Shared and Unique Disease Pathways in Amyotrophic Lateral Sclerosis and Parkinson's Disease Unveiled in Peripheral Blood Mononuclear Cells.

Recent evidence supports an association between amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Indeed, prospective population-based studies demonstrated that about one-third of ALS patients develop parkinsonian (PK) signs, even though different neuronal circuitries are involved. In this context, proteomics represents a valuable tool to identify unique and shared pathological pathways. Here, we used two-dimensional electrophoresis to obtain the proteomic profile of peripheral blood mononuclear cells (PBMCs) from PD and ALS patients including a small cohort of ALS patients with parkinsonian signs (ALS-PK). After the removal of protein spots correlating with confounding factors, we applied a sparse partial least square discriminant analysis followed by recursive feature elimination to obtain two protein classifiers able to discriminate (i) PD and ALS patients (30 spots) and (ii) ALS-PK patients among all ALS subjects (20 spots). Functionally, the glycolysis pathway was significantly overrepresented in the first signature, while extracellular interactions and intracellular signaling were enriched in the second signature. These results represent molecular evidence at the periphery for the classification of ALS-PK as ALS patients that manifest parkinsonian signs, rather than comorbid patients suffering from both ALS and PD. Moreover, we confirmed that low levels of fibrinogen in PBMCs is a characteristic feature of PD, also when compared with another movement disorder. Collectively, we provide evidence that peripheral protein signatures are a tool to differentially investigate neurodegenerative diseases and highlight altered biochemical pathways.

Ligand-Based Regulation of Dynamics and Reactivity of Hemoproteins.

Hemoproteins include several heme-binding proteins with distinct structure and function. The presence of the heme group confers specific reactivity and spectroscopic properties to hemoproteins. In this review, we provide an overview of five families of hemoproteins in terms of dynamics and reactivity. First, we describe how ligands modulate cooperativity and reactivity in globins, such as myoglobin and hemoglobin. Second, we move on to another family of hemoproteins devoted to electron transport, such as cytochromes. Later, we consider heme-based reactivity in hemopexin, the main heme-scavenging protein. Then, we focus on heme-albumin, a chronosteric hemoprotein with peculiar spectroscopic and enzymatic properties. Eventually, we analyze the reactivity and dynamics of the most recently discovered family of hemoproteins, i.e., nitrobindins.

Neurodegenerative disorders: From clinicopathology convergence to systems biology divergence.

Neurodegenerative diseases are multifactorial. This means that several genetic, epigenetic, and environmental factors contribute to their emergence. Therefore, for the future management of these highly prevalent diseases, it is necessary to change perspective. If a holistic viewpoint is assumed, the phenotype (the clinicopathological convergence) emerges from the perturbation of a complex system of functional interactions among proteins (systems biology divergence). The systems biology top-down approach starts with the unbiased collection of sets of data generated through one or more -omics techniques and has the aim to identify the networks and the components that participate in the generation of a phenotype (disease), often without any available a priori knowledge. The principle behind the top-down method is that the molecular components that respond similarly to experimental perturbations are somehow functionally related. This allows the study of complex and relatively poorly characterized diseases without requiring extensive knowledge of the processes under investigation. In this chapter, the use of a global approach will be applied to the comprehension of neurodegeneration, with a particular focus on the two most prevalent ones, Alzheimer's and Parkinson's diseases. The final purpose is to distinguish disease subtypes (even with similar clinical manifestations) to launch a future of precision medicine for patients with these disorders.

The endogenous HBZ interactome in ATL leukemic cells reveals an unprecedented complexity of host interacting partners involved in RNA splicing.

