSA, an inhibitor of cell proliferation and inducer of differentiation, HSP 60, a protein folding chaperone and GRP78, an unfolding protein response signal. Additionally, we observed an induction of EF1D, a transcription factor for genes that contain heat-shock responsive elements. Conversely, RAD23, a protein involved in regulating ER-associated protein degradation was down-regulated. All these modifications indicated an ER stress response, which in turn activated the unfolded protein response signaling pathway through ATF4, as confirmed both by the modulation of amino acids metabolism genes, such as XBP1, PDI and GPR78, and by the metabolomic analysis. Finally, we detected a reduction of PDI protein, as confirmed by the increase of oxidized glutathione. Metabolome analysis indicated that glycolysis failed to fuel the Krebs cycle, which continued to decrease during differentiation, at glance with the PPP pathway, allowing NADH production and glutathione reduction. Since unfolded protein response is linked to keratinization, these results may be useful for studying pathological mechanisms as well as potential treatments for different pathological conditions. Abbreviation: UPR, unfolded protein response; HEK, human epidermal keratinocytes; HKGS, human keratinocytes growth factor.

The concomitant lower concentrations of vitamins B6, B9 and B12 may cause methylation deficiency in autistic children.

Autism spectrum disorder (ASD) is characterized by severe and persistent difficulties in social communication and social interaction at multiple levels. Recently, metabolic disorders have been associated with most cases of patients with ASD. The aim of this study was to investigate, through a new and more sophisticated mass technique, such as UHPLC-mass spectrometry (Q-exactive analyzer), alteration in metabolisms analyzing ASD children urine samples from children showing simultaneous vitamin B6, B9 and B12 deficiencies. This in order to study how these concurrent deficiencies may influence some phenotypic aspects of autistic disorder. Thus, urinary metabolic patterns specific to ASD were explored at an early age in 60 children with ASD, showing lower three vitamins levels, and 60 corresponding controls (age group 3-8, M: F=42:18). The results showed significant block of cystathionine formation with consequent accumulation of homocysteine. A lower glutathione levels (GSH), with reduction of essential intracellular reducing environment required for normal immune function, detoxification capacity and redox-sensitive enzyme activity. Increased concentration of 5-methyltetrahydrofolate, which leads to a lower availability of methyl group and significant decrease in urinary methionine and S-adenosyl-L-methionine (SAM) concentrations, the major methyl donor. The latter justify the well-known reduction in protein and DNA methylation reported in autistic children. As a final consideration, the concomitant deficiencies of all three B vitamins, recorded in a significant number of autistic children, suggests that intestinal dysbiosis in these patients may be the main cause of a reduction in their absorption, in addition to the genetic mutation of a specific gene.

Untargeted Metabolomics of Plant Leaf Tissues.

Untargeted metabolomics is a useful approach for the simultaneous analysis of a vast array of compounds from a single extract. Metabolomic profiling is the relative multi-parallel quantification of a mixture of low molecular weight compounds, or classes of compounds, and it is most often performed by using ultra performance liquid chromatography (UPLC) coupled with mass spectrometry (MS). Being an extension of the classical targeted methods, this approach allows a broader view of the main biochemical events within a particular sample. This chapter exemplifies and provides experimental details on the basic steps to perform a non-targeted metabolomic analysis on plant leaf tissues: sample collection and homogenization, extraction of metabolites, raw data acquisition, and processing into formats for data mining and informatics. In particular, the approach was applied to two spring wheat varieties with different level of drought tolerance (Kavir, drought-resistant; Bahar, drought-sensitive) developed by the CIMMYT (International Center for the Improvement of Corn and Wheat).

Deoxynivalenol Detoxification in Transgenic Wheat Confers Resistance to Fusarium Head Blight and Crown Rot Diseases.

Fusarium diseases, including Fusarium head blight (FHB) and Fusarium crown rot (FCR), reduce crop yield and grain quality and are major agricultural problems worldwide. These diseases also affect food safety through fungal production of hazardous mycotoxins. Among these, deoxynivalenol (DON) acts as a virulence factor during pathogenesis on wheat. The principal mechanism underlying plant tolerance to DON is glycosylation by specific uridine diphosphate-dependent glucosyltransferases (UGTs), through which DON-3-ß-d-glucoside (D3G) is produced. In this work, we tested whether DON detoxification by UGT could confer to wheat a broad-spectrum resistance against Fusarium graminearum and F. culmorum. These widespread Fusarium species affect different plant organs and developmental stages in the course of FHB and FCR. To assess DON-detoxification potential, we produced transgenic durum wheat plants constitutively expressing the barley HvUGT13248 and bread wheat plants expressing the same transgene in flower tissues. When challenged with F. graminearum, FHB symptoms were reduced in both types of transgenic plants, particularly during early to mid-infection stages of the infection progress. The transgenic durum wheat displayed much greater DON-to-D3G conversion ability and a considerable decrease of total DON+D3G content in flour extracts. The transgenic bread wheat exhibited a UGT dose-dependent efficacy of DON detoxification. In addition, we showed, for the first time, that DON detoxification limits FCR caused by F. culmorum. FCR symptoms were reduced throughout the experiment by nearly 50% in seedlings of transgenic plants constitutively expressing HvUGT13248. Our results demonstrate that limiting the effect of the virulence factor DON via in planta glycosylation restrains FHB and FCR development. Therefore, ability for DON detoxification can be a trait of interest for wheat breeding targeting FHB and FCR resistance.

Ribosomal RACK1 promotes proliferation of neuroblastoma cells independently of global translation upregulation.

Neuroblastoma is the most frequent solid tumor among those diagnosed during infancy and like most tumors, it is characterized by elevated rates of cell proliferation, migration and invasion. RACK1 is among the top 10 genes identified for unfavorable prognosis at 5 years in neuroblastoma cases and its depletion negatively affects proliferation, invasion and migration. Here, we show that the ribosomal localization of RACK1 modulates the proliferation of SH-SY5Y neuroblastoma cells by regulating the expression of cell cycle genes, such as Cyclin D1, D3 and B1 independently of global translation increase. Ribosomal RACK1 is not involved in general protein synthesis, which is instead dependent on total RACK1 and PKC but independent from mTOR. Thus, ribosomal RACK1 may represent a new target to develop more efficient therapies for neuroblastoma treatment.

Urinary Metabolic Signature of Primary Aldosteronism: Gender and Subtype-Specific Alterations.

PURPOSE: The current clinical investigation for primary aldosteronism (PA) diagnosis requires complex expensive tests from the initial suspicion to the final subtype classification, including invasive approaches; therefore, appropriate markers for subtype definition are greatly desirable. The present study performs a metabolomics analysis to further examine specific molecular signatures of PA urines EXPERIMENTAL DESIGN: The study considered PA subtype and gender-related differences using two orthogonal advanced UHPLC-MS metabolomics approaches. Patients with essential hypertension (n = 36) and PA (n = 50) who were referred to the outpatient hypertension clinic and matched healthy subjects (n = 10) are investigated. RESULTS: Statistically significant changes (p < 0.05 ANOVA, Fc > 1.5) of metabolites involved in central carbon, energy, and nitrogen metabolism are identified, especially purine and pyrimidine nucleosides and precursors, and free amino acids. PLS-DA interpretation provides strong evidence of a disease-specific metabolic pattern with dAMP, diiodothyronine, and 5-methoxytryptophan as leading factors, and a sex-specific metabolic pattern associated with orotidine 5-phosphate, N-acetylalanine, hydroxyproline, and cysteine. The results are verified using an independent sample set, which confirms the identification of specific signatures. CONCLUSIONS AND CLINICAL RELEVANCE: Metabolomics is used to identify low molecular weight molecular markers of PA, which paves the way for follow-up validation studies in larger cohorts.

Metabolic patterns in insulin-resistant male hypogonadism.

Male hypogonadism associated with insulin resistance (IR) very often leads to metabolic syndrome, at variance with hypogonadism in its first stadium of insulin sensitivity (IS). A plasma metabolomic investigation of these patients can provide useful information in comparison with the values of IS patients. To this aim plasma from insulin-resistant males with hypogonadism were analysed by using ultra high-performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (HRMS). Thus, metabolites were compared to the controls through multivariate statistical analysis and grouped by metabolic pathways. Metabolite database searches and pathway analyses identified imbalances in 18-20 metabolic pathways. Glucose metabolism (e.g., glycolysis and the Krebs cycle) is fuelled by amino acids degradation, in particular of branched amino acids, in individuals with lean body mass. Gluconeogenesis is strongly activated. Some crucial pathways such as glycerol are skewed. Mitochondrial electron transport is affected with a reduction in ATP production. Beta-oxidation of short and medium chain fatty acids did not represent an energy source in hypogonadism, at variance with long and branched fatty acids, justifying the increase in fat mass. Carnosine and ß-alanine are strongly reduced resulting in increased fatigue and mental confusion. A comparison of IR with IS male hypogonadism will contribute to a better understanding of how these two hormones work in synergy or antagonise each other in humans. It could also help to select patients who will respond to hormone treatment, and provide accurate biomarkers to measure the response to treatment eventually leading to better strategies in preventing systemic complications in patients not fit for hormone replacement therapy.