A High-Resolution LED Stimulator for Steady-State Visual Stimulation: Customizable, Affordable, and Open Source.

Visually evoked steady-state potentials (SSVEPs) are neural responses elicited by visual stimuli oscillating at specific frequencies. In this study, we introduce a novel LED stimulator system explicitly designed for steady-state visual stimulation, offering precise control over visual stimulus parameters, including frequency resolution, luminance, and the ability to control the phase at the end of the stimulation. The LED stimulator provides a personalized, modular, and affordable option for experimental setups. Based on the Teensy 3.2 board, the stimulator utilizes direct digital synthesis and pulse width modulation techniques to control the LEDs. We validated its performance through four experiments: the first two measured LED light intensities directly, while the last two assessed the stimulator's impact on EEG recordings. The results demonstrate that the stimulator can deliver a stimulus suitable for generating SSVEPs with the desired frequency and phase resolution. As an open source resource, we provide comprehensive documentation, including all necessary codes and electrical diagrams, which facilitates the system's replication and adaptation for specific experimental requirements, enhancing its potential for widespread use in the field of neuroscience setups.

Source space connectomics of neurodegeneration: One-metric approach does not fit all.

Brain functional connectivity in dementia has been assessed with dissimilar EEG connectivity metrics and estimation procedures, thereby increasing results' heterogeneity. In this scenario, joint analyses integrating information from different metrics may allow for a more comprehensive characterization of brain functional interactions in different dementia subtypes. To test this hypothesis, resting-state electroencephalogram (rsEEG) was recorded in individuals with Alzheimer's Disease (AD), behavioral variant frontotemporal dementia (bvFTD), and healthy controls (HCs). Whole-brain functional connectivity was estimated in the EEG source space using 101 different types of functional connectivity, capturing linear and nonlinear interactions in both time and frequency-domains. Multivariate machine learning and progressive feature elimination was run to discriminate AD from HCs, and bvFTD from HCs, based on joint analyses of i) EEG frequency bands, ii) complementary frequency-domain metrics (e.g., instantaneous, lagged, and total connectivity), and iii) time-domain metrics with different linearity assumption (e.g., Pearson correlation coefficient and mutual information). <10% of all possible connections were responsible for the differences between patients and controls, and atypical connectivity was never captured by >1/4 of all possible connectivity measures. Joint analyses revealed patterns of hypoconnectivity (patientsHCs) in both groups was mainly identified in frontotemporal regions. These atypicalities were differently captured by frequency- and time-domain connectivity metrics, in a bandwidth-specific fashion. The multi-metric representation of source space whole-brain functional connectivity evidenced the inadequacy of single-metric approaches, and resulted in a valid alternative for the selection problem in EEG connectivity. These joint analyses reveal patterns of brain functional interdependence that are overlooked with single metrics approaches, contributing to a more reliable and interpretable description of atypical functional connectivity in neurodegeneration.

Unraveling Protein Interactions between the Temperate Virus Bam35 and Its Bacillus Host Using an Integrative Yeast Two Hybrid-High Throughput Sequencing Approach.

Bacillus virus Bam35 is the model Betatectivirus and member of the family Tectiviridae, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. Interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, which are known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein-protein interactions (PPIs) network for a tectivirus-host system by studying the Bam35-Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and high-throughput sequencing (Y2H-HTS). We generated and thoroughly analyzed a genomic library of Bam35's host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait-prey couples. Initial analysis of the raw data enabled the identification of over 4000 candidate interactions, which were sequentially filtered to produce 182 high-confidence interactions that were defined as part of the core virus-host interactome. Overall, host metabolism proteins and peptidases were particularly enriched within the detected interactions, distinguishing this host-phage system from the other reported host-phage PPIs. Our approach also suggested biological roles for several Bam35 proteins of unknown function, including the membrane structural protein P25, which may be a viral hub with a role in host membrane modification during viral particle morphogenesis. This work resulted in a better understanding of the Bam35-B. thuringiensis interaction at the molecular level and holds great potential for the generalization of the Y2H-HTS approach for other virus-host models.

Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins.

The family Tectiviridae comprises a group of tailless, icosahedral, membrane-containing bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well-characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria. In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing knowledge about the viral proteome, we carried out a comprehensive yeast two-hybrid analysis of all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprised 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggest that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, with the latter finding also being supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving nonstructural proteins, such as the DNA-binding protein P1 and the genome terminal protein (P4), which was confirmed by coimmunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle.IMPORTANCE Tailless viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown function. Comprehensive protein-protein interaction (PPI) analysis of viral proteins can eventually disclose biological mechanisms and thus provide new insights into protein function unattainable by studying proteins one by one. Here we comprehensively describe intraviral PPIs among tectivirus Bam35 proteins determined using multivector yeast two-hybrid screening, and these PPIs were further supported by the results of coimmunoprecipitation assays and protein structural models. This approach allowed us to propose new functions for known proteins and hypothesize about the biological role of the localization of some viral ORFan proteins within the viral particle that will be helpful for understanding the biology of tectiviruses infecting Gram-positive bacteria.

Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35.

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in a number of linear genomes of viruses, linear plasmids and mobile elements. By this mechanism, a so-called terminal protein (TP) primes replication and becomes covalently linked to the genome ends. Bam35 belongs to a group of temperate tectiviruses infecting Gram-positive bacteria, predicted to replicate their genomes by a protein-primed mechanism. Here, we characterize Bam35 replication as an alternative model of protein-priming DNA replication. First, we analyze the role of the protein encoded by the ORF4 as the TP and characterize the replication mechanism of the viral genome (TP-DNA). Indeed, full-length Bam35 TP-DNA can be replicated using only the viral TP and DNA polymerase. We also show that DNA replication priming entails the TP deoxythymidylation at conserved tyrosine 194 and that this reaction is directed by the third base of the template strand. We have also identified the TP tyrosine 172 as an essential residue for the interaction with the viral DNA polymerase. Furthermore, the genetic information of the first nucleotides of the genome can be recovered by a novel single-nucleotide jumping-back mechanism. Given the similarities between genome inverted terminal repeats and the genes encoding the replication proteins, we propose that related tectivirus genomes can be replicated by a similar mechanism.

DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity.

DNA polymerases (DNAPs) responsible for genome replication are highly faithful enzymes that nonetheless cannot deal with damaged DNA. In contrast, translesion synthesis (TLS) DNAPs are suitable for replicating modified template bases, although resulting in very low-fidelity products. Here we report the biochemical characterization of the temperate bacteriophage Bam35 DNA polymerase (B35DNAP), which belongs to the protein-primed subgroup of family B DNAPs, along with phage Φ29 and other viral and mobile element polymerases. B35DNAP is a highly faithful DNAP that can couple strand displacement to processive DNA synthesis. These properties allow it to perform multiple displacement amplification of plasmid DNA with a very low error rate. Despite its fidelity and proofreading activity, B35DNAP was able to successfully perform abasic site TLS without template realignment and inserting preferably an A opposite the abasic site (A rule). Moreover, deletion of the TPR2 subdomain, required for processivity, impaired primer extension beyond the abasic site. Taken together, these findings suggest that B35DNAP may perform faithful and processive genome replication in vivo and, when required, TLS of abasic sites.

NFAT transcription factors regulate survival, proliferation, migration, and differentiation of neural precursor cells.

The study of factors that regulate the survival, proliferation, and differentiation of neural precursor cells (NPCs) is essential to understand neural development as well as brain regeneration. The Nuclear Factor of Activated T Cells (NFAT) is a family of transcription factors that can affect these processes besides playing key roles during development, such as stimulating axonal growth in neurons, maturation of immune system cells, heart valve formation, and differentiation of skeletal muscle and bone. Interestingly, NFAT signaling can also promote cell differentiation in adults, participating in tissue regeneration. The goal of the present study is to evaluate the expression of NFAT isoforms in NPCs, and to investigate its possible role in NPC survival, proliferation, migration, and differentiation. Our findings indicate that NFAT proteins are active not only in neurogenic brain regions such as hippocampus and subventricular zone (SVZ), but also in cultured NPCs. The inhibition of NFAT activation with the peptide VIVIT reduced neurosphere size and cell density in NPC cultures by decreasing proliferation and increasing cell death. VIVIT also decreased NPC migration and differentiation of astrocytes and neurons from NPCs. In addition, we identified NFATc3 as a predominant NFAT isoform in NPC cultures, finding that a constitutively-active form of NFATc3 expressed by adenoviral infection reduces NPC proliferation, stimulates migration, and is a potent inducer of NPC differentiation into astrocytes and neurons. In summary, our work uncovers active roles for NFAT signaling in NPC survival, proliferation and differentiation, and highlights its therapeutic potential for tissue regeneration.

DIVA Meets EEG: Model Validation Using Formant-Shift Reflex.

The neurocomputational model 'Directions into Velocities of Articulators' (DIVA) was developed to account for various aspects of normal and disordered speech production and acquisition. The neural substrates of DIVA were established through functional magnetic resonance imaging (fMRI), providing physiological validation of the model. This study introduces DIVA_EEG an extension of DIVA that utilizes electroencephalography (EEG) to leverage the high temporal resolution and broad availability of EEG over fMRI. For the development of DIVA_EEG, EEG-like signals were derived from original equations describing the activity of the different DIVA maps. Synthetic EEG associated with the utterance of syllables was generated when both unperturbed and perturbed auditory feedback (first formant perturbations) were simulated. The cortical activation maps derived from synthetic EEG closely resembled those of the original DIVA model. To validate DIVA_EEG, the EEG of individuals with typical voices (N = 30) was acquired during an altered auditory feedback paradigm. The resulting empirical brain activity maps significantly overlapped with those predicted by DIVA_EEG. In conjunction with other recent model extensions, DIVA_EEG lays the foundations for constructing a complete neurocomputational framework to tackle vocal and speech disorders, which can guide model-driven personalized interventions.

Harmonized multi-metric and multi-centric assessment of EEG source space connectivity for dementia characterization.

Introduction: Harmonization protocols that address batch effects and cross-site methodological differences in multi-center studies are critical for strengthening electroencephalography (EEG) signatures of functional connectivity (FC) as potential dementia biomarkers. Methods: We implemented an automatic processing pipeline incorporating electrode layout integrations, patient-control normalizations, and multi-metric EEG source space connectomics analyses. Results: Spline interpolations of EEG signals onto a head mesh model with 6067 virtual electrodes resulted in an effective method for integrating electrode layouts. Z-score transformations of EEG time series resulted in source space connectivity matrices with high bilateral symmetry, reinforced long-range connections, and diminished short-range functional interactions. A composite FC metric allowed for accurate multicentric classifications of Alzheimer's disease and behavioral variant frontotemporal dementia. Discussion: Harmonized multi-metric analysis of EEG source space connectivity can address data heterogeneities in multi-centric studies, representing a powerful tool for accurately characterizing dementia.

Modelling neural entrainment and its persistence: influence of frequency of stimulation and phase at the stimulus offset.

Neural entrainment, the synchronization of brain oscillations to the frequency of an external stimuli, is a key mechanism that shapes perceptual and cognitive processes.Objective.Using simulations, we investigated the dynamics of neural entrainment, particularly the period following the end of the stimulation, since the persistence (reverberation) of neural entrainment may condition future sensory representations based on predictions about stimulus rhythmicity.Methods.Neural entrainment was assessed using a modified Jansen-Rit neural mass model (NMM) of coupled cortical columns, in which the spectral features of the output resembled that of the electroencephalogram (EEG). We evaluated spectro-temporal features of entrainment as a function of the stimulation frequency, the resonant frequency of the neural populations comprising the NMM, and the coupling strength between cortical columns. Furthermore, we tested if the entrainment persistence depended on the phase of the EEG-like oscillation at the time the stimulus ended.Main Results.The entrainment of the column that received the stimulation was maximum when the frequency of the entrainer was within a narrow range around the resonant frequency of the column. When this occurred, entrainment persisted for several cycles after the stimulus terminated, and the propagation of the entrainment to other columns was facilitated. Propagation also depended on the resonant frequency of the second column, and the coupling strength between columns. The duration of the persistence of the entrainment depended on the phase of the neural oscillation at the time the entrainer terminated, such that falling phases (fromπ/2 to 3π/2 in a sine function) led to longer persistence than rising phases (from 0 toπ/2 and 3π/2 to 2π).Significance.The study bridges between models of neural oscillations and empirical electrophysiology, providing insights to the mechanisms underlying neural entrainment and the use of rhythmic sensory stimulation for neuroenhancement.

Persistence of EEG Alpha Entrainment Depends on Stimulus Phase at Offset.

Neural entrainment is the synchronization of neural activity to the frequency of repetitive external stimuli, which can be observed as an increase in the electroencephalogram (EEG) power spectrum at the driving frequency, -also known as the steady-state response. Although it has been systematically reported that the entrained EEG oscillation persists for approximately three cycles after stimulus offset, the neural mechanisms underpinning it remain unknown. Focusing on alpha oscillations, we adopt the dynamical excitation/inhibition framework, which suggests that phases of entrained EEG signals correspond to alternating excitatory/inhibitory states of the neural circuitry. We hypothesize that the duration of the persistence of entrainment is determined by the specific functional state of the entrained neural network at the time the stimulus ends. Steady-state visually evoked potentials (SSVEP) were elicited in 19 healthy volunteers at the participants' individual alpha peaks. Visual stimulation consisted of a sinusoidally-varying light terminating at one of four phases: 0, π/2, π, and 3π/2. The persistence duration of the oscillatory activity was analyzed as a function of the terminating phase of the stimulus. Phases of the SSVEP at the stimulus termination were distributed within a constant range of values relative to the phase of the stimulus. Longer persistence durations were obtained when visual stimulation terminated towards the troughs of the alpha oscillations, while shorter persistence durations occurred when stimuli terminated near the peaks. Source localization analysis suggests that the persistence of entrainment reflects the functioning of fronto-occipital neuronal circuits, which might prime the sensory representation of incoming visual stimuli based on predictions about stimulus rhythmicity. Consequently, different states of the network at the end of the stimulation, corresponding to different states of intrinsic neuronal coupling, may determine the time windows over which coding of incoming sensory stimulation is modulated by the preceding oscillatory activity.

A Method for Tracking the Time Evolution of Steady-State Evoked Potentials.

Neural entrainment refers to the synchronization of neural activity to the periodicity of sensory stimuli. This synchronization defines the generation of steady-state evoked responses (i.e., oscillations in the electroencephalogram phase-locked to the driving stimuli). The classic interpretation of the amplitude of the steady-state evoked responses assumes a stereotypical time-invariant neural response plus random background fluctuations, such that averaging over repeated presentations of the stimulus recovers the stereotypical response. This approach ignores the dynamics of the steady-state, as in the case of the adaptation elicited by prolonged exposures to the stimulus. To analyze the dynamics of steady-state responses, it can be assumed that the time evolution of the response amplitude is the same in different stimulation runs separated by sufficiently long breaks. Based on this assumption, a method to characterize the time evolution of steady-state responses is presented. A sufficiently large number of recordings are acquired in response to the same experimental condition. Experimental runs (recordings) are column-wise averaged (i.e., runs are averaged but epoch within recordings are not averaged with the preceding segments). The column-wise averaging allows analysis of steady-state responses in recordings with remarkably high signal-to-noise ratios. Therefore, the averaged signal provides an accurate representation of the time evolution of the steady-state response, which can be analyzed in both the time and frequency domains. In this study, a detailed description of the method is provided, using steady-state visually evoked potentials as an example of a response. Advantages and caveats are evaluated based on a comparison with single-trial methods designed to analyze neural entrainment.

Context dependency, co-introductions, novel mutualisms, and host shifts shaped the ectomycorrhizal fungal communities of the alien tree Eucalyptus globulus.

The identity and relevance of the ectomycorrhizal (ECM) fungal partners of Eucalyptus globulus was investigated in NW Spain, to detect which symbionts mainly support its invasiveness. Root tips of E. globulus and of three common native plant species (Quercus robur, Pinus pinaster and Halimium lasianthum) were collected in eucalypt plantations, Q. robur forests, P. pinaster plantations and shrublands. Fungal taxonomical identity was ascertained by use of rDNA and direct sequencing. We studied diversity, composition and colonization rate of the ECM fungal communities of E. globulus to determine if fungal assemblages are host specific (i.e. similar in different habitats) or more dependent on the neighbourhood context. We also identified the type of associations formed (i.e. co-introductions, familiar or novel associations). Twenty-six ECM taxa were associated with E. globulus. Most of them engaged in novel associations with eucalypts, whereas only three fungal species were co-introduced Australian aliens. Eucalypt fungal richness, diversity and colonization rate differed between habitats, being higher in native oak forests, whereas in shrublands E. globulus showed the lowest colonization rate and diversity. The Australian fungus Descolea maculata dominated the eucalypt fungal assemblage and also spread to the native host plants, in all the habitats, posing the risk of further co-invasion.

The dual lifestyle of genome-integrating virophages in protists.

DNA viruses with efficient host genome integration capability were unknown in eukaryotes until recently. The discovery of virophages, satellite-like DNA viruses that depend on lytic giant viruses that infect protists, revealed a genetically diverse group of viruses with high genome mobility. Virophages can act as strong inhibitors of their associated giant viruses, and the resulting beneficial effects on their unicellular hosts resemble a population-based antiviral defense mechanism. By comparing various aspects of genome-integrating virophages, in particular the virophage mavirus, with other mobile genetic elements and parasite-derived defense mechanisms in eukaryotes and prokaryotes, we show that virophages share many features with other host-parasite systems. Yet, the dual lifestyle exhibited by mavirus remains unprecedented among eukaryotic DNA viruses, with potentially far-reaching ecological and evolutionary consequences for the host.

Characterization of Retinal Functionality at Different Eccentricities in a Diurnal Rodent.

Although the properties of the neurons of the visual system that process central and peripheral regions of the visual field have been widely researched in the visual cortex and the LGN, they have scarcely been documented for the retina. The retina is the first step in integrating optical signals, and despite considerable efforts to functionally characterize the different types of retinal ganglion cells (RGCs), a clear account of the particular functionality of cells with central vs. peripheral fields is still wanting. Here, we use electrophysiological recordings, gathered from retinas of the diurnal rodent Octodon degus, to show that RGCs with peripheral receptive fields (RF) are larger, faster, and have shorter transient responses. This translates into higher sensitivity at high temporal frequencies and a full frequency bandwidth when compared to RGCs with more central RF. We also observed that imbalances between ON and OFF cell populations are preserved with eccentricity. Finally, the high diversity of functional types of RGCs highlights the complexity of the computational strategies implemented in the early stages of visual processing, which could inspire the development of bio-inspired artificial systems.

Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay.

This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules out nucleic acid-mediated indirect interaction. Then, a second immunoprecipitation step is performed using the purified putative partner protein. Co-immunoprecipitated proteins can be detected either by Coomassie Blue staining and/or Western blotting (WB) if a specific antibody is available. Moreover, many DNA/RNA binding proteins are highly electropositive, which can hinder WB under standard conditions, as has been shown in histones and histone-like proteins. In this case, we show that the high isoelectric point of the putative partner results in a poor transfer. Tips to troubleshot WB transfer of highly electropositive DNA-binding proteins are provided.

Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements.

Family B DNA polymerases (PolBs) play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB), that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance.

Tercer molar inferior ectópico asociado a un quiste dentígero: caso / Ectopic lower third molar associated to a dentigerous cyst: a case report

La exodoncia de piezas dentariass ectópicas no es frecuente; tampoco lo es la asociación de dicha pieza a un quiste folicular. Se presenta un caso de una paciente de 50 años de edad con una pieza retenida en la rama ascendente del maxilar inferior; se efectúa un diagnóstico clínico y radiográfico preciso de manera tal de no lesionar las estructuras anatómicas vecinas de gran importancia y se extrae la pieza mediante abordaje intraoral y bajo anestesia general.

Cáncer de labio inferior: reconstrucción con técnica de Karapandjic / Lower lip cancer: reconstruction with the Karapandjic technique

La técnica de Karapandjic fue inicialmente descripta en el año 1974 y se indica en resecciones del labio inferior, donde la pérdida de sustancia no es muy importante. La característica principal es su fácil realización y los resultados son aceptables, tanto en estética como en funcionalidad. En este trabajo se presenta la aplicación de la misma en un caso de carcinoma espinocelular

Reconstrucción de defectos intraorales con la bolsa adiposa de Bichat / Reconstruction of intraoral defects with the buccal fat pad

El propósito de este estudio es evaluar la utilización de la bosa adiposa de Bichat para el cierre de defectos bucales. Durane los años 2000 a 2004 se utilizó la bola adiposa en 19 pacientes, para la reconstrucción de distintos defectos: 13 comunicaciones bucosinusales, 5 defectos resultantes de escisiones tumorales y 1 quiste maxilar. Las resecciones neoplásicas fueron 3 adenomas pleomorfos y 2 carcinomas mucoepidermoides de bajo grado de malignidad. Se evaluó la epitelización del colgajo, infección, recurrencia fistular y deficiencias del controno facial. El proceso de epitelización se completó luego de 30 a 40 días. Tan sólo un paciente sufrió la persistencia de una fístula oronasal, la cual debió luego ser tratada mediante un colgajo palatino. Los resultados obtenidos de este estudio indican que la utilización de la bola adiposa de Bichat para el cierre de defectos intraorales pequeños o medianos es un método confiable, sencillo y seguro