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Atypical sensory processing is common in autism, but how neural coding is disrupted in sensory cortex is unclear. We evaluate whisker touch coding in L2/3 of somatosensory cortex (S1) in Cntnap2-/- mice, which have reduced inhibition. This classically predicts excess pyramidal cell spiking, but this remains controversial, and other deficits may dominate. We find that c-fos expression is elevated in S1 of Cntnap2-/- mice under spontaneous activity conditions but is comparable to that of control mice after whisker stimulation, suggesting normal sensory-evoked spike rates. GCaMP8m imaging from L2/3 pyramidal cells shows no excess whisker responsiveness, but it does show multiple signs of degraded somatotopic coding. This includes broadened whisker-tuning curves, a blurred whisker map, and blunted whisker point representations. These disruptions are greater in noisy than in sparse sensory conditions. Tuning instability across days is also substantially elevated in Cntnap2-/-. Thus, Cntnap2-/- mice show no excess sensory-evoked activity, but a degraded and unstable tactile code in S1.
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The formation and analysis of amyloid fibers by two ß-glucosidases, BglA and BglB, belonging to the GH1 enzyme family, are reported. Both proteins have the (ß/α)8 TIM-barrel fold, which is characteristic of this family and is also the most common protein structure. BglA is an octamer, whereas BglB is a monomer. Amyloid fibrillation using pH and temperature as perturbing agents was investigated using fluorescence spectroscopy as a preliminary approach and corroborated using wide-field optical microscopy, confocal microscopy, and field-emission scanning electron microscopy. These analyses showed that both enzymes fibrillate at a wide range of acidic and alkaline conditions and at several temperature conditions, particularly at acidic pH (3-4) and at temperatures between 45 and 65 °C. Circular dichroism spectroscopy corroborated the transition from an α-helix to a ß-sheet secondary structure of both proteins in conditions where fibrillation was observed. Overall, our results suggest that fibrillation is a rather common phenomenon caused by protein misfolding, driven by a transition from an α-helix to a ß-sheet secondary structure, that many proteins can undergo if subjected to conditions that disturb their native conformation.
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Amiloide , Amiloide/química , Amiloide/metabolismo , Concentración de Iones de Hidrógeno , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Dicroismo Circular , Temperatura , Estructura Secundaria de Proteína , Pliegue de ProteínaRESUMEN
The barrel fill level is defined as the fraction of the free available volume for a given screw configuration that is occupied by the wet material and is an interplay of the material throughput, screw speed, screw setup, barrel length of the twin-screw granulator used and the properties of the starting material. The fill level has a major impact on mixing and densification of the wetted mass and thus on the granules produced. It influences the twin-screw granulation process accordingly. In the current study, a model has been developed which is predictive in terms of material hold-ups in the barrel at various process settings by considering the geometries of the different screw elements in a configuration and the conveying velocity of the wet mass through the barrel. The model was checked on two granulators of different dimensions with various screw configurations, different materials and at different process settings. The model represents a step forward in predicting the barrel fill level but further research with a broader spectrum of materials, screw configurations and process settings is still needed and additional twin-screw granulators of other dimensions must be investigated.
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Early life experiences shape physical and behavioral outcomes throughout lifetime. Sensory circuits are especially susceptible to environmental and physiological changes during development. However, the impact of different types of early life experience are often evaluated in isolation. In this mini review, we discuss the specific effects of postnatal sensory experience, sleep, social isolation, and substance exposure on barrel cortex development. Considering these concurrent factors will improve understanding of the etiology of atypical sensory perception in many neuropsychiatric and neurodevelopmental disorders.
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Corteza Somatosensorial , Corteza Somatosensorial/fisiología , Corteza Somatosensorial/crecimiento & desarrollo , Animales , Humanos , Aislamiento Social/psicología , Sueño/fisiologíaRESUMEN
Biological nanopores are ultrasensitive and highly attractive platforms for disease diagnostics, including the sequencing of viral and microbial genes and the detection of biomarkers and pathogens. To utilize biological nanopores as diagnostic sensors, they have been engineered through various methods resulting in the accurate and highly sensitive detection of biomarkers and disease-related biomolecules. Among diverse biological nanopores, the ß-barrel-containing nanopores have advantages in nanopore engineering because of their robust structure, making them well-suited for modifications. In this review, we highlight the engineering approaches for ß-barrel-containing nanopores used in single-molecule sensing for applications in early diagnosis and prognosis. In the highlighted studies, ß-barrel nanopores can be modified by genetic mutation to change the structure; alter charge distributions; or add enzymes, aptamers, and protein probes to enhance sensitivity and accuracy. Furthermore, this review discusses challenges and future perspectives for advancing nanopore-based diagnostic sensors.
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Técnicas Biosensibles , Nanoporos , Humanos , Biomarcadores/análisisRESUMEN
PURPOSE: Trigonocephaly is the most common craniosynostosis involving orbits. Although some degree of agreement has been reached regarding surgical timing and indications for treatment, there is no consensus regarding the ideal operative technique to guarantee an optimal morphological outcome. The purpose of this study is to describe both strategies and to compare morphological outcomes by means of morphological surface analysis obtained from three-dimensional (3D) stereophotogrammetry, with two different techniques. METHODS: We retrospectively investigated 43 patients with metopic synostosis surgically treated between 2004 and 2020. Two different techniques were applied, addressed as technique A and B. Ten patients undergone postoperative 3d stereophotogrammetry were enrolled, and cephalometric measurements were taken and compared to a cohort of unaffected patients matched by age and gender. RESULTS: Comparison of the groups demonstrated a hypercorrection of the metopic angle of the second technique, associated with a slightly lower correction of the interfrontoparietal diameter. The metopic angle showed to be significantly undercorrected with the first method. CONCLUSIONS: Alternated barrel staving technique appears to be a quick and satisfactory method in cranial remodelling for metopic synostosis. It guarantees an optimal aesthetic result in the first years after surgery.
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Sensory adaptation is the process whereby brain circuits adjust neuronal activity in response to redundant sensory stimuli. Although sensory adaptation has been extensively studied for individual neurons on timescales of tens of milliseconds to a few seconds, little is known about it over longer timescales or at the population level. We investigated population-level adaptation in the barrel field of the mouse somatosensory cortex (S1BF) using in vivo two-photon calcium imaging and Neuropixels recordings in awake mice. Among stimulus-responsive neurons, we found both adapting and facilitating neurons, which decreased or increased their firing, respectively, with repetitive whisker stimulation. The former outnumbered the latter by 2:1 in layers 2/3 and 4; hence, the overall population response of mouse S1BF was slightly adapting. We also discovered that population adaptation to one stimulus frequency (5 Hz) does not necessarily generalize to a different frequency (12.5 Hz). Moreover, responses of individual neurons to repeated rounds of stimulation over tens of minutes were strikingly heterogeneous and stochastic, such that their adapting or facilitating response profiles were not stable across time. Such representational drift was particularly striking when recording longitudinally across 8-9 days, as adaptation profiles of most whisker-responsive neurons changed drastically from one day to the next. Remarkably, repeated exposure to a familiar stimulus paradoxically shifted the population away from strong adaptation and toward facilitation. Thus, the adapting vs. facilitating response profile of S1BF neurons is not a fixed property of neurons but rather a highly dynamic feature that is shaped by sensory experience across days.
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Adaptación Fisiológica , Corteza Somatosensorial , Vibrisas , Animales , Corteza Somatosensorial/fisiología , Ratones , Vibrisas/fisiología , Adaptación Fisiológica/fisiología , Masculino , Neuronas/fisiología , Ratones Endogámicos C57BL , Femenino , Estimulación FísicaRESUMEN
Spontaneous and sensory-evoked activity sculpts developing circuits. Yet, how these activity patterns intersect with cellular programs regulating the differentiation of neuronal subtypes is not well understood. Through electrophysiological and in vivo longitudinal analyses, we show that C-X-C motif chemokine ligand 14 (Cxcl14), a gene previously characterized for its association with tumor invasion, is expressed by single-bouquet cells (SBCs) in layer I (LI) of the somatosensory cortex during development. Sensory deprivation at neonatal stages markedly decreases Cxcl14 expression. Additionally, we report that loss of function of this gene leads to increased intrinsic excitability of SBCs-but not LI neurogliaform cells-and augments neuronal complexity. Furthermore, Cxcl14 loss impairs sensory map formation and compromises the in vivo recruitment of superficial interneurons by sensory inputs. These results indicate that Cxcl14 is required for LI differentiation and demonstrate the emergent role of chemokines as key players in cortical network development.
