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Background: Molecular understanding of muscle-invasive (MIBC) and non-muscle-invasive (NMIBC) bladder cancer is currently based primarily on transcriptomic and genomic analyses., Objective: To conduct proteogenomic analyses to gain insights into bladder cancer (BC) heterogeneity and identify underlying processes specific to tumor subgroups and therapeutic outcomes., Design, Setting, and Participants: Proteomic data were obtained for 40 MIBC and 23 NMIBC cases for which transcriptomic and genomic data were already available. Four BC-derived cell lines harboring FGFR3 alterations were tested with interventions., Intervention: Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), second mitochondrial-derived activator of caspases mimetic (birinapant), pan-FGFR inhibitor (erdafitinib), and FGFR3 knockdown., Outcome Measurements and Statistical Analysis: Proteomic groups from unsupervised analyses (uPGs) were characterized using clinicopathological, proteomic, genomic, transcriptomic, and pathway enrichment analyses. Additional enrichment analyses were performed for FGFR3-mutated tumors. Treatment effects on cell viability for FGFR3-altered cell lines were evaluated. Synergistic treatment effects were evaluated using the zero interaction potency model., Results and Limitations: Five uPGs, covering both NMIBC and MIBC, were identified and bore coarse-grained similarity to transcriptomic subtypes underlying common features of these different entities; uPG-E was associated with the Ta pathway and enriched in FGFR3 mutations. Our analyses also highlighted enrichment of proteins involved in apoptosis in FGFR3-mutated tumors, not captured through transcriptomics. Genetic and pharmacological inhibition demonstrated that FGFR3 activation regulates TRAIL receptor expression and sensitizes cells to TRAIL-mediated apoptosis, further increased by combination with birinapant., Conclusions: This proteogenomic study provides a comprehensive resource for investigating NMIBC and MIBC heterogeneity and highlights the potential of TRAIL-induced apoptosis as a treatment option for FGFR3-mutated bladder tumors, warranting a clinical investigation., Patient Summary: We integrated proteomics, genomics, and transcriptomics to refine molecular classification of bladder cancer, which, combined with clinical and pathological classification, should lead to more appropriate management of patients. Moreover, we identified new biological processes altered in FGFR3-mutated tumors and showed that inducing apoptosis represents a new potential therapeutic option., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

This study evaluates the performance of two convolutional neural networks (CNNs) in a brain-machine interface (BMI) based on motor imagery (MI) by using a small dataset collected from five participants wearing a lower-limb exoskeleton. To address the issue of limited data availability, transfer learning was employed by training models on EEG signals from other subjects and subsequently fine-tuning them to specific users. A combination of common spatial patterns (CSP) and linear discriminant analysis (LDA) was used as a benchmark for comparison. The study's primary aim is to examine the potential of CNNs and transfer learning in the development of an automatic neural classification system for a BMI based on MI to command a lower-limb exoskeleton that can be used by individuals without specialized training.Clinical Relevance- BMI can be used in rehabilitation for patients with motor impairment by using mental simulation of movement to activate robotic exoskeletons. This can promote neural plasticity and aid in recovery.

