Robust brain-computer interfaces

A brain-computer interface (BCI) enables direct communication from the brain to devices, bypassing the traditional pathway of peripheral nerves and muscles. Current BCIs aimed at patients require that the user invests weeks, or even months, to learn the skill to intentionally modify their brain signals. This can be reduced to a calibration session of about half an hour per session if machine learning (ML) methods are used. The laborious recalibration is still needed due to inter-session differences in the statistical properties of the electroencephalography (EEG) signal. Further, the natural variability in spontaneous EEG violates basic assumptions made by the ML methods used to train the BCI classifier, and causes the classification accuracy to fluctuate unpredictably. These fluctuations make the current generation of BCIs unreliable. In this dissertation,we will investigate the nature of these variations in the EEG distributions, and introduce two new, complementary methods to overcome these two key issues. To confirm the problem of non-stationary brain signals, we first show that BCIs based on commonly used signal features are sensitive to changes in the mental state of the user. We proceed by describing a method aimed at removing these changes in signal feature distributions. We have devised a method that uses a second-order baseline (SOB) to specifically isolate these relative changes in neuronal firing synchrony. To the best of our knowledge this is the first BCI classifier that works on out-of-sample subjects without any loss of performance. Still, the assumption made by ML methods that the training data consists of samples that are independent and identically distributed (iid) is violated, because EEG samples nearby in time are highly correlated. Therefore we derived a generalization of the well-known support vector machine (SVM) classifier, that takes the resulting chronological structure of classification errors into account. Both on artificial data and real BCI data, overfitting is reduced with this dependent samples support vector machine (dSVM), leading to BCIs with an increased information throughput

Genetic modifiers of Duchenne muscular dystrophy and dilated cardiomyopathy

OBJECTIVE: Dilated cardiomyopathy (DCM) is a major complication and leading cause of death in Duchenne muscular dystrophy (DMD). DCM onset is variable, suggesting modifier effects of genetic or environmental factors. We aimed to determine if polymorphisms previously associated with age at loss of independent ambulation (LoA) in DMD (rs28357094 in the SPP1 promoter, rs10880 and the VTTT/IAAM haplotype in LTBP4) also modify DCM onset. METHODS: A multicentric cohort of 178 DMD patients was genotyped by TaqMan assays. We performed a time-to-event analysis of DCM onset, with age as time variable, and finding of left ventricular ejection fraction 70 mL/m2 as event (confirmed by a previous normal exam < 12 months prior); DCM-free patients were censored at the age of last echocardiographic follow-up. RESULTS: Patients were followed up to an average age of 15.9 \ub1 6.7 years. Seventy-one/178 patients developed DCM, and median age at onset was 20.0 years. Glucocorticoid corticosteroid treatment (n = 88 untreated; n = 75 treated; n = 15 unknown) did not have a significant independent effect on DCM onset. Cardiological medications were not administered before DCM onset in this population. We observed trends towards a protective effect of the dominant G allele at SPP1 rs28357094 and recessive T allele at LTBP4 rs10880, which was statistically significant in steroid-treated patients for LTBP4 rs10880 (< 50% T/T patients developing DCM during follow-up [n = 13]; median DCM onset 17.6 years for C/C-C/T, log-rank p = 0.027). CONCLUSIONS: We report a putative protective effect of DMD genetic modifiers on the development of cardiac complications, that might aid in risk stratification if confirmed in independent cohorts

2-Deoxy-D-glucose couples mitochondrial DNA replication with mitochondrial fitness and promotes the selection of wild-type over mutant mitochondrial DNA

Pathological variants of human mitochondrial DNA (mtDNA) typically co-exist with wild-type molecules, but the factors driving the selection of each are not understood. Because mitochondrial fitness does not favour the propagation of functional mtDNAs in disease states, we sought to create conditions where it would be advantageous. Glucose and glutamine consumption are increased in mtDNA dysfunction, and so we targeted the use of both in cells carrying the pathogenic m.3243A>G variant with 2-Deoxy-D-glucose (2DG), or the related 5-thioglucose. Here, we show that both compounds selected wild-type over mutant mtDNA, restoring mtDNA expression and respiration. Mechanistically, 2DG selectively inhibits the replication of mutant mtDNA; and glutamine is the key target metabolite, as its withdrawal, too, suppresses mtDNA synthesis in mutant cells. Additionally, by restricting glucose utilization, 2DG supports functional mtDNAs, as glucose-fuelled respiration is critical for mtDNA replication in control cells, when glucose and glutamine are scarce. Hence, we demonstrate that mitochondrial fitness dictates metabolite preference for mtDNA replication; consequently, interventions that restrict metabolite availability can suppress pathological mtDNAs, by coupling mitochondrial fitness and replication.publishedVersio