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Acetic acid bacteria are used in many industrial processes such as the production of vinegar, vitamin C, the antidiabetic drug miglitol, and various artificial flavorings. These industrially important reactions are primarily carried out by an arsenal of periplasmic-facing membrane-bound dehydrogenases that incompletely oxidize their substrates and shuttle electrons directly into the respiratory chain. Among these dehydrogenases, GOX1969 in Gluconobacter oxydans was predicted to be a pyrroloquinoline quinone-dependent dehydrogenase of unknown function. However, after multiple analysis by a number of labs, no dehydrogenase activity has been detected. Reanalysis of GOX1969 sequence and structure reveals similarities to Escherichia coli BamB, which functions as a subunit of the ß-barrel assembly machinery complex that is responsible for the assembly of ß-barrel outer membrane proteins in Gram-negative bacteria. To test if the physiological function of GOX1969 is similar to BamB in E. coli, we introduced the gox1969 gene into an E. coli ∆bamB mutant. Growth deficiencies in the ∆bamB mutant were restored when gox1969 was expressed on the plasmid pGox1969. Furthermore, increased membrane permeability conferred by bamB deletion was restored upon gox1969 expression, which suggests a direct link between GOX1969 and a role in maintaining outer membrane stability. Together, this evidence strongly suggests that GOX1969 is functionally acting as a BamB in G. oxydans. As such, functional information on uncharacterized genes will provide new insights that will allow for more accurate modeling of acetic acid bacterial metabolism and further efforts to design rational strains for industrial use.IMPORTANCEGluconobacter oxydans is an industrially important member of the acetic acid bacteria. Experimental characterization of putative genes is necessary to identify targets for further engineering of rational acetic acid bacteria strains that can be used in the production of vitamin C, antidiabetic compounds, artificial flavorings, or novel compounds. In this study, we have identified an undefined dehydrogenase GOX1969 with no known substrate and defined structural similarities to outer membrane biogenesis protein BamB in E. coli K12. Furthermore, we demonstrate that GOX1969 is capable of complementing bamB knockout phenotypes in E. coli K12. Taken together, these findings enhance our understanding of G. oxydans physiology and expand the list of potential targets for future industrial strain design.
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Escherichia coli , Gluconobacter oxydans , Gluconobacter oxydans/metabolismo , Gluconobacter oxydans/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
In the last decade, "expressions" of grape marc spirits aged in wooden barrels of characteristic amber color and complex sensory attributes have been introduced. Yet studies on constituents migrating from the barrel to the beverage are scarce, and their metabolic profile remains unexplored. Furthermore, the literature on the assessment of their antioxidant activity is limited. NMR metabolomics and spectrophotometry have been implemented in 38 samples to elucidate the impact of the aging procedure on the metabolites' composition and establish whether these beverages exhibit antioxidant activity. Provenance was related to fusel alcohols, esters, acetaldehyde, methanol, saccharides, and 2-phenylethanol, while ethyl acetate and ethyl lactate contributed to discriminating samples of the same winery. Identified metabolites such as vanillin, syringaldehyde, and sinapaldehyde were related to the aging procedure. The maturation in the barrel was also associated with an increase in xylose, glucose, fructose, and arabinose. The antioxidant potential of the aged Greek grape marc spirits resulting from their maturation in oak barrels was highlighted. The metabolic profiling and antioxidant potential of aged Greek grape marc spirits were assessed for the first time. Finally, the enrichment of the aromatic region was noted with the presence of metabolites with a furanic and phenolic ring derived, respectively, from the polysaccharides' degradation or the thermal decomposition of lignin.
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The effect of halide substitution in Grubbs-Hoveyda II catalysts (GHII catalysts) embedded in the engineered ß-barrel protein nitrobindin (NB4exp) on metathesis activity in aqueous media was studied. Maleimide tagged dibromido and diiodido derivates of the GHII catalyst were synthesized and covalently conjugated to NB4exp. The biohybrid catalysts were characterized spectroscopically confirming the structural integrity. When the two chloride substituents at ruthenium center were exchanged against bromide and iodide, the diiodo derivative was found to show significantly higher catalytic activity in ring-closing metathesis of α,ω-diolefins, whereas the dibromido derivative was less efficient when compared with the parent dichlorido catalyst. Using the diiodido catalyst, high turnover numbers of up to 75 were observed for ring-closing metathesis (RCM) yielding unsaturated six- and seven-membered N-heterocycles.