Adult T-cell leukemia/lymphoma (ATL) is a T-cell lymphoproliferative neoplasm caused by the human T-cell leukemia virus type 1 (HTLV-1). Two viral proteins, Tax-1 and HBZ play important roles in HTLV-1 infectivity and in HTLV-1-associated pathologies by altering key pathways of cell homeostasis. However, the molecular mechanisms through which the two viral proteins, particularly HBZ, induce and/or sustain the oncogenic process are still largely elusive. Previous results suggested that HBZ interaction with nuclear factors may alter cell cycle and cell proliferation. To have a more complete picture of the HBZ interactions, we investigated in detail the endogenous HBZ interactome in leukemic cells by immunoprecipitating the HBZ-interacting complexes of ATL-2 leukemic cells, followed by tandem mass spectrometry analyses. RNA seq analysis was performed to decipher the differential gene expression and splicing modifications related to HTLV-1. Here we compared ATL-2 with MOLT-4, a non HTLV-1 derived leukemic T cell line and further compared with HBZ-induced modifications in an isogenic system composed by Jurkat T cells and stably HBZ transfected Jurkat derivatives. The endogenous HBZ interactome of ATL-2 cells identified 249 interactors covering three main clusters corresponding to protein families mainly involved in mRNA splicing, nonsense-mediated RNA decay (NMD) and JAK-STAT signaling pathway. Here we analyzed in detail the cluster involved in RNA splicing. RNAseq analysis showed that HBZ specifically altered the transcription of many genes, including crucial oncogenes, by affecting different splicing events. Consistently, the two RNA helicases, members of the RNA splicing family, DDX5 and its paralog DDX17, recently shown to be involved in alternative splicing of cellular genes after NF-κB activation by HTLV-1 Tax-1, interacted and partially co-localized with HBZ. For the first time, a complete picture of the endogenous HBZ interactome was elucidated. The wide interaction of HBZ with molecules involved in RNA splicing and the subsequent transcriptome alteration strongly suggests an unprecedented complex role of the viral oncogene in the establishment of the leukemic state.

Whole Exome Sequencing in 16p13.11 Microdeletion Patients Reveals New Variants Through Deductive and Systems Medicine Approaches.

The 16p13.11 microdeletion, whose prevalence in the general population is about 0.04%, is known in literature as a predisposition factor to neurodevelopmental disorders, being found in about 0.13% of patients with schizophrenia, in 0.5-0.6% of patient with epilepsy, cognitive impairment, autism spectrum disorder (ASD) and aggressiveness. The goal of this study was to identify a specific gene set pattern unique for the affected patients in comparison with other familial components. Due to the incomplete penetrance of this copy number variant (CNV), we studied by whole exome sequencing (WES), with particular regard of 850 SFARI genes, three families with an affected member carrier of inherited 16p13.11 and 16p13.11p12.3 microdeletion and one family with an affected member with a de novo 16p13.11 microdeletion. By combining a deductive approach together with personalized network models, we identified gene signatures potentially capable of explaining the clinical phenotype. Candidate variants in genes of interest were identified as possibly involved in determining the neurological phenotype of the four patients, such as compound heterozygosity in CECR2, variants in MTOR and RICTOR genes, compound heterozygous single nucleotide variants in the LRRK2 gene. Moreover, genes present in the microdeletion region were partially present as central nodes, with a focus on NDE1. No additional pathogenetic or uncertain CNVs were found in all four patients. No significant variants were detected in genes included in the microdeletion in patients 1, 2 and 3, excluding the finding of unmasked recessive variants. In conclusion, WES is a fundamental tool in the genetic investigation of patients having a predisposing variant, which is not sufficient to define the clinical phenotype. Moreover, the analysis of WES data using Systems medicine tools, such as personalized network models, led to the prioritization of genes on a high throughput scale and to discover variants in genes that were not prioritized at first.

Looking at COVID-19 from a Systems Biology Perspective.

The sudden outbreak and worldwide spread of the SARS-CoV-2 pandemic pushed the scientific community to find fast solutions to cope with the health emergency. COVID-19 complexity, in terms of clinical outcomes, severity, and response to therapy suggested the use of multifactorial strategies, characteristic of the network medicine, to approach the study of the pathobiology. Proteomics and interactomics especially allow to generate datasets that, reduced and represented in the forms of networks, can be analyzed with the tools of systems biology to unveil specific pathways central to virus-human host interaction. Moreover, artificial intelligence tools can be implemented for the identification of druggable targets and drug repurposing. In this review article, we provide an overview of the results obtained so far, from a systems biology perspective, in the understanding of COVID-19 pathobiology and virus-host interactions, and in the development of disease classifiers and tools for drug repurposing.

C9ORF72 Repeat Expansion Affects the Proteome of Primary Skin Fibroblasts in ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of the corticospinal motor neurons, which ultimately leads to death. The repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) represents the most common genetic cause of ALS and it is also involved in the pathogenesis of other neurodegenerative disorders. To offer insights into C9ORF72-mediated pathogenesis, we quantitatively analyzed the proteome of patient-derived primary skin fibroblasts from ALS patients carrying the C9ORF72 mutation compared with ALS patients who tested negative for it. Differentially expressed proteins were identified, used to generate a protein-protein interaction network and subjected to a functional enrichment analysis to unveil altered molecular pathways. ALS patients were also compared with patients affected by frontotemporal dementia carrying the C9ORF72 repeat expansion. As a result, we demonstrated that the molecular pathways mainly altered in fibroblasts (e.g., protein homeostasis) mirror the alterations observed in C9ORF72-mutated neurons. Moreover, we highlighted novel molecular pathways (nuclear and mitochondrial transports, vesicle trafficking, mitochondrial bioenergetics, glucose metabolism, ER-phagosome crosstalk and Slit/Robo signaling pathway) which might be further investigated as C9ORF72-specific pathogenetic mechanisms. Data are available via ProteomeXchange with the identifier PXD023866.

Features Selection and Extraction in Statistical Analysis of Proteomics Datasets.

"Omics" techniques (e.g., proteomics, genomics, metabolomics), from which huge datasets can nowadays be obtained, require a different way of thinking about data analysis that can be summarized with the idea that, when data are enough, they can speak for themselves. Indeed, managing huge amounts of data imposes the replacement of the classical deductive approach (hypothesis-driven) with a data-driven hypothesis-generating inductive approach, so to generate mechanistical hypotheses from data.Data reduction is a crucial step in proteomics data analysis, because of the sparsity of significant features in big datasets. Thus, feature selection/extraction methods are applied to obtain a set of features based on which a proteomics signature can be drawn, with a functional significance (e.g., classification, diagnosis, prognosis). Despite big data generated almost daily by proteomics studies, a well-established statistical workflow for data analysis in proteomics is still lacking, opening up to misleading and incorrect data analysis and interpretation. This chapter will give an overview of the methods available for feature selection/extraction in proteomics datasets and how to choose the most appropriate one based on the type of dataset.

Low noncarbonic buffer power amplifies acute respiratory acid-base disorders in patients with sepsis: an in vitro study.

Patients with sepsis have typically reduced concentrations of hemoglobin and albumin, the major components of noncarbonic buffer power (ß). This could expose patients to high pH variations during acid-base disorders. The objective of this study is to compare, in vitro, noncarbonic ß of patients with sepsis with that of healthy volunteers, and evaluate its distinct components. Whole blood and isolated plasma of 18 patients with sepsis and 18 controls were equilibrated with different CO2 mixtures. Blood gases, pH, and electrolytes were measured. Noncarbonic ß and noncarbonic ß due to variations in strong ion difference (ßSID) were calculated for whole blood. Noncarbonic ß and noncarbonic ß normalized for albumin concentrations (ßNORM) were calculated for isolated plasma. Representative values at pH = 7.40 were compared. Albumin proteoforms were evaluated via two-dimensional electrophoresis. Hemoglobin and albumin concentrations were significantly lower in patients with sepsis. Patients with sepsis had lower noncarbonic ß both of whole blood (22.0 ± 1.9 vs. 31.6 ± 2.1 mmol/L, P < 0.01) and plasma (0.5 ± 1.0 vs. 3.7 ± 0.8 mmol/L, P < 0.01). Noncarbonic ßSID was lower in patients (16.8 ± 1.9 vs. 24.4 ± 1.9 mmol/L, P < 0.01) and strongly correlated with hemoglobin concentration (r = 0.94, P < 0.01). Noncarbonic ßNORM was lower in patients [0.01 (-0.01 to 0.04) vs. 0.08 (0.06-0.09) mmol/g, P < 0.01]. Patients with sepsis and controls showed different amounts of albumin proteoforms. Patients with sepsis are exposed to higher pH variations for any given change in CO2 due to lower concentrations of noncarbonic buffers and, possibly, an altered buffering function of albumin. In both patients with sepsis and healthy controls, electrolyte shifts are the major buffering mechanism during respiratory acid-base disorders.NEW & NOTEWORTHY Patients with sepsis are poorly protected against acute respiratory acid-base derangements due to a lower noncarbonic buffer power, which is caused both by a reduction in the major noncarbonic buffers, i.e. hemoglobin and albumin, and by a reduced buffering capacity of albumin. Electrolyte shifts from and to the red blood cells determining acute variations in strong ion difference are the major buffering mechanism during acute respiratory acid-base disorders.

Gene Set Enrichment Analysis of Interaction Networks Weighted by Node Centrality.

Gene set enrichment analysis (GSEA) is a powerful tool to associate a disease phenotype to a group of genes/proteins. GSEA attributes a specific weight to each gene/protein in the input list that depends on a metric of choice, which is usually represented by quantitative expression data. However, expression data are not always available. Here, GSEA based on betweenness centrality of a protein-protein interaction (PPI) network is described and applied to two cases, where an expression metric is missing. First, personalized PPI networks were generated from genes displaying alterations (assessed by array comparative genomic hybridization and whole exome sequencing) in four probands bearing a 16p13.11 microdeletion in common and several other point variants. Patients showed disease phenotypes linked to neurodevelopment. All networks were assembled around a cluster of first interactors of altered genes with high betweenness centrality. All four clusters included genes known to be involved in neurodevelopmental disorders with different centrality. Moreover, the GSEA results pointed out to the evidence of "cell cycle" among enriched pathways. Second, a large interaction network obtained by merging proteomics studies on three neurodegenerative disorders was analyzed from the topological point of view. We observed that most central proteins are often linked to Parkinson's disease. The selection of these proteins improved the specificity of GSEA, with "Metabolism of amino acids and derivatives" and "Cellular response to stress or external stimuli" as top-ranked enriched pathways. In conclusion, betweenness centrality revealed to be a suitable metric for GSEA. Thus, centrality-based GSEA represents an opportunity for precision medicine and network medicine.

Demonstration of fibrinogen-FcRn binding at acidic pH by means of Fluorescence Correlation Spectroscopy.

The neonatal Fc receptor (FcRn) interacts with IgG and albumin at acidic pH within endosomes, thus protecting these plasma proteins from degradation. Recently, we proposed fibrinogen as a new binding partner of FcRn. This work was aimed at providing a direct demonstration of FcRn-fibrinogen binding at acidic pH by Fluorescence Correlation Spectroscopy. The increase in diffusion time between free and fibrinogen-bound FITC-labelled FcRn was assumed as the binding indicator. We observed that, at acidic pH (pH = 5.3), FcRn diffusion time shifted from ≈730 µs (FITC-labelled FcRn alone) to >1200 µs (FITC-labelled FcRn added with fibrinogen). A similar trend was exhibited by albumin, a known FcRn interactor, while no significant variations in diffusion time were observed upon incubation with catalase as negative control. Our results demonstrate a binding interaction between fibrinogen, one of the most abundant plasma proteins, and FcRn, a receptor involved in the regulation of the levels of IgG and albumin. This interaction is likely responsible for fibrinogen protection from intracellular degradation and recycling in plasma. Fibrinogen is crucial not only in haemostasis but also in acute inflammatory response and in some pathological conditions. The interaction with FcRn can influence not only the levels of fibrinogen in plasma and other tissues, but also the levels of other FcRn binding partners, among which are some plasma proteins of clinical relevance.

Proteostasis and Proteotoxicity in the Network Medicine Era.

Neurodegenerative proteinopathies are complex diseases that share some pathogenetic processes. One of these is the failure of the proteostasis network (PN), which includes all components involved in the synthesis, folding, and degradation of proteins, thus leading to the aberrant accumulation of toxic protein aggregates in neurons. The single components that belong to the three main modules of the PN are highly interconnected and can be considered as part of a single giant network. Several pharmacological strategies have been proposed to ameliorate neurodegeneration by targeting PN components. Nevertheless, effective disease-modifying therapies are still lacking. In this review article, after a general description of the PN and its failure in proteinopathies, we will focus on the available pharmacological tools to target proteostasis. In this context, we will discuss the main advantages of systems-based pharmacology in contrast to the classical targeted approach, by focusing on network pharmacology as a strategy to innovate rational drug design.

Exploring the Impact of PARK2 Mutations on the Total and Mitochondrial Proteome of Human Skin Fibroblasts.

Mutations in PARK2 gene are the most frequent cause of familial forms of Parkinson's disease (PD). This gene encodes Parkin, an E3 ubiquitin ligase involved in several cellular mechanisms, including mitophagy. Parkin loss-of-function is responsible for the cellular accumulation of damaged mitochondria, which in turn determines an increment of reactive oxygen species (ROS) levels, lower ATP production, and apoptosis activation. Given the importance of mitochondrial dysfunction and mitophagy impairment in PD pathogenesis, the aim of the present study was to investigate both total and mitochondrial proteome alterations in human skin fibroblasts of PARK2-mutated patients. To this end, both total and mitochondria-enriched protein fractions from fibroblasts of five PARK2-mutated patients and five control subjects were analyzed by quantitative shotgun proteomics to identify proteins specifically altered by Parkin mutations (mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD015880). Both the network-based and gene set enrichment analyses pointed out pathways in which Rab GTPase proteins are involved. To have a more comprehensive view of the mitochondrial alterations due to PARK2 mutations, we investigated the impact of Parkin loss on mitochondrial function and network morphology. We unveiled that the mitochondrial membrane potential was reduced in PARK2-mutated patients, without inducing PINK1 accumulation, even when triggered with the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Lastly, the analysis of the mitochondrial network morphology did not reveal any significant alterations in PARK2-mutated patients compared to control subjects. Thus, our results suggested that the network morphology was not influenced by the mitochondrial depolarization and by the lack of Parkin, revealing a possible impairment of fission and, more in general, of mitochondrial dynamics. In conclusion, the present work highlighted new molecular factors and pathways altered by PARK2 mutations, which will unravel possible biochemical pathways altered in the sporadic form of PD.

Mycobacterial and Human Nitrobindins: Structure and Function.

Aims: Nitrobindins (Nbs) are evolutionary conserved all-ß-barrel heme-proteins displaying a highly solvent-exposed heme-Fe(III) atom. The physiological role(s) of Nbs is almost unknown. Here, the structural and functional properties of ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) and ferric Homo sapiens Nb (Hs-Nb(III)) have been investigated and compared with those of ferric Arabidopsis thaliana Nb (At-Nb(III), Rhodnius prolixus nitrophorins (Rp-NP(III)s), and mammalian myoglobins. Results: Data here reported demonstrate that Mt-Nb(III), At-Nb(III), and Hs-Nb(III) share with Rp-NP(III)s the capability to bind selectively nitric oxide, but display a very low reactivity, if any, toward histamine. Data obtained overexpressing Hs-Nb in human embryonic kidney 293 cells indicate that Hs-Nb localizes mainly in the cytoplasm and partially in the nucleus, thanks to a nuclear localization sequence encompassing residues Glu124-Leu154. Human Hs-Nb corresponds to the C-terminal domain of the human nuclear protein THAP4 suggesting that Nb may act as a sensor possibly modulating the THAP4 transcriptional activity residing in the N-terminal region. Finally, we provide strong evidence that both Mt-Nb(III) and Hs-Nb(III) are able to scavenge peroxynitrite and to protect free l-tyrosine against peroxynitrite-mediated nitration. Innovation: Data here reported suggest an evolutionarily conserved function of Nbs related to their role as nitric oxide sensors and components of antioxidant systems. Conclusion: Human THAP4 may act as a sensing protein that couples the heme-based Nb(III) reactivity with gene transcription. Mt-Nb(III) seems to be part of the pool of proteins required to scavenge reactive nitrogen and oxygen species produced by the host during the immunity response.

Saliva is a reliable tool to detect SARS-CoV-2.

OBJECTIVES: This study analyzed salivary samples of COVID-19 patients and compared the results with their clinical and laboratory data. METHODS: Salivary samples of 25 COVID-19 patients were analyzed by rRT-PCR. The following data were collected: age, sex, comorbidities, drugs. Lactate dehydrogenase (LDH) and ultrasensitive reactive C protein (usRCP) values were registered on the same day when a salivary swab was collected. Prevalence of positivity in saliva and association between clinical data and the cycle threshold as a semiquantitative indicator of viral load were considered. RESULTS: Twenty-five subjects were recruited into this study, 17 males and 8 females. The mean age was 61.5 +/- 11.2 years. Cardiovascular and/or dysmetabolic disorders were observed in 65.22% of cases. All the samples tested positive for the presence of SARS-CoV-2, while there was an inverse association between LDH and Ct values. Two patients showed positive salivary results on the same days when their pharyngeal or respiratory swabs showed conversion. CONCLUSIONS: Saliva is a reliable tool to detect SARS-CoV-2. The role of saliva in COVID-19 diagnosis could not be limited to a qualitative detection of the virus, but it may also provide information about the clinical evolution of the disease.

Mitochondrial Proteins in the Development of Parkinson's Disease.

Parkinson's disease (PD) is a multifactorial disorder whose etiology is not completely understood. Strong evidences suggest that mitochondrial impairment and altered mitochondrial disposal play a key role in the development of this pathology. Here we show this association in both genetic and sporadic forms of the disease. Moreover, we describe the mitochondrial dysfunctions in toxin-induced models of PD, thus highlighting the importance of environmental factors in the onset of this pathology. In particular, we focus our attention on mitochondrial dynamics, mitochondrial biogenesis, and mitophagy and explain how their impairment could have a negative impact on dopaminergic neurons function and survival. Lastly, we aim at clarifying the important role played by proteomics in this field of research, proteomics being a global and unbiased approach suitable to unravel alterations of the molecular pathways in multifactorial diseases.

Exploring the Mitochondrial Degradome by the TAILS Proteomics Approach in a Cellular Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disease worldwide and the availability of early biomarkers and novel biotargets represents an urgent medical need. The main pathogenetic hallmark of PD is the specific loss of nigral dopaminergic neurons, in which mitochondrial dysfunction plays a crucial role. Mitochondrial proteases are central to the maintenance of healthy mitochondria and they have recently emerged as drug targets. However, an exhaustive characterization of these enzymes and their targets is still lacking, due to difficulties in analyzing proteolytic fragments by bottom-up proteomics approaches. Here, we propose the "mitochondrial dimethylation-TAILS" strategy, which combines the isolation of mitochondria with the enrichment of N-terminal peptides to analyze the mitochondrial N-terminome. We applied this method in a cellular model of altered dopamine homeostasis in neuroblastoma SH-SY5Y cells, which recapitulates early steps of PD pathogenesis. The main aim was to identify candidate mitochondrial proteases aberrantly activated by dopamine dysregulation and their cleaved targets. The proposed degradomics workflow was able to improve the identification of mitochondrial proteins if compared to classical shotgun analysis. In detail, 40% coverage of the mitochondrial proteome was obtained, the sequences of the transit peptides of two mitochondrial proteins were unveiled, and a consensus cleavage sequence for proteases involved in the processing of mitochondrial proteins was depicted. Mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD013900. Moreover, sixty-one N-terminal peptides whose levels were affected by dopamine treatment were identified. By an in-depth analysis of the proteolytic peptides included in this list, eleven mitochondrial proteins showed altered proteolytic processing. One of these proteins (i.e., the 39S ribosomal protein L49 - MRPL49) was cleaved by the neprilysin protease, already exploited in clinics as a biotarget. We eventually demonstrated a mitochondrial subcellular localization of neprilysin in human cells for the first time. Collectively, these results shed new light on mitochondrial dysfunction linked to dopamine imbalance in PD and opened up the possibility to explore the mitochondrial targets of neprilysin as candidate biomarkers.

Deinococcus radiodurans' SRA-HNH domain containing protein Shp (Dr1533) is involved in faithful genome inheritance maintenance following DNA damage.

BACKGROUND: Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn2+ protection of DNA repair enzymes. METHODS: Possible annotated domains of the DR1533 locus protein (Shp) were searched by bioinformatic analysis. The gene was cloned and expressed as fusion protein. Band-shift assays of Shp or the SRA and HNH domains were performed on oligonucleotides, genomic DNA from E. coli and DR. shp knock-out mutant was generated by homologous recombination with a kanamycin resistance cassette. RESULTS: DR1533 contains an N-terminal SRA domain and a C-terminal HNH motif (SRA-HNH Protein, Shp). Through its SRA domain, Shp binds double-strand oligonucleotides containing 5mC and 5hmC, but also unmethylated and mismatched cytosines in presence of Mn2+. Shp also binds to Escherichia coli dcm+ genomic DNA, and to cytosine unmethylated DR and E. coli dcm- genomic DNAs, but only in presence of Mn2+. Under these binding conditions, Shp displays DNAse activity through its HNH domain. Shp KO enhanced >100 fold the number of spontaneous mutants, whilst the treatment with DNA double strand break inducing agents enhanced up to 3-log the number of survivors. CONCLUSIONS: The SRA-HNH containing protein Shp binds to and cuts 5mC DNA, and unmethylated DNA in a Mn2+ dependent manner, and might be involved in faithful genome inheritance maintenance following DNA damage. GENERAL SIGNIFICANCE: Our results provide evidence for a potential role of DR Shp protein for genome integrity maintenance, following DNA double strand breaks induced by genotoxic agents.

Statistical analysis of proteomics data: A review on feature selection.

The spread of "-omics" strategies has strongly changed the way of thinking about the scientific method. Indeed, managing huge amounts of data imposes the replacement of the classical deductive approach with a data-driven inductive approach, so to generate mechanistical hypotheses from data. Data reduction is a crucial step in the process of proteomics data analysis, because of the sparsity of significant features in big datasets. Thus, feature selection methods are applied to obtain a set of features based on which a proteomics signature can be drawn, with a functional significance (e.g., classification, diagnosis, prognosis). In this frame, the aim of the present review article is to give an overview of the methods available for proteomics data analysis, with a focus on biomedical translational research. Suggestions for the choice of the most appropriate standard statistical procedures are presented to perform data reduction by feature selection, cross-validation and functional analysis of proteomics profiles. SIGNIFICANCE: The proteome, including all so-called "proteoforms", represents the highest level of complexity of biomolecules when compared to the other "-omes" (i.e., genome, transcriptome). For this reason, the use of proper data reduction strategies is mandatory for proteomics data analysis. However, the strategies to be employed for feature selection must be carefully chosen, since many different approaches exist based on both input data and desired output. So far, a well-established decision-making workflow for proteomics data analysis is lacking, opening up to misleading and incorrect data analysis and interpretation. In this review article many statistical approaches are described and compared for their application in the field of biomedical research, in order to suggest the reader the most suitable analysis pathway and to avoid mistakes.