DMPK prevents ROS-induced cell death by assembling a HK II-Src complex on mitochondrial surface

DMPK is a serine/threonine protein kinase that was initially proposed to be the cause of the most frequent adult muscular dystrophy, myotonic dystrophy 1 (DM1). Recently, it has been shown that DMPK is not the primary determinant of the DM1, but its deletion causes late onset myopathy and cardiac abnormalities in knock-out mice. Evidence present in the literature suggests a mitochondrial localization of high MW DMPK isoforms in muscle and cardiac tissue. However, to date, there is not a single association of mitochondria-anchored isoforms with the respective function of the organelle in the affected tissues. Therefore, we have examined the role of mitochondria-anchored isoform A, either by stably expressing it in cells lacking endogenous protein, or by stably silencing the endogenous one. DMPK significantly decreased levels of mitochondrial superoxide and consequently increased cell survival in prolonged serum and glucose depletion, both in SAOS-2 and rhabdomyosarcoma cells. At the molecular level we have found DMPK to interact with HK II and Src, increasing the HK II association to mitochondria. Detachment of HK II from mitochondria abolished differences in superoxide levels, while a HK II inhibitor 5-TG protected cells from death by stabilizing HK II on the OMM and by decreasing mitochondrial ROS in the absence of DMPK. Src activity was also important for HK II maintenance on OMM since its inhibition sensitized only DMPK-expressing cells to detachment of HK II. These data attribute an antiapoptotic role to DMPK due to an unprecedented link to HK II and its protective effect against mitochondrial ROS.DMPK è la serina/treonina protein kinasi, la quale è stata inizialmente proposta come la causa della più frequente distrofia muscolare negli adulti, la distrofia miotonica del tipo 1 (DM1). Recentemente si è visto che la DMPK non è la causa principale della DM1, ma la sua delezione causa miopatia ad insorgenza tardiva e anomalie cardiache nei topi knock-out. I dati presenti in letteratura attribuiscono la localizzazione mitocondriale alle isoforme ad alto peso molecolare nel muscolo e nel tessuto cardiaco. Comunque, finora non vi sono stati studi volti ad associare il ruolo delle isoforme mitocondriali della DMPK alla funzione dell’organulo nei tessuti in questione. Perciò, abbiamo deciso di esaminare il ruolo dall’isoforma A associata ai mitocondri, sia esprimendola stabilmente nelle cellule prive della DMPK endogena, sia silenziando stabilmente quella endogena. DMPK ha significativamente diminuito i livelli del superossido mitocondriale e, di conseguenza, ha aumentato la sopravvivenza delle cellule SAOS-2 e rabdomiosarcoma in deplezione di siero e glucosio. A livello molecolare, abbiamo trovato che la DMPK interagisce con HK II e Src aumentando l’associazione dell’HK II ai mitocondri. Il distacco dell’HK II dai mitocondri ha cancellato le differenze nei livelli di superossido, mentre l’inibitore dell’HK II 5-TG ha protetto le cellule dalla morte stabilizzando l’HK II sulla membrana mitocondriale esterna e diminuendo i livelli di ROS mitocondriali in assenza della DMPK. Src aveva la funzione di mantenere HK II sulla membrana mitocondriale esterna, in quanto la sua inibizione ha sensibilizzato le cellule al distacco dell’HK II solo se esprimevano la DMPK. Questo studio attribuisce un ruolo anti-apoptotico alla DMPK grazie all’interazione con HK II e la sua funzione protettiva contro i ROS di origine mitocondriale

Signal transduction to the permeability transition pore

The permeability transition pore (PTP) is an inner mitochondrial membrane channel that has been thoroughly characterized functionally, yet remains an elusive molecular entity. The best characterized PTP-regulatory component, cyclophilin (CyP) D, is a matrix protein that favors pore opening. CyP inhibitors, CyP-D null animals, and in situ PTP readouts have established the role of PTP as an effector mechanism of cell death, and the growing definition of PTP signalling mechanisms. This review briefly covers the functional features of the PTP and the role played by its dysregulation in disease pathogenesis. Recent progress on PTP modulation by kinase/phosphatase signal transduction is discussed, with specific emphasis on hexokinase and on the Akt-ERK-GSK3 axis, which might modulate the PTP through CyP-D phosphorylation

