Search

About 71% of healthcare-associated infections are due to antibiotic-resistant bacteria, such as carbapenem-resistant A. baumannii , classified by World Health Organization into a critical priority group of pathogens. The antimicrobial resistance profile of A. baumannii relies on its ability to produce several virulence factors, including biofilm formation. Its ability to adhere and persist on surfaces as biofilm has contributed to its pathogenicity and drug resistance. In this study, the ability of an antimicrobial peptide (a chionodracine-derived peptide named KHS-Cnd) to inhibit or reduce biofilm formation was investigated as an example of a potential strategy to counteract infections caused by biofilm-forming pathogens. To this aim, the antimicrobial profiles were first analyzed in selected A. baumannii strains, two reference and six clinical strains, all biofilm-forming with different capability, regardless of whether they are drug resistant or sensitive. Successively, we investigated the bactericidal activity of the peptide that showed MIC values ranging from 5 to 10 µM and a significative antibiofilm activity on all tested strains at sub-inhibitory concentrations. In fact, KHS-Cnd can hinder biofilm A. baumannii strains formation with an inhibition percentage ranging between 65% and 10%. Also a statistically significant reduction of mature biofilm ranging from 20% to 50% was observed in four out of eight tested A. baumannii strains. KHS-Cnd impacts various stages of biofilm formation, including the inhibition of surface-associated and twitching motilities depending on the different strain. In particular, our results showed that only two strains possessed surface-associated motility that was strongly impaired by KHS-Cnd treatment; three clinical strains, instead, showed twitching motility, whose inhibition for two of them was evident after 24 h of incubation with peptide. Moreover, the invasion of pulmonary cells by A. baumannii was significantly impaired with a reduction of about 32% after treatment with 1.25 µM KHS-Cnd. Finally, when the peptide was used together with ceftazidime/avibactam against resistant A. baumannii strains, it was able to reduce the minimal inhibitory concentration of antibiotics needed to inhibit the microorganism growth., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Artini, Paris, Imperlini, Buonocore, Vrenna, Papa and Selan.)

Antarctic fishes, living in an extreme environment and normally exposed to pathogens, are a promising source of antimicrobial peptides (AMPs). These are emerging as next-generation drugs due to their activity against multidrug resistant (MDR) bacteria. To infect hosts, beyond intrinsic/acquired resistance, MDR species also use virulence factors such as protease secretion. Hence, AMPs targeting virulence factors could represent a novel strategy to counteract the antimicrobial resistance (AMR). In this paper, we focused on a mutant peptide, named KHS-Cnd, that was obtained from the scaffold of the chionodracine (Cnd), a natural peptide identified in the icefish Chionodraco hamatus. We studied different effects caused by the peptide interaction with the cell membrane of two model bacteria, E. coli and B. cereus. First, we investigated its membranolytic activity revealing that the peptide action is more evident on E. coli, with a 69 % uptake of the used dye at 3 μM, whereas for B. cereus we found only a 65 % uptake at 6 μM. Successively, we determined the impact of this lysis on total protein concentration in the medium and an increase was estimated for both bacteria (84 % after 1 h for E. coli and 90 % for B. cereus, respectively). Moreover, we evaluated the changes in the proteolytic activity of the supernatant, that is an important aspect of bacterial resistance, showing that there was a significant reduction for both bacteria, although at higher level in the case of E. coli. The membranolytic activity was evidenced also morphologically with TEM analysis and a different alteration was evidenced for the two bacteria. Moreover, NMR metabolomics analysis showed that peptide induces changes in E. coli and B. cereus extracellular metabolites especially at the higher tested concentrations: this metabolic variation could be used as a fingerprinting of the peptide action on bacteria physiology due to its interaction with cell wall. Finally, we determined the KHS-Cnd cytotoxicity on human primary cell lines to verify its selectivity toward bacterial cell membranes and we found low toxicity until a concentration of 5 μM. Considering that the peptide exerts both membranolytic and anti-virulence activity on E. coli at 1.5 μM, we confirmed the interesting potential of this AMP as a new drug to counteract AMR., Competing Interests: Declaration of Competing Interest The authors have nothing to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Nanoparticles (NPs) interact with biological fluids after being injected into the bloodstream. The interactions between NPs and plasma proteins at the nano-bio interface affect their biopharmaceutical properties and distribution in the organ and tissues due to protein corona (PrC) composition, and in turn, modification of the resulting targeting capability. Moreover, lipid and polymer NPs, at their interface, affect the composition of PrC and the relative adsorption and abundance of specific proteins. To investigate this latter aspect, we synthesized and characterized different liposomal formulations (LFs) with lipids and polymer-conjugated lipids at different molar ratios, having different sizes, size distributions and surface charges. The PrC composition of various designed LFs was evaluated ex vivo in human plasma by label-free quantitative proteomics. We also correlated the relative abundance of identified specific proteins in the coronas of the different LFs with their physicochemical properties (size, PDI, zeta potential). The evaluation of outputs from different bioinformatic tools discovered protein clusters allowing to highlight: (i) common as well as the unique species for the various formulations; (ii) correlation between each identified PrC and the physicochemical properties of LFs; (iii) some preferential binding determined by physicochemical properties of LFs; (iv) occurrence of formulation-specific protein patterns in PrC. Investigating specific clusters in PrC will help decode the multivalent roles of the protein pattern components in the drug delivery process, taking advantage of the bio-nanoscale recognition and identification for significant advances in nanomedicine., Competing Interests: All authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

During tumorigenesis, miRNAs with unbalanced expression profiles can increase the threat of disease progression. Here, we focus on the role of miR-331-5p in the pathogenesis of thyroid cancer (TC). In vitro studies were conducted using TC cell lines after the forced expression and silencing of miR-331-5p. Cell proliferation and viability were analyzed via cell counts and colorimetric assays. Cell motility was analyzed via wound healing assays, Transwell migration and invasion assays, and Matrigel Matrix assays. The putative targets of miR-331-5p were unveiled via label-free proteomic screening and then verified using Western blot and luciferase assays. Expression studies were conducted by interrogating The Cancer Genome Atlas (TCGA). We found that ectopic miR-331-5p expression reduces TC cell motility, while miR-331-5p silencing induces the opposite phenotype. Proteomic screening revealed eight putative downregulated targets of miR-331-5p, among which BID was confirmed as a direct target. TCGA data showed the downregulation of miR-331-5p and the upregulation of BID in TC tissues. In summary, deregulation of the miR-331-5p/BID axis could enhance the aggressiveness of TC cell lines, providing new insights into the mechanisms of the progression of this disease and suggesting a potential role of the component factors as possible biomarkers in TC tissues.