This study explores the use of a brain-computer interface (BCI) based on motor imagery (MI) for the control of a lower limb exoskeleton to aid in motor recovery after a neural injury. The BCI was evaluated in ten able-bodied subjects and two patients with spinal cord injuries. Five able-bodied subjects underwent a virtual reality (VR) training session to accelerate training with the BCI. Results from this group were compared with a control group of five able-bodied subjects, and it was found that the employment of shorter training by VR did not reduce the effectiveness of the BCI and even improved it in some cases. Patients gave positive feedback about the system and were able to handle experimental sessions without reaching high levels of physical and mental exertion. These results are promising for the inclusion of BCI in rehabilitation programs, and future research should investigate the potential of the MI-based BCI system., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Introduction: Brain-machine interfaces (BMIs) attempt to establish communication between the user and the device to be controlled. BMIs have great challenges to face in order to design a robust control in the real field of application. The artifacts, high volume of training data, and non-stationarity of the signal of EEG-based interfaces are challenges that classical processing techniques do not solve, showing certain shortcomings in the real-time domain. Recent advances in deep-learning techniques open a window of opportunity to solve some of these problems. In this work, an interface able to detect the evoked potential that occurs when a person intends to stop due to the appearance of an unexpected obstacle has been developed., Material and Methods: First, the interface was tested on a treadmill with five subjects, in which the user stopped when an obstacle appeared (simulated by a laser). The analysis is based on two consecutive convolutional networks: the first one to discern the intention to stop against normal walking and the second one to correct false detections of the previous one., Results and Discussion: The results were superior when using the methodology of the two consecutive networks vs. only the first one in a cross-validation pseudo-online analysis. The false positives per min (FP/min) decreased from 31.8 to 3.9 FP/min and the number of repetitions in which there were no false positives and true positives (TP) improved from 34.9% to 60.3% NOFP/TP. This methodology was tested in a closed-loop experiment with an exoskeleton, in which the brain-machine interface (BMI) detected an obstacle and sent the command to the exoskeleton to stop. This methodology was tested with three healthy subjects, and the online results were 3.8 FP/min and 49.3% NOFP/TP. To make this model feasible for non-able bodied patients with a reduced and manageable time frame, transfer-learning techniques were applied and validated in the previous tests, and were then applied to patients. The results for two incomplete Spinal Cord Injury (iSCI) patients were 37.9% NOFP/TP and 7.7 FP/min., 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 © 2023 Quiles, Ferrero, Iáñez, Ortiz, Gil-Agudo and Azorín.)

The monitoring of machine conditions is very important from the viewpoints of productivity, economic benefits, and maintenance. Several techniques have been proposed in which sensors are the key to providing relevant information to verify the system. Recently, the smart sensor concept is common, in which the sensors are integrated with a data processing unit executing dedicated algorithms used to generate meaningful information about the system in situ. Additionally, infrared thermography has gained relevance in monitoring processes, since the new infrared cameras have more resolution, smaller dimensions, reliability, functionality, and lower costs. These units were firstly used as secondary elements in the condition monitoring of machines, but thanks to modern techniques for data processing, the infrared sensors can be used to give a first, or even a direct, diagnosis in a nonintrusive way in industrial applications. Therefore, in this manuscript, the structure and development of an infrared-thermography-based smart sensor for diagnosing faults in the elements associated with induction motors, such as rolling bearings and the gearbox, is described. The smart sensor structure includes five main parts: an infrared primary sensor, a preprocessing module, an image processing module, classification of faults, and a user interface. The infrared primary sensor considers a low-cost micro thermal camera for acquiring the thermal images. The processing modules and the classification module implement the data processing algorithms into digital development boards, enabling smart system characteristics. Finally, the interface module allows the final users to require the smart sensor to perform processing actions and data visualization, with the additional feature that the diagnosis report can be provided by the system. The smart sensor is validated in a real experimental test bench, demonstrating its capabilities in different case studies.

In the EEG literature, there is a lack of asynchronous intention models that realistically propose interfaces for applications that must operate in real time. In this work, a novel BMI approach to detect in real time the intention to turn is proposed. For this purpose, an offline, pseudo-online and online analysis is presented to validate the EEG as a biomarker for the intention to turn. This article presents a methodology for the creation of a BMI that could differentiate two classes: monotonous walk and intention to turn. A comparison of some of the most popular algorithms in the literature is conducted. To filter the signal, two relevant algorithms are used: H∞ filter and ASR. For processing and classification, the mean of the covariance matrices in the Riemannian space was calculated and then, with various classifiers of different types, the distance of the test samples to each class in the Riemannian space was estimated. This dispenses with power-based models and the necessary baseline correction, which is a problem in realistic scenarios. In the cross-validation for a generic selection (valid for any subject) and a personalized one, the results were, on average, 66.2% and 69.6% with the best filter H∞. For the pseudo-online, the custom configuration for each subject was an average of 40.2% TP and 9.3 FP/min; the best subject obtained 43.9% TP and 2.9 FP/min. In the final validation test, this subject obtained 2.5 FP/min and an accuracy rate of 71.43%, and the turn anticipation was 0.21 s on average.

Spinal Cord Injury (SCI) refers to damage to the spinal cord that can affect different body functionalities. Recovery after SCI depends on multiple factors, being the rehabilitation therapy one of them. New approaches based on robot-assisted training offer the possibility to make training sessions longer and with a reproducible pattern of movements. The control of these robotic devices by means of Brain-Machine Interfaces (BMIs) based on Motor Imagery (MI) favors the patient cognitive engagement during the rehabilitation, promoting mechanisms of neuroplasticity. This research evaluates the acceptance and feedback received from patients with incomplete SCI about the usage of a MI-based BMI with a lower-limb exoskeleton. Clinical Relevance- Patients experienced satisfaction when using the exoskeleton and levels of mental and physical workload were withing reasonable limits. In addition results from the BMI were promising for the inclusion of this type of systems in rehabilitation programs.

In this paper, the paradigm of the intention of speed changes from EEG signals with Riemannian classifiers methods is studied in 10 subjects. In addition, the best frequency band and how different electrode configurations affect the accuracy of the model are analyzed. In the prediction of the intention to change speed, results of 68.6% were obtained, in the one of only Increase, results of 64.41 % were obtained, and in the one of only Decrease, results of 71.5% were obtained.

This article presents an exhaustive analysis of the works present in the literature pertaining to transcranial direct current stimulation(tDCS) applications. The aim of this work is to analyze the specific characteristics of lower-limb stimulation, identifying the strengths and weaknesses of these works and framing them with the current knowledge of tDCS. The ultimate goal of this work is to propose areas of improvement to create more effective stimulation therapies with less variability.

Brain-Computer Interfaces (BCIs) are becoming an important technological tool for the rehabilitation process of patients with locomotor problems, due to their ability to recover the connection between brain and limbs by promoting neural plasticity. They can be used as assistive devices to improve the mobility of handicapped people. For this reason, current BCIs have to be improved to allow an accurate and natural use of external devices. This work proposes a novel methodology for the detection of the intention to change the direction during gait based on event-related desynchronization (ERD). Frequency and temporal features of the electroencephalographic (EEG) signals are characterized. Then, a selection of the most influential features and electrodes to differentiate the direction change intention from the walking is carried out. Best results are obtained when combining frequency and temporal features with an average accuracy of [Formula: see text]%, which are promising to be applied for future BCIs.

Large-scale multi-omic analysis allows a thorough understanding of different physiological or pathological conditions, particularly cancer. Here, an extraction method simultaneously yielding DNA, RNA and protein (thereby referred to as "triple extraction", TEx) was tested for its suitability to unbiased, system-wide proteomic investigation. Largely proven efficient for transcriptomic and genomic studies, we aimed at exploring TEx compatibility with mass spectrometry-based proteomics and phospho-proteomics, as compared to a standard urea extraction. TEx is suitable for the shotgun investigation of proteomes, providing similar results as urea-based protocol both at the qualitative and quantitative levels. TEx is likewise compatible with the exploration of phosphorylation events, actually providing a higher number of correctly localized sites than urea, although the nature of extracted modifications appears somewhat distinct between both techniques. These results highlight that the presented protocol is well suited for the examination of the proteome and modified proteome of this bladder cancer cell model, as efficiently as other more widely used workflows for mass spectrometry-based analysis. Potentially applicable to other mammalian cell types and tissues, TEx represents an advantageous strategy for multi-omics on scarce and/or heterogenous samples., (© 2021 Wiley-VCH GmbH.)

Spinal cord injury (SCI) limits life expectancy and causes a restriction of patient's daily activities. In the last years, robotics exoskeletons have appeared as a promising rehabilitation and assistance tool for patients with motor limitations, as people that have suffered a SCI. The usability and clinical relevance of these robotics systems could be further enhanced by brain-machine interfaces (BMIs), as they can be used to foster patients' neuroplasticity. However, there are not many studies showing the use of BMIs to control exoskeletons with patients. In this work we show a case study where one SCI patient has used a BMI based on motor imagery (MI) in order to control a lower limb exoskeleton that assists their gait.

This paper studies the direction changes during the gait by means of two different distributions of electrodes located in the motor, premotor and occipital areas. The objective is analyzing which areas are involved in the detection of the intention of turning while the person is walking. The signals in both options are characterized with frequency and temporal features and classified following a cross-validation process. A 95% of success rate is achieved when the electrodes are disposed along the motor, premotor and occipital areas.Clinical Relevance- The objective of this study is applying the acknowledgements obtained in the designing of a brain-machine interface (BMI) based in the detection of the intention of the direction change during the gait. This BMI has clinical relevance in the rehabilitation of the gait in patients with motor injuries, assisting the patient to perform the movements as realistic as it is possible.

Bone marrow-derived mesenchymal stem cells (MSCs) differentiate into osteoblasts upon stimulation by signals present in their niche. Because the global signaling cascades involved in the early phases of MSCs osteoblast (OB) differentiation are not well-defined, we used quantitative mass spectrometry to delineate changes in human MSCs proteome and phosphoproteome during the first 24 h of their OB lineage commitment. The temporal profiles of 6252 proteins and 15,059 phosphorylation sites suggested at least two distinct signaling waves: one peaking within 30 to 60 min after stimulation and a second upsurge after 24 h. In addition to providing a comprehensive view of the proteome and phosphoproteome dynamics during early MSCs differentiation, our analyses identified a key role of serine/threonine protein kinase D1 (PRKD1) in OB commitment. At the onset of OB differentiation, PRKD1 initiates activation of the pro-osteogenic transcription factor RUNX2 by triggering phosphorylation and nuclear exclusion of the histone deacetylase HDAC7., (© 2020 Barrio-Hernandez et al.; Published by Cold Spring Harbor Laboratory Press.)

The aim of this paper is to describe new methods for detecting the appearance of unexpected obstacles during normal gait from EEG signals, improving the accuracy and reducing the false positive rate obtained in previous studies. This way, an exoskeleton for rehabilitation or assistance of people with motor limitations commanded by a Brain-Machine Interface (BMI) could be stopped in case that an obstacle suddenly appears during walking. The EEG data of nine healthy subjects were collected during their normal gait while an obstacle appearance was simulated by the projection of a laser line in a random pattern. Different approaches were considered for selecting the parameters of the BMI: subsets of electrodes, time windows and classifier probabilities, which were based on a linear discriminant analysis (LDA). The pseudo-online results of the BMI for detecting the appearance of obstacles, with an average percentage of 63.9% of accuracy and 2.6 false positives per minute, showed a significant improvement over previous studies.

The present data article corresponds to the proteomic data of the involvement of Cylindromatosis protein (CYLD) in the ubiquitination signaling initiated by EGF stimulation. CYLD tumor suppressor protein has Lys63-chain deubiquitinase activity that has been proved essential for the negative regulation of crucial signaling mechanisms, namely the NFkB pathway. Previous results have suggested the involvement of CYLD in the EGF-dependent signal transduction as well, showing its engagement within the tyrosine-phosphorylated complexes formed following the addition of the growth factor. EGFR signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis. We carried out the substitution of the endogenous pool of ubiquitin for a His-FLAG-tagged ubiquitin (Stable Ubiquitin Exchange, StUbEx), in combination with the shRNA silencing of CYLD and SILAC-labeling on HeLa cells. The subsequent tandem affinity purification of ubiquitinated proteins in control and CYLD-depleted cells was followed by mass spectrometric analysis. Therefore, we present an unbiased study investigating the impact of CYLD in the EGF-dependent ubiquitination. The data supplied herein is related to the research article entitled "Cylindromatosis tumor suppressor protein (CYLD) deubiquitinase is necessary for proper ubiquitination and degradation of the epidermal growth factor receptor" (Sanchez-Quiles et al., 2017) [1]. We provide the associated mass spectrometry raw files, excel tables and gene ontology enrichments. The data have been deposited in the ProteomeXchange with the identifier PXD003423.

FGFR3 alterations (mutations or translocation) are among the most frequent genetic events in bladder carcinoma. They lead to an aberrant activation of FGFR3 signaling, conferring an oncogenic dependence, which we studied here. We discovered a positive feedback loop, in which the activation of p38 and AKT downstream from the altered FGFR3 upregulates MYC mRNA levels and stabilizes MYC protein, respectively, leading to the accumulation of MYC, which directly upregulates FGFR3 expression by binding to active enhancers upstream from FGFR3 Disruption of this FGFR3/MYC loop in bladder cancer cell lines by treatment with FGFR3, p38, AKT, or BET bromodomain inhibitors (JQ1) preventing MYC transcription decreased cell viability in vitro and tumor growth in vivo A relevance of this loop to human bladder tumors was supported by the positive correlation between FGFR3 and MYC levels in tumors bearing FGFR3 mutations, and the decrease in FGFR3 and MYC levels following anti-FGFR treatment in a PDX model bearing an FGFR3 mutation. These findings open up new possibilities for the treatment of bladder tumors displaying aberrant FGFR3 activation., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Cylindromatosis tumor suppressor protein (CYLD) is a deubiquitinase, best known as an essential negative regulator of the NFkB pathway. Previous studies have suggested an involvement of CYLD in epidermal growth factor (EGF)-dependent signal transduction as well, as it was found enriched within the tyrosine-phosphorylated complexes in cells stimulated with the growth factor. EGF receptor (EGFR) signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis. Here, using a combination of mass spectrometry-based quantitative proteomic approaches with biochemical and immunofluorescence strategies, we demonstrate the involvement of CYLD in the regulation of the ubiquitination events triggered by EGF. Our data show that CYLD regulates the magnitude of ubiquitination of several major effectors of the EGFR pathway by assisting the recruitment of the ubiquitin ligase Cbl-b to the activated EGFR complex. Notably, CYLD facilitates the interaction of EGFR with Cbl-b through its Tyr15 phosphorylation in response to EGF, which leads to fine-tuning of the receptor's ubiquitination and subsequent degradation. This represents a previously uncharacterized strategy exerted by this deubiquitinase and tumors suppressor for the negative regulation of a tumorigenic signaling pathway., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

We provide detailed datasets from our analysis of the proteins that associate with IL-2Rβ and IL-2Rγ in T-cells stimulated with IL-2 or IL-15 compared with resting T-cells, as identified by SILAC-based quantitative proteomics. We also include quantitative data regarding site-specific phosphorylation events observed both in IL-2Rβ and IL-2Rγ. Moreover, we provide results demonstrating the specific protein recruitment capacity of four of those site-specific phosphorylations. The proteomics and phosphoproteomics data described in this article is associated with a research article entitled "Characterization of receptor-associated protein complex assembly in Interleukin (IL)-2- and IL-15-activated T-lymphocytes" (Osinalde et al., 2016 [1]). The mass spectrometry data have been deposited to the ProteomeEXchange Constorium with the identifier PXD002386.

It remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. We have previously dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells, unveiling subtle differences that may contribute to their functional dichotomy. In this study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is highly orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptor-associated complex discovering novel constituents. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and -independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2 receptor β subunit (IL-2Rβ). We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Overall, our study sheds new light into the initial molecular events triggered by IL-2 and IL-15 and constitutes a further step toward a better understanding of the early signaling aspects of the two closely related cytokines in T-lymphocytes.

S-nitrosoglutathione reductase (GSNOR) represents the best-documented denitrosylase implicated in regulating the levels of proteins posttranslationally modified by nitric oxide on cysteine residues by S-nitrosylation. GSNOR controls a diverse array of physiologic functions, including cellular growth and differentiation, inflammation, and metabolism. Chromosomal deletion of GSNOR results in pathologic protein S-nitrosylation that is implicated in human hepatocellular carcinoma (HCC). Here we identify a metabolic hallmark of aberrant S-nitrosylation in HCC and exploit it for therapeutic gain. We find that hepatocyte GSNOR deficiency is characterized by mitochondrial alteration and by marked increases in succinate dehydrogenase (SDH) levels and activity. We find that this depends on the selective S-nitrosylation of Cys(501) in the mitochondrial chaperone TRAP1, which mediates its degradation. As a result, GSNOR-deficient cells and tumors are highly sensitive to SDH inhibition, namely to α-tocopheryl succinate, an SDH-targeting molecule that induced RIP1/PARP1-mediated necroptosis and inhibited tumor growth. Our work provides a specific molecular signature of aberrant S-nitrosylation in HCC, a novel molecular target in SDH, and a first-in-class therapy to treat the disease. Cancer Res; 76(14); 4170-82. ©2016 AACR., (©2016 American Association for Cancer Research.)

Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4(+) T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Interleukin-2 (IL-2) stimulation results in T-cell growth as a consequence of activation of highly sophisticated and fine-tuned signaling pathways. Despite lacking intrinsic enzymatic activity, scaffold proteins such as Gab2, play a pivotal role in IL-2-triggered signal transduction integrating, diversifying and amplifying the signal by serving as a platform for the assembly of effectors proteins. Traditionally, Gab2-mediated protein recruitment was believed to solely depend on cytokine-induced phosphotyrosine moieties. At present, phosphorylation on serine/threonine residues is also emerging as a key mediator of Gab2-dependent signal regulation. Despite its relevance, IL-2-triggered regulation on Gab2 phosphorylation is yet poorly understood. Combining antibody- and TiO2-based enrichment of the scaffold protein with SILAC quantitative mass spectrometry we disclose the prominent regulation IL-2 exerts on Gab2 serine/threonine phosphorylation by showing that at least 18 serines and 1 threonine, including previously non-reported ones, become phosphorylated in response to cytokine stimulation. Additionally, we decipher the interactome of the docking protein in resting and cytokine-treated T-lymphocytes and besides well-known Gab2 interactors we discover three novel cytokine-inducible Gab2-binding proteins. Thus, our data provide novel insights and a wealth of candidates for future studies that will shed light into the role of Gab2 in IL-2-initiated signal transduction.

This data article presents the first large-scale quantitative phosphoproteomics dataset generated to decipher the signaling networks initiated by IL-2 and IL-15 in T-lymphocytes. Data was collected by combining immunoprecipitation of tyrosine phosphorylated proteins and TiO2-based phosphopeptide enrichment with SILAC-based quantitative mass spectrometry. We report all the proteins and phosphotyrosine-containing peptides identified and quantified in IL-2- and IL-15-stimulated T-lymphocytes. The gene ontology analysis of IL-2 and IL-15 effector proteins detected in the present work is also included. The data supplied in this article is related to the research work entitled "Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics" [1]. All mass spectrometry data have been deposited in the ProteomeXchange with the identifier PXD001129.

Common γ-chain family of cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, where IL stands for interleukin) are key regulators of the immune homeostasis that exhibit pleiotropic biological activities and even sometimes redundant roles as a result of the utilization of the same receptor subunit. However, they also exert distinct functions that make each of them to be indispensable. For instance, all family members can act as T-cell growth factors; however, we found that IL-15 but not IL-7 can replace IL-2 to promote and sustain the proliferation of Kit225T cells. In addition to the γ-chain, IL-2 and IL-15 share the β-chain, which creates the paradox of how they can trigger diverse phenotypes despite signaling through the same receptors. To investigate this paradigm, we combined SILAC with enrichment of tyrosine-phosphorylated proteins and peptides followed by mass spectrometric analysis to quantitatively assess the signaling networks triggered downstream IL-2/IL-2R and IL-15/IL-15R. This study confirmed that the transduction pathways initiated by both cytokines are highly similar and revealed that the main signaling branches, JAK/STAT, RAS/MAPK and PI3K/AKT, were nearly equivalently activated in response to both ILs. Despite that, our study revealed that receptor internalization rates differ in IL-2- and IL-15-treated cells indicating a discrete modulation of cytokine signaling. All MS data have been deposited in the ProteomeXchange with identifier PXD001129 (http://proteomecentral.proteomexchange.org/dataset/PXD001129)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Liver diseases are the fifth cause of mortality in Western countries, and as opposed to other major causes of mortality, their incidence is increasing. Understanding the molecular background contributing to the progression of liver ailments will surely open new perspectives for the better management of patients. The aim of this study is to elucidate mechanisms underlying the progression of liver injury associated with deficient prohibitin 1, an essential protein to maintain mitochondrial homeostasis and gene expression. PHB1+/- mice developed a more severe steatohepatitis than WT littermates when exposed to a choline and methionine deficient diet. The increased sensitivity was mediated by mitochondrial dysfunction and metabolic impairment in PHB1+/- livers, including inactivation of AMP kinase, measured under a non-restricted diet. Moreover, pro-inflammatory challenges induced higher mortality and liver injury in PHB+/- mice. The increased proliferative capacity of PHB+/- splenocytes, resulting from constitutive defects in central molecular pathways as stated by deregulation of GSK3β, Erk, Akt or SHP-1, and the concomitant overproduction of pro-inflammatory mediators in Phb1 deficient mice, might account for these effects. In light of these results it might be concluded that Phb1 deficiency is a potential driver of chronic liver diseases by inducing hepatocyte damage and inflammation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Like other DNA viruses, herpes simplex virus type 1 (HSV-1) replicates and proliferates in host cells continuously modulating the host molecular environment. Following a sophisticated temporal expression pattern, HSV-1 encodes at least 89 multifunctional proteins that interplay with and modify the host cell proteome. During the last decade, advances in mass spectrometry applications coupled to the development of proteomic separation methods have allowed to partially monitor the impact of HSV-1 infection in human cells. In this review, we discuss the current use of different proteome fractionation strategies to define HSV-1 targets in two major application areas: (i) viral-protein interactomics to decipher viral-protein interactions in host cells and (ii) differential quantitative proteomics to analyze the virally induced changes in the cellular proteome. Moreover, we will also discuss the potential application of high-throughput proteomic approaches to study global proteome dynamics and also post-translational modifications in HSV-1-infected cells that will greatly improve our molecular knowledge of HSV-1 infection.

Reversible protein phosphorylation is a ubiquitous posttranslational modification that regulates cellular signaling pathways in multiple biological processes. A comprehensive analysis of protein phosphorylation patterns can only be achieved by employing different complementary experimental strategies all aiming at selective enrichment of phosphorylated proteins/peptides. In this chapter, we describe a method that utilizes a phosphoprotein affinity chromatography (Qiagen) to isolate intact phosphoproteins. These are subsequently detected by difference in two-dimensional gel electrophoresis and identified by mass spectrometry techniques. Additional experiments using a specific stain for phosphoproteins demonstrated that phosphoprotein affinity column was an effective method for enriching phosphate-containing proteins. Further validating the method, this workflow was applied to probe changes in the activation patterns of intermediates involved in different signaling pathways, such as NDRG1 and stathmin, in liver progenitor cells (MLP-29) upon proteasome inhibition.

Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal(+) infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal(+) induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27(Kip1) protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16-24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator.

Prohibitin is a multifunctional protein participating in a plethora of essential cellular functions, such as cell signaling, apoptosis, survival and proliferation. In the liver, deficient prohibitin activity participates in the progression of non-alcoholic steatohepatitis and obesity, according to mechanisms that still must be elucidated. In this study, we have used a combination of transcriptomics and proteomics technologies to investigate the response of human hepatoma PLC/PRF/5 cells to prohibitin silencing to define in detail the biological function of hepatic Phb1 and to elucidate potential prohibitin-dependent mechanisms participating in the maintenance of the transformed phenotype. Abrogation of prohibitin reduced proliferation and induced apoptosis in human hepatoma cells in a mechanism dependent on NF kappaB signaling. Moreover, down-regulation of ERp29 together with down-regulation of Erlin 2 suggests ER stress. In agreement, increased C/EBP homologous protein levels, poly-ADP ribose polymerase cleavage and activation of caspase 12 and downstream caspase 7 evidenced ER stress-induced apoptosis. Down-regulation of proteasome activator complex subunit 2 and stathmin as well as accumulation of ubiquitinated proteins suggest interplay between ER stress and proteasome malfunction. Taken together, our results provide evidences for prohibitin having a central role in the maintenance of the transformed and invasive phenotype of human hepatoma cells and may further support previous studies suggesting prohibitin as a potential clinical target.

Proteasome inhibitors are potential therapeutic agents in the treatment of hepatocarcinoma and other liver diseases. The analysis of alternative protein phosphorylation states might contribute to elucidate the underlying mechanisms of proteasome inhibitor-induced apoptosis. We have investigated the response of mouse liver progenitor-29 (MLP-29) cells to MG132 using a combination of phosphoprotein affinity chromatography, DIGE, and nano LC-MS/MS. Thirteen unique deregulated phosphoproteins involved in chaperone activity, stress response, mRNA processing and cell cycle control were unambiguously identified. Alterations in NDRG1 and stathmin suggest new mechanisms associated to proteasome inhibitor-induced apoptosis in MLP-29 cells. Particularly, a transient modification of the phosphorylation state of Ser(16), Ser(25) and Ser(38), which are involved in the regulation of stathmin activity, was detected in three distinct isoforms upon proteasome inhibition. The parallel deregulation of calcium/calmodulin-activated protein kinase II, extracellular regulated kinase-1/2 and cyclin-dependent kinase-2, might explain the modified phosphorylation pattern of stathmin. Interestingly, stathmin phosphorylation profile was also modified in response to epoxomicin treatment, a more specific proteasome inhibitor. In summary, we report here data supporting that regulation of NDRG1 and stathmin by phosphorylation at specific Ser/Thr residues may participate in the cellular response induced by proteasome inhibitors.

MTAP (5'-methylthioadenosine phosphorylase) catalyses the reversible phosphorolytic cleavage of methylthioadenosine leading to the production of methylthioribose-1-phosphate and adenine. Deficient MTAP activity has been correlated with human diseases including cirrhosis and hepatocellular carcinoma. In the present study we have investigated the regulation of MTAP by ROS (reactive oxygen species). The results of the present study support the inactivation of MTAP in the liver of bacterial LPS (lipopolysaccharide)-challenged mice as well as in HepG2 cells after exposure to t-butyl hydroperoxide. Reversible inactivation of purified MTAP by hydrogen peroxide results from a reduction of V(max) and involves the specific oxidation of Cys(136) and Cys(223) thiols to sulfenic acid that may be further stabilized to sulfenyl amide intermediates. Additionally, we found that Cys(145) and Cys(211) were disulfide bonded upon hydrogen peroxide exposure. However, this modification is not relevant to the mediation of the loss of MTAP activity as assessed by site-directed mutagenesis. Regulation of MTAP by ROS might participate in the redox regulation of the methionine catabolic pathway in the liver. Reduced MTA (5'-deoxy-5'-methylthioadenosine)-degrading activity may compensate for the deficient production of the precursor S-adenosylmethionine, allowing maintenance of intracellular MTA levels that may be critical to ensure cellular adaptation to physiopathological conditions such as inflammation.