Acknowledgements

Laughter is a highly variable signal, and can express a spectrum of emotions. This makes the automatic detection of laughter a challenging but interesting task. We perform automatic laughter detection using audio-visual data from the AMI Meeting Corpus. Audio-visual laughter detection is performed by combining (fusing) the results of a separate audio and video classifier on the decision level. The video-classifier uses features based on the principal components of 20 tracked facial points, for audio we use the commonly used PLP and RASTA-PLP features. Our results indicate that RASTA-PLP features outperform PLP features for laughter detection in audio. We compared hidden Markov models (HMMs), Gaussian mixture models (GMMs) and support vector machines (SVM) based classifiers, and found that RASTA-PLP combined with a GMM resulted in the best performance for the audio modality. The video features classified using a SVM resulted in the best single-modality performance. Fusion on the decision-level resulted in laughter detection with a significantly better performance than single-modality classification. 3

Myotonic dystrophy protein kinase (DMPK) prevents ROS-induced cell death by assembling a hexokinase II-Src complex on the mitochondrial surface

The biological functions of myotonic dystrophy protein kinase (DMPK), a serine/threonine kinase whose gene mutations cause myotonic dystrophy type 1 (DM1), remain poorly understood. Several DMPK isoforms exist, and the long ones (DMPK-A/B/C/D) are associated with the mitochondria, where they exert unknown activities. We have studied the isoform A of DMPK, which we have found to be prevalently associated to the outer mitochondrial membrane. The kinase activity of mitochondrial DMPK protects cells from oxidative stress and from the ensuing opening of the mitochondrial permeability transition pore (PTP), which would otherwise irreversibly commit cells to death. We observe that DMPK (i) increases the mitochondrial localization of hexokinase II (HK II), (ii) forms a multimeric complex with HK II and with the active form of the tyrosine kinase Src, binding its SH3 domain and (iii) it is tyrosine-phosphorylated by Src. Both interaction among these proteins and tyrosine phosphorylation of DMPK are increased under oxidative stress, and Src inhibition selectively enhances death in DMPK-expressing cells after HK II detachment from the mitochondria. Down-modulation of DMPK abolishes the appearance of muscle markers in in vitro myogenesis, which is rescued by oxidant scavenging. Our data indicate that, together with HK II and Src, mitochondrial DMPK is part of a multimolecular complex endowed with antioxidant and pro-survival properties that could be relevant during the function and differentiation of muscle fibers

Apoptosis regulation by the mitochondrial chaperone TRAP-1/HSP-75

Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues. Shigella spp. are human pathogens that invade colonic mucosa, where they provoke lesions caused by their ability to manipulate the host cell responses. Shigella spp. induce various types of cell death in different cell populations. However, they are equally able to protect host cells from death. Here, we have investigated on the molecular mechanisms and cell effectors governing the balance between survival and death in epithelial cells infected with Shigella. To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis. Our results definitely show that Shigella induces a rapid intrinsic apoptosis of infected cells, via mitochondrial depolarization and the ensuing caspase-9 activation. Moreover, for the first time we identify the eukaryotic stress-response factor growth arrest and DNA damage 45a as a key player in the induction of the apoptotic process elicited by Shigella in epithelial cells, revealing an unexplored role of this molecule in the course of infections sustained by invasive pathogens. Type II hexokinase (HKII) is overexpressed in the outer mitochondrial membrane of most neoplastic cells. Current work postulates that HKII release from its mitochondrial interactor, the voltage-dependent anion channel, prompts outer mitochondrial membrane permeabilization and the ensuing release of apoptogenic proteins, and that these events are inhibited by growth factors. Here we show that a HKII Nterminal peptide selectively detaches HKII from mitochondria transduces a permeability transition pore opening signal that results in cell death, does not require the voltage-dependent anion channel and is not affected by insulin stimulation. These findings have implications for our understanding of the pathways of outer mitochondrial membrane permeabilization and their inactivation in tumors. TRAP1 is a mitochondrial chaperone also known as heat shock protein 75, which is overexpressed in several tumor cell types. Herewe analyze whether mitochondrial TRAP1 elicits cytoprotective functions in a model of human osteosarcoma, SAOS-2 cells, either wild-type or in which TRAP1 expressionwas knocked down by RNA interference. Cells were exposed to different kinds of pro-apoptotic stimuli: chemotherapeutics, oxidative stress or death ligands, and several apoptotic parameters were measured in order to dissect whether and how TRAP1 impacts on these stress-induced transduction pathways. TRAP1 displays a general antiapoptotic role in all the examined conditions, whereas TRAP1 interference increases cell sensitivity to death. Serine phosphorylation and mitochondrial localization are required for TRAP1 cytoprotective function. In fact, a deletion mutant lacking the mitochondrial import sequence is not phosphorylated and is unable to counteract apoptosis induction in all conditions. Preliminary data showthat TRAP1 interacts with Bcl-2 family proteins and is involved in the regulation of the mitochondrial permeability transition pore opening. Altogether, these results suggest that TRAP1 acts as a key anti-apoptotic molecule in mitochondria of neoplastic cells

Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA.

Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants