RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition.

BACKGROUND: The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. METHODS: After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. RESULTS: Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. CONCLUSIONS: Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.

Cockayne syndrome group A and ferrochelatase finely tune ribosomal gene transcription and its response to UV irradiation.

CSA and CSB proteins are key players in transcription-coupled nucleotide excision repair (TC-NER) pathway that removes UV-induced DNA lesions from the transcribed strands of expressed genes. Additionally, CS proteins play relevant but still elusive roles in other cellular pathways whose alteration may explain neurodegeneration and progeroid features in Cockayne syndrome (CS). Here we identify a CS-containing chromatin-associated protein complex that modulates rRNA transcription. Besides RNA polymerase I (RNAP1) and specific ribosomal proteins (RPs), the complex includes ferrochelatase (FECH), a well-known mitochondrial enzyme whose deficiency causes erythropoietic protoporphyria (EPP). Impairment of either CSA or FECH functionality leads to reduced RNAP1 occupancy on rDNA promoter that is associated to reduced 47S pre-rRNA transcription. In addition, reduced FECH expression leads to an abnormal accumulation of 18S rRNA that in primary dermal fibroblasts from CS and EPP patients results in opposed rRNA amounts. After cell irradiation with UV light, CSA triggers the dissociation of the CSA-FECH-CSB-RNAP1-RPs complex from the chromatin while it stabilizes its binding to FECH. Besides disclosing a function for FECH within nucleoli, this study sheds light on the still unknown mechanisms through which CSA modulates rRNA transcription.

Proteomics Reveals How the Tardigrade Damage Suppressor Protein Teaches Transfected Human Cells to Survive UV-C Stress.

The genome sequencing of the tardigrade Ramazzottius varieornatus revealed a unique nucleosome-binding protein named damage suppressor (Dsup), which was discovered to be crucial for the extraordinary abilities of tardigrades in surviving extreme stresses, such as UV. Evidence in Dsup-transfected human cells suggests that Dsup mediates an overall response in DNA damage signaling, DNA repair, and cell cycle regulation, resulting in an acquired resistance to stress. Given these promising outcomes, our study attempts to provide a wider comprehension of the molecular mechanisms modulated by Dsup in human cells and to explore the Dsup-activated molecular pathways under stress. We performed a differential proteomic analysis of Dsup-transfected and control human cells under basal conditions and at 24 h recovery after exposure to UV-C. We demonstrate via enrichment and network analyses, for the first time, that even in the absence of external stimuli, and more significantly, after stress, Dsup activates mechanisms involved with the unfolded protein response, the mRNA processing and stability, cytoplasmic stress granules, the DNA damage response, and the telomere maintenance. In conclusion, our results shed new light on Dsup-mediated protective mechanisms and increases our knowledge of the molecular machineries of extraordinary protection against UV-C stress.

BAL Proteomic Signature of Lung Adenocarcinoma in IPF Patients and Its Transposition in Serum Samples for Less Invasive Diagnostic Procedures.

Idiopathic pulmonary fibrosis (IPF) is a form of chronic and irreversible fibrosing interstitial pneumonia of unknown etiology. Although antifibrotic treatments have shown a reduction of lung function decline and a slow disease progression, IPF is characterize by a very high mortality. Emerging evidence suggests that IPF increases the risk of lung carcinogenesis. Both diseases show similarities in terms of risk factors, such as history of smoking, concomitant emphysema, and viral infections, besides sharing similar pathogenic pathways. Lung cancer (LC) diagnosis is often difficult in IPF patients because of the diffuse lung injuries and abnormalities due to the underlying fibrosis. This is reflected in the lack of optimal therapeutic strategies for patients with both diseases. For this purpose, we performed a proteomic study on bronchoalveolar lavage fluid (BALF) samples from IPF, LC associated with IPF (LC-IPF) patients, and healthy controls (CTRL). Molecular pathways involved in inflammation, immune response, lipid metabolism, and cell adhesion were found for the dysregulated proteins in LC-IPF, such as TTHY, APOA1, S10A9, RET4, GDIR1, and PROF1. The correlation test revealed a relationship between inflammation- and lipid metabolism-related proteins. PROF1 and S10A9, related to inflammation, were up-regulated in LC-IPF BAL and serum, while APOA1 and APOE linked to lipid metabolism, were highly abundant in IPF BAL and low abundant in IPF serum. Given the properties of cytokine/adipokine of the nicotinamide phosphoribosyltransferase, we also evaluated its serum abundance, highlighting its down-regulation in LC-IPF. Our retrospective analyses of BAL samples extrapolated some potential biomarkers of LC-IPF useful to improve the management of these contemporary pathologies. Their differential abundance in serum samples permits the measurement of these potential biomarkers with a less invasive procedure.

Antithrombin III as predictive indicator of survival in idiopathic pulmonary fibrosis (IPF) patients treated with nintedanib: a preliminary study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease often managed with nintedanib, a tyrosine kinase inhibitor targeting several profibrotic pathways. Although clotting processes are involved in wound healing and repair in the lung, there are no data on the role of antithrombin III (ATIII) in IPF patients treated with nintedanib. A previous proteomic analysis of serum of IPF patients before and after 1 year of nintedanib treatment showed differential protein expression of ATIII. AIMS: Here we used quantitative methods to evaluate differential ATIII concentrations in IPF patients before and after 1 year of nintedanib treatment and to assess the potential of ATIII as a prognostic biomarker in IPF patients. METHODS: Serum levels of ATIII were measured by enzyme-linked immunosorbent assay in 14 IPF patients before and after 1 year of nintedanib treatment. RESULTS: A statistically significant inverse correlation was found between serum ATIII concentrations and pulmonary function test parameters in all patients at baseline and follow up. Baseline serum ATIII and bronchoalveolar lavage (BAL) neutrophils proved to be reliable predictors of poor prognosis. A baseline ATIII threshold of 126.5 µg/mL discriminated survivors from non-survivors. CONCLUSIONS: After 12 months of antifibrotic treatment, IPF patients with high serum ATIII concentrations and high BAL neutrophil percentages had a poor prognosis and increased survival risk. The results of this preliminary study suggest that ATIII has potential as a biomarker of IPF severity and in predicting response to nintedanib therapy. As a marker, ATIII showed several advantages over BAL neutrophil percentage.

Depletion of the RNA binding protein HNRNPD impairs homologous recombination by inhibiting DNA-end resection and inducing R-loop accumulation.

DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD binds chromatin, although this binding occurred independently of DNA damage. However, upon damage, HNRNPD re-localized to γH2Ax foci and its silencing impaired CHK1 S345 phosphorylation and the DNA end resection process. Indeed, HNRNPD silencing reduced: the ssDNA fraction upon camptothecin treatment; AsiSI-induced DSB resection; and RPA32 S4/8 phosphorylation. CRISPR/Cas9-mediated HNRNPD knockout impaired in vitro DNA resection and sensitized cells to camptothecin and olaparib treatment. We found that HNRNPD interacts with the heterogeneous nuclear ribonucleoprotein SAF-A previously associated with DNA damage repair. HNRNPD depletion resulted in an increased amount of RNA:DNA hybrids upon DNA damage. Both the expression of RNase H1 and RNA pol II inhibition recovered the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage, suggesting that RNA:DNA hybrid resolution likely rescues the defective DNA damage response of HNRNPD-depleted cells.

Differential Proteomic Analysis of Astrocytes and Astrocytes-Derived Extracellular Vesicles from Control and Rai Knockout Mice: Insights into the Mechanisms of Neuroprotection.

Reactive astrocytes are a hallmark of neurodegenerative disease including multiple sclerosis. It is widely accepted that astrocytes may adopt alternative phenotypes depending on a combination of environmental cues and intrinsic features in a highly plastic and heterogeneous manner. However, we still lack a full understanding of signals and associated signaling pathways driving astrocyte reaction and of the mechanisms by which they drive disease. We have previously shown in the experimental autoimmune encephalomyelitis mouse model that deficiency of the molecular adaptor Rai reduces disease severity and demyelination. Moreover, using primary mouse astrocytes, we showed that Rai contributes to the generation of a pro-inflammatory central nervous system (CNS) microenvironment through the production of nitric oxide and IL-6 and by impairing CD39 activity in response to soluble factors released by encephalitogenic T cells. Here, we investigated the impact of Rai expression on astrocyte function both under basal conditions and in response to IL-17 treatment using a proteomic approach. We found that astrocytes and astrocyte-derived extracellular vesicles contain a set of proteins, to which Rai contributes, that are involved in the regulation of oligodendrocyte differentiation and myelination, nitrogen metabolism, and oxidative stress. The HIF-1α pathway and cellular energetic metabolism were the most statistically relevant molecular pathways and were related to ENOA and HSP70 dysregulation.