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Alquenos , Catálisis , Alquenos/química , Rutenio/química , Conformación Proteica en Lámina betaRESUMEN
Background: Mandibular defects resulting from oncological treatment pose significant aesthetic and functional challenges due to the involvement of bone and soft tissues. Immediate reconstruction is crucial to address complications such as malocclusion, mandibular deviation, temporomandibular joint (TMJ) changes, and soft tissue retraction. These issues can lead to functional impairments, including difficulties in chewing, swallowing, and speech. The fibula flap is widely used for mandibular reconstruction due to its long bone segment and robust vascular supply, though it may not always provide adequate bone height for optimal dental rehabilitation. This systematic review aims to determine if the double-barreled fibula flap (DBFF) configuration is a viable alternative for mandibular reconstruction and to evaluate the outcomes of dental implants placed in this type of flap. Materials and Methods: This study adhered to the Cochrane Collaboration criteria and PRISMA guidelines and was registered on the International Platform of Registered Systematic Review and Meta-Analysis Protocols Database (INPLASY2023120026). We included clinical studies published in English, Spanish, or French that focused on adult patients undergoing segmental mandibulectomy followed by DBFF reconstruction and dental rehabilitation. Data sources included Medline/PubMed, the Cochrane Library, EMBASE, Scopus, and manual searches. Two reviewers independently screened and selected studies, with discrepancies resolved by a third reviewer. Data extraction captured variables such as publication year, patient demographics, number of implants, follow-up duration, flap survival, implant failure, and aesthetic outcomes. The risk of bias was assessed using the JBI appraisal tool, and the certainty of evidence was evaluated using the GRADE approach. Results: A total of 17 clinical studies were included, evaluating 245 patients and 402 dental implants. The average patient age was 43.7 years, with a mean follow-up period of 34.3 months. Flap survival was high, with a 98.3% success rate and only four flap losses. The implant failure rate was low at 1.74%. Esthetic outcomes were varied, with only three studies using standardized protocols for evaluation. The overall certainty of evidence for flap survival was moderate, low for implant failure, and very low for aesthetics due to the subjective nature of assessments and variability in reporting. Conclusions: The primary limitations of the evidence included in this review are the observational design of the studies, leading to an inherent risk of bias, inconsistency in reporting methods, and imprecision in outcome measures. Additionally, the subjective nature of aesthetic evaluations and the variability in assessment tools further limit the reliability of the findings. The DBFF technique demonstrates excellent outcomes for mandibular reconstruction, with high flap survival and low implant failure rates, making it a viable option for dental rehabilitation. However, the evidence for aesthetic outcomes is less certain, highlighting the need for more rigorous and standardized research. This review supports the DBFF as a good alternative for mandibular reconstruction with successful dental implant integration, although further studies are needed to enhance the reliability of aesthetic evaluations.
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Dopamine critically influences reward processing, sensory perception, and motor control. Yet, the modulation of dopaminergic signaling by sensory experiences is not fully delineated. Here, by manipulating sensory experience using bilateral single-row whisker deprivation, we demonstrated that gene transcription in the dopaminergic signaling pathway (DSP) undergoes experience-dependent plasticity in both granular and supragranular layers of the primary somatosensory (barrel) cortex (S1). Sensory experience and deprivation compete for the regulation of DSP transcription across neighboring cortical columns, and sensory deprivation-induced changes in DSP are topographically constrained. These changes in DSP extend beyond cortical map plasticity and influence neuronal information processing. Pharmacological regulation of D2 receptors, a key component of DSP, revealed that D2 receptor activation suppresses excitatory neuronal excitability, hyperpolarizes the action potential threshold, and reduces the instantaneous firing rate. These findings suggest that the dopaminergic drive originating from midbrain dopaminergic neurons, targeting the sensory cortex, is subject to experience-dependent regulation and might create a regulatory feedback loop for modulating sensory processing. Finally, using topological gene network analysis and mutual information, we identify the molecular hubs of experience-dependent plasticity of DSP. These findings provide new insights into the mechanisms by which sensory experience shapes dopaminergic signaling in the brain and might help unravel the sensory deficits observed after dopamine depletion.
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Dopamina , Plasticidad Neuronal , Transducción de Señal , Corteza Somatosensorial , Corteza Somatosensorial/metabolismo , Corteza Somatosensorial/fisiología , Animales , Transducción de Señal/fisiología , Dopamina/metabolismo , Plasticidad Neuronal/fisiología , Neuronas Dopaminérgicas/fisiología , Neuronas Dopaminérgicas/metabolismo , Vibrisas/fisiología , Receptores de Dopamina D2/metabolismo , Privación Sensorial/fisiología , Ratones , MasculinoRESUMEN
OBJECTIVE: Chronic constriction injury (CCI) of the infraorbital nerve induces neuropathic pain, such as allodynia and hyperalgesia, in the orofacial area. However, the changes in the local circuits of the central nervous system following CCI remain unclear. This study aimed to identify the changes following CCI in Thy1-GCaMP6s transgenic mice. METHODS: Neural activity in the primary somatosensory cortex (S1) and motor cortex (M1) following whisker stimulation was assessed using in vivo Ca2+ imaging. CCI-induced changes in responses were analyzed. RESULTS: Before CCI, whisker stimulation induced a greater Ca2+ response in the contralateral S1 than in the ipsilateral S1 and contralateral M1. The peak Ca2+ response amplitude in the bilateral S1 and contralateral M1 decreased two days after CCI compared to before CCI. Decreased Ca2+ response amplitude in these regions was observed until four days after CCI. Seven days after CCI, the Ca2+ response amplitude in the contralateral S1 decreased, whereas that in the ipsilateral S1 and contralateral M1 recovered to control levels. CONCLUSION: These results suggest that neural activity in regions receiving excitatory inputs via corticocortical pathways recovers earlier than in regions receiving thalamocortical inputs. (185/250 words).
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Calcio , Modelos Animales de Enfermedad , Ratones Transgénicos , Corteza Motora , Corteza Somatosensorial , Vibrisas , Animales , Corteza Motora/fisiopatología , Corteza Somatosensorial/fisiopatología , Vibrisas/inervación , Vibrisas/fisiología , Ratones , Calcio/metabolismo , Masculino , Neuralgia/fisiopatología , Neuralgia del Trigémino/fisiopatología , Neuralgia del Trigémino/metabolismoRESUMEN
For neural circuit construction in the brain, coarse neuronal connections are assembled prenatally following genetic programs, being reorganized postnatally by activity-dependent mechanisms to implement area-specific computational functions. Activity-dependent dendrite patterning is a critical component of neural circuit reorganization, whereby individual neurons rearrange and optimize their presynaptic partners. In the rodent primary somatosensory cortex (barrel cortex), driven by thalamocortical inputs, layer 4 (L4) excitatory neurons extensively remodel their basal dendrites at neonatal stages to ensure specific responses of barrels to the corresponding individual whiskers. This feature of barrel cortex L4 neurons makes them an excellent model, significantly contributing to unveiling the activity-dependent nature of dendrite patterning and circuit reorganization. In this review, we summarize recent advances in our understanding of the activity-dependent mechanisms underlying dendrite patterning. Our focus lays on the mechanisms revealed by in vivo time-lapse imaging, and the role of activity-dependent Golgi apparatus polarity regulation in dendrite patterning. We also discuss the type of neuronal activity that could contribute to dendrite patterning and hence connectivity.
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Dendritas , Corteza Somatosensorial , Vibrisas , Animales , Dendritas/fisiología , Corteza Somatosensorial/fisiología , Corteza Somatosensorial/crecimiento & desarrollo , Corteza Somatosensorial/citología , Vibrisas/fisiología , Animales Recién NacidosRESUMEN
Neocortex is a complex structure with different cortical sublayers and regions. However, the precise positioning of cortical regions can be challenging due to the absence of distinct landmarks without special preparation. To address this challenge, we developed a cytoarchitectonic landmark identification pipeline. The fluorescence micro-optical sectioning tomography method was employed to image the whole mouse brain stained by general fluorescent nucleotide dye. A fast 3D convolution network was subsequently utilized to segment neuronal somas in entire neocortex. By approach, the cortical cytoarchitectonic profile and the neuronal morphology were analyzed in 3D, eliminating the influence of section angle. And the distribution maps were generated that visualized the number of neurons across diverse morphological types, revealing the cytoarchitectonic landscape which characterizes the landmarks of cortical regions, especially the typical signal pattern of barrel cortex. Furthermore, the cortical regions of various ages were aligned using the generated cytoarchitectonic landmarks suggesting the structural changes of barrel cortex during the aging process. Moreover, we observed the spatiotemporally gradient distributions of spindly neurons, concentrated in the deep layer of primary visual area, with their proportion decreased over time. These findings could improve structural understanding of neocortex, paving the way for further exploration with this method.
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Aprendizaje Profundo , Neocórtex , Neuronas , Animales , Neocórtex/citología , Ratones , Ratones Endogámicos C57BL , Masculino , Imagenología Tridimensional/métodos , Tomografía Óptica/métodosRESUMEN
Rapid eye movement (REM) sleep, also referred to as paradoxical sleep for the striking resemblance of its electroencephalogram (EEG) to the one observed in wakefulness, is characterized by the occurrence of transient events such as limb twitches or facial and rapid eye movements. Here, we investigated the local activity of the primary somatosensory or barrel cortex (S1) in naturally sleeping head-fixed male mice during REM. Through local field potential recordings, we uncovered local appearances of spindle waves in the barrel cortex during REM concomitant with strong delta power, challenging the view of a wakefulness-like activity in REM. We further performed extra- and intracellular recordings of thalamic cells in head-fixed mice. Our data show high-frequency thalamic bursts of spikes and subthreshold spindle oscillations in approximately half of the neurons of the ventral posterior medial nucleus which further confirmed the thalamic origin of local cortical spindles in S1 in REM. Cortical spindle oscillations were suppressed, while thalamus spike firing increased, associated with rapid mouse whisker movements and S1 cortical activity transitioned to an activated state. During REM, the sensory thalamus and barrel cortex therefore alternate between high (wake-like) and low (non-REM sleep-like) activation states, potentially providing a neuronal substrate for mnemonic processes occurring during this paradoxical sleep stage.
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Electroencefalografía , Sueño REM , Corteza Somatosensorial , Tálamo , Animales , Ratones , Sueño REM/fisiología , Corteza Somatosensorial/fisiología , Masculino , Tálamo/fisiología , Ratones Endogámicos C57BL , Vibrisas/fisiología , Vibrisas/inervación , Vigilia/fisiología , Vías Nerviosas/fisiologíaRESUMEN
De novo protein design expands the protein universe by creating new sequences to accomplish tailor-made enzymes in the future. A promising topology to implement diverse enzyme functions is the ubiquitous TIM-barrel fold. Since the initial de novo design of an idealized four-fold symmetric TIM barrel, the family of de novo TIM barrels is expanding rapidly. Despite this and in contrast to natural TIM barrels, these novel proteins lack cavities and structural elements essential for the incorporation of binding sites or enzymatic functions. In this work, we diversified a de novo TIM barrel by extending multiple ßα-loops using constrained hallucination. Experimentally tested designs were found to be soluble upon expression in Escherichia coli and well-behaved. Biochemical characterization and crystal structures revealed successful extensions with defined α-helical structures. These diversified de novo TIM barrels provide a framework to explore a broad spectrum of functions based on the potential of natural TIM barrels.
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Modelos Moleculares , Escherichia coli/genética , Escherichia coli/metabolismo , Cristalografía por Rayos X , Pliegue de Proteína , Ingeniería de Proteínas/métodos , Proteínas/química , Proteínas/metabolismoRESUMEN
The biogenesis of outer membrane proteins is mediated by the ß-barrel assembly machinery (BAM), which is a heteropentomeric complex composed of five proteins named BamA-E in Escherichia coli. Despite great progress in the BAM structural analysis, the molecular details of BAM-mediated processes as well as the exact function of each BAM component during OMP assembly are still not fully understood. To enable a distinguishment of the function of each BAM component, it is the aim of the present work to examine and identify the effective minimum form of the E. coli BAM complex by use of a well-defined reconstitution strategy based on a previously developed versatile assay. Our data demonstrate that BamADE is the core BAM component and constitutes a minimum functional form for OMP assembly in E. coli, which can be stimulated by BamB and BamC. While BamB and BamC have a redundant function based on the minimum form, both together seem to cooperate with each other to substitute for the function of the missing BamD or BamE. Moreover, the BamAE470K mutant also requires the function of BamD and BamE to assemble OMPs in vitro, which vice verse suggests that BamADE are the effective minimum functional form of the E. coli BAM complex.
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Proteínas de la Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genéticaRESUMEN
Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a ß-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the ß-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods. In addition, we describe recent evidence that molecular machines that were thought to function independently might interact to form dynamic intermembrane supercomplexes. Finally, we discuss new results that suggest that OMPs are inserted primarily near the middle of the cell and packed into supramolecular structures (OMP islands) that are distributed throughout the OM.