The airways of cystic fibrosis (CF) patients are colonized by many pathogens and the most common is Pseudomonas aeruginosa , an environmental pathogen that is able to infect immunocompromised patients thanks to its ability to develop resistance to conventional antibiotics. Over 12% of all patients colonized by P. aeruginosa harbour multi-drug resistant species. During airway infection in CF, P. aeruginosa adopts various mechanisms to survive in a hostile ecological niche characterized by low oxygen concentration, nutrient limitation and high osmotic pressure. To this end, P. aeruginosa uses a variety of virulence factors including pigment production, biofilm formation, motility and the secretion of toxins and proteases. This study represents the first report that systematically analyzes the differences in virulence features, in normoxia and anoxia, of clinical P. aeruginosa isolated from CF patients, characterized by multi- or pan-drug antibiotic resistance compared to antibiotic sensitive strains. The virulence features, such as biofilm formation, protease secretion and motility, are highly diversified in anaerobiosis, which reflects the condition of chronic CF infection. These findings may contribute to the understanding of the real-world lifestyle of pathogens isolated during disease progression in each particular patient and to assist in the design of therapeutic protocols for personalized medicine.

In cancer research today, one of the major challenges is the eradication of cancer stem cells (CSCs) within the tumor mass. These cells play a crucial role in initiating, growing, and maintaining the tumor. Evidence has demonstrated the presence and significance of CSCs in the development and progression of osteosarcoma (OS). However, our understanding of the specific markers for OS stem cells remains limited. In this study, we aim to identify distinct biomarkers for this cell population by conducting a proteomic analysis comparing OS stem cells to their non-stem counterparts. Our investigation focuses on a particular cell line called 3AB-OS, which exhibits stem-like characteristics, and its differentiated parental cell line, MG63. Through this research, we discovered 63 proteins exclusively expressed in 3AB-OS cells. Applying an in silico bioinformatics approach, we determined that the majority of these proteins are associated with RNA metabolism. Additionally, we identified a potential correlation between the insulin-like growth factor-binding proteins (IGF2BPs) signaling pathway and the tumorigenic and stemness features observed in 3AB-OS cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The introduction of antibiotics has revolutionized the treatment and prevention of microbial infections. However, the global spread of pathogens resistant to available antibiotics is a major concern. Recently, the WHO has updated the priority list of multidrug-resistant (MDR) species for which the discovery of new therapeutics is urgently needed. In this scenario, antimicrobial peptides (AMPs) are a new potential alternative to conventional antibiotics, as they show a low risk of developing antimicrobial resistance, thus preventing MDR bacterial infections. However, there are limitations and challenges related to the clinical impact of AMPs, as well as great scientific efforts to find solutions aimed at improving their biological activity, in vivo stability, and bioavailability by reducing the eventual toxicity. To overcome some of these issues, different types of nanoparticles (NPs) have been developed for AMP delivery over the last decades. In this review, we provide an update on recent nanosystems applied to AMPs, with special attention on their potential pharmaceutical applications for the treatment of bacterial infections. Among lipid nanomaterials, solid lipid NPs and lipid nanocapsules have been employed to enhance AMP solubility and protect peptides from proteolytic degradation. In addition, polymeric NPs, particularly nanogels, are able to help in reducing AMP toxicity and also increasing AMP loading. To boost AMP activity instead, mesoporous silica or gold NPs can be selected due to their easy surface functionalization. They have been also used as nanocarriers for different AMP combinations, thus synergistically potentiating their action against pathogens.

Background: Aging and sedentary behavior are independent risk factors for non-communicable diseases. An active lifestyle and structured physical activity are positively associated with a healthier quality of life in the elderly. Here, we explored the proteomic/metabolomic muscular signature induced by lifelong football training associated with successful aging., Methods: The study was performed on nine lifelong football players (67.3 ± 2.8 yrs) and nine aged-matched untrained subjects. We performed a proteomic/metabolomic approach on V. lateralis muscle biopsies; the obtained data were analyzed by means of different bioinformatic tools., Results: Our results indicated that lifelong football training is able to enhance the muscles' oxidative capacity in the elderly by promoting fatty acids as preferential energetic substrates and hence determining a healthier body composition and metabolic profile; furthermore, we showed that the total polyamine content is higher in lifelong football players' muscle, enforcing the involvement of polyamines in muscle growth and hypertrophy., Conclusions: Lifelong football training, as a structured physical activity, significantly influences the expression of the proteins and metabolites involved in oxidative metabolism and muscle hypertrophy associated with successful aging.

Pseudomonas aeruginosa is an opportunistic pathogen causing several chronic infections resistant to currently available antibiotics. Its pathogenicity is related to the production of different virulence factors such as biofilm and protease secretion. Pseudomonas communities can persist in biofilms that protect bacterial cells from antibiotics. Hence, there is a need for innovative approaches that are able to counteract these virulence factors, which play a pivotal role, especially in chronic infections. In this context, antimicrobial peptides are emerging drugs showing a broad spectrum of antibacterial activity. Here, we tested the anti-virulence activity of a chionodracine-derived peptide (KHS-Cnd) on five P. aeruginosa clinical isolates from cystic fibrosis patients. We demonstrated that KHS-Cnd impaired biofilm development and caused biofilm disaggregation without affecting bacterial viability in nearly all of the tested strains. Ultrastructural morphological analysis showed that the effect of KHS-Cnd on biofilm could be related to a different compactness of the matrix. KHS-Cnd was also able to reduce adhesion to pulmonary cell lines and to impair the invasion of host cells by P. aeruginosa . A cytotoxic effect of KHS-Cnd was observed only at the highest tested concentration. This study highlights the potential of KHS-Cnd as an anti-biofilm and anti-virulence molecule against P. aeruginosa clinical strains.

This study analyses the influence of different area per player (A P ; 75, 98 and 131 m 2 ) on the average metabolic power (MP) and other soccer-related performance variables in relation to the positional roles. We recruited 19 non-professional male soccer players (25.2 ± 6.3 y; 23.7 ± 2.3 kg/m 2 ; 16.4 ± 6.3 y soccer experience) to play three different small-sided games (SSGs): SSG1 (5 vs. 5; 30 × 30 m; 5 min), SSG2 (5 vs. 5; 35 × 45 m; 5 min) and SSG3 (7 vs. 7; 35 × 45 m; 8 min). Specific playing rules were applied. GPS-assessed soccer-related variables were: average MP (AMP), distance covered in 1 min (DIS); % time spent at high speed (v > 16 km/h; % hst) or MP (>20 W/kg; % hmpt); % distance covered at high positive/negative speed (2 < v < 4 m/s 2 , % ACC; -6 < v < -2 m/s 2 , % DEC); and number of actions at high MP (hmpa). All recorded variables differed when each SSG was compared to the others ( p < 0.05), but for hmpa for attackers. Most performance variables were positively associated with increasing A P ( p < 0.05), but for % ACC and % DEC, and differed among positional roles within the same SSG ( p < 0.05). Here the general applicability of SSGs, regardless the physical/technical skills of the group of players, to enhance performance is confirmed; furthermore, quantitative advices on AMP and other performance variables are provided to achieve significant improvements in all soccer players of the team.

Unraveling the proteins interacting with nanoparticles (NPs) in biological fluids, such as blood, is pivotal to rationally design NPs for drug delivery. The protein corona (PrC), formed on the NP surface, represents an interface between biological components and NPs, dictating their pharmacokinetics and biodistribution. PrC composition depends on biological environments around NPs and on their intrinsic physicochemical properties. We generated different formulations of non-ionic surfactant/non-phospholipid vesicles, called niosomes (NIOs), using polysorbates which are biologically safe, cheap, non-toxic and scarcely immunogenic. PrC composition and relative protein abundance for all designed NIOs were evaluated ex vivo in human plasma (HP) by quantitative label-free proteomics. We studied the correlation of the relative protein abundance in the corona with cellular uptake of the PrC-NIOs in healthy and cancer human cell lines. Our results highlight the effects of polysorbates on nano-bio interactions to identify a protein pattern most properly aimed to drive the NIO targeting in vivo, and assess the best conditions of PrC-NIO NP uptake into the cells. This study dissected the biological identity in HP of polysorbate-NIOs, thus contributing to shorten their passage from preclinical to clinical studies and to lay the foundations for a personalized PrC.

Searching for new cancer-related biomarkers is a key priority for the early detection of solid tumors, such as colorectal cancer (CRC), in clinically relevant biological fluids. The cell line and/or tumor tissue secretome represents a valuable resource for discovering novel protein markers secreted by cancer cells. The advantage of a secretome analysis is the reduction of the large dynamic range characterizing human plasma/serum, and the simultaneous enrichment of low abundance cancer-secreted proteins, thereby overcoming the technical limitations underlying the direct search in blood samples. In this review, we provided a comprehensive overview of recent studies on the CRC secretome for biomarker discovery, focusing both on methodological and technical aspects of secretome proteomic approaches and on biomarker-independent validation in CRC patient samples (blood and tissues). Secretome proteomics are mainly based on LC-MS/MS analyses for which secretome samples are either in-gel or in-solution trypsin-digested. Adequate numbers of biological and technical replicates are required to ensure high reproducibility and robustness of the secretome studies. Moreover, another major challenge is the accuracy of proteomic quantitative analysis performed by label-free or labeling methods. The analysis of differentially expressed proteins in the CRC secretome by using bioinformatic tools allowed the identification of potential biomarkers for early CRC detection. In this scenario, this review may help to follow-up the recent secretome studies in order to select promising circulating biomarkers to be validated in larger screenings, thereby contributing toward a complete translation in clinical practice.

Mesoporous silicon microparticles (MSMPs) can incorporate drug-carrying nanoparticles (NPs) into their pores. An NP-loaded MSMP is a multistage vector (MSV) that forms a Matryoshka-like structure that protects the therapeutic cargo from degradation and prevents its dilution in the circulation during delivery to tumor cells. We developed an MSV constituted by 1 µm discoidal MSMPs embedded with PEGylated liposomes containing oxaliplatin (oxa) which is a therapeutic agent for colorectal cancer (CRC). To obtain extra-small liposomes able to fit the 60 nm pores of MSMP, we tested several liposomal formulations, and identified two optimal compositions, with a prevalence of the rigid lipid 1,2-distearoyl-sn-glycero-3-phosphocholine and of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. To improve the MSV assembly, we optimized the liposome-loading inside the MSMP and achieved a five-fold increase of the payload using an innovative lyophilization approach. This procedure also increased the load and limited dimensional changes of the liposomes released from the MSV in vitro. Lastly, we found that the cytotoxic efficacy of oxa-loaded liposomes and-oxa-liposome-MSV in CRC cell culture was similar to that of free oxa. This study increases knowledge about extra-small liposomes and their loading into porous materials and provides useful hints about alternative strategies for designing drug-encapsulating NPs.

We aimed to compare the effects of a personalized short-term high-intensity interval training (HIIT) vs. standard moderate intensity continuous training (MICT) on body fat percentage, abdominal circumference, BMI and maximal oxygen uptake (VO 2max ) in overweight volunteers. Twenty overweight sedentary volunteers (24.9 ± 2.9y; BMI: 26.1 ± 1 kgm -2 ) were randomly assigned to 2 groups, HIIT or MICT. HIIT trained 6 weeks (3-days/week), 40-min sessions as follows: 6-min warm-up, 20-min resistance training (RT) at 70% 1-RM, 8-min HIIT up to 90% of the predicted Maximal Heart Rate (HR max ), 6-min cool-down. MICT trained 6 weeks (3-days/week) 60-min sessions as follows: 6-min warm-up, 20-min RT at 70% 1-RM, 30-min MICT at 60-70% of the predicted HR max , 4-min cool-down. Two-way ANOVA was performed in order to compare the efficacy of HIIT and MICT protocols, and no significant interaction between training x time was evidenced ( p > 0.05), indicating similar effects of both protocols on all parameters analyzed. Interestingly, the comparison of Δ mean percentage revealed an improvement in VO 2max ( p = 0.05) together with a positive trend in the reduction of fat mass percentage ( p = 0.06) in HIIT compared to MICT protocol. In conclusion, 6 weeks of personalized HIIT, with reduced training time (40 vs. 60 min)/session and volume of training/week, improved VO 2max and reduced fat mass percentage more effectively compared to MICT. These positive results encourage us to test this training in a larger population.

Exercise represents the most important integrative therapy in metabolic, immunologic and chronic diseases; it represents a valid strategy in the non-pharmacological intervention of lifestyle linked diseases. A large body of evidence indicates physical exercise as an effective measure against chronic non-communicable diseases. The worldwide general evidence for health benefits are both for all ages and skill levels. In a dysregulated lifestyle such as in the obesity, there is an imbalance in the production of different cytokines. In particular, we focused on Adiponectin, an adipokine producted by adipose tissue, and on Orexin-A, a neuropeptide synthesized in the lateral hypothalamus. The production of both Adiponectin and Orexin-A increases following regular and structured physical activity and both these hormones have similar actions. Indeed, they improve energy and glucose metabolism, and also modulate energy expenditure and thermogenesis. In addition, a relevant biological role of Adiponectin and Orexin A has been recently highlighted in the immune system, where they function as immune-suppressor factors. The strong connection between these two cytokines and healthy status is mediated by physical activity and candidates these hormones as potential biomarkers of the beneficial effects induced by physical activity. For these reasons, this review aims to underly the interconnections among Adiponectin, Orexin-A, physical activity and healthy status. Furthermore, it is analyzed the involvement of Adiponectin and Orexin-A in physical activity as physiological factors improving healthy status through physical exercise., (Copyright © 2020 Polito, Monda, Nigro, Messina, Di Maio, Giuliano, Orrù, Imperlini, Calcagno, Mosca, Mollica, Trinchese, Scarinci, Sessa, Salerno, Marsala, Buono, Mancini, Monda, Daniele and Messina.)

This narrative review aims to critically analyze the effects of exercise on health in aging. Here we discuss the main clinical and biomolecular modifications induced by long-term recreational football training in older subjects. In particular, the effects induced by long-term recreational football training on cardiovascular, metabolic and musculo-skeletal fitness, together with the modifications in the muscle expression of hallmarks related to oxidative metabolism, DNA repair and senescence suppression pathways and protein quality control mechanisms will be provided. All these topics will be debated also in terms of preventing non-communicable metabolic diseases, in order to achieve successful aging over time.

Purpose: Aberrant expression of miRNAs is crucial in several tissues tumorigenesis including thyroid. Recent studies demonstrated that miR-650 plays different role depending on the cancer type. Herein, we investigated the role of miR-650 in thyroid carcinoma., Methods: The expression of miR-650 was analyzed in human thyroid tissues by q-RT-PCR. Anaplastic (8505C, CAL62, SW1736) and papillary (TPC-1) thyroid cancer cell lines were used to dissect the role of miR-650 on malignant hallmarks of transformation. Label-free proteomic analysis was exploited to unravel the targets of miR-650, while luciferase reporter assay and functional experiments were performed to confirm a selected target. Spearman's rank correlation test was used to assess the association between miR-650 and its target in human thyroid cancer tissues., Results: miR-650 is over-expressed in anaplastic (ATC) thyroid carcinoma where it enhances cell migration and invasion. Proteomic label-free and bioinformatics analysis revealed that the serine-threonine protein phosphatase 2 catalytic subunit alpha (PPP2CA) is a target of miR-650; these finding were confirmed by luciferase assay. Restoration of PPP2CA mRNA, deprived of its 3'UTR, is able to revert the malignant phenotype induced by miR-650 in HEK-293 cells. Importantly, PPP2CA is down-regulated in ATC tissues and is inversely correlated with miR-650., Conclusions: miR-650 displayed oncogenic activity in ATC cells through targeting PPP2CA phosphatase. These results suggest that miR-650/PPP2CA axis could be modulated to interfere with motile ability of thyroid carcinoma cells.

Aging is a physiological process characterized by a progressive decline of biological functions and an increase in destructive processes in cells and organs. Physical activity and exercise positively affects the expression of skeletal muscle markers involved in longevity pathways. Recently, a new mechanism, autophagy, was introduced to the adaptations induced by acute and chronic exercise as responsible of positive metabolic modification and health-longevity promotion. However, the molecular mechanisms regulating autophagy in response to physical activity and exercise are sparsely described. We investigated the long-term adaptations resulting from lifelong recreational football training on the expression of skeletal muscle markers involved in autophagy signaling. We demonstrated that lifelong football training increased the expression of messengers: RAD23A, HSPB6, RAB1B, TRAP1, SIRT2, and HSBPB1, involved in the auto-lysosomal and proteasome-mediated protein degradation machinery; of RPL1, RPL4, RPL36, MRLP37, involved in cellular growth and differentiation processes; of the Bcl-2, HSP70, HSP90, PSMD13, and of the ATG5-ATG12 protein complex, involved in proteasome promotion and autophagy processes in muscle samples from lifelong trained subjects compared to age-matched untrained controls. In conclusion, our results indicated that lifelong football training positively influence exercise-induced autophagy processes and protein quality control in skeletal muscle, thus promoting healthy aging.

Methylmalonic acidemias (MMAs) are inborn errors of metabolism due to the deficient activity of methylmalonyl-CoA mutase (MUT). MUT catalyzes the formation of succinyl-CoA from methylmalonyl-CoA, produced from propionyl-CoA catabolism and derived from odd chain fatty acids β-oxidation, cholesterol, and branched-chain amino acids degradation. Increased methylmalonyl-CoA levels allow for the presymptomatic diagnosis of the disease, even though no approved therapies exist. MMA patients show hyperammonemia, ketoacidosis, lethargy, respiratory distress, cognitive impairment, and hepatomegaly. The long-term consequences concern neurologic damage and terminal kidney failure, with little chance of survival. The cellular pathways affected by MUT deficiency were investigated using a quantitative proteomics approach on a cellular model of MUT knockdown. Currently, a consistent reduction of the MUT protein expression was obtained in the neuroblastoma cell line (SH-SY5Y) by using small-interfering RNA (siRNA) directed against an MUT transcript (MUT siRNA). The MUT absence did not affect the cell viability and apoptotic process in SH-SY5Y. In the present study, we evaluate and quantify the alterations in the protein expression profile as a consequence of MUT-silencing by a mass spectrometry-based label-free quantitative analysis, using two different quantitative strategies. Both quantitative methods allowed us to observe that the expression of the proteins involved in mitochondrial oxido-reductive homeostasis balance was affected by MUT deficiency. The alterated functional mitochondrial activity was observed in siRNA&#95;MUT cells cultured with a propionate-supplemented medium. Finally, alterations in the levels of proteins involved in the metabolic pathways, like carbohydrate metabolism and lipid metabolism, were found.

New competencies may be learned through active experience (experiential learning or learning by doing) or observation of others' experiences (learning by observation). Observing another person performing a complex action facilitates the observer's acquisition of the same action. The present research is aimed at analyzing if the observation of specific explorative strategies adopted in a constrained environment, such as the Radial Arm Maze (RAM), could help young children to explore the maze and to build a cognitive spatial map of the explored environment. To this aim young children were randomly assigned to three groups: children who performed the RAM task following the observation of an actor solving the same maze by putting into action a highly structured exploratory strategy; children who performed the RAM task following the observation of the actor solving the same maze by putting into action a less structured exploratory strategy; children who directly performed the RAM task without any observation. The main result of the present research is that the children who observed the highly structured and correct exploratory strategy spent less time, made fewer errors, exhibited a longer spatial span, and thus they explored the maze more efficiently than the children who directly performed the RAM task without any observation. This finding indicates that when the observed explorative procedure is structured, sequential and repetitive the action understanding and information storage processes are more effective. Importantly, the observation of specific spatial strategies helped the children to build the cognitive spatial map of the explored environment and consequently to acquire/enrich the declarative knowledge of the environment.

Functional beverages represent a palatable and efficient way to hydrate and reintegrate electrolytes, carbohydrates, and other nutrients employed and/or lost during physical training and/or competitions. Bodily hydration during sporting activity is one of the best indicators of health in athletes and can be a limiting factor for sport performance. Indeed, dehydration strongly decreases athletic performance until it is a risk to health. As for other nutrients, each of them is reported to support athletes' needs both during the physical activity and/or in the post-workout. In this study, we review the current knowledge of macronutrient-enriched functional beverages in sport taking into account the athletes' health, sports performance, and recovery.

The insulin-like growth factor (IGF) system, which is constituted by the IGF-1 and IGF-2 peptide hormones, their corresponding receptors and several IGF binding proteins, is involved in physiological and pathophysiological processes. The IGF system promotes cancer proliferation/survival and its signaling induces the epithelial-mesenchymal transition (EMT) phenotype, which contributes to the migration, invasiveness, and metastasis of epithelial tumors. These cancers share two major IGF-1R signaling transduction pathways, PI3K/AKT and RAS/MEK/ERK. However, as far as we could review at this time, each type of cancer cell undergoes EMT through tumor-specific routes. Here, we review the tumor-specific molecular signatures of IGF-1-mediated EMT in breast, lung, and gastric cancers., Competing Interests: The authors declare no conflict of interest.

The position of the fatty acids ( sn -1, sn -2 and sn -3) (stereospecific numbering ( sn )) in triacylglycerol (TAG) molecules produces a characteristic stereospecificity that defines the physical properties of the fats and influences their absorption, metabolism and uptake into tissues. Fat interesterification is a process that implies a positional distribution of fatty acids (FAs) within the TAG molecules, generating new TAG species, without affecting the FA cis - trans natural balance. The interesterified (IE) fats, frequently used in the food industry comprise fats that are rich in long-chain saturated FAs, such as palmitic acid (16:0) and stearic acid (18:0). Within the interesterified fats, a critical role is played by FA occupying the sn -2 position; in fact, the presence of an unsaturated FA in this specific position influences early metabolic processing and postprandial clearance that in turn could induce atherogenesis and thrombogenesis events. Here, we provide an overview on the role of TAG structures and interesterified palmitic and stearic acid-rich fats on fasting and postprandial lipemia, focusing our attention on their physical properties and their effects on human health., Competing Interests: All the authors declare that they have no conflicts of interest.

Voltage-dependent anion channels (VDACs) are a family of three mitochondrial porins and the most abundant integral membrane proteins of the mitochondrial outer membrane (MOM). VDACs are known to be involved in metabolite/ion transport across the MOM and in many cellular processes ranging from mitochondria-mediated apoptosis to the control of energy metabolism, by interacting with cytosolic, mitochondrial and cytoskeletal proteins and other membrane channels. Despite redundancy and compensatory mechanisms among VDAC isoforms, they display not only different channel properties and protein expression levels, but also distinct protein partners. Here, we review the known protein interactions for each VDAC isoform in order to shed light on their peculiar roles in physiological and pathological conditions. As proteins associated with the MOM, VDAC opening/closure as a metabolic checkpoint is regulated by protein-protein interactions, and is of pharmacological interest in pathological conditions such as cancer. The interactions involving VDAC1 have been characterized more in depth than those involving VDAC2 and VDAC3. Nevertheless, the so far explored VDAC-protein interactions for each isoform show that VDAC1 is mainly involved in the maintenance of cellular homeostasis and in pro-apoptotic processes, whereas VDAC2 displays an anti-apoptotic role. Despite there being limited information on VDAC3, this isoform could contribute to mitochondrial protein quality control and act as a marker of oxidative status. In pathological conditions, namely neurodegenerative and cardiovascular diseases, both VDAC1 and VDAC2 establish abnormal interactions aimed to counteract the mitochondrial dysfunction which contributes to end-organ damage.

AL amyloidosis is characterized by widespread deposition of immunoglobulin light chains (LCs) as amyloid fibrils. Cardiac involvement is frequent and leads to life-threatening cardiomyopathy. Besides the tissue alteration caused by fibrils, clinical and experimental evidence indicates that cardiac damage is also caused by proteotoxicity of prefibrillar amyloidogenic species. As in other amyloidoses, the damage mechanisms at cellular level are complex and largely undefined. We have characterized the molecular changes in primary human cardiac fibroblasts (hCFs) exposed in vitro to soluble amyloidogenic cardiotoxic LCs from AL cardiomyopathy patients. To evaluate proteome alterations caused by a representative cardiotropic LC, we combined gel-based with label-free shotgun analysis and performed bioinformatics and data validation studies. To assess the generalizability of our results we explored the effects of multiple LCs on hCF viability and on levels of a subset of cellular proteins. Our results indicate that exposure of hCFs to cardiotropic LCs translates into proteome remodeling, associated with apoptosis activation and oxidative stress. The proteome alterations affect proteins involved in cytoskeletal organization, protein synthesis and quality control, mitochondrial activity and metabolism, signal transduction and molecular trafficking. These results support and expand the concept that soluble amyloidogenic cardiotropic LCs exert toxic effects on cardiac cells.

A functional relationship is suggested between two well-known protein hormones, insulin-like growth factor 1 (IGF-1) and adiponectin. In the last two decades in fact, different experimental evidence has indicated a non-random link between them. Here, we describe briefly the IGF-1 and adiponectin systems, and we then focus on their putative interplay in relation to several pathological conditions, including obesity, diabetes, insulin resistance, cardiovascular disease, and cancer. Although the existing studies are hardly comparable, they definitely indicate a functional connection between these two protein hormones. In conclusion, the current knowledge strongly encourages further research into the common, as well as novel, mechanisms through which IGF-1 and adiponectin exert their concerted action., Competing Interests: The authors declare no conflict of interest.

The aim of this study was to evaluate the effects of 6 different areas per player (AP) on exercise intensity (EI) measured during small-sided games (SSGs) and expressed as percentage of maximal heart rate (%MHR) and technical actions (TAs) involvement with the ball, crosses, headers, tackles, shots on goal, dribbling, passing, and target passing-in U-12 and U-14 soccer players during SSGs. Seventeen male U-12 soccer players (age 10.0 ± 0.5 years, body mass 39.3 ± 5.3 kg, and height 143.8 ± 4.6 cm) and 16 male U-14 soccer players (age 13.2 ± 0.3 years, body mass 46.6 ± 11.9 kg, and height 154.8 ± 8.5 cm) performed SSGs with different AP: 40, 50, 66.7, 90, 112.5, and 150 m. Our results indicate that at larger AP, the U-12 group's mean EI values were significantly higher than those at smaller AP (p ≤ 0.05); in addition, intergroup comparison showed that EI was higher in U-12 than that in U-14 players when AP of 112.5 and 150 m were considered (p ≤ 0.05). Technical action analysis evidenced that moving from smaller to larger AP, U-14 players adapted better to AP changes. In conclusion, these results suggest that AP influences differently EI and TAs in U-12 and U-14 players. Our results could be taken into account by conditioning coaches to better tailor the physiological and technical training in young players through the modulation of AP.

Methylmalonic acidemia (MMA) is a heterogeneous and severe autosomal recessive inborn error of metabolism most commonly caused by the deficient activity of the vitamin B12 dependent enzyme, methylmalonyl-CoA mutase (MUT). The main treatment for MMA patients is the dietary restriction of propiogenic amino acids and carnitine supplementation. Despite treatment, the prognosis for vitamin B12 non-responsive patients remains poor and is associated with neonatal lethality, persistent morbidity and decreased life expectancy. While multi-organ pathology is a feature of MMA, the liver is severely impacted by mitochondrial dysfunction which likely underlies the metabolic instability experienced by the patients. Liver and/or combined liver/kidney transplantation is therefore sometimes performed in severely affected patients. Using liver specimens from donors and MMA patients undergoing elective liver transplantation collected under a dedicated natural history protocol (clinicaltrials.gov: NCT00078078), we employed proteomics to characterize the liver pathology and impaired hepatic metabolism observed in the patients. Pathway analysis revealed perturbations of enzymes involved in energy metabolism, gluconeogenesis and Krebs cycle anaplerosis. Our findings identify new pathophysiologic and therapeutic targets that could be valuable for designing alternative therapies to alleviate clinical manifestations seen in this disorder., Competing Interests: Marianna Caterino declares that she has no conflict of interest. Randy J. Chandler declares that she has no conflict of interest. Jennifer L. Sloan declares that she has no conflict of interest. Kenneth Dorko declares that she has no conflict of interest. Kristina Cusmano-Ozog declares that she has no conflict of interest. Laura Ingenito declares that she has no conflict of interest. Stephen C. Strom declares that she has no conflict of interest. Esther Imperlini declares that she has no conflict of interest. Emanuela Scolamiero declares that he has no conflict of interest. Charles P. Venditti declares that he has no conflict of interest. Margherita Ruoppolo declares that she has no conflict of interest.

Organic acidemias (OAs) are inherited metabolic disorders caused by deficiency of enzymatic activities in the catabolism of amino acids, carbohydrates, or lipids. These disorders result in the accumulation of mono-, di-, or tricarboxylic acids, generally referred to as organic acids. The OA outcomes can involve different organs and/or systems. Some OA disorders are easily managed if promptly diagnosed and treated, whereas, in others cases, such as propionate metabolism-related OAs (propionic acidemia, PA; methylmalonic acidemia, MMA), neither diet, vitamin therapy, nor liver transplantation appears to prevent multiorgan impairment. Here, we review the recent developments in dissecting molecular bases of OAs by using integration of mass spectrometry- (MS-) based metabolomic and proteomic strategies. MS-based techniques have facilitated the rapid and economical evaluation of a broad spectrum of metabolites in various body fluids, also collected in small samples, like dried blood spots. This approach has enabled the timely diagnosis of OAs, thereby facilitating early therapeutic intervention. Besides providing an overview of MS-based approaches most frequently used to study the molecular mechanisms underlying OA pathophysiology, we discuss the principal challenges of metabolomic and proteomic applications to OAs.

In immunoglobulin (Ig) light-chain (LC) (AL) amyloidosis, AL deposition translates into life-threatening cardiomyopathy. Clinical and experimental evidence indicates that soluble cardiotoxic LCs are themselves harmful for cells, by which they are internalized. Hypothesizing that interaction of soluble cardiotoxic LCs with cellular proteins contributes to damage, we characterized their interactome in cardiac cells. LCs were purified from patients with AL amyloidosis cardiomyopathy or multiple myeloma without amyloidosis (the nonamyloidogenic/noncardiotoxic LCs served as controls) and employed at concentrations in the range observed in AL patients' sera. A functional proteomic approach, based on direct and inverse coimmunoprecipitation and mass spectrometry, allowed identifying LC-protein complexes. Findings were validated by colocalization, fluorescence lifetime imaging microscopy (FLIM)-fluorescence resonance energy transfer (FRET), and ultrastructural studies, using human primary cardiac fibroblasts (hCFs) and stem cell-derived cardiomyocytes. Amyloidogenic cardiotoxic LCs interact in vitro with specific intracellular proteins involved in viability and metabolism. Imaging confirmed that, especially in hCFs, cardiotoxic LCs (not controls) colocalize with mitochondria and spatially associate with selected interactors: mitochondrial optic atrophy 1-like protein and peroxisomal acyl-coenzyme A oxidase 1 (FLIM-FRET efficiencies 11 and 6%, respectively). Cardiotoxic LC-treated hCFs display mitochondrial ultrastructural changes, supporting mitochondrial involvement. We show that cardiotoxic LCs establish nonphysiologic protein-protein contacts in human cardiac cells, offering new clues on the pathogenesis of AL cardiomyopathy., (© FASEB.)

Methylmalonic acidemia with homocystinuria, cobalamin deficiency type C (cblC) (MMACHC) is the most common inborn error of cobalamin metabolism. Despite a multidrug treatment, the long-term follow-up of early-onset patients is often unsatisfactory, with progression of neurological and ocular impairment. Here, the in-vivo proteome of control and MMACHC lymphocytes (obtained from patients under standard treatment with OHCbl, betaine, folate and L-carnitine) was quantitatively examined by two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty three proteins were found up-regulated and 38 proteins were down-regulated. Consistent with in vivo studies showing disturbance of glutathione metabolism, a deregulation in proteins involved in cellular detoxification, especially in glutathione metabolism was found. In addition, relevant changes were observed in the expression levels of proteins involved in intracellular trafficking and protein folding, energy metabolism, cytoskeleton organization and assembly. This study demonstrates relevant changes in the proteome profile of circulating lymphocytes isolated from treated cblC patients. Some results confirm previous observations in vivo on fibroblast, thus concluding that some dysregulation is ubiquitous. On the other hand, new findings could be tissue-specific. These observations expand our current understanding of the cblC disease and may ignite new research and therapeutic strategies to treat this disorder.

Performance-enhancing drugs (PEDs) gained wide popularity not only among sportsmen but also among specific subsets of population, such as adolescents. Apart from their claimed effects on athletic performance, they are very appealing due to the body shaping effect exerted on fat mass and fat-free mass. Besides the "underestimated" massive misuse of PEDs, the short- as well as long-term consequences of such habits remain largely unrecognized. They have been strictly associated with serious adverse effects, but molecular mechanisms are yet to be elucidated. Here, we analyze the current understanding of the molecular effects of supraphysiological doses of doping agents in healthy biological systems, at genomic and proteomic levels, in order to define the molecular sensors of organ/tissue impairment, determined by their misuse. The focus is put on the anabolic androgenic steroids (AASs), specifically testosterone (T) and its most potent derivative dihydrotestosterone (DHT), and on the peptide hormones, specifically the growth hormone (GH) and the insulin-like growth factor-1 (IGF-1). A map of molecular targets is defined and the risk incidence for human health is taken into account.

The abuse of mixed or combined performance-enhancing drugs is widespread among athletes and amateurs, adults and adolescents. Clinical studies demonstrated that misuse of these doping agents is associated with serious adverse effects to many organs in human. Previously, we demonstrated in human peripheral blood lymphocytes that high doses of anabolic androgenic steroids, such as dihydrotestosterone (DHT) and growth factors, such as insulin-like growth factor-1 (IGF-1), have effects at gene and protein levels. Supraphysiological treatments of DHT and IGF-1 affected the expression of genes involved in skeletal muscle disorders as well as in cell-mediated immunological response. At protein level, DHT hyperdosage affects cell motility and apoptosis; IGF-1 hyperstimulation triggers an active cytoskeletal reorganization and an overproduction of immune response- and inflammation-related cytokines. In this study, we investigate the combined effects of DHT and IGF-1 hyperdosage in peripheral blood lymphocytes using a differential proteomic approach. DHT and IGF-1 combined treatment affects cell adhesion, migration, and survival through modulation of expression levels of cytokines and paxillin-signaling-related proteins, and activation of several pathways downstream focal adhesion kinase. Our results indicate a synergistic effect of DHT and IGF-1 which has potential implications for health risk factors., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Diamond-Blackfan anemia, characterized by defective erythroid progenitor maturation, is caused in one-fourth of cases by mutations of ribosomal protein S19 (RPS19), which is a component of the ribosomal 40S subunit. Our previous work described proteins interacting with RPS19 with the aim to determine its functions. Here, two RPS19 mutants, R62W and R101H, have been selected to compare their interactomes versus the wild-type protein one, using the same functional proteomic approach that we employed to characterize RPS19 interactome. Mutations R62W and R101H impair RPS19 ability to associate with the ribosome. Results presented in this paper highlight the striking differences between the interactomes of wild-type and mutant RPS19 proteins. In particular, mutations abolish interactions with proteins having splicing, translational and helicase activity, thus confirming the role of RPS19 in RNA processing/metabolism and translational control. The data have been deposited to the ProteomeXchange with identifier PXD000640 (http://proteomecentral.proteomexchange.org/dataset/PXD000640)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Insulin-like growth factor-1 (IGF-1) mediates some of growth hormone anabolic functions through its receptor, IGF-1R. Following ligand binding, intracellular signaling pathways are activated favouring proliferation, cell survival, tissue growth, development, and differentiation. IGF-1 is included in the World Anti-Doping Agency Prohibited List. While the evidence for IGF-1 as performance-enhancing substrate in healthy humans is still weak, clinical studies demonstrated that the endogenous growth hormone/IGF-1 excess is associated with cardiovascular implications. Previously, we demonstrated that human peripheral blood lymphocytes represent a suitable system to identify a gene signature, related to dihydrotestosterone or IGF-1 abuse, independent from the type of sport. In addition, in a proteomic study, we demonstrated that dihydrotestosterone hyperdosage affects cell motility and apoptosis. Here, we investigate the doping action of IGF-1 by means of a differential proteomic approach and specific protein arrays, revealing an active cytoskeletal reorganization mediated by Stat-1; moreover, IGF-1 stimulation produces a sustained activation of different signaling pathways as well as an overproduction of cytokines positively related to immune response and inflammation. In conclusion, these data indicate that, following IGF-1 hyperdosage, circulating peripheral blood lymphocytes could be more prone to transendothelial migration., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phenylketonuria (PKU), if not detected and treated in newborns, causes severe neurological dysfunction and cognitive and behavioral deficiencies. Despite the biochemical characterization of PKU, the molecular mechanisms underlying PKU-associated brain dysfunction remain poorly understood. The aim of this study was to gain insights into the pathogenesis of this neurological damage by analyzing protein expression profiles in brain tissue of Black and Tan BRachyury-PahEnu2 mice (a mouse model of PKU). We compared the cerebral protein expression of homozygous PKU mice with that of their heterozygous counterparts using two-dimensional difference gel electrophoresis analysis, and identified 21 differentially expressed proteins, four of which were over-expressed and 17 under-expressed. An in silico bioinformatic approach indicated that protein under-expression was related to neuronal differentiation and dendritic growth, and to such neurological disorders as progressive motor neuropathy and movement disorders. Moreover, functional annotation analyses showed that some identified proteins were involved in oxidative metabolism. To further investigate the proteins involved in the neurological damage, we validated two of the proteins that were most strikingly under-expressed, namely, Syn2 and Dpysl2, which are involved in synaptic function and neurotransmission. We found that Glu2/3 and NR1 receptor subunits were over-expressed in PKU mouse brain. Our results indicate that differential expression of these proteins may be associated with the processes underlying PKU brain dysfunction, namely, decreased synaptic plasticity and impaired neurotransmission. We identified a set of proteins whose expression is affected by hyperphenylalaninemia. We think that phenylketonuria (PKU) brain dysfunction also depends on reduced Syn2 and Dpysl2 levels, increased Glu2/3 and NR1 levels, and decreased Pkm, Ckb, Pgam1 and Eno1 levels. These findings finally confirm that alteration in synaptic function, in transmission and in energy metabolism underlie brain damage provoked by hyperphenylalaninemias., (© 2014 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

The definition of the secretome signature of a cancer cell line can be considered a potential tool to investigate tumor aggressiveness and a preclinical exploratory study required to optimize the search of cancer biomarkers. Dealing with a cell-specific secretome limits the contamination by the major components of the human serum and reduces the range of dynamic concentrations among the secreted proteins, thus favouring under-represented tissue-specific species. The aim of the present study is to characterize the secretome of two human colon carcinoma cell lines, CaCo-2 and HCT-GEO, in order to evaluate differences and similarities of two colorectal cancer model systems. In this study, we identified more than 170 protein species, 64 more expressed in the secretome of CaCo-2 cells and 54 more expressed in the secretome of HCT-GEO cells; 58 proteins were shared by the two systems. Among them, more than 50% were deemed to be secretory according to their Gene Ontology annotation and/or to their SignalP or SecretomeP scores. Such a characterization allowed corroborating the potential of a cell culture-based model in order to describe the cell-specific invasive properties and to provide a list of putative cancer biomarkers., (© 2013 Wiley Periodicals, Inc.)

We performed a pilot study using human peripheral blood lymphocytes (PBL) as a novel system to identify new biomarkers of dihydrotestosterone (DHT) and insulin-like growth factor-1 (IGF-1) abuse in sport. First, to obtain a gene signature, we treated cultures of lymphocytes from sedentary males with three doses of 0.237 microg/ml DHT, each of which is 80-fold the physiological concentration in young adult male serum, at days 0, 2 and 4, or with a single dose of 1.25 microg/ml IGF-1, which is 5-fold the physiological concentration in young adult male serum. We then used the Human Genome U133 Plus 2.0 microarray to identify a gene signature related to DHT or IGF-1 administration. Gene expression was evaluated after 7 and 21 days of DHT treatment, and after 24 h, 72 h and 7 days of IGF-1 treatment. Microarray analysis yielded a list of genes whose expression was altered after DHT or IGF-1 treatment. Among these we selected the genes that are most representative of the pathways associated with skeletal and muscular disorders using the IPA bioinformatics tool. We identified six (IDO1, CXCL13, CCL1, GZMB, VDR and IL2RA) and two (FN1 and RAB31) genes that were up-regulated in lymphocytes from sedentary subjects after 7 days of DHT and IGF-1 treatment, respectively. The expression of these genes in lymphocytes from differently trained athletes was either down-regulated or similar to that in lymphocytes from sedentary subjects. This finding suggests that up-regulation was due to the drug and not to physical exercise. In conclusion, we demonstrate that PBL can be useful in anti-doping checks, and we describe new biomarkers of DHT and IGF-1 abuse which can be included in the Athlete's Biological Passport.

The biochemical phenotype of cells affected by ribosomal stress has not yet been studied in detail. Here we report a comparative proteomic analysis of cell lines silenced for the RPS19 gene versus cell lines transfected with scramble shRNA cells performed using the DIGE technology integrated to bioinformatics tools. Importantly, to achieve the broadest possible understanding of the outcome, we carried out two independent DIGE experiments using two different pH ranges, thus, allowing the identification of 106 proteins. Our data revealed the deregulation of proteins involved in cytoskeleton reorganization, PTMs, and translation process. A subset (26.9%) of these proteins is translated from transcripts that include internal ribosome entry site motifs. This supports the hypothesis that during ribosomal stress translation of specific messenger RNAs is altered., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cancer stem cell (CSC) theory represents a breakthrough in cancer research. We characterized the protein pattern of CSCs to identify specific intracellular pathways in this subpopulation of tumor cells. We studied colon CSCs using two different colon cancer cell lines: CaCo-2 and HCT-116. Putative CSCs were separated from non-CSCs by flow cytometry using CD133 as stemness marker. Total protein extracts of CD133+ cells were then compared to protein extracts of CD133- cells by 2D DIGE. The protein spots differentially expressed in the two subpopulations of cells were analyzed by mass spectrometry. Bioinformatics analysis of the identified proteins indicated alteration of two main processes: energy metabolism and the Wnt pathway. Interestingly, we observed upregulation of the splicing factor SRp20, a newly identified target gene of the Wnt/β-catenin pathway, and we demonstrated a direct cause-effect relationship between Wnt pathway activation and the increased SRp20 expression. Our results also show that SRp20 influences cell proliferation, which suggests it plays a role in the tumorigenicity of CD133+ cells. In conclusion, activation of the Wnt pathway in CD133+ cells and upregulation of SRp20, which is implicated in tumorigenesis, raises the possibility of a sequential series of molecular events occurring in connection with this process., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Anabolic androgenic steroids, a class of steroid hormones related to testosterone, are natural ligands of androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. AR binds specific DNA elements, known as androgen-response elements. Testosterone, the main male sexual hormone, binds AR directly and indirectly, through conversion into dihydrotestosterone (DHT), its more active metabolite. Anabolic androgenic steroids are frequently detected in the urine of doped athletes; their consumption is also growing among sport amateurs and adolescents. The effects of androgens can differ depending on the target cells and/or tissues. To gain insight into transcription activation mechanisms of AR, we investigated AR protein signaling in human peripheral blood lymphocytes treated with supraphysiological doses of DHT. We performed a comparative proteomic analysis and we identified about 30 differentially expressed proteins. At least five species contained a consensus androgen-response elements sequence in the promoter region of related coding genes. The analysis also revealed that high doses of DHT activate the drug detoxification process, could stimulate an increase in cell motility and exert a prosurvival effect rather than an apoptotic one.

We introduced into Rhizobium leguminosarum bv. viciae LPR1105 a new pathway for the biosynthesis of the auxin, indole-3-acetic acid (IAA), under the control of a stationary phase-activated promoter active both in free-living bacteria and bacteroids. The newly introduced genes are the iaaM gene from Pseudomonas savastanoi and the tms2 gene from Agrobacterium tumefaciens. Free-living bacteria harbouring the promoter-iaaMtms2 construct release into the growth medium 14-fold more IAA than the wild-type parental strain. This IAA overproducing R. l. viciae, the RD20 strain, elicits the development of vetch root nodules containing up to 60-fold more IAA than nodules infected by the wild-type strain LPR1105. Vetch root nodules derived from RD20 are fewer in number per plant, heavier in terms of dry weight and show an enlarged and more active meristem. A significant increase in acetylene reduction activity was measured in nodules elicited in vetch by RD20.