Proteome Characterization of BALF Extracellular Vesicles in Idiopathic Pulmonary Fibrosis: Unveiling Undercover Molecular Pathways.

In the longtime challenge of identifying specific, easily detectable and reliable biomarkers of IPF, BALF proteomics is providing interesting new insights into its pathogenesis. To the best of our knowledge, the present study is the first shotgun proteomic investigation of EVs isolated from BALF of IPF patients. Our main aim was to characterize the proteome of the vesicular component of BALF and to explore its individual impact on the pathogenesis of IPF. To this purpose, ultracentrifugation was chosen as the EVs isolation technique, and their purification was assessed by TEM, 2DE and LC-MS/MS. Our 2DE data and scatter plots showed considerable differences between the proteome of EVs and that of whole BALF and of its fluid component. Analysis of protein content and protein functions evidenced that EV proteins are predominantly involved in cytoskeleton remodeling, adenosine signaling, adrenergic signaling, C-peptide signaling and lipid metabolism. Our findings may suggest a wider system involvement in the disease pathogenesis and support the importance of pre-fractioning of complex samples, such as BALF, in order to let low-abundant proteins-mediated pathways emerge.

Proteostasis network alteration in lysosomal storage disorders: Insights from the mouse model of Krabbe disease.

In Krabbe disease, a mutation in GALC gene causes widespread demyelination determining cell death by apoptosis, mainly in oligodendrocytes and Schwann cells. Less is known on the molecular mechanisms induced by this deficiency. Here, we report an impairment in protein synthesis and degradation and in proteasomal clearance with a potential accumulation of the misfolded proteins and induction of the endoplasmic reticulum stress in the brain of 6-day-old twitcher mice (TM) (model of Krabbe disease). In particular, an imbalance of the immunoproteasome function was highlighted, useful for shaping adaptive immune response by neurological cells. Moreover, our data show an involvement of cytoskeleton remodeling in Krabbe pathogenesis, with a lamin meshwork disaggregation in twitcher oligodendrocytes in 6-day-old TM. This study provides interesting protein targets and mechanistic insight on the early onset of Krabbe disease that may be promising options to be tested in combination with currently available therapies to rescue Krabbe phenotype.

Cognitive impairment and CSF proteome modification after oral bacteriotherapy in HIV patients.

OBJECTIVE: To investigate whether a probiotic supplementation to cART patients modifies the cerebrospinal fluid (CSF) proteome and improves neurocognitive impairment. METHODS: 26 CSF samples from 13 HIV-positive patients [six patients living with HIV (PLHIV) and seven patients with a history of AIDS (PHAIDS)] were analyzed. All patients underwent to neurocognitive evaluation and blood sampling at baseline and after 6 months of oral bacteriotherapy. Immune phenotyping and activation markers (CD38 and HLA-DR) were evaluated on peripheral blood mononuclear cells (PBMC). Plasma levels of IL-6, sCD14, and MIP-1ß were detected, by enzyme-linked immunosorbent assay (ELISA). Functional proteomic analysis of CSF sample was conducted by two-dimensional electrophoresis; a multivariate analysis was performed by principal component analysis (PCA) and data were enriched by STRING software. RESULTS: Oral bacteriotherapy leads to an improvement on several cognitive test and neurocognitive performance in both groups of HIV-positive subjects. A reduction in the percentage of CD4+CD38+HLA-DR+ T cells was also observed at peripheral level after the probiotic intake (p = 0.008). In addition, the probiotic supplementation to cART significantly modifies protein species composition and abundance at the CSF level, especially those related to inflammation (ß2-microglobulin p = 0.03; haptoglobin p = 0.06; albumin p = 0.003; hemoglobin p = 0.003; immunoglobulin heavy chains constant region p = 0.02, transthyretin p = 0.02) in PLHIV and PHAIDS. CONCLUSIONS: Our results suggest that oral bacteriotherapy as a supplement to cART could exert a role in the amelioration of inflammation state at peripheral and CNS level.

Mepolizumab and Benralizumab in Severe Eosinophilic Asthma: Preliminary Results of a Proteomic Study.

Benralizumab and mepolizumab are new therapies for severe eosinophilic asthma. They are both humanized IgG antibodies, targeting the IL-5 receptor and IL-5, respectively, suppressing the corresponding pathways. No specific biomarkers have been proposed to evaluate treatment response to benralizumab or mepolizumab. The aim of this proteomic study was to compare serum protein profiles of patients with severe eosinophilic asthma before and after anti-IL5 or anti-IL5R therapies. Proteomic analysis highlighted 22 differently abundant spots. Among the proteins identified, CAYP1, A1AT and A2M expression was significantly modified in both groups of patients after therapies while ceruloplasmin showed a significant modification in the group of benralizumab treatment. These differentially expressed proteins could be potential biomarkers of response to mepolizumab and benralizumab treatments and need further evaluation.

Metabolic Dysregulation in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative disorder limited to the lung. New findings, starting from our proteomics studies on IPF, suggest that systemic involvement with altered molecular mechanisms and metabolic disorder is an underlying cause of fibrosis. The role of metabolic dysregulation in the pathogenesis of IPF has not been extensively studied, despite a recent surge of interest. In particular, our studies on bronchoalveolar lavage fluid have shown that the renin-angiotensin-aldosterone system (RAAS), the hypoxia/oxidative stress response, and changes in iron and lipid metabolism are involved in onset of IPF. These processes appear to interact in an intricate manner and to be related to different fibrosing pathologies not directly linked to the lung environment. The disordered metabolism of carbohydrates, lipids, proteins and hormones has been documented in lung, liver, and kidney fibrosis. Correcting these metabolic alterations may offer a new strategy for treating fibrosis. This paper focuses on the role of metabolic dysregulation in the pathogenesis of IPF and is a continuation of our previous studies, investigating metabolic dysregulation as a new target for fibrosis therapy.

Proteomic characterization of idiopathic pulmonary fibrosis patients: stable versus acute exacerbation.

Acute exacerbations (AEs) are among the main causes of death in idiopathic pulmonary fibrosis (IPF) patients. In this study proteomic comparative analysis of bronchoalveolar lavage (BAL) fluid samples was performed in stable IPF patients versus AEs IPF group to identify AE pathogenetic mechanisms and novel potential predictive biomarkers. A functional proteomic analysis of BAL fluid samples from stable and AE-IPF patients was conducted in a population of 27 IPF patients. Fifty-one differentially abundant spots were observed and identified by mass spectrometry. Enrichment analysis found proteins of interest involved in the regulation of macrophages and lipid metabolism receptors. In acute exacerbation IPF group, differentially abundant proteins were involved in propagation of the ß-catenin WNT transduction signal, and proteins up-regulated in lung carcinogenesis (IGKC, S100A9, PEDF, IGHG1, ALDOA, A1AT, HPT, CO3 and PIGR) and acute phase proteins involved in protease-antiprotease imbalance (such as A1AT fragments). Dot-blot analysis of A1AT C-36 peptide allowed validating our findings, confirming up-regulation in AE IPF patients and suggesting its potential pathogenetic role. A crucial role of protease/antiprotease imbalance, clathrin-mediated endocytosis signalling and carcinogenesis emerged in IPF patients developing acute exacerbations.

Bronchoalveolar lavage proteomic analysis in pulmonary fibrosis associated with systemic sclerosis: S100A6 and 14-3-3ε as potential biomarkers.

Objective: SSc is a rare severe connective tissue disorder. Its prognosis is mainly related to the development of pulmonary fibrosis (PF)-SSc and pulmonary arterial hypertension. No known therapy for PF-SSc modifies progressive lung fibrotic involvement. Research is therefore aimed at a deeper understanding of complex pathogenetic mechanisms and the possibility of new prognostic biomarkers and therapeutic targets. Methods: Towards the first of these aims, we conducted functional proteomic analysis of bronchoalveolar lavage samples from PF-SSc patients and smoker and non-smoker controls. Results: The differential expression pattern revealed by principal component analysis highlighted a specific protein profile of PF-SSc with respect to control samples, and enrichment analysis shed light on process networks involved in pathogenesis. The proteins identified are known to be involved in lung inflammation of PF-SSc-induced IL6 signalling, the complement system, innate immunity, Jak-STAT, the kallikrein-kinin system, blood coagulation, the immune response mediated by phagocytosis and phagosomes in antigen presentation. In particular, our MetaCore network suggested C3a, APOAI, 14-3-3ε, SPFA2 and S100A6 as potential biomarkers; these are upstream molecules involved in lung fibrosis, innate immunity and vascular damage occurring in PF-SSc. Conclusion: This report provides a molecular overview of pathological processes in PF-SSc, pinpointing possible new disease biomarkers and therapeutic targets.

Sarcoidosis: proteomics and new perspectives for improving personalized medicine.

INTRODUCTION: Through synergistic approaches integrating biomedical data from omics sciences to the clinical practice, precision medicine aims at more accurate identification of risk factors, characterization of endotypes, patient stratification, establishment of individualized therapy, and prediction of outcomes. Areas covered: This review evaluates the potential role of different omics approaches for the development and application of precision medicine to sarcoidosis patients. This systemic and heterogeneous inflammatory disease is of unknown etiology, affects people of any age, and requires genotypic and phenotypic characterization. The latter can be achieved through the integration of genomic (i.e. information about genes and their mutations potentially involved in sarcoidosis), transcriptomic (reflecting the dynamic state of a cell and measuring the transcribed genes over time), and proteomic data (i.e. proteins in bronchoalveolar lavage, lung tissues, lung cells, serum and immunity system). Expert commentary: Genomic studies have revealed numerous aspects of sarcoidosis; however, for precision medicine, it is necessary to implement genomics with other omic approaches. The improving reliability of omics data, their storage, and their bioinformatics processing represents the next step to recapitulate in silico biological systems, with the final aim to simulate potential molecular pathways involved in the pathology and useful for clinical purposes.

Altered cytoskeletal organization characterized lethal but not surviving Brtl+/- mice: insight on phenotypic variability in osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable bone disease with dominant and recessive transmission. It is characterized by a wide spectrum of clinical outcomes ranging from very mild to lethal in the perinatal period. The intra- and inter-familiar OI phenotypic variability in the presence of an identical molecular defect is still puzzling to the research field. We used the OI murine model Brtl(+/-) to investigate the molecular basis of OI phenotypic variability. Brtl(+/-) resembles classical dominant OI and shows either a moderately severe or a lethal outcome associated with the same Gly349Cys substitution in the α1 chain of type I collagen. A systems biology approach was used. We took advantage of proteomic pathway analysis to functionally link proteins differentially expressed in bone and skin of Brtl(+/-) mice with different outcomes to define possible phenotype modulators. The skin/bone and bone/skin hybrid networks highlighted three focal proteins: vimentin, stathmin and cofilin-1, belonging to or involved in cytoskeletal organization. Abnormal cytoskeleton was indeed demonstrated by immunohistochemistry to occur only in tissues from Brtl(+/-) lethal mice. The aberrant cytoskeleton affected osteoblast proliferation, collagen deposition, integrin and TGF-ß signaling with impairment of bone structural properties. Finally, aberrant cytoskeletal assembly was detected in fibroblasts obtained from lethal, but not from non-lethal, OI patients carrying an identical glycine substitution. Our data demonstrated that compromised cytoskeletal assembly impaired both cell signaling and cellular trafficking in mutant lethal mice, altering bone properties. These results point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment.

Proteomics analysis of a long-term survival strain of Escherichia coli K-12 exhibiting a growth advantage in stationary-phase (GASP) phenotype.

The aim of this work was the functional and proteomic analysis of a mutant, W3110 Bgl(+) /10, isolated from a batch culture of an Escherichia coli K-12 strain maintained at room temperature without addition of nutrients for 10 years. When the mutant was evaluated in competition experiments in co-culture with the wild-type, it exhibited the growth advantage in stationary phase (GASP) phenotype. Proteomes of the GASP mutant and its parental strain were compared by using a 2DE coupled with MS approach. Several differentially expressed proteins were detected and many of them were successful identified by mass spectrometry. Identified expression-changing proteins were grouped into three functional categories: metabolism, protein synthesis, chaperone and stress responsive proteins. Among them, the prevalence was ascribable to the "metabolism" group (72%) for the GASP mutant, and to "chaperones and stress responsive proteins" group for the parental strain (48%).

Protein pathways working in human follicular fluid: the future for tailored IVF?

The human follicular fluid (HFF) contains molecules and proteins that may affect follicle growth, oocyte maturation and competence acquiring. Despite the numerous studies, an integrated broad overview on biomolecular and patho/physiological processes that are proved or supposed to take place in HFF during folliculogenesis and oocyte development is still missing. In this review we report, for the first time, all the proteins unambiguously detected in HFF and, applying DAVID (Database for Annotation, Visualization and Integrated Discovery) and MetaCore bioinformatic resources, we shed new lights on their functional correlation, delineating protein patterns and pathways with reasonable potentialities for oocyte quality estimation in in vitro fertilisation (IVF) programs. Performing a rigorous PubMed search, we redacted a list of 617 unique proteins unambiguously-annotated as HFF components. Their functional processing suggested the occurrence in HFF of a tight and highly dynamic functional-network, which is balanced by specific effectors, primarily involved in extracellular matrix degradation and remodelling, inflammation and coagulation. Metalloproteinases, thrombin and vitamin-D-receptor/retinoid-X-receptor-alpha resulted as the main key factors in the nets and their differential activity may be indicative of ovarian health and oocyte quality. Despite future accurate clinical investigations are absolutely needed, the present analysis may provide a starting point for more accurate oocyte quality estimation and for defining personalised therapies in reproductive medicine.

Subclinical inflammatory status in Rett syndrome.

Inflammation has been advocated as a possible common central mechanism for developmental cognitive impairment. Rett syndrome (RTT) is a devastating neurodevelopmental disorder, mainly caused by de novo loss-of-function mutations in the gene encoding MeCP2. Here, we investigated plasma acute phase response (APR) in stage II (i.e., "pseudo-autistic") RTT patients by routine haematology/clinical chemistry and proteomic 2-DE/MALDI-TOF analyses as a function of four major MECP2 gene mutation types (R306C, T158M, R168X, and large deletions). Elevated erythrocyte sedimentation rate values (median 33.0 mm/h versus 8.0 mm/h, P < 0.0001) were detectable in RTT, whereas C-reactive protein levels were unchanged (P = 0.63). The 2-DE analysis identified significant changes for a total of 17 proteins, the majority of which were categorized as APR proteins, either positive (n = 6 spots) or negative (n = 9 spots), and to a lesser extent as proteins involved in the immune system (n = 2 spots), with some proteins having overlapping functions on metabolism (n = 7 spots). The number of protein changes was proportional to the severity of the mutation. Our findings reveal for the first time the presence of a subclinical chronic inflammatory status related to the "pseudo-autistic" phase of RTT, which is related to the severity carried by the MECP2 gene mutation.

Proteomics of human primary osteoarthritic chondrocytes exposed to extremely low-frequency electromagnetic fields (ELF EMFs) and to therapeutic application of musically modulated electromagnetic fields (TAMMEF).

Osteoarthritis (OA) is the most frequent joint disease, characterized by degradation of extracellular matrix and alterations in chondrocyte metabolism. Some authors reported that electromagnetic fields (EMFs) can positively interfere with patients affected by OA, even though the nature of the interaction is still debated. Human primary osteoarthritic chondrocytes isolated from the femoral heads of OA-patients undergoing to total hip replacement, were cultured in vitro and exposed 30 min/day for two weeks to extremely-low-frequency electromagnetic field (ELF) with fixed frequency (100 Hz) and to therapeutic application of musically modulated electromagnetic fields (TAMMEF) with variable frequencies, intensities and waveforms. Sham-exposed (S.E.) cells served as control group. Cell viability was measured at days 2, 7 and 14. After two weeks, cell lysates were processed using a proteomic approach. Chondrocyte exposed to ELF and TAMMEF system demonstrated different viability compared to untreated chondrocytes (S.E.). Proteome analysis of 2D-Electrophoresis and protein identification by mass spectrometry showed different expression of proteins derived from nucleus, cytoplasm and organelles. Function analysis of the identified proteins showed changes in related-proteins metabolism (glyceraldeyde-3-phosphate-dehydrogenase), stress response (Mn-superoxide-dismutase, heat-shock proteins), cytoskeletal regulation (actin), proteinase inhibition (cystatin-B) and inflammation regulatory functions (S100-A10, S100-A11) among the experimental groups (ELF, TAMMEF and S.E.). In conclusion, EMFs do not cause damage to chondrocytes, besides stimulate safely OA-chondrocytes and are responsible of different protein expression among the three groups. Furthermore, protein analysis of OA-chondrocytes treated with ELF and the new TAMMEF systems could be useful to clarify the pathogenetic mechanisms of OA by identifying biomarkers of